The PD-1/PD-L1 Axis Contributes to T Cell Dysfunction in Chronic Lymphocytic Leukemia

Abstract Abstract 1778 Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of mature B lymphocytes and it is marked by profound defects in T cell function. The mechanisms responsible for T cell dysfunction remain unclear, even if several observations show that T cells from CLL patients express markers of chronic activation. One of this marker is Programmed death-1 (PD-1), a cell surface molecule that inhibits activation of immune cells and it is involved in tumor escape mechanisms through binding of the specific PD-L1 ligand. The aim of this work is to evaluate the expression and function of the PD-1/PD-L1 axis in the CLL context. Using multiparameter flow cytometry, we showed that CD4+ and CD8+ T lymphocytes from CLL patients (n=117) express significantly higher levels of the PD-1 receptor, as compared to the same cell subpopulations purified from age- and sex-matched normal donors (n=33; 52% vs 34%, p <0.001). In keeping with the notion that PD-1 is a marker of cell exhaustion, CD4+ and CD8+ T lymphocytes from CLL patients displayed increased numbers of effector memory and terminally differentiated cells, respectively, with a concomitant decrease in naïve and central memory cells, when compared to controls. The number of effector memory and terminally differentiated cells positively associated with a more advanced stage of disease, treatment requirements and unfavorable genomic aberrations. Moreover, leukemic lymphocytes expressed higher levels of PD-L1 than circulating B lymphocytes from normal donors. PD-1 and PD-L1 expression significantly increased when T or B lymphocytes were treated with mitogenic signals, suggesting that this interaction might work efficiently in an activated environment. This hypothesis was tested by immunohistochemical analyses determining PD-1 and PD-L1 expression in the proliferation centers of lymph nodes sections from CLL patients. The results obtained indicate that PD-L1+ proliferating CLL cells are in close contact with CD4+/PD-1+ T lymphocytes. Lastly, functional experiments performed using anti-PD-1 antibodies or recombinant soluble PD-L1 clearly indicate that the PD-1/PD-L1 axis contributes to driving IL-4 secretion and to the inhibition of IFN-g production by CD8+ T cells. In conclusion, these results show that CD4+ and CD8+ T lymphocytes from CLL patients express high levels of the surface marker PD-1 and exhibit an exhausted phenotype, while B leukemic cells express the PD-L1 ligand. Functional data suggest that PD-1/PD-L1 interactions are critical in skewing the T cell compartment towards a Th2 phenotype, by impairing IFN-g secretion by CD8+ cells. Taken together, these observations suggest that pharmacological manipulation of the PD-1/PD-L1 axis might be relevant in restoring T cell functions in the CLL microenvironment. Disclosures: Inghirami: OncoEthix SA: Research Funding.

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Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+T cell dysfunction and maintain memory phenotype

Science Immunology ◽

10.1126/sciimmunol.aat7061 ◽

2018 ◽

Vol 3 (29) ◽

pp. eaat7061 ◽

Cited By ~ 44

Author(s):

Bei Wang ◽

Wen Zhang ◽

Vladimir Jankovic ◽

Jacquelynn Golubov ◽

Patrick Poon ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Programmed Cell Death ◽

Cancer Immunotherapy ◽

Effector Memory ◽

Cell Phenotype ◽

Checkpoint Inhibition ◽

Cell Dysfunction ◽

T Cell Dysfunction

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor–related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1–Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

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Lenalidomide Promotes The Expansion Of CD8 T Cells With An Effector Memory Phenotype In a Murine Xenograft Model Of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.119.119 ◽

2013 ◽

Vol 122 (21) ◽

pp. 119-119

Author(s):

