WO2012112079A1

WO2012112079A1 - IMPROVING CELLULAR IMMUNOTHERAPEUTIC VACCINES EFFICACY WITH GENE SUPPRESSION IN DENDRITIC CELLS AND T-LYMPHOCYTES USING SiRNA

Info

Publication number: WO2012112079A1
Application number: PCT/RU2012/000101
Authority: WO
Inventors: Mikhail Valentinovich Filatov; Anna Il'inichna GUTKINA
Original assignee: VITASPERO Inc
Current assignee: VITASPERO Inc
Priority date: 2011-02-14
Filing date: 2012-02-14
Publication date: 2012-08-23
Anticipated expiration: 2013-08-14

Abstract

The present invention relates to medicine and immunology and can be used for improving the efficacy of cellular immimotherapeutic vaccines through manipulation of gene expression in the vaccine cells. The present invention provides specific immunogenic composition comprising dendritic cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, TIM -2, TIΜ-3, and TIM-4. The present invention also provides methods for producing the composition comprising modified dendritic cells wherein the said composition can be used for the treatment of cancer or infectious diseases.

Description

IMPROVING CELLULAR IMMUNOTHERAPEUTIC VACCINES EFFICACY WITH GENE SUPPRESSION IN DENDRITIC CELLS AND T-LYMPIIOCYTES USING

SiRNA

FIELD OF THE INVENTION

The present invention relates to medicine and immunology and can be used for methods of improving the efficacy of cellular immunotherapeutic vaccines through manipulation of gene expression in the vaccine cells.

BACKGROUND OF THE INVENTION

There is considerable interest in developing therapeutic vaccines based on immune system cells. For example, in recent years, there has been interest in the establishment of anticancer vaccines based on immune system cells. Modern immunological approaches to treat malignant tumors involve the use of immune system cells comprising tumor antigens, such cells including dendritic cells and/or activated lymphocytes (Janikashvili et al., Immunotherapy (2010) 2(1):57; Eshhar, Curr Opin Mol Ther. (2010) 12(l):55-63).

A number of immunotherapy treatments are currently being developed, which can potentially be less toxic and/or more effective for treatment at different stages of disease. It is known that the immune system is able to distinguish between the "self and the "foreign", and under normal conditions, an immune response against such antigens is occurring.

For example, virus infected cells are recognized by the immune system as foreign, though such a natural immune response is often not strong enough to block the development of viruses. One purpose of immunotherapy is to enhance the ability of the immune system to recognize cells infected with viruses, for example, and to establish effective mechanisms to reduce viral load. One important issue for any immunotherapy is the selection of antigens for directional effects and opt imal presentation of antigens to the immune system.

Prerequisites for the development of vaccines that have therapeutic value became possible, in part, by way of the development of anti-cancer vaccines for antitumor immunization, based on the dendritic cells that contain antigenic material. Dendritic cells' use as immuno-stimulatmg agents is of growing interest for both in vivo and in vitro applications. For example, a method used in oncology for the treatment of tumors includes the administration of dendritic cells induced in vitro for the beginning of maturation and characterized by their ability to capture and process an antigen in vivo. Pharmaceutical compositions used in these methods include the dendritic cells in combination with a pharmaceutically acceptable carrier for their administration. This method and pharmacological composition provide induction of antitumor immune response by efficiently capturing and processing, by dendritic cells, of tumor antigens at the site of a tumor, secretion of the cytokines and contact with T-cells in the lymph node.

For example, the use of dendritic cells and activated lymphocytes for immunotherapy of malignant tumors has been investigated (Janikashvili et al., Immunotherapy (2010) 2(1):57). A number of studies in this area generates reasonable prospect of success for biotherapeutic approaches in medicine. Progress in this area is observed in Russia (Moiseenko et al., Medical Academic Journal, 7 (2007), 4:17-35; Akhmatova et al., Oncology and Immunology in Pediatrics (2006) 2:35-41; Mikhailova et al, Russian Biotherapeutic Journal (2007) 2:39-44; Lcplina et al., Cell Technologies in Biology and Medicine (2007) 2:92-98; Tarasov et al, Hybridoma (1999) 18:99-102; Ostreiko et al., Neurosurgery, (2003) 4:40-44; Bazhanov et al, Bulletin of the Siberian Medicine (2008) 5:46-50; Ostreiko et al, Medical Immunology (2008) 6:593-596; Olyushin et al, Russian Journal of Neurosurgery (2009) 1:58-64; and Russian Patent Application 2000122041/14 (023265) filed on 8/17/2000).

There arc methods of differentiation of monocytic precursors of dendritic cells into immature dendritic cells that are known for treatment of cancer. For example, the production of mature dendritic cells using granulocyte-macrophage colony stimulating factor GM-CSF without other cytokines under conditions that prevent cell adhesion to the flask, providing contact of differentiated dendritic cells' precursors with an antigen of interest for a period of time sufficient to absorb; the antigen. An antigen can be a tumor-specific antigen, tumor- associated antigen, viral antigen, bacterial antigen, a tumor cell, a nucleic acid encoding the antigen isolated from tumor cells, a bacterial cell, recombinant cell expressing the antigen, a cell lysate, a membrane. preparation, a recombinant antigen, a peptide antigen or an isolated antigen.

There is an autologous vaccine for treating oncological diseases, which includes lymphocytes that have been activated with intei eukin-2 and dendritic cells that have been prepared by incubating the immature dendritic cells with a tumor lysate. The vaccine has T- lymphocytes that were specifically activated by mature dendritic cells and is characterized by the manner in which it was produced. The method of preparation includes the isolation of mononuclear lymphocytes (MNK) from the peripheral blood of a subject, cultivation of MNKs in the DMLM medium, separation of cells into monocytes that adhere to the substrate and lymphocytes that do not adhere to the substrate, placing of the MNKs into a culture medium, isolation of non-adhering lymphocytes and subsequent addition of IL-2 to obtain lymphokin-activatcd killer cells (LAK-cells), adding to a remanding adhering monocytes of a growth factor, stimulating DC cells' maturation by an autologous tumor lysate in vitro and the subsequent addition of maturation factors for the duration of 1 day. For the isolation of adherent monocytes, MNKs are cultivated in the medium augmented with 10% FSC for 1 hour, after which immature dendritic cells are obtained. The adhering monocytes are cultivated in a medium with neupogen at a 50 ng/inl concentration during 48 hours, and after obtaining mature dendritic cells, the immature DCs are cultivated in a medium with 2000 MU/ml of Reofcron and 50 ng/ml beta-leukin. At the same time, LAK cells from the non- adhering fraction of MNK cells are cultivated in a medium containing 100 U/ml Roncoleukin for 72 hours. The mDCs and LAKs are washed by centrifugation in saline solution for 10 min at 1500 rpm, after which mDCs and LAKs are cultivated together in a medium with 100 U/ml of roncoleukin for 24 hours, after which the adhering fraction and non-adhering fraction are washed separately by centrifugation in saline solution for 10 min at 1,500 rpm. The resultant population comprises the vaccine.

