WO2015081235A1 - Methods of inducing t cell polyfunctionality - Google Patents

Abstract

The ability of individual T cells to perform multiple effector functions is crucial for protective immunity against viruses and cancer. This polyfunctionality is frequently lost during chronic infections. This disclosure provides methods of inducing T cell polyfunctionality to treat chronic viral infections and cancer

Description

METHODS OF INDUCING T CELL POLYFUNCTIONALITY

[1] Each reference cited in this disclosure is incorporated herein by reference in its entirety.

[2] This invention was made with government support under NIH grants NCI ROl CA 108835, NIAID POl AI072677 AI 080313, and AI 36219. The government has certain rights in the invention.

FIELD OF THE INVENTION

[3] The invention is in the field of immunology. BACKGROUND

[4] A growing consensus indicates that T cells capable of simultaneously producing multiple effector functions, referred to as "polyfunctional" T cells, are a key subset in the development of effective immune responses against pathogens and cancer (1-7). These T cells, in addition to producing cytokines such as IL-2,TNF-a, and IFN-γ, produce chemokines and also display cytolytic function. In contrast to acute infection, optimal polyfunctional memory T cell responses are lost in chronic infections and cancer, both in humans and mice (5, 8, 9). These cells have been characterized as "exhausted" (8, 10) and factors promoting the development of T cell exhaustion include persistent and high levels of antigen stimulation (11, 12), DC inhibition (3),and upregulation of inhibitory receptors, such as PD-1, on Tells (13-17). While recent work has highlighted a role for PD-land other inhibitory receptors in T cell exhaustion, blockade of inhibitory receptor signaling in HlV-specific T cells had only a moderate effect in reversing the exhaustion phenotype and increasing T cell polyfunctionality (14, 15), thus indicating that additional molecular mechanisms are involved in the inhibition of T cell polyfunctionality.

[5] Differences in T cell polyfunctionality correlate with differences in memory T cell formation in response to immunization as well as to viral infection. Regulation of vaccine-induced adaptive immune responses is complex and, in part, dependent on antigen dose (l).In animal models, high-dose vaccination results in inferior T cell polyfunctionality, poor memory formation and weaker immune protection as compared with optimal dose vaccination (4, 18). As a result, polyfunctional T cells are more than just a "marker" of protective immune response. The molecular mechanisms linking high-dose antigenic stimulation with inferior polyfunctionality and poor memory formation remain largely unclear.

[6] Collectively, these data indicate the need to understand the molecular control of

T cell polyfunctionality, especially in the context of antigen dose. Elucidating the molecular details underlying T cell polyfunctionality could provide additional insights into T cell exhaustion and serve as a basis for vaccine design by optimizing T cell activity against virus infection or cancer.

[7] Polyfunctionality is crucial for protective immunity against viruses and cancer (Seder et al, 2008; Wherry, 2011). It is widely accepted that a robust T cell immune response is not only dependent on the absolute cell number, but is also critically dependent on cell quality. Polyfunctional T cells, characterized by the ability to execute multiple effector functions, are a hallmark of healthy, non-exhausted memory T cell response against acute infection, such as influenza infection (Joshi and Kaech, 2008; Weng et al, 2012; Wherry, 2011; Wherry and Ahmed, 2004). On the other hand, during chronic infectious diseases such as HIV and HCV, dysfunctional antigen-specific T cells are frequently formed due to repetitive stimulation and are associated with worse disease outcomes (Betts et al, 2006; Ciuffreda et al, 2008; Frebel et al, 2010; Rehr et al, 2008). Besides infectious diseases, loss of polyfunctional T cell responses is also reported in human tumors (Baitsch et al, 2011; Tran et al.) and successful anti-tumor immunotherapy treatments correlate with the induction of polyfunctional T cells (Ding et al, 2012; Yee, 2010; Yuan et al, 2008).

BRIEF DESCRIPTION OF THE FIGURES

Figures for Examples 1-8

[8] FIGS. 1A-D. Results of experiments demonstrating that activation of Wnt signaling reprograms memory T cells to a polyfunctional state.

[9] FIGS 2A-F. Results of experiments demonstrating that Wnt pathway activation with TWS119 induces polyfunctional influenza-specific T cells with a central memory phenotype. Human CD8+ T cells were stimulated with autologous, Ml -peptide loaded moDCs for two weeks (stimulations were given on DO and D7). The culture was also treated with different amount of TWS119, as indicated. Tetramer staining and cell counts were determined on D7 and D14. FIG. 2C and FIG. 2D, D14 CD8+ T Cells induced with different concentration of TWS119 were harvested and re-challenged with target T2 cells loaded with Ml peptide for six hours and analyzed for their effector functions. FIG. 2C, Representative intracellular cytokine staining for polyfunctionality analysis. Top row: Intracellular cytokine staining for IL-2 versus CD 107. Numbers in the right upper quadrant represent the percentage of IL-2 and CD 107a double-positive cell population. Bottom row: Intracellular cytokine staining for TNFa versus MIP-1 β. Numbers in the right upper quadrant represent the percentage of TNFa and MIP-1 β double-positive cell population. FIG. 2D, Polyfunctionality pie charts. Each slice of pie represents the percentage of cells expressing between 1-5 effector functions. To compare between pie charts, permutation test with 10,000 repetitions was used using SPICE. FIG. 2E, TWSl 19 treatment significantly increased the absolute number of 5+ polyfunctional cells during antigen-specific expansion. Numbers were calculated based on a starting CD8+ culture composed of 100,000 cells. FIG. 2F, D14 CD8+M1+ Cells were also analyzed for their surface marker expression. TWSl 19 3mM, blue; 1.5mM, green; 0.7mM, orange; OmM, red. Histograms were gated on CD8+tetramer+ cells. *: P value<0.05. **: P value<0.01. The results shown are from five different donors.

[10] FIGS. 3A-E. Results of experiments demonstrating that TWSl 19 treatment enhances polyfunctionality in terminally differentiated CMV-specific T cells. FIG. 3A, Ex vivo CMV-pp65 -specific response. FIG. 3B, Representative tetramer staining of Human CD8+ T cells stimulated with CMV-pp65-NLV loaded moDCs for two weeks (D14).

TWSl 19 were added in culture during stimulation at the indicated concentration. FIG. 3C and FIG. 3D, D14 cells were rechallenged with pp65-loaded T2 target cells for six hours to assess their polyfunctional response. Total numbers of pp65-specific 5+ polyfunctional cells were calculated as in FIG. 2. Similar to what has been observed with Ml -specific response, there is enhanced polyfunctional response in TWSl 19 treated cells. FIG. 3E, TWSl 19 treated cells also express higher level of CD62L and CD28. TWSl 19 3mM, blue; 1.5mM, green; 0.7mM, orange; OmM, red. Histograms were gated on CD8+CMV-tetramer+ cells. *: P value<0.05. **: P valueO.01.

[11] FIGS. 4A-D. Results of experiments demonstrating that the effects TWSl 19 on CD62L expression and polyfunctionality are seen in divided cells and are independent of dendritic cells. Human CD8+ T cells were stimulated with Ml or CMV-peptide loaded moDCs for two weeks in the presence or absence of TWSl 19. Before the second moDC stimulation, T cells were labeled with CFSE to monitor population divisions. On D14, cells were either harvested and stained with CD8, tetramer and CD62L, or rechallenged with Ml- labeled T2 cells for six hours for intracellular cytokine staining. FIG. 4A, Representative staining showing CD62L by CFSE dilution among CD8+ Ml-tetramer+ cells. TWSl 19 decreased the proliferation of tetramer+ cells, but almost all tetramer+ cells divided. FIG. 4B, Representative result of cytokine production by CFSE dilution among CD8+ Ml-tetramer+ cells. T cells induced with TWSl 19 exhibited higher level of IL-2 or TNFa in divided cells. FIG. 4C and FIG. 4D, The effect of Wnt pathway activation was observed when T cells were expanded with aAPCs. Purified CD8+ T cells were stimulated with Ml -loaded or pp65- loaded aAPCs weekly for two weeks. Tetramer specificity (FIG. 4C) and T cell

polyfunctionality (FIG. 4D) were determined weekly. *: P value<0.05.

[12] FIGS. 5A-E. Results of experiments demonstrating that Wnt pathway activation induced a long-standing polyfunctional phenotype associated with other stemness features. FIG. 5A, Ml -tetramer positive T cells induced by moDCs in the presence (Blue) or absence of TWSl 19 (Red) were studied for their anti-apoptotic protein expression levels on D14. Numbers shown indicate the MFI value for each staining. FIGS. 5B-5E, D14 Ml- specific cells were further cultured in the presence of IL-15 (25ng/ml) for 7 days. CFSE dilution, phenotype and polyfunctionality were analyzed on D21. FIG. 5B, Representative CFSE dilution of Ml-tetramer specific cells measured by flow cytometry. PI: proliferation index. FIG. 5C and FIG. 5D, Phenotype analysis performed after homeostatic proliferation. A significant higher percentage of cells previously treated with TWSl 19 maintained the preferred CD28+CD62L+ phenotype. FIG. 5E, After homeostatic proliferation, superior polyfunctionality is maintained in cells previously treated with TWSl 19. In contrast, cells untreated with TWSl 19 remain poorly polyfunctional.

[13] FIGS. 6A-B. Results of experiments demonstrating that TWSl 19 induces Wnt pathway activation in memory T cells after ex vivo activation. Memory T cells were isolated from fresh collected PBMCs and stimulated with plate-bound anti-CD3/CD28 for 2h or 6h, in the presence (triangle) and absence (half square) of 3mM TWSl 19. FIG. 6A, At 6h, intracellular level of β-catenin was measured by flow cytometry (3mM TWSl 19, blue;

DMSO, red; isotype control, shaded grey). FIG. 6B, qPCR performed at 6h showed TWSl 19 treatment upregulated all five Wnt pathway target genes (solid triangle: TWSl 19; open square: DMSO). [14] FIG. 7A-C. Results of experiments demonstrating that activation of Wnt signaling inhibits the overall expansion of influenza-specific cell but increases the tetramer specificity in culture. Human CD8+ T cells were stimulated with autologous, Ml -peptide loaded moDCs for two weeks (stimulations were given on DO and D7). The culture was also treated with different amount of TWSl 19, as indicated. Tetramer staining and cell counts were determined on D7 and D14. Cell numbers shown were presented based on a starting culture composed of 100,000 CD8+ T cells. FIG. 7A and 7B, Despite higher antigen specificity in the culture as shown in FIG. 2, TWSl 19 inhibits the expansion of all cells in the culture as well as the Ml-specific cells being generated. FIG. 7C, CD8+ T cells were labeled with CFSE before second moDC stimulation. On D7, CFSE dilution was analyzed within CD8+Ml-tetramer+ or CD8+M1 -tetramer- populations. TWSl 19 inhibited the overall proliferation of Ml-specific T cells but more inhibitory effect is seem in non-specific CD8+ T cells. *: P value<0.05.

