WO2009149539A1

WO2009149539A1 - Enhancing antigen-specific cd8+ t cell response using irf-7 mrna

Info

Publication number: WO2009149539A1
Application number: PCT/CA2009/000450
Authority: WO
Inventors: Rafick-Pierre SÉKALY; Bader Yassine-Diab; Oleg Yegorov
Original assignee: Université de Montréal
Priority date: 2008-06-10
Filing date: 2009-04-09
Publication date: 2009-12-17

Abstract

The invention provides compositions and methods useful for treating a subject in need thereof by stimulating an immune response. In some embodiments, the compositions and methods increase the population size of at least one type of CD8⁺ T cells in vivo or in vitro, such as transitory effector memory cells. The compositions are antigen-presenting cells that have been transfected with RNA encoding the regulatory factor IRF-7 as well as T cell populations induced by those APCs. In some embodiments, the APCs of the invention further comprise RNA encoding at least one antigen. The compositions of the invention can be administered to a subject to stimulate an immune response.

Description

ENHANCING ANTIGEN-SPECIFIC CD8⁺T CELL RESPONSE USING IRF-7 mRNA

STATEMENT REGARDING FEDERALLY-SPONSORED

RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Contract No. HHSN2662006600019C awarded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.

FIELD OF THE INVENTION

The present invention relates to improved methods of treating a subject in need thereof by stimulating an immune response against one or more antigens. The methods comprise the use of antigen-presenting cells that have been transfected with RNA encoding the regulatory factor IRF-7. The methods can be used to stimulate an immune response in a subject.

BACKGROUND OF THE INVENTION

Cytotoxic CD8⁺ T cells perform an important role in the immune response by destroying cells infected by intracellular pathogens such as viruses or bacteria (see, e.g., Chapter 8 of Murphy et al. (2007), Janeway's Immunobiology, 7^th Ed., Garland Science, London). Cytotoxic T cells kill target cells by inducing them to undergo apoptosis, or programmed cell death {Id. at Chapter 8). Cytotoxic CD8⁺ T cells act to kill other cells mainly through their release of specialized lytic granules which are modified lysosomes containing at least two types of cytotoxic proteins (perforin and granzymes), although they also express Fas ligand. {Id.) Perforin acts by making transmembrane pores in the membranes of target cells, while granzymes are powerful serine proteases. Granzyme B is believed to activate cell death by cleaving cellular enzyme CPP-32. Ligation of Fas by Fas ligand also leads to apoptosis by activating caspases in the target cell. (Id.) Fortunately, the destructive actions of CD8⁺ T cells are only exerted on target cells expressing specific antigens, and other nearby cells generally are not harmed. (Id.)

In HIV disease, cytotoxic CD8⁺ T cells are defective and are often unable to lyse HIV-I- infected cells (see, e.g., Lieberman et al. (2001) Blood 98: 1667-1677). Defects in the CD8⁺ T cell response have also been observed in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) (see, e.g., Kang et al. (2004) J. Immunol. 172: 1287-94) and in patients with cancer (see, e.g., Critchley-Thorne et al. (2007) PLoS Med. 4: 897-911).

Research on CD8⁺ T cells has increased understanding of the roles played by these cells and led to the identification of new subpopulations of CD8⁺ T cells (see, e.g., Wood et al. (2007) Crit Rev. Immunol. 27: 527-537; Klebanoffef al (2006) Immunol. Rev. 211 : 214-224; Zhang et al. (2006) J. Immunol. Ill: 6730-6737). However, the exact cause of the defects in the CD8⁺ T cell response to these diseases remains unknown.

SUMMARY OF THE INVENTION

The invention provides compositions and methods useful for treating a subject in need thereof by stimulating an immune response. In some embodiments, the compositions and methods increase the population size of at least one type of T cells in vivo or in vitro, such as, for example, central memory ("CM") cells, transitory effector memory ("TEM") cells, and/or effector memory ("EM") cells. The compositions of the invention include antigen-presenting cells that have been transfected with RNA encoding the regulatory factor IRF-7 as well as CD8⁺ T cell types or populations stimulated and/or expanded by these antigen-presenting cells. For example, the compositions of the invention include TEM cells induced by IRF-7-transfected APCs. In some embodiments, the compositions further comprise RNA encoding at least one antigen. The compositions of the invention can be administered to a subject to stimulate an immune response. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (see Example 1) shows expression of IRF-7 in DCs electroporated with different amounts of mRNA encoding IRF-7. Western blot analysis is shown in the left panel, and FACS analysis is shown in the right panel.

Figure 2 (see Example 1) shows the expression of various cell surface markers on DCs transfected with mRNA encoding CD40L and IRF-7. For comparison, data is shown from immature DCs (solid lines, shaded) as well as data from DCs transfected with mRNA encoding CD40L (solid lines, no shading) and data from DCs transfected with mRNA encoding both CD40L and IRF-7 (dashed lines, no shading). Data on the horizontal ("x") axis is shown on a logarithmic scale.

Figure 3 (see Example 1) shows increased expression of MHC class I in mature DCs transfected with mRNA encoding IRF7. For comparison, data is shown from immature DCs (solid lines, shaded) as well as data from DCs transfected with mRNA encoding CD40L (solid lines, no shading) and data from DCs transfected with mRNA encoding both CD40L and IRF-7 (dashed lines, no shading). Data on the horizontal ("x") axis is shown on a logarithmic scale.

Figure 4 (see Example 2) shows that DCs from a healthy patient when transfected with mRNA encoding IRF-7 increase the capacity of CMV-specific CD8⁺ T cells to proliferate, but do not significantly affect CD4⁺ T cell proliferation. "Co-stimulator RNA" is IRF-7 mRNA; "NS" means no exogenous stimulus.

Figure 5 (see Example 2) shows that DCs from an HIV-infected patient when transfected with IRF-7 increase the capacity of HIV-specific CD8⁺(but not CD4⁺) T cells to proliferate. "Co-stimulator RNA" is IRF-7 mRNA; "NS" means no exogenous stimulus.

Figure 6 (see Example 3) shows that DCs transfected with mRNA encoding IRF-7 increase Gag-specific responses by CD8⁺ T cells as detected by CFSE and tetramer assays. The panels in the left-hand column indicate for each treatment the percentage of the overall cell population which is proliferating (as measured by CFSE assay). The panels in the right-hand column indicate for each treatment the percentage of the overall CD8⁺ T-cell population which is Gag-specific (as measured by tetramer assay).

Figure 7 (see Example 3) shows that DCs transfected with mRNA encoding IRF-7 cause certain populations of CD8⁺ T cells to proliferate as measured by CFSE assay. On this "heat map," increased proliferation is shown by darker colors. Surface markers for each population are indicated on the left-hand side: "28" = CD28; "RA" = CD45RA; "R7" = CCR7; and "27" = CD27. "Co-stimulator RNA" is IRF-7 niRNA; "NS" means no exogenous stimulus.

Figure 8 (see Example 4) shows that the CD8⁺ T-cell populations produced following co- culture with DCs transfected with IRF-7 mRNA also exhibit functions including expression of CD 107 and secretion of IFN-γ. Notably, the "TEM 107a⁺" population was not produced in the absence of IRF-7.

Figure 9 (see Example 4) shows a heteroduplex mobility assay (HMA) and demonstrates that IRF-7 enhances the HIV-specific T-cell repertoire.

Figure 10 shows a diagram of CD8⁺ T-cell differentiation as well as cell surface markers and typical functional attributes associated with each type of CD8⁺ T cells. CD8⁺ T-cell maturation can be examined and defined using a combination of the phenotypic markers CCR7, CD27, and CD45RA. This diagram also indicates the expression of CD28 for intermediate stages in this differentiation pathway. This diagram is provided to assist in understanding current theories in the art about the relationship between and among different types of CD8⁺ T cells, but is not in any way to be construed as a limitation on the invention.

Figure 11 shows a comparison of Gag-specific CD8⁺ T cells on day 5 and day 6 after stimulation with DCs transfected with Gag mRNA or Gag and IRF-7 mRNA. The stimulus for each group of cells is shown on the horizontal axis, and the percentage of CFSE^low CD8⁺ T cells is shown on the vertical axis.

Figure 12 the effect of anti-IFN-α antibodies in growth medium on proliferative activity of CD8⁺ cells stimulated by DCs transfected as indicated.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods useful for treating a subject in need thereof by enhancing the immune response to at least one antigen. In some embodiments, the compositions are antigen-presenting cells ("APCs") that have been transfected with exogenous RNA encoding the regulatory factor IRF-7. In some embodiments, the compositions are antigen- presenting cells that have been transfected with exogenous RNA comprising RNA encoding the regulatory factor IRF-7 and RNA encoding at least one antigen. In some embodiments, these antigen-presenting cells have also been transfected with exogenous RNA encoding CD40 ligand ("CD40L"). Alternatively, in some embodiments, the compositions are antigen-presenting calls that have been transfected with exogenous RNA comprising RNA encoding the regulatory factor IRF-7 and CD40L and, optionally, RNA encoding at least one antigen. Thus, the invention provides compositions which are antigen-presenting cells comprising RNA encoding IRF-7 and, optionally, further comprising RNA encoding at least one antigen and/or RNA encoding CD40L.

The methods of the invention can be used to stimulate an immune response in a subject and, in some embodiments, involve the use of antigen-presenting cells that have been transfected with exogenous RNA encoding the regulatory factor IRF-7 and optionally also with RNA encoding at least one antigen of interest and/or with RNA encoding CD40L. Thus, in some embodiments, the methods comprise the use of antigen-presenting cells that have been transfected to contain exogenous RNA encoding the regulatory factor IRF-7 and exogenous RNA encoding at least one antigen of interest, while in other embodiments the methods comprise the use of antigen-presenting cells that have been transfected to contain exogenous RNA encoding IRF-7 and CD40L and optionally RNA encoding at least one antigen of interest. The transfected APCs of the invention can be administered to a subject to stimulate an immune response, or they can be cultured in vitro with a population of cells comprising PBMCs or CD8⁺ cells, which can then be administered to a subject either separately or together with the APCs. In this manner, the compositions and methods of the invention provide increased populations of effector and memory CD8⁺ T cells in vivo and in vitro.

Where desired, other polynucleotides such as DNA or polynucleotides containing synthetic nucleic acids may be used instead of RNA. Thus, in some embodiments, the compositions and/or methods of the invention involve antigen-presenting cells that have been transfected with exogenous RNA or DNA or polynucleotide(s) comprising synthetic nucleic acids encoding the regulatory factor IRF-7 and optionally also with RNA or DNA or polynucleotide(s) comprising synthetic nucleic acids encoding at least one antigen of interest and/or with RNA encoding CD40L. Thus, in some embodiments, the methods comprise the use of antigen-presenting cells that have been transfected to contain exogenous RNA or DNA or polynucleotide(s) comprising synthetic nucleic acids encoding the regulatory factor IRF-7 and exogenous RNA or DNA or polynucleotide(s) comprising synthetic nucleic acids encoding at least one antigen of interest, while in other embodiments the methods comprise the use of antigen-presenting cells that have been transfected to contain exogenous RNA or DNA or polynucleotide(s) comprising synthetic nucleic acids encoding IRF-7 and CD40L and optionally RNA or DNA or polynucleotide(s) comprising synthetic nucleic acids encoding at least one antigen of interest.

In some embodiments, antigen-presenting cells may be transfected with RNA (e.g., RNA encoding IRF-7 and optionally also RNA encoding CD40L and optionally also RNA encoding one or more antigens) and also loaded with at least one antigen for presentation by "pulsing" the cells with at least one antigen. As used herein, "pulsing" or pulsed" indicates that the APCs have been incubated in the presence of the antigen. In embodiments where the APCs are "pulsed" with antigen, the antigen may be a lysate or extract of tissue or cells, such as, for example, a lysate or extract prepared by sonication or freeze/thawing of tissue or cells. As used herein, "lysate" refers to material produced by the lysis of a cell, which may be, for example, a pathogenic bacteria or a mammalian cell infected with a virus. An "extract" is a fraction of a lysate, such as, for example, a pellet or supernatant of a centrifuged lysate or a fraction enriched by size or affinity from the lysate. An antigen or mRNA encoding an antigen may be or encode an isolated or individually-prepared antigen (such as, for example, a selected, previously-known antigen); thus, where more than one antigen is used, a mixture of isolated, individually-prepared, and/or selected antigens or RNAs encoding them may be used.

