WO2001078748A2 - Immunomodulatory methods using carbohydrate antigens - Google Patents

Abstract

Methods for modulating immune responses, such as IgE responses and autoimmune responses, are provided. The methods involve contacting a cell with an agent comprising multivalent lacto-N-neotetraose (LNnT), which modulates an immune response. The methods are useful for enhancing production of non-specific polyclonal IgE, inhibiting production of antigen-specific IgE responses, inducing cytokine production, and stimulating proliferation of splenocytes. In a preferred embodiment, the invention provides methods for modulating an immune response to an antigen (e.g., an allergan) in vivo. Pharmaceutical compositions for modulating immune responses comprising the agents of the invention are also provided. The invention also provides for treatment or prevention of shock in a subject using multivalent LNnT and methods for treating an autoimmune disease in a subject using multivalent LNnT. Still further the invention provides for treatment of cancer in a subject using monovalent LNnT.

Description

IMMUNOMODULATORY METHODS USING CARBOHYDRATE ANTIGENS

Background of the Invention

Helminth infection is strongly associated with the production of large amounts of serum IgE in humans and experimental animals (King, CL. et al., (1993), J. Immunol, vol. 150 pp. 1873-1880; Ogilvie BM, Nature 1964 204. 91-92; Sadun, EH. et al. (1970) Exp. Parasitol. vol. 28 pp. 435-449). The majority of serum IgE in infected host is nonspecific and polyclonal (Dessaint, JP. et al. (1975) Clin. Exp. Immunol, vol. 20 pp. 427-436; Jarret, et al. (1976) Clin. Exp. Immunol, vol. 24 pp. 346-351). Such IgE has several roles in immunity against helminth parasites. Ag-specific IgE, especially anti- adult worm Ag, is involved with the resistance to reinfection (Hagan, P. et al. (1991) Nature vol. 349 pp. 243-245; Viana, J-RC. et al. (1995) Parasite Immunol, vol. 17 pp. 297-304), and cell-mediated cytotoxicity against parasites (Capron, A. et al. (1980) Am. J. Trop. Med. Hyg. vol. 29 pp. 849-857; Gounni, AS. et al. (1994) Nature vol. 367 pp. 183-186; Cutts, L. et al. (1997) Parasite Immunol, vol. 19 pp. 91-102). On the other hand, non-specific, polyclonal IgE seems to be beneficial for both host and parasites to reduce the risk of the potentially lethal anaphylaxic reaction to parasitic antigens by saturating the available Fc_εRs on effector cells, although IgE plays a detrimental role for the host in primary infection of schistosomiasis (Hagan, P. (1993) Parasite Immunol. vol. 15 p. 14; Pritchard, DL (1993) Parasite Immunol, vol. 15 pp. 5-9; Amiri, P. et al. (1993) J. Exp. Med. vol.180 pp. 43-51).

Several allergic components in schistosomal Ag have been reported to react with IgE from humans and rodents at various stages of infections. (Damonneville, M. et al. (1984) Int. Arch. Allergy appl. Immunol, vol. 73 pp. 248-255; Owhashi, M. et al. (1986) Int. Arch.

Allergy appl. Immunol, vol. 81 pp. 129-135). However little is known about the molecules that induce polyclonal IgE production from host. Toxocara canis adult worm antigen has B cell mitogenic activity to induce proliferation, IgG and IgE production although the allergenic molecule remains undefined (Wang, MQ. et al. (1995) Parasite Immunol, vol. 17 pp. 609- - 2 - HUI-038CPPC

615Namashita, U. et al. (1993) Jap. J. Parasitol. vol. 42 pp. 211-219). Ascaris body fluid also contains a B cell mitogen, however, this itogen needs the help of IL4 delivered by the other factors to induce polyclonal IgE production (McGibbon, AM. et al. (1990) Mol. Biochem. Parasitol. vol. 39 pp. 163-172; Lee, TDG. et al. (1995) J. Allergy Clin. Immunol. vol. 95 pp. 124-1254.Lee, TDG. et al. (1993) Int. Arch. Allergy Immunol, vol. 102 pp. 185- 190). Two recombinant filarial proteins are capable of inducing polyclonal IgE production in vitro, however, they also induce Ag-specific IgE (Garrud, O. et al. (1995) J. Immunol, vol. 155 pp. 131-1325).

Schistosoma mansoni synthesize glycoproteins containing polylactosamine sugars (Srivastan, J. et al. (1992) J. Biol. Chem. vol. 267 pp. 14730-14737; Nan Dam, GJ. et al.

(1994) Eur. J. Biochem. vol. 225 pp. 467-482). Lacto-Ν-fucopentaose m (LΝFJJI) found on adult worm and egg of S. mansoni has been found to be an antigenic determinant (KO, AL et al. (1990) Proc. Natl. Acad. Sci. USA vol. 87 pp. 4159-4163). LNFIH stimulates splenic B cells from parasite-infected mice to proliferate and produce J-L-10, a cytokine that downregulates Thl immune responses (W Nelupillai, P. et al. Proc. Natl. Acad. Sci. USA vol. 91 pp. 18-22; Palanivel, N. et al. (1996) Exp. Parasitol. vol. 84 pp. 168-177; Nelupillai, P. et al. (1997) J. Immunol, vol. 158 pp. 338-344). In addition, this sugar has been found to have an adjuvant effect in inducing Th2 immune reaction in both Ab and cytokine production against protein with which the sugar is physically conjugated. Additional insight into molecules which bias the immune response towards specific cytokine profiles will be important in developing methods of regulating immune responses. In particular, identification of molecules which induce polyclonal IgΕ synthesis will be of tremendous benefit in treating allergy.

Summary of the Invention

The instant invention provides novel methods of regulating the immune response. The invention is based, at least in part, on the discovery of the functional characteristics of LΝnT, a nonfucosylated homologue of LΝFHt, that is converted to LΝFIH by αl-3 fucosyltransf erase in adult worms. Multivalent LΝnT induces polyclonal IgΕ production and - 3 - HUI-038CPPC

cytokine production (e.g., IL-10, JJ -5, IL-4, IL-13 and TGF-β) from mice by JP inoculation. This molecule can be used to modulate the IgE response, not only to parasite antigens, but to environmental allergens by saturating Fcεreceptors on the effector cells.

The invention further pertains to other uses of LNnT, either in a multivalent form or as a free sugar (i.e., monovalent form), in the modulation of immune responses. For example, multivalent LNnT (e.g., LNnT conjugated to dextran) has been found to render spleen cells less responsive to lipopolysaccharide (LPS) and Con-A (e.g., as measured by spleen cell proliferation and the production of the Thl -associated cytokines interleukin-12 (J-L-12), interleukin-18 (IL-18) and interferon-gamma (IFN-γ)). Accordingly, multivalent LNnT can be used to protect a subject from the effects of shock (e.g., toxic shock), for example as a pretreatment in subjects susceptible to shock (e.g., surgery patients at risk for shock). Moreover, the ability of multivalent LNnT to downmodulate Thl-associated cytokines allows for use of such agents in any clinical setting in which it is desireable to downmodulate type 1 immune responses, such as in autoimmune diseases (including inflammatory bowel disease, diabetes and rheumatoid arthritis).

Furthermore, multivalent LNnT has been found to induce a population of suppressor cells that are Grl+, CDl lb+. These suppressor cells, which also can be induced by sugars expressed on tumor cells, are able to suppress T cell proliferative responses and furthermore can make cytokines that may downregulate immune responses or that may promote angiogenesis (such as TGFβ), Thus, in the context of tumor treatment, it would be desirable to inhibit the induction of this population of suppressor cells. The invention provides for the use of free (i.e., unconjugated, monovalent) LNnT to inhibit the generation of these Grl+, CDl lb+ suppressor cells, for example in the treatment of cancer. In contrast, in other clinical settings it may be desireable to induce this Grl+, CDllb+ suppressor cell population, for example in situations where one wants to downmodulate immune responses, such as in autoimmune diseases (e.g., inflammatory bowel disease, diabetes, rheumatoid arthritis, allergic asthma, multiple sclerosis). Accordingly, other embodiments of the invention provide for use of multivalent LNnT to induce (e.g., stimulate, recruit) the generation of Grl+, CDllb+ suppressor cells, for example in the treatment of autoimmune diseases, and - 4 - HUI-038CPPC

other contexts wherein suppression of the immune system is desireable (e.g., transplantation, allergy)

Brief Description of the Drawings

Figures la-lc: Serum Ig production in BALB/C mice following JP immunization. Figure 1(a): Female BALB/C mice were JP immunized with saline, HSA, Le^v-HSA, LNnT- HSA or HSA- Alum. All the antigens were inoculated at the dose of 10 μg protein weight of HSA. 6 and 5 days following first (dotted bar) and second (closed bar) boosting immunization, respectively, blood samples were taken and serum total IgE was measured. Results shown are the mean + 1 SE of four individual serum. Figure 1(b): Course of serum total IgE following JP immunization. BALB/C mice were JP immunized with saline (open square), HSA (lOμg; open triangle) or LNnT-HSA (10 μg of HSA; closed circle) at day 0. Two weeks later, the boosting immunization was followed in a same fashion. Blood was taken at day 10, 20, 27, 41, and 69, and serum total IgE was determined. Results shown are the mean + 1 SE of four individual serum. Figure 1(c): Serum IgG isotypes 5 days following third JP immunization with saline (dotted bar), HSA (open bar), or LNnT-HSA (closed bar). Results shown are the mean + 1 SE of four individual serum. All the results are representative of three experiments.

Figures 2a-2d Antigen-specific Ab production following JP immunization with multivalent sugars. Female BALB/C mice were immunized and bled as described in Fig. 1. Following first (dotted bar) and second (closed bar) immunization, HSA-specific IgE (Figure 2a), HSA-specific IgG (Figure 2b), LNnT-HSA-specific IgE (Figure 2c), and LNnT-HSA- specific IgG (Figure 2d) were measured by ELISA as described in materials and methods. Specific IgE were determined as the absorbance at 450 nm of sera diluted 4 times. Specific IgG were determined as the endpoint titer. Results shown are the mean + 1 SE of four individual serum. Results are representative of three experiments. - 5 - HUI-038CPPC

Figures 3a-3d: Serum total IgE in CBA/J (Figure 3a) and C57BL/6 (Figure 3b) mice following JP immunization with saline, HSA or LNnT-HSA. 6 and 5 days following first (dotted bar) and second (closed bar) boosting immunization, respectively, blood samples were taken and serum total IgE were determined. Results shown are the mean + 1 SE of four individual serum. Figure 3c: Course of serum total IgE following SC immunization.

BALB/C mice were SC immunized with saline (open square), HSA (10 μg; open triangle) or LNnT-HSA (10 μg of HSA; closed circle) at day 0. Two weeks later, the boosting immunization was followed in a same fashion. Blood was taken at day 10, 20, 27, 41, and 69, and serum total IgE was determined. Results shown are the mean + 1 SE of four individual serum. Figure 3D: Serum total IgE in CBA/CaJ (open bar) and CBA/CaJ xid (hatched bar) following second boosting JP immunization with saline, HSA or LNnT-HSA. Results shown are the mean + 1 SE of four individual serum. Results are representative of two experiments.

Figure 4 Proliferative responses of splenocytes. BALB/C mice were JP immunized with saline, HSA ( 10 μg) or LNnT-HSA ( 10 μg weight of HSA). 2 and 3 weeks later, boosting immunization was done in the same fashion. 5 days following the final immunization, spleens were removed. 2.5 x lO^{0^} splenocytes were stimulated with ConA (2 μg/ml; dotted bar), HSA (lOμg/ml; hatched bar), LNnT-HSA ( lOμg/ml of HSA; closed bar) or no restimulation (open bar). Results shown are the mean cpm (experimental-medium control) + 1 SE of four individual mice per group. Results are representative of three experiments.

Figures 5a-5h: B7-1 and B7-2 expression on B220+ cells. Splenocytes from mice JP immunized with saline (Figures 5a, 5d), HSA (Figures 5b, 5e), or LNnT-HSA (Figures 5c, 5f) were incubated for 24 hrs without additional stimulants. Cell pellets were stained with PE- conjugated mAb against either B7-1 (Figures 5a, 5b, 5c) or B7-2 (Figures 5d, 5e, 5f). Numbers expressed in upper right quadrant show the mean + 1SE of percentage of cells expressing both B220 and B7 molecules from six individual sample. Figures 5g, 5h: Effect - 6 - HUI-038CPPC

of the duration of culture incubation on B7 expression in mice JP immunized with saline (open square), HSA (open triangle) or LNnT-HSA (closed circle). Percentage of B220+ cells expressing B7-1 (Figure 5g) or B7-2 (Figure 5h) were monitored at preincubation, 24, 72, and 120 hrs postincubation without restimulation. Results were representative for two experiments.

Figure 6: Levels of total IgE (ng/ml) in mice treated two (open bars) or three (closed bars) times intraperitoneally with the LNnT-dextran conjugate LNnT35.

Figure 7: Levels of total IgE (ng/ml) in mice treated first with RMPI (saline), ovalbumin, the LNnT-dextran conjugate LNnT45, or dextran followed by booster treatment with ovalbumin.

Figure 8: Levels of ovalbumin-specific IgE in mice treated first with RMPI (saline), ovalbumin, the LNnT-dextran conjugate LNnT45, or dextran followed by booster treatment with ovalbumin.

Figure 9: Levels of total IgE (ng/ml) over time (up to 70 days post-immunization) in mice treated with either saline, HSA or LNnT-HSA.

Figures lOa-lOc: Levels of Th2-type cytokine production by total spleen cells of mice immunized with vehicle (dextran) or LNnT-dextran conjugate. Figure 10a shows JL-13 levels (pg/ml). Figure 10b shows JL-4 levels (pg/ml). Figure 10c shows JL-10 levels (pg/ml).

Figure 11: Level of production of the Thl -type cytokine interferon-gamma (pg/ml) by total spleen cells of mice immunized with vehicle (dextran) or LNnT-dextran conjugate. - 7 - HUI-038CPPC

Figure 12: Proliferative responses of total spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with LPS in vitro.

Figure 13: Level of production of the Thl -type cytokine interferon-gamma (pg/ml) by spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with Con A in vitro.

Figure 14: Level of production of the Thl-type cytokine JL-12 (pg/ml) by spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with LPS or LNnT-dextran in vitro.

Figure 15: Level of production of the Th2-type cytokine JL-13 (pg/ml) by spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with Con A or LNnT-dextran in vitro.

Figure 16: Level of production of the Th2-type cytokine JL-13 (pg/ml) by CD4+ cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with Con A in vitro.

Figure 17: Proliferative responses of naive spleen cells stimulated with anti-CD3 in the presence of peritoneal exudate cells (PECs) from mice treated with vehicle (Dex) or LNnT-dex conjugate (LNnT) or in the presence of PECs from LNnT-dex injected mice which had the Grl+ cells removed from the PEC population (LNnT(-)).

Figure 18: Proliferative responses of CD4+ cells from Balb/c mice stimulated with anti-CD3 in the presence of peritoneal exudate cells (PECs) from mice treated with vehicle (Dex), saline (control), or LNnT-dex conjugate (LNnT) or Grl+ enriched PECs (Grl+) from LNnT-dex injected mice. - 8 - HU1-038CPPC

Figure 19: LNnT-Dex injection recruits greater numbers of peritoneal cells expressing

Gr-1+/CD1 lb+ F4/80+ surface markers, (a) Number of PECs recruited at different times after

a single LNnT-Dex injection, (b) FACS analysis of PECs recruited by LNnT-Dex, dextran or

saline. Mice injected with LNnT-Dex showed an increase in the markers for Gr-l/CDl lb and

F4/80. No changes are observed in B7-2, CD40 and CDl lc (data not shown) between the

different groups. PECs were obtained at different times post-sugar injection, analysis was

performed in individual mice, 4 mice per group. Data are representative of five separate,

independently performed experiments with BALB/c and C57BL/6 strains.

Figure 20: PECs recruited by LNnT-Dex inhibit the proliferative response of naive

CD4 cells stimulated with plate-bound anti-CD3/CD28 antibodies, (a) PECs from C57BL/6

mice were obtained 2h or (b) from BALB/c mice 18h post-injection of LNnT-Dex or dextran, co-cultured at different ratios with 1X10⁵ previously stimulated naive CD4⁺ cells. 72 h later

H-Thymidine was added to the cultures and after 18h cells were harvested and processed for

radioactivity uptake. Individual mice were assayed and data are shown as mean + SE. Results

are representative of four independent experiments performed with BALB/c and C57BL/6

strain of mice at both time points. * p<0.05.