Rita Simone ◽

Sonia Marsilio ◽

Piers E.M. Patten ◽

Gerardo Ferrer ◽

Shih-Shih Chen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

T Lymphocytes ◽

B Cell ◽

Xenograft Model ◽

Lymphocytic Leukemia ◽

Effector Memory ◽

Cell Engraftment

Abstract Lenalidomide (Revlimid®), a thalidomide analogue, is an orally administered second generation immunomodulator with anti-angiogenic and anti-neoplastic properties. Initial studies treating patients with chronic lymphocytic leukemia (CLL) suggest that lenalidomide can have considerable efficacy and that its mode of action is mainly indirect, affecting non-malignant cells in the microenvironment, in particular T lymphocytes. Because a recently described xenograft model for CLL has highlighted the importance of CLL-derived, autologous T cells in promoting leukemic B-cell engraftment and growth in vivo, we have studied the influence of lenalidomide on the expansion of CLL B- and T-lymphocytes in this model. After an initial 12 day culture of FACS-isolated CLL-derived T cells with or without anti-CD3/CD28 beads plus IL-2 (30 IU/ml), T lymphocytes were transferred into alymphoid NSG mice via the retro-orbital plexus (day 0). On day 7, CLL cells were delivered retro-orbitally. These recipient animals are referred to as “T + PBMC mice”. Mice that did not receive T cells on day 0 but were given CLL PBMCs at day 7, with or without lenalidomide, served as controls (“PBMC only mice”). Recipient mice received lenalidomide (10mg/kg/day) or vehicle control daily by gavage starting at day 0. All mice were sacrificed at day 28 (28 days after T-cell and 21 days after B-cell transfer), and blood, spleen, and bone marrow were collected. On this material, four analyses were performed: [1] level of human CD45+ cell engraftment; [2] numbers and types of CLL-derived T cells; [3] numbers of CLL B cells; and [4] levels of cytokines reflective of Th1 and Th2 immune responses. There was a clear enhancement in human hematopoietic (CD45+) cell engraftment in those mice exposed to lenalidomide. This was most marked for the PBMC only mice (vehicle: 10.64%; lenalidomide: 38.53%), although it was also evident for T + PBMC mice (vehicle: 55.96%; lenalidomide: 69.65%). T-cell phenotyping was carried out, before and after cell culture and also at sacrifice. Prior to culture, CLL samples contained on average ∼96% CD5+CD19+ cells and ∼3% CD5+CD19- cells; for the latter, ∼67% were CD4+ and ∼33% CD8+. After 12-day culture, these percentages remained largely unchanged. However, the numbers and types of T cells recovered from the spleens at sacrifice were quite different after in vivo exposure to lenalidomide. For the PBMC only, the percentages of CD4+ and CD8+ cells in the spleens differed somewhat based on lenalidomide exposure (CD4: Vehicle 86% vs. Lenalidomide 61%; CD8: Vehicle 10% vs. Lenalidomide 28%). However, this change was dramatic for the T + PBMC mice (CD4: Vehicle 64.1% vs. Lenalidomide 28.9%; CD8: Vehicle 34% vs. Lenalidomide 62%). Furthermore, when the CD8+ cells from these animals were subsetted based on antigen-experience and function, it appeared that lenalidomide exposure had led to the outgrowth of a greater number of effector memory (CD45RO+ CD62L-) than central memory (CD45RO+ CD62L+) T-cells. For CLL-derived B cells, the numbers differed, based not only on lenalidomide exposure but also on prior in vitro activation. Specifically, in PBMC only mice, the addition of lenalidomide led to increased numbers of CLL B cells in the spleen (Vehicle: 7.81% vs. Lenalidomide: 14%). Conversely, in the T + PBMC mice, the numbers of B cells decreased (Vehicle: 2.36% vs. Lenalidomide: 0.34%). An analysis of Th1 and Th2-related cytokines in the plasmas of the mice at sacrifice revealed a fall in IL-4, IL-5, and IL-10 and a marked increase in IFNg, consistent with a Th2 to Th1 transition. The above data suggest that administration of lenalidomide permits greater engraftment of human hematopoietic cells in alymphoid mice. Although this enhancement involves all members of the hematopoietic lineage, T cells, in particular CD8+ effector memory T cells, emerge in excess over time. This CD8 expansion is associated with diminished levels of CLL B cells suggesting that the decrease is due to T-cell mediated cytolysis. In contrast, in the absence of prior T-cell activation, CLL T cells appear to support better CLL B-cell growth. These findings suggest that lenalidomide alters B-cell expansion in vivo depending on the activation and differentiation state of the autologous T-cell compartment. They also implicate the generation of cytolytic T cells as one mechanism whereby lenalidomide leads to clinical improvement in CLL. Disclosures: Allen: Celgene Corporation: Honoraria.

