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WO2015081235A1 - Méthodes d'induction de la polyfonctionnalité des lymphocytes t - Google Patents

Méthodes d'induction de la polyfonctionnalité des lymphocytes t Download PDF

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WO2015081235A1
WO2015081235A1 PCT/US2014/067674 US2014067674W WO2015081235A1 WO 2015081235 A1 WO2015081235 A1 WO 2015081235A1 US 2014067674 W US2014067674 W US 2014067674W WO 2015081235 A1 WO2015081235 A1 WO 2015081235A1
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cells
cell
specific
polyfunctionality
spry2
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Jonathan Schneck
Yen-Ling CHIU
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Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled

Definitions

  • the invention is in the field of immunology. BACKGROUND
  • T cells capable of simultaneously producing multiple effector functions are a key subset in the development of effective immune responses against pathogens and cancer (1-7).
  • These T cells in addition to producing cytokines such as IL-2,TNF-a, and IFN- ⁇ , produce chemokines and also display cytolytic function.
  • cytokines such as IL-2,TNF-a, and IFN- ⁇
  • chemokines and also display cytolytic function.
  • optimal polyfunctional memory T cell responses are lost in chronic infections and cancer, both in humans and mice (5, 8, 9).
  • T cell polyfunctionality correlates with differences in memory T cell formation in response to immunization as well as to viral infection. Regulation of vaccine-induced adaptive immune responses is complex and, in part, dependent on antigen dose (l).In animal models, high-dose vaccination results in inferior T cell polyfunctionality, poor memory formation and weaker immune protection as compared with optimal dose vaccination (4, 18). As a result, polyfunctional T cells are more than just a "marker" of protective immune response. The molecular mechanisms linking high-dose antigenic stimulation with inferior polyfunctionality and poor memory formation remain largely unclear.
  • T cell polyfunctionality especially in the context of antigen dose. Elucidating the molecular details underlying T cell polyfunctionality could provide additional insights into T cell exhaustion and serve as a basis for vaccine design by optimizing T cell activity against virus infection or cancer.
  • Polyfunctionality is crucial for protective immunity against viruses and cancer (Seder et al, 2008; Wherry, 2011). It is widely accepted that a robust T cell immune response is not only dependent on the absolute cell number, but is also critically dependent on cell quality. Polyfunctional T cells, characterized by the ability to execute multiple effector functions, are a hallmark of healthy, non-exhausted memory T cell response against acute infection, such as influenza infection (Joshi and Kaech, 2008; Weng et al, 2012; Wherry, 2011; Wherry and Ahmed, 2004).
  • FIGS. 1A-D Results of experiments demonstrating that activation of Wnt signaling reprograms memory T cells to a polyfunctional state.
  • FIGS 2A-F Results of experiments demonstrating that Wnt pathway activation with TWS119 induces polyfunctional influenza-specific T cells with a central memory phenotype.
  • Human CD8+ T cells were stimulated with autologous, Ml -peptide loaded moDCs for two weeks (stimulations were given on DO and D7). The culture was also treated with different amount of TWS119, as indicated. Tetramer staining and cell counts were determined on D7 and D14.
  • FIG. 2C and FIG. 2D D14 CD8+ T Cells induced with different concentration of TWS119 were harvested and re-challenged with target T2 cells loaded with Ml peptide for six hours and analyzed for their effector functions.
  • FIG. 2C Representative intracellular cytokine staining for polyfunctionality analysis. Top row: Intracellular cytokine staining for IL-2 versus CD 107. Numbers in the right upper quadrant represent the percentage of IL-2 and CD 107a double-positive cell population. Bottom row: Intracellular cytokine staining for TNFa versus MIP-1 ⁇ . Numbers in the right upper quadrant represent the percentage of TNFa and MIP-1 ⁇ double-positive cell population.
  • FIG. 2D Polyfunctionality pie charts. Each slice of pie represents the percentage of cells expressing between 1-5 effector functions. To compare between pie charts, permutation test with 10,000 repetitions was used using SPICE. FIG.
  • TWSl 19 treatment significantly increased the absolute number of 5+ polyfunctional cells during antigen-specific expansion. Numbers were calculated based on a starting CD8+ culture composed of 100,000 cells.
  • FIG. 2F D14 CD8+M1+ Cells were also analyzed for their surface marker expression. TWSl 19 3mM, blue; 1.5mM, green; 0.7mM, orange; OmM, red. Histograms were gated on CD8+tetramer+ cells. *: P value ⁇ 0.05. **: P value ⁇ 0.01. The results shown are from five different donors.
  • FIGS. 3A-E Results of experiments demonstrating that TWSl 19 treatment enhances polyfunctionality in terminally differentiated CMV-specific T cells.
  • FIG. 3A Ex vivo CMV-pp65 -specific response.
  • FIG. 3B Representative tetramer staining of Human CD8+ T cells stimulated with CMV-pp65-NLV loaded moDCs for two weeks (D14).
  • TWSl 19 were added in culture during stimulation at the indicated concentration.
  • FIG. 3C and FIG. 3D D14 cells were rechallenged with pp65-loaded T2 target cells for six hours to assess their polyfunctional response. Total numbers of pp65-specific 5+ polyfunctional cells were calculated as in FIG. 2. Similar to what has been observed with Ml -specific response, there is enhanced polyfunctional response in TWSl 19 treated cells.
  • FIG. 3E TWSl 19 treated cells also express higher level of CD62L and CD28. TWSl 19 3mM, blue; 1.5mM, green; 0.7mM, orange; OmM, red. Histograms were gated on CD8+CMV-tetramer+ cells. *: P value ⁇ 0.05. **: P valueO.01.
  • FIGS. 4A-D Results of experiments demonstrating that the effects TWSl 19 on CD62L expression and polyfunctionality are seen in divided cells and are independent of dendritic cells.
  • Human CD8+ T cells were stimulated with Ml or CMV-peptide loaded moDCs for two weeks in the presence or absence of TWSl 19. Before the second moDC stimulation, T cells were labeled with CFSE to monitor population divisions. On D14, cells were either harvested and stained with CD8, tetramer and CD62L, or rechallenged with Ml- labeled T2 cells for six hours for intracellular cytokine staining.
  • FIG. 4A Representative staining showing CD62L by CFSE dilution among CD8+ Ml-tetramer+ cells.
  • TWSl 19 decreased the proliferation of tetramer+ cells, but almost all tetramer+ cells divided.