Figure 21; Depletion of Gr-1⁺ cells from PECs recruited by LNnT-Dex restores the

proliferative response to anti-CD3/CD28 antibodies in naive CD4⁺ cells. PECs recruited by

LNnT-Dex or dextran were harvested, pooled and depleted of Grl⁺ cells by MACS and added

in a ratio 1:2 to naive CD4⁺ cells previously stimulated with anti-CD3/CD28. Cells were co-

cultured for 72h and incorporation of ³H-Thymidine was measured. Results are the average of triplicates and are representative of three separate experiments performed in BALB/c and - 9 - HUI-038CPPC

C57BL/6 mice. * p< 0.05.

Figure 22: Proliferative inhibition by PECs recruited by LNnT-Dex can be mediated

by both cell to cell contact and by soluble factors. PECs harvested at 2h or 18h post-sugar

injection either fixed with 0.5% of paraformaldehyde before being directly co-cultured with

CD4 naive cells (a) or placed in a separate transwell plate separated by a 0.4 μm membrane

(b). PECs were obtained from individual mice and triplicate wells set up. Proliferation was

measured by H-Thymidine uptake. Data are shown as mean + SE from 4 animals per group

and are representative of three independent experiments. * p<0.05.

Figure 23: PECs recruited by LNnT-Dex modify the cytokine profile of CD4⁺ cells

stimulated with anti-CD3/CD28 antibodies. PECs from animals injected with LNnT-Dex or

dextran were co-cultured with CD4⁺ naive cells, after 72h the J-FN-γ (a) and IL-13 production

(b) were assayed by ELISA.

Figure 24: Suppressive PECs recruited by LNnT-Dex commit CD4 cells to produce

more JL-13 in a secondary stimulation. CD4 naive cells were previously stimulated with plate

bound anti-CD3/CD28 and 3 h later PECs (ratio 1:4) recruited by LNnT-Dex or dextran were

added to cultures in the absence (a-b) or presence (c-d) of JL-12 (50 ng/ml). After 3 days,

primed CD4 cells were washed, re-purified, rested for 3 more days and re-stimulated with

anti-CD3/CD28 antibodies. 24 h after secondary stimulation supernatants were assayed for

J-FN-γ(a-c) and JL-13 (c-d) production. Data are representative of three experiments

performed in BALB/c mice, and show mean + SE of triplicate cultures of 4 mice assayed individually. *p<0.05. - 10 - HUI-038CPPC

Figure 25: JL-10 is involved in the soluble-mediated suppressive activity of PECs

recruited by LNnT-Dex. Co-cultures of PECs and CD4⁺ (1 :4 ratio) were performed as

described in material and methods and neutralizing monoclonal anti-JL-10 or anti-TGF-β

antibodies were added to the in vitro co-cultures. After 72 h proliferation was measured by

³H-Thymidine uptake. Data are representative of three experiments performed in BALB/c

mice, and show mean + SE of triplicate cultures of 4 mice assayed individually. *p<0.05

compared to PECs-dextran.

Figure 26: LNFPIJI-Dex expands Grl⁺ cells in the peritoneal cavity of BALB/c mice.

Twenty hours after being injected with Dextran or LNFPJH-Dex, peritoneal cells were

harvested by Saline lavage. A) Cells were stained with antibodies to CDl lb and Grl. B) Cells

were stained with antibodies to F4/80 and Grl. Q In the left hand panel, CDl lb/F4/80

double positive cells were gated (R2); the right hand panel shows a histogram of the % Grl⁺ cells of the R2 gated population. The plots shown in this figure are representative of 7

different experiments. Arrow indicates presence of Grl ^lgh cells.

Figure 27: PECs from LNFPJJ -Dex injected mice suppress proliferation of naive

CD4⁺ T cells. Naive splenocytes were labeled with CFSE dye (as in Materials and Methods)

then plated (0.5xl0⁶/well) onto 48-well plates coated with antibodies to CD3 (1 μg/ml) and

CD28 (5 μg/ml). Three hours later, PECs from control or LNFPIJI-Dex injected mice were

added at the indicated ratios to the wells containing the splenocytes. Cells were harvested

after 72 hours of co-culture and stained for CD4. During FACScan analysis, gating was on

the CD4⁺ population of cells. Data are representative of 5 separate experiments.

Figure 28: Nitric oxide (NO) production is associated with suppression of splenocyte - 11 - HUI-038CPPC proliferation by PECs from LNFPUI-Dex injected mice. A) iNos inhibition by L-nMMA

abrogates PEC suppression of naive splenocytes. Left hand panel shows CFSE fluorescence

intensity in cultures without L-nMMA, and the right hand panel shows cultures with L-

nMMA (0.5 mM). B) Levels of NO produced in co-cultures with or without L-nMMA.

Figure 29. IFN-γ is required for PEC suppression of T cell proliferation. Histograms

show CFSE fluorescence intensity in T cells from co-cultures containing isotype control

antibodies (left) or antibodies to IFN-γ (10 μg/ml, right). Data are representative of three

separate experiments.

Figure 30: The Grl^4" subpopulation of F4/80⁺ PECs is responsible for T cell

suppression in an IFN-γ dependent mechanism. A) The Grl⁺/F4/80⁺ and the Grl^low F4/80⁺

PEC populations shown in the left and right hand panels, respectively, were purified as

described in materials in methods. B) CFSE fluorescence intensity of T cells co-cultured with

total PECs (left hand panel), F4/80⁺/Grl⁺ PECs (center), or F4/807Grl^lo PECs (right). Q

Suppression of co-cultured T cells by F4/80⁺/Grl⁺ PECs is dependent on J-FN-γ. Data are

representative of three separate experiments.

Figure 31 : LNFPUI-Dex drives an NO-dependent suppressor PEC population in SCUD

mice. Histograms show CFSE fluorescence intensity in T cells co-cultured with PECs from

control mice (left hand panel), LNFPUI-Dex injected mice (center), and LNFPJU-Dex

injected mice in the presence of L-nMMA (right). Data shown are representative of 3 separate experiments. - 12 - HUI-038CPPC

Detailed Description of the Invention

This invention provides immunomodulatory methods in which a cell (e.g., a human immune cell) is contacted with an agent which modulates an immune response (e.g., nonspecific polyclonal IgE production, immune cell mitogenesis, or production by the cell of one or more cytokines). The invention is based, at least in part, on the discovery that when animals are immunized with multivalent lacto-N-neotetraose (LNnT), a carbohydrate that is putatively expressed on helminth parasite Schistosoma mansosi, both BALB/C and CB A J mice produced significantly higher amounts of total serum IgE following two intraperitoneal (JP) immunizations with multivalent LNnT conjugated to human serum albumin (LNnT- HSA) compared to groups immunized with saline or HSA alone. Interestingly, no specific IgE against the carbohydrate or the carrier protein was detected in ELISA, suggesting an induction of polyclonal nonspecific IgE production in vivo by multivalent LNnT. Moreover, this neo-glycoprotein did not promote significant production of IgG against either the carbohydrate or the carrier protein. C57BL/6 mice only showed the elevation of serum total IgE after three times immunization with LNnT-HSA, reflecting strain-dependent reactions against LNnT-HSA. Spleen cells from mice JP immunized with LNnT-HSA produced in vitro significant amount of JL-4, JL-5, and JL-10 as well as JL-2 and J-FN-γ compared to controls. In addition, cultured B220+ cells had increased expression of B7-2 (CD86) but not B7- 1 (CD80), suggesting that B7-2 expression is strongly associated with polyclonal production of IgE. Further, JL-4 gene deficient BALB/C mice did not produce polyclonal IgE following JP immunization with LNnT-HSA. Spleen cells from these mice produced lower but significant amounts of JL-5 and JL-10, and same amounts of IFN-γ compared to the wild type, demonstrating that JL-4 is critical for promoting polyclonal IgE production induced by the multivalent carbohydrate naturally expressed on helminth parasite. Additional experiments demonstrated that LNnT conjugated to dextran also stimulates polyclonal IgE responses and that pretreatment with multivalent LNnT (e.g., LNnT conjugated to dextran), prior to immunization with an antigen, inhibits the production of antigen-specific IgE responses. The enhanced levels of polyclonal IgE stimulated by multivalent LNnT are persistent (e.g., sustained for at least 70 days). In addition to JL-4, the - 13 - HUI-038CPPC

production of Th2-type cytokines JL-10 and IL-13 is stimulated by treatment with LNnT conjugated to dextran, whereas levels of the Thl-type cytokine interferon gamma are inhibited by treatment with LNnT conjugated to dextran.

Thus, the methods of the invention allow for IgE production to be modulated (e.g., stimulation of non-specific IgE and/or inhibition of antigen-specific IgE), as well as allowing for modulation of cytokine production. Accordingly, the immunomodulatory methods of the invention allow for an immune response to be biased towards a specific cytokine secretion profile, for example, a Th2 response. The ability to influence the production of non-specific, polyclonal IgE using the immunomodulatory methods of the invention can be used in the prevention of detrimental host reaction against parasite infection and is further applicable to the protection against environmental allergens by saturating FcεRs on effector cells. Moreover, the ability to influence the development of, for example, a Th2 response using the immunomodulatory methods of the invention is applicable to the treatment of a wide variety of disorders, including cancer, infectious diseases (e.g., HJN and tuberculosis), allergies and autoimmune diseases.

Another aspect of the invention pertains to use of multivalent LΝnT is the treatment or prevention of shock. As demonstrated in Example 3, administration of multivalent LΝnT (e.g., LΝnT conjugated to dextran) renders spleen cells less responsive to LPS, as measured by proliferative responses and production of type 1 cytokines such as JL-12 and J-FΝ-γ. Thus, multivalent LΝnT treatment can be used to inhibit the shock response in a patient, either in a patient suffering from shock or, more preferably, in a patient at risk of (susceptible to) shock, such as a surgery patient who is susceptible to shock. For at risk patients, these patients can be pretreated with a multivalent LΝnT composition of the invention to render them less susceptible to shock. Yet another aspect of the invention pertains to methods for inhibiting the induction of suppressor cells, in particular a population of Grl+, CDl lb+ suppressor cells, by blocking their induction using monovalent (free, unconjugated) LΝnT. As demonstrated in Example 4, administration of multivalent LΝnT induces this suppressor cell population, which is capable of inhibiting T cell proliferative responses. This suppressor cell population may be induced - 14 - HUI-038CPPC

in a clinical setting in a patient by tumor cells that express multivalent LNnT, and the induction of such a suppressor population could promote tumor growth and expansion, by suppression of immune responses against the tumor and/or by production by the suppressor population of factors (such as TGF-β) that promote tumor angiogenesis. Accordingly, inhibition of the induction of this suppressor population can be used in the treatment of cancer, by adminstration of monovalent LNnT to thereby competitively block the induction of the suppressor population.

Ln order that the present invention may be more readily understood, certain terms are first defined. Standard abbreviations for sugars are used herein. As used herein, the term " lacto-N-neotetraose" ("LNnT") is intended to refer to a polylactosamine sugar which is a non-fucosylated homologue of lacto-N-fucopentaose UI, which is found at least on the parasite Schistosoma mansoni.

As used herein, the term "multivalent lacto-N-neotetraose" (multivalent LNnT) is intended to refer to a form of LNnT comprising multiple moieties of the carbohydrate, such as a form in which multiple LNnT carbohydrates are conjugated to a carrier molecule.

As used herein, the term "monovalent lacto-N-neotetraose" (monovalent LNnT) is intended to refer to a form of LNnT comprising a single moiety of the carbohydrate, such as a free, unconjugated form of the sugar.

As used herein, the term "agent comprising LNnT" is intended to refer to a molecule or molecules that includes the LNnT carbohydrate moiety. In certain embodiments, the agent comprises LNnT with the proviso that the agent is not an antigen from S. mansoni, or an antigen from Toxocara canis, or is not Ascaris body fluid, or is not a filarial protein capable of inducing polyclonal IgE.

As used herein, the term "LewisY oligosaccharide" refers to a lacto type U carbohydrate comprising the structure: {Fuc( l-2)Gal(βl-4)[Fuc(αl-3)]GlcNac}.

As used herein, the term "human immune cell" is intended to include cells of the human immune system which are capable of producing cytokines. Examples of human immune cells include human T cells, human macrophages and human B cells. - 15 - HUI-038CPPC

As used herein, the term "T cell" (i.e., T lymphocyte) is intended to include all cells within the T cell lineage, including thymocytes, immature T cells, mature T cells and the like, from a mammal (e.g., human or mouse).

As used herein, a "T helper type 2 response" (Th2 response) refers to a response by CD4+ T cells that is characterized by the production of one or more cytokines selected from IL-4, JL-5, IL-6 and JL-10, and that is associated with efficient B cell "help" provided by the Th2 cells (e.g., enhanced IgGl and/or IgE production).

As used herein, the term "a cytokine that regulates development of a Th2 response" is intended to include cytokines that have an effect on the initiation and/or progression of a Th2 response, in particular, cytokines that promote the development of a Th2 response. Preferred cytokines that are produced by the methods of the invention are JL-4, JL-5 and J-L-10.

As used herein, the various forms of the term "modulation" are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity). As used herein, the term "contacting" (i.e., contacting an agent with a cell) is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) and administering the agent to a subject such that the agent and cells of the subject are contacted in vivo.

Various aspects of the invention are described in further detail in the following subsections.

I. Immunomodulatory Agents

In the immunomodulatory methods of the invention, a cell (e.g., a human immune cell, macrophage or T cell) is contacted either in vitro or in vivo with an agent such that an immune response is modulated. Preferably, the agent itself comprises LNnT, as described in further detail below. In one embodiment, the agent stimulates production by the cell of at least one cytokine (e.g., a cytokine that regulates development of a Th2 response). In another embodiment, the agent stimulates production of IL-4. In another embodiment, the agent - 16 - HUI-038CPPC

stimulates cellular proliferation (e.g., B cell proliferation). I-n yet another embodiment, the agent stimulates production of non-specific polyclonal IgE.

A. Stimulatory Agents The agents of the invention stimulate cytokine production by cells, stimulate production of non-specific polyclonal IgE by cells, and/or stimulate proliferation of cells. Accordingly, in one embodiment, the agent is a stimulatory form of a compound comprising LNnT. A "stimulatory form of a compound comprising LNnT" typically is one in which the carbohydrate structure (e.g., the LNnT) is present in a multivalent, crosslinked form. I-n a preferred embodiment, the stimulatory form of a compound comprising LNnT is a conjugate of a carrier molecule and multiple carbohydrate molecules (e.g., the LNnT). For example, carbohydrate molecules can be conjugated to a protein carrier, such as a conjugate of human serum albumin (HSA). When a sugar-carrier protein conjugate is to be administered to a subject, the carrier protein should be selected such that an immunological reaction to the carrier protein is not stimulated in the subject (e.g., a human carrier protein should be used with a human subject, etc). Alternative to a carrier protein, multivalent LNnT can be conjugated to other carrier molecules, for example carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Other preferred carriers include polymers, such as carbohydrate or polysaccharide polymers. A preferred carbohydrate polymer is dextran. - 17 - HUI-038CPPC

The degree of stimulatory ability of the conjugate is influenced by the density of sugars conjugated to the carrier. Preferably, the sugar molecules comprise at least 10% of the conjugate by weight, more preferably at least 15% of the conjugate by weight, even more preferably at least 20% of the conjugate by weight and even more preferably at least 25% of the conjugate by weight or at least 30% of the conjugate by weight or at least 35% of the conjugate by weight or at least 40% of the conjugate by weight or at least 45% of the conjugate by weight. In certain embodiments, the sugar molecules comprise about 10-25 % of the conjugate by weight, about 15-25% of the conjugate by weight or about 20-25% of the conjugate by weight or about 30-35% by weight or about 35-40% by weight or about 40-45% by weight. In a preferred embodiment, the stimulatory form of a compound comprising LNnT is a conjugate of multiple carbohydrate molecules. More preferably, the conjugates comprise 10-11, 12-13, 14-15, 6-17, 18-19, or 20 or more sugars/conjugate. Agents for use in the methods of the invention can be purchased commercially or can be purified or synthesized by standard methods. Conjugates of LNnT and a carrier protein (e.g., HSA) are available from Accurate Chemical and Scientific Corporation, Westbury, NY.

In addition to conjugates comprising LNnT described above, another form of a stimulatory agent comprising LNnT is an isolated protein that naturally expresses LNnT in a form suitable for stimulatory activity.