[15] FIG. 8. Results of experiments demonstrating that individual effector function changes in response to Wnt pathway activation in Ml-specific T cells. On D14, after two Ml -loaded moDC stimulations, expanded Ml-specific cells were rechallenged with Ml- loaded T2 cells for six hours to assess their polyfunctionality. IL-2 and TFNa production are significantly improved when cells were treated with TWS 119. * : P value<0.05.

[16] FIG. 9. Results of experiments demonstrating that CMV p65 -specific T cells are terminally differentiated memory cells. Freshly isolated CD8+ T cells from healthy donors were stained with anti-CD8 and Ml - or CMV-tetramer along with other surface markers to determine the ex vivo phenotype of antigen-specific T cells. FIG. 9A, Majority of Ml-specific cells was CD28+CD27+ but majority of pp65-specific cells was CD28-CD27. FIG. 9B, Percentage of tetramer-specific cells within each memory differentiation subset according to the definition of Sallusto et al. (Sallusto et al., 2004). Ml-specific cells were either CCR7+CD45RA+ (T_N) or CCR7+CD45RA-(T_CM) or CCR7-CD45RA- (T_EM) phenotype. In contrast, the majority of CM V-pp65 -specific cells were CCR7-CD45RA+ (T_EMRA) phenotype. *: P value<0.05.

Figures for Examples 9-17

[17] FIG. 10. Influenza virus Ml -pulsed moDCs induce concentration-dependent proliferation of Ml-specific CD8+ T cells with variable levels of polyfunctionality. (FIGS. lOA-C) Percentage of Ml -specific T cells and Ml -specific T cell expansion. Autologous moDCs pulsed with variable amounts of the HLA-A*0201 restricted immunodominant Ml peptide (10 μΜ~ 10 fM) were used to stimulate HLA*A201+ CD8+ T cells ex vivo weekly for 2 weeks. Cultures were analyzed on D14. (FIGS. 10D-H) Functional assessment of Mi- specific T cells: polyfunctionality assessment of Ml -specific T cells reveals significant impairment of T cells induced with a high antigen concentration (10 μΜ peptide-pulsed moDCs). (FIG. 10D) Top row: intracellular cytokine staining for IL-2 versus CD 107.

Numbers in red represent the percentage of IL-2-producing (right upper quadrant) versus IL- 2-negative cells (right lower quadrant) in the CD107a-positive cell population. Bottom row: intracellular cytokine staining for TNF-a versus MIP-Ιβ. Numbers in red represent the percentage of TNF-a-producing(right upper quadrant) versus TNF-a-negative cells (right lower quadrant) in the ΜΙΡ-Ιβ-positive cell population. (FIG. 10E) Percentage of individual effector function expression out of total antigen specific response. (FIG. 10F)

Polyfunctionality pie charts. Each slice of pie represents the percentage of cells expressing between 1~5 effector functions. To compare different pie charts, permutation test with 10,000 repetitions was used. (G) The percentage of 5+ function cells in each T cell culture. (FIG> 10H) Amount of IL-2 produced by 5+ function cells. IL-2 production was quantified by measuring MFI and plotted for 5+ function cells. * < 0.05. The data are representative of at least 3 assessments with 5 different donors.

[18] FIG. 11. Microarray analysis reveals unique molecular signatures associated with T cell polyfunctionality. (FIG. 11 A) Heat map of differentially expressed genes among T cells induced by 10 μΜ moDCs or 10 nM moDCs (adjusted P < 0.05, fold change > 1.6). Red and blue indicate increased and decreased gene expression, respectively. (FIG. 11B) Expression level of EOMES among T cells induced by 10M moDCs (red) or 10 nM moDCs (blue), determined by flow cytometry. (FIG. 11C) Real-time qPCR validation of TCF7 level. T cells induced with 10 μΜ or 10 nM pulsed moDCs were tetramer sorted and analyzed by qPCR. *P < 0.05. (29. (FIG. 11D) GSEA shows that high antigen concentration-induced T cells are enriched with exhaustion signature from LCMV or HIV. In contrast, optimal concentration-induced T cells were enriched with the genetic signature of memory T cells. Molecular signature from PD-1 ligation was not able to distinguish between T cells stimulated with high and optimal antigen concentrations.

[19] FIG. 12. Polyfunctionality regulation by antigen concentration is independent of inhibitory receptor signaling. (FIG. 12A) On D14, T cells were harvested and stained with anti-CD8 and Ml tetramer, then assayed for inhibitory receptor expression. Representative histograms shown were gated on CD8+, Ml tetramer+ population. Red: 10 μΜ. Blue: 10 nM. Yellow: 10 fM. High antigen concentration-induced Ml-specific T cells (10 μΜ) showed higher inhibitory receptor expression except for BTLA. (FIG. 12B) Real-time qPCR analysis of BATF expression level in D14 sorted Ml-specific cells. (FIG. 12C) Blockade of PD-1 signaling by anti-PD-1 antibody (clone EH12.2H7) during high antigen concentration stimulation did not improve polyfunctionality. The lack of any effect of anti-PD-1 treatment was seen at the standard dose used of 10 μg/ml (see above) as well as at 10-fold higher concentrations of anti-PD-1 treatment. Isotype control antibody (clone MG1-45) was added at the same concentration. (FIGS. 12D-F) Ex vivo CD8+ T cells stimulated with different concentrations of aAPCs weekly for 2 weeks showed different levels of T cell

polyfunctionality. aAPCs were made by conjugating biotinylated HLA-A2-M1 and biotinylated anti-CD28 complex onto anti-biotin microbeads (Miltenyi Biotec). (FIG. 12D) Percentage of tetramer-positive cells and (FIG. 12E) total Ml-specific cell expansion. Higher concentrations of aAPCs induced more robust cell expansion, similar to results seen in FIG. 1 with peptide -pulsed moDC-induced T cells. (FIG. 12F) Polyfunctionality assessment of aAPC-stimulated Ml-specific T cells. High concentration aAPCs induced Ml-specific T cells with lower polyfunctionality even in the absence of inhibitory receptors on the aAPCs. The results are representative of 3 independent experiments performed on at least 3 different subjects. *P < 0.05.

[20] FIGS. 13A-D. MAPK/ERK pathway controls T cell polyfunctionality. (FIG.

13 A) D14 tetramer-sorted high and optimal antigen concentration-stimulated T cells were restimulated with PMA/ionomycin for 10 minutes and analyzed for expression of pERK by flow cytometry. Red, high concentration; blue, optimal concentration; Gray, unstimulated. (FIG. 13B) ER inhibitor U0126 inhibits pERK expression in a concentration-dependent fashion. T cells stimulated by the optimal concentration of antigen were restimulated with PMA/ionomycin in the presence or absence of varying concentrations of the ERK inhibitor U0126 for 10 minutes. Cells were then stained for pERK expression. Red, 100 μΜ U0126; orange: 1 μΜ U0126; blue, no U0126; gray, unstimulated. (FIGS. 13C-D) Optimal concentration-induced T cells were incubated with T2 target cells for 6 hours in the presence of various amounts of U0126 before intracellular cytokine staining and polyfunctionality analysis. U0126 selectively inhibited cytokine secretion but not CD 107a upregulation and MIP-1 expression. (FIG. 13E) T cells still mediated effective lysis of peptide-pulsed T2 target cells in the presence of 100 μΜ U0126.

[21] FIG. 14. Upregulation of SPRY2 in high antigen concentration- induced T cells inhibits polyfunctionality. (FIG. 14A) qPCR comparison of SPRY2 expression levels on high and optimal antigen concentration-stimulated T cells. (FIG. 14B) The knockdown efficiency of lentiviral particles containing shRNA targeting SPRY2. Twenty- four hours after the second high antigen concentration moDC stimulation, cells were transduced with either SPRY2 knockdown virus or NT sequence virus. qPCR, flow cytometry staining of SPRY2 (red, NT virus control; blue, SPRY2 knockdown virus; gray, FMO control) and polyfunctionality assessment were performed on D14. (FIG. 14C) Representative flow plots of effector functions in virus-transduced T cells. T cells transduced with SPRY2 knockdown virus exhibit enhanced cytokine production of IL-2 and TNF-a as compared with control NT virus. SPRY2 knockdown had no impact on either CD 107a upregulation or MIP-1 β production. (FIG. 14D) SPRY2 knockdown virus-transduced T cells showed greater percentage of 5+ polyfunctional T cells than control virus-transduced T cells. *P < 0.05. The data are representative of more than 3 independent experiments performed on 3 different subjects.

[22] FIGS. 15A-F. SPRY2 inhibition enhances HIV-specific T cell

polyfunctionality. (FIGS. 15A-B) SPRY2 expression was studied in 12 HIV-infected HLA- A2+ patients. HIV-Gag-specific T cells and influenza Ml -specific T cells were sorted by pentamer/tetramer staining and mRNA extracted from Gag- and Ml -specific cells for qPCR analysis. (FIG. 15A) An example of flow cytometry-based simultaneous analysis of Gag- and Ml-specific T cells from PBMCs of an HIV-infected donor. (FIG. 15B) qPCR and flow cytometry analysis of SPRY2 and PD-1 expression in Gag-specific, Ml-specific, and total CD8+ T cells from HLA-A2+ HIV donors. Statistical analyses were performed by nonparametric Wilcoxon matched-pairs signed rank test. (FIGS. 15C-F) PBMCs from 19 HIV-infected patients were activated with soluble anti-CD3, anti-CD28, and a mix of CEF/HIV peptide pools. Some cultures were also treated with anti-PD-1 (10 μg/ml) to block PD-1 signaling during activation. 24 hours later, cells were transduced with SPRY2 knockdown (KD) or NT control lentivirus. On D7 after virus transduction, PBMCs were stimulated with CEF or HIV Gag, Nef, and Tat peptide pools for 6 hours and analyzed for polyfunctionality. (FIGS. 15C-E) Inhibition of SPRY2 expression led to augmented HIV- specific T cell polyfunctionality above and beyond that seen by inhibition of PD-1 pathway alone. (FIG. 15F) PD-1 blockade alone or in combination with SPRY2 inhibition had no significant effect on the CEF-specific response.

[23] FIG. 16. Polyfunctionality profile of T cells induced with different antigen concentrations. Complete polyfunctionality profile of Ml -specific T cells on D14. For simplicity, only T cells induced with ΙΟμΜ moDCs, lOnM moDCs, and lOfM moDCs are shown. ΙΟμΜ moDCs induced T cells with a dominant subpopulation producing MIP-Ι β and CDl-7a, but none of the tested cytokines (CD107a+, ΜΙΡ-1β+, IL-1-, TNFa-, ΙΕΝγ-). In contrast, lOnM moDCs induced highest percentage of cells capable of five effector functions simultaneously (CD107a+, ΜΙΡ=1β+, IL-2+, TNFa+, IFNy+).