  • FIG. 4B Representative result of cytokine production by CFSE dilution among CD8+ Ml-tetramer+ cells. T cells induced with TWSl 19 exhibited higher level of IL-2 or TNFa in divided cells.
  • FIG. 4C and FIG. 4D The effect of Wnt pathway activation was observed when T cells were expanded with aAPCs. Purified CD8+ T cells were stimulated with Ml -loaded or pp65- loaded aAPCs weekly for two weeks. Tetramer specificity (FIG. 4C) and T cell
  • FIGS. 5A-E Results of experiments demonstrating that Wnt pathway activation induced a long-standing polyfunctional phenotype associated with other stemness features.
  • FIG. 5A Ml -tetramer positive T cells induced by moDCs in the presence (Blue) or absence of TWSl 19 (Red) were studied for their anti-apoptotic protein expression levels on D14. Numbers shown indicate the MFI value for each staining.
  • FIGS. 5B-5E D14 Ml- specific cells were further cultured in the presence of IL-15 (25ng/ml) for 7 days. CFSE dilution, phenotype and polyfunctionality were analyzed on D21.
  • FIG. 5A Ml -tetramer positive T cells induced by moDCs in the presence (Blue) or absence of TWSl 19 (Red) were studied for their anti-apoptotic protein expression levels on D14. Numbers shown indicate the MFI value for each staining.
  • FIGS. 5B-5E D14 M
  • FIG. 5B Representative CFSE dilution of Ml-tetramer specific cells measured by flow cytometry. PI: proliferation index.
  • FIG. 5C and FIG. 5D Phenotype analysis performed after homeostatic proliferation. A significant higher percentage of cells previously treated with TWSl 19 maintained the preferred CD28+CD62L+ phenotype.
  • FIG. 5E After homeostatic proliferation, superior polyfunctionality is maintained in cells previously treated with TWSl 19. In contrast, cells untreated with TWSl 19 remain poorly polyfunctional.
  • FIGS. 6A-B Results of experiments demonstrating that TWSl 19 induces Wnt pathway activation in memory T cells after ex vivo activation.
  • Memory T cells were isolated from fresh collected PBMCs and stimulated with plate-bound anti-CD3/CD28 for 2h or 6h, in the presence (triangle) and absence (half square) of 3mM TWSl 19.
  • FIG. 6A At 6h, intracellular level of ⁇ -catenin was measured by flow cytometry (3mM TWSl 19, blue;
  • FIG. 7A-C Results of experiments demonstrating that activation of Wnt signaling inhibits the overall expansion of influenza-specific cell but increases the tetramer specificity in culture.
  • Human CD8+ T cells were stimulated with autologous, Ml -peptide loaded moDCs for two weeks (stimulations were given on DO and D7). The culture was also treated with different amount of TWSl 19, as indicated. Tetramer staining and cell counts were determined on D7 and D14.
  • FIG. 7A and 7B Despite higher antigen specificity in the culture as shown in FIG. 2, TWSl 19 inhibits the expansion of all cells in the culture as well as the Ml-specific cells being generated.
  • FIG. 7C CD8+ T cells were labeled with CFSE before second moDC stimulation. On D7, CFSE dilution was analyzed within CD8+Ml-tetramer+ or CD8+M1 -tetramer- populations. TWSl 19 inhibited the overall proliferation of Ml-specific T cells but more inhibitory effect is seem in non-specific CD8+ T cells. *: P value ⁇ 0.05.
  • FIG. 8 Results of experiments demonstrating that individual effector function changes in response to Wnt pathway activation in Ml-specific T cells.
  • D14 after two Ml -loaded moDC stimulations, expanded Ml-specific cells were rechallenged with Ml- loaded T2 cells for six hours to assess their polyfunctionality.
  • IL-2 and TFNa production are significantly improved when cells were treated with TWS 119.
  • * P value ⁇ 0.05.
  • FIG. 9 Results of experiments demonstrating that CMV p65 -specific T cells are terminally differentiated memory cells. Freshly isolated CD8+ T cells from healthy donors were stained with anti-CD8 and Ml - or CMV-tetramer along with other surface markers to determine the ex vivo phenotype of antigen-specific T cells.
  • FIG. 9A Majority of Ml-specific cells was CD28+CD27+ but majority of pp65-specific cells was CD28-CD27.
  • FIG. 9B Percentage of tetramer-specific cells within each memory differentiation subset according to the definition of Sallusto et al. (Sallusto et al., 2004).
  • Ml-specific cells were either CCR7+CD45RA+ (T N ) or CCR7+CD45RA-(T CM ) or CCR7-CD45RA- (T EM ) phenotype.
  • T N CCR7+CD45RA+
  • T CM CCR7+CD45RA-(T CM )
  • T EM CCR7-CD45RA-
  • FIG. 10 Influenza virus Ml -pulsed moDCs induce concentration-dependent proliferation of Ml-specific CD8+ T cells with variable levels of polyfunctionality.
  • FIG. lOA-C Percentage of Ml -specific T cells and Ml -specific T cell expansion.
  • Autologous moDCs pulsed with variable amounts of the HLA-A*0201 restricted immunodominant Ml peptide (10 ⁇ 10 fM) were used to stimulate HLA*A201+ CD8+ T cells ex vivo weekly for 2 weeks. Cultures were analyzed on D14.
  • Numbers in red represent the percentage of IL-2-producing (right upper quadrant) versus IL- 2-negative cells (right lower quadrant) in the CD107a-positive cell population.
  • Bottom row intracellular cytokine staining for TNF-a versus MIP- ⁇ .
  • Numbers in red represent the percentage of TNF-a-producing(right upper quadrant) versus TNF-a-negative cells (right lower quadrant) in the ⁇ - ⁇ -positive cell population.
  • FIG. 10E Percentage of individual effector function expression out of total antigen specific response.
  • FIG. 10F Percentage of individual effector function expression out of total antigen specific response.
  • FIG. 11 Microarray analysis reveals unique molecular signatures associated with T cell polyfunctionality.
  • FIG. 11 A Heat map of differentially expressed genes among T cells induced by 10 ⁇ moDCs or 10 nM moDCs (adjusted P ⁇ 0.05, fold change > 1.6). Red and blue indicate increased and decreased gene expression, respectively.
  • FIG. 11B Expression level of EOMES among T cells induced by 10M moDCs (red) or 10 nM moDCs (blue), determined by flow cytometry.