The ability of an agent of the invention to stimulate production by immune cells of a cytokine can be evaluated using an in vitro culture system such as that described in the Examples. Cells are cultured in the presence of the agent to be evaluated in a medium suitable for culture of the chosen cells. After a period of time (e.g., 24, 48, 72, or 120 hours), production of the cytokine is assessed by determining the level of the cytokine in the culture supernatant. Preferably, the cytokine assayed is IL-4. Additionally or alternatively, IL-2, JL- 5, JL-10, JL-13 and/or JPN-γ levels can be assessed. Cytokine levels in the culture supernatant can be measured by standard methods, such as by an enzyme linked immunosorbent assay (ELISA) utilizing a monoclonal antibody that specifically binds the cytokine. The ability of the agent to stimulate cytokine production is evidenced by a higher level of cytokine (e.g., JL-4) in the supernatants of cells cultured in the presence of the agent - 18 - HUI-038CPPC

compared to the level of cytokine in the supernatant of cells cultured on the absence of the agent.

The ability of an agent of the invention to stimulate production of non-specific polyclonal IgE by cells (e.g., immune cells) can be evaluated in vitro utilizing methods such as those described in the Examples. For example, serum isolated from a subject can be analyzed by sandwich ELISA for the presence of total, as well as antigen-specific, IgE. Briefly, plates are coated with anti-IgE antibodies, washed extensively, blocked to prevent non-specific adsorption of reagents to the plate, then incubated with serum samples isolated from subjects. Labeled antibody (e.g., biotinylated anti-IgE antibody) can be used to detect antigen, for example, by detection with peroxidase-congugated strepavidin. The reactions can be subsequently developed using, for example, tetramethyl-benzidine substrate. Such methods are further useful for detection of, for example, Ag-specific IgG, HSA-specific IgE, LNnT-HS A-specific IgE, as well as specific IgG subtypes, by altering the specificity of the primary antibody (e.g., that used in initial coating of the plate). The ability of an agent of the invention to stimulate proliferation of cells (e.g., proliferation responses) can be evaluated in vitro utilizing methods such as those described in the Examples. For example, spleen cells can be isolated from sacrificed mice, cultured in vitro in appropriate culture medium, and labeled with ^H thymidine as an indicator of DNA replication.

B. Inhibitory Agents

The inhibitory agents of the invention can inhibit induction of a Grl+, CDl lb+ suppressor cell population that recruited by multivalent LNnT treatment. Accordingly, in one embodiment, the agent is an inhibitory form of a compound comprising LNnT. An "inhibitory form of a compound comprising LNnT" typically is one in which the carbohydrate structure (e.g., the LNnT) is present in a monovalent, non-crosslinked form. LNnT is commercially available (e.g., as a custom order from GlycoTech, Rockville MD). - 19 - HUI-038CPPC

U. Pharmaceutical Compositions

Another aspect of the invention pertains to pharmaceutical compositions of the agents (e.g., stimulatory agents) of the invention. The pharmaceutical compositions of the invention typically comprise an agent of the invention and a pharmaceutically acceptable carrier. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. I-n various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, transdermal or oral administration. In a preferred embodiment, the composition is formulated such that it is suitable for intraperitoneal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. I-n many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the modulators can be administered in a time release formulation, for example in a - 20 - HUI-038CPPC

composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Depending on the route of administration, the agent may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent. For example, the agent can be administered to a subject in an appropriate carrier or diluent co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The active agent in the composition (i.e., a stimulatory or inhibitory agent of the invention) preferably is formulated in the composition in a therapeutically effective amount. - 21 - HUI-038CPPC

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as the production of sufficient levels of non-specific polyclonal IgE to thereby influence the therapeutic course of a particular disease state. A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. In another embodiment, the active agent is formulated in the composition in a prophylactically effective amount. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, for example, influencing the production of sufficient levels of non-specific polyclonal IgE for prophylactic purposes. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

A non-limiting range for a therapeutically or prophylactically effective amounts of a stimulatory or inhibitory agent of the invention is 0.01 nM-20 mM. Alternatively, a stimulatory or inhibitory agent can be used in an amount between 500 μg to 100 mgs. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. - 22 - HUI-038CPPC

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. An agent of the invention can be formulated into a pharmaceutical composition wherein the agent is the only active compound therein. Alternatively, the pharmaceutical composition can contain additional active compounds. For example, two or more agents may be used in combination. Moreover, an agent of the invention can be combined with one or more other agents that have immunomodulatory properties. For example, a stimulatory agent may be combined with one or more cytokines or adjuvants.

A pharmaceutical composition of the invention, comprising a stimulatory or inhibitory agent of the invention, can be administered to a subject to modulate immune responses (e.g., production of non-specific polyclonal IgE) in the subject. As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

A pharmaceutical composition of the invention can be formulated to be suitable for a particular route of administration. For example, in various embodiments, a pharmaceutical composition of the invention can be suitable for injection, inhalation or insufflation (either through the mouth or the nose), or for intranasal, mucosal, oral, buccal, parenteral, rectal, intramuscular, intravenous, intraperitoneal, and subcutaneous delivery.

In certain embodiments, a pharmaceutical composition of the invention can be packaged with instructions for using the pharmaceutical composition for a particular purpose, - 23 - HUI-038CPPC

such as to modulate an immune response, for use as an adjuvant, to modulate an allergic response or to modulate an autoimmune disease.

UI. Modulation of Immune Responses The invention provides immunomodulatory methods that can be used modulate various immune responses. In the methods of the invention, a cell is contacted with an agent (e.g., an agent comprising LNnT) with the cell such that the immune response is modulated (e.g.., stimulated). The methods of the invention can be practiced either in vitro or in vivo. For practicing the method of the invention in vitro, cells can be obtained from a subject by standard methods and incubated (i.e., cultured) in vitro with an agent of the invention to modulate, for example, the production of a cytokine, the production of non-specific, polyclonal IgE, proliferation of an immune cell (e.g., a splenocyte), or the development of a Th2 response. For example, peripheral blood mononuclear cells (PBMCs) can be obtained from a subject and isolated by density gradient centrifugation, e.g., with Ficoll/Hypaque. Specific cell populations can be depleted or enriched using standard methods. For example, monocytes/macrophages can be isolated by adherence on plastic. T cells or B cells can be enriched or depleted, for example, by positive and/or negative selection using antibodies to T cell or B cell surface markers, for example by incubating cells with a specific mouse monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using anti- mouse-Ig coated magnetic beads. Monoclonal antibodies to cell surface markers are commercially available.

For practicing the methods of the invention in vivo, an agent is administered to a subject in a pharmacologically acceptable carrier (as described in the previous section) in amounts sufficient to achieve the desired effect, such as to modulate, for example, the production of a cytokine, the production of non-specific, polyclonal IgE, proliferation of an immune cell, or the development of a Th2 response in the subject or to prevent a detrimental host reaction against parasite infection or to protect against environmental allergens by saturating FcεRs on effector cells in the subject or to inhibit a disease or disorder (e.g., an allergy or an autoimmune disease) in the subject. Any route of administration suitable for - 24 - HUI-038CPPC

achieving the desired immunomodulatory effect is contemplated by the invention. One preferred route of administration for the agent is intraperitoneal. Another preferred route of administration is orally. Yet another preferred route of administration is intravenous. Application of the methods of the invention to the treatment of disease conditions may result in cure of the condition, a decrease in the type or number of symptoms associated with the condition, either in the long term or short term (Le., amelioration of the condition) or simply a transient beneficial effect to the subject.

Numerous disease conditions associated with a predominant Th2-type response have been identified and could benefit from modulation of the type of response mounted in the individual suffering from the disease condition. Application of the immunomodulatory methods of the invention to such diseases as cancer, infectious disease, allergies, autoimmune disease, and inflammatory bowel disease. In addition to the foregoing disease situations, the immunomodulatory methods of the invention also are useful for other purposes. For example, the methods of the invention (i.e., methods using a stimulatory agent) can be used to stimulate production cytokines (such as JL-4) in vitro for commercial production of these cytokines (e.g., cells can be cultured with a stimulatory agent in vitro to stimulate IL-4 production and the IL-4 can be recovered from the culture supernatant, further purified if necessary, and packaged for commercial use).

Another aspect of the invention pertains to use of a stimulatory agent of the invention in the treatment or prevention of shock in a subject. As demonstrated in Example 3, administration of multivalent LNnT (e.g., LNnT conjugated to dextran) renders spleen cells less responsive to LPS, as measured by proliferative responses and production of type 1 cytokines such as JL-12 and J-FN-γ. Thus, multivalent LNnT treatment can be used to inhibit the shock response in a patient. For at risk patients, these patients can be pretreated with a multivalent LNnT composition of the invention to render them less susceptible to shock. Accordingly, the invention provides a method for inhibiting or preventing shock in a subject comprising administering an agent comprising multivalent lacto-N-neotetraose (LNnT), such that shock is inhibited or prevented in the subject. The subject may be a patient already - 25 - HUI-038CPPC

experiencing at least one symptom of shock or, more preferably, is a patient at risk of (susceptible to) shock, such as a surgery patient who is susceptible to shock. For at risk patients, the multivalent LNnT agent can be administered to the patient at least 1 hour prior to a time when shock may develop in the patient or at least 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours or more prior to a time when shock may develop in the patient.

Yet another aspect of the invention pertains to a method of inhibiting induction of Grl+, CDl lb+ suppressor cells in a subject. This method can be used to inhibit induction of the suppressor cells in a clinical setting where such inhibition is desirable, such as in the treatment of cancer. The metho comprises: administering to the subject an agent comprising monovalent lacto-N-neotetraose (LNnT), such that induction of Grl+, CDllb+ suppressor cells in the subject is inhibited. As demonstrated in Example 4, administration of multivalent LNnT induces this suppressor cell population, which is capable of inhibiting T cell proliferative responses. This suppressor cell population may be induced in a clinical setting in a patient by tumor cells that express multivalent LNnT, and the induction of such a suppressor population could promote tumor growth and expansion, by suppression of immune responses against the tumor and/or by production by the suppressor population of factors (such as TGF-β) that promote tumor angiogenesis. Accordingly, inhibition of the induction of this suppressor population can be used in the treatment of cancer, by adminstration of an agent comprising monovalent LNnT to thereby competitively block the induction of the suppressor population. In one embodiment, the agent is administered intraperitoneally. I-n another embodiment, the agent is administered intravenously.

Preferably, the method for inhibiting induction of the suppressor population is carried out in a subject suffering from cancer.

The stimulatory methods of the invention (i.e., methods using the stimulatory agents of the invention) also can be used therapeutically for treating type 1 autoimmune diseases

(i.e., autoimmune diseases that are associated with Thl -type dysfunction). Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and that promote the production of cytokines and autoantibodies involved - 26 - HUI-038CPPC in the pathology of the diseases. It has been shown that modulation of T helper-type responses can either have a beneficial or detrimental effect on an autoimmune disease. For example, in experimental allergic encephalomyelitis (EAE), stimulation of a Th2-type response by administration of IL-4 at the time of the induction of the disease diminishes the intensity of the autoimmune disease (Paul, W.E., et al. (1994) Cell 76:241-251).

Furthermore, recovery of the animals from the disease has been shown to be associated with an increase in a Th2-type response as evidenced by an increase of Th2-specific cytokines (Koury, S. J., et al. (1992) J. Exp. Med. 176:1355-1364). Moreover, T cells that can suppress EAE secrete Th2-specific cytokines (Chen, C, et al. (1994) Immunity 1: 147-154). Since stimulation of a Th2-type response in EAE has a protective effect against the disease, stimulation of a Th2 response (and/or downmodulation of a Thl response) in subjects with multiple sclerosis (for which EAE is a model) may be beneficial therapeutically.

Similarly, stimulation of a Th2-type response in type I diabetes in mice provides a protective effect against the disease. Indeed, treatment of NOD mice with IL-4 (which promotes a Th2 response) prevents or delays onset of type I diabetes that normally develops in these mice (Rapoport, M.J., et al. (1993) J. Exp. Med. 178:87-99). Thus, stimulation of a Th2 response (and/or downmodulation of a Thl response) in a subject suffering from or susceptible to diabetes may ameliorate the effects of the disease or inhibit the onset of the disease. Yet another autoimmune disease in which stimulation of a Th2-type response may be beneficial is rheumatoid arthritis (RA). Studies have shown that patients with rheumatoid arthritis have predominantly Thl cells in synovial tissue (Simon, A.K., et al., (1994) Proc. Natl. Acad. Sci. USA £1:8562-8566). By stimulating a Th2 response in a subject with RA, the detrimental Thl response can be concomitantly downmodulated to thereby ameliorate the effects of the disease.

To treat a type 1 autoimmune, a stimulatory agent of the invention (e.g., an agent comprising multivalent LNnT) can be administered to the subject, for a variety of therapeutically beneficial purposes, including downmodulating the production of the Thl- associated cytokines IL-12 and J-FN-γ, and induction of Grl+, CDl lb+ suppressor cells. The stimulatory agent can be used alone, or in combination with one or more additional agents that promote Th2 responses (e.g., Th2-promoting cytokines, such as JL-4 or JL-10) and/or downmodulate Thl responses (e.g., antibodies to Thl-promoting cytokines such as anti-IL-2, anti-IL-12, anti-JEN-γ). Depending on the disease, the stimulatory agent may be administered - 27 - HUI-038CPPC either systemically or locally. For example in the case of rheumatoid arthritis, the agent may be administered directly into the joints. For systemic treatment, the stimulatory agent preferably is administered intravenously. Alternative to direct administration of the stimulatory agent to the subject, autoimmune diseases may be treated by an ex vivo approach. In this case, cells (e.g., T cells, macrophages, B cells, peritoneal exudate cells) are obtained from a subject having an autoimmune disease, cultured in vitro with a stimulatory agent of the invention, for example, to stimulate generation of the Grl+, CDl lb+ suppressor cell population and/or to inhibit production of Thl -associated cytokines (e.g., JL-12, IFN-γ) and/or to stimulate production of Th2-associated cytokines (e.g., IL-13), followed by readministration of the cells to the subject.

Non-limiting examples of autoimmune diseases and disorders having an autoimmune component that may be treated according to the invention include diabetes mellitus, inflammatory bowel disease, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjδgren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

The invention also provides pharmaceutical compositions for carrying out the methods of the invention. For example, in one embodiment, the invention provides a pharmaceutical composition comprising an agent comprising multivalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition as a modulator of IgE responses in a subject. In another - 28 - HUI-038CPPC

embodiment, the invention provides a pharmaceutical composition comprising an agent comprising multivalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition for the treatment or prevention of shock in a subject. For either of these compositions, the agent can comprise LNnT conjugated to a protein carrier, such as human serum albumin, or LNnT conjugated to a carbohydrate polymer, such as dextran.

In yet another embodiment, the invention provides a pharmaceutical composition comprising an agent comprising monovalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition for the treatment of cancer in a subject.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLES

Materials and methods used in the Examples

Animals

Young adult (7-9 weeks old) CBA/J, BALB/C, and C57BL/6 strain female mice were purchased from Harlan (Indianapolis, IN). Female CBA/CaJ xid and age-matched control female CBA CaJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). JL-4 deficient BALB/C mice were generated as described (). This deficient mice were bred and maintained at Harvard School of Public Health according to the guidelines set forth by the Harvard Medical Area Research Committee. Antigens and inoculations

Human serum albumin (HSA) was selected as a carrier protein for multivalent carbohydrate because HSA is a simple protein and does not contain any carbohydrate motif. Multivalent LNnT or LewisΥ were conjugated with HSA (LNnT-HSA and LeY-HS A) by Accurate Chemical and Scientific Corporation (NY). In both neoglycoproteins, 13 molecules - 29 - HUI-038CPPC

of sugar conjugated to 1 HSA molecule. As controls, HSA (Sigma Chemical Co., MO), HSA adsorbed to alum (Intergen Company, NY; HSA-alum), or Dulbecco's PBS (Gibco BRL, NY) were prepared for immunization. Groups of four to six mice were immunized intraperitoneally or subcutaneously with Ags (lOμg of HSA) or saline. First and second boosting immunization were performed in the same fashion 2 and 3 weeks later, respectively. Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce, JL).