A prototypical method of treatment of malignant brain tumors is known, comprising the isolation of a patient's blood monocytes, cultivation with growth factors, and addition of the monocytes to ihose dendritic cells obtained from monocytes of the antigenic material from patients' tumor. The resulting dendritic cells are injected back into the patient subcutaneously, in conjunction with additional immune modulation by activated lymphocytes. This method purportedly allows activation of specific antitumor immunity and is characterized by the fact that the patients' peripheral blood mononuclear cells are obtained and cultivated with growth factors, then added to a medium with dendritic cells obtained from patient monocytes. The antigenic material prepared from a patient's tumor is added by electroporation, then the dendritic cells are injected intradermally, followed by immunomodulation using activation by phytohemagglutinin lymphocytes of a patient. At this point, in addition to dendritic cells, autologous activated lymphocytes are injected, providing production of cytokines that can catalyze the immunological antitumor response.

With such a method, a fragment of a patient's tumor that has been. taken during the surgery is dissociated into protein extract, to be used as the antigen in later work. The patient's peripheral blood monocytes are cultivated with growth factors. The antigenic material is added first to monocytes, and then to the dendritic cells. Next, dendritic cells are injected intradermally into a subject. In parallel, immunomodulation of the patient is performed with activated lymphocytes, wherein the lymphocytes were derived from mononuclear leukocytes' of the subject, and activated by phytohemagglutinin; Activated lymphocytes are then also injected intradermally.

Thus, adequately presenting a tumor antigen to immunocompetent cells in combination with a nonspecific immunity (due to activated lymphocytes) can activate specific antitumor immunity and enables the use of this method as an adjuvant treatment of complex antineoplastic treatments. In that way, the method provides treatment of malignant brain tumors, reducing side effects and complications, increases the life expectancy of patients, and adds to the- quality of life of the patient by offering T-lymphocytes of tumor antigen "professional" antigen-presenting cells of the body, i.e., dendritic cells which contain the tumor antigen.

Therefore, the establishment of effective drugs, vaccines and medical treatment is essential, and, despite notable achievements in this area, more progress is needed. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an image depicting a method of measuring the proportion of cell populations at various stages in the cell cycle. For cells that have incorporated bromodeoxyuridine during DNA replication, fluorescence is much weaker when stained with fluorescent dye Hocchsl 33258. As a result, the flow cytofluorometry histogram such cells after the division form a "peak" to the left (closer to the origin) than non-proliferating cells having diploid DNA amount (see dark area). The fraction of cells caught in this peak enables the estimation of the percentage of cells that were incubated with bromodeoxyuridine. The abscissa: fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.

Figure 2 is an image illustrating the suppression of gene expression in cells of the immune system (for example, the gene SOCS1) by transfection of specific short interfering RNA. The effectiveness of suppressing the expression was evaluated by the contents of protein, detectable by enzyme immunoassay of the proteins after electrophorelic separation and immobilization on a nitrocellulose membrane. Left lane: molecular weight markers, lane Average: Socsl protein in control cells, right-hand lane: protein Socsl in cells previously transfected with short interference RNA, anti-SOCSI.

Figure 3 is a series of graphs illustrating that transfection of anti-FAS and anti- CTLA4 short interfering RNA dramatically increases the proportion of proliferative capacity of T lymphocytes. Upper histogram: phytohemagglutinin stimulation of proliferation of T lymphocytes of cancer patients. Average histogram: the same, but after transfection mixture of anti-FAS and anti-CTLA4 short interfering RNA. Lower histogram: the same, but after transfection to study the non-specific short interfering RNA genes. The abscissa: fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.

Figure 4 is an image depicting that transfection of anti-FOXP3 short interfering RNA increases the proportion of proliferating T lymphocytes. Upper histogram: phytohemagglutinin stimulation of proliferation of T lymphocytes of cancer patients. Lower histogram: the same as upper histogram, with the addition that after transfection, anti-FOXP3 short interfering RNA was used. The abscissa: fluorescence intensity in arbitrary units, ordinate: number of analyzed cells. Figure 5 is an image depicting that addition of a lysate of tumor cells can suppress the proliferation of T lymphocytes, and transfection of anti-SOCSI short interfering RNA can abolish this effect. Upper histogram: phytohemagglutinin stimulation of proliferation of T lymphocytes of cancer patients. Average histogram: the same as the upper histogram, after adding the lysatcs of tumor cells. Lower histogram: the same, but at the point after the combined action of tumor cell lysate and transfection of anti-SOCSI short interfering RNA. The abscissa: fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the present disclosure presents compositions and methods useful to overcome the immunosuppressive state of immune system cells due to a temporary suppression of gene expression that may limit the proliferative capacity of T lymphocytes. In an aspect, the disclosure presents compositions and methods for overcoming suppression of the immune system by temporarily switching off the expression of one or more genes including FAS, CTLA4, ΓΌΧΡ3, S0CS1, CD200, CDS I (PECA -1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, TIM- 2, TIM-3, and ΤΊΚ -Τ

In an embodiment, by directing the temporary suppression of the expression of one or more genes in dendritic cells and/or activated lymphocytes, as disclosed herein, the properties of the cells can be manipulated. In an embodiment, suppression of dendritic cells to the induction of tolerance when interacting with lymphocytes increases the viability and immunogenicity of such dendritic cells. In an embodiment, this suppression can be used to overcome the immunosuppressive properties of T lymphocytes, preventing their activation. In another embodiment, it is demonstrated herein that suppression of gene expression of one or more of FAS, ( I I ..VI. SOCS1 and/or FOSP3 genes can be used to prepare such dendritic cells.