  • FIG. 11C Real-time qPCR validation of TCF7 level. T cells induced with 10 ⁇ or 10 nM pulsed moDCs were tetramer sorted and analyzed by qPCR.
  • GSEA shows that high antigen concentration-induced T cells are enriched with exhaustion signature from LCMV or HIV. In contrast, optimal concentration-induced T cells were enriched with the genetic signature of memory T cells. Molecular signature from PD-1 ligation was not able to distinguish between T cells stimulated with high and optimal antigen concentrations.
  • FIG. 12 Polyfunctionality regulation by antigen concentration is independent of inhibitory receptor signaling.
  • FIG. 12A On D14, T cells were harvested and stained with anti-CD8 and Ml tetramer, then assayed for inhibitory receptor expression. Representative histograms shown were gated on CD8+, Ml tetramer+ population. Red: 10 ⁇ . Blue: 10 nM. Yellow: 10 fM. High antigen concentration-induced Ml-specific T cells (10 ⁇ ) showed higher inhibitory receptor expression except for BTLA.
  • FIG. 12B Real-time qPCR analysis of BATF expression level in D14 sorted Ml-specific cells.
  • aAPCs were made by conjugating biotinylated HLA-A2-M1 and biotinylated anti-CD28 complex onto anti-biotin microbeads (Miltenyi Biotec).
  • FIG. 12D Percentage of tetramer-positive cells and
  • FIG. 12E total Ml-specific cell expansion. Higher concentrations of aAPCs induced more robust cell expansion, similar to results seen in FIG. 1 with peptide -pulsed moDC-induced T cells.
  • FIG. 12F Polyfunctionality assessment of aAPC-stimulated Ml-specific T cells. High concentration aAPCs induced Ml-specific T cells with lower polyfunctionality even in the absence of inhibitory receptors on the aAPCs. The results are representative of 3 independent experiments performed on at least 3 different subjects. *P ⁇ 0.05.
  • FIGS. 13A-D MAPK/ERK pathway controls T cell polyfunctionality.
  • ER inhibitor U0126 inhibits pERK expression in a concentration-dependent fashion. T cells stimulated by the optimal concentration of antigen were restimulated with PMA/ionomycin in the presence or absence of varying concentrations of the ERK inhibitor U0126 for 10 minutes. Cells were then stained for pERK expression. Red, 100 ⁇ U0126; orange: 1 ⁇ U0126; blue, no U0126; gray, unstimulated.
  • FIGS. 13C-D Optimal concentration-induced T cells were incubated with T2 target cells for 6 hours in the presence of various amounts of U0126 before intracellular cytokine staining and polyfunctionality analysis. U0126 selectively inhibited cytokine secretion but not CD 107a upregulation and MIP-1 expression.
  • FIG. 13E T cells still mediated effective lysis of peptide-pulsed T2 target cells in the presence of 100 ⁇ U0126.
  • FIG. 14 Upregulation of SPRY2 in high antigen concentration- induced T cells inhibits polyfunctionality.
  • FIG. 14A qPCR comparison of SPRY2 expression levels on high and optimal antigen concentration-stimulated T cells.
  • FIG. 14B The knockdown efficiency of lentiviral particles containing shRNA targeting SPRY2. Twenty- four hours after the second high antigen concentration moDC stimulation, cells were transduced with either SPRY2 knockdown virus or NT sequence virus. qPCR, flow cytometry staining of SPRY2 (red, NT virus control; blue, SPRY2 knockdown virus; gray, FMO control) and polyfunctionality assessment were performed on D14.
  • FIG. 14C Representative flow plots of effector functions in virus-transduced T cells.
  • T cells transduced with SPRY2 knockdown virus exhibit enhanced cytokine production of IL-2 and TNF-a as compared with control NT virus.
  • SPRY2 knockdown had no impact on either CD 107a upregulation or MIP-1 ⁇ production.
  • FIG. 14D SPRY2 knockdown virus-transduced T cells showed greater percentage of 5+ polyfunctional T cells than control virus-transduced T cells. *P ⁇ 0.05. The data are representative of more than 3 independent experiments performed on 3 different subjects.
  • FIGS. 15A-F SPRY2 inhibition enhances HIV-specific T cell
  • FIGS. 15A-B SPRY2 expression was studied in 12 HIV-infected HLA- A2+ patients. HIV-Gag-specific T cells and influenza Ml -specific T cells were sorted by pentamer/tetramer staining and mRNA extracted from Gag- and Ml -specific cells for qPCR analysis.
  • FIG. 15A An example of flow cytometry-based simultaneous analysis of Gag- and Ml-specific T cells from PBMCs of an HIV-infected donor.
  • FIG. 15B qPCR and flow cytometry analysis of SPRY2 and PD-1 expression in Gag-specific, Ml-specific, and total CD8+ T cells from HLA-A2+ HIV donors.
  • PBMCs from 19 HIV-infected patients were activated with soluble anti-CD3, anti-CD28, and a mix of CEF/HIV peptide pools. Some cultures were also treated with anti-PD-1 (10 ⁇ g/ml) to block PD-1 signaling during activation. 24 hours later, cells were transduced with SPRY2 knockdown (KD) or NT control lentivirus. On D7 after virus transduction, PBMCs were stimulated with CEF or HIV Gag, Nef, and Tat peptide pools for 6 hours and analyzed for polyfunctionality. (FIGS.
  • FIGS. 17A-B Highly polyfunctional T cells are equipped with superior proliferative capacity. T cells previously stimulated for 2 weeks with either high (10 ⁇ ) or optimal (10 nM) antigen concentration-pulsed moDCs were re-stimulated with different concentrations of antigen-pulsed moDCs. After seven days, cells were harvested, counted, analyzed by tetramer staining and assayed for polyfunctionality. FIG. 17A, Ml-tetramer staining, D21. FIG. 17B, absolute cell expansion after third stimulation. Despite various antigen concentration conditions used for third stimulation, T cells previously stimulated with optimal (10 nM) antigen concentration exhibited superior third week cell number expansion (for all restimulation doses, P ⁇ .05).
  • FIG. 18 Naive T cells exhibit impaired polyfunctional response in response to high concentration of anti-CD3/CD28 stimulations. Naive T cells were isolated using
  • Miltenyi naive CD8+ T cell isolation kit Cells were stimulated with plate-bound antibodies at the indicated concentrations (equal concentrations of anti-CD3, clone HIT31, and anti- CD28, clone 28.2). Cells were harvested on D7 and restimulated with PMH/ionomycin for 5 hours before staining for intracellular cytokines. Consistent with what was observed with Ml -specific T cells, strong stimulation of na ' ive T cells induced poorly polyfunctional T cells.