[24] FIGS. 17A-B. Highly polyfunctional T cells are equipped with superior proliferative capacity. T cells previously stimulated for 2 weeks with either high (10 μΜ) or optimal (10 nM) antigen concentration-pulsed moDCs were re-stimulated with different concentrations of antigen-pulsed moDCs. After seven days, cells were harvested, counted, analyzed by tetramer staining and assayed for polyfunctionality. FIG. 17A, Ml-tetramer staining, D21. FIG. 17B, absolute cell expansion after third stimulation. Despite various antigen concentration conditions used for third stimulation, T cells previously stimulated with optimal (10 nM) antigen concentration exhibited superior third week cell number expansion (for all restimulation doses, P<.05).

[25] FIG. 18. Naive T cells exhibit impaired polyfunctional response in response to high concentration of anti-CD3/CD28 stimulations. Naive T cells were isolated using

Miltenyi naive CD8+ T cell isolation kit. Cells were stimulated with plate-bound antibodies at the indicated concentrations (equal concentrations of anti-CD3, clone HIT31, and anti- CD28, clone 28.2). Cells were harvested on D7 and restimulated with PMH/ionomycin for 5 hours before staining for intracellular cytokines. Consistent with what was observed with Ml -specific T cells, strong stimulation of na^'ive T cells induced poorly polyfunctional T cells.

[26] FIG. 19. Phenotypic characterization of Ml -specific T cells induced by different antigen concentrations. On D14, T cells were harvested and stained with anti-CD8, Ml tetramer and differentiation markers (FIG. 19 A) or transcription factors (FIG. 19B). Representative histograms shown were gated on CD8+, Ml tetramer+ population. Red, 10 μΜ; blue 10 nM; yellow lOfM. All antigen concentrations induced Ml -specific T cells with a CD45RO⁺, CCR&-, CD28+, CD27+ effector memory phenotype. High (10μΜ) antigen concentration-induced Ml -specific CD8+ T cells expressed significantly lower level of eomesodermin (FIG. 19B) but maintained high levels of T-bet and bcl-2.

[27] FIG. 20. Upregulation of Spry2 in high concentration antigen-induced cells depends on the prior activation of MAPK/ERK pathway. D7 CD8+ T cells were restimulated with either high (ΙΟμΜ) or optimal (lOnM) concentration Ml peptide-pulsed moDCs in the presence of different amounts of the ERK inhibitor, U0126. On D14, T cells were harvested and analyzed for SPRY2 levels. FIG. 20A, CD8+ T cells induced with 10 μΜ antigen were analyzed for SPRY2 levels after treatment with varying concentrations of U0126 (1 μΜ green; 100 nM, blue; no U0126, red). SPRY2 levels in T cells induced with optimal concentration of antigen (10 nM, shaded teal). FIG. 20B, inhibition of ERK pathway during prior T cell activation significantly inhibited the subsequent upregulation of SPRY2 (*P value <0.05).

[28] FIG. 21. Representative HlV-specific T cell polyfunctional response and ex vivo polyfunctionality. PBMCs from HIV-infected patients were thawed, rested overnight and stimulated with peptide pools for six hours prior to cytokine analysis. FIG. 20A, the direct ex vivo Gag and CEF-specific polyfunctional responses. Gag-specific T cells exhibit much a much lower level of polyfunctionality. FIG. 20B, representative flow cytometry results of Gag-specific polyfunctional response in anti-PD-1 and SPR Y2 -knockdown virus treated cells.

Table Legends

[29] Supplementary Table 1. Functional clustering of differentially expressed genes between high (10 μΜ moDCs) and optimal concentration (10 nM moDCs)-induced T cells (P<0.05, fold change >1.6). Functional clustering was performed by DAVID/BP-FAT platform. For simplicity, only certain functional groups highly related to immune response are shown. Red and blue colored numbers indicate gene expression fold change as compared between different concentration-induced T cells. Refer to Table 1 for a full list of

differentially expressed genes and Supplementary Table 2 for a full list of functional groups.

[30] Supplementary Table 2. Full list of differentially expressed genes identified by the microarray experiment. Group A, high antigen concentration ( 10 μΜ); group B, optimal antigen concentration (10 nM). [31] Supplementary Table 3. A full list of functional groups generated from the full list of differentially expressed genes by GO-BP-FAT (Gene Ontology Biological Process, FAT term) tool on the DAVID platform.

DETAILED DESCRIPTION

[32] Human T cells stimulated by a high concentration of antigen lack

polyfunctionality and express a transcription profile similar to exhausted T cells. Several specific pathways were implicated by the transcription profile in control of T cell

polyfunctionality.

MAPK/ERK pathway

[33] As described below, we investigated the control of polyfunctionality in primary human virus-specific CD8+ T cells in response to antigen. In our model system, memory influenza-specific T cells repetitively stimulated with a high antigen

concentration, 10 μΜ of the influenza Ml peptide on monocyte-derived DCs (moDCs), showed robust influenza antigen-specific CD8+ T cell proliferation, but low levels of polyfunctionality. In contrast, an optimal antigen concentration was determined, which induced highly polyfunctional influenza- specific T cells. An antigen concentration- dependent effect on polyfunctionality could also be demonstrated in naive human CD8+ T cells. Genomic gene set enrichment analysis (GSEA) revealed that the global transcriptome of high antigen concentration-induced T cells was similar, but not identical, to that of exhausted T cells observed in chronic infections. Importantly, low-level polyfunctionality induced by high antigen concentration stimulation led to increased expression of inhibitory receptors without evidence of inhibitory receptor signaling. Genetic and biochemical studies indicated that high antigen concentration impaired CD 8+ T cell polyfunctionality through inhibition of the MAPK/ERK pathway via upregulation of sprouty-2 (SPRY2), a negative regulator of the MAPK ERK pathway. Based on these findings, we analyzed a cohort of HIV-infected patients and found that SPRY2is involved in HIV-specific T cell exhaustion. HlV-specific T cells showed marked increased levels of SPRY2 mRNA and SPRY2 protein compared with influenza-specific T cells from the same donors. Furthermore, shRNA- mediated inhibition of SPRY2 enhanced HIV-specific polyfunctionality independently of PD- 1 blockade. When shRNA-mediated inhibition of SPRY2 was analyzed in the presence of anti-PD-1, HIV Gag-specific CTL had levels of polyfunctionality similar to the non- exhausted CEF responses. SPRY2 thus appears to mediate inhibition of HIV-specific T cell polyfunctionality independently of the PD-1 pathway. These findings advance our understanding of the molecular control of T cell polyfunctionality and indicate what we believe to be a novel therapeutic target to reverse T cell dysfunction associated with chronic viral infection or cancer.

[34] T cells stimulated with a high concentration of antigen upregulated sprouty-2 (SPRY2), a negative regulator of the MAPK/ERK pathway. The clinical relevance of SPRY2 was confirmed by examining SPRY2 expression in HIV-specific T cells, where high levels of SPRY2 were seen in HIV-specific T cells and inhibition of SPRY2 expression enhanced the HIV-specific polyfunctional response. Our findings indicate that increased SPRY2 expression during chronic viral infection reduces T cell polyfunctionality and identify SPRY2 as a potential target for immunotherapy.

Decreasing SPRY2 Expression

[35] Spry2 expression can be inhibited by any method known in the art. In some embodiments, Spry2 expression is inhibited by a Spry2 -targeted ribozyme, a Spry2 -targeted antisense oligonucleotide, or a Spry2 -targeted siRNA. In some embodiments, Spry2 expression is inhibited by an aptamer, a protein, a peptide, a cyclic peptide, a peptidomimetic or a small molecule. In some embodiments, Spry2 expression is inhibited by a polypeptide fragment or mutant form of a Spry2 protein, which may function as a competitive inhibitor of a Spry2-interacting polypeptide (e.g., the c-Cbl E3 ubiquitin ligase, EGFR, Ras, or CIN85). See, e.g., O 2007/0066522.

Wnt signaling pathway

[36] A second pathway identified was the Wnt signaling pathway. The Wnt signaling pathway was identified in the microarray pathway analysis suggesting it may be involved in polyfunctionality regulation.

[37] Wnt signaling pathway has been extensively studied in embryonic

development (MacDonald et al., 2009) and regulation of self-renewal of pluripotent stem cells (Kalani et al., 2008; Zeng et al., 2005). Earlier reports of the importance of Wnt signaling in the immune system comes from studies in the context of T cell development in the thymus (Fleming et al., 2008; Schilham et al., 1998; Staal et al., 2008). More recently, accumulating studies indicate that Wnt pathway also controls peripheral immune responses. For example, Wnt pathway activation inhibits naive T cell proliferation and effector differentiation, preferentially generating a novel T cell subset with stem-cell features (TSCM, T memory stem cells) (Gattinoni et al., 2009), was preferentially generated. Compared with other memory subsets, TSCM cells lack immediate effector functions but are multipotent and have enhanced self-renewal capacity to persist as memory cells in vivo (Gattinoni et al., 2011; Gattinoni et al., 2009; Lugli et al., 2013). Other roles of Wnt signaling in mediating mature T cell response have also been reported. TCF1 is essential for generating memory CD8+ T cells (Jeannet et al., 2010). Enforced expression of β-catenin and TCF1 inhibits the effector phase of the immune response and enhances the generation of memory T cells in response to Listeria monocytogenes infection in mice (Zhao et al., 2009). Wnt signaling activation also arrests the naive to effector differentiation in human peripheral and cord blood-derived T cells (Muralidharan et al., 2011).

[38] We found that during antigen-driven expansion of human virus-specific T cells, Wnt pathway activation enhances the CD62L+, CD28+, and KLRGl - central memory T cell phenotype. This effect is novel and distinct from the effects of Wnt pathway on naive T cells and not predicted from that previous work. Such effects are not only seen in influenza- specific response but can also be seen in dysfunctional HlV-specific T cells. These findings provide the first evidence that dysfunctional virus-specific human T cell response could be chemically reprogrammed to polyfunctionality and have strong implications for chronic viral infections and cancer.

[39] The ability to artificially change the program of somatic cell differentiation and function has been gradually revealed in the past decade. Mounting evidence, including this current report, suggests that Wnt pathway controls cell fate in various tissues beyond the stage of embryonic stem cells. For example, Wnt pathway activation promotes epithelial- mesenchymal transdifferentiation (EMT)(Anson et al.; Wu et al.), cardiac hypertrophy (Gessert and Kuhl) and wound healing (Whyte et al . In the immune system, forced expression of Wnt pathway proteins are associated superior memory T cell formation

(Jeannet et al., 2010; Zhao et al., 2009; Zhou et al., 2010) while Wnt pathway negatively regulates regulatory T cell functions (van Loosdregt et al., 2013). Activation of Wnt pathway also programs dendritic cells and NKT cells to tolerance induction (Deng et al.; Oderup et al.).