Determination of serum Ab titers

Immunized mice were bled from the tail 10, 6, and 5 days following primary, first, and second boosting immunization, respectively. Total and HSA-specific IgE were determined by sandwich ELISA. In brief, ELISA plates (Corning Inc., NY) were coated overnight at 4°C with 100 μl of 5μg/ml rat anti -mouse IgE mAb (Biosource, CA) in carbonate-bicarbonate buffer, pH 9.6. After washing four times with PBS containing 0.05% Tween20 (PBS-T), plates were blocked with 200 μl PBS containing 10% FCS and 0.3% Tween20 for 2 hrs at 37°C. After washing as above, 100 μl samples of serially-diluted serum or standard mouse IgEmAb (Pharmingen, CA) were added in duplicate wells and incubated for 2 hrs at 37°C. Thereafter, 100 μl biotinylated anti-mouse IgEmAb (0.5 μg/ml, Pharmingen) or biotinylated HSA (1 μg/ml) were added to detect total IgE and HSA-specific IgE, respectively. After 2 hrs incubation at 37°C, plates were washed and 100 μl peroxidase- conjugated streptavidin (Sigma) diluted 1/1000 was added to each well and incubated for 1 hr at 37°C. Finally plates were washed eight times, the reactions were developed by addition of tetramethyl-benzidine substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and stopped by addition of phosphoric acid (0.4M). The absorbance was measured at 450 nm in UNMax automated plate reader (Molecular Devices Corp., Menlo Park, CA). For biotinylation, HSA (2 mg/ml) in sodium bicarbonate buffer pH 8.5 was incubated with biotin (long arm) Ν-hydroxy succumide ester (Vector Lab., CA) for 2 hrs at room temperature, stopped the reaction by addition of 5 μl ethanolamine, and dialyzed overnight with PBS/0.05% sodium azide. - 30 - HUI-038CPPC

Ag-specific IgG ELISA were determined. Briefly, ELISA plates were coated with lOOμl Ag (2μg/ml) overnight at 4°C in carbonate-bicarbonate buffer, and blocked as described above. Then plates were incubated with samples from individual serum in two-fold serial dilution from 100 times for 2 hrs at 37°C, followed by goat anti-mouse IgG mAb- peroxidase conjugate (Boehringer-Mannheim, J- ) for 1 hr at 37°C. Thereafter, plates were developed and terminated as described above. Finally the absorbance at 450 nm was measured using a UNMax automatic microplate reader. Results were expressed as endpoint titers where the endpoint was determined as the final serum dilution which yields a higher absorbance than twice of the background absorbance. Optimum dilutions of anti-mouse IgG mAb-horseradish peroxidase-labeled conjugates were determined to be 1/1000. Plasma IgE specific for LΝnT-HS A was also tested for in the same fashion as this method using LΝnT- HSA (2μg/ml) as a coating Ag and biotinylated anti-mouse IgEmAb (1 μg/ml, Pharmingen) followed by avidin-peroxidase conjugate (Sigma) as a detection Ab.

Serum total IgG isotypes were also determined by ELISA. Plates were coated with

100 μl of 2 μg/ml rat anti-mouse IgGl, IgG2a, IgG2b, and IgG3 mAb (Pharmingen) overnight at 4°C. After washing and blocking described as above, 100 μl samples of serially-diluted serum or standard mouse IgG isotypes (Pharmigen) were added in duplicate wells and incubated for 2 hrs at 37°C. Thereafter, 100 μl peroxidase-conjugated polyclonal goat anti- mouse Ig (Pharmingen) diluted in 1/1000 were added and incubated for 1 hr at 37°C. After the washing, the reactions were developed and stopped as described above, and the absorbance was measured at 450 nm.

Proliferative responses of splenocytes Groups of mice were killed by carbon dioxide 5 days following second boosting immunization. Spleens were removed aseptically and proliferation assays were performed as described previously (Velupillai, P. et al. (1997) J. Immunol, vol. 158 pp. 338-344). In brief, cell suspensions were prepared in RPMI 1640 supplemented with 10% FCS (Gibco), 2 mM L-glutamine, 5 x 10"⁵ M 2-mercaptoethanol, 100 Unit/ml penicillin, and 100 μg/ml - 31 - HUI-038CPPC

streptomycin (Sigma). Red blood cells were removed by incubation in Boyle's solution, and 2.5 x lθ6 cells per ml were added to 96-well flat-bottom tissue culture plates (Corning) in triplicate for 72 hrs at 37°C, 5% CO2 in air, with 2 μg/ml ConA (Vector), 10 μg/ml HSA or LNnT-HSA. For the final 8 hrs, cells were incubated with 1 μCi ^*^H thymidine (Amersham Life Science Inc., JL) and then harvested onto filter paper for scintillation counting.

Cytokine assays

In flat-bottom 24-well culture plates (Corning), suspensions of 2.5 x 10° cells per ml were cultured with ConA (2μg/ml) or without restimulation at 37°C in supplemented RPMI 1640 medium as described above. 24, 72 and 120 hrs later, the cell cultures were then centrifuged, and the culture supernatants were harvested and kept frozen (-80°C) until assayed. Pelleted cells were resuspended then stained with mAbs coupled to FITC or PE for immunofluorescent staining. JL-2, JL-4, IL-5, JL-10 and J-FN-γ levels in supernatants from ConA-stimulated or unstimulated splenocytes were measured by capture ELISA. In brief, Maxisorp microtitre plates (Nunc Laboratories Ltd., Denmark) were coated with 50μl of capture Ab at 2.0 μg/ml (rat anti- mouse JL-5, JL-10 and JPN-γ from Pharmingen and rat anti- mouse JL-4 from Endogen, MA ) in Carbonate-bicarbonate buffer by overnight incubation at 4°C. Wells were then washed with PBS-T four times and blocked by addition of 10% FCS in PBS (2 hours, 37°C). 50 μl of culture supernatants and appropriate recombinant standards were then added to individual well in duplicate. For standard curves, recombinant JL-5 (0 to 5,000 pg/ml), JL-10 (0 to 25,000 pg/ml), JPN-γ (0 to 20,000 pg/ml) (Pharmingen, CA) and EL- 4 (0 to 1,500 pg/ml) (Endogen, MA) were used in duplicate. Following overnight incubation at 4°C, the wells were washed and appropriate 50 μl/well biotinylated detection Ab at lμ g/ml (rat anti-mouse IL-5, EL- 10 or JPN-γfrom Pharmingin and rat anti-mouse JL-4 from Endogen) were added. For the detection of bound biotinylated Ab, 50 μl of streptavidin- alkaline phosphatase conjugate (1/2000, Pharmingen) was added to each well for 45 minutes, then washed. Level of cytokine in the wells was visualized by addition of p-nitrophenyl phosphate (Sigma) in glycine buffer. Absorbance was measured at 405 nm using a UVMax automatic microplate reader. - 32 - HUI-038CPPC

Immunofluorescent staining

Following in vitro culture without restimulation, splenocytes were resuspended and incubated on ice for 15 min with mAbs as follows: anti-CD45R/B220 (RA3-6B2), anti-B7-l (16-lOA 1), and anti-B7-2 (GL-1) or isotype-matched controls. All mAbs were either FJTC- or PE-conjugated (Pharmingen). After washing with Hanks' balanced salt solution (Gibco) containing 0.05% sodium azide, flow cytometry analysis was performed by using a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson, CA). Dead cells were excluded from analysis on the basis of propidium iodide (Molecular Probes, OR) staining. Lymphocytes were gated according to the physical characteristics of forward and side scatter and at least 10,000 events were acquired. Statistics

Statistical analysis was performed using the Student's unpaired t test. A value of p<0.05 was considered significant.

EXAMPLE 1:

Induction of polyclonal IgE by multivalent LNnT in vivo

Following first boosting JP immunization with LNnT-HSA, BALB/C mice produced significantly higher amounts of serum total IgE than those JP immunized with saline, HSA, LeY-HSA, and HSA- Alum (Fig. la). Serum IgE was significantly elevated following second JP immunization. This elevation was not seen following primary inoculation, however, high elevation of serum IgE lasted at least 8 weeks following second JP immunization with LNnT (Fig. lb). Serum IgGl was also significantly increased in mice JP immunized with multivalent LNnT compared with those immunized with saline or HSA alone, whereas the amount of other isotypes were not significantly different (Fig. Ic).

HSA-specific IgE and IgG were determined in sera in mice immunized JP with HS A- Alum. On the contrary, those signals in mice immunized IP with LNnT-HSA were not detected and were statistically no different from the control groups immunized with HSA - 33 - HUI-038CPPC

alone, LeY-HSA, or saline (Fig. 2a,b). Similar findings were seen in Ab titers against LNnT- HSA. Mice IP immunized with LNnT-HSA did not produce significant amounts of IgG or IgE specific for LNnT-HSA compared with the controls (fig. 2c,d). Moreover, specific signal against Gal (βl-4) GlcNAc and Gal (βl-4) Glc, the components of LNnT, were also not detected when biotinylated Gal (βl-4) GlcNAc and Gal (βl-4) Glc (Glycotech, MD) were used, respectively, instead of biotinylated HSA in specific IgE ELISA. These results suggested that multivalent LNnT induced nonspecific polyclonal production of IgE in BALB/C mice following boosting immunization.

Specificity of induction of polyclonal IgE by multivalent LNnT

As in the BALB/C strain, CB A/J mice showed significant elevation of serum IgE following first boosting JP immunization with LNnT-HSA when compared to the control groups (Fig. 3a). On the other hand, C57BL/6 mice did not show the increase of serum IgE following first JP immunization with LNnT-HSA. However, this strain induced the increase of serum IgE following second IP immunization (Fig. 3b). Both strains did not elicit either IgE or IgG against HSA. These results indicate that the induction of polyclonal IgE by multivalent LNnT was genetically affected.

Elevation of serum total IgE was not seen in SC immunization with LNnT-HSA even following the boost (Fig.3c). It has previously been suggested that LNnT-HSA does not induce such an elevation of serum IgE following intranasal sensitization, implying that the route of inoculation of multivalent LNnT is critical for the induction of nonspecific IgE. CBA/CaJ xid mice, which are deficient CD5+ B-l cells, showed significantly higher amounts of IgE following IP immunization with LNnT-HSA compared with those IP immunized with saline or HSA, although the amounts were significantly lower than the control CBA/CaJ mice (Fig.3d). This result indicates that B-l cells, which is one of the specific components of peritoneal cavity, seems to be in part involved but not critical for the induction of polyclonal IgE by multivalent LNnT.

Proliferative responses against multivalent LNnT - 34 - HUI-038CPPC

Splenocytes of BALB/C mice were prepared 5 days following second boosting JP immunization with saline, HSA or LNnT-HSA. Mice JP immunized with HSA or saline showed moderate but significant in vitro proliferative responses to LNnT-HSA compared to HSA or unrestimulation, suggesting that LNnT induces the proliferative responses in naive splenocytes. Mice immunized JP with LNnT-HSA showed the significant responses even without restimulation compared to the control mice (Fig.4). J-n addition, the response was significantly enhanced with restimulation of LNnT-HSA In ConA stimulation, LNnT- inoculated mice also responded significantly more than the controls. These results suggested that splenocytes specific for LNnT-HSA were spontaneously activated following IP immunization with multivalent LNnT.

Cytokine production by LNnT-inoculated mice

Splenocytes of mice IP immunized with LNnT-HSA also produced cytokines without restimulation. Significant amounts of JL-2, JL-4, and JL-5 were detected as early as 24 hours incubation in culture supernatants without restimulation. IL-10 and IFN-γ were detected as early as 72 hrs incubation. The results are summarized below in Table 1. Interestingly, in response to ConA, splenocytes of mice immunized JP with LNnT-HSA produced significantly more U- , JL-5, and JL-10, but not IFN-γ compared to those JP immunized with saline or HSA, suggesting that LNnT skewed splenocytes into polarized Th2 responses in response to ConA stimulation.

^■ 35 - HUI-038CPPC

Table 1 in vitro stumulation (hrs)

Cytokine Immunization

(IP) None None None ConA

(24hrs) (72hrs) (120hrs) (72hrs)

IL-2 (pg/ml) Saline ND ND ND ND

HSA ND ND ND ND

LNnT-HSA 209+68* 2,060+286* 63+1* ND

IL-4 (pg/ml) Saline ND ND ND 76.56+18.28

HSA ND ND ND 31.81+3.64

LNnT-HSA 63.74+24.11* 479.07+86.80* 1,363.33+259.70* 234.00+28.15*

IL-5 (pg/ml) Saline 48+4 48±2 49±1 81+2

HSA 43+2 42+1 45+6 78±6

LNnT-HSA 178+5* 808+93* 3,018+604* 671+34*

IL-10 (pg/ml) Saline ND ND ND ND

HSA ND ND ND ND

LNnT-HSA ND 1,068+223* 7,064+1,381* . 1,529+361*

IFN-g (pg/ml) Saline ND ND ND 3,577+510

HSA ND ND ND 3,358+471 LNnT-HSA ND 2,090+891* 12,220+1,251* 5,917+1,132

Selective in vitro B7-2 (CD86) expression on B220+ cells in LNnT-inoculated mice The expression of the costimulatory molecules, B7-1 and B7-2 on B220 positive splenocytes was also investigated. In freshly isolated splenocytes, the percentage of the cells positive for both B7-1 and B220 molecules were 1.02+0.08, 0.95+0.07, and 0.88±0.06 in mice IP immunized with saline, HSA, and LNnT-HSA, respectively (n=6). The percentage of the cells positive for both B7-2 and B220 molecules were 0.59±0.04, 0.59+0.05, and 0.56+0.04 in mice IP immunized with saline, HSA, and LNnT-HSA, respectively, suggesting no difference of the expression of these molecules in freshly isolated splenocytes. B7-1 expression was not altered in cultured splenocytes (Fig. 5a,c,e). However, following 24 hrs culture incubation without restimulation in vitro, B220 positive cells expressing B7-2 molecule were increased in mice JP immunized with LNnT-HSA compared to the control mice immunized with saline or HSA (Fig. 5b.d.f). This increase was found following 6 hr - 36 - HUI-038CPPC

incubation, and lasted at least 120 hrs, although B7-1 expression on B220 positive cells was not altered throughout the period observed (Fig. 5g,h).

IL-4 is required for the induction of polyclonal IgE production by multivalent LNnT Because Th-2 cytokines, especially JL-4, are involved with IgE production, the role of

IL-4 for the induction of polyclonal IgE production by multivalent LNnT in vivo was investigated using JL-4 gene deficient mice. JL-4 deficient mice did not induce polyclonal IgE production following repeated JP immunization with LNnT-HSA. J-n this experiment, total serum IgE. J-L-4 deficient mice or wild type BALB/.C mice were immunized with saline, HSA or LNnT-HSA. Following second boosting immunization, sera were sampled and serum total IgE was measured. Following second boosting immunization, 2.5 x 10"/ml splenocytes from wild type or J-L-4 deficient mice were cultured without additional stimulants and the production of IL-4, IL-5, IL-10, and JEN-γwere measured at 24, 72, and 120 hrs postincubation. Interestingly, splenocytes from JL-4 deficient mice with the immunization of LNnT-HSA produced JL-5, JL-6, and JPN-γ without restimulation. However, the amounts of IL-5 and IL-6 were significantly lower than wild type BALB/C mice (Fig. 6b,c,d,e). These cytokines were not detected in JL-4 deficient mice IP immunized with saline or HSA, indicating that JL-4 is required for the induction of polyclonal IgE by multivalent LNnT.

EXAMPLE 2:

Additional experiments demonstrating the induction of polyclonal IgE by multivalent LNnT were performed, as described below.

J-n a first series of experiments, mice were injected intraperitoneally either two or three times with either dextran alone, RPMI media or LNnT conjugated to dextran in saline

(LNnT-dextran). The sugar conjugate was referred to as LNnT35, wherein the 35 refers to the degree of LNnT substitution on each dextran molecule. The total amount of IgE elicited in the mice was determined. The results are shown in Figure 6, wherein the numbers after LNnT (i.e., 200, 100 or 50) refer to the amount injected (dextran weight). The results - 37 - HUI-038CPPC

demonstrate that the LNnT-dextran construct is able to raise large amounts of total IgE in vivo.

L another series of experiments, the effect of pre-treatment with multivalent LNnT on the production of antigen-specific IgE was investigated. Mice were injected either with ovalbumin (as a specific antigen) followed by a second injection of ovalbumin, or with the dextran conjugate LNnT45 followed by ovalbumin. Controls were mice receiving RPMI or dextran, followed by ovalbumin. Figure 7 shows the total IgE in the mice and Figure 8 shows the ovalbumin-specific IgE in the mice. Figure 7 demonstrates that there was no discernible difference in levels of total IgE in the mice. Figure 8 demonstrates, however, that prior treatment with the LNnT dextran conjugate does in fact reduce the amount of OVA-specific IgE by 40-50%. Thus, the LNnT conjugate functions to reduce the allergan specific IgE in vivo.