  • FIG. 19 Phenotypic characterization of Ml -specific T cells induced by different antigen concentrations.
  • T cells were harvested and stained with anti-CD8, Ml tetramer and differentiation markers (FIG. 19 A) or transcription factors (FIG. 19B). Representative histograms shown were gated on CD8+, Ml tetramer+ population. Red, 10 ⁇ ; blue 10 nM; yellow lOfM. All antigen concentrations induced Ml -specific T cells with a CD45RO + , CCR&-, CD28+, CD27+ effector memory phenotype. High (10 ⁇ ) antigen concentration-induced Ml -specific CD8+ T cells expressed significantly lower level of eomesodermin (FIG. 19B) but maintained high levels of T-bet and bcl-2.
  • FIG. 20 Upregulation of Spry2 in high concentration antigen-induced cells depends on the prior activation of MAPK/ERK pathway.
  • D7 CD8+ T cells were restimulated with either high ( ⁇ ) or optimal (lOnM) concentration Ml peptide-pulsed moDCs in the presence of different amounts of the ERK inhibitor, U0126.
  • T cells were harvested and analyzed for SPRY2 levels.
  • FIG. 20A CD8+ T cells induced with 10 ⁇ antigen were analyzed for SPRY2 levels after treatment with varying concentrations of U0126 (1 ⁇ green; 100 nM, blue; no U0126, red).
  • FIG. 20B inhibition of ERK pathway during prior T cell activation significantly inhibited the subsequent upregulation of SPRY2 (*P value ⁇ 0.05).
  • FIG. 21 Representative HlV-specific T cell polyfunctional response and ex vivo polyfunctionality.
  • PBMCs from HIV-infected patients were thawed, rested overnight and stimulated with peptide pools for six hours prior to cytokine analysis.
  • FIG. 20A the direct ex vivo Gag and CEF-specific polyfunctional responses.
  • Gag-specific T cells exhibit much a much lower level of polyfunctionality.
  • FIG. 20B representative flow cytometry results of Gag-specific polyfunctional response in anti-PD-1 and SPR Y2 -knockdown virus treated cells.
  • Supplementary Table 2 Full list of differentially expressed genes identified by the microarray experiment. Group A, high antigen concentration ( 10 ⁇ ); group B, optimal antigen concentration (10 nM). [31] Supplementary Table 3. A full list of functional groups generated from the full list of differentially expressed genes by GO-BP-FAT (Gene Ontology Biological Process, FAT term) tool on the DAVID platform.
  • GO-BP-FAT Gene Ontology Biological Process, FAT term
  • T cells stimulated with a high concentration of antigen upregulated sprouty-2 (SPRY2), a negative regulator of the MAPK/ERK pathway.
  • SPRY2 antigen upregulated sprouty-2
  • the clinical relevance of SPRY2 was confirmed by examining SPRY2 expression in HIV-specific T cells, where high levels of SPRY2 were seen in HIV-specific T cells and inhibition of SPRY2 expression enhanced the HIV-specific polyfunctional response.
  • Our findings indicate that increased SPRY2 expression during chronic viral infection reduces T cell polyfunctionality and identify SPRY2 as a potential target for immunotherapy.
  • Spry2 expression can be inhibited by any method known in the art.
  • Spry2 expression is inhibited by a Spry2 -targeted ribozyme, a Spry2 -targeted antisense oligonucleotide, or a Spry2 -targeted siRNA.
  • Spry2 expression is inhibited by an aptamer, a protein, a peptide, a cyclic peptide, a peptidomimetic or a small molecule.
  • Spry2 expression is inhibited by a polypeptide fragment or mutant form of a Spry2 protein, which may function as a competitive inhibitor of a Spry2-interacting polypeptide (e.g., the c-Cbl E3 ubiquitin ligase, EGFR, Ras, or CIN85).
  • a Spry2-interacting polypeptide e.g., the c-Cbl E3 ubiquitin ligase, EGFR, Ras, or CIN85.
  • the Wnt signaling pathway was identified in the microarray pathway analysis suggesting it may be involved in polyfunctionality regulation.
  • Wnt pathway activation inhibits naive T cell proliferation and effector differentiation, preferentially generating a novel T cell subset with stem-cell features (TSCM, T memory stem cells) (Gattinoni et al., 2009), was preferentially generated.
  • TSCM stem-cell features
  • TSCM cells lack immediate effector functions but are multipotent and have enhanced self-renewal capacity to persist as memory cells in vivo (Gattinoni et al., 2011; Gattinoni et al., 2009; Lugli et al., 2013).
  • Other roles of Wnt signaling in mediating mature T cell response have also been reported.
  • TCF1 is essential for generating memory CD8+ T cells (Jeannet et al., 2010). Enforced expression of ⁇ -catenin and TCF1 inhibits the effector phase of the immune response and enhances the generation of memory T cells in response to Listeria monocytogenes infection in mice (Zhao et al., 2009). Wnt signaling activation also arrests the naive to effector differentiation in human peripheral and cord blood-derived T cells (Muralidharan et al., 2011).
  • Wnt pathway activation promotes epithelial- mesenchymal transdifferentiation (EMT)(Anson et al.; Wu et al.), cardiac hypertrophy (Gessert and Kuhl) and wound healing (Whyte et al .
  • EMT epithelial- mesenchymal transdifferentiation
  • Gessert and Kuhl cardiac hypertrophy
  • wound healing wound healing
  • Wnt pathway negatively regulates regulatory T cell functions (van Loosdregt et al., 2013). Activation of Wnt pathway also programs dendritic cells and NKT cells to tolerance induction (Deng et al.; Oderup et al.).
  • T S C M cells are shown to be superior to central memory cells for adoptive therapy against cancer in preclinical mice models (Gattinoni et al., 2011), the number of TSC M cells from human peripheral blood is extremely limited, thus limiting its potential in clinical use.
  • CD62L+ T cells are capable of entering secondary lymphoid organ and are superior in controlling pathogen challenge (Gattinoni et al., 2005; Hengel et al., 2003).