As used herein, "antigen-presenting cell" or "APC" refers to specialized cells that can process antigens into peptide fragments and display those peptide fragments on the cell surface together with molecules required for T-cell activation. An antigen-presenting cell of the invention can be any cell which is capable of presenting an antigen to a T cell; for example, an antigen-presenting cell can be a dendritic cell, a B cell, a macrophage, or an artificial antigen- presenting cell. An "artificial antigen-presenting cell" is a cell which has been engineered to express MHC class I and/or II molecules and/or other molecules required for costimulating CD4⁺ and/or CD8⁺ T cells (see, e.g., Kim et al. (2004) Nat. Biotechnol. 22: 403-410). Artificial antigen-presenting cells can include, but are not limited to: genetically-engineered insect cells, mouse fibroblasts and human leukemia cell lines. Generally, when an APC has been transfected with exogenous RNA, the APC expresses the product of that RNA and at least one function of the APC is altered by the product. For example, when an APC is transfected with exogenous RNA encoding an antigen, the transfected RNA is typically expressed and the APC can present the encoded antigen to a T cell. The term "dendritic cell" ("DC") refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues in mammals (see, e.g., Steinman (1991) Ann. Rev. Immunol. 9: 271-296). Dendritic cells are the most potent APCs in vivo, and can be isolated from a mammal or differentiated from CD14⁺ monocytes or CD34⁺ hematopoietic stem cells isolated from a mammal. The maturation state of DCs can be followed by monitoring the change of the DC surface markers during the maturation process. While surface markers can vary depending on the maturation process, typically, mature DCs are characterized as CD14^~ CD83⁺, CD86⁺, and CD80⁺.

Methods for preparing antigen-presenting cells and methods for transfecting antigen- presenting cells are known in the art (for example, as described in U.S. Pat. No. 5,853,719; 7,198,948; and WO 2008/055354). Briefly, for example, to prepare dendritic cells (DCs), adherent monocytes can be obtained from a leukapheresis product of a subject, incubated for about 6 days in the presence of GM-CSF and IL-4, and then matured by incubation in the presence of TNFα, IFN-γ, IL-6, and/or PGE₂ for about 24 hours. Other maturation methods are known in the art. These cells can then be transfected by electroporation with RNA and/or pulsed with at least one antigen. In other exemplary methods, dendritic cell precursors or immature dendritic cells can be transfected with RNA and/or pulsed with at least one antigen and then matured in vitro by incubation in media containing GM-CSF and IL-4, if necessary, followed by a maturation process such as incubation in monocyte-conditioned medium or a synthetic substitute thereof. APCs of the invention can also be produced by transfecting immature DCs with RNA and/or pulsing with antigen as discussed above, followed by administration of the cells to a subject and maturation of the cells in vivo to mature DCs.

Methods known in the art and suitable for use in the invention also include the "CD40L base process" and the "Post-Maturation Electroporation-CD40L process" ("PME process") for DC maturation, for example, as described in International Application WO 2006/042177. In the CD40L base process, immature DCs are transfected with CD40L-encoding mRNA and antigen- encoding mRNA, and then treated with IFN-γ (1000 U/ml) or TNF-α (10 ng/ml) or a combination of IFN- γ and PGE₂ (1 μg/ml). The cytokine levels may be increased or decreased. In the PME-CD40L process, typically, after 4-7 days after beginning culture of monocytes with GM-CSF and IL-4, the resulting monocyte-derived immature DCs are matured by culture with TNF-α (10 ng/ml), IFN-γ (1000 U/ml), and PGE₂ (1 μg /ml) for between 12 and 30 hours, preferably about 18 hours. DCs are then harvested from the culture and electroporated with antigen-encoding RNA and CD40L mRNA and cultured in X-VIVO 15 media containing 800 U/ml GM-CSF and 500 U/ml IL-4 for 4 or more hours. It is known in the art that culture conditions (including cytokine concentrations) for culturing and maturing or differentiating cells including monocytes and dendritic cells are approximate and can readily be adjusted to optimize culture conditions. Cytokines from different suppliers and lots often vary in activity, and one of skill in the art can readily perform titration experiments to determine the optimal dose for any particular cytokine or combination of cytokines.

Other variations of these methods may also be used in preparing APCs of the invention, so long as APCs of the invention are produced (i.e., APCs that contain exogenous RNA encoding IRF-7 and optionally RNA encoding at least one antigen of interest and/or RNA encoding CD40L). For example, APCs can be derived from CD34⁺ cells from peripheral blood or from cord blood. The RNA with which the APCs are transfected can be a pure preparation (i.e., containing only one species of RNA) or it may be a mixture of different RNAs. APCs can be transfected with RNA once or multiple transfections can be performed, either with pure preparations that are the same or different from each other, or with mixtures of RNA.

DCs may be transfected with RNA and/or loaded with antigen when they are immature or mature. In some embodiments, DCs are pulsed with antigen when they are immature and then transfected with RNA following maturation. Alternatively, DCs can be transfected with RNA when they are immature and pulsed with antigen after maturation. APCs that are prepared by transfecting APCs with RNA and also pulsing them with antigen can efficiently present antigen to both CD4⁺ T cells and to CD8⁺ T cells; in these embodiments, the compositions and methods of the invention also provide stimulation of the CD4⁺ T-cell compartment (in addition to the stimulation of CD8⁺ T cell types and populations as specifically discussed herein for embodiments in which APCs are not pulsed with antigen).

Thus, in some embodiments of the invention, APCs (such as, for example, DCs) are produced that express exogenous RNA with which they have been transfected, so that a protein encoded by the RNA is produced by the APC. These APCs can then be used to stimulate the development of T cell populations either in vivo following administration of transfected dendritic cells of the invention to a subject or, alternatively, by culture of the APCs in vitro with T cells or PBMCs. In some embodiments, the APCs of the invention and methods of use increase at least one population of CD8⁺ T cells, such as, for example, the transitory effector memory ("TEM") cell population. That is, in some embodiments, the populations (i.e., number of various types) of CD8⁺ T cells increases as a result of exposure of CD8⁺ T cells to the APCs of the invention, either in a subject (i.e., in vivo) or in in vitro culture. In this manner, at least one population or various populations of CD8⁺ T cells are increased by the methods of the invention.

In some embodiments, transfected APCs are used to produce antigen-specific CD8⁺ T cells in vitro. In these embodiments, T-cell populations comprising CD8⁺ T cells may be isolated from a subject or may be cultured, and either PBMCs or subpopulations of T cells may also be used, such as, for example, populations sorted by cell surface marker so as to comprise enriched populations of particular cells, such as CD8⁺ cells. If the T cells are being produced in vitro, the APCs may be allogeneic to or syngeneic with the T cells. In some embodiments, DCs transfected with RNA encoding IRF-7 and optionally other RNAs are also pulsed with antigen and present the antigen to T cells, resulting in the production of antigen-specific T cells. In this manner, the invention also provides an increase in antigen-specific T cells, which can be one or more type of CD8⁺ T cell. For example, the compositions and methods of the invention can be combined with the compositions and methods taught in WO 2008/055354.

During production of T cells, the T cells may be exposed to and/or cultured with the APCs once or more than once. For example, the T cells may be cultured with APCs for hours, days, or weeks, and the APCs in the mixed culture may be replenished if necessary. In some embodiments, culture will continue until a therapeutic amount of CD8⁺ T cells has been obtained. Other culture techniques and/or additives may be used to improve the results obtained; for example, the culture media may also contain cytokines such as, for example, IL-2. After compositions of the invention which are APCs or T cells are made, they can be administered to a subject without further modification or they can be further processed and/or frozen for subsequent use. In some embodiments, the transfected antigen-presenting cells and/ or T cells are further cultured in vitro prior to administration to a patient.

Thus, in some embodiments, compositions of the invention are CD8⁺ cells produced using such APCs; in this manner, the compositions of the invention include TEM CD8⁺ T cells generated in vivo following injection of APCs into a subject or generated in vitro by culturing PBMCs or T cells with APCs transfected with exogenous RNA encoding IRF-7. Accordingly, the methods of the invention also provide methods for stimulating and/or expanding certain populations or types of CD8⁺ T cells. In some embodiments, the resulting TEM cells are purified or isolated using techniques known in the art, such as, for example, FACS or magnetic separation using antibodies directed to relevant cell surface markers and positive or negative selection, as appropriate.

Cell-surface markers can be used to isolate or separate cell types from other cell types. In this manner, cells necessary to practice the methods of the invention or to make the compositions of the invention can be isolated and/or purified. For example, human CD34⁺ stem cells express CD34 antigen, while DCs express MHC molecules and costimulatory molecules (e.g., B7-1 and B7-2), and lack markers specific for granulocytes, NK cells, B cells, and T cells; TEM cells express CD8 and CD27 but do not express CD45RA and CCR7. The expression of surface markers facilitates identification and purification of these cells. For example, positive and negative selection can be used to separate cells of interest from a mixture. Generally, positive selection separates a group of cells of interest from a mixture of cells based on the expression by the cells of interest of at least one particular cell surface marker, while negative selection removes cells that are not of interest from a mixture based on their expression of at least one particular cell surface marker that is not expressed by the cells of interest. That is, for example, TEM cells (which are CD45RA^", CD27⁺, and CCR7^") can be removed from a CD8⁺ cell mixture using an antibody that binds to CD27 coupled to magnetic beads; other cells can then be removed or separated from the TEM cells using antibodies that bind to CD45RA and to CCR7 and are coupled to magnetic beads; in this manner, a purified or isolated TEM cell population can be prepared from a cell mixture. It will be understood from this description that corresponding procedures can be used to purify or isolate the other CD8 cell types described herein.

CD8⁺ T-cell maturation can be examined and described and CD8⁺ T-cell types and/or populations can be identified using the phenotypic cell-surface markers CCR7, CD27, and CD45RA. As used herein, CD8⁺ T-cell types and/or populations have the following characteristics or pattern of expression of cell surface markers: Naive T cells are characterized as CD45RA⁺, CD27⁺, and CCR7⁺; Central Memory T cells ("CM cells") have the phenotype CD45RA^~, CD27⁺, and CCR7⁺; Transitory Effector Memory T cells ("TEM cells") are characterized as CD45RA^", CD27⁺, and CCRr ; Effector Memory T cells ("EM cells") are defined by the lack of expression of these three markers (CD45RA , CD27 , and CCR7^~); and terminally differentiated Effector T cells are characterized as CD45RA⁺, CD27^", and CCRT.

Thus, different types and/or populations of CD8⁺ T cells are defined herein by the expression profile of the cell surface markers CD45RA, CCR7, and CD27 as indicated above. These different CD8⁺ T cell types can also exhibit particular functions, including, for example: secretion of IFN-γ; secretion of IL-2; production of Granzyme B; and expression of CD 107. However, while the expression pattern of cell surface markers is considered diagnostic of each particular CD8⁺ T cell type and/or population as described herein, the functional attributes of each cell type and/or population may vary depending on the amount of stimulation the cell(s) has or have received (see, for example, Zhang et al. (2006) J. Immunol. 177: 6730-37; see also data presented in Figure 8 and related discussion in the working Examples).

Other markers known in the art to differ among CD8⁺ T cell populations that may be helpful in identifying, distinguishing, purifying, or isolating different CD8⁺ T cell populations or subtypes include, but are not limited to, CD28, CD62L, CD45RO, and KLRG-I; these cells may also differ in the length of their telomeres (see, e.g., Sallusto et al. (2004) Ann. Rev. Immunol. 22: 745-63). Thus, the five cell types shown in Figure 10 and listed above can be separated from other cells and/or from each other based on their expression or lack of expression of at least one marker selected from the group consisting of: CD45RA, CD27, and CCR7. In this manner, the invention provides methods of separating different populations of CD8⁺ T cells and also separated or isolated populations of CD8⁺ T cells. These cell types may also be separated or isolated from other cells based on their expression or lack of expression of at least one marker selected from the group consisting of: CD28, CD62L, CD45RO, and KLRG-I . The CD8⁺ T cell types described herein may also be isolated by any other suitable method known in the art; for example, if a particular antigen or antigens are used to produce antigen-specific CD8⁺ T cells, those cells can be separated or isolated from other cells by affinity purification using that antigen or antigens; appropriate protocols are known in the art.

Figure 10 shows a diagram of a simplified differentiation pathway for CD8⁺ T cells; this diagram is provided to assist in understanding current theories in the art about the relationship between and among different types of CD8⁺ T cells, but is not to be construed as a limitation on the invention, which is not bound by any particular mechanism of operation. As indicated in Figure 10, the different CD8⁺ T-cell types or populations also typically have different functions (also referred to as "functional attributes"), either prior to or following antigenic stimulation. For example, in the absence of TCR stimulation (also referred to as "antigenic stimulation"), Transitory Effector Memory (TEM) cells generally do not secrete IFN-γ or IL-2, and may or may not also express the cell surface marker CD28; however, following stimulation with an antigen (such as, for example, a model CMV antigen), some of the TEM cells will secrete IFN-γ, and following stimulation with a "superantigen" such as SEB {Staphylococcus aureus enterotoxin B) will show dramatic increases in IFN-γ and IL-2 secretion (that is, a significant portion of the cells having the TEM cell surface marker profile will secrete at least some IFN-γ and IL-2, with different TEM cells expressing different amounts of IFN-γ and/or IL-2). Effector Memory (EM) cells in the absence of TCR or antigen stimulation show some low level of IFN-γ secretion and very little IL-2 secretion and may or may not also express the cell surface marker CD28, but following TCR or antigenic stimulation with a model CMV antigen will show increased IFN-γ secretion, a small increase in IL-2 secretion, and increased expression of CD 107a, while following stimulation with a "superantigen" such as SEB these cells will show dramatic increases in secretion of IFN-γ and IL-2 and also in expression of CD 107a. The increase in the variety and strength of these functions provided by the invention can be referred to as "polyfunctionality."