In another experiment, the length of time that non-specific IgE levels remain elevated after treatment with multivalent LNnT was investigated. LNnT-HSA was used as the conjugate, with saline and HSA alone serving as controls. The results are shown in Figure 9, which demonstrates that mice treated with LNnT-HSA exhibited high levels of non-specific IgE for at least 70 days (the longest time point in the study). Thus, treatment with multivalent LNnT leads to persistent non-specific IgE.

J-n another experiment, the effect of treatment with LNnT-dextran on production of either Th2-type cytokines or Thl -type cytokines was examined. Mice were immunized intraperitoneally with LNnT-dextran, total splenocytes were harvested and stimulated with ConA, followed by measurement of cytokine levels at 48 or 72 hours of culture. The results for Th2-type cytokines are shown in Figure 10, which demonstrates that LNnT-dextran treatment leads to significantly elevated levels of JL-10, JL-4 and IL-13 at 72 hours culture compared to vehicle (dextran) immunized controls. The results for the Thl -type cytokine interferon-gamma are shown in Figure 11, which demonstrates that LNnT-dextran treatment reduces the level of interferon-gamma at both 48 and 72 hours. - 38 - HUI-038CPPC

Discussion

These examples teach that multivalent LNnT induces polyclonal IgE production in mice by repeating IP immunization in vivo. Despite the fact that several reports have demonstrated polyclonal IgE production in mice and human in vivo and in vitro by the extracts from the parasites, the mechanism for promoting polyclonal IgE production by parasites is yet unclear (Wang, MQ. et al. (1995) Parasite Immunol, vol. 17 pp. 609-615; McGibbon, AM. et al. (1990) Mol. Biochem. Parasitol. vol. 39 pp. 163-172; Lee, TDG. et al. (1995) /. Allergy Clin. Immunol, vol. 95 pp. 124- 1254 Yamashita, T. et al. (1993) Immunology vol. 79 pp. 185-195Namashita, U. et al. (1993) Jap. J. Parasitol. vol. 42 pp. 211-219). In fact, B cells are nonspecifically activated in parasite-infected host (Fisher, E. et al. (1981) Clin. Exp. Immunol, vol. 46 pp. 89-97). And carbohydrate moieties on schistosoma japonicum egg antigen is reported to activate not only schistosome-primed but also naive B cells (Yamashita, T. et al. (1993) Immunology vol. 79 pp. 185-195). Therefore it seems that schistosomal antigens, especially carbohydrate moieties, have a mitogenic effect against B cells to induce nonspecific activation. Indeed, there are reports that the extracts from nematodes contain a B cell mitogen that regulate nonspecific B cell activation to induce polyclonal IgE production (Wang, MQ. et al. (1995) Parasite Immunol, vol. 17 pp. 609-615; Lee, TDG. et al. (1995) /. Allergy Clin. Immunol, vol. 95 pp. 124-1254). However, B cell mitogenic activity is not enough to promote polyclonal IgE production. It is known that IL-4 induces B cells to develop polyclonal IgE producing cells in mice in vivo and in vitro (Coffman, RL. et al. (1986) J. Immunol, vol. 136 pp. 949-954; Finkelman, FD et al. (1990) Annu. Rev. Immunol, vol. 8 pp. 303-333; Tepper, RI. et al. (1990) Cell vol. 62 pp. 457; Snapper, CM. et al. (1991) J.Immunol, vol. 147 pp. 1163-1170; Nakanishi, K. et al. (1995) Int. Immunol, vol. 7 pp. 259-268). Therefore the help of IL-4 derived from IL-4 producing cells such as T cells (including NK1+ CD4+ T cells), eosinophils, and cells of the mast cell/basophil lineage may be required (Coffman, RL. et al. (1997) J. Exp. Med. vol. 185 pp. 373-375; Sabin, EA. et al. (1996) J. Exp. Med. vol. 184 pp. 1871-1878). Thus, at least two factors in parasite antigens may be required to induce polyclonal IgE production: B cell mitogenic activity and induction of IL-4 production. Lee et - 39 - HUI-038CPPC

al. showed that the body fluid of Ascaris is capable of increasing total IgE levels in mice by a single subcutaneous injection (McGibbon, AM. et al. (1990) Mol. Biochem. Parasitol. vol. 39 pp. 163-172; Lee, TDG. et al. (1995) J. Allergy Clin. Immunol, vol. 95 pp. 124-1254; Lee, TDG. et al. (1993) Int. Arch. Allergy Immunol, vol. 102 pp. 185-190). However, they did not isolate the molecule that induce IgE production, but showed that ABA-1, the purified major allergen of Ascaris did not induce the increase of total IgE. Thus they suggested the model of polyclonal IgE induction by nematode that nematode products contain a B-cell mitogen that polyclonally activates B cells, which is converted into a polyclonal IgE response when these stimulated B cells come under the influence of IL-4 or an IL-4 like molecule activated by other factors.

Splenocytes from mice JP immunized with LNnT produced IL-4, JL-5, and JL- 10 without restimulation in vitro, although they also produced detectable amount of IL-2 and JPN-γ. In addition, JL-4 deficient mice did not induce polyclonal IgE production following JP immunization with this carbohydrate although they produced significant amount of JL-5, JL- 10, and JFN-γ. Development of IL-5 and JL-10 production following S. mansoni infection was also seen in JL-4 deficient mice (Pearce, EJ. et al. (1996) Int. Immunol, vol. 8 pp. 435- 444; King., CL. et al. (1996) Exp. Parasitol. vol. 84 pp. 245-252). These results indicate that JL-4 is required for inducing polyclonal IgE production by multivalent LNnT in vivo. Moreover, splenocytes from mice IP immunized with multivalent LNnT were found to produce significantly more IL-4 in response to ConA, the T cell mitogen. This result is consistent with the reports that schistosome-infected host shows mitogen-driven JL-4 production and the production is correlated to serum total IgE (King, CL. et al., (1993), J. Immunol, vol. 150 pp. 1873-1880; Ogilvie BM Nature 1964 204. 91-92; Zwingenberger, K. et al. (1991) Scand. J. Immunol, vol. 34 pp. 243-251). Therefore, multivalent LNnT likely skews host susceptible to produce JL-4. The source of IL-4 was analyzed by intracellular staining in in vitro culture, and it was demonstrated that CD4+ T cells are responsible for the production of JL-4.

Collecting these findings, multivalent LNnT may possess at least two functions, B cell mitogenic activity and induction of JL-4 production, to induce polyclonal IgE production. In - 40 - HUI-038CPPC

fact, splenocyte from control mice IP immunized with saline also showed the significant proliferative responses against LNnT, suggesting that this carbohydrate possesses the mitogenic activity.

CBA J and BALB/C mice produce the polyclonal IgE following second immunization, on the other hand, C57BL/6 mice produce it following third immunization. It is known that host response against S. mansoni infection is strain dependent. CBA/J, C3H/HeJ, and BALB/C mice developed bigger liver granulomas and higher portal hypertension whereas C57BL/6 mice developed relatively smaller granulomas and lower portal hypertension (Fanning, MM. et al. (1981) J. Inf. Dis. vol. 144 148-153; Hernandez, NJ. et al. (1997) Eur. J. Immunol, vol. 27 pp. 666-670). Immunization with LNnT seems to have a same characteristics as S. mansoni infection in terms of the susceptible strain of mice, suggesting that this carbohydrate may be a dominant putative antigen in S. mansoni. Amiri et al. demonstrated that complete suppression of the total IgE response resulted in the decreases in worm burden and egg production in S. mansoni-infected normal and JPN-γ knockout mice (Amiri, P. et al. (1993) J. Exp. Med. vol.180 pp. 43-51). The present result may relate with these reports that in primary S. mansoni infection, C57BL/6 mice produced relatively lower amount of polyclonal IgE production in response to carbohydrate antigen in S. mansoni, results in the decreased egg production and worm burden (Amiri, P. et al. (1992) Nature vol. 356 pp. 604). In addition, it has been previously demonstrated that peritoneal B-l cell outgrowth due to S. mansoni infection was strain dependent, occurring in CBA/J, C3H HeJ, and BALB/C mice but not in C57BL/6 mice (Palanivel, V. et al. (1996) Exp. Parasitol. vol. 84 pp. 168-177). B-l cell subset is a major source of B cell JL-10 that downregulate Thl responses (Amiri, P. et al. (1992) Nature vol. 356 pp. 604). The present result that peritoneal B-l cells seem to be involve in part in the induction of polyclonal IgE by multivalent LNnT (Fig.2d) is consist with the report.

Polyclonal IgE production in response to multivalent LNnT is not due to LPS contamination, because mice JP immunized with LNnT-HSA produced same amount of serum IgG3 as compared with controls. It is known that LPS induced IgG3 production both in - 41 - HUI-038CPPC

vivo and in vitro (Coffman, RL. et al. (1986) /. Immunol, vol. 136 pp. 949-954; Finkelman, FD et al. (1990) Annu. Rev. Immunol, vol. 8 pp. 303-333). And LPS itself did not induce JL-4 production in vitro, whereas splenocytes from mice JP immunized with LNnT-HSA did produce JL-4. Following in vitro culture incubation without restimulation, B7-2 positive cells were increased in B220+ cell population in mice IP immunized with LNnT-HSA compared to the control mice immunized with saline or HSA. B7-1 and B7-2 costimulatory molecules are ligands for CD28/CTLA-4 and involved in T cell activation, cytokine production, and regulation of tolerance (McKnight, AJ. et al. (1994) J. Immunol, vol. 152 pp. 5220-5225; Perez, VL. et al. (1997) Immunity vol. 6 pp. 411-417). Costimulation by B7-1 and B7-2 can differentially regulate Thl cell differentiation, although the effect of these molecules are dependent on the status of immune reaction, doses and routes of antigen inoculation, types of APC, and the experimental model of diseases (Thompson, CB. (1995). Cell vol. 81 pp. 979- 982). For example, in studies of experimental allergic encephalomyelitis (EAE) in mice, administration with anti-B7-l diminished the severity of neurologic disease, which is mediated by Thl cells, while anti-B7-2 administration enhanced the severity (Kuchroo, VK. et al. (1995) Cell vol. 80 pp. 707-718). Recent reports suggest that B7-2 may play a critical role in the ability to initiate a Th2 response (Thompson, CB. (1995). Cell vol. 81 pp. 979- 982; McArthur, JG. et al. (1993) /. Exp. Med. vol. 178 pp. 1645-1653). The present results are consistent with these reports and suggest that B7-2 expression is closely associated with polyclonal IgE production by multivalent LNnT. However, freshly isolated B220+ cells from mice JP immunized with multivalent LNnT did not express the significant levels of B7-2 compared with those from control mice. This result means that LNnT indirectly induce the B7-2 expression on B220+ cells. This may be due to IL-4 secreted by Th2 cells, because IL-4 deficient mice did not induce B7-2 expression. Stack et al. demonstrated that JL-4 treatment of small splenic B cells induced both B7-2 and B7-2 molecules (Stack, RM. et al. (1994) /. Immunol, vol. 152 pp. 5723-5733. They also reported that B7-2 expression was detected at 6 hr and appeared to be maximal at 24 hr, whereas B7-1 was not observed until 48 hr and was maximal at 72 hr. B7-1 expression could not be detected until 120 hr. This difference may - 42 - HUI-038CPPC

be due to the status of B cells. This result represents the expression of primed B220+ cells by multivalent LNnT, on the contrary, the authors investigated the resting B cells.

The current findings suggest that in vivo induction of polyclonal IgE production by multivalent LNnT may be useful for immunotherapy or prophylaxis of allergic and anaphylactic reaction in which detrimental chemical mediators are released from effector cells by the crosslink of antigen-specific IgE on the cell surface. Further, these results encourage us to apply for the reduction of anaphylactic reaction in not only parasite infection but also environmental allergy (Hagel, I. et al. (1993) Parasite Immunol, vol. 15 pp. 311- 315).

EXAMPLE 3: In Vivo Stimulation with LNnT-Dextran Conjugate

Promotes Th2 and Suppresses Thl Responses

Ln this example, mice were injected intraperitoneally with either media alone (RPMI), vehicle alone (dextran), 50 μg of LNnT-dextran conjugate (LNnT 50) or 100 μg of LNnT- dextran conjugate weekly for three weeks. The spleen cells were then harvested from the mice and either total spleen cells or CD4+ cells were stimulated in vitro with ConA or LPS. Following in vitro stimulation, the proliferative and cytokine responses of the cells were measured. The results are illustrated in Figures 12-16. Figure 12 demonstrates that the proliferative response of total spleen cells from the

LNnT-dextran treated mice following in vitro LPS stimulation was significantly decreased as compared to mice treated only with vehicle (dextran). Figure 13 demonstrates that interferon- gamma production (after 48 or 72 hours) by total spleen cells from the LNnT-dextran treated mice (treated in vivo with either 200, 100 or 50 μg of conjugate) following in vitro ConA stimulation was significantly decreased as compared to mice treated only with vehicle (dextran).

In the experiment for which the results are depicted in Figure 14, the mice were treated with media alone (RPMI), vehicle (dextran) or LNnT-Dex at either 100 μg or 50 μg doses. The spleen cells were taken from these groups of mice and then stimulated in vitro - 43 - HUI-038CPPC

with either LNnT-dextran or LPS. The results illustrated in Figure 14 demonstrate that in virtually naϊve animals there is an initial burst of JL-12 production, which goes away and is actually suppressed in LNnT injected animals, especially at lower doses (compare RPMI and LNnT-Dex 50 groups). In the experiment for which the results are depicted in Figure 15, the mice were treated with media alone (RPMI), vehicle (dextran) or LNnT-Dex at either 100 μg or 50 μg doses. The spleen cells were taken from these groups of mice and then stimulated in vitro with either LNnT-dextran or ConA. Figure 15 demonstrates that JL-13 production by total spleen cells from the LNnT-dextran treated mice following in vitro stimulation with either LNnT-dex or ConA was significantly greater than JL-13 production by mice treated with vehicle (dextran) alone and then stimulated in vitro with LNnT-dex or ConA.

Figure 16 demonstrates that IL-13 production by CD4+ cells from the LNnT-dextran treated mice following in vitro stimulation with ConA was significantly greater than JL-13 production by mice treated with vehicle (dextran) alone and then stimulated in vitro with ConA.

In summary, the data presented in Figures 12-16 demonstrate that spleen cells from LNnT-dex injected mice have suppressed responses to LPS and ConA as measured by reduced proliferation, reduced IL-12 production and reduced JPN-γ production. In contrast, production of JL-13 is elevated.

EXAMPLE 4: LNnT-Dex Conjugates Recruit Peritoneal Exudate Cells that Resemble "Natural Suppressors"

Mice were treated with LNnT-dextran conjugate as described in Example 3 and peritoneal exudate cells (PECs) were recovered and analyzed by FACS analysis to characterize the surface markers expressed on this population of cells. The results demonstrated that intraperitoneal injection of the LNnT-Dex conjugate recruits a population of cells that are Grl+, CDl lb+ (but are CDl la- and CDl lc-). J-n one experiment, these cells - 44 - HUI-038CPPC

accounted for 30-40% of PECs in immunized mice compared to less than 4% in saline or dextran injected controls. These cells are seen as early as 2 hours post-injection.

To further characterize this population of PECs, similar FACS analyses were performed on PECs from both wild-type mice and Stat 6 knockout (KO) mice treated with LNnT-dextran. The data showed that injection of LNnT-Dex recruited about 19% Grl+, CDl lb+ cells in the peritoneal cavity after two hours in the Stat 6 KO mice and about 18.0- 24% Grl+, CDl lb+ cells in the peritoneal cavity after two hours in the wild type mice. In contrast, injection of dextran alone in wild-type mice yielded only 5.5% Grl+, CDl lb+ cells. The ability to recruit the Grl+, CDl lb+ population of cells in the Stat 6 KO mice indicates that this induction is not JL-4 or JL-13 dependent. Furthermore, while the Grl+ phenotype suggests granulocytes, this antibody is not very specific and cytospin analysis showed the population of cells to be less than 8% granulocytes, the remainder appearing as mononuclear cells. Staining with the murine macrophage specific monoclonal antibody F4/80 was negative. This cumulative data distinguish the population of cells that are recruited by LNnT- dex treatment from another population of cells called "alternatively activated macrophages." It has been shown that the induction of "alternatively activated macrophages" requires IL-4 and that the cells are F4/80+, whereas the population of cells that are recruited by LNnT-dex treatment are F4/80- and do not require JL-4 for their induction.