[40] In this disclosure, we demonstrate that enhancing Wnt signaling promotes the reprogramming of human virus-specific T cells to a polyfunctional status associated with many other sternness features. Polyfunctionality is a feature of early memory T cells. Such finding, nevertheless, contrasts the results obtained from naive T cells. When Wnt pathway activation is activated in na^'ive T cells, a population of na^'ive-like, TSC_M cells (CD45RO- CCR7+CD45RA+CD62L+CD27+CD28+ IL7Ra+CD95+) is generated (Gattinoni et al., 2011). TSCM cells have limited effector functions but TWSl 19-treated memory T cells have substantially better polyfunctionality. This discrepancy could be explained by the fact that, the majority of virus-specific precursor cells in healthy donors have already been exposed to antigen and thus have acquired most, if not all effector functions. Despite T_SC_M cells are shown to be superior to central memory cells for adoptive therapy against cancer in preclinical mice models (Gattinoni et al., 2011), the number of TSC_M cells from human peripheral blood is extremely limited, thus limiting its potential in clinical use.

[41] We have also found that Wnt pathway activation enhanced the expression of CD62L on T cells. Increased expression of CD62L is an important characteristic since CD62L+ T cells are capable of entering secondary lymphoid organ and are superior in controlling pathogen challenge (Gattinoni et al., 2005; Hengel et al., 2003). TWSl 19-treated cells also express higher level of CD28 and lower level of KLRG1, again indicating those cells are of less-differentiated state and are preferable for adoptive immunotherapy

{Klebanoff, 2012 #588; Ebert, 2012 #603} .

[42] The feasibility of reprogramming terminal differentiated T cells offers many practical implications. Because TWSl 19-treated cells express many central memory T cell associated sternness features, we believe these cells are of superior quality for adoptive T cell therapy. Since the numbers of donor-derived moDCs are very limiting clinically, large number of polyfunctional T cells could be potentially achieved by combining aAPC technology with TWSl 19. Finally, the enhancement of polyfunctionality could provide a therapeutic possibility for curing chronic virus infections.

[43] This disclosure describes the impact of Wnt pathway activation on

polyfunctionality in memory T cells. Unexpectedly, instead of limiting T cell effector functions (as happens in na^'ive T cells), Wnt pathway activation dramatically enhances memory T cell polyfunctionality. This effect not only is observed with the less-differentiated memory T cell specific for influenza virus, but also occurs in the terminally differentiated CMV virus-specific response. Importantly, the effect of TWS119 does not result from cell proliferation arrest but can be observed in cells already underwent multiple divisions.

Moreover, the TWS119-treated cells exhibit many features of "sternness" because treated cells are capable of maintaining the CD62L+ polyfunctional phenotype during homeostatic proliferation and express higher level of anti-apoptotic proteins. These results provide strong evidence that polyfunctionality reprogramming in memory cells can be achieved with Wnt pathway activation. Finally, using the acellular aAPCs, we demonstrated that combing aAPC technology with Wnt pathway modulation is a promising strategy to induce large number of highly functional human antigen-specific T cells for adoptive immunotherapy.

Activators of the Wnt/p-Catenin Pathway

[44] The Wnt/p-catenin pathway can be activated by any method known in the art Activators of the Wnt/p-catenin pathway are disclosed, e.g., in Table 1 of US 2012/0046242 and in US 2013/0143227. Examples of Wnt signaling pathway activators include Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, Wntl 0b, Wntl 1, and Wntl 6b. In some embodiments, the Wnt/β- catenin pathway is activated by inhibiting serine -threonine kinase glycogen synthase kinase- 3β (GSK3); see, e.g., US 2001/0052137. Inhibitors of GSK3 include lithium, LiCl, bivalent zinc, BIO, SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119, Kenpaullone, alsterpaullone, indirubin-3'-oxime, TDZD-8 and Ro 31-8220 methanesulfonate salt; Axin inhibitors, APC inhibitors, norrin, and R-spondin 2; see US 2011/0223660.

Pharmaceutical Compositions and Therapeutic Methods

[45] An inhibitor of SPRY2 expression and/or an activator of the Wnt/p-catenin pathway can be formulated in a composition suitable for therapeutic administration to treat chronic infections (e.g., HIV, HCV) and cancer. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the inhibitor of SPRY2 expression and/or an activator of the Wnt/p-catenin pathway and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. EXAMPLE 1. Methods for Examples 2-8

[46] Study subjects and cell isolation. Fresh human CD14+ and CD8+ cells were isolated from PBMCs of seven HLA*0201 positive healthy donors using Miltenyi isolation kits. Monocyte-derived dendritic cells (moDCs) were generated as previously described (Ndhlovu et al., 2010; Oelke et al., 2003). Briefly, CD14+ cells were cultured in the presence of 50ng/ml IL-4 and lOOng/ml GM-CSF for six days and then matured with lOng/ml TNFa, lOng/ml IL-1, lOOOU/ml IL-6 and ^g/ml prostaglandin E2.

[47] Antigens-specific cell activation and expansion by moDCs or aAPCs. moDCs were pulsed with ^g/ml Ml (58-66; GILGFVFTL, SEQ ID NO: 1) or CMV pp65 (NLVPMVATV; SEQ ID NO:2) peptide for an hour at 37°C. Peptide-loaded aAPCs were produced as previously described (Chiu et al., 2011; Oelke et al., 2003). For each stimulation, 1 million peptide-pulsed moDCs or 3 million aAPCs were cocultured with 3 million CD8+ cells in a 96 well, U-bottom plate. The culture media was complete RPMI media

supplemented with antibiotics, 5% autologous plasma, and 3% T cell factor (Oelke et al., 2003).

[48] Antibodies and flow cytometry. The following antibodies were used: anti- CD8 pacific blue (Biolegend, HIT 8 a), anti-CCR7 PE (Biolegend, G043H7), anti-CD45RA APC (Biolegend, HI 100), anti-CD62L Alexa 647 (Biolegend, DREG-56), anti-CD27 Pe-Cy7 (Biolegend, M-T271), anti-CD28 pacific blue (Biolegend, CD28.2), anti-IL-2 Percp-Cy5.5 (MQ1-17H12), anti-ΜΙΡ-Ιβ PE (BD, D21-1351), anti-TNFa Pe-Cy7 (BD, Mabl l), anti-IFNy APC (BD, 25723.11), anti-CD107a FITC (BD, H4A3), anti-IL15Ra APC (Biolegend, JM7A4), anti-CD 122 BV421 (Biolegend, TU27), anti-CD 132 APC (Biolegend, TUGh4). Alexa647-conjugated anti-KLRGl antibody (Clone 13F12F2) was kindly provided by professor Hans-Peter Pircher (University of Freiburg, Germany). Intracellular staining for β- catenin was performed using alexa488-conjugated anti-P-catenin antibody (eBioscience, clone 15B8) according to the manufacturer's protocol.

[49] Intracellular cytokine detection and polyfunctionality analyses. 3xl0⁵ Ml or CMV-specific CD8+ T cells were incubated with 1.5xl0⁵ target cell (HLA*0201 positive T2 cell line) at 37°C in the presence of monensin, brefeldin A and anti-CD28/CD49d costimulation (all from BD Biosciences). For experiments about CD 107a, Anti-CD 107a was added at the start of stimulation. Target T2 cells were loaded with Ml peptide at a concentration of 1 μΜ for at least an hour and washed extensively before use. Unloaded target cells were used as background stimulation. After 6 hours, cells were washed twice with FACS wash buffer and then stained with viability dye, anti-CD3 and anti-CD8 for 20 minutes. Cells were then fixed and permeabilized at 4°C with the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer's protocol. A cocktail of fluorophore-conjugated antibodies containing anti-IL-2, anti-TNFoc, anti-IFNy and anti-ΜΙΡ-Ιβ were added to the cells and stained for two hours. Subsequently cells were washed twice before acquisition by a LSRII flow cytometer. The polyfunctionality analysis was performed by the Boolean gate platform of Flow Jo version 9.3.1 software (TreeStar, Ashland, Oregon).

[50] Cell proliferation analyses. T cells were labeled with ImM

Carboxyfluorescein diacetate Succinimidyl Ester (CFSE, Invitrogen, C34554) in culture media at 37°C for 10 minutes. CFSE were quenched by adding 5 times more cold media, washed twice and let sit at 37°C for an additional 30 minutes. After one more wash with medium cells were then cocultured with moDCs. On selected days as indicated, cells were harvested for tetramer staining and other assays. After gated on CD8+, tetramer-specific cells, the progressive dilution of CFSE corresponding to cell division cycles was analyzed on the Flowjo's proliferation platform. Proliferation index (P.I.) was calculated as the total number of cell divisions divided by the number of cells that have divided.

[51] Quantitative RT-PCR. Cells were lysed with Trizol and stored at -80°C. cDNA synthesis was performed by using Taqman Gold RT-PCR kit. Measurements of mRNA level of Wnt target genes (Tcf7, Lefl, Nik, Fzd7 and Jun) were done by Real-time qPCR (Taqman gene expression assay) using Taqman gene expression kits. 18S was used as internal controls to determine the differential mRNA expression between samples.

[52] Statistical Analyses. Results were shown as mean+/-SD. Student's t test was used to determine the statistical significance of difference between treatment conditions. For comparison between pie charts, permutation test was performed using SPICE (Data Mining & Visualization Software for Multicolor Flow Cytometry) version 5.2. (NIAID, NIH, available at exon.niaid.nih.gov/)(Roederer et al., 2011). EXAMPLE 2. TWSl 19 has distinct effect on polyfunctionality in memory versus naive T cells.

[53] In the literature, a GSK3P (serine-threonine kinase glycogen synthase kinase- 3β) inhibitor, TWSl 19, has been widely used to mimic the activation of canonical Wnt- signaling pathway (Forget et al., 2012; Gattinoni et al., 2011; Gattinoni et al., 2009;

Muralidharan et al., 2011; Staal et al., 2008). Inhibition of GSK3P prevents the degradation of β-catenin promotes the translocation of β-catenin into the nucleus where it binds to other transcription factors that promote the transcriptional activities of Wnt target genes. Upon TWSl 19 treatment, all the Wnt target genes tested were upregulated in memory T cells (FIG. 6).