  • TWSl 19-treated cells also express higher level of CD28 and lower level of KLRG1, again indicating those cells are of less-differentiated state and are preferable for adoptive immunotherapy
  • TWSl 19-treated cells express many central memory T cell associated sternness features, we believe these cells are of superior quality for adoptive T cell therapy. Since the numbers of donor-derived moDCs are very limiting clinically, large number of polyfunctional T cells could be potentially achieved by combining aAPC technology with TWSl 19. Finally, the enhancement of polyfunctionality could provide a therapeutic possibility for curing chronic virus infections.
  • Wnt pathway activation dramatically enhances memory T cell polyfunctionality. This effect not only is observed with the less-differentiated memory T cell specific for influenza virus, but also occurs in the terminally differentiated CMV virus-specific response. Importantly, the effect of TWS119 does not result from cell proliferation arrest but can be observed in cells already underwent multiple divisions.
  • the TWS119-treated cells exhibit many features of "sternness" because treated cells are capable of maintaining the CD62L+ polyfunctional phenotype during homeostatic proliferation and express higher level of anti-apoptotic proteins. These results provide strong evidence that polyfunctionality reprogramming in memory cells can be achieved with Wnt pathway activation. Finally, using the acellular aAPCs, we demonstrated that combing aAPC technology with Wnt pathway modulation is a promising strategy to induce large number of highly functional human antigen-specific T cells for adoptive immunotherapy.
  • the Wnt/p-catenin pathway can be activated by any method known in the art Activators of the Wnt/p-catenin pathway are disclosed, e.g., in Table 1 of US 2012/0046242 and in US 2013/0143227.
  • Wnt signaling pathway activators include Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, Wntl 0b, Wntl 1, and Wntl 6b.
  • the Wnt/ ⁇ - catenin pathway is activated by inhibiting serine -threonine kinase glycogen synthase kinase- 3 ⁇ (GSK3); see, e.g., US 2001/0052137.
  • GSK3 serine -threonine kinase glycogen synthase kinase- 3 ⁇
  • Inhibitors of GSK3 include lithium, LiCl, bivalent zinc, BIO, SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119, Kenpaullone, alsterpaullone, indirubin-3'-oxime, TDZD-8 and Ro 31-8220 methanesulfonate salt; Axin inhibitors, APC inhibitors, norrin, and R-spondin 2; see US 2011/0223660.
  • An inhibitor of SPRY2 expression and/or an activator of the Wnt/p-catenin pathway can be formulated in a composition suitable for therapeutic administration to treat chronic infections (e.g., HIV, HCV) and cancer.
  • a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the inhibitor of SPRY2 expression and/or an activator of the Wnt/p-catenin pathway and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • EXAMPLE 1 Methods for Examples 2-8
  • CD14+ and CD8+ cells were isolated from PBMCs of seven HLA*0201 positive healthy donors using Miltenyi isolation kits.
  • Monocyte-derived dendritic cells (moDCs) were generated as previously described (Ndhlovu et al., 2010; Oelke et al., 2003). Briefly, CD14+ cells were cultured in the presence of 50ng/ml IL-4 and lOOng/ml GM-CSF for six days and then matured with lOng/ml TNFa, lOng/ml IL-1, lOOOU/ml IL-6 and ⁇ g/ml prostaglandin E2.
  • Antibodies and flow cytometry The following antibodies were used: anti- CD8 pacific blue (Biolegend, HIT 8 a), anti-CCR7 PE (Biolegend, G043H7), anti-CD45RA APC (Biolegend, HI 100), anti-CD62L Alexa 647 (Biolegend, DREG-56), anti-CD27 Pe-Cy7 (Biolegend, M-T271), anti-CD28 pacific blue (Biolegend, CD28.2), anti-IL-2 Percp-Cy5.5 (MQ1-17H12), anti- ⁇ - ⁇ PE (BD, D21-1351), anti-TNFa Pe-Cy7 (BD, Mabl l), anti-IFNy APC (BD, 25723.11), anti-CD107a FITC (BD, H4A3), anti-IL15Ra APC (Biolegend, JM7A4), anti-CD 122 BV421 (Biolegend, TU27), anti-CD 132 APC (
  • Alexa647-conjugated anti-KLRGl antibody (Clone 13F12F2) was kindly provided by professor Hans-Peter Pircher (University of Freiburg, Germany). Intracellular staining for ⁇ - catenin was performed using alexa488-conjugated anti-P-catenin antibody (eBioscience, clone 15B8) according to the manufacturer's protocol.
  • CFSE Carboxyfluorescein diacetate Succinimidyl Ester
  • GSK3P serine-threonine kinase glycogen synthase kinase- 3 ⁇
  • TWSl 19 a GSK3P (serine-threonine kinase glycogen synthase kinase- 3 ⁇ ) inhibitor, TWSl 19, has been widely used to mimic the activation of canonical Wnt- signaling pathway (Forget et al., 2012; Gattinoni et al., 2011; Gattinoni et al., 2009;
  • TWSl 19 treatment of naive T cells inhibits their acquisition of effector functions (Gattinoni et al., 2009).
  • To determine the effect of Wnt pathway activation on memory T cell function we isolated naive and memory CD8+ T cells and activated them with anti-CD3/CD28 in the presence of TWSl 19. The effector functions were studied on D7. As shown in FIG. 1 A, effector functions of naive T cells were severely impaired by
  • TWSl 19 memory T cells activated in the presence of TWSl 19 expressed much higher level of IFNy and TNFa.
  • all the cytokines tested including IL-2, TNFa and IFNy as well as the polyfunctionality (as determined by the percentage of cell making all three cytokine simultaneously) were significantly upregulated by TWSl 19 treatment (FIG. 1C). This is in sharp contrast to what is observed in naive T cells, in which all the effector functions and polyfunctionality were inhibited by TWSl 19 (FIG. IB).
  • T CM CCR7+CD45RA-
  • T EM CCR7-CD45RA-
  • T EMR A CCR7-CD45RA+
  • TWSl 19 When co-expression of five effector functions were analyzed, highest dose of TWSl 19 induced 28% of responding cells capable of executing five effector function simultaneously (IL-2+TNFa+INFg+CD107a+MIP-ip+; "5+”), as opposed to only 9% in cells expanded without TWSl 19 treatment (FIG. 2D). TWSl 19 also enhanced the absolute number of 5+ polyfunctional cell being generated (FIG. 2E).
  • Influenza-specific T cells are known to be in early memory differentiation. T cells specific for chronic infections are constantly exposed to antigen stimulation and frequently persist in vivo as advanced differentiated cells. To determine if Wnt pathway activation can also reprogram virus-specific memory T cells with advanced differentiation to a polyfunctional state, we tested the effect of TWSl 19 on CMV pp65 -specific T cells. In humans, CMV-specific T cells are known to be highly-differentiated, have limited effector functions and proliferative capability, and are constantly driven to a immunosenescence state (Fletcher et al., 2005; Hertoghs et al.; Mekker et al., 2012).