Assays for expression of cell surface markers and assays for various T cell functions are known in the art and are also illustrated in the working examples provided herein. For example, effector functions of T cells are determined by the effector molecules that they release in response to specific binding of their T-cell receptor ("TCR") with antigen:MHC complex on the target cell. Cytotoxic effector molecules that can be released by cytotoxic CD8⁺ T cells include perforin, granzymes, granulysin and Fas ligand. Generally, among other functions, perform forms transmembrane pores in the target cell, granzymes are serine proteases which can trigger apoptosis, granulysin induces apoptosis in target cells, and Fas ligand can also induce apoptosis. Generally, these cytotoxic effector molecules are stored in lytic granules in the cell prior to release. Other effector molecules that can be released by cytotoxic T cells include IFN-γ, TNF-β and TNF-α. Among other functions, IFN-γ can inhibit viral replication and activate macrophages, while TNF-β and TNF-α can participate in macrophage activation and in killing target cells. Suitable assays for expression of cell surface markers are known in the art and can measure expression directly or indirectly. These assays include, but are not limited to, ELISA, FACS analysis, and ICS (intracellular cytokine staining; see, e.g., Meddows-Taylor et al. (2007) J. Virol. Meth. 144: 115-121). Suitable functional assays are known in the art and can measure function directly or indirectly. These assays include, but are not limited to: cytotoxicity assays; assays for the production of IL-2; assays for the production of interferon-gamma ("IFN-γ") and Granzyme B; and assays for the expression of CD 107.

In accordance with the present invention, techniques known in the art may be used, including conventional molecular biology, microbiology, and cell culture techniques. Such techniques are explained fully in the literature; see, e.g., Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.) (herein, "Sambrook"); Ausubel et al, eds. (2008) Current Protocols in Molecular Biology, Supplement 82 (John Wiley & Sons, Inc., Hoboken, N.J.).

Methods of identification and isolation of particular cells include FACS, column chromatography, panning with magnetic beads, Western blots, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, as well as various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like. For a review of immunological and immunoassay procedures in general, see, e.g., Stiles and Terr (eds.) (1991) Basic and Clinical Immunology (7^th ed.); for a discussion of how to make antibodies to selected antigens see Harlow and Lane (1989) Antibodies: A Laboratory Manual (Cold Spring Harbor Press).

Cell isolation or immunoassays for detection of cells during cell purification can be performed in any of several configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay (CRC Press, Boca Raton, Florida); Tijan (1985) "Practice and Theory of Enzyme Immunoassays," in Laboratory Techniques in Biochemistry and Molecular Biology (Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane (1989), supra; Chan (ed.) (1987) Immunoassay: A Practical Guide (Academic Press, Orlando, Florida); Price and Newman (eds.) (1991) Principles and Practice of Immunoassays (Stockton Press, NY); and Ngo (ed.) (1988) Non-isotopic Immunoassays (Plenum Press, NY). Cells can be isolated and characterized by flow cytometry methods and FACS analysis. A wide variety of flow-cytometry methods are known. For a general overview of fluorescence activated flow cytometry, see, for example, Abbas et al. (1991) Cellular and Molecular Immunology (W.B. Saunders Company) (especially Chapter 3), and Kuby (1992) Immunology (W.H. Freeman and Co.) (especially Chapter 6). Labeling agents which can be used in these methods include, but are not limited to: monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any known method, such as immunoblotting, Western blot analysis, tracking of radioactive or bioluminescent markers, capillary electrophoresis, or other methods which can be used to track a molecule based on size, charge, or affinity.

Experiments described in the working examples demonstrated that antigen-specific CD8⁺ T cells produced following in vitro coculture with IRF-7-transfected APCs can have different phenotypic profiles (i.e., patterns of cell-surface marker expression and/or functional attributes) from those produced in the absence of IRF-7 (see, for example, data shown in Figure 7 and described in working Example 3). In this manner, the methods and compositions of the invention can provide increased polyfunctionality of T cells as well as increased numbers of CD8⁺ T cells and T cells expressing CD28. Such cells are expected to improve the clinical outcome for a subject (see, e.g., Berts et al. (2006) Blood 107: 4781-4789; Halwani et al. (2006) Springer Semin. Immun. 28: 197-208).

It is known in the art that various cell types generally exhibit a characteristic array of cell surface markers, as discussed above. Thus, for example, a mature DC (whether a DC produced in vivo or a DC produced in vitro) will exhibit no or little expression of CD 14 but will typically express the cell surface markers CD80, CD83, and CD86, and will secrete IL- 12. Similarly, some types of CD8⁺ T cells are described herein as having a particular array of cell surface markers. It further known in the art that sometimes commonly-used techniques for the analysis of expression of cell surface markers (e.g., FACS analysis) can yield data that represents mixed populations of cells, and care should be taken to identify which populations may be represented in a particular data set. In many instances, a sample of cells (whether directly isolated from a subject or whether obtained following in vitro culture) will contain a number of different cell types. Attention should be paid to the level of expression of a particular marker as well as to the percentage of a population expressing that marker. Fortunately, one of skill can avoid confusion by making use of an appropriate control.

In some embodiments of the methods of the invention, an increase in a first particular CD8⁺ T cell type and/or population may result from an initial increase in a different type of CD8⁺ T cells followed by differentiation of those cells into cells of the first particular CD8⁺ T cell type, thereby increasing the size of that population. For example, an increase in the TEM cell population may result in an increase in the EM (effector memory) cell population and/or the CM (central memory) population, following the differentiation of at least some of the TEM cells into EM and/or CM cells. In other words, TEM cells can be produced and then can become, for example, EM or CM cells. These changes from one cell type into another can take a period of time during which various markers and functional attributes may change at different rates, producing cells that do not fit exactly into one of the cell type designations described herein (i.e., Naϊve, CM, TEM, EM, or Effector), or that fit one of these cell type designations but that have different functional attributes. However, the recognition of new types of cells or of cells in transition between one cell type and another does not negate the invention, which in some embodiments provides an increase in at least one of the cell types as defined herein but may also have other effects on other cell types.

Generally, the T cells stimulated by the antigen-presenting cells of the invention will recognize at least one antigen presented by the APCs, including, for example, an antigen encoded by the exogenous RNA with which the APCs were transfected. In this manner, the invention also provides compositions which are T cells that recognize a particular antigen or antigens, and methods of use thereof. In some embodiments, a further benefit of the invention is that it enhances and broadens the antigen- specific T cell repertoire (i.e., the number of different T-cell receptors specific to a particular antigen, as illustrated, for example, by the data presented in Figure 9). In this manner, the invention provides antigen-specific CD8⁺ T cells (i.e., increased populations of antigen-specific CD8⁺ T cells) with increased functionality and also provides increased population(s) of long-lasting memory CD8⁺ T cells (such as, for example, CM cells).

Antigens for use in the compositions and methods of the invention can be any antigen against which an immune response is desired. Thus, exemplary antigens include any protein or other composition to which an immune response can be raised, and can be from or derived from any cell or pathogen or other composition. As used herein, "antigen" encompasses polypeptides, proteins and peptides that consist of or comprise at least one epitope, which when presented as an MHC/peptide complex can specifically bind to a particular T-cell antigen receptor (also called "T-cell receptor," or "TCR"). As used herein, "peptide" refers to five or more amino acids co valently joined by peptide bonds. Antigens for use in the compositions and methods of the invention can be derived from HIV and related viruses and/or from any kind of tumor or cancer, including, for example, a solid or liquid tumor. A "tumor-associated antigen" ("TAA") refers to an antigen that is associated with a tumor. Examples of TAAs known in the art include gplOO, survivin, MART, and MAGE. An antigen is "derived from" or "associated with" a virus, cell, or other organism if it is encoded by a gene of that organism or cell, even though an antigen is generally a fragment of a native gene product. An antigen may be modified by inclusion in a fusion protein with another protein.

Antigens for use in the compositions and methods of the invention that are tumor antigens or pathogen antigens can, but need not be, specifically and/or preferentially expressed in the tumor cell or pathogen as compared to expression in other types of cells. Antigens are "specifically expressed" by a tumor or pathogen (or pathogen-infected cell) if they are not normally expressed at the same time point in other cells of a subject. Antigens are "preferentially expressed" by a tumor or pathogen or pathogen-infected cell if they are expressed at a level that is at least 50% higher than the level of expression of the antigen in other cells of a subject (which cells may be a selected population of cells or cell types).

In some embodiments of the compositions and/or methods of the invention, more than one antigen can be used. An antigen can be administered to the subject separately from the APCs of the invention or can be administered via transfection of the APCs with RNA encoding the antigen. That is, in some embodiments, one or more protein antigens or one or more antigen- encoding RNAs can be administered to the subject separately from the APCs of the invention (see, e.g., U.S. Pat. No. 7,015,204). In some embodiments, an antigen-presenting cell comprises RNA encoding IRF-7 and one or more antigens, such as, for example, 2, 3, 4, 5, or 10 or more antigens. The antigens can be selected individually or can be a mixture of antigens; for example, the antigens can be a mixture of antigens the exact proportions and/or identities of which are unknown, such as, for example, antigens encoded by total RNA from a particular tumor. Thus, the antigen-encoding RNA used to transfect an APC can encode one selected antigen, a mixture of selected antigens, or can comprise total RNA from a suitable source, such as a tumor or pathogen-infected cells. In some embodiments, it is not important to know how many antigens are encoded by the RNA used, so long as at least one antigen of interest is encoded by the RNA. The term "antigen of interest" refers to an antigen to which an immune response is desired. Antigens can be either wild type or mutant (mutated). RNA encoding an antigen may be identical in sequence to RNA found in nature, or it may differ, so long as it encodes the same antigen.

In some embodiments, where an APC is transfected with RNA encoding at least one antigen, the RNA is total RNA obtained from cells expressing an antigen against which an immune response is sought, such as, e.g., cells of a tumor of interest, pathogenic bacteria, virions, or cells containing a virus. Generally, total RNA is obtained by lysing such cells by homogenization or sonication in suitable buffers followed by extraction and precipitation of the RNA fraction from the cell homogenate. Alternatively, total RNA can be prepared using RNA purification methods known in the art, such as, for example, methods utilizing guanidinium isothiocyanate and/or oligo-dT chromatography methods, which enrich for poly- A⁺ RNA. The RNA-containing preparation can optionally be fractionated to decrease the concentration of other components in the preparation (such as, for example, lipids, proteins or DNA), and can also optionally be treated with proteases or RNAse-free DNAses.

RNA for use in the compositions and methods of the invention can be obtained using a variety of methods known in the art. It is not necessary that the RNA be in purified form; in some embodiments, the RNA sample contains at least 80%, at least 90%, or at least 95% RNA (wt/wt). As used herein, the terms "total RNA" and "RNA" encompass messenger RNA ("mRNA") and/or poly- A⁺ RNA, and refer to RNA that includes RNA other than mRNA. Generally, total RNA will contain mRNA encoding at least one antigen against which an immune response is desired. Where appropriate, total RNA may be obtained, for example, from cells of a tumor of interest or from pathogenic bacteria or from cells containing a virus. Total RNA from any source can also be amplified using standard techniques to prepare sufficient amounts of RNA for transfection of antigen-presenting cells; the term "total RNA" as used herein also encompasses such amplified RNA. RNA can be synthetically manufactured or amplified from an RNA or DNA template, including, for example, total RNA that has been amplified from reverse-transcribed cDNA representing total RNA from a cell. For use in the compositions and methods of the invention, mRNA (poly- A⁺ RNA) can be obtained using a poly-T column, as is known in the art. In order to obtain amounts of mRNA sufficient for use in the compositions and methods of the invention, conventional amplification techniques may be used. For example, cellular, total, or messenger RNA can be reverse- transcribed in vitro to produce cDNA for amplification by PCR; the cDNA can then be transcribed in vitro to produce mRNA for use in transfecting antigen-presenting cells. As used herein, "mRNA" means a translatable RNA.