[54] TWSl 19 treatment of naive T cells inhibits their acquisition of effector functions (Gattinoni et al., 2009). To determine the effect of Wnt pathway activation on memory T cell function, we isolated naive and memory CD8+ T cells and activated them with anti-CD3/CD28 in the presence of TWSl 19. The effector functions were studied on D7. As shown in FIG. 1 A, effector functions of naive T cells were severely impaired by

TWSl 19. In contrast, memory T cells activated in the presence of TWSl 19 expressed much higher level of IFNy and TNFa. In fact, among memory T cells, all the cytokines tested including IL-2, TNFa and IFNy as well as the polyfunctionality (as determined by the percentage of cell making all three cytokine simultaneously), were significantly upregulated by TWSl 19 treatment (FIG. 1C). This is in sharp contrast to what is observed in naive T cells, in which all the effector functions and polyfunctionality were inhibited by TWSl 19 (FIG. IB).

[55] To determine if the impact on memory T cell polyfunctionality by TWS 119 was driven by a specific memory subset, we sorted cells from individual memory subset (T_CM: CCR7+CD45RA-; T_EM: CCR7-CD45RA-; T_EMRA: CCR7-CD45RA+) and activated them in the presence and absence of TWSl 19. We observed significant improvement in polyfunctionality with TWSl 19 treatment in all three sorted memory subsets, indicating that Wnt pathway activation affects all the subsets similarly.

[56] Because TWSl 19 also affects other signaling pathways, we examined the effect of other Wnt pathway modulators on polyfunctionality. Induction of polyfunctionality can be achieved by using another Wnt pathway activator, SKL2001 (FIG. IE). Furthermore, when AVN361 (a small molecule that inhibits β-catenin downstream of TWSl 19) was added to TWSl 19-treated culture, the induction of polyfunctionality was largely inhibited.

EXAMPLE 3. Wnt pathway activation increases the antigen specificity of human T cell culture during ex vivo expansion

[57] We next tested whether if Wnt pathway activation will affect the

polyfunctionality of antigen-specific memory T cells. Autologous CD8+ T cells were stimulated with Ml peptide-pulsed monocyte-derived dendritic cells (moDCs) in the presence of different concentration of TWSl 19 weekly for two weeks to test the effect of TWSl 19 on polyfunctionality. First we determined the percentage of Ml -specific T cells on D7 and D14. As shown in FIG. 2A and FIG. 2B, a higher percentage of Ml -specific T cells was induced in the cultures treated with TWSl 19. However, TWSl 19 treatment impaired the expansion of all cells in the culture as well as the total number of Ml -specific cells (FIGS. 7A-B). This effect was due to the fact that proliferation of non-specific cells was arrested in TWSl 19 treated cultures (FIG. S3C). Overall, TWSl 19 inhibition of Ml -specific cell expansion was dose-dependent, but substantial proliferation was observed even with the highest dose tested (3mM).

EXAMPLE 4. Influenza Ml-specific T cells expanded in the presence of Wnt pathway activation exhibit higher CD62L and better polyfunctionality

[58] To study the effect of TWSl 19 in an antigen-specific fashion, expanded Ml- specific cells were rechallenged with Ml -peptide-pulsed target cells and investigated for their ability to upregulate various effector functions (IL-2, TNFa, INFy, CD 107a and ΜΙΡ-1 β. Expression of IL-2 is a feature early memory T cells and the first function to be lost when virus-specific T cells are repetitively stimulated to terminal differentiation or exhaustion (Wherry, 2011). The co-expression of these five effector functions, or polyfunctionality, is critical for optimal immunity against pathogens and correlates with memory T cell formation (Betts et al., 2006; Joshi and Kaech, 2008; Seder et al., 2008; Wherry, 2011). Compared with T cells expanded without TWSl 19, cells generated in the presence of 3mM TWSl 19 produced more IL-2 and TNFa when rechallenged with antigen (FIG. 2C). In contrast, the expression level of INFy, CD 107a and MIP-Ιβ were not changed by Wnt pathway activation (FIG. 8). When co-expression of five effector functions were analyzed, highest dose of TWSl 19 induced 28% of responding cells capable of executing five effector function simultaneously (IL-2+TNFa+INFg+CD107a+MIP-ip+; "5+"), as opposed to only 9% in cells expanded without TWSl 19 treatment (FIG. 2D). TWSl 19 also enhanced the absolute number of 5+ polyfunctional cell being generated (FIG. 2E).

[59] We next performed phenotypic analysis on the expanded Ml -specific cells. Consistent with previous studies (Gattinoni et al., 2011; Muralidharan et al., 2011), CD62L expression on Ml -specific cells increased under TWSl 19 treatment. However, TWSl 19 also induced higher level of CD28 surface expression and lower level of KLRG1 expression (FIG. 2F). KLRG1 signaling has been shown to inhibit Akt phosphorylation and is associated with immunosenescence of T cells (Henson et al., 2009).

EXAMPLE 5. Wnt pathway activation increases CMV-pp65-specific T cell

polyfunctionality

[60] Influenza-specific T cells are known to be in early memory differentiation. T cells specific for chronic infections are constantly exposed to antigen stimulation and frequently persist in vivo as advanced differentiated cells. To determine if Wnt pathway activation can also reprogram virus-specific memory T cells with advanced differentiation to a polyfunctional state, we tested the effect of TWSl 19 on CMV pp65 -specific T cells. In humans, CMV-specific T cells are known to be highly-differentiated, have limited effector functions and proliferative capability, and are constantly driven to a immunosenescence state (Fletcher et al., 2005; Hertoghs et al.; Mekker et al., 2012). When analyzed directly ex vivo, CMV-specific T cells have low levels of CD28 and CD27 and the majority of them are of the CD45RA+CCR7- phenotype (terminal effector, T_EMRA) (FIG. 9). Consistent with the terminal differentiation phenotype, upon stimulation with pp65 peptide, they produce little IL-2 and thus are not polyfunctional (FIG. 3A).

[61] To show that Wnt pathway activation could also modulate CMV-specific T cell polyfunctionality, CM V-pp65 -specific T cells from healthy donors were expanded with autologous moDCs in the presence or absence of TWSl 19. After two weeks of pp65-pulsed moDCs stimulation, similar to what has been observed in the influenza-Mi response, TWSl 19-treated culture also had substantially improved CM V-pp65 -specificity in culture (FIG. 3B). CMV-specific cells expanded without TWSl 19 still do not produce significant amount of IL-2. In sharp contrast, CMV-specific cells are reprogrammed to a much more polyfunctional state with TWSl 19 treatment and express higher level of CD62L and CD28 (FIGS. 3C-3E). These results indicate that highly differentiated, poorly polyfunctional CMV-specific T cells can also be reprogrammed to a polyfunctional state by Wnt pathway activation.

EXAMPLE 6. Effect of Wnt pathway activation on polyfunctionality and CD62L expression are seen in dividing cells

[62] Previous studies on TWS 119 effect on naive T cell differentiation were questioned on whether such effect is largely due to arresting cellular proliferation (Prlic and Bevan, 2011). In fact, naive cells treated with the same dose of TWSl 19 (3mM) in a previous study were largely undivided (Muralidharan et al, 2011). To further clarify if Wnt pathway activation only improves polyfunctionality in cells did not proliferate, we labeled Ml -specific T cells with CFSE on D7 before restimulation and followed the expression of CD62L and polyfunctionality among different division generations on D 14. As shown in FIG. 4 A, the enhancement of CD62L and effector function expression in TWSl 19-treated cells clearly was seen even in cells that underwent a significant number of divisions.

EXAMPLE 7. The effect of Wnt pathway activation on polyfunctionality is not dependent on dendritic cells

[63] To test whether TWS 119 acts directly on the T cells or rather through modulation of the stimulatory capacity of the dendritic cells, we tested the impact of TWSl 19 on polyfunctionality in a dendritic cell-free system. We used our previously described, HLA- Ig-based aAPCs to stimulate CD8+ T cells (Chiu et al, 2011; Oelke et al, 2003). Briefly, Briefly, HLA-A2-Ig and anti-CD28 were coupled to cell-sized (4.5μΜ) dextran-coated particles and loaded with either Flu-Mi or CMV-pp65-NLV peptide to generate aAPCs for CD8 T cell stimulation. As shown in FIGS. 4C-D, TWSl 19 significantly increased the antigenic specificity of the culture similarly to what had been observed with stimulation of moDCs. TWSl 19 also enhanced the polyfunctionality of both Ml and CMV-specific cells. Thus the effect of TWSl 19 on polyfunctionality does not seem to be dependent on dendritic cells and is largely intrinsic to T cells. EXAMPLE 8. Wnt pathway activation-induced polyfunctionality is associated with many sternness features

[64] Finally, we sought to determine if Wnt pathway-induced highly polyfunctional T cells will be equipped with stem cell-like properties suggestive of superior survival. First we studied the expression level of anti-apoptotic proteins, Bcl-2 and Bcl-XL, in expanded Ml -specific T cells. Bcl-2 and Bcl-XL overexpression has been shown to increase human embryonic stem cell expansion and survival (Bai et al.; Goff et al.). As shown in FIG. 5A, Ml -specific T cells expanded in the presence of TWSl 19 expressed higher level of Bcl-2 and Bcl-XL. Similar results were seen with CMV-specific cells. In addition to superior survival, stem cells are characterized by their ability to self-renewal. Thus if TWSl 19-treated cells are reprogrammed to polyfunctionality, the polyfunctional phenotype should be maintained even after the treatment was stopped. We thus harvested TWSl 19-treated or non-treated Mi- specific cells on D14 and further cultured these cells in the presence of IL-15 for another week (without TWSl 19). As shown in FIG. 5B, we found that TWSl 19-treated cells proliferated better than untreated cells. In addition, T cells previously treated with TWSl 19 also maintained higher CD62L and CD28 expression after homeostatic proliferation (FIG. 5C, FIG. 5D). Finally, cells previously treated with TWSl 19 still exhibited higher polyfunctionality after homeostatic proliferation (FIG. 5E). These results indicate that Wnt pathway activation by Wnt pathway activation reprograms human virus-specific T cells to a long-standing, young, polyfunctional phenotype.

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Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1.

Immunity 33, 229-240.

EXAMPLE 9. Methods for Examples 10-17

[65] Study subjects and purification of primary human PBMCs and CD14+ and CD8+ T cells. PBMCs from 5 healthy HLA* 0201 -positive donors and 15 HIV-infected patients were isolated by Ficoll-Paque PLUS gradient centrifugation following the manufacturer's protocol (GE Healthcare). The HIV-infected, HAART-treated aviremic patients were recruited from Johns Hopkins and Case Western Reserve University. The average age of donors was 49 years old. For some experiments, CD 14+ and CD8+ T cells were further purified from fresh PBMCs using the CD 14+ cell-positive selection and CD8+ T cell-negative selection kits according to the manufacturer's instructions (Miltenyi Biotec).