  • CMV-specific T cells When analyzed directly ex vivo, CMV-specific T cells have low levels of CD28 and CD27 and the majority of them are of the CD45RA+CCR7- phenotype (terminal effector, T EMR A) (FIG. 9). Consistent with the terminal differentiation phenotype, upon stimulation with pp65 peptide, they produce little IL-2 and thus are not polyfunctional (FIG. 3A).
  • CM V-pp65 -specific T cells from healthy donors were expanded with autologous moDCs in the presence or absence of TWSl 19.
  • TWSl 19-treated culture also had substantially improved CM V-pp65 -specificity in culture (FIG. 3B).
  • CMV-specific cells expanded without TWSl 19 still do not produce significant amount of IL-2.
  • CMV-specific cells are reprogrammed to a much more polyfunctional state with TWSl 19 treatment and express higher level of CD62L and CD28 (FIGS. 3C-3E).
  • TWS 119 acts directly on the T cells or rather through modulation of the stimulatory capacity of the dendritic cells
  • TWSl 19 we tested the impact of TWSl 19 on polyfunctionality in a dendritic cell-free system.
  • HLA- Ig-based aAPCs to stimulate CD8+ T cells (Chiu et al, 2011; Oelke et al, 2003). Briefly, Briefly, Briefly, Briefly, Briefly, Briefly, Briefly, HLA-A2-Ig and anti-CD28 were coupled to cell-sized (4.5 ⁇ ) dextran-coated particles and loaded with either Flu-Mi or CMV-pp65-NLV peptide to generate aAPCs for CD8 T cell stimulation. As shown in FIGS.
  • TWSl 19 significantly increased the antigenic specificity of the culture similarly to what had been observed with stimulation of moDCs. TWSl 19 also enhanced the polyfunctionality of both Ml and CMV-specific cells. Thus the effect of TWSl 19 on polyfunctionality does not seem to be dependent on dendritic cells and is largely intrinsic to T cells. EXAMPLE 8. Wnt pathway activation-induced polyfunctionality is associated with many sternness features
  • TWSl 19-treated cells are reprogrammed to polyfunctionality, the polyfunctional phenotype should be maintained even after the treatment was stopped.
  • TWSl 19-treated cells proliferated better than untreated cells.
  • T cells previously treated with TWSl 19 also maintained higher CD62L and CD28 expression after homeostatic proliferation (FIG. 5C, FIG. 5D).
  • FIG. 5E shows that Wnt pathway activation by Wnt pathway activation reprograms human virus-specific T cells to a long-standing, young, polyfunctional phenotype.
  • Bcl-xL enhances single-cell survival and expansion of human embryonic stem cells without affecting self-renewal. Stem Cell Res 8, 26-37.
  • nonprogressors preferentially maintain highly functional HlV-specific CD8+ T cells.
  • Ciuffreda D., Comte, D., Cavassini, M., Giostra, E., Buhler, L., Perruchoud, M., Heim, M.H., Battegay, M., Genne, D., Mulhaupt, B., et al. (2008). Polyfunctional HCV-specific T- cell responses are associated with effective control of HCV replication. Eur J Immunol 38, 2665-2677.
  • a Pan-BCL2 inhibitor renders bone- marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition.
  • KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells. Blood 113, 6619-6628.
  • CTLA-4 blockade enhances polyfunctional NY- ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit. Proc Natl Acad Sci U S A 105, 20410-20415.
  • PBMCs from 5 healthy HLA* 0201 -positive donors and 15 HIV-infected patients were isolated by Ficoll-Paque PLUS gradient centrifugation following the manufacturer's protocol (GE Healthcare).
  • the HIV-infected, HAART-treated aviremic patients were recruited from Johns Hopkins and Case Western Reserve University. The average age of donors was 49 years old.
  • CD 14+ and CD8+ T cells were further purified from fresh PBMCs using the CD 14+ cell-positive selection and CD8+ T cell-negative selection kits according to the manufacturer's instructions (Miltenyi Biotec).
  • GILGFVFTL SEQ ID NO: l
  • Peptide-pulsed moDCs were then washed extensively to remove free peptide in solution before use.
  • Ml peptide-loaded HLA-A2-Ig and anti-CD28 (clone 9.3) were biotinylated and coupled to anti-biotin coated microbeads (Miltenyi Biotec) and stored at 4°C (29) before use.
  • OligoMicroarrays (Agilent Technologies). Gene expression data were analyzed using Partek Genomic Suite. Each sample was normalized and log2 transformed before being compared by t test (P ⁇ 0.05). Genes with fold changes of more than 1.6 were selected for further analysis. Differential expressed genes were functionally grouped and annotated using DAVID Bioinformatics Resources 6.7 (NIAID, NIH; available at
  • GSEA Gene Ontology
  • GSEA calculates a running sum of the statistic for each gene set.
  • the test statistic is the maximum of the running sum (enrichment score), and permutation tests are performed to evaluate the empirical P value of the enrichment.
  • Gene sets for LCMV exhaustion versus normal effector and memory T cells (25) were provided by John Wherry.
  • Gene sets for HIV progressor and PD-1 signaling (26) were provided by Nicolas Haining.
  • pERK staining and cytotoxicity assay were based on protocols as previously described (47). Briefly, Ml -specific T cells were stimulated with PMA/ionomycin for 10 minutes before fixation with 2% formaldehyde and permeabilized with 90% methanol. Staining was performed with anti-phospho-p44/42 MAPK (ER 1/2) antibody from Cell Signaling(clone E10). Flow cytometry-based cytotoxicity assay (48) was used to investigate the effect of ERK inhibition on T cell-mediated HLA-A2-positiveT2 cell killing.
  • shRNA sequence (5'-CTGAACAGAGACTGCTAGGATCATCCTTC-3'; SEQ ID NO:3) targeting the human SPRY2 gene was cloned into pLKO.
  • l puro plasmid (provided by Joel Pomerantz.
  • Viral particles were made by co-transfecting 20 ng of the PLKO plasmid plus 15 ng delta 8.9 plasmid and 10 ng pCMV-VSV-G into HEK293T cells (plated 24 hours earlier at 2.5 x 10 6 cells/dish) via calcium phosphate precipitation as previously described (49).