Generally, an mRNA will contain a ribosome binding site and at least one start codon, and may also optionally contain a 5' cap, stop codon, and/or poly-A⁺ tail. In some embodiments, RNA may be modified in a number of different ways to enhance transcription. RNA may be capped co-transcriptionally or post-transcriptionally with a Type 0 or Type 1 cap, as is known in the art. For example, a Type I cap indistinguishable from the cap formed on RNA transcripts in eukaryotic cells can be added using vaccinia virus capping enzyme and 2'-O-methyltransferase, using methods known in the art and also implemented in commercially-available kits such as, for example, the Epicentre Biotechnologies ScriptCap™ Capl Capping Kit, as described in the product literature and also, for example, in WO 2007/117682. In some embodiments, RNA may have a poly-A⁺ tail which is 100 or more nucleic acids in length, or the poly- A⁺ tail may be shorter, for example, the poly-A⁺ tail may contain about or exactly 50, 60, 64, 70, 80, or 90 nucleic acids. In some embodiments, the poly-A⁺ tail can be included in a desired RNA by including a poly-T tract at the end of the DNA which is transcribed to produce the RNA. In some embodiments, the RNA has a Type 1 cap and a poly- A⁺ tail which is 64 nucleic acids in length.

DNA and/or RNA for use in the compositions and methods of the invention may also be modified so that translation begins at a different position than in a native form of the RNA. For example, DNA and/or RNA may be modified so that translation of the RNA begins from the methionine which in the native RNA is the second encoded methionine (see, e.g., CD40L Δ XE - Met#l, as described in U.S. App. No. 11/400,774 and set forth herein as SEQ ID NO: 17; the corresponding translated protein sequence is set forth herein as SEQ ID NO: 18). DNA and/or RNA may also be modified to include a non-native 5' or 3' UTR; for example, CD40L mRNA can be modified to include a rotavirus gene 6 3' UTR as described in U.S. App. No. 11/400,774 and as set forth in SEQ ID NO: 19 and 20 herein. DNA and/or RNA may also be modified to substitute optimized codons to enhance transcription and/or translation. For example, CD40L RNA may be modified as described in U.S. App. No. 11/400,774 and set forth in SEQ ID NO: 16 herein; another example is the modified, codon-optimized IRF-7 RNA set forth in SEQ ID NO:22 herein. SEQ ID NO:22 includes a T7 promoter sequence so that RNA transcription would begin at nucleotide 18; the IRF-7 coding sequence begins at nucleotide 48 ("ATG") and ends at the "TAG" stop codon at nucleotide 1,510.

Where RNA is isolated from tumor cells, the tumor cells may be obtained in any suitable manner; e.g., cells can be obtained from tumors surgically resected in the course of a treatment for a cancer. The tumor cells can be processed in any suitable manner, for example, as described by: Berd et al. (1986) Cancer Res. 46: 2572; Sato et al. (1997) Cancer Invest. 15: 98; or U.S. Pat. No. 5,290,551. Briefly, in some embodiments, the tumor cells are extracted by dissociation, such as: enzymatic dissociation with collagenase and DNAse; mechanical dissociation in a blender; teasing apart with tweezers; using mortar and pestle; cutting into small pieces using a scalpel blade, and the like. If a tumor is liquid (such as a tumor of blood or bone marrow), samples may be collected and tumor cells isolated by density gradient centrifugation or any other suitable method. By "tumor" or "cancer" is meant the presence of abnormal cells which exhibit aberrant growth. The aberrant growth is often characterized by a significant loss of cell proliferation control. Tumor cells can be benign or malignant. As used herein, "tumor cell" includes not only a primary tumor cell, but also any cell derived from an ancestor which was a tumor cell; thus, "tumor" encompasses metastasized cancer cells, in vitro cultures, and cell lines derived from cancer cells. When RNA is prepared from cells of a tumor, the tumor cells may be from the type of tumor which is to be treated. The tumor cells may be but are not limited to autologous cells dissociated from biopsy or surgical resection specimens, or from tissue culture of such cells. In other embodiments, RNA is prepared from allogeneic cells or stem cells.

The compositions and method of the present invention may be used to treat any type of cancer, including metastatic and primary cancers. Accordingly, cancers treatable with the present invention include solid tumors (such as, for example, carcinomas), and non-solid tumors, (such as, for example, hematologic malignancies). Generally, as used herein, the terms "cancer" and "tumor" are interchangeable. Examples of cancers that can be treated according to the invention include but are not limited to: renal cell cancer, melanoma, breast cancer, prostate cancer, testicular cancer, bladder cancer, ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, colon cancer, neuroblastoma, glioblastoma, retinoblastoma, leukemia (including chronic lymphocytic leukemia), myeloma (including multiple myeloma), lymphoma, hepatoma, adenoma, sarcoma, carcinoma, and blastoma. Selected antigens of interest from tumors include, but are not limited to: telomerase, prostate specific antigen (PSA), MARTl, MAGE, and various angiogenesis factors known in the art.

The compositions and methods may also be used to treat any kind of viral, bacterial, or other infection, including infection with HIV. When RNA is isolated from cells of a pathogen or from cells infected with a virus, the pathogen or virus can be, for example: multiple-drug- resistant bacteria, Heliobacter pylori, Salmonella, Shigella, Enterobacter, Campylobacter, Mycobacterium spp., Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella species, Leptospira interrogans, Staphylococcus aureus, Streptococcus, Clostridium, Candida albicans, Plasmodium, Leishmania, Trypanosoma, human immunodeficiency virus (HIV), cytomegalovirus (CMV), HCV (hepatitis C virus), hepatitis B virus (HBV), human papilloma virus (HPV), human T-cell lymphotropic virus (HTLV), herpes simplex virus type 1 (HSV-I), herpes simplex virus type 2 (HSV-2), coronavirus, varicella-zoster virus, Epstein-Barr virus (EBV), influenza virus, poliomyelitis virus, measles virus, mumps virus, or rubella virus. Thus, in some embodiments, the virus can be a retrovirus (such as, for example, HIV). As used herein, "pathogen" refers to any organism which is capable of causing disease, or capable of living as a parasite on or in another organism (the "host"), even if it does not cause detectable symptoms or abnormalities in its host.

The antigen-presenting cells of the invention can be administered to a subject. A subject may be a human patient or a non-human animal patient, such as, for example, an ape, cow, horse, dog or cat, or any other mammal. A subject may be treated for multiple conditions, such as, for example, HIV infection and a tumor. By "patient" is intended a human or animal subject being treated by a medical or veterinary professional, as appropriate.

The antigen-presenting cells administered to a subject may be "autologous" to that subject; that is, they may be cells that were originally isolated from the same subject, or may be cells that were descended from cells originally isolated from the same subject and expanded using in vitro culture. Alternatively, the antigen-presenting cells administered to a subject may be "heterologous" to the subject, which as used herein means that the cells are from a source other than the subject. Heterologous cells may be cells from or derived or descended from cells originally isolated from another subject, including but not limited to stem cells.

"Activated," when used in reference to a T cell, implies that the cell is no longer in GO phase, and begins to produce one or more cytotoxins, cytokines and/or other membrane- associated markers characteristic of the cell type (e.g., CD8⁺) and is capable of recognizing and binding any target cell that displays the particular peptide:MHC complex on its surface and releasing its effector molecules. As used herein, the term "educated, antigen-specific immune effector cell" refers to an immune effector cell that has previously encountered an antigen. In contrast to its naive counterpart, an educated, antigen-specific, immune effector cell does not require a costimulatory signal for activation; recognition of the peptide: MHC complex is sufficient.

By "effective amount" of a substance or a therapeutic composition is intended that the amount is at least sufficient to achieve at least one object of the invention when administered to a subject. Thus, for example, an "effective amount" of a therapeutic composition which is antigen-presenting cells of the invention or CD8⁺ T cells of the invention is at least sufficient to achieve at least one object of the invention as further discussed elsewhere herein. An effective amount may be determined by one of skill in the art with regard to symptoms exhibited by an individual subject or it may be determined from clinical studies or extrapolated from an appropriate study in a model system. Thus, in some embodiments, an effective amount of a therapeutic composition of the invention is sufficient to change a measurement of a symptom or a response of the subject in an amount that is statistically significant. An effective amount can be administered in one or more applications or dosages. Suitable administrations, applications, and dosages can be determined by one of skill in the art and are known to vary depending on a number of factors, which include but are not limited to: specific activity of the compositions; the formulation of the compositions; the body weight, age, health, disease and condition of the subject to be treated; and the route of administration of the compositions into the subject.

As used herein, "immune response" refers broadly to the antigen-specific responses of lymphocytes to foreign substances. An immune response can be humoral and/or cell mediated. An immune response to an antigen or epitope includes but is not limited to production of an antigen-specific or epitope-specific antibody and/or production of an immune cell expressing on its surface a molecule which specifically binds to an antigen or epitope. Methods of determining whether an immune response to a given antigen or epitope has been induced are well known in the art. For example, an antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including but not limited to ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen or epitope is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).

CD8⁺ T cells can also be induced to expand and mature by immunization of a subject with peptides or dendritic cells other than those which are compositions of the present invention. Thus, in some embodiments, the compositions and/or methods of the invention can be used in combination with other compositions and/or methods. Thus, for example, in addition to administering transfected antigen-presenting cells to the patient as described above, some embodiments of methods of the invention include further stimulation of the immune response by immunizing a subject with peptides or dendritic cells that have not been transfected with IRF-7.

IRF-7 encodes a member of the interferon regulatory transcription factor (IRF) family (see, e.g., Sgarbanti et al. (2007) Ann. N. Y. Acad. Sci. 1095: 325-33). In some embodiments, an IRF-7 protein encoded by an IRF-7 mRNA will have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more sequence identity to the corresponding portion of the sequence of the human IRF-7 protein (for example, SEQ ID NO:2, which is the human IRF-7 protein set forth as IRF-7A in Figure IB of Zhang and Pagano (1997) (Afo/. Cell. Biol. 17: 5748-5757, hereby incorporated by reference, and is encoded by the nucleotide sequence set forth in SEQ ID NO:1, as also taught in the Zhang and Pagano reference) and/or any IRF-7 sequence as described by SwissProt Ace. No. Q92985, all of which are hereby incorporated by reference and including, for example, the sequences set forth in SEQ ID NOs: 3, 4, 5, 6, and 7. Isoforms, splice isoforms, and variants of IRF-7 are also known in the art and are suitable for use in the methods and compositions of the invention so long as the desired result is achieved (see, e.g., isoforms and variants described in association with SwissProt Ace. No. Q92985, such as, for example, K179E and Q412R (i.e., a variant in which the lysine at position 179 is changed to glutamic acid and a variant in which the glutamine at position 412 is changed to arginine).

In some embodiments, an IRF-7 mRNA encodes a protein which comprises or consists of a fragment of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, or more consecutive amino acids from the native, full-length IRF-7 protein, or comprises an amino acid sequence which shares at least 50%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to such a fragment. Generally, "sequence identity" as used herein refers to the sequence identity between two sequences as determined using the well-known BLAST alignment program with default parameters as appropriate for the type of sequences (i.e., nucleotide or amino acid). Alternative programs are known in the art. In some embodiments, the comparison of sequences and determination of percent identity between two sequences can be determined using the Gapped BLAST or PSI-BLAST algorithm, for example, as described in Altschul et al. (1997) Nucl. Acids Res. 25: 3389-3402, or a software program implementing one of these algorithms and using default parameters.

The activity of IRF-7 for use in the present invention can be evaluated by methods known in the art. An IRF-7 protein is said to have "IRF-7 activity" if it exhibits at least one activity of the native, full-length IRF-7 protein as measured by any suitable assay. A IRF-7 protein is said to have "IRF-7 activity" if in such an assay it has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the activity of the native, full-length IRF-7 protein as measured in the same assay. Preferably, IRF-7 protein for use in the present invention is capable of stimulating interferon production. IRF-7 activity can be evaluated directly or indirectly and can be measured in vivo or in vitro; suitable assays are known in the art. IRF-7 proteins useful in the compositions and/or methods of the invention will exhibit at least one activity of the native, full- length IRF-7 protein. Assays for IRF-7 activity are known in the art and include, for example: assays for binding to interferon-stimulated response element (ISRE) and/or repression of transcriptional activation by interferon or IRF-I (see, e.g., Zhang and Pagano (1997) MoI. Cell. Biol. 17: 5748-5757); or FACS analysis of IRF-7 expression by cells transfected with IRF-7- encoding RNA (as described, for example, in working Example 1); or an increase in at least one T-cell populations following coculture with APCs transfected with IRF-7-encoding RNA (as described, for example, in working Example 3), such as a detectable or increased TEM cell population; or increased polyfunctionality of T cells following APC coculture.