[66] Generation of peptide-loaded moDCs and aAPCs. The generation of moDCs was done by following the standard protocol as previously described (28,43). Briefly, CD 14+ cells were cultured in complete RPMI supplemented with5% autologous plasma, 100 ng/ml human granulocyte -macrophage colony stimulating factor, and 50 ng/ml IL-4. After 6 days of culture, a maturation cocktail containing 10 ng/ml TNF-a, 10 ng/ml IL-1, 1000 U/ml IL-6 and ^g/ml prostaglandin E2 was added to culture. MoDCs were harvested on D7, and the mature DC phenotype was confirmed by flow cytometric analysis of CD80, CD86, and HLA-DR. moDCs were then pulsed with influenza virus matrix peptide Ml (58-66;

GILGFVFTL; SEQ ID NO: l) at different concentrations in serum- free media for an hour at 37°C. Peptide-pulsed moDCs were then washed extensively to remove free peptide in solution before use. For generation of aAPCs, Ml peptide-loaded HLA-A2-Ig and anti-CD28 (clone 9.3) were biotinylated and coupled to anti-biotin coated microbeads (Miltenyi Biotec) and stored at 4°C (29) before use.

[67] In vitro expansion of Ml-specific CD8+ T cells. Three million freshly isolated CD 8+ T cells were co-cultured with 1 million moDCs in complete RPMI medium containing 5% autologous plasma, 3% T cell growth factor (28) and antibiotics. For micro- aAPC stimulation, different amounts of microaAPCs were co-cultured with CD8+ T cells. On D7 and weekly thereafter, Tells were harvested, counted, and replated at the same T cell/APC density. Influenza Ml-antigen specificity was determined by using HLA-M1 -specific A*0201 PE or APC tetramers (GILGFVFTL; SEQ ID NO: l , Beckman Coulter).

[68] Detection of intracellular cytokines to assess polyfunctionality. For experiments involving Ml -specific T cells, 2 x 105 CD8+ T cells were incubated with 1 x l05 target cells (HLA* 0201 -positive T2 cell line at 37°C in the presence of monensin, brefeldin A andanti-CD28/CD49d costimulation (all from BD Biosciences). Anti-CD 107a was added at the start of stimulation. Target cells were pulsed with Ml peptide at a concentration of 1 μΜ for an hour and washed twice before use. Unpulsed target cells were used as background stimulation. After 6 hours, cells were washed twice with FACS wash buffer and then stained with viability dye, anti-CD3, and anti-CD8 for 20 minutes. Cells were then fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer's protocol. A cocktail of fluorophore-conjugated antibodies containing anti-IL- 2, anti-TNF-a, anti-IFN-γ, and anti-ΜΙΡ-Ι β was added to the cells and stained for an hour. For experiments involving HlV-specific T cell responses, 2 x 10⁵ PBMCs were stimulated with different peptide pools at a concentration of 0.5 μg/ml per peptide(Gag, Nef, Tat peptide pools, NIH AIDS reagent program; CEF peptide pool, Anaspec) for 6 hours for effector function detection. Complete combinations of different effector function were performed using the Boolean gate platform of Flow Jo version 9.3.1 software (TreeStar). Reported data were further adjusted by background subtraction. Polyfunctionality pie charts were generated by SPICE (Data Mining and Visualization Software for Multicolor Flow Cytometry) version 5.2. (National Institute of Allergy and Infectious Diseases [NIAID], NIH) (44).

[69] Microarray experiment, data analysis, and qPCR validation. More than half a million Ml -specific T cells induced by 10 μΜ or 10 nM moDCs from5 experiments on 3 donors were tetramer sorted and preserved in TRI reagent (Molecular Research Center) at -80°C. RNA extraction and microarray experiments were performed by Miltenyi Biotec. Hybridization was performed with single-colored Whole Human Genome 8^χ 60K

OligoMicroarrays (Agilent Technologies). Gene expression data were analyzed using Partek Genomic Suite. Each sample was normalized and log2 transformed before being compared by t test (P < 0.05). Genes with fold changes of more than 1.6 were selected for further analysis. Differential expressed genes were functionally grouped and annotated using DAVID Bioinformatics Resources 6.7 (NIAID, NIH; available at

david.abcc.ncifcrf.gov/home.jsp) (45). Functional annotation and pathway identification were performed using the Gene Ontology (GO) fat category, as provided by the DAVID platform. Validations of genes were done by real-time qPCR(TaqMan gene expression assay) using ACTB or HPRTI as internal controls. The complete microarray dataset is available from GEO {G§E5916X).GSEA. GSEA (46) was used to identify gene sets that are significantly enriched in either high concentration (10 μΜ) or optimal concentration(10 nM) stimulation. The input to GSEA is the log2 transformed expression ratio for each gene. When expression of one gene was measured by multiple probes, we used the maximum expression ratio of all probes for that gene. GSEA (46) calculates a running sum of the statistic for each gene set. The test statistic is the maximum of the running sum (enrichment score), and permutation tests are performed to evaluate the empirical P value of the enrichment. Gene sets for LCMV exhaustion versus normal effector and memory T cells (25) were provided by John Wherry. Gene sets for HIV progressor and PD-1 signaling (26) were provided by Nicolas Haining.

[70] pERK staining and cytotoxicity assay. pERK detection was based on protocols as previously described (47). Briefly, Ml -specific T cells were stimulated with PMA/ionomycin for 10 minutes before fixation with 2% formaldehyde and permeabilized with 90% methanol. Staining was performed with anti-phospho-p44/42 MAPK (ER 1/2) antibody from Cell Signaling(clone E10). Flow cytometry-based cytotoxicity assay (48) was used to investigate the effect of ERK inhibition on T cell-mediated HLA-A2-positiveT2 cell killing.

[71] Production of shRNA-containing lentiviral particles and T cell

transduction. shRNA sequence (5'-CTGAACAGAGACTGCTAGGATCATCCTTC-3'; SEQ ID NO:3) targeting the human SPRY2 gene was cloned into pLKO. l puro plasmid (provided by Joel Pomerantz. Viral particles were made by co-transfecting 20 ng of the PLKO plasmid plus 15 ng delta 8.9 plasmid and 10 ng pCMV-VSV-G into HEK293T cells (plated 24 hours earlier at 2.5 x 10⁶ cells/dish) via calcium phosphate precipitation as previously described (49). Transduction of primary Ml -specific T cells was performed 24 hours after DC stimulation by spinning at 300 g for 90 minutes in the presence of 8 μg/ml polybrene (Sigma-Aldrich). 24 hours after transduction, culture medium containing 1.5 μg/ml puromycin was used to select for virus-transduced T cells. RNA isolation (Cells-to-CT kit; Invitrogen) and qPCR (TaqMan gene expression assay) for knockdown efficiency and polyfunctionality analysis were performed 6 days after viral transduction. Similar

transduction procedures were performed with HlV-specific patients' PBMCs. PBMCs were thawed and rested overnight before activation with soluble anti-CD3 (clone HIT3a, 1 μg/ml; Biolegend) and anti-CD28 (1 μg/ml, clone CD28.2; Biolegend) in the presence of Gag/Nef/Tat/CEF peptide pools and transduced with lentivirus. Anti-PD-1 antibody (10 μ /ηι1, clone EH12.2H7; Biolegend) was used to block PD-1 signaling. Isotype control antibody (clone MG1-45; Biolegend) was added to control cultures.

[72] HIV-specific T cell sorting and measurement of SPRY2 expression. HIV

Gag antigen specificity was determined by using HLA-A2 restricted PE pentamer

(SLYNTVATL; SEQ ID NO:4; Proimmune). CD8+ T cells were purified from 7 HLA- A2+HIV patients from the study subjects and stained with HIV-Gag-pentamer (PE) and Ml- tetramer (APC). For each donor, more than 1000 antigen-specific T cells were sorted for mR A extraction and cDNA conversion using the Cells-to-Ct kit (Invitrogen). After preamplification using the TaqMan PreAmp Mastermix Kit (Invitrogen), qPCR was performed using TaqMan gene expression assays, and SPRY2 expression level were adjusted to HPRT1 expression. For some donors, unsorted PBMCs were stained with Gag-specific pentamer or Ml -specific tetramers, anti-PD-1, and anti-CD8 for an hour. Cells were fixed, permeabilized, and stained with APC-conjugated anti-human SPRY2 antibody (clone ab60719; Abeam) for an additional 2 hours. Cells were then washed twice before acquisition by flow cytometry.

[73] Statistics. Statistical comparisons between different polyfunctionality pie charts were performed by permutation test set at 10,000 repetitions using SPICE. Other statistical analyses were performed by Prism version 5 (Graph-Pad). For comparison between 2 groups of related samples, the nonparametric Wilcoxon matched-pairs signed rank test was used. The Mann- Whitney U test was used to detect the differences between 2 independent groups of samples. A P value of less than 0.05 was considered statistically significant.

EXAMPLE 10. Antigen concentration regulates polyfunctionality of human T cells.

[74] Autologous moDCs were pulsed with various concentrations of the immunodominant, HLA-A*0201 restricted influenza peptide Ml 58-66 and used to stimulate CD8+ T cells ex vivo. On day 14 (D14), after 2 rounds of peptide-pulsed moDC stimulation, cell counts and percentage of Ml -specific CD8+ T cells were determined. There was a direct correlation between the amount of antigen used, from 10 fM to 10 μΜ, to pulse DCs and percentage ofMl -specific CD8+ T cells as well as the overall cell number (FIGS. lOA-C). Percentage of Ml -specific T cells varied over 40-fold, from approximately 2% to 85%, and a similar impact was seen on total number of Mi-specific CD8+ T cells. [75] Antigen concentration also controlled T cell effector function. On D14, T cells were incubated with Ml peptide-pulsed target cells and analyzed for multiple effector functions. Compared with T cells induced by 10 nM peptide-pulsed moDCs, T cells induced by high antigen concentration (10 μΜ) had a significantly reduced ability to simultaneously produce multiple cytokines or effector functions (FIGS. 10D-E). Only 5.6% of the Ml- specific cells induced with 10 μΜ peptide-pulsed moDCs produced both IL-2and CD 107, and 15.1% produced both TNF-a and MIP-Ι β. In contrast, when T cells were induced with 10 nM moDCs, there were significantly larger percentages of T cells with multiple effector functions. 25.4% of the Ml -specific T cells produced both IL-2 andCD107, and

approximately 50% produced both TNF-a and MIP-Ι β. A low antigen concentration, 10 fM, also induced a lower percentage of dual-function Ml -specific T cells when compared with the 10 nM antigen concentration, but a higher percentage of dual function Ml -specific T cells compared with the 10 μΜ antigen. The effector function most sensitive to antigen

concentration was IL-2 production, and functions least sensitive were CD 107a andMIP-Ι β (FIG. 10E). The loss of IL-2 responses during high antigen concentration stimulation is also observed during chronic infections such as HIV (9), where IL-2 production is the most sensitive and first function lost out of other effector functions.