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EBV-Specific CD8+ T-Cells Are Not Functionally Impaired in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.1723.1723 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1723-1723

Author(s):

Tom Hofland ◽

Iris de Weerdt ◽

Sanne Terpstra ◽

Ester B.M. Remmerswaal ◽

Ineke J.M. ten Berge ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Cd8 T Cells ◽

Synapse Formation ◽

T Cell Differentiation ◽

Lymphocytic Leukemia ◽

Immune Synapse ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to increased susceptibility to infections and a decreased immunosurveillance (Görgün et al. JCI, 2005). Furthermore, T-cell dysfunction impairs novel treatment strategies that rely on T-cell mediated effects. The dysfunction of T-cells in CLL is characterized by an inability to form immune synapses, increased expression of exhaustion markers and impaired cytotoxicity and proliferative capacity (Ramsay et al. JCI 2008; Ramsay et al. Blood 2012; Riches et al. Blood 2013). However, we recently found that CMV-specific CD8+ T-cells from CLL patients are functionally intact with respect to cytokine production, cytotoxicity and immune synapse formation when compared to age-matched healthy controls (HC)(te Raa et al. Blood 2014). The finding that specific subsets of T-cells in CLL patients are functionally intact challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown. Aim To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8+ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection. Methods EBV-specific CD8+ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 42 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation based on surface markers CD45RA, CCR7, CD27 and CD28 and 2 master regulators of T-cell differentiation, the transcription factors T-bet and Eomes. We also measured expression of exhaustion markers (PD-1, CD244 and CD160), functional markers (such as KLRG1, CD127, granzyme B, granzyme K and Ki-67) and homing markers (CXCR3 and CX3CR1). To study the functionality of EBV-specific CD8+ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides. Results Using a comprehensive T-cell differentiation staining we found that when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of "early" effector memory cells (also called EM1, CD45RA- CCR7- CD27+ CD28+; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression patterns of the transcription factors T-bet and Eomes; 25.79% EBV-specific T-cells of CLL patients display a T-bethigh Eomeshigh phenotype vs 17.44% in HC. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160 (MFI 4896.42 vs 3130.56 and 2320.09 vs 1097.38, respectively), but not PD-1. However, there were no significant differences in granzyme B and K expression in EBV-specific T-cells, suggesting an unaltered cytotoxic potential. On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β of EBV-specific T-cells after peptide stimulation. Also, degranulation (measured as CD107a+ cells) was similar between CLL patients and healthy controls after peptide stimulation. Finally, polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. We are currently determining cytotoxicity and immune synapse formation. Conclusion So far, although the phenotype may suggest an increased exhaustive state, we have not observed signs of dysfunction of EBV-specific T-cells in CLL patients when compared to HC. We are currently performing experiments to test cytotoxicity and ability to produce immune synapses of EBV-specific T-cells (which we will be able to present during the ASH meeting). Based on these results, we will be able to conclude if EBV-specific CD8+ T-cells are also functionally intact in CLL patients, and whether this population joins CMV-specific T-cells as a subset that eludes CLL induced T-cell dysfunction. T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects. T-cell populations that escape suppression may be good targets for future therapies to build around. Disclosures No relevant conflicts of interest to declare.