In some embodiments, APCs of the invention are transfected with RNA encoding IRF-7 and also with RNA encoding CD40 ligand (also called "CD40L" or CD 154), a molecule involved in development of the acquired immune response (see, e.g., Quezada et al. (2004) Ann. Rev. Immunol. 22: 307-328). In some embodiments, a CD40L protein encoded by a CD40L mRNA will have at least 50%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more sequence identity to the corresponding portion of the sequence of the human CD40 ligand protein, for example, a human CD40 ligand amino acid sequence in U.S. Pat. No. 5,981,724 (hereby incorporated by reference, and as set forth, for example, in SEQ ID NO:9 of the present sequence listing), or any other CD40 ligand sequence set forth in U.S. Pat. No. 5,981,724; and/or the hCD40-L sequence set forth in Figure 1 of Spriggs et al. (1992) J Exp. Med. 176: 1543-1550, hereby incorporated by reference, and set forth in SEQ ID NO:11. This protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 10. CD40L proteins and nucleic acids encoding them are also taught in U.S. App. No. 11/400,774 (also set forth herein as SEQ ID NO: 14 (nucleic acid) and SEQ ID NO: 15 (protein)). Other CD40L proteins and nucleic acids are discussed, for example, in WO 2008/055354, which is hereby incorporated by reference, and are set forth in SEQ ID NO: 12 (nucleic acid) and SEQ ID NO: 13 (protein). Any CD40L is suitable for use in the invention so long as it provides the desired results. In some embodiments, a CD40L mRNA encodes a protein which comprises or consists of a fragment of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, or more consecutive amino acids from the native, full- length CD40L protein, or comprises an amino acid sequence which shares at least 50%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to such a fragment.

Fragments of full-length CD40L protein and the nucleic acids encoding them are taught, for example, in U.S. Pat. No. 5,981,724, and include nucleotides 46 through 828, nucleotides 184 through 828; nucleotides 196 through 828; nucleotides 403 through 828; and nucleotides 382 through 828 of SEQ ID NO:8 of the present sequence listing (disclosed in U.S. Pat. No. 5,981,724). Fragments of the nucleic acids encoding CD40L also include nucleotides 133 through 855 of the sequence set forth in SEQ ID NO: 12, which was also disclosed in WO 2008/055354. Fragments of CD40L protein also include amino acids 47 through 261; 120 through 261; and 113 through 261 of SEQ ID NO:9 (disclosed in U.S. Pat. No. 5,981,724) and amino acids 21 through 261 of SEQ ID NO: 13 (also disclosed in WO 2008/055354). Known specific variants of CD40L may also be used, such as, for example, a variant taught in pending U.S. App. No. 1 1/400,774, which is hereby incorporated by reference, such as the variant set forth in SEQ ID NO: 18; other variants are encoded by the nucleic acid sequences disclosed in U.S. App. No. 11/400,774 and set forth herein in SEQ ID NOs: 16, 17, 19, 20, 21. Other variants include CD40L and fragments thereof in which the cysteine at nucleotides 625-627 of the full-length CD40L coding sequence is replaced with nucleotides encoding tryptophan, and a variant of the CD40L amino acid sequence which is C 194 W (i.e., the cysteine at position 194 is changed to tryptophan).

CD40 ligand ("CD40L") proteins useful in the compositions and/or methods of the invention will exhibit at least one activity of the native, full-length CD40L protein. A CD40L protein is said to have "CD40L activity" if in such an assay it has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the activity of the native, full-length CD40L protein as measured in the same assay. Assays for CD40L activity are known in the art and include, for example: assays for binding to soluble CD40 (as described, for example, in Graf et al. (1992) Eur. J. Immunol. 22: 3191-94); FACS analysis of CD40L expression by cells transfected with CD40L-encoding RNA; an increase in proliferation of B cells following coculture with APCs transfected with CD40L-encoding RNA; or an induction of immunoglobulin E secretion by B cells following coculture with APCs transfected with CD40L- encoding RNA (as described, for example, in Spriggs et al. (1992) J. Exp. Med. 176: 1543-50).

The objects of the invention can vary depending on the embodiment. In some embodiments, the objects of the invention include the prevention, cure, reduction, and/or alleviation of at least one symptom of a disease or disorder. Thus, in some embodiments, at least one symptom of a disease or disorder is prevented, cured, reduced, or alleviated in comparison to an untreated control or other appropriate control (e.g., in comparison to the symptom prior to treatment or to the expected severity of the symptom without treatment). Those of skill in the art are familiar with the selection and application of methods of measurement and evaluation of symptoms as well as with the selection of appropriate controls. For example, where the subject is being treated for a tumor, symptoms include, for example, the size or estimated size of the tumor and the grade of the tumor, as well as the formation of new tumors following metastasis. Accordingly, objects of the invention in such a subject can include a reduction of the size or estimated size of the tumor(s), the grade of the tumor(s), and/or the rate or degree of metastasis. Where the subject is being treated for infection with a pathogen such as, for example, a virus, objects of the invention include reduction in the amount and/or viability of pathogen in the subject, increasing the number and/or functionality of T cell types that recognize an antigen associated with the pathogen, and increasing the titer in the blood of antibodies that recognize an antigen associated with the pathogen. Other objects of the invention may also be appropriate and can be identified by those of skill in the art. A symptom of a disease or disorder in a subject is considered to be reduced or alleviated by a treatment if that symptom is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in comparison to an appropriate control, such as, for example, the symptom prior to treatment or the expected change in the symptom if it were left untreated. This assessment may be based on a statistical analysis of a population of subjects. For example, the ability of a method of the invention to reduce or inhibit the formation of new tumors in a subject can be determined by measuring the tumor growth over a period of time before during and after treatment. Measurements of tumor size or growth can be made after surgical excision of a tumor, e.g., by CAT scan, MRI, PET scan, and the like.

In some embodiments, an object of the invention is the production and/or increase of particular populations of cells, such as, for example, Naive, CM, TEM, EM, and/or Effector cells. That is, in some embodiments, at least one of these populations is increased; in some embodiments, any pairwise combination of these populations is increased; and in some embodiments, at least three, four, or five of these populations are increased in any combination. These cells can be produced and assessed either in vivo or in vitro. Such increase(s) in the population is (are) statistically significant according to a suitable statistical analysis known in the art. A population is increased when the cells are present in an amount which is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% higher in comparison to an appropriate control such as, for example, the size of the population prior to treatment with a method of the invention. "Functionality" is increased when cells have a function which is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% higher or lower, as appropriate, than an appropriate control, such as, for example, the performance of a sample of cells in a particular assay in the absence of a particular event or condition. Where appropriate, in vivo function or the presence of a cell population in vivo may be measured using cells isolated from a subject in in vitro assays.

One of skill in the art is familiar with techniques and criteria for evaluating changes in symptoms, or changes in the amount or functionality of a particular cell population. Symptoms and/or cell populations can be evaluated after a suitable interval following treatment according to a method of the invention or with a composition of the invention. Generally, a suitable interval is an amount of time which is expected to or has been shown to be sufficient for an effect of treatment to be seen. For example, a symptom of a subject treated according to a method of the invention or with a composition of the invention can be evaluated and show an effect of treatment on or about the first day after treatment, or on or about the second, third, fourth, fifth, or sixth day after treatment, or at or about a week after treatment, or at or about two, three, or four weeks after treatment, or at or about two, three, four, five, or six months after treatment. In this context, "treatment" can refer to the entire course of treatment or it can refer to a particular administration, such as the first administration of a composition of the invention to a subject.

In some embodiments, the methods of the invention provide the benefit of a synergistic effect produced by the APCs expressing IRF-7 and also presenting an antigen of interest to T cells. In this manner, the invention provides an unexpected benefit. In some embodiments, the benefit provided by a composition or method of the invention is greater than the benefit provided by previously known treatments by at least 10%, 20%, 25%, 30%, 50%, 75%, 100%, 200%, or more, or by 1.5-fold, 2-fold, 3-fold, 4-fold, or more.

Methods of the invention include methods of treatment in which the subject is treated during only one stage of therapy or treatment interval, or is treated during at least one stage of therapy or treatment interval. Stages of therapy and/or treatment intervals as well as what adjustments to treatment parameters such as doses, timing, and monitoring of progress of the treatment are understood and can readily be performed and adjusted as necessary by those of skill in the art. In some embodiments, a treatment interval is a period of time during which treatment is conducted and/or evaluated, such as a day, a week, a month, or two, three, four, five, or six months, or a year, or an interval during or following which the subject is shown to have achieved certain treatment landmark(s). In some embodiments, a subject may be treated with a method and/or composition of the invention at regular intervals (e.g., approximately every two years, every year, every six months, every two to four months, every month, every two weeks, or every week). However, generally, the methods and compositions of the invention are administered to a subject that has been identified as having a particular disease or disorder that could benefit from treatment using a method of the invention. Generally, a course of treatment ends when the subject is no longer being treated for a particular disease or medical condition.

The compositions and methods of the invention can be used alone (i.e., they can be used to treat a subject that is not receiving other treatments) or they can be administered to a subject that is receiving or has received other treatments. For example, a subject afflicted with a particular disease or disorder could be treated only with the methods and compositions of the invention, or they could be treated with the methods and compositions of the invention as well as another treatment or therapy commonly administered to subjects that are or may become affected by the particular disease or disorder, such as, for example, conventional chemotherapy or pharmaceutical treatment. Thus, in some embodiments, the methods of the invention can also include administration of at least one other immunostimulatory compound or pharmaceutical, such as, for example, an immunostimulatory cytokine such as IL-2.

A substance is "coadministered" with another substance (i.e., "coadministration" occurs) when substances are simultaneously administered to the subject, or when a substance is administered to the subject within 2, 4, 6, 8, 10, 12, or 14 hours, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 days before or after another substance is administered to the subject. When substances are administered "simultaneously," they can be combined prior to administration, or separately administered at the same time. The methods of the invention can include one dose or administration or multiple doses or administrations of each compound or composition. In some embodiments of the invention, APCs of the invention can be coadministered with antigens and/or with other APCs.

For use in the assays or methods of the invention, T cells can be prepared or isolated from a subject by methods known to those of skill in the art, such as, for example, the following method. Ficoll-Hypaque density gradient centrifugation is used to separate PBMCs from red blood cells and neutrophils according to established procedures. Cells are washed with modified AIM-V (which consists of AIM-V (GIBCO) with 2 mM glutamine, 10 μg/ml gentamicin sulfate, 50 μg/ml streptomycin) supplemented with 1% fetal bovine serum (FBS). T cells are enriched by negative or positive selection with appropriate monoclonal antibodies coupled to columns or magnetic beads according to standard techniques. An aliquot of cells is analyzed for cell surface markers such as CD4, CD8, and CD14. For the purpose of illustration only, cells are washed and resuspended at a concentration of about 5 x 10⁵ cells/ml of AIM-V modified as above and containing 5% FBS and 100 U/ml recombinant IL-2 (rIL-2) (supplemented AIM-V). Where the cells are isolated from an HIV⁺ patient, HIV-infected cells are generally selectively removed from the culture, for example, using a cytotoxic molecule treatment such as 25 nM CD4-PE40, which is a recombinant protein consisting of the HIV-1-binding CD4 domain linked to the translocation and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A. CD4- PE40 has been shown to inhibit p24 production in HIV-infected cell cultures and to selectively kill HIV-I -infected cells. Other suitable methods for isolating, culturing, and expanding T cells are known in the art. For example, to stimulate T-cell proliferation, an antibody directed against the CD3 molecule (such as the OKT3 monoclonal antibody from Ortho Diagnostics) can be added to a concentration of 10 ng/ml; the cells are plated in 24-well plates with 0.5 ml of media per well and cultured at a temperature of about 37°C in a humidified incubator with 5% CO₂.

Various functions of the T cells produced by the methods of the invention can be evaluated by well known methodologies including but not limited to the following:

⁵¹Cr-release lysis assay for CTL function. Cytotoxic T cells can kill cells that present the particular peptide:MHC class I complex that they specifically recognize. CTL function is typically determined by measuring the release of radioactive isotope by a target cell (e.g., an APC, tumor cell, pathogen cell, etc.). In a non-limiting example of this assay, target cells are incubated with 100 μCi OfNa₂ ⁵¹CrO₄ for approximately 90 minutes at 37°C. Excess ⁵¹Cr is washed away and 5000 labeled targets are incubated with various ratios of CD8⁺ cells for one or more specific time intervals (e.g., 4 hours). Non-specific lysis can be reduced by the addition of unpulsed T2 cells at 25,000 cells per well. ⁵¹Cr released by lysed target cells is measured in the supernatant by scintillation counting. Total release is calculated by addition of 1% Triton X-IOO to the targets, while spontaneous release is calculated by addition of media alone. Percent lysis is calculated using the formula: (sample cpm released minus spontaneous cpm) divided by (total cpm released minus spontaneous cpm released) (see, e.g., Ware et al. (1983) J. Immunol. 131: 1312).