To examine the suppressor activity of the LNnT-activated PECs , mice were injected i.p. with either LNnT-dex, dextran alone or saline and two hours later PECs were harvested. Total naϊve spleen cells were planted on wells coated with anti-CD3 antibodies and then the LNnT-activated PECs were added to the culture. The proliferative response of the naϊve spleen cells was determined as a measure of their activation by anti-CD3. The results are illustrated in Figures 17 and 18. Figure 17 demonstrates that PECs obtained 2 hours post-injection of LNnT-dex are able to inhibit the proliferative responses of naive spleen cells stimulated with anti-CD3. (The data labeled CD3 + PEC LNnT(-) represents PECs from LNnT-dex injected mice which have had the Grl+ cells removed; i.e., this data represents a Grl- population of PECs). - 45 - HUI-038CPPC

Figure 18 demonstrates that anti-CD3 proliferative responses by CD4+ cells from Balb/c mice are inhibited by PECs from LNnT-dex treated mice. (The data labeled CD4+ aCD3 + Grl+ represents PECs from LNnT-dex injected mice which have had enriched for Grl+ cells). In summary, the above data demonstrates that multivalent LNnT recmits a population of cells that are Grl+, CDl lb+, but that do not require JL-4 or JL-13 for their induction and that are F4/80-, wherein this population of cells has suppressor activity as evidenced by their ability to inhibit anti-CD3 proliferative responses.

EXAMPLE 5: LNn-T Expands Gr-1⁺ Suppressor Cells That Secrete Anti-

Jjiflammatory Cytokines and Inhibit Proliferation of CD4+ cells.

The inhibition of T cell responses has been attributed to the presence of a suppressor cell population identified phenotypically as Gr-1⁺/CD1 lb⁺. These cells have been termed natural suppressor cells or immature myeloid cells and appear to be mediating immunosuppressive effects in a wide variety of unrelated pathological conditions (23-27). To investigate whether the immune biasing provoked by polyvalent LNnT-Dex injection is related to a specific cell population, a series of ex- vivo experiments were conducted to decipher the early response to LNnT-Dex glycoconjugates. The early response to LNn-T-Dex in naϊve mice was examined using a constract having 21 or more molecules of LNn-T-Dex conjugated to dextran.

Mice

Six-Ten week old female BALB/c and C57BL/6 mice were purchased from Jackson

Laboratories (Bar Harbor, ME) and were maintained in a pathogen free environment at the

Harvard Medical School animal facility in accordance with institutional guidelines.

Preparation and Injection of carbohydrates. - 46 - HUI-038CPPC

The Neo-glycoconjugate Lacto-N-neotetraose (LNnT-Dex) conjugated to a lOKDa

dextran backbone was supplied by Neose Technologies I-nc (Horsham, PA). The level of

LNnT-Dex substitution varied from 21 to 45 LNnT-Dex residues per molecule of dextran.

LNnT-Dex 21, 35 or 45, diluted in saline, was administered at a dose of 100

μg/mouse in all experiments. Naive BALB/c or C57BL/6 mice were injected i.p. with the

LNnT-Dex, dextran or saline and animals were sacrificed at 2 and 18 h later by CO₂

inhalation.

Peritoneal exudate cells (PECs) were obtained at 2 and 18h post-injection by

peritoneal lavage with 5 ml of ice-cold Hank's balanced salt solution (HBSS, Gibco). PECs were washed 2 times and red blood cells were lysed by hypotonic shock with amonium

chloride. Viable cells were counted and adjusted to 5xl0⁵ cells/ml. Viability measured by

trypan blue exclusion was routinely over 95%.

PECs were analyzed for surface markers, cytokine production and for suppressor activity in co-cultures with naive CD4 cells. Flow Cytometric Analysis.

Peritoneal exudate cells were blocked with anti-mouse FcγR antibody (CD16/CD32)

and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies

against Mac-1 (CDl lb), F4/80, Gr-1 (Ly-6G), M HC-U, B7-2 or phycoeritrin (PE)-

conjugated antibodies against Gr-1 (Ly-6G), and IL-10R. All antibodies were purchased from

Pharmingen (San Diego, CAL), except anti-F4/80, which was obtained from Serotec

(England). Stained cells were analyzed on a FACSCalibur using Cell Quest software (Becton

Dickinson). Live cells were electronically gated using forward and side scatter parameters.

Cell Culture and Co-culture of PECs-CD4+ cells. - 47 - HUI-038CPPC

All cultures and co-cultures were maintained in RPMI 1640 (Gibco BRL)

supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma),

5xl0^"5 M 2-β-mercaptoethanol (GIBCO BRL) and 10 % fetal bovine serum (FBS, Hyclone).

PECs were adjusted to a concentration of 5x10 /ml, plated in 24 well plates (Costar) and

were maintained at 37 C and 5% CO₂ for 72h and the spontaneous production of cytokines

was evaluated by ELISA. Supernatants were harvested, centrifuged and examined for IFN-γ,

IL-12, JL-10 (antibodies and cytokines were obtained from Pharmingen), JL-lβ, JL-18, IL-13,

and TGF-β (obtained from R&D).

Co-culture of PECs with naive CD4⁺ cells was performed as follows: PECs were

obtained as described and adjusted to 5xl0⁵PECs/ml. Splenocytes were prepared from naive

mice, and enriched for CD4+ cells (>95% by FACs analysis) using CD4 magnetic cell sorter beads (MACS, Miltenyi Biotec, Germany). CD4⁺ cells were plated in 96 well flat bottom

plates (Costar, Cambridge, MA) which were pre-coated with anti-CD3 and anti-CD28

antibodies (Pharmingen) at 1 μg/ml. Three hours later PECs were added at ratios of 1:4-1:16

(PECs:CD4⁺) into cultures. Cultures were maintained at 37 C and 5 % CO2 for 72h, then ³H-

Thymidine (185 GBb/mmol activity, Amersham, England) lμCi/well was added and

incubated for a further 18h. Cells were harvested on a 96 well harvester (Tomtec, Toku,

Finland) then counted using a β-plate counter. Values are represented as CPM from triplicate

wells. I-n some experiments supernatants from co-cultures were harvested and analyzed for

IL-4, JL- 13 and J-FN-γ production.

Removal of Gr-1⁺ cells.

The Gr-1⁺ cell population of PECs was removed using MACS beads. Briefly, PECs

pooled from 4 mice were incubated with monoclonal anti-Gr-1 antibody (Pharmingen), for 30

min at 4 C, washed two times and re-incubated with paramagnetic beads (MiniMAcs, Miltenyi Biotec) coupled to goat anti-rat IgG (isotype of the anti-Gr-1 antibody) for 15 min at - 48 - HUI-038CPPC

4 C. PECs were washed twice and passed through a magnetic column (Miltenyi Biotec) to

retain Gr-1⁺ cells. The eluted cell fraction (< 3% Gr-1⁺ according to FACS analysis) was

adjusted to 5X10⁵ cells/ml and used in the co-cultures as described above.

Fixed PECs, Transwells cultures and blocking antibody experiments.

In some co-cultures PECs were fixed with 0.5% paraformaldehyde for 5-10 min,

washed extensively with RPMI, adjusted to 5X10⁵/ml and added in varying concentrations to

96-well plates in the presence of CD4+ naive cells previously stimulated with plate-bound

anti-CD3/CD28 antibodies. These co-cultures were processed as described.

J-n others cultures 24 well (0.4 μm pore) transwell cell culture plates (Costar) were

used to separate PECs from naive CD4⁺ cells. PECs were plated on the superior chamber at a

ratio 1:4 to CD4⁺ cells, the inferior chamber was coated with anti-CD3/CD28 antibodies and

naive CD4⁺ cells were added. Cultures were maintained for 72h, then lμCi/well ³H-

Thymidine added (Amersham) and incubated for an additional 18h. CD4⁺ cells were transferred to a 96 well plate for harvesting as described.

The role of soluble factors in PEC suppression was assayed by using varying

concentrations of isotype control or blocking antibodies ((anti-IL-10 (1, 2 and 5 μg/ml) and

anti-TGF-β (2-5 μg/ml)) in 96-well plate co-cultures, where antibodies were added at the

same time as PECs.

LNnT-Dex injection expands a greater number of PECs than injection with control dextran.

The peritoneal cell response in mice to the polyvalent sugar LNnT-Dex was examined

at early (2h) or late (18h) time points after injection. The peritoneal cavity of animals was

rinsed with 5 ml of cold HBSS and PECs harvested. Figure 19a shows that at both time

points animals receiving polyvalent LNnT-Dex rapidly expanded peritoneal cells compared to

animals that received dextran, and that at 18h after injection more PECs were recruited to the

peritoneal cavity than at 2h in LNnT-Dex injected mice. - 49 - HUI-038CPPC

Gr-l+/F4/80+/ CDl lb+ is the predominant cell type expanded by LNnT-Dex.

The composition of the PECs was analyzed by two-color flow cytometry. Figure 19b

summarizes the main surface markers detected in the population expanded by polyvalent

LNnT-Dex. The majority of the PECs are Gr-1+/CD1 lb+ (ranging from 30 to 40 % of the

total cells, Fig. 19b) and F4/80+/Gr-l+ (in lower percentage, ranging 25-37%). In contrast,

PECs recruited by dextran or saline did not show high levels of cells positive for Gr-1 (5-7

%), but did express CDl lb (28% or higher) and F4/80 (30-50 %). Gr-1⁺ PECs from LNnT-

Dex expressed low levels of B7-2, MHC-U and CD40, but did not express CDl lc, JL-10

receptor or NK surface marker. PECs expanded by LNnT-Dex exhibit "Natural Suppressor Cell" activity.

Recent reports in animals receiving chronic injections of virus (Bronte, et al. J.

Immunol. 165:163:5728, 1999), or tumor bearing mice (Kusmartsev, et al., J. Immunol. 165:779, 2000) have shown the accumulation of Gr-1⁺/CD1 lb⁺ cell populations called

"Natural Suppressors". To determine the suppressive activity in PECs, co-cultures were

performed with naive CD4⁺ T cells and PECs expanded by polyvalent LNnT-Dex, dextran, or

saline. Naive CD4⁺ cells were stimulated by plate-bound anti-CD3 and anti-CD28 antibodies,

and three hours later PECs in varying ratios were added to the cultures. Figure 20 shows that

CD4⁺ T cells in the absence of PECs, or in the presence of control PECs (saline or dextran)

exhibited high levels of proliferation in response to anti-CD3/CD28 stimulation. In contrast,

in the presence of PECs from LNnT-Dex injected mice, the CD4⁺ cells proliferated poorly,

especially at higher PEC concentrations (Fig 20a-b). This phenomenon was observed in both

strains of mice tested (BALB/c and C57BL/6) and was characteristic of both early (2h) and

late (18h) times post-sugar injection. There was a significant inhibition in the proliferative

response even when the PECs:CD4⁺ ratio was 1:8, with a 50 % reduction of proliferation - 50 - HUI-038CPPC compared to controls. However, the clearest inhibition was detected using a ratio 1 :4 where

greater than 90% suppression was observed (Fig 20a-b).

Gr-1^4" cells mediate suppression induced by PECs elicited by LNnT-Dex.

As shown in Figure 20, polyvalent LNnT-Dex recruits PECs that specifically inhibits

naive CD4⁺ cells proliferative response to anti-CD3/CD28 stimulation. To identify the cell

type responsible for this suppressive activity, we depleted PECs of Gr-1⁺ cells (the most

prominent cell marker detected) using magnetic cell sorter, and the negative fraction was used

in PEC:CD4⁺ co-cultures. It was found that the suppressive activity previously observed in

PECs recruited by LNnT-Dex was significantly abolished (p<0.01) in the absence of Gr-1⁺

cells (Fig. 21), indicating an important role for cell populations bearing this marker in suppression.

Suppression is mediated by both cell to cell contact and by soluble factors.

Fixed cells were tested to determine whether they could retain their suppressor activity. PECs from LNnT-Dex or dextran injected mice were harvested, adjusted to

5X10 /ml and fixed with 0.5 % paraformaldehyde for 5-10 min, PECs were then washed and

added to previously (3h) stimulated naive CD4⁺ cells. Fixed LNnT-Dex recruited PECs

retained some ability to inhibit the proliferation of CD4⁺ cells stimulated by anti-CD3/CD28

antibodies (Fig 22a). As expected, the inhibition was not as potent as the observed by live

PECs, but was significantly greater than that observed for control fixed-PECs (p<0.05), thus

demonstrating that cell-to-cell interactions are partially responsible for the observed

proliferative suppression.

The role of soluble factors in PECs medieated suppression was also examined. In

these experiments transwells were used for PECs:CD4⁺ co-culture. PECs were plated in the

upper chamber in a ratio 1:4 with respect to the naive CD4⁺ cells, which were previously

stimulated with anti-CD3/CD28 plate bound antibodies. It was found that, even in the - 51 - HUI-038CPPC absence of cell to cell contact, there was significant inhibition in the proliferation of anti-

CD3/CD28 antibodies stimulated CD4⁺ cells (approximately 50% inhibition, Fig 4b, p<0.05),

strongly implicating a role for soluble factors in the Gr-1⁺ PEC mediated suppression of T

cell proliferation.

PECs expanded by LNnT-Dex secrete a different profile of cytokines than control PECs.

To determine if the PECs expanded by polyvalent LNnT-Dex produced soluble

factors involved in suppressive activity, the peritoneal cells were havrvested 2h or 18h after

injection of LNnT-Dex or dextran and cultured then for 72h. Supernatants were harvested and

used for analysis of cytokines or other soluble factors. The results demonstrated that the

pattern of cytokines spontaneously released by LNnT-Dex expanded or dextran expanded

PECs was different. Notably PECs expanded by LNnT-Dex produced significantly lower

levels of pro-inflammatory cytokines such as IL-lβ, JL-12, JL-18 and JPN-γthan PECs

recruited by dextran (Table U). In addition, PECs expanded by LNnT-Dex produced

significantly greater quantities of JL-10 and TGF-β than dextran recruited PECs (Table JH).

PECs expanded by LNnT-Dex inhibit IFN-γ production in co-cultures with naive CD4⁺ T

cells while enhancing the production of IL-13.

To exclude the possibility that the CD4⁺ cells in co-cultures had reduced proliferation

due to cell death, cytokine production was measured in response to anti-CD3/CD28

stimulation in the presence of PECs. The results demonstrate that there was a significant

decrease in IFN-γ production in supernatants from co-cultures containing PECs expanded by

LNnT-Dex, compared to PECs from dextran or non-injected mice (Fig 23a, p<0.05). Parallel

testing in these LNnT-Dex-PECs:CD4⁺ supernatants revealed that levels of IL-13 were

significantly elevated, compared to cultures containing dextran-recruited PECs (Fig. 23b,

p<0.01). This, finding demonstrates that co-cultured CD4+ cells are alive and their cytokine

profile is differentially regulated depending on the source of PECs. - 52 - HUI-038CPPC

Co-cultured CD4⁺ cells were then examined to determine whether the cells would

respond to secondary stimulation. PECs:CD4⁺ cells were co-cultured as previously described,

but after 72h in culture, cells were removed, washed and CD4⁺ cells re-purified. Purified

CD4⁺ cells were then plated and incubated in RPMI for another 72 h (rested). CD4+ cells

were then re-stimulated with anti-CD3/CD28 plate bound antibodies in the absence of PECs

and their supernatants harvested 24 h later. Interestingly, rested and re-stimulated CD4 cells

produced an JEN-γ profile in the secondary stimulation that was similar to those seen in the

primary stimulation (Fig. 24a). Conversely, the production of JL-13 was enhanced in the

secondary stimulation of CD4⁺ cells that came from primary co-cultures where the LNnT-

Dex-PECs were present (Fig 24b). These data indicate that CD4⁺ cells in co-cultures are alive

and furthermore suggest that the majority of T cells isolated after primary co-culture with

LNnT-Dex-PECs are Th2 committed, because the greatest amounts of IL-13 are secreted

following secondary stimulation (Constant, et al, Annu. Rev. Immunol. 15:297, 1997). Other

experiments showed that the addition of IL-12 could restore JEN-γ production in the co-

culture LNnT-Dex/PECs:CD4⁺, confirming that CD4⁺ cells are alive and responding to

external stimuli (Fig 24c). J-n addition, JL-12 in the co-cultures diminished the previously

highly detected IL-13 production (Fig. 24d), thereby suggesting that the absence, or low

levels, of JL-12 in PECs expanded by LNnT-Dex could be a determining factor in the

outcome of the response to anti-CD3/CD28 stimulation by naive CD4⁺ cells.

Blockade of JL-10 but not of TGF-β abrogates suppression activity in PECs recruited by

LNnT-Dex.