[76] By analyzing overall T cell polyfunctionality, we determined an antigen concentration that induced the maximal amount of polyfunctional T cells. When individual T cell cultures were analyzed for all 5 functions simultaneously, there was a modal distribution for induction of polyfunctional T cells (FIGS. 10F-G). 10 nM antigen pulsed moDCs induced the greatest polyfunctional response; 26.4% of all Ml -specific T cells had all 5 functions (5+ polyfunctional T cells). This was significantly higher than the response induced by the high antigen concentration, 10 μΜ, 4.5% 5+ polyfunctionalMl -specific T cells (P = 0.002), or by the low antigen concentration, 10 fM, 13.7% 5+ polyfunctional Mi-specific T cells (P = 0.001). There was no further inhibition of T cell polyfunctionality response to an even higher antigen concentration, 100 μΜ. Within the population of 5+ polyfunctional Tells, T cells induced with 10 nM moDCs also produced more IL-2 on a per cell basis (FIG. 10H). T cells with the lowest polyfunctionality, induced by 10 μΜ moDCs, were dominated by a population of cells capable of producing MIP-i pand degranulation, but did not produce any cytokines (FIG. 16). Upon rechallenge with antigen, T cells induced with 10 nM Ml antigen- pulsed moDCs also exhibited superior cell expansion (FIG. 17). In the remainder of this disclosure, we refer to 10 nM as the "optimal antigen concentration" and 10 μΜ as the "high antigen concentration" of peptide used to pulse moDCs. While these studies analyzed the memory Ml -specific T cell responses, we also analyzed the impact of antigen concentration on naive CD8+ T cells. When naive T cells were stimulated with different concentrations of anti-CD3and anti-CD28, a similar modal distribution for induction of polyfunctional T cells was observed (FIG. 18). Optimal concentration of anti-CD3/anti-CD28 resulted in 28% polyfunctional T cells, while the highest concentration of anti-CD3/anti-CD28 led to only 1.8% polyfunctional T cells. Thus, antigen concentration controls T cell polyfunctionality both memory and naive CD8+ T cell populations.

EXAMPLE 11. Regulation of polyfunctionality is independent of the T cell differentiation markers.

[77] It is possible that different Ml -specific T cell clones are hardwired with different polyfunctionality potential and selectively expanded in response to particular concentrations of antigen. This seemed unlikely as more than 95% of the Ml tetramer- positive cells were positive for νβ17 TCR, independent of the amount of antigen used for stimulation. In addition, it is possible that the T cell differentiation state could also have an impact on their effector functions (19). We characterized T cells stimulated with a range of peptide concentrations using a panel of differentiation markers including CD45RO,

CD45RA, CCR7,CD28, and CD27 and found no significant differences (FIG. 19A) among the populations. T cells from all stimulation conditions also expressed similar levels of bcl-2 and T-bet (FIG. 19B).

EXAMPLE 12. Molecular signature of T cell polyfunctionality.

[78] Based on these results, we hypothesized that distinct molecular mechanisms regulate T cell polyfunctionality. To establish the molecular signature of polyfunctionality, antigen-specific T cells were induced by either high or optimal antigen concentration, tetramer sorted on D 14, and analyzed for global gene expression. Cut-off point analysis was set at 1.6-fold change and an adjusted P value of less than 0.05. optimal antigen

concentration-induced T cells, and 302 genes were down-regulated (FIG. 11 A). Functional grouping performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) revealed that most differentially regulated genes are central to lymphocyte function, including inhibitory receptor expression, lymphocyte activation, chemotaxis, cytotoxicity, and antigen presentation (Supplementary Table 1). A full list of differentially expressed genes can be found in Supplementary Table 2. Compared with high antigen concentration-induced T cells, highly polyfunctional T cells induced by the optimal antigen concentration expressed less inhibitory receptors at the mR A level, including PD-1, 2B4 (CD244), and KLRG1. Microarray data showed that transcription factors that promote CD8+ T cell memory formation, such as EOMES (20) and TCF7 (21), were higher in highly polyfunctional T cells, and differences were confirmedly flow cytometry or quantitative PCR (qPCR) (FIGS. 11B-C). We also found differential expression of multiple chemokines and chemokine receptors, which have also been shown to regulate T cell differentiation and memory formation (22, 23), and a large group of differentially expressed genes were involved in signal transduction.

EXAMPLE 13. High antigen concentration-induced T cells are enriched with the molecular signature of T cell exhaustion.

[79] GSEA has been used to compare genomic signatures between microarray experiments in order to integrate different phenotypic cellular states (24). GSEA showed that highly polyfunctional T cells were significantly enriched with the genetic signature for memory T cells (ref. 25 and FIG. llD).In contrast, there was a significant enrichment of the exhausted gene signature (25) among high antigen concentration-induced T cells, indicating that these T cells share aspects of global gene expression patterns with T cells that are exhausted due to chronic LCMV infection (FIG. 11D). Similar enrichment was found while using the differentially expressed gene list from HIV progressors versus controllers (26). Interestingly, while it has been established that the PD-1/PD-L1 pathway is involved in T cell exhaustion during chronic viral infections (27), GSEA analysis of the PD-1 ligation signature (26) showed no significant enrichment.

EXAMPLE 14. Inhibitory receptor signaling is not required for high antigen

concentration-induced polyfunctionality inhibition.

[80] To specifically study the role of inhibitory receptor signaling in the generation of polyfunctional T cells, we analyzed the inhibitory receptor expression on T cells induced by different antigen concentrations. Significantly higher levels of multiple inhibitory molecules such as PD-1, CTLA-4, and TIM-3 were seen in Ml -specific T cells induced by high versus optimal concentration of antigen (FIG. 12A). However, no difference was seen in expression of another inhibitory receptor, BTLA. We took several approaches to analyzing the potential role of signaling through inhibitory receptors, such as PD-1. Expression of basic leucine transcription factor, ATF-like (BATF), an important PD-1 downstream signaling molecule (26),was not upregulated by qPCR, in high antigen concentration-induced T cells (FIG. 12B). We further analyzed the potential role of PD-1 during high antigen concentration stimulation using anti-PD-1 antibody to block PD-1/PD-L1 interaction (10 μg/ml, clone EH12.2H7). Nevertheless, no increase in polyfunctionality was seen in high antigen concentration-induced T cells treated with anti-PD-1 (FIG. 12C). In addition to PD-1, there are other inhibitory ligands present on DCs that could potentially modulate polyfunctionality. To analyze the impact of antigen concentration on polyfunctionality in the absence of potential inhibitory molecules, we used artificial APCs (aAPCs), which have no inhibitory receptor ligands. This is a reductionist system in which aAPCs are made by conjugatingHLA- A2-Ig-Ml complex (signal 1) and anti-CD28 (signal 2) onto particles (28, 29). Higher concentrations of aAPCs with signal 1 plus signal 2 induced significantly more antigen- specific T cell proliferation (FIGS. 12D-E), but lower T cell polyfunctionality (FIG. 12F). Similar to moDC stimulation, there was an optimal antigen concentration for aAPC stimulation. Therefore, while signaling through PD-1 and other inhibitory receptors is widely involved in T cell exhaustion, polyfunctionality regulation due to antigen concentration requires only signal 1 and signal 2.

EXAMPLE 15. MAPK/ERK pathway controls T cell poly functionality in response to antigen concentration.

[81] To further investigate the molecular mechanism regulating polyfunctionality, we performed GO-BP-FAT analysis on the microarray result and found that the MAPK/ERK pathway is altered in T cells with different levels of polyfunctionality (Supplementary Table 3). The MAPK ERK pathway is an important signaling pathway that critically regulates a variety of physiological processes, such as cell growth, differentiation, and survival. This pathway also plays important roles in many aspects of lymphocyte biology, such as thymocyte selection, CD4+ T cell differentiation, and T cell activation (30). However, the role of the MAPK/ERK pathway in T cell polyfunctionality and exhaustion has not been reported. Differences were seen in the level of phospho-ERK (pERK)upregulation in T cells induced by either high or optimal antigen concentration. 67% of the optimal antigen concentration-induced T cells upregulated pERK in response to stimulation, while only 33% of the poorly polyfunctional T cells showed increased levels of pERK (FIG. 13A).

Upregulation of pERK was shown to be specific, as treatment of cells with U0126, a MEKl/2 specific inhibitor, inhibited ERK phosphorylation in a concentration-dependent fashion, with complete inhibition of pERK seen with 100 μΜΙΙ0126 (FIG. 14B). If the MAPK/ERK pathway differentially controls effector functions involved in polyfunctionality, such as cytokine production, one should be able to convert 5+ polyfunctional antigen-specific Tells to low polyfunctional T cells by inhibiting ER phosphorylation. In the presence of U0126, there was a dose-dependent inhibition of cytokine production (IL-2, TNF, and IFN-γ), with little effect on CD 107 and MIP-Ι β expression in optimal antigen concentration-induced T cells. There was an approximately 10-fold reduction in the amount of IL-2 and TNF-a made by Ml-specific CD8+ T cells (FIGS. 11C-D). However, CD8+ T cell-mediated lysis of target cells was largely insensitive to U0126 treatment (FIG. 13E). Overall, the data indicate that cytokine secretion and polyfunctionality but not lytic ability or MIP-Ιβ expression is dependent on the MAPK/ERK pathway. Thus, the differential consequences of optimal and high antigen concentrations could be replicated by biochemical inhibition of the MAPK ERK pathway.

EXAMPLE 16. Upregulation ofSPRY2 in high antigen concentration-induced T cells inhibits polyfunctionality.

[82] One gene identified in the microarray gene list (Supplementary Table 1) known to affect the MAPK/ERK pathway is SPRY2. Sprouty proteins are a well-conserved family known to mediate the negative feedback regulation of the MAPK pathway (31 , 32). Sprouty proteins bind to Grb2 and other components of the MAPK pathway, preventing the upregulation of pERK (33, 34). qPCR analysis confirmed that SPRY2 is upregulated in high antigen concentration-induced CD8+ T cells (FIG. 14A) and upregulation of SPRY2 is downstream of ERK activation, as it is inhibited by U0126 treatment added during high antigen concentration stimulation (FIG. 20). To determine whether SPRY2 mediates suppression of polyfunctionality in high antigen concentration-induced T cells, we engineered lentiviral particles containing shRNA-targeting SPRY2 in order to inhibit its expression. SPRY2 knockdown virus efficiently inhibited mRNA expression of SPRY2, by approximately 80%, while control non-target (NT) virus had no effect (FIG. 14B). SPRY2 knockdown also led to a significant decrease in SPRY2 protein levels as determined by flow cytometry (FIG. 14B). The MFI of SPRY2 in the SPRY2knockdown cells decreased from 1860 to 910. Upon antigen stimulation, the SPRY2 knockdown T cells produced more cytokines than the control lentivirus-infected T cells (FIG. 14C), and overall, the percentage of 5+ polyfunctional T cells increased from approximately21% to 37% (FIG. 14D). Thus, high antigen concentration stimulation leads to lower CD 8+ T cell polyfunctionality through upregulation of SPRY2, which inhibits the MAPK/ERK pathway. EXAMPLE 17. SPRY 2 controls HlV-specific T cell poly functionality.