Cvtokine-release assay. Analysis of the types and quantities of cytokines secreted by T cells upon contacting modified APCs is generally a measure of functional activity. Methods for measuring cytokines include ELISA or ELISPOT assays to determine the rate and total amount of cytokine production (see, e.g., Fujihashi et al. (1993) J Immunol. Meth. 160: 181; Tanquay and Killion (1994) Lymphokine Cytokine Res. 13: 259).

In a non-limiting example of an ELISpot assay for IFN-γ or IL-2 secretion by PBMCs or T cells, PVDF membrane ELIspot plates (Millipore, Ballerica, MA) are coated with 5 μg/mL monoclonal anti-IFN-γ or anti-IL-2 capture antibody (BD Pharmingen, San Diego, CA) and incubated at 4°C for 24 hours. After incubation, plates are washed with PBS/0.05% Tween 20 and blocked with 5% human AB serum/ RPMI 1640 medium for 1 hour. PBMCs, T-cells, or CD8⁺-enriched T cells are plated at 1 x 10⁵ cells/well with mRNA-transfected, optionally antigen-pulsed DC targets at 1 x 10⁴ cells/well for a 10:1 effector: target ratio and incubated at 37°C, 5% CO₂ for a minimum of 16 hours. Following incubation, plates are washed 6 times, and anti-IFN-γ detection antibody (BD Pharmingen) or anti-IL-2 detection antibody (BD Pharmingen) is added to the appropriate plates at 1 μg/ml for 2 hours. After six more washes, Streptavidin-HRP (BD Pharmingen) is added to each well for 1 hour. Finally, after another wash cycle, color is developed with AEC Peroxidase Substrate for 5-15 minutes and the reaction is stopped with water. The plates are left to air dry prior to analysis on a CTL Immunospot Plate Reader (CTL, Cleveland, OH).

ELISA: In a non-limiting example of an ELISA assay, ELISA plates (BD Biosciences) can be coated with ELISA capture antibody, specific for a marker or other antigen or epitope of interest, in coating buffer for 24 hours at 4°C. Plates can be blocked with 200 μl per well 10% FCS/PBS for one hour prior to the addition of standards (BD Pharmingen) and supernatant samples, in duplicate, at 100 μl per well and incubated at room temperature for 2 hours. Plates are washed and anti-capture antibody detection antibody added and incubated for one hour. The plates are then washed and solutions replaced with 100 μl of streptavidin-HRP; the plates are further incubated for one hour at room temperature. Plates are washed again and color development substrates applied for 10-20 minutes, followed by the addition of stop solution to halt color development. Plate analysis can be undertaken using Bio-Tek instruments ELx800 plate reader with KC junior software (Winooski, VT).

Monitoring TCR Signal Transduction Events. Several intracellular signal transduction events (e.g., phosphorylation) are associated with successful TCR engagement by MHC-ligand complexes. The qualitative and quantitative characteristics of these events have been correlated with the relative abilities of compositions to activate effector cells through TCR engagement, as taught, for example, in Salazar et al. (2000) Int. J. Cancer 85: 829-35 and Isakov et al. (1995) J. Exp. Med. 181 : 375.

In vitro T-cell education. The APC compositions of the invention can be assayed for the ability to elicit reactive T-cell populations from normal donor or patient-derived PBMCs, and elicited T cells can be tested for lytic activity, cytokine-release, polyclonality, and cross- reactivity to the antigenic epitope (see, e.g., Parkhurst et al. (1996) Immunol. 157: 2539). For example, CD8⁺ T cells can be activated by coculture with antigen-loaded dendritic cells {i.e., DCs that have been pulsed with antigen or transfected with antigen-encoding RNA and which therefore express the antigen). CD8⁺ cells can be purified from non-adherent cells harvested from the monocyte adherence step using the CD8⁺ T cell isolation kit II (Miltenyi Biotec, Auburn, CA). The mature, antigen-loaded dendritic cells are then co-cultured with the purified CD8⁺ T cells at a ratio of 10: 1 CD8⁺: DC. Co-cultures can be performed in R-IO media (10% FBS, RPMI- 1640 supplemented with 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM sodium glutamate, 55 μM β-mercaptoethanol), or other media. For the first seven days, the cells can be cultured in media supplemented with 0.2 U/ml IL-2 (R&D Systems, Minneapolis, MN) and then aliquoted into 24-well tissue culture dishes at 1 ml/ well (e.g., 1 x 10⁶ CD8⁺ cells/ well). Following this initial seven-day incubation, the CD8⁺ T cells can be harvested, counted, and re-cultured with fresh DC stimulators at a ration of 10: 1 in media supplemented with 5 U/ml IL-2. The cells are then cultured for about one week and then restimulated with fresh DC and 20 U/ml IL-2. CTL assays can be performed 3 or 7 days following the third stimulation.

In vitro T-cell education. The APCs of the invention can be assayed for the ability to elicit reactive T-cell populations from normal donor or subject-derived PBMC. In this system, elicited T cells can be tested for lytic activity, cytokine release, polyclonality, and cross- reactivity to the antigenic epitope (see, e.g., Parkhurst et al. (1996) Immunol. 157: 2539).

For example, CD8⁺ T cells can be activated to become CTL by coculture with antigen- loaded dendritic cells. CD8⁺ cells can be purified from non-adherent cells harvested from the monocyte adherence step using the CD8⁺ T-cell Isolation Kit II (Miltenyi Biotec, Auburn, CA). Mature dendritic cells loaded with antigen (e.g., by transfection with antigen encoding mRNA and/or pulsing with antigen) are co-cultured with the CD8⁺ purified T cells at 10:1 CD8⁺ T cells:DC. Co-cultures can be performed in R-10 media (10% FBS, RPMI-1640 media supplemented with 10 mM HEPES pH 7.4, ImM sodium pyruvate, 0.1 mM non-essential amino acids, 2mM sodium glutamate, 55 μM β-mercaptoethanol) or other media. For the first seven days, the cells can be cultured in media supplemented with 0.2 U/ml IL-2 (R&D Systems, Minneapolis, MN) and then aliquoted into 24-well tissue culture dishes at 1 ml/ well (e.g., 1 x 10⁶ CD8⁺ cells/well). Following this initial seven-day incubation, the CD8⁺ T cells can be harvested, counted, and re-cultured with fresh DC stimulators at a 10:1 ratio in media supplemented with 5 U/ml IL-2. The cells are then cultured for about one week and then restimulated with fresh DC and 20 U/ml IL-2. CTL assays can be performed 3 or 7 days following the third stimulation.

Proliferation Assays. T cells will proliferate in response to reactive compositions. Proliferation can be measured, for example, by determining ³H-thymidine uptake (e.g., as in Caruso et al. (1997) Cytometry 27: 71) or with a CFSE assay as illustrated in working Examples 2 and 3 (see also, e.g., Lyons (2000) J. Immunol. Meth. 243: 147). CFSE consists of a fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties. Lymphocytes are first incubated with membrane-permeable, non-fluorescent CFSE which passively diffuses into cells; intracellular esterases cleave the acetate groups converting it to a fluorescent, membrane-impermeant dye. Excess dye is washed away and quiescent cells are induced to proliferate by in vitro mitogenic or antigenic stimulation. The cells are maintained in culture for six days. During each round of cell division, the CFSE fluorescence is halved, allowing the identification of successive cell generations. CFSE is detected using standard fluorescein filters (e.g., excitation = 492 run, emission = 517 nm). Staining with fluorescence- labeled antibodies for cell surface molecules (such as CD4 and CD8) and intracellular markers allows examination of the proliferation of specific cell types as well as characterization of the phenotypic and functional properties of proliferating cells using flow cytometry. Propidium iodide (PI) can also be used to assess cell viability. CFSE flow kits are available through Renovar, Inc. (Madison, WI) and other sources.

Transgenic animal models. Immunogenicity can be assessed in vivo by vaccinating HLA transgenic mice with compositions and determining the nature and magnitude of the induced immune response. Alternatively, the hu-PBL-SCID mouse model allows reconstitution of a human immune system in a mouse by adoptive transfer of human PBL. These animals may be vaccinated with the compositions and analyzed for immune response (for example, as in Shirai et al. (1995) J Immunol. 154: 2733 and Mosier et al. (1993) Proc. Nat'l. Acad. ScL USA 90: 2443).

Primate models. A non-human primate (chimpanzee) model system can be used to monitor in vivo immunogenicities of HLA-restricted ligands. It has been demonstrated that chimpanzees share overlapping MHC-ligand specificities with human MHC molecules, thus allowing tests of HLA-restricted ligands for relative in vivo immunogenicity (see, e.g., Bertoni et al. (1998) J. Immunol. 161 : 4447-55). By an "enriched" or "purified" population of cells is meant that the ratio of particular cells to other cells is increased, for example, in comparison to the cells as found in a subject's body, or in comparison to the ratio prior to at least one enrichment or purification step. In some embodiments, in an enriched or purified population of cells, the particular cells comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the total cell population. A population of cells may be defined by one or more cell surface markers and/or properties. Thus, in some embodiments, a cell population is defined by the presence or absence of a single cell surface markers, such as, for example, CD8, while in some embodiments, a cell population is defined by the presence of certain markers and/or the absence of other markers, such as, for example, TEM cells, which express CD8 and CD27, but do not express CD45RA or CCR7. By "isolated" is intended that a composition (such as, for example, a cell type or an antigen) is removed from its native context in vivo. Thus, an isolated composition, for example, may be an enriched or purified cell population, or an antigen which has been prepared using standard molecular biology techniques so that it is present in a solution or as a percentage by weight or volume in a mixture at higher amounts than it would be in its native setting in vivo.

A cell type or population is "stimulated" when the cells of that type or population exhibit a phenotypic change following an event (e.g., exposure to an agent such as another cell type or chemical activator such as a cytokine, or exposure to a culture condition). For example, a cell type or population is stimulated following an event when it exhibits a change in expression or level of expression of at least one cell surface marker or a change in expression or level of expression of at least one cytokine, or a change in at least one other function. A cell population is "expanded" when it is increased following an event; for example, an expanded cell population will be increased in number by at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 500%, or more in comparison to the number of cells in the population prior to the event.

The term "vector" refers to a plasmid, virus, or other vehicle known in the art that can be manipulated by insertion or incorporation of a polynucleotide. Such vectors can be used for genetic manipulation (i.e., "cloning vectors") or can be used to transcribe and/or translate the inserted polynucleotide ("expression vectors"). A vector generally contains at least an origin of replication for propagation in a cell and a promoter. Control elements present within an expression vector, including expression control elements as set forth herein, are included to facilitate proper transcription and translation (e.g., splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA, stop codons, etc). The term "control element" includes, at a minimum, one or more components whose presence can influence expression; the term "expression control element" refers to one or more nucleic acid sequences that regulates the expression of a nucleic acid sequence to which it is operably linked. An expression control element operably linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence. Thus an expression control element can include, as appropriate, promoters, enhancers, transcription terminators, and/or a start codon {e.g., ATG) in front of a protein-encoding gene. Vectors can also include additional components such as, for example, leader sequences and fusion protein sequences. "Operably linked" refers to a juxtaposition wherein components are in a relationship permitting them to function in their intended manner.

By "promoter" is meant at least a minimal sequence that is sufficient to direct transcription. Promoters for use in or with the invention can be constitutive or inducible, as appropriate (see, e.g. Bitter et al. (1987) Methods in Enzymology 153: 516-544). Inducible promoters are activated by external signals or agents. Other promoter elements can include those which are sufficient to provide control of promoter-dependent gene expression for specific cell- types, tissues or physiological conditions; such elements may be located in the 5', 3', or intronic regions of the gene. Useful promoters also include "conditional promoters," which are active only under certain conditions. For example, a conditional promoter may be inactive or repressed when a particular agent is present (e.g., a chemical compound), but may be active or derepressed when the agent is no longer present.

As used herein, "culturing" refers to the in vitro maintenance, differentiation, and/or propagation of cells in a suitable liquid medium. As used herein, "to transfect" or "transfection" refers to the introduction of one or more exogenous nucleic acids or polynucleotides into a eukaryotic cell. Transfection includes introduction in such a manner that a protein encoded by the nucleic acid or polynucleotide can be expressed. Transfection methods are known in the art and include a variety of techniques, such as: electroporation, methods using protein-based, lipid- based, and cationic-ion-based nucleic acid delivery complexes, transduction using viral vectors, "gene gun" delivery, passive uptake, microinjection, calcium phosphate-based methods, and various other techniques known in the art. In some embodiments, the APC is transfected using electroporation. DCs can be transfected when they are immature or mature. In an exemplary method, monocytes are cultured at 1 x 10⁶ cells/ml for six days in AIM- V medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4 to generate immature DCs. On the sixth day, a maturation formulation in AIM-V medium is added directly to the immature DC to give a final concentration of 10 ng/ml TNF-α, 1000 U/ml IFN-γ, and 1 μg/ml PGE₂. The cells are cultured overnight and mature DC are harvested and electroporated with 1 μg of antigen-encoding RNA and 4 μg of CD40L RNA per 10⁶ cells. Post-electroporation, the cells are cultured at 1 x 10⁶ cells/ml in AIM-V medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4. These cells can be cultured, preferably for at least 30 minutes, and optionally an aliquot may be removed for the preparation of a cell lysate or extract. The remaining cells can then be pulsed with the lysate or extract made from the aliquot, or can be pulsed with a different lysate or extract of cells or virions.