  • Transduction of primary Ml -specific T cells was performed 24 hours after DC stimulation by spinning at 300 g for 90 minutes in the presence of 8 ⁇ g/ml polybrene (Sigma-Aldrich). 24 hours after transduction, culture medium containing 1.5 ⁇ g/ml puromycin was used to select for virus-transduced T cells.
  • RNA isolation Cells-to-CT kit; Invitrogen
  • qPCR TraqMan gene expression assay
  • PBMCs were thawed and rested overnight before activation with soluble anti-CD3 (clone HIT3a, 1 ⁇ g/ml; Biolegend) and anti-CD28 (1 ⁇ g/ml, clone CD28.2; Biolegend) in the presence of Gag/Nef/Tat/CEF peptide pools and transduced with lentivirus.
  • Anti-PD-1 antibody (10 ⁇ / ⁇ 1, clone EH12.2H7; Biolegend) was used to block PD-1 signaling.
  • Isotype control antibody (clone MG1-45; Biolegend) was added to control cultures.
  • HIV-specific T cell sorting and measurement of SPRY2 expression HIV-specific T cell sorting and measurement of SPRY2 expression.
  • Gag antigen specificity was determined by using HLA-A2 restricted PE pentamer
  • CD8+ T cells were purified from 7 HLA- A2+HIV patients from the study subjects and stained with HIV-Gag-pentamer (PE) and Ml- tetramer (APC). For each donor, more than 1000 antigen-specific T cells were sorted for mR A extraction and cDNA conversion using the Cells-to-Ct kit (Invitrogen). After preamplification using the TaqMan PreAmp Mastermix Kit (Invitrogen), qPCR was performed using TaqMan gene expression assays, and SPRY2 expression level were adjusted to HPRT1 expression.
  • PE HIV-Gag-pentamer
  • APC Ml- tetramer
  • unsorted PBMCs were stained with Gag-specific pentamer or Ml -specific tetramers, anti-PD-1, and anti-CD8 for an hour.
  • Cells were fixed, permeabilized, and stained with APC-conjugated anti-human SPRY2 antibody (clone ab60719; Abeam) for an additional 2 hours. Cells were then washed twice before acquisition by flow cytometry.
  • EXAMPLE 10 Antigen concentration regulates polyfunctionality of human T cells.
  • T cells induced by high antigen concentration (10 ⁇ ) had a significantly reduced ability to simultaneously produce multiple cytokines or effector functions (FIGS. 10D-E).
  • IL-2 concentration was IL-2 production, and functions least sensitive were CD 107a andMIP- ⁇ ⁇ (FIG. 10E).
  • the loss of IL-2 responses during high antigen concentration stimulation is also observed during chronic infections such as HIV (9), where IL-2 production is the most sensitive and first function lost out of other effector functions.
  • T cells induced with 10 nM moDCs also produced more IL-2 on a per cell basis (FIG. 10H).
  • T cells with the lowest polyfunctionality, induced by 10 ⁇ moDCs were dominated by a population of cells capable of producing MIP-i pand degranulation, but did not produce any cytokines (FIG. 16).
  • T cells induced with 10 nM Ml antigen- pulsed moDCs Upon rechallenge with antigen, T cells induced with 10 nM Ml antigen- pulsed moDCs also exhibited superior cell expansion (FIG. 17).
  • FIG. 19A T cells from all stimulation conditions also expressed similar levels of bcl-2 and T-bet (FIG. 19B).
  • Microarray data showed that transcription factors that promote CD8+ T cell memory formation, such as EOMES (20) and TCF7 (21), were higher in highly polyfunctional T cells, and differences were confirmedly flow cytometry or quantitative PCR (qPCR) (FIGS. 11B-C).
  • qPCR quantitative PCR
  • EXAMPLE 13 High antigen concentration-induced T cells are enriched with the molecular signature of T cell exhaustion.
  • GSEA has been used to compare genomic signatures between microarray experiments in order to integrate different phenotypic cellular states (24). GSEA showed that highly polyfunctional T cells were significantly enriched with the genetic signature for memory T cells (ref. 25 and FIG. llD).In contrast, there was a significant enrichment of the exhausted gene signature (25) among high antigen concentration-induced T cells, indicating that these T cells share aspects of global gene expression patterns with T cells that are exhausted due to chronic LCMV infection (FIG. 11D). Similar enrichment was found while using the differentially expressed gene list from HIV progressors versus controllers (26). Interestingly, while it has been established that the PD-1/PD-L1 pathway is involved in T cell exhaustion during chronic viral infections (27), GSEA analysis of the PD-1 ligation signature (26) showed no significant enrichment.
  • aAPCs are made by conjugatingHLA- A2-Ig-Ml complex (signal 1) and anti-CD28 (signal 2) onto particles (28, 29).
  • Higher concentrations of aAPCs with signal 1 plus signal 2 induced significantly more antigen- specific T cell proliferation (FIGS. 12D-E), but lower T cell polyfunctionality (FIG. 12F).
  • FIG. 12F Similar to moDC stimulation, there was an optimal antigen concentration for aAPC stimulation. Therefore, while signaling through PD-1 and other inhibitory receptors is widely involved in T cell exhaustion, polyfunctionality regulation due to antigen concentration requires only signal 1 and signal 2.
  • MAPK/ERK pathway controls T cell poly functionality in response to antigen concentration.
  • the MAPK ERK pathway is an important signaling pathway that critically regulates a variety of physiological processes, such as cell growth, differentiation, and survival. This pathway also plays important roles in many aspects of lymphocyte biology, such as thymocyte selection, CD4+ T cell differentiation, and T cell activation (30). However, the role of the MAPK/ERK pathway in T cell polyfunctionality and exhaustion has not been reported.
  • Upregulation of pERK was shown to be specific, as treatment of cells with U0126, a MEKl/2 specific inhibitor, inhibited ERK phosphorylation in a concentration-dependent fashion, with complete inhibition of pERK seen with 100 ⁇ 0126 (FIG. 14B). If the MAPK/ERK pathway differentially controls effector functions involved in polyfunctionality, such as cytokine production, one should be able to convert 5+ polyfunctional antigen-specific Tells to low polyfunctional T cells by inhibiting ER phosphorylation. In the presence of U0126, there was a dose-dependent inhibition of cytokine production (IL-2, TNF, and IFN- ⁇ ), with little effect on CD 107 and MIP- ⁇ ⁇ expression in optimal antigen concentration-induced T cells.