In the recent past, others have used activated interleukin-2 (IL-2) lymphocytes and natural killer cells. However, the ability to control cell behavior, relying only on existing cytokines and growth factors, was very limited. Among the factors limiting the effectiveness of immunotherapy using dendritic cells are: 1) the ability of dendritic cells, depending on the variety of circumstances, to effect not only activation of T lymphocytes and activation of the immune response, but also the state of tolerance of T lymphocytes and associated with this immunosuppression; and 2) states of immunosuppression that were previously developed in cancer patients, combined with the inability of T lymphocytes to proliferate in response to antigenic stimulation.

Such obstacles can now be overcome, as set forth herein, through the controlled manipulation of gene expression in the cells of immune system responsible for the above complicating properties (e.g., SOCS-1 CTLA4, FAS and FOXP3, among others disclosed herein). By trans ecting cells with short interfering NA (siRNA) specific for these genes, vaccines specific for various types of cancer or infectious diseases can be prepared using the cells. Using the compositions and methods set forth herein provides for the development of more effective protocols of immunotherapy of diseases such as, but not limited to, malignant tumors based on the use of dendritic cells and activated T-lymphocytes.

In an embodiment, it is an objective to develop a new composition, e.g., a cell-based vaccine, and to develop a method of treatment for infectious diseases, including, but not limited to, hepatitis C, hepatitis B, papilloma virus, AIDS. In an embodiment, it is an objective to develop a new composition, e.g., a cell-based vaccine, and to develop a method of treatment for cancers, including, but not limited to, hepatocellular carcinoma, glioma; ovarian cancer, peritoneal cancer, lung cancer, breast cancer, uterine cancer, and cervical cancer.

As used herein, the following terms have the following meanings:

"Modified to suppress expression", as used herein, refers to a cell that has been treated in any manner to suppress the expression of one or more genes. The suppression can be temporary, for any length of time, or can be permanent. The suppression may partially decrease the expression of the target gene, or it may completely shut off the expression of the target gene. Suppression of the expression of desired genes begins before administration of the vaccine to a subject. Suppression of expression may or may not continue after administration of the vaccine to a subject. By way of example, a dendritic cell may be modified to suppress the expression of SOCS1 by transfecting the dendritic cell with siRNA specific to suppress the expression of the SOCS 1 gene.

"Dendritic cells" (DCs) refers to a heterogeneous population of antigen-presenting cells of the bone marrow origin. Morphologically, the dendritic cel ls are large cells (1 5-20 microns) round, of oval or polygonal shape, with off-center located nucleus, numerous branched processes of the membrane. Dendritic cel ls express a set of surface molecules characteristic of other antigen presenting cel ls: receptors for cel l wall components and nucleic acids of m icroorganisms, including the receptors for complement components and toll-like receptors, the molecules of class II major histocompatibi lity complex (MHC); costimulatory molecules CD40, B7 Y₂ (CD80, CD86), 137 -DC, B7-H 1 ; intercel lular adhesion molecules (ICAM- 1 ). Dendritic cells can be produced easily from peripheral blood monocytes and can effectively present the antigen of T-lymphocytes. To date, many studies on the modulation of immune response in patients with chronic infectious diseases and cancer using dendritic cel ls primed with antigen were done.

"Antigen" refers to substances that cause specific to them cellular or humoral immune response.

"Monocytes" refers to a large group of mature single-core agranu locytic leukocytes 12-20 microns in d iameter with an eccentrically located polymorph ic nucleus with loose chromatin network, and azurophi lic granules in the cytoplasm. Monocytes have an unsegmented nucleus, and arc the most active phagocytes in peripheral blood. The cells have an oval shape with a large bean-shaped, chromatin-rich nucleus and a large cytoplasm having many lysosomes. Normal ly, monocytes comprise from 3% to 1 1 % of the total number of blood leukocytes. I n an embod iment, there are approximately 450 monocytes in ^'l ul of blood. Monocytes are resident i n the blood for 2-3 days, after which they go into the surrounding- tissues, where, having reached maturity, they become tissue macrophages or dendritic cells.

"T-lymphocytes" or "T-cells" refer to lymphocytes, the developing in the thymus of mammals from precursors, prc-thymocytes, derived from the red bone marrow. In the thymus T-lymphocytes di fferentiate, acquiring T-cell receptor (TCR) and surface markers. They play an important role i n adaptive, that is, acquired immune response. Provide recognition and destruction of cel ls bearing foreign antigen, enhances the action of monocytes, N -cells, as well as take part in the switching of immunoglobulin isotypes from early immunoglobulin IgM, the later production of IgG, IgE, IgA in B-cells.

"Activated T-lymphocytes" refers to T-lymphocytes exposed to inflammatory cytokines and agents that stimulate their proliferation (phytohemagglutinin) for the implementation of Thl response as well as T-lymphocytes obtained as above where the expression of genes CTLA4 and/or FAS and/or FOXP3 has been suppressed.

"Composition for treatment" generally refers to a therapeutic cell vaccine (also referred to herein as an "immunogenic composition") designed to treat a particular disease or disorder, prepared on the basis of the loading antigenic (immunogenic) material on dendritic cells and phytohemagglutin-activated T lymphocytes, used directly as such or in an amplified version - in conjunction with the temporary arrest of the genes SOCS1, FAS , CTLA4, and FOXP3, for example, by introducing into cells of siRNA or any modified form of siRNA, for example chemically modified form of siRNA, including asymmetric siRNAs and self-deliverable siRNA.

"Small interfering RNA" (siRNA) refers to short double-stranded RNA molecules capable of RNA i nter ferencc.

"Asymmetric siRNA" refers to siRNA with single stranded overhangs either on the 3' or 5' end of the duplex, or combination of both.

Modified siRNA refers to chemically modified siRNA enabling more specific , less toxic and longer lasting gene knockdown effect than unmodified siRNA. Chemical modifications include but not limited to 2'-flouro, phosphorothioates (PS), 2'-0-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), neutral methoxyethyl (MEA), phosphoramidales, cationic Ν,Ν-dimethylethylenediamine (DMED), morpholinos.

"Self-deliverable" siRNA refers to chemically modified siRNA that penetrates cells without additional transfection agents, formulations, delivery vehicles and transfection procedures.

"RNA interference" (also referred to as born RNA interference, RNAi) refers to small RNA molecules mediating the suppression of gene expression at the stage of transcription, translation or degradation of mRNA. Small interfering RNA. (small interfering RNA, siRNA), representing a short double-stranded RNA molecules that bind to specific sequences of mRNA (usually in the coding region), leading to degradation of mRNA. Selective effect of RNA interference on gene expression makes RNAi a useful tool for studies using cell cultures and in living organisms, as synthetic double-stranded RNA introduced into cells, causing suppression of specific genes. For example, RNA interference is used to systematically "turn off genes in the cells.