The term "exogenous RNA" as used herein means that the RNA was prepared outside of the cell into which it is being introduced {e.g., transfected). Exogenous RNA may be autologous or heterologous, and it may or may not be amplified and/or partially purified, e.g., to isolate poly-A⁺ RNA, as further discussed elsewhere herein.

In some embodiments of the invention, a polynucleotide {e.g., RNA, DNA, or polynucleotide comprising synthetic nucleic acids) introduced into a cell {e.g., an APC) does not genetically modify the cell and is not stably maintained. The term "genetically modified" or "transformed" means containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype of the cell or its progeny; in some embodiments, the phenotype of the cell is also altered. "Genetically modified" or "transformed" also refers to any addition, deletion, or disruption to a cell's endogenous nucleotides. Stable maintenance of an introduced polynucleotide typically requires that the polynucleotide either contains an origin of replication compatible with the host cell or that it integrates into a replicon of the cell, such as an extrachromosomal replicon {e.g. , a plasmid) or a nuclear or mitochondrial chromosome.

The term "transiently transfected" refers to a cell that has been transfected but which is not genetically modified and so progeny of the cell do not inherit the transformed genetic material {e.g., nucleic acid or polynucleotide). The genetic material may be RNA or it may be transcribed into RNA, and a protein encoded by the genetic material may be expressed. Such expression is referred to herein as "transient expression." Normally, transient expression is accomplished by not incorporating the transfected genetic material into the chromosome. Thus, in some embodiments of the invention, antigen-presenting cells are transiently transfected using RNA electroporation. Methods of RNA electroporation are well-known in the art. Generally, mRNA does not become a permanent part of the genome of the cell, either chromosomal or extrachromosomal. Any other methods that could be used to transiently express a desired protein are also contemplated within the scope of the invention. The methods do not involve permanent alteration of the genome (i.e., do not result in heritable genetic change to the cell) and thus avoid the disadvantages associated with the use of viral vectors, such as, for example, genetic vectors which are retroviruses and adenoviruses.

If desired, transformation of a cell with a polynucleotide (e.g., RNA, DNA, or a polynucleotide comprising synthetic nucleic acids) may be carried out by other conventional techniques known to those skilled in the art. For example, when the cell is a eukaryote, methods of DNA transformation include, for example, calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, and viral vectors. Eukaryotic cells also can be cotransformed with DNA sequences encoding a nucleic acid of interest and/or a second foreign DNA molecule encoding a selectable phenotype, such as those described herein. Another method is to use a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus to transiently infect or transform eukaryotic cells and express the protein.

Compositions, medicaments, and methods of treatment provided by the invention include APCs and T cells produced according to the methods of the invention and the use thereof to induce an immune response in a subject in vivo. In this manner, the invention provides a medicament or "therapeutic composition" for increasing the population of transitory effector memory cells in a subject, wherein said medicament comprises a dendritic cell which has been transfected with and contains exogenous mRNA encoding IRF-7.

As used herein, the term "therapeutic composition" encompasses pure preparations (i.e., preparations consisting of a single, essentially pure substance or compound) as well as mixtures of substances or compounds. In some embodiments, a therapeutic composition comprises at least one active agent which is a composition of the invention and a carrier (which may be inert or active); the therapeutic composition is suitable for diagnostic or therapeutic use in vitro or in vivo. Thus, the therapeutic compositions of the invention which are APCs can be provided as an essentially pure composition or an isolated or purified population of cells, or they can be provided in a mixture, for example, with CD8⁺ T cells and/or with a carrier, such as, for example, saline solution. As used herein, the term "therapeutic purposes" includes an effort to prevent, cure, reduce, or alleviate at least one symptom of a disease or disorder, or to prevent parasitism by a pathogen in the absence of symptoms. In this manner, "therapeutic purposes" include both therapeutic and prophylactic uses of the compositions and/or methods of the invention.

A therapeutic composition that is a protein or polypeptide may be administered as a protein or polypeptide or it may be administered by providing a nucleic acid encoding it to a cell or to a subject. Thus, in some embodiments, a therapeutic composition comprises RNA (see, e.g., U.S. Pat. No. 7,015,204). In some embodiments, more than one therapeutic composition is administered to a subject. A therapeutic composition that is a population of cells (such as, for example, a population of transfected antigen-presenting cells produced in vitro or a population of TEM cells produced in vitro) may be administered to a subject by any suitable means, such as, for example, by intravenous ("IV"), intradermal, or subcutaneous injection or by injection into the peritoneal cavity {i.e., intraperitoneal injection). . In some embodiments, APCs and/or CD8⁺ T cells of the invention are injected into a subject near or in a tumor. Administration can also be accomplished by any suitable means, such as, for example, by injection or via particle bombardment. Objective clinical responses have been reported following intravenous, subcutaneous, and intradermal dosing of other APC vaccines (see, e.g., Santin et al. (2008) J. Virol. 82: 1968-79). Other routes of administration that can be used to administer a composition to a subject include, but are not limited to: intranodal {i.e., administration into or near a lymph node) injection; intratumoral injection; oral, pulmonary, other parenteral routes {e.g., intramuscular or intra-articular); by inhalation (via a fine powder formulation or a fine mist (aerosol), if the composition is capable of being provided in such a formulation, such as, for example, an antigen or a cytokine); or by any other suitable route of administration.

While the invention is not bound by any particular mechanism of operation, intradermal administration, for example, is believed to be effective in part because the dermis is a normal residence for dendritic cells from which they are known to migrate to draining lymph nodes. In some murine models, subcutaneously-injected DCs are later found in T-cell areas of draining lymph nodes, where they are thought to trigger protective antitumor immunity superior to that following IV immunization (see, e.g., Sevko et al. (2007) Adv. Exp. Med. Biol. 601 : 257-64). There is evidence from work in mice that DC injection directly into a lymph node is superior to other delivery routes in generating protective antitumor immunity or cytotoxic T-lymphocytes (CTLs) (see, e.g., Lambert et al. (2001) Cancer Res. 61 : 641-646, the contents of which are hereby incorporated by reference). This suggests that, in some embodiments, it may be desirable to deliver an entire DC dose so that it impacts a single draining lymph node or basin rather than dividing the dose among multiple sites to engage as many nodes as possible.

The cells of the present invention {i.e., the APCs and/or T cells produced by methods of the invention) and other substances {e.g., antigens, if administered separately, and/or other immunostimulatory compounds) may be administered with a carrier and they may be administered in the same formulation and via the same route of administration or they may be administered in different formulations and/or via different routes of administration. For example, in some embodiments, transfected APCs are administered via intraperitoneal injection, while antigens and/or cytokines are administered via intravenous injection.

Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions and/or medicaments of the present invention. In some embodiments, quality controls are performed {e.g., microbiology, clonogenic assays, viability tests) and the cells are reinfused back to the subject, preceded by the administration of diphenhydramine and hydrocortisone (see, for example, Korbling et al. (1986) Blood 67: 529-532 and Haas et al. (1990) Exp. Hematol. 18: 94- 98). Formulations and carriers suitable for administration can include aqueous isotonic sterile injection solutions, which can further contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

Thus, a carrier for cells or antigen(s) for use in the methods of the invention can comprise any suitable physiological solution or dispersant or the like, such as, for example, saline or buffered saline. The carrier may also comprise antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like. Except insofar as any conventional media, carrier or agent is incompatible with the active ingredient, its use is contemplated. The carrier may further comprise one or more additional compounds for administration to the subject. In some embodiments, a carrier comprises or consists of 85% heat-inactivated autologous serum, 10% DMSO, and 5% dextrose as well as, optionally, at least one stabilizer and/or preservative. In embodiments where the antigen(s) are administered separately from the APCs, they can be administered in a suitable carrier and formulation that differs from the carrier and/or formulation used for cells.

Methods of formulating and administering APCs to subjects are known in the art. See, for example, Nicolette et al. (2007) Vaccine 25 Suppl. 2: B47-60 (the content of which is hereby incorporated by reference in its entirety); Fay et al. (2000) Blood 96: 3487; Fong et al. (2001)b J. Immunol. 166: 4254-59; Ribas et al. (2001) Proc. Am. Soc. Clin. One. 20: 1069; Schuler-Thurner et al. (2002) J Exp. Med. 195: 1279-88 (and erratum in (2003) J. Exp. Med. 197 395); and Stift et al. (2003) J CHn. Oncol. 21: 135-142.

The dose of cells {e.g., APCs or CD8⁺ T cells) administered to a subject is in an amount that is an effective amount or is an amount that is expected to be an effective amount, even if no favorable result is achieved. In embodiments where compositions of the invention are coadministered with each other or with other compositions, preferably, each of the substances is administered at its optimal dosage so as to obtain optimal therapeutic effect of the coadministration. As is familiar to those of skill in the art, dosages of the cells of the present invention to be administered to a subject in vivo are determined with reference to various parameters, such as, for example, the species of the host, the age, weight, disease status, and location to be targeted within the host. Generally, dosages of cells to a typical human subject of about 70 kg may range from about at least 1 x 10⁴ cells to about at least 1 x 10⁹ cells per administration. In some embodiments, the dosage ranges from about 5 x 10⁵ cells to about 5 x 10⁷ cells per administration. To achieve maximal therapeutic effect, several doses may be required. The dose level selected for vaccination is expected to be safe and well-tolerated by the subject.

For administration, cells of the present invention can be administered at a rate determined by the effective dose, the LD₅₀ of the cell type (or other measure of toxicity), and the side effects of the cell type at various concentrations in view of the mass and overall health of the subject. Administration can be accomplished via single or divided doses. One skilled in the art can determine whether repeated administration is necessary and the frequency at which administration should be repeated. In some embodiments, patients are vaccinated five times with between 1 x 10⁶ to 1 x 10⁷ viable APCs or T cells per dose.

The compositions of the invention can supplement other treatments for a condition by known conventional therapy, including cytotoxic agents, nucleotide analogues and biologic response modifiers. Thus, biological response modifiers are optionally included for treatment of the subject with the APCs and/or T cells of the invention. For example, the cells are optionally administered with at least one adjuvant, immunomodulating agent, or cytokine, including but not limited to: IL- 15, anti-CTLA-4 antibodies, TLR agonists, interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-12 (IL-12), G-CSF, and/or GM- CSF . As used herein, the term "cytokine" refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. Cytokines are commercially available from many vendors, including Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems (Minneapolis, MN), and Immunex (Seattle, WA).

To assess the vaccine's immunogenicity, immune responses in vaccinated individuals can be monitored by following the maturation profiles of CD4⁺ and CD8⁺ T cells. For example, restoration of HIV-specific effector cell function can be determined by the presence of cells expressing the phenotype of effector T-cells and secreting elevated levels of IFN-γ and granzyme B. Restoration of HIV-specific proliferative responses can be determined by the cells' capacity to produce IL-2 and to become CFSE^low following stimulation with dendritic cells transfected with HIV-RNAs, such as, for example, Gag-encoding RNA. For example, restoration of the HIV-specific memory T-cell compartment can be assessed as follows: Maturation of specific T cells induced by the vaccine can be measured using surface and intracellular markers (e.g., using a flow cytometry assay). CDS⁺ T cells can be monitored by staining for surface markers including, for example, αβTCR, CD45RA, CCR7, CD27, CD28, and CD 107 or intracellular molecules such as granzyme B or IFN-γ, and/or by secretion of IL-2. CD3, CD4, CCR7, and IL-2, among other markers, can be used to monitor CD4⁺ T cells. Such assays can be used to monitor immune response following incubation with peptides encompassing the autologous HIV sequences from the subject. Comparison of the cellular immune responses at baseline (i.e., prior to treatment) and monthly prior to each new vaccination enables determination of the vaccine's impact on the breadth of the cellular immune response. The breadth of the immune response can also be measured using the CFSE proliferation assay.