  • IL-2, TNF, and IFN- ⁇ IL-2, TNF, and IFN- ⁇
  • EXAMPLE 16 Upregulation ofSPRY2 in high antigen concentration-induced T cells inhibits polyfunctionality.
  • SPRY2 One gene identified in the microarray gene list (Supplementary Table 1) known to affect the MAPK/ERK pathway is SPRY2.
  • Sprouty proteins are a well-conserved family known to mediate the negative feedback regulation of the MAPK pathway (31 , 32). Sprouty proteins bind to Grb2 and other components of the MAPK pathway, preventing the upregulation of pERK (33, 34).
  • qPCR analysis confirmed that SPRY2 is upregulated in high antigen concentration-induced CD8+ T cells (FIG. 14A) and upregulation of SPRY2 is downstream of ERK activation, as it is inhibited by U0126 treatment added during high antigen concentration stimulation (FIG. 20).
  • SPRY2 knockdown virus efficiently inhibited mRNA expression of SPRY2, by approximately 80%, while control non-target (NT) virus had no effect (FIG. 14B).
  • SPRY2 knockdown also led to a significant decrease in SPRY2 protein levels as determined by flow cytometry (FIG. 14B).
  • the MFI of SPRY2 in the SPRY2knockdown cells decreased from 1860 to 910.
  • the SPRY2 knockdown T cells produced more cytokines than the control lentivirus-infected T cells (FIG.
  • HlV-specific CTL responses in infected patients are known to have significantly lower levels of
  • HlV-specific T cells also upregulate SPRY2 and that upregulation of SPRY2 is responsible for the low levels of T cell
  • PBMCs from 7 HLA-A2-positive HIV- infected patients were sorted on the basis of pentamer/tetramer staining into either HIV Gag- or influenzaMl -specific CTL (FIG. 15A).
  • qRT-PCR on these populations showed that SPRY2 was upregulated in the Gag-specific T cells compared with both theM-1 -specific T cells and nonspecific CD8+ T cells from the same donors.
  • the molecular profile of highly polyfunctional T cells is enriched with memory signature genes and is consistent with the idea that the polyfunctional state is associated with memory T cell development (1, 19, 36).
  • Two key genes identified as part of the polyfunctional signature were EOMES and TCF7, both known to be indispensable for optimal CD8+ T cell memory formation (20, 37).
  • the mechanisms governing their expression levels are not entirely clear, but here we identified antigen concentration as an important factor. The finding that optimal antigen concentration induced higher levels of TCF7 and EOMES could identify the molecular mechanism linking optimal antigen concentration to superior T cell memory formation.
  • Chronic viral infection is also associated with high antigen load, T cell exhaustion, and the loss of polyfunctionality.
  • the loss of each individual function in our system follows the hierarchical order for loss of functions observed in HIV and LCMV infections (8).
  • IL-2 a homeostatic cytokine that promotes T cell proliferation, is lost first.
  • effector cytokines such as TNF-a and IFN- ⁇ secretion are impaired.
  • upregulation of CD 107a and MIP- ⁇ are least affected (9).
  • Enrichment of the T cell exhaustion signature in the high antigen concentration-induced T cells indicates that these T cells share certain biological pathways with exhausted T cells seen in HIV (26) and chronic LCMV (25) infection.
  • high antigen concentration-induced T cells may represent cells "on their way" to becoming completely exhausted.
  • Inhibitory receptor upregulation is a key feature of T cell exhaustion (10) and often characterized by active suppression mediated through the PD-1/PD-L1 pathway (13, 14).
  • the poorly polyfunctional T cells expressed more inhibitory receptors, the poorly polyfunctional phenotype was independent of inhibitory receptor signaling. This is evident by the fact that PD-1/PD-L1 blockade did not attenuate high antigen concentration- induced loss of polyfunctionality and that the effect of high antigen concentration was seen even when using a signal 1/2 only-aAPC system to induce poorly polyfunctional T cells. Additional evidence comes from the lack of enrichment of the PD-1 molecular signature, such as upregulation of BATF, in the high antigen concentration-induced T cells. Overall, our data show that TCR signaling strength alone determines T cell polyfunctionality.
  • SPRY2 as a regulator of T cell polyfunctionality and showed that SPRY2 expression level is dependent on antigen-concentration.
  • Sprouty proteins were originally identified in Drosophila as regulators for embryonic development, and mutations in sprouty proteins were found in various human cancers (31). All sprouty proteins were found to be negative regulators of growth factor signaling, especially the MAPK ER pathway (32, 33).
  • CD8+CXCR1-CD27-CD28- T cells capable of producingIL-2 (40).
  • SPRY2 the physiological role of SPRY2 was not validated in these studies. Given the importance of the MAPK pathway in immune system signaling, it is likely that sprouty proteins could also regulate the outcome of diverse immune responses. During immune response against infection, upregulation of sprouty proteins might be beneficial to the host as a strategy for preventing immune system overactivation and subsequent immunopathology. On the other hand, modulation of SPRY2 expression could be a potential way to enhance T cell polyfunctionality and immunological memory during chronic infections.
  • the human T cell polyfunctionality signature and the molecular mechanisms involving SPRY2 and the MAPK/ERK pathway can have important clinical applications such as identifying desirable immune responses in response to immunization and infection.
  • Rodrigue-Gervais IG et al. Dendritic cell inhibition is connected to exhaustion of CD8+ T cell polyfunctionality during chronic hepatitis C virus infection. J Immunol. 2010;184(6):3134-3144.4.
  • Ahmadzadeh M et al. Tumor antigen-specificCD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood.2009;l 14(8): 1537— 1544.6.

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Abstract

Selon l'invention, la capacité de lymphocytes T individuels à réaliser des fonctions d'effecteurs multiples est essentielle à l'immunité protectrice contre les virus et le cancer. Cette polyfonctionnalité est fréquemment perdue en cas d'infections chroniques. La présente invention concerne des méthodes d'induction de la polyfonctionnalité des lymphocytes T pour traiter les infections virales chroniques et le cancer.
PCT/US2014/067674 2013-12-01 2014-11-26 Méthodes d'induction de la polyfonctionnalité des lymphocytes t Ceased WO2015081235A1 (fr)

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WO2025227702A1 (fr) * 2024-04-29 2025-11-06 深圳泽医细胞治疗集团有限公司 Milieu de culture pour cellules immunitaires et utilisation associée

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