"Gene expression" refers to a process in which genetic information from genes (DNA nucleotide sequence) is transformed into a functional product (RNA or protein).

"Immunosuppression" refers to suppression of immunity of an organism.

"SOCSl" refers to the gene coding a protein called a suppressor of cytokine signaling 1. This protein is a negative regulator of activation of macrophages and plays an important role in regulating autoimmune reactions involving dendritic cells. The suppression of gene expression by SOCSl breaks the tolerance of the immune system to its own antigens and may enhance the immune response.

"FAS gene" refers to a gene which encodes a receptor on the cell surface (FasR), which upon activation, causes programmed cell death. The emergence of FasR on dendritic cells and activated lymphocytes limits their viability.

"CTLA4" refers to a gene that encodes Cytotoxic T-Lymphocyte Antigen 4 - a protein of immunoglobulin family whose appearance on the surface of activated T- lymphocytes leads to a suppression of cellular proliferation.

"FOXP3" refers to forkhead box P3, a gene that encodes the coding transcriptional regulator of T-lymphocytes that is necessary for the formation and functioning of regulatory cells that negatively regulate immune response.

Dendritic cells are a heterogeneous cell population with characteristic morphology and a widespread distribution in tissues, including blood. The cell surface of dendritic cells is unusual, with characteristic veil-like buds. Mature dendritic cells are usually identified as CD3-, CD1 lc I , CD 19-, CD83 -I-, CD86 + and HLA-DR +. Dendritic cells process and present antigens and stimulate the activation of T-cells and T-cell memory. Dendritic cells have a high ability to effectively present antigens to T-cells as major histocompatibility complex (MHC), and contribute to the initiation of the immune response by releasing cytokines that stimulating the activity of lymphocytes and macrophages.

In an embodiment, an immunogenic composition is provided. In an aspect, the immunogenic composition is a cell-based vaccine. In an embodiment, an immunogenic composition is provided for use as an individual therapeutic cellular vaccine.

In another embodiment, the immunogenic composition is used as a cellular vaccine in conjunction with one or more other compositions, complementary to conventional treatment. In an embodiment, a cellular vaccine is used in conjunction with one or more other compositions known in the art to be useful for treating a particular disease or disorder. In an embodiment, a cellular vaccine set forth herein is administered in conjunction with chemotherapeutic therapy. In an embodiment, a cellular vaccine set forth herein is administered in conjunction with therapy for hepatitis C. In an embodiment, a cellular vaccine is used in conjunction with one or more other compositions previously unknown in the art to be useful for treating a particular disease or disorder.

In an embodiment, a cellular vaccine is administered as a sole therapeutic agent to a patient having cancer and/or an infectious disease. In an aspect, the cellular vaccine is administered in a dosing regimen that is similar to a dosing regimen for a previously-known therapeutic agent for the particular cancer or infectious disease. By way of a non-limiting example, a cellular vaccine as described generally herein may be administered to patient having hepatitis C, wherein the cellular vaccine is administered once a week for a time course of five weeks.

In an embodiment, disclosed herein is an immunogenic composition comprising dendritic cells modified to suppress the expression of at least one of the genes selected from the group consisting of PAS, CTLA4, FOXP3, SOCSI, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TTM-1 , TIM-2, TIM-3, and TIM-4.

In an embodiment, disclosed herein is an immunogenic composition comprising T cells that were previously activated for Thl response and further comprising dendritic cells modified to suppress the expression of at least one of the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECA -1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein FIuR, amphiregulin (A REG), TIM-1, TIM-2, TIM-3, and ΊΊ -4. In an embodiment, the T cells are modified to suppress of the expression of at least one genes selected from the group consisting of CTL4, FAS, FOXP3 CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, TIM-2, TIM-3, and T1M-4,

In an embodiment, disclosed herein is an immunogenic composition comprising T cells modified to suppress the expression of at least one of the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein FluR, amphiregulin (AREG), TIM-1, TIM-2, TIM-3, andTIM-4.

In an embodiment, the number of cells administered in a vaccine is an effective amount of cells for bringing about the desired treatment. In the treatment of cancer, an effective amount of cells is that amount which can slow down or stop the progression of the cancer. In another embodiment, an effective amount of cells is that amount which can send the cancer into remission, and in other cases, even cure the cancer. In the treatment of an infectious disease, an effective amount of cells is that amount which can slow down or stop the progression of the in ectious disease. In another embodiment, an effective amount of cells is that amount which can send the partially or wholly eliminate the infectious disease in the patient. Based on the disclosure herein, the skilled artisan will understand how to determine the effective amount of cells in a cellular vaccine. The effective amount may comprise dendritic cells, T cells, or a combination of both.

By way of a non-limiting example, an effective amount of cells for treating hepatitis C is an amount of dendritic cells, T cells, or a combination of both, wherein aftcr^'administration of a vaccine comprising the effective amount of such cells to a patient having hepatitis C, the level of hepatitis C in the patient is diminished and/or eliminated. In other words, the administration of the cellular vaccine improves the condition of the patient and/or partially or completely eliminates the virus from the patient's body. In an embodiment, dendritic cells in an immunogenic composition are present at a concentration of about 5xl0³ to about 5xl0⁷ cells/ml. In an embodiment, the T cells in an immunogenic composition are present at a concentration of about Ixl O⁷ cells/ml.

In an embodiment, the T-lymphocytes in an immunogenic composition are prepared from the monocytes from a subject's blood.

In an embodiment, the compositions disclosed herein can be used by introducing into cells short interfering RNAs that temporarily switch off genes, suppressing the immune response. siRNAs are directed to one or more of the genes FAS, CTLA4, FOXP3, SOCSl, CD200, CD31 (PECAM-l), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphircgulin (A EG), TJM-1, TIM-2, TIM -3, and ΊΊΜ-4. As described herein, enhanced efficacy of the disclosed methods and compositions (therapeutic vaccines based on dendritic cells and/or activated lymphocytes) may be obtained by employing siRNA techniques to obtain a temporary shutdown of certain genes that are limiting the development of the immune response in the patient.