[83] As compared with acute infection such as influenza, HlV-specific CTL responses in infected patients are known to have significantly lower levels of

polyfunctionality, and CTL dysfunction is not restored by antiretroviral therapy (9, 35). Recent studies indicate that inhibition of the PD-1 pathway partially reverses low

polyfunctionality associated with chronic viral infection (13). However, the effects of anti- PD-1 treatment were modest (14, 15) and indicated that additional mechanisms control HlV- specific T cell polyfunctionality. We hypothesized that HlV-specific T cells also upregulate SPRY2 and that upregulation of SPRY2 is responsible for the low levels of T cell

polyfunctionality seen in HlV-specific responses. PBMCs from 7 HLA-A2-positive HIV- infected patients were sorted on the basis of pentamer/tetramer staining into either HIV Gag- or influenzaMl -specific CTL (FIG. 15A). Within CD8+ T cells, the average percentage of Gag-specific T cells was 0.45% as compared withO.04% of Ml -specific T cells (P = 0.03,). qRT-PCR on these populations showed that SPRY2 was upregulated in the Gag-specific T cells compared with both theM-1 -specific T cells and nonspecific CD8+ T cells from the same donors. Flow cytometry staining for SPRY2 showed that Gag-specific T cells express higher levels of SPRY2 protein and PD-1 (FIG. 15B). Consistent with previous findings (9), these cells are less polyfunctional when analyzed directly ex vivo (FIG. 21). We next studied the effect of SPRY2 knockdown on HIV Gag-,Nef-, and Tat-specific CTL responses (FIG. 15C-F). Inhibition of SPRY2 enhanced all the HlV-specific polyfunctional responses independently of PD-1 blockade. In addition, inhibition of SPRY2 in combination with anti- PD-1 treatment further augmented polyfunctional responses (FIG. 15C-F). By combining anti-PD-1 with SPRY2 inhibition, HIV Gag-specific polyfunctionality improved to levels seen in the CEF-specific (CMV,EBV, and influenza) responses. In contrast, neither SPRY2 inhibition nor PD-1 blockade had a significant effect on CEF-specific responses from the same donors (FIG. 15F). These findings indicate that SPRY2 upregulation inhibited HlV- specific T cell polyfunctionality independently of the PD-1 pathway.

Discussion

[84] Polyfunctionality has emerged as a highly significant predictor of protective immunity (1, 2, 7, 9, 18). In this report, we generated antigen concentration-dependent influenza-specific CD8+T cells with differing levels of polyfunctionality. An optimal concentration of antigen, resulting in highly polyfunctional T cells, was determined, and microarray analysis showed that these T cells were enriched for a memory T cell signature. In contrast, high antigen concentration-induced T cells that were significantly less polyfunctional were enriched for a T cell exhaustion signature. These poorly polyfunctional T cells had high levels of inhibitory receptor expression, but inhibitory receptor signaling did not regulate polyfunctionality in this system. By utilizing the aAPC system, we demonstrated that high antigen concentration alone could regulate polyfunctionality. Through these findings, we identified the MAPK/ERK pathway and SPRY2 as regulators of

polyfunctionality in high antigen concentration stimulation and validated the importance of SPRY2 in chronic HIV infection.

[85] The molecular profile of highly polyfunctional T cells is enriched with memory signature genes and is consistent with the idea that the polyfunctional state is associated with memory T cell development (1, 19, 36). Two key genes identified as part of the polyfunctional signature were EOMES and TCF7, both known to be indispensable for optimal CD8+ T cell memory formation (20, 37). The mechanisms governing their expression levels are not entirely clear, but here we identified antigen concentration as an important factor. The finding that optimal antigen concentration induced higher levels of TCF7 and EOMES could identify the molecular mechanism linking optimal antigen concentration to superior T cell memory formation.

[86] High antigen concentration induced the most robust proliferation, but the lowest levels of T cell polyfunctionality. The dominant subpopulation of high antigen concentration-induced Tells did not produce any cytokines, but these T cells efficiently mediated target cell recognition and killing. Thus, high antigen concentration-induced T cells were not anergized. These T cells, which exhibit poor cytokine production, low proliferative potential, and high levels of KLRGl, may represent short-lived effector T cells driven toward terminal differentiation during antigenic stimulation, albeit they did not yet exhibit the CD28- and CD27- phenotype (FIG. 19).

[87] Chronic viral infection is also associated with high antigen load, T cell exhaustion, and the loss of polyfunctionality. The loss of each individual function in our system follows the hierarchical order for loss of functions observed in HIV and LCMV infections (8). In brief, IL-2, a homeostatic cytokine that promotes T cell proliferation, is lost first. Subsequently, effector cytokines such as TNF-a and IFN-γ secretion are impaired. In contrast, upregulation of CD 107a and MIP-Ιβ are least affected (9). Enrichment of the T cell exhaustion signature in the high antigen concentration-induced T cells indicates that these T cells share certain biological pathways with exhausted T cells seen in HIV (26) and chronic LCMV (25) infection. Thus, high antigen concentration-induced T cells may represent cells "on their way" to becoming completely exhausted.

[88] Inhibitory receptor upregulation is a key feature of T cell exhaustion (10) and often characterized by active suppression mediated through the PD-1/PD-L1 pathway (13, 14). Although the poorly polyfunctional T cells expressed more inhibitory receptors, the poorly polyfunctional phenotype was independent of inhibitory receptor signaling. This is evident by the fact that PD-1/PD-L1 blockade did not attenuate high antigen concentration- induced loss of polyfunctionality and that the effect of high antigen concentration was seen even when using a signal 1/2 only-aAPC system to induce poorly polyfunctional T cells. Additional evidence comes from the lack of enrichment of the PD-1 molecular signature, such as upregulation of BATF, in the high antigen concentration-induced T cells. Overall, our data show that TCR signaling strength alone determines T cell polyfunctionality.

[89] Our study identified SPRY2 as a regulator of T cell polyfunctionality and showed that SPRY2 expression level is dependent on antigen-concentration. Sprouty proteins were originally identified in Drosophila as regulators for embryonic development, and mutations in sprouty proteins were found in various human cancers (31). All sprouty proteins were found to be negative regulators of growth factor signaling, especially the MAPK ER pathway (32, 33).

[90] A potential role of sprouty proteins has been seen in T cell activation where both SPRY1 and SPRY2 inhibit T cell activation and IL-2 production (34, 38). SPRY1- deficient mice have been shown to have enhanced antitumor T cell responses (39).

Interestingly, a recent microarray analysis indicated that SPRY2 is upregulated in

CD8+CXCR1-CD27-CD28- T cells capable of producingIL-2 (40). However, the physiological role of SPRY2 was not validated in these studies. Given the importance of the MAPK pathway in immune system signaling, it is likely that sprouty proteins could also regulate the outcome of diverse immune responses. During immune response against infection, upregulation of sprouty proteins might be beneficial to the host as a strategy for preventing immune system overactivation and subsequent immunopathology. On the other hand, modulation of SPRY2 expression could be a potential way to enhance T cell polyfunctionality and immunological memory during chronic infections.

[91] Authors of several previous reports have tried to improve HIV or HCV virus- specific polyfunctional responses by blocking inhibitory receptor interaction with the receptors' ligands, such as PD-1/PD-L1 (13-15, 41). To date, the improvement in T cell function has been modest and probably is limited only to less terminally differentiated cells (42). Here, we show that inhibition of SPRY2 expression was associated with improvement of HlV-specific polyfunctional responses above and beyond the effects ofPD-1 blockade. As a result, SPRY2 could be a novel therapeutic target for reversing T cell exhaustion to be used in combination with inhibitory receptor blockade. The finding that CEF-specific Tells, which remain highly polyfunctional in HIV patients, did not respond to SPRY2 inhibition indicates that the expression level of SPRY2 could be related to different T cell outcomes of viral infections and additional studies on the regulatory mechanisms of its expression are warranted.

[92] Our study mechanistically reveals the need to choose optimal antigen doses for immunization to achieve effective immunological memory and protection. While it is hard to extrapolate from the doses of antigen used in our in vitro studies to specific vaccine doses, simply increasing the dose of antigen could result in generation of poorly

polyfunctional T cells with less memory potential and might not be an appropriate approach for at-risk populations who exhibit inferior responses to standard vaccinations. Thus, the human T cell polyfunctionality signature and the molecular mechanisms involving SPRY2 and the MAPK/ERK pathway, as identified in the current study, can have important clinical applications such as identifying desirable immune responses in response to immunization and infection.

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Claims

1. A method of inducing T cell polyfunctionality, comprising inhibiting SPRY2 expression.

2. The method of claim 1, wherein SPRY2 expression is inhibited by an agent selected from the group consisting of a Spry2 -targeted ribozyme, a Spry2 -targeted antisense oligonucleotide, a Spry2 -targeted siR A, an aptamer, a protein, a peptide, a cyclic peptide, a peptidomimetics, a small molecule, and a polypeptide fragment or mutant form of a Spry2 protein which functions as a competitive inhibitor of a Spry2 -interacting polypeptide.

3. A method of inducing T cell polyfunctionality, comprising activating the Wnt/β- catenin signaling pathway.

4. The method of claim 2, wherein the Wnt/p-catenin signaling pathway is activated by inhibiting serine-threonine kinase glycogen synthase kinase-3p.

5. A method of treating chronic infection or cancer, comprising administering to a patient in need thereof a therapeutic amount of an agent that inhibits SPRY2 expression.

6. A method of treating chronic infection or cancer, comprising administering to a patient in need thereof a therapeutic amount of an agent that activates the Wnt/p-catenin signaling pathway.

7. Use of an agent that inhibits SPRY2 expression in the manufacture of a

medicament for inducing T cell polyfunctionality.

8. Use of an agent that activates the Wnt/p-catenin signaling pathway in the manufacture of a medicament for inducing T cell polyfunctionality.

9. Use of an agent that inhibits SPRY2 expression to treat chronic infection or cancer.

10. Use of an agent that activates the Wnt/p-catenin signaling pathway to treat chronic infection or cancer.