Because a significant inhibitory effect was shown in experiments where PECs:CD4⁺

co-cultures were performed with trans wells, and because important differences in the release

of IL-10 and TGF-β from PECs expanded by polyvalent LNnT-Dex versus those recruited by

dextran were observed, neutralization experiments were conducted to determine if either of - 53 - HUI-038CPPC these molecules classically associated with inhibitory effects were involved in the suppressive

activity. As shown in Figure 25, neutralization of IL-10 with 2 μg/ml of blocking monoclonal

antibodies resulted in a significant decrease (95%, p<0.01) in the ability of the PECs

expanded by LNnT-Dex to induce their suppressive effect as measured by cellular

proliferation. In contrast, neutralizing antibodies against TGF-β 1,2 and 3 had no significant

effect on cellular proliferation (Fig 25). This shows that JL-10 plays a key role as a soluble

factor in the suppressive activity associated with polyvalent LNnT-Dex expanded PECs.

In summary, this study demonstrated that as early as 2 h after injection of the neo-

glycoconjugate, the peritoneal cell population was significantly expanded in Gr-

l⁺/CDllb⁺/F4/80⁺ cells. This population was maintained in vivo for at least 24h post- injection of LNnT-Dex. The cells expanded by LNnT-Dex adoptively suppress a primary

response to powerful stimuli such as anti-CD3 and anti-CD28 antibodies in naive CD4⁺ cells.

Removal of Gr-1⁺ cells from this suppressor population renders the remaining PECs unable

to suppress CD4⁺ cell proliferation in response to the same stimulation, even at the 1:2 ratio

of PECs:CD4⁺ cell. These findings are evidence that the Gr-1⁺ cells are critical to the

suppression activity. Similarly, it was demonstrated that this suppressive population can

exert its action by both cell to cell contact (e.g., involving a membrane-asociated factor) and

through the release of soluble factors.

These experiments also demonstrated that CD4⁺ cells are still viable after rest and re¬

stimulation. Supernatants obtained from co-cultures where there was an inhibition of 90% in

the proliferative response of CD4⁺ cells, contained high levels of cytokines, indicating that

the cells were alive. Furthermore, when CD4⁺ cells in co-cultures were separated and given a

secondary stimulation they maintained the profile of cytokine production seen in the initial

co-cultures, that is, lower levels of JFN-γand higher production of JL-13 compared with their - 54 - HUI-038CPPC respective controls. Together, these data support the fact that CD4⁺ cells remain viable in the

presence of PECs recruited by LNnT-Dex.

Surprisingly, the blockade of TGF-β, a cytokine known to induce suppression in

activated T cells (29, 35) did not restore the CD4⁺ proliferative response. Tin contrast to

TGF-β, IL-10 was a major regulatory factor involved in the suppressive activity of PECs

recruited by LNnT-Dex. As shown in the blocking Ab experiments, blockade of IL-10 was

enough to restore the proliferative response of CD4⁺ cells co-cultured with PECs recruited by

LNnT-Dex. IL-10 is a potent suppressor of cell mediated immune responses ( involved in

restricting cellular proliferation in S. mansoni infected individuals. This correlates with

results which indicate that some of the complex carbohydrates structurally related to S.

mansoni are critical in the down-regulation of the early events that favor the development of a Thl -type response. Another significant biological function of IL-10 is inhibition of both JPN-

γ secretion by T cells and NO production in activated macrophages. Taken together, PECs

expanded by polyvalent LNnT-Dex produce two key cytokines with suppressive or anti-

inflammatory activity, IL-10 and TGF-β, which are capable of counterbalancing the pro-

inflammatory cytokines IL-lβ, IL-12, JL-18 and JEN-γ .

The findinga are interesting in light of the fact that other groups have identified

similar populations of suppressor cells (Gr-l⁺/CDllb⁺) recruited by virus inoculation

(Bronte, et al, J. Immunol. 161, 5313, 1998) or by tumor implantation (Bronte, et al. J.

Immunol, 162:5728, 1999). In either case, the suppressor cell populations are expanded

approximately 10 days post-challenge compared with the 2 hours seen in the system

described herein. The rapid and local responses to the polysaccharide LNnT-Dex reported

indicate that these sugars can be used therapeutically when an effective and fast anti-

inflammatory response is needed, or as a potential adjuvant to induce Th2 responses later in disease progression. - 55 - HUI-038CPPC

EXAMPLE 6: LNnT Expands GR1+ Macrophages That Supress CD4+ T Cell

Proliferation Via an INF-γ and NO Dependent Mechanism

All suppressor macrophages resemble one another phenotypically and share a similar

suppressor function. Despite these similarities, mechanistically there are two different

subpopulations of suppressor macrophages: classically activated (CA) macrophages, which

are JEN-γ dependent, and alternatively activated (AA) macrophages, which are IL-4

dependent. IFN-γ dependent Grl⁺ suppressors are found in the bone marrow and peripheral

lymphoid organs in cancer patients or tumor-bearing mice and during viral infection (Young

et al, J. Immunol. 156, 1916, 1996; Kusmartev, et al, J. Immunol, 165:779, 2000; Cauley, et

al, J. Immunol, 165:6065, 2000). A A macrophages are found in the peritoneal cavity (PC) of mice in response to the filiarial nematode Brugia malayi as well as in other cases (Goerdt, et

al, Immunity 10:137, 1999; MacDonaldet al, J. Immunol. 160:1304, 1998; MacDonald, et

al, Pathobiology 67:265, 1999: Allen, et al, J. Immunol. 165:6723, 2000; Goerdt et al,

Pathobiology 67;222, 1999).

Mice

Female BALB/c and SCID:SCID mice between 6 and 8 weeks of age were used in

these studies and were purchased from (Taconic Farms, Germantown, NY and The Jackson

Laboratory, Bar Harbor, ME).

Media and reagents

LNFPJH-Dex and Dextran were obtained from Neose Technologies Inc., Horsham,

PA. The glyco-conjugate consisted of 12 LNFPJU molecules conjugated to a 10 kDa

molecule of dextran. RPMI 1640 medium was supplemented with 10% fetal bovine serum

(FBS; Hyclone, Logan, UT), 100 U/mg penicillin, 100 μg/ml streptomycin, 0.05 mM 2- - 56 - HUI-038CPPC

Mercaptoethanol, and 2 mM glutamine (Sigma, St. Louis, MO). L-nMMA (Λ^-monomethyl-

L-arginine) and MnTBAP (manganese [U J tetrakis [4-benzoic acid] porphyrin) were

obtained from Calbiochem (San Diego, CA).

Monoclonal Antibodies

Purified anti-mouse CD3e (clone 145-2C11), anti-CD28, anti-mouse JEN-γ, CDl lb-

FJTC, Gr-l-PE (RB6-8C5), CD4-PE, purified rat IgGl and IgG2a isotype control IgG were

purchased from PharMingen (San Diego, CA). Rat anti-mouse F4/80-Cy5 mAbs was

purchased from Serotec (Raleigh, NC). Anti-FJTC and anti-PE microbeads were obtained

from Miltenyi Biotec (Auburn, CA).

Cell preparation

Mice were injected intraperitoneally with 50 μg of LNFPUI-Dex or Dextran in Hanks'

Balanced Salt Solution (HBSS; Life Technologies, Frederick,, MD ). Approximately 20 hrs

post-injection, mice were euthanized by CO₂ inhalation and peritoneal cells (PECs) were

obtained by lavage under sterile conditions by injection of 5 ml HBSS into the peritoneal

cavity. I-n some experiments, Grl⁺ and F4/80⁺ cells were separated from PECs of LNFPUI-

Dex or Dextran injected mice. PECs were incubated for 30 min at 4°C with anti-F4/80-FITC

and anti-Grl-PE antibodies. Cells were washed to remove unbound antibodies then incubated

with anti-FJTC and/or anti-PE microbeads for 20 min at 4°C. Positive and negative cell

populations were then separated on a MACS column according to the manufacturer's

instructions. Purity of the various populations was determined via FACScan.

T cell activation and proliferation assay - 57 - HUI-038CPPC

Spleen cell preparations were prepared from naϊve mice. Following lysis of red blood

cells with Boyle's solution, splenocytes were washed and resuspended at 5-10 x 10⁶/ml in

PBS. CFSE (5- [and -6]-carboxyfluorescein diacetate succinimidyl ester; Molecular Probes,

Eugene, OR) was added to a final concentration of 5 μM and incubated at room temperature

for 8 min. Next, FBS was added to a final concentration of 20%. Cells were washed three

times in cold RPMJ-/10% FBS and plated. CFSE labeled splenocytes (at a concentration of

lxl0⁶/ml and a volume of 0.5 ml) were plated in 48 well plates coated with 1 μg/ml anti-CD3

and 5 μg/ml anti-CD28 mAbs. Splenocytes were cultured for three hours on antibody coated

plates then 0.5 ml of PECs were added such that the ratios of PECs to splenocytes/T cells

were 1:2, 1:4, 1:8, and 1:16. The co-cultures were incubated for 72 hours, then harvested and

analyzed by flow cytometry. In some experiments, L-nMMA, MnTBAP, or anti-IFN-γ mAbs

(final concentrations 0.5 mM, 10 μM, and 10 μg/ml, respectively) were added to co-culture of

splenocytes and PECs. In the same experiments, co-cultured supernatants were harvested and

levels of NO measured by mixing equal volumes of culture supernatants (50 μl) and Greiss

reagent. After 5 min incubation at room temperature, the absorbance was read at 550 nm

using a Spectramax plate reader (Molecular Devices, Sunnyvale, CA). Nitrite concentrations

were determined by comparing absorbance values of the test samples to a standard curve

generated by serial dilution of 62.5 μM sodium nitrite.

Flow cytometry

FJTC or PE labeled positive and negative populations of Grl and F4/80 PECs,

isolated as described in Cell preparation, or CFSE labeled co-cultured cells (2-5 x 10⁵) were

transferred to 12 x 75-mm polystyrene tubes and washed with FACs buffer (PBS containing

0.1% BSA and 0.1% sodium azide). Cό-cultured cells or freshly isolated PECs were stained with various combinations of mAbs for 30 min on ice in the dark and washed twice in FACs - 58 - HUI-038CPPC buffer. Acquisition of cells was preformed using a FACScalibur flow cytometer (Becton

Dickinson, San Jose, CA). A minimum of 20,000 events was required for analysis. Cell

populations were analyzed using CELLQUEST software (Becton Dickinson).

Injection of LNFPUI-Dex expands Grl⁺ macrophages in the peritoneal cavities of mice.

BALB/c mice were used to study the expansion and function of PEC subpopulations

within 20 hours post-injection of 50 μg of LNFPUI-Dex or Dextran alone. As shown in

Figure 26A, PECs from LNFPUI-Dex injected mice contained higher numbers of

Grl⁺/CDllb⁺ cells than control (uninjected and Dextran injected) mice. The presence of

GrlYF4/80⁺ PECs in LNFPUI-Dex injected mice is shown in Figure 26B. Analysis of double- positive cells from sugar-injected mice showed two subpopulations characterized as Grl^4"

high or low (arrows on Figure 26). Both Grl⁺ subpopulations express CDl lb and F4/80

surface markers. To study the expression of Grl by macrophages, we gated double-positive

CDl lb/F4/80 cells (Figure 26C, left panel). The results, showing Grl-positive cells, are given in the histogram in Figure 26C (right panel).

The percentages of Grl⁺/CDl lb⁺ cells and Grl⁺ cells of CDllb/F4/80-positive PECs

are summarized in Table IN. Injection of LΝFPUI-Dex resulted in a significant (p< 0.05)

increase in the percentage of CDl lb⁺/F4/80⁺ macrophages expressing the Grl⁺ marker

(75.6+2.1%), compared to control (uninjected and Dex-injected mice, 18.5+1.8% and

18.4+1.9 %, respectively). It is clear that not only the percentage but also the absolute number

of Grl⁺ macrophages was dramatically increased in mice injected with LNFPUI-Dex.

PECs from mice injected with LNFPUI-Dex conjugate suppress the proliferation of CD4⁺ T

cells stimulated with anti-CD3 and anti-CD28. - 59 - HUI-038CPPC

Further experiments were perforemed to determine if Grl^4", F4/80^4" PECs from

LNFPUI-Dex injected mice were able to suppress proliferation of naive splenocytes. Naϊve

splenocytes were stained with the proliferation marker carboxyfluorescein diacetate

succinimidyl ester (CFSE) before stimulation with anti-CD3 and anti-CD28 antibodies

(Lyons, et al, Jimmunol Methods 171:131, 1994). CFSE-labeled splenocytes were incubated

in 48 well plates coated with anti-CD3/CD28 mAbs for three hours then mixed with varying

numbers of PECs from uninjected, Dex-injected or LNFPUI-Dex injected mice. In this

example, CD4⁺ T cells were gated and analyzed their proliferation. Figure 27 shows data

from a representative experiment where PECs from mice injected with LNFPUI-Dex, at ratios

of 1:4 and 1:8 (PECs to splenocytes), significantly suppressed CD4⁺ T cell proliferation compared with PECs from control mice at the same ratios.

Suppression caused by PECs from LNFPJH-Dex injected mice is NO dependent.

Activated macrophages often suppress T cell activity via nitric oxide (NO)

production. Measurement of NO production in supernatants of co-cultured PECs and naϊve

splenocytes is shown in Table V. In all experiments, at all ratios of PECs to splenocytes,

slightly higher nitrite levels were detected in co-culture supernatants containing PECs from LNFPUI-injected mice. Neither PECs nor splenocytes alone generated NO.

To confirm that NO was involved in the suppression of naϊve splenocytes, L-nMMA,

an inhibitor of inducible NO synthase (iNOS) was added to co-cultures. The effects of not

only L-nMMA but also a superoxide dismutase (SOD) mimetic (MnTBAP). MnTBAP did

not reverse immune suppression at any concentrations tested. However, addition of L-nMMA

to co-cultures completely restored the ability of CD4 T cells to proliferate in response to

stimulation with anti-CD3/CD28 mAbs at all ratios of suppressors to responders examined

(Figure 28 A). NO production in supernatants obtained from the same co-cultures is shown in - 60 - HUI-038CPPC

Figure 28B. The concentration of NO was dependent on the numbers of PECs in co-culture

with naϊve splenocytes. L-nMMA decreased NO in cell cultures to background level.

An IFN-γ-dependent mechanism of inhibition of T cell proliferation in vitro.

Natural suppressor macrophages have been shown to suppress via JEN-γ or JL-4

dependent mechanisms (Holda et al., 1988; Huchet et al., 1993; Rodriguez et al., 1994;

Klimpel et al., 1990; Angulo et al., 1995). In addition, NO production by suppressor cells has

been linked with an JFN-γ dependent mechanism (Angulo et al., 1995). To further define the

suppressive mechanism employed by LNFPUI-Dex PECs, co-culture experiments with

neutralizing antibodies to JEN-γ or JL-4 were conducted.

Figure 29 demonstrates that addition of anti-JEN-γ Abs to co-cultures completely

restored the proliferative activity of naϊve CD4⁺ T cells activated with anti-CD3/CD28 mAbs.

In contrast, CD4⁺ T cells in co-cultures containing anti-JL-4 (not shown) or isotype control Abs did not restore proliferation, demonstrating that LNFPUI-Dex expanded suppressor

macrophages were functioning via an JEN-γ but not JL-4 dependent mechanism. NO

production of co-cultured PECs and splenocytes was reduced to 2.5 μM at all concentrations

of PECs in wells where anti-IFN-γ was added. Thus, JFN-γ is a critical factor for development

of LNFPUI-Dex induced PEC suppressor activity.

Grl^hlgh positive cells are responsible for the suppression of anti-CD3/CD28-induced T cell

proliferation.

Previous studies have shown that suppression of T cell proliferation is linked to the

presence of Grl⁺ cells which also express CDl lb and/or F4/80 (Kusmartev, supra; Cauley,

supra). To identify whether suppression was due to Grl^4" macrophages, positively selected - 61 - HUI-038CPPC

Grl/F4/80 cells were used. In these experiments, PECs were separated into Grl^4"/- and

F4/80^4"/- populations as described. FACS staining (Figure 30A) confirmed the efficiency of

the selection. The Grl^4" subpopulation contained 99.7% positive cells. The ability of

Grl⁺/F4/80⁺ and the various depleted subpopulations to suppress naϊve splenocytes were

tested in co-culture experiments. The population that was depleted of F4/80⁺/Grl^hlgh cells still

contained Grl^4" cells, although the overall fluorescence intensity was low (Figure 30A, right

panel). In co-culture experiments it was found that the Grl^hlgh, F4/80^4" purified subpopulation

was responsible for suppression. The representative pattern in Figure 5B shows that double-

positive cells blocked T cell proliferation even at a ratio of 1:16, PECs to splenocytes. In

contrast, the PEC population that contained Grl^low/F4/80^" cells did not inhibit splenocyte proliferation nor did the F4/80⁺/Grl^low population.