Therefore, in an embodiment, the cells in a cellular vaccine or immunogenic composition have some degree of suppression of expression of one or more genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCSl, CD200, CD31 (PECAM-l), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TlM-1, TIM-2, ΊΊΜ-3, and T1M-4. The suppression may be in dendritic cells, T cells, or both. In an embodiment, when both cells are present in an immunogenic composition, one cells or both cells may have suppression of one or more genes. In an embodiment, the same genes may be suppressed in both cells. In another embodiment, different genes may be suppressed in each type of cell. In an embodiment, a cell may have the expression of all of FAS, CTLA4, FOXP3, SOCSl, CD2'00, CD31 (PECAM-l), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), ΊΜ-1, TIM-2, ΊΊΜ-3, and TIM -4 suppressed. In an embodiment, the suppression is a partial suppression. In another embodiment, the suppression is nearly total suppression. In yet another embodiment, the suppression is 100%) suppression. The level or degree of suppression of any such gene is measured in comparison to the level of expression of the same gene in the absence of any suppression of the gene. In an embodiment, one or more of such genes is suppressed at least 5%>, at least 1 ()%>, at least 1 5%, at least 20%>, at least 25%., at least 30%», at least 35%, at least 40%), at least 45%, at least 50%., at least 55%, at least 60%>, at least 65%, at least 70%., at least 75%., at least 80%., at least 85%, at least 90%, at least 95%., at least 96%., at least 97%., at least 98%., at least 99%., and 100%. In another embodiment, one or more of such genes is suppressed from about 1 %. to about 99%., from about 5%. to about 95%., from about 1 0%. to about 90%., from about 20%. to about 80%., from about 25%) to about 75%>, or from about 40%> to about 60%). In another embodiment, one or more of such genes is suppressed about 5%>, about 10%>, about 1 5%, about 20%), about 25%>, about 30%>, about 35%., about 40%), about 45%, about 50%), about 55%., about 60%), about 65%,, about 70%>, about 75%, about 80%), about 85%, about 90%, about 95%), about 96%, about 97%, about 98%, about 99%., and 1 00%.. In an embodiment, disclosed herein is a method of treating cancer and/or an infectious disease comprising administering to a patient in need thereof a composition comprising dendritic cel ls modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS 1 , CD200, CD31 (PECA - 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), ΙΙΝΛ-binding protein FIuR, amph iregul in (A EG), ΊΊΜ- 1 , TIM -2, TIM-3, and TIM-4.

In an embodiment, disclosed herein is a method of treating cancer/or an infectious disease comprising adm in istering to a patient in need thereof a composition comprising: dendritic cells modi fied to suppress the expression of at least one gene selected from the group consisting of I AS, CTLA4, FOXP3, SOCS 1 , CD200, CD3 1 (PECAM- 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein Hull, amphiregul in (AREG), TIM- 1 , TIM-2, TIM-3, and TIM-4, and T-lymphocytes that were activated for Th- J type response. In an embodiment, the T-lymphocytes were modified to suppress at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, CD200, CD3 1 (PECAM- 1 ), Cylindromatosis gene (CY LD), Fas l igand (FasL), RNA-binding protein Hull, amphiregulin (AREG), TIM- 1 , TI M-2, TIM-3, andTIM-4. In an embodiment, disclosed herein is a method of treating cancer/or an infectious disease comprising adm inistering to a patient in need thereof a composition comprising T cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS 1 , CD200, CD31 (PECAM- 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein HuR, amph iregul in (AREG), TIM- 1 , TIM-2, T1M-3, and TI M -4.

In an embodi ment, disclosed herein is a method of making of a composition for treatment of canccr/or an in lectious disease comprising preparing dendritic cel ls modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3. SOCS 1 , CD200, CD31 (PECAM-1 ), Cyl indromatosis gene (CYLD), Fas ligand (FasL), RNA-bind ing protein HuR, amphiregul in (AREG), ΊΊ Μ- 1 , ΊΊΜ-2,· TI -3, and TIM-4, preparin T-lym phocytes modi fied to suppress the expression of at least one gene selected from the group consisting of CTLA4, FAS FOXP3, CD200, CD3 1 (PECAM- 1 ), Cylindromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein HuR, amphiregul in (AREG), TIM- 1 , TI M-2, TI M -3, and TIM-4, and combining the dendritic cel ls and the T- lymphocytes.

In an aspect, the suppression of gene expression can be achieved by any means known in the art. In an embodiment, suppression of gene expression is achieved using short interfering RNA techniques. In an embodiment, the suppression of expression of a gene is achieved by transfecting dendritic cells and/or T-lymphocytes with the respective correspond ing short interfering double-stranded RNAs. In another embodiment, transfection of the cells with leiitivi rus, targeting a gene of interest, can be used to suppress expression of the target gene.

In an embod iment, the gene selected for suppression of expression is a gene that would otherwise place the cel l (e.g., dendritic cell, T cell) in an i mmunosuppressive state. In an embodiment, such a gene is CT.LA4, FAS FOXP3, CD200, CD3 1 (PECAM- 1 ), Cylindromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM- 1 . TI M-2, TIM-3, and TIM-4, among others. In an embodiment, the expression of one or more of such genes is suppressed to overcome the immunosuppressive state.

The invention may be further described by the following examples. It should be recognized that variations based on the inventive features are within the skill of the ordinary artisan, and that the scope of the invention should not be limited by the examples. To properly determine the scope of the present disclosure, an interested party should consider the claims herein, and any equivalent thereof. In addition, all citations herein are incorporated by reference, and unless otherwise expressly stated, all percentages are by weight.

EXPERIMENTAL EXAMPLES Example 1: Gene

suppression experiments

Materials and methods. The system of RNAi used to suppress gene expression was the Ki-RNA transfection (ON-^'f ARGETplus SMARTpool, Dharmacon). The RNAi was used at a concentration of 100 nM / ml in IX transfection buffer (Dharmacon) was performed using lipofectaminc (Dharmacon-FECT), in combination with clectroporalion (350V, 50 microseconds). Given the possible toxicity of lipofectamine, each experiment included a negative control transfection without ki-RNA.

Immunodetection Assay. For the immunnodeteclion assay (ELISA), the level of expression of proteins was assayed by fixing the cells of interest with 4% formaldehyde (PBS) for 10 minutes, followed by incubation in 5.1% solution of BSA (bovine serum albumin (Biolot)), in phosphate-buffered saline (PBS), for 1 hour. After each treatment, the cell suspension was washed three times with PBS. Cells were incubated overnight at 4 °C with monoclonal antibodies to the corresponding protein in a dilution of 1 :200, washed three times with the FSB and incubated at room temperature for 1 hour with Alexa 488 fluorochrome labeled secondary antibodies against mouse immunoglobulins at a dilution of 1:2000 (Sigma). After a triple rinse in FSB, the proteins in the cells were visualized by fluorescent microscopy (OPTON-AXIOPLAN).

Quantifying the level of protein expression. The efficiency of suppression of gene expression was evaluated at the protein level, detected by enzyme immunoassay. The proteins were clcctrophorctically separated and immobilized on a nitrocellulose membrane. For this, cells (l-2hl()6) were subjected to electroporation in the presence of Ki-RNA and lipofectamine followed by the incubation with a mixture of kinetic and NA lipofectamine within 48 hours. Cells were washed twice with a solution of the FSB, the number of cells counted in each sample

for further 10 °C for 30 minutes with lysis buffer (balance the load paths, incubated at 4 mM Tris-HCl ph 7.4, 0.1% Triton X-100, 5 mm PMSF, 5 mm MgCl₂, 5 units / ml DNase I, 20 mM p'-mercaploethanol) obtained cell lysates was boiled in a standard buffer for the application for 5 minutes (0.25 M Tris ph 6.8, 8% SDS, 40% glycerol, 20% P- mercaptoethanol, 0.2% bromophenol blue). Electrophoretic protein separation was performed in 12% polyacrylamidc gel containing 0.1%) SDS, followed by protein transfer onto nitrocellulose membrane. Visualization of the protein on the membrane was performed with monoclonal antibodies at a dilution of 1:500 (Invitrogen) and followed by incubation with antibodies against mouse immunoglobulin (IgG), conjugated with horseradish peroxidase.

To assess changes in immune status of mononuclear cells, the "reaction blast" transformation of T lymphocytes was used. The ability off lymphocytes to respond to the impact of proliferation with phytohemagglutinin was assayed. To this end, mononuclear leukocytes isolated from peripheral blood were incubated 4 days at 37 °C in RPMI-1640 medium supplemented with 10% of human serum and 10 (ig / ml PHA. It is well known that under these conditions, T lymphocytes enter into the proliferative phase of the cell cycle in massive numbers. In the case of the presence of specific immunosuppression, a decrease in the proportion of lymphocytes capable of proliferation was observed. Thus, measuring the proportion of PI 1 A -activated lymphocytes, the potential readiness immune system to respond to antigenic stimulation of the proliferation of T lymphocytes was assessed. Shared their estimate of the proportion of incubation time of cells was carried out using the technique of flow cytometry. In this case, we used a method based on fluorescence quenching of binding of the dye Hoechst33258 to cellular DNA. Scoring included DNA replication and bi iTiodeoxyuridinc incorporation. For reference to techniques, see Kroll, Histochemistry (1984) 80:493-496; ubbies et al, Cell Tissue Ki net. (1985) 18:551-562.

In cells that have incorporated bromodeoxyuridine during DNA replication, fluorescence is much weaker when staining with fluorescent dye Hoechst33258. As a result, flow cytometry shows such cells after the division form a "peak" to the left (closer to the origin) than neproli crativc cells (Figure 1). The fraction of cells caught in this peak enables estimation of the proportion of cells that shared incubation time with bromodeoxyuridine.

In this experimental model, the ability of T lymphocyte proliferation to respond to nonspecific stimulation by PHA was monitored. Using How cytometry, the relative proportion of

lymphocytes passing through the cell cycle in the preceding 4 days was assessed. Fluorescence quenching showed the dye Hoechst 33258 binding to cellular DNA.

The essence of the method used is that for cells which have incorporated bromodeoxyuridine during DNA replication, fluorescence is much weaker when staining with the fluorescent dye l-Ioechst 33258. The flow cytometry histogram of the cells after division shows a "peak" to the left (closer to the origin) than the peak for non-proliferating cells (i.e., those having diploid DMA amount) (Figure 1). The fraction of cells caught in this peak allows estimation of the percentage of cells that shared incubation time with bromodeoxyuridine. Reaction of blast transformation based on the aggregation of T cell receptors, lectins, which mimics a similar aggregation in the interaction with a specific set of recognizable antigenic peptide and major histocompatibility complex in the normal response to an antigen. As is the case with normal antigen-dependent activation of T lymphocytes, proliferation of the cells is not enough to transmit the signal through the T cell receptor, and activation requires interaction with the subsidiary cells, and in particular, peripheral blood monocytes in the case of blast transformation reactions. In other words, signal transduction via the T-cell receptor is not enough for inducing the proliferation of T- cells, and the interaction with helper cells is required, just as in the case of the blast transformation reaction, where the peripheral blood monocytes are playing the role of these helper cells.

For technical description of such methods, see, eg., Folch et al, .1 Immunol. (1973), 110:835-9; Heilman ct al., Immunol Commun. (1974) 3:97-107; Schmidtke et al., .1 Immunol. (1976) 116:357-62; Schmidtke et al, Infect Immun. (1976) 13:1061-8, Mookerjee, Transplantation (1977) 23:22-8; Taniguchi et al, J Immunol. (1977) 118:193-7.

Thus, the model used can be regarded as a close analogue of the normal process of activation of T cell responses, based not only on activation via the T cell receptor, but also because it requires other co-stimulating signals received in the interaction of T lymphocytes and antigen presenting cells.

Measurement of the proportion of cells capable of responding to the proliferation of phytohemagglutinin impact varies greatly among different donors. Since T lymphocytes are usually about 60% of the original mononuclear blood cells used in the reaction of blast transformation, and by dividing the number of partitions of cells is doubled (assuming that the incubation time of cells carry no more than one division), the maximum possible share of the last cell cycle of f cells, presented in a flow-cytometry histogram, will not exceed 75%.

The actual proportion capable of activating T lymphocytes, as a rule, is substantially less, resulting in significantly fewer turns and share the descendants of this division, represented by reducible cytometric histograms as a peak with fluorescence intensity less than non-proliferating cells in phase Gl. According to our estimates of the average healthy person, it varies from 30 to 40%.

However, a significant number of people, especially patients suffering from cancer or other diseases, the percentage of activated T cells may be substantially lower, due to a lack of cells capable of proliferation under such conditions, which may be regarded as a state of i m m u n o s u p p re s s i o n .

In another aspect, the possibility to increase the proportion of activated T lymphocytes through the temporary shutdown of gene expression CTLA4, FAS, FOXP3, and SOCS1 was investigated. This negative regulation of immune response is known to be carried out through various mechanisms. For examples of experimental techniques known in the art, see Strasser et al, Immunity (2009) 30:180-92; Khattar et al., Arch Immunol Ther Exp (Warsz) (2009) 57:199-204; Bettini et al, Curr Opin Immunol (2009) 21 :612-8. Epub (2009) Oct 23; Baker et al, Trends Immunol (2009) 30:392-400.

To suppress the expression of genes, RNA interference methods were used. Short interfering RNA (siRNA), homologous to the studied genes in mononuclear cells, was transfected by clcctroporation of RNA preparations associated with lipofectamine. Transfected and control cells were activated with phytohemagglutinin. On the 4th day after transfection, flow cytometry was used to estimate the amount of cells proceeding through the cell cycle. The suppression of gene expression was revealed in a comparison, by electrophoresis, of the decrease in level and/or presence of the control content of the cells, versus the codin of proteins. Electrophoresis was followed by immunob lotting or immunofluorescence detection of the cell in situ. Suppressing the expression of genes studied on the example of the gene SOCS1, presented in Figure 2.

It is known that proliferative activity of T lymphocytes may be limited by the appearance on the surface of cytotoxic T-lymphocyte antigen 4 (Cytotoxic T-Lymphocyte Antigen 4, CTLA4). CTLA4 is a protein of the immunoglobulin superfamily, which proteins are expressed on the surface of activated T lymphocytes, and transmit inhibitory signals to stop the proliferation. Another factor that may inhibit the proliferation of activated lymphocytes is the appearance, on their surface, of significant amounts of FAS antigens, i.e., cell surface receptors that induce apoptosis during their

The results herein demonstrate that the decreased expression of genes CTLA4 and FAS can significantly increase the proportion of f lymphocytes responsible for stimulating proliferation of PHA (Figure 3), while transfection of non-specific short double-stranded RNA did not affect the result, but in high doses even suppressed the proliferation.

Another way to suppress the proliferation of T lymphocytes may be through modulation of their interaction with the regulatory CD4 + CD25 + FOXP3 cells belonging to a specialized subpopulalion off cells, which suppress the immune system and thus participate in maintaining homeostasis and tolerance to their own antigens. Regulatory T cells modulated the expression of wishbone-head transcription factor Foxp3 (forkhead box p3). Expression of FOXP3 is required for the development of regulatory T cells and for monitoring program that determines their destiny. Figure 4 illustrates that the inhibitory effect of regulatory CD4 i CD25 I FOXP3 cells can make a significant contribution to limiting the proliferative activity of phytohemaggkitinin stimulated T lymphocytes. Suppressing the expression of FOXP3 leads to a marked increase in the proportion of dividing cells.

Another important factor in the development of cellular technologies for immunotherapy is the ability of many tumors to suppress the potential immune response. The lysates of many (but not all) tumors can suppress the reaction of blast transformation of T lymphocytes. Figure 5 illustrates an example of this phenomenon. While not wishing to be bound by any particular theory, the gene SOCS1, which encodes a negative regulator of cytokine signals, and which can suppress the activation of macrophages and plays an important role in suppressing autoimmune reactions induced by dendritic cells, may be responsible for the above-mentioned phenomenon. It is known that a gene defect SOCS1 breaks tolerance to its own antigens and may contribute to an effective antitumor immune response (Hong el al., Cancer Res. (2009) 69:8076-84).

In some embodiments, the use of short interfering RTMA as set forth herein to specifically suppress the expression of the gene SOCS1 was able to overcome the inhibitory effect of lysates of tumor cells. However, while not wishing to be bound by any particular theory, it should be noted that such a rejuvenating effect may be due to a variety of factors in lysates of tumors that may inhibit the proliferation of T lymphocytes.

Example 2: Methods of treating viral infection.

In an embodiment, a therapeutic cell vaccine is prepared and used as follows:

Before each round of treatment for hepatitis C, 40-60 ml of peripheral blood of a

patient is extracted into a syringe containing heparin at 50 U / ml. Blood must be received for processing no later than 36 hours after collection. Using the known methods, monocytes are obtained from blood and are cultivated at 37 ° C in RPMI1640 medium supplemented with 10%

human serum obtained from donors with a 4-th group of blood. Isolation of monocytes and lymphocytes is done by standard centrifugation on a stepwise gradient ficol-urografin (density

solution 1.077 g / ml), followed by adhesion of monocytes to the surface of culture flasks. For induction of formation of dendritic cells to the culture medium, growth factors are added as follows: 1) granulocyte-macrophage colony-stimulating factor in the concentration of 3000 U / ml, (as is possible to use pharmacological agents "Leukomax" and "Sargramostim") and 2), intcrlcukin 4 (IL4) in a concentration of 500 U / ml, which can be replaced by interlcukin 15 or interferon alpha.

On the second day after the addition of growth factors to the medium, a mixture of NA homologous to the genes SOCS1 and FAS is added, as well as an agent promoting its penetration into the cells - Lipofectamin (Dharmacon), prepared in accordance with the manufacturer's protocol. The remainder of the procedure for making of dendritic cells may be any method known in the art.

At 4-5 days, antigenic material is added to dendritic cells, mainly in the form of fragments of viral protein 1MS3 and / or core protein (in case of hepatitis C) /or the combination of a tumor lysate with antigens (in the case of tumors) or another specific antigens in other cases. On the same day, to further strengthen the capture of antigenic material by dendritic cells, antigenic material is injected into the dendritic cells by electric charge (e I ectropo rat i o n ) ., '

For the induction of full maturation of dendritic cells after the addition of antigen, proinflammatory signals are added: a conditioned medium derived from autologous mononuclear cultivation, or trace amounts of bacterial lipopolysaccharide (0.2 microgram per ml), or a mixture of cytokines TNFa + I Lib.

As a proof of obtaining true dendritic cells as a result of the above method, the following criteria may be used:

a) presence of growth in non-adhering to the substrate state (in contrast to macrophages, which are lightly adhered to the substrate);

b) a characteristic morphology of dendritic cells presenting multiple dendrites;

a) the emergence of a large number of surface markers characteristic of dendritic cells (HLA-DR, HLA-ABC, CD80, CD83) is determined using a fluorescent microscope or flow cytometry technique.

In parallel with the preparation of dendritic cells, the activated T-lymphocytes are also prepared from the same portion of blood, for which the mononuclear leukocytes are isolated by centrifugation in a density gradient by a standard procedure, and T-lymphocytes from the cell mixture are activated to proliferate by adding phytohemagglutinin (20 micrograms / ml). The procedure is carried out as standard blast transformation of T-lymphocytes, except that for the primary stimulation of Thl-cell immune response, the bacterial lipopolysaccharide (0.2 micrograms per ml) is added to the cells.

In an embodiment, the siRNA specific for the genes FAS, CTLA4 and FOXP3 is injected into cells by clcctroporation prior to before adding phytohemagglutinin.

The remainder of the procedure of preparing the T-lympocytes is conducted in any manner known in the art.

On the sixth day, the dendritic cells loaded with antigen and T-lymphocytes activated to implement the Thl response are combined together in 1.5 ml of medium in which they are cultured, and injected into the patient mainly paravertebral ly in the interscapular region, intradermally, in the form of lemon-peel in 2 or 3 points on the back.

The preferential treatment of chronic hepatitis lasts for 5 weeks with weekly blood sampling and preparation of portions of dendritic cells loaded with antigen, mainly gene-engineered fragments of protein NS3 and/or core protein. Parallel to immunotherapy, a standard treatment of hepatitis, such as the one using interferon and ribavirin, may be employed. Upon completion of the treatment, the standard polymerase chain reaction (PCR) is used to estimate the virus titer in the blood. If necessary, the treatment can be repeated.

In other embodiments, a similar approach can also be used to treat other viral infections, particularly hepatitis B, herpes and papilloma virus infections. In this case, the antigen can be used in standard commercial vaccine against hepatitis B, herpes simplex virus and papilloma virus.

It will be appreciated by those skilled in the art that changes could be made to the exemplary embodiments shown and described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the exemplary embodiments shown and described, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the claims. For example, specific features of the exemplary embodiments may or may not be part of the claimed invention and features of the disclosed embodiments may be combined. Unless specif cally set forth herein, the terms "a", "an" and "the" are not limited to one element but instead should be read as meaning "at least one".

It is to be understood that at least some of the descriptions of the invention have been simplified to focus on elements that are relevant for a clear understanding of the invention, while eliminating, for purposes of clarity, other elements that those of ordinary skill in the ait will appreciate may also comprise a portion of the invention. However, because such elements are well known in the art, and because they do not necessarily facilitate a better understanding of the invention, a description of such elements is not provided herein.

Further, to the extent that the method does not rely on the particular order of steps set forth herein, the particular order of the steps should not be construed as limitation on the claims. The claims directed to the method of the present invention should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the steps may be varied and still remain within the spirit and scope of the present invention.

Claims

1. An immunogenic composition comprising dendritic cells modified to suppress the expression of at least one of the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCSL CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein Hull, amphiregulin (A REG), TIM-1, TIM -2, TIM -3, and T1M-4.

2. The composition of claim 1, further comprising T cells that were previously activated for ^"fhl response.

3. The composition of claim 2, wherein the T cells modified to suppress of the expression of at least one genes selected from the group consisting of CTL4, FAS, FOXP3 CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein Hull, amphiregulin (A EG), TlM-1, T1M-2, TJM-3, and TIM-4.

4. An immunogenic composition comprising T cells modified to suppress the expression of at least one of the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCSL CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein Hull, amphiregulin (AREG), TIM-1, ΊΊΜ-2, ΊΊΜ-3, and TI.M- 4.

5. The composition of claim 3 or claim 4, wherein the T-lymphocytes are prepared from the monocytes from a subject's blood.

6. A method of making of a composition for treatment of cancer comprising: a. ) preparing dendritic cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TlM-1, TIM-2, TIM-3, and TIM-4,

b. ) preparing T-lym hocytes modified to suppress the expression of at least one gens selected from the group consisting of CTLA4, FAS FOXP3, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein Hull, amphiregulin (AREG), TIM- 1, TIM-2, ΊΊΜ-3, and TTM-4 and c.) combining the dendritic cells and the T-lymphocytes.

7. The method of claim 6, wherein the suppressed expression of genes CTLA4, FAS FOXP3, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, ΊΊΜ-2, ΊΊΜ-3, and TIM-4 is achieved by transfecting the T-lymphocytes with the corresponding short interfering double- stranded RNAs.

8. A method of treating cancer comprising administering to a patient in need thereof a composition comprising dendritic cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM- 1 , ΊΊΜ-2, TIM-3, and TIM-4.

9. A method of treating cancer comprising administering to a patient in need thereof a composition comprising:

a. ) dendritic cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1),

Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1. ΊΊΜ-2, TIM-3, and TIM-4, and

b. ) T-lymphocytes that were activated for Th-1 type response.

10. The method of claim 9, further wherein the T-lymphocytes were modified to suppress at least one gene selected from the group consisting of FAS, CTLA4, FOXP3,

CD200,

CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1 , TIM-2, TIM-3. and TIM-4,

11. A method of making of a composition for treatment of an infectious disease comprising:

a.) preparing dendritic cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), ΊΊΜ-1 , T1M-2, TIM-3, and ΤΪΜ-4,

b. ) preparing T-!ymphocytes modified to suppress the expression of at least one gene selected from the group consisting of CTLA4, FAS FOXP3, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), ΊΊΜ-1 , ΊΊΜ-2, TIM-3, and TIM-4 and

c. ) combining the dendritic cells and the T-lymphocytes.

12. The method of claim 11, wherein the suppressed expression of genes CTLA4, FAS FOXP3, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1 , T1M-2, TIM-3, and TIM-4 is achieved by trans feet ing the T-lymphocytes with the corresponding short interfering double- stranded RNAs.

13. A method of treating an infectious disease comprising administering to a patient in need thereof a composition comprising dendritic cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCSl, CD200, CD3I (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR_; amphiregulin (AREG), TIM-1, TIM -2, TIM -3, and TIM-4.

14. A method of treating an infectious disease comprising administering to a patient in need thereof a composition comprising:

a. ) dendritic cells modified to suppress the expression of at least one gene selected from the group consistin of FAS, CTLA4, FOXP3, SOCSl, CD200, CD31 (PECAM-1),

Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR. amphiregulin (AREG), TIM-1, TIM-2, TIM-3, and TIM-4, and

b. ) T-lymphocytes that were activated for Th-1 type response.

15. The method of claim 14, further wherein the T-lymphocytes were modified to suppress at least one gene selected from the group consisting of FAS, CTLA4, FOXP3,

CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, TJM-2, TIM-3, and TIM-4.