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BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2021-153921 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3715-3715

Author(s):

Audrey L Smith ◽

Alexandria P Eiken ◽

Sydney A Skupa ◽

Dalia Y Moore ◽

Avyakta Kallam ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Ex Vivo ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

T Cell Proliferation ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Introduction : Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies. Methods : Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (>10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h). Results : T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P<0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P<0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P<0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P<0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME. Conclusion : Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. Figure 1 Figure 1. Disclosures Lunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

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Alloreactivity Directed Against the Widely Distributed HY Antigen Impairs Antitumor Immunity and Results in T-Cell Dysfunction

Blood ◽

10.1182/blood.v118.21.2964.2964 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2964-2964

Author(s):

Jessica C Shand ◽

Christian M. Capitini ◽

Haiying Qin ◽

Nicole Nasholm ◽

Brynn B Duncan ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Dendritic Cell Vaccine ◽

Effector Memory ◽

Cell Vaccine ◽

Antitumor Response ◽

Cell Dysfunction ◽

Tumor Bearing ◽

T Cell Dysfunction

Abstract Abstract 2964 INTRODUCTION: The curative potential of allogeneic transplant for high-risk malignancy is based on the observation that alloreactivity can result in a clinically significant graft-versus-tumor (GVT) effect. However, we have observed that alloreactivity directed against non-tumor restricted miHA's reduces quantitative responses to vaccines targeting tumor-specific antigens. The relative impact of the GVHD-mediating antigen on the potency of the GVT response when the antigen is shared has not been well studied. METHODS: A murine allotransplant system in which the clinically relevant GVHD antigen HY drives both graft-versus-host disease and the antitumor response was utilized. Following lethal irradiation, combinations of B6 male (HY-expressing) and female (HY-naïve) donors and recipients were used in T-cell depleted bone marrow transplants to control for HY expression in hematopoetic and non-hematopoetic compartments. Delayed donor lymphocyte infusion (DLI) with female HY-specific transgenic T-cells was then performed which allowed tracking of antigen-specific cells. Mice were subsequently challenged with an immunogenic HY-expressing tumor (MB49). In tumor protection studies, transplant recipients received a male dendritic cell vaccine at the time of DLI. Recipients were monitored for clinical GVHD scoring, weight loss, tumor-free and overall survival. Surface phenotyping of HY-specific CD8 T cells from recipient bone marrow, tumor-draining lymph node (LN) and spleen was performed serially by flow cytometry using congenic markers. Statistical analyses were performed using paired Student t-tests and Kaplan-Meier survival estimates. RESULTS: Transplantation of female marrow and HY-specific T cells into male recipients produces a mild HY-targeted GVHD, indicated by weight loss and skin GVHD scores. Female recipients of female marrow and HY-specific DLI had 100% survival following HY-expressing tumor challenge. In contrast, male recipients had only 20 +/− 4.7% tumor-free survival (p<0.0001), despite receiving HY-reactive female marrow and HY-specific DLI. Administration of an HY-expressing male dendritic cell vaccine did not improve either tumor growth velocity or tumor-free survival in male recipients. Despite a poor antitumor response in males, expression of HY on nonhematopoetic tissues produced a significant expansion of HY specific T-cells following DLI, regardless of tumor-bearing status (30.5 −77.4% total CD8 from spleen, draining LN and marrow, vs 0.01–1% from female recipient controls, p<0.0001). This suggested that impaired tumor control was due to dysfunction, rather than deletion, of HY-specific T cells. Indeed, nearly 100% of HY-specific CD8 isolated from the spleen, tumor-draining lymph node, and bone marrow of male recipients expressed high levels of PD-1, a phenomenon observed at all time points in tumor-bearing and non tumor-bearing male recipients with HY-directed GVHD. Non-HY specific CD8 cells did not express PD-1 (p<0.0001). Further, HY-specific CD8 from spleen and tumor-draining LN of male recipients display a significantly increased percentage of CD44+CD62L- effector memory (72.4 +/− 17.2%) vs. CD44+CD62L+ central memory (15.9 +/− 9.7%, p= 0.006) cells compared to non-HY specific CD8 cells (26.5% +/− 2.8 % vs. 28.2 +/− 12.7%, p= 0.52) from male and female recipient controls. CONCLUSIONS: In an experimental system where HY is expressed on both recipient nonhematopoetic tissue and tumor, HY-directed alloreactivity impairs the antitumor response despite antigen-specific DLI and effective vaccination. Characterization of alloreactive CD8 T cells in this setting reveals a persistence