To confirm the role for F4/80^4" cells in suppression, F4/80^4" cells were isolated using

anti-FJTC microbeads. Grl^4" cells were isolated from this same population of F4/80^4" cells by

anti-PE microbeads and MACS column separation. It was noted that following purification, all F4/80^4" cells expressed the Grl marker, yet in contrast, not all Grl^4" cells express the F4/80

marker. Similar to what was observed with total PECs from LNFPJH-Dex injected mice,

suppression by purified Grl^4" cells was also IFN-γ-dependent, as anti-IFN-γ mAbs completely

abrogated suppression at all ratios of suppressor/responder (Figure 30 C). Supernatants from

co-cultures of Grl7F4/80^" PECs with splenocytes which contained small amounts of NO

products, as opposed to co-cultures of splenocytes and Grl⁺/F4/80⁺ PECs, containing high

amounts of NO. Thus, F4/80⁺/Grl⁺ double-positive macrophages are responsible for the

observed suppressive activity of PECs from LNFPUI-Dex injected mice.

Mechanism of PEC suppression is T cell-independent. - 62 - HUI-038CPPC

In normal mice, resident PECs include macrophages, neutrophils and NK cells, as

well as T and B cells. LNFPUI-Dex or dextran were injected into SCUD mice to determine if

the LNFPUI-Dex induced accumulation of Grl^4" macrophages was dependent on the presence

of T cells in the PEC population. Table VI compares the percentage Grl⁺ cells of the

CDllb⁺/F4/80⁺ population in both SCUD and BALB/c mice 20 hours after LNFPUI-Dex or

Dex injection. In LNFPUI-Dex injected SCUD mice, 92.5±2.5% of the CDllb⁺/F4/80⁺ cells

were Grl^4" vs 75.6+2.1% in BALB/c, though absolute numbers of PECs from SCUD mice

were substantially less than those from BALB/c. The greater percentage of Grl^4" cells seen in

SCIDs compared to BALB/c may be due to immuno-compensatory mechanisms in SCUD

mice.

The PECs from SCUD mice injected with LNFPUI-Dex were functional suppressors,

as shown in Figure 31. The degree of suppression was higher than in wild-type mice, even when the ratio of PECs to splenocytes was 1:16, and inhibition was also NO dependent, as

addition of L-nMMA to co-cultures completely restored proliferation of CD4^4" T cells at all

ratios of suppressor :responder. For both strains of mice, the amount of NO products present

was found to be dependent on the number of PECs in culture, although the difference in the

amount of NO products between control and SCJ-D mice was not significant. This may be due

to the existence of a "ceiling" concentration of NO products.

Because expansion of natural suppressor cell populations in helminth infected or

tumor bearing mice has been reported to take days to weeks, this phenomenon has not been

considered an innate response. However, in contrast to these studies, these experiments

demonstrated that injection of LNFPJH-Dex significantly expanded the peritoneal suppressor

macrophage population within 20 hours. This observation, coupled with the results from a - 63 - HUI-038CPPC study showing that suppressor macrophage populations could be increased within 48 hours by

injection of superantigen, suggest that expansion of this cell type may occur through an innate

immune response triggered by ligation of pattern recognition receptors (Cauley, supra).

The rapid expansion of suppressor macrophages observed here, following injection of

LNFPUI-Dex, has not previously been observed. Twenty hours after injection of LNFPUI-

Dex, the percentage of Grl ^" cells as a subpopulation of F4/80⁺/CDllb⁺ PECs was expanded

more than four-fold, to 75%, compared with 18% in uninjected or Dex-injected mice. It was

demonstrated that these cells were functional suppressors, as they inhibited the proliferation

of anti-CD3/CD28 stimulated naϊve T cells in vitro at ratios of 1:4 and 1:8

(PECs:splenocytes), whereas PECs from control mice did not. The suppressor population

described in our study suppressed via NO and was JEN-γ dependent and JL-4 independent.

Thus, in comparison to the studies that generated JL-4 dependent, AA suppressor populations including those examining suppressor cells from filaria harboring mice, injection of LNFPUI-

Dex led to expansion of CA, IFN-γ dependent suppressors. In addition to demonstrating that

the mechanism of suppression was NO and JPN-γ dependent, these experiments also

demonstrated that Grl⁺/F4/80⁺ PECs depleted of Grl^4" cells were no longer able to suppress

naϊve T cells, functionally showing that the Grl^4" macrophages are the suppressors and that

Grl^low/F4/80^" cells are not. Injection of LNFPJH-Dex gave rise to two populations of Grl^4"

cells which were termed high and low. In this study, depletion of either Grl^hlgh or

F4/80⁺/Grl⁺ cells from PEC populations of LNFPUI-Dex injected mice eliminated the ability

of the PECs to suppress naϊve T cell proliferation.

Considering the rapid time frame in which the suppressor population expanded

following LNFPUI-Dex injection, it is unlikely that these cells required other cell

populations, and they likely directly interacted with the LNFPUI-Dex conjugate, leading to

upregulation of Grl . Inject of LNFPUI-Dex into SCUD mice resulted in similar kinetics of the - 64 - HUI-03βCPPC

PEC suppressor population, showing that this phase was T cell independent. Further, Grl^4"

suppressor cells from SCUD mice were functional suppressors and the mechanism of

suppression was also NO-dependent. In the current study it was demonstrated that increased

numbers of Grl⁺/F4/80⁺ cells were accompanied by strong suppression and NO production

during co-culture of PECs and naϊve splenocytes, and the effects of suppression and NO

production were abrogated by addition of an inhibitor of NO synthase (L-nMMA) .

Taken together, injection of the S. mansoni expressed immunoregulatory

oligosaccharide as the neo-glyco-conjugate LNFPUI-Dex rapidly expanded a peritoneal

suppressor macrophage population that phenotypically and mechanistically resembles the CA

suppressor macrophages defined in viral, tumor and superantigen exposed murine systems.

The kinetics of expansion of the suppressor macrophages reported here are the most rapid

observed to date, and suggest an innate response.

EXAMPLE 7 : NOD-Model of Insulin-Dependent Diabetes Mellitus

In the NOD-Model, mice develop pancreatitis starting around 4 weeks of age, and

75% of the femal mice develop J-nsulin-dependent diabetes mellitus (JJDDM) between 25-30

weeks of age. While some interventions admistered between 4-6 weeks of age have been

found to be effective in preventing onset of the disease in these mice, few interventions

initiated after 12 weeks are effective.

In this example, age matched female NOD mice were allowed to progress until 22

weeks of age. Blood sugars were examined, and found to be elevated at this age, but not

technically diabetic. Mice were then given a single JP injection of LNn-T(25)-Dex or dextran

(100 ug). Eight weeks later, mice were observed for symptoms and blood glucose levels were

measured by standard methods. - 65 - HUI-038CPPC

The mice injected with dextran were completely wet, and the bedding soaked,

indicating the presence of large amounts of dilute urine. In contrast, the mice injected with

LNn-T-Dex were dry and the bedding was dry. The blood glucose levels in mg/dL for the

mice are shown in Table NU.

Table NU

Treatment Mouse 1 Mouse 2 Mouse 3 Mouse 4

DEX 796 732 191 271

LΝnT 190 195 149 144

These results demonstrate that LΝnT is effective in lowerin the blood glucose levels

in a art-recognized animal model of UDDM.

EXAMPLE 8 SCUD T Cell Transfer Model of IBD

In this example, one million wild-type naϊve (CD45Rb^hlgh) T cells into SCUD mice.

The onset of inflammatory bowel disease occurs 3-5 weeks following cell transfer, and visual

symptoms (diarrhea, weight loss, prolapsed rectum) occur from 8-12 weeks post transfer.

Animals were treated one week after cell transfer with IP injection of 100 ug of LΝnt(35)-

Dex or Dextran, followed by weekly injections. Experiments were terminated when mice

demonstrated serious diarrhea or prolapsed rectum. Animals were necropsied, examined for

gross lesions, and the tissues were examined using standard histological methods.

Li one experiment, 75% (3/4) of the mice treated with LNnT were demonstrated no

diarrhea or prolapsed rectum. In a second experiment, 100% (4/4) of the LNnT treated mice

were free of IBD symptoms. These results demonstrate that administration of LNnt is

effective in preventing onset of JBD in an art-recognized model of IBD. - 66 - HUI-038-β.PP

.T B LE IT

Strain BALB/c C57BL/6

PECs' origin Dextran LNnT Dextran LNnT

Cytokines

IFN-γ 2h 497+35 22.6+22* ND ND

18h 427+78 68.8+26* ND ND

DL-18 2 2148+146 418+150* 882+112 195±65*

lδh 591+89 365+37* 1247+446 68.2+30*

IL-12 2h 350+66 43+34* 761+227 250+132*

18h 1883+434 332+123* 1428+39 429+71*

IL-lβ 2h 253+29 68+11* <20 <20

18h 241+94 <20* 60+10 31.5+8*

TABLE It Spontaneous pro-inflammatory cytokine production by PECs recruited by

LNnT or Dextran in BALB/c or C57BL/6 mice- PECs were obtained at 2h or 18h after

injection, adjusted to

and 1 ml added in 24-wells plate. Supernatants were

harvested 96h later. Data are representative of 5 independent experiments, using 4 mice

per group individually assayed. Data shows Mean+SE. *p value <0.05 compared with

control values. ND= Not determined. (o - )al- 038&PPc

ThBLE WL

Strain BALB/c CS7BL/6

PECs¹ origin Dextran LNnT Dextran LNnT

Cytokines

IL-10 2h 108+20 337+39* 188.1+37 438.4+31*

18h 397+32.5 1140+327* 255.8+64 933-5+65*

TGF-β 2h 434±10.7 607±34* 706.8+22 995.8+154

18h <20 7000+375* 397+33 1433+409*

TABLE πr-Spontaneous production of anti-inflammatory cytokines by PECs recruited by

LNnT or Dextran in BALB/c or C57BL/6 mice. PECs were obtained and cultured under

the same conditions as described for table I. Data are representative of 5 independent

experiments, using 4 mice per group individually assayed. Data are shown as Mean±SE.

p value <0.05 compared with control values.

TTtøiε S

Table I. Percentage of GrJ⁺ cells between groups of control, Dextran and LNFPUI-Dex injected mice

PECs obtained from control, Dextran. or LNFPIII-Dex injected mice

stained with antibodies against Grl/CDllb and F4/80. The results shown are representative of several different .experiments. *p<0.001 between control (uninjected and Dex-iηjected) and LNFPJJI-Dex injected mice.

/ ABLE 3T

Table II. Nitric oxide (NO) production (μM) by co-cultures of CD3/CD28 stimulated naive splenocytes and PECs

Naϊve splenocytes were preincubated for 3 hours with anti-CD3/CD28 mAbs. then PECs from control (uninjected or Dex injected) and LNFPIII-Dex injected mice were added at the above ratios. After 72 hours of co-culture- supernatants were harvested and NO products assayed.

^♦Between uninjected and LNFPIII-Dex injected mice. p<0.05 fBetweenDEX injected and LNFPJJI-Dex injected mice, p<0.05

TABLE £L

Table IQ. Percent Grl * of CDUb^* IF O* cells from BALB/c and SCJD mice

PECs obtained from control, Dextran, or LNFPHI-injected mice were stained with antibodies against Grl/CDl lb and F4/80 ^♦Between SCID and BALB/c, p<0.05

- 71 - HUI-038CPPC

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

- 72 - HUI-038CPPC CLAIMS We claim:

1. A method of modulating an immune response in a subject comprising: administering to the subject an agent comprising multivalent lacto-N-neotetraose (LNnT), such that an immune response is modulated in the subject.

2. The method of claim 1, wherein the agent comprises LNnT conjugated to a protein carrier.

3. The method of claim 2, wherein the agent comprises LNnT conjugated to human serum albumin.

4. The method of claim 1, wherein the agent comprises LNnT conjugated to a carbohydrate polymer.

5. The method of claim 4, wherein the agent comprises LNnT conjugated to dextran.

6. The method of claim 1, wherein the agent is administered intraperitoneally.

7. The method of claim 1, wherein the agent is administered intravenously.

8. The method of claim 1, wherein the immune response that is modulated is an IgE response.

9. The method of claim 8, wherein a polyclonal IgE response is stimulated. - 73 - HUI-038CPPC

10. The method of claim 8, wherein the method comprises administering the agent to the subject prior to exposure of the subject to an allergan and wherein production of an allergan-specific IgE response in the subject is inhibited.

11. The method of claim 1 , wherein the immune response that is modulated is shock.

12. The method of claim 11 , wherein the method comprises administering the agent to a subject susceptible to shock.

13. The method of claim 12, wherein the subject susceptible to shock is a surgery patient.

14. The method of claim 1, wherein the immune response is an autoimmune response in a subject suffering from an autoimmune disease.

15. The method of claim 14, wherein the autoimmune disease is inflammatory bowel disease.

16. The method of claim 14, wherein the autoimmune disease is diabetes.

17. The method of clailm 14, wherein the autoimmune disease is arthritis.

18. A method of inhibiting induction of Grl+, CDl lb+ suppressor cells in a subject comprising: administering to the subject an agent comprising monovalent lacto-N- neotetraose (LNnT), such that induction of Grl+, CDl lb+ suppressor cells in the subject is inhibited.

1 . The method of claim 18, wherein the agent is administered intraperitoneally. - 74 - HUI-038CPPC

20. The method of claim 18, wherein the agent is administered intravenously.

21. The method of claim 18, wherein the subject is suffering from cancer.

22. A method of stimulating production of a Th2-type cytokine comprising contacting a cell capable of producing a Th2-type cytokine with an agent comprising multivalent lacto-N-neotetraose (LNnT) such that a Th2-type cytokine is produced by the cell.

23. The method of claim 22, wherein the cytokine is IL-4.

24. The method of claim 22, wherein the cytokine is IL-10 or IL-13.

25. The method of claim 22, wherein the agent comprises LNnT conjugated to a protein carrier.

26. The method of claim 25, wherein the agent comprises LNnT conjugated to human serum albumin.

27. The method of claim 22, wherein the agent comprises LNnT conjugated to a carbohydrate polymer.

28. The method of claim 27, wherein the agent comprises LNnT conjugated to dextran.

29. A method of stimulating proliferation of splenocytes comprising contacting splenocytes with an agent comprising multivalent lacto-N-neotetraose (LNnT) such that proliferation of the splenocytes is stimulated. - 75 - HUI-038CPPC

30. . The method of claim 29, wherein the agent comprises LNnT conjugated to a protein carrier.

5 31. The method of claim 30, wherein the agent comprises LNnT conjugated to human serum albumin.

32. The method of claim 29, wherein the agent comprises LNnT conjugated to a carbohydrate polymer. ισ

33. The method of claim 32, wherein the agent comprises LNnT conjugated to dextran.

34. A pharmaceutical composition comprising an agent comprising multivalent 15 lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition as a modulator of IgE responses in a subject.

35. The pharmaceutical composition of claim 34, wherein the agent comprises LNnT conjugated to a protein carrier.

20

36. The pharmaceutical composition of claim 35, wherein the agent comprises LNnT conjugated to human serum albumin.

37. The pharmaceutical composition of claim 34, wherein the agent comprises 25 LNnT conjugated to a carbohydrate polymer.

38. The pharmaceutical composition of claim 37, wherein the agent comprises LNnT conjugated to dextran. - 76 - HUI-038CPPC

39. A pharmaceutical composition comprising an agent comprising multivalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition for the treatment or prevention of shock in a subject.

40. The pharmaceutical composition of claim 39, wherein the agent comprises

LNnT conjugated to a protein carrier.

41. The pharmaceutical composition of claim 40, wherein the agent comprises LNnT conjugated to human serum albumin.

42. The pharmaceutical composition of claim 39, wherein the agent comprises LNnT conjugated to a carbohydrate polymer.

43. The pharmaceutical composition of claim 42, wherein the agent comprises LNnT conjugated to dextran.

44. A pharmaceutical composition comprising an agent comprising monovalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition for the treatment of cancer in a subject.

45. A method of inhibiting inflammation in a subject comprising administering to the subject and agent comprising LNnT such that inflammation is inhibited.

46. A method for expanding a Gr-1+ cell population in a subject comprising administering an effective amount of and agent comprising LNnT to the subject.