In an exemplary method, monitoring of a subject is practiced by obtaining and saving blood samples from the subject prior to infusion for subsequent analysis and comparison. Generally at least about 10⁴to 10⁶ and typically, between 1 x 10⁶ and 1 x 10¹⁰ cells are infused intravenously or intraperitoneally into a 70 kg patient over roughly 60-120 minutes. In one aspect, administration is by intratumoral injection. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples can be obtained at intervals (e.g., 5 minutes and 1 hour following infusion) and saved for analysis. In some embodiments, cell re-infusions are repeated roughly every month for a total of 10-12 treatments in a one year period. After the first treatment, infusions can be performed on an outpatient basis at the discretion of the clinician.

In some embodiments, another benefit of the invention is that the compositions and methods of the invention do not affect CD4⁺ T cells, so that adverse consequences of suppression of CD4⁺ T cells are generally avoided. That is, in some embodiments, the compositions and/or methods of the invention have no measurable and/or statistically significant effect on CD4⁺ T cells in vivo or in vitro, and do not induce a CD4⁺ T cell response. In some embodiments, the compositions and/or methods may have a slight effect on CD4⁺ T cells; that is, treatment of a subject or of cells in vitro with a composition or method of the invention may decrease a population or function of a type of CD4⁺ T cell by no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in comparison to an appropriate control population or function, or may induce a CD4⁺ T cell response that is about or no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% higher than an appropriate control response.

Each patent, patent application, scientific article, book or book chapter, and other reference cited in the specification is hereby incorporated by reference in its entirety to the same extent as if each individual citation was particularly and individually indicated to be incorporated by reference.

This specification should be read and considered as a whole. Because the invention is complicated and because it must be described in words in this specification, it is necessary for the sake of clarity to focus on one or a few aspects of the invention at a time before moving on to discuss other aspect of the invention. This necessarily results in different aspects of the invention being discussed in different parts of the specification. However, this is merely a reflection of the weaknesses of using language to describe ideas and should not be misconstrued as limiting the invention, or as indicating that ideas or embodiments described in different portions of the specification are unrelated. Rather, as will be understood by one of skill in the art, the concepts and variations discussed herein are generally applicable to all the embodiments of the invention unless specifically stated otherwise, and different embodiments of the invention can generally be combined unless specifically stated otherwise or unless an inoperable embodiment would clearly result.

In the following, various aspects of the invention are more particularly described via examples. However, the invention is not limited merely to the embodiments illustrated by these examples but rather provides benefits as more fully described herein in the application as a whole.

EXPERIMENTAL

Example 1 - DCs Electroporated with IRF-7 mRNA Enhance Proliferative Capacity of CD8⁺ T Cells But Not of CD4⁺ T Cells

DCs were electroporated with three different concentrations of IRF-7 mRNA (0.25, 1 and 5 μg mRNA per million electroporated DCs). Expression of the IRF-7 mRNA was confirmed using Western blot analysis of cells collected four hours post-transfection (shown in Figure 1, left panel). Cells were also assayed using intracellular flow cytometry (FACS) staining with an IRF-7 specific monoclonal antibody (shown in Figure 1, right panel). This FACS analysis demonstrated that when DCs were electroporated with mRNA at a concentration of 5 μg/million electroporated DCs, more than 80% of the electroporated DCs expressed IRF-7.

The DCs were then examined for any effect of IRF-7 on DC maturation. Adherent monocytes were obtained from CMV⁺ or HIV⁺ leukapheresis products, incubated for 6 days in the presence of GM-CSF and IL-4, and then matured with the "PME maturation process" by incubation in the presence of TNFα, IFNγ and PGE2 for 24 hours. The cells were then electroporated with CD40L and Gag or CMV with or without IRF-7 mRNA and analyzed four hours later by FACS. As shown in Figure 2, mature DCs electroporated with IRF-7 and CD40L were virtually identical to mature DCs electroporated with CD40L in their expression patterns of cell surface markers CD80, CD83, CD86, HLA-DR, PD-Ll and PD-L2. These experiments demonstrated that IRF-7 mRNA did not affect DCs matured from the three CMV⁺ and the five HIV⁺ (infected) patients.

However, as shown in Figure 3, DCs electroporated with IRF-7 mRNA expressed high levels of MHC class I molecules. Thus, MHC class I expression by DCs is upregulated by IRF-7 expression in those DCs.

Example 2 -CD8⁺ T Cell Response is Enhanced by mRNA Encoding IRF-7 Mature DCs from a healthy patient and from an HIV-infected patient were electroporated with RNA encoding GFP (negative control), CMV pp65, or consensus HIV GAG, with or without increasing concentrations of mRNA encoding IRF-7. These DCs were then used to stimulate autologous PBMCs. Following a six-day stimulation with a 1 :40 ratio of DC:PBMC, proliferation of CD8⁺ and CD4⁺ cells was measured using a CFSE assay. The assay was performed in triplicate; representative results are shown in Figures 4 (DCs from healthy patient) and 5 (DCs from HIV-infected patient). The negative control (GFP) showed minimal background levels of CFSE^low cells. These data clearly demonstrate that the presence of IRF-7 enhances the proliferative capacity of CMV- and HIV-specific CD8⁺ T cells in a dose-dependent manner. Moreover, no significant (detectable) CD4⁺ responses were seen, indicating that the presence of IRF-7 does not affect the CD4⁺ compartment.

Example 3 — -DCs Electroporated with IRF-7 mRNA Induce the Proper Maturation of Antigen-Specific CD8⁺ T Cells

Experiments were then conducted to evaluate whether the effect on CD8⁺ T-cell proliferation was antigen-specific or due to bystander activation. These experiments combined a CFSE assay with tetramer staining to evaluate the specificity of the proliferating cells. Experiments were performed in triplicate, and representative results are shown in Figure 6. Figure 6 shows results of the CFSE assay (left column) and combined CFSE and tetramer assays (right column). The numbers next to each panel in the right column indicate the size of the Gag- specific, proliferating CD8⁺ T cell population as a percentage of the total CD8⁺ T cell population. IRF-7 expression caused an increase of over two-fold in the number of tetramer-binding T cells, demonstrating that the increase in CD8⁺ T-cells is antigen-specific. Thus, these experiments demonstrated that IRF-7 induces the proliferation of antigen-specific CD8⁺ T cells.

These antigen-specific CD8⁺ T cells were then further examined using multi-parametric assays to obtain comprehensive information on the immune responses generated by IRF-7. Figure 7 shows results of a typical experiment in which antigen-specific CD8⁺ T cells were evaluated. In these experiments, CD8⁺ T cells were co-cultured for six days with DCs transfected (as indicated at the bottom of the figure) with GFP (negative control) or CMV mRNA and with or without different amounts of IRF-7 mRNA ("co-stimulator" RNA). In one sample, rather than transfecting the DCs with CMV mRNA prior to co-culture, CMV was added as an exogenous stimulus (i.e., an antigen) (see second column from left in "heat map"). Darker colors on the 10-level "heat map" indicate increasing numbers of CMV-specific CD8⁺ T cells. Cell surface marker expression was determined for CD28, CD45RA, CCR7, and CD27, and is shown on the left-hand side of Figure 7.

These experiments demonstrated that antigen-specific CD8⁺ T cells have four different major phenotypic profiles (i.e., patterns of cell-surface marker expression), designated "EM," "TEM," "EM CD28⁺," and "TEM CD28⁺." In this manner, these experiments demonstrate that the methods and compositions of the invention can provide increased polyfunctionality of T cells as well as increased numbers of T cells expressing CD28. Moreover, these experiments demonstrated that different cell types are obtained in the presence of CD4⁺ T-cell help ("heat map" column second from left, showing results from DCs transfected with GFP and including CMV as an exogenous stimulus). In contrast, IRF-7 caused a major shift in the distribution of cell types obtained, as can be seen by comparing the "heat map'"s right-most column (showing results from DCs transfected with CMV mRNA and 5 μg IRF-7 mRNA) to the cell types obtained in the presence of CD4⁺ T-cell help. This effect of IRF-7 was also found in cells from an HIV patient, but to a lesser degree.

Example 4 — IRF-7 Increases the Polyfunctionality of Antigen-Specific CD8+ T cells Gag-specific CD8⁺ T cells were examined in vitro (as CFSE^low proliferating T cells) after 8 days of incubation with Gag- and IRF-7-transfected DCs and then were further stimulated overnight with Gag peptides and further analyzed for particular functions, including secretion of IFN-γ and IL-2 and expression of CD 107; results are shown in Figure 8. This "heat map" analysis reveals striking differences in the distribution of Gag-specific CD8⁺, IFNy⁺ T cells obtained by coculture with DCs electroporated with or without IRF-7 mRNA. The results demonstrate that CD8⁺ IFNy⁺ T cells obtained by coculture with IRF-7-electroporated DCs show higher polyfunctionality and include not only an effector memory (EM) cell population but also a population of transitory effector memory (TEM) cells.

This TEM cell population was not produced in the absence of IRF-7. Thus, coculture of CD8⁺ T cells with IRF-7-electroporated DCs produces a population of TEM cells with the expected functional attributes. TEM cells were also found in a subject infected with HIV who was a "long-term non-progressor" ("LTNP") patient). While the existence of a TEM population in other HIV-infected patients could not be excluded, the CMV-specifϊc CD8⁺ T cell population in LTNP patients effectively disappeared following in vitro expansion.

Experiments were then conducted to determine whether IRF-7-mRNA-transfected DCs can enhance the repertoire of CD8⁺ T cells generated by coculture. Following co-culture with IRF-7-transfected or control DCs, proliferating, A2-tetramer⁺ CD8⁺ T cells were isolated and analyzed using a TCR-heteroduplex mobility assay ("HMA"), as shown in Figure 9. Similar TCR-HMA patterns were seen for Vβ5 and Vβ8, but additional patterns were found in cells that had been cocultured with DCs transfected with IRF-7. These data demonstrate that IRF-7- transfected DCs enhance and broaden the antigen-specific T cell repertoire.

Example 5 — Effect of anti-IFN-α antibody on IRF-7 stimulation

Dendritic cells were transfected with GFP- or GAG-encoding mRNA and with or without IRF-7-encoding mRNA; transfected DCs were then cultured in vitro with CD8⁺ T cells. The CD8+ T cells were then analyzed with CFSE and tetramer analysis on days 5 and 6, as shown in Figure 11. To determine the effect of anti-IFN-α antibody on this stimulation, experiments were performed in which DCs were transfected with mRNA encoding GAG or GAG and IRF-7. DCs transfected with mRNA encoding GAG and IRF-7 were also incubated with growth medium containing anti-IFN-α at 1 μg/ml or 25 μg/ml; results are shown in Figure 12.

Claims

WHAT IS CLAIMED:

1. A method of increasing a population of CD8⁺ T cells in a subject comprising the steps of: a) transfecting dendritic cells with RNA that encodes IRF-7; and b) administering the cells of step (a) to said subject, whereby the population of CD8⁺ T cells in the subject is significantly increased.

2. The method of claim 1, wherein said population of CD8⁺ T cells is a population of cells selected from the group consisting of: TEM cells, CM cells, and EM cells.

3. The method of claim 1, further comprising transfecting dendritic cells with RNA that encodes CD40 ligand.

4. A method of increasing a population of antigen-specific CD8⁺ T cells in a subject comprising the steps of: a) transfecting dendritic cells with RNA that encodes IRF-7 and at least one antigen of interest; and b) administering the cells of step (a) to said subject, whereby the population of antigen-specific CD8⁺ T cells in the subject is significantly increased.

5. The method of claim 4, wherein said population of CD8⁺ T cells is a population of TEM cells.

6. A method of increasing a population of transitory effector memory cells in an in vitro cell culture comprising the steps of: a) transfecting dendritic cells with RNA encoding IRF-7; and b) culturing the cells of step(a) in vitro with PBMCs or a purified population of CD8⁺ T cells, whereby the population of transitory effector memory cells in the in vitro culture is significantly increased.

7. A method of increasing a population of antigen-specific transitory effector memory cells in an in vitro cell culture comprising the steps of: a) transfecting dendritic cells with RNA that encodes IRF-7 and at least one antigen of interest; and b) culturing the cells of step(a) in vitro with PBMCs or a purified population of CD8+ T cells, whereby the population of transitory effector memory cells in the in vitro culture is significantly increased.

8. A dendritic cell which has been transfected to contain exogenous RNA encoding IRF- 7, wherein said dendritic cell expresses IRF-7.

9. The dendritic cell of claim 8, further comprising a second exogenous mRNA encoding an antigen, wherein said dendritic cell is capable of presenting said antigen to a T cell.

10. The dendritic cell of claim 8, further comprising a second exogenous mRNA encoding CD40 ligand, wherein said dendritic cell expresses CD40 ligand.

1 1. A TEM cell produced by a method of any of claims 1-7.

12. A medicament for increasing CD8⁺ T cells in a subject, wherein said medicament comprises a dendritic cell which has been transfected with and contains exogenous mRNA encoding IRF-7, wherein said CD8⁺ T cells are selected from the group consisting of TEM cells, CM cells, and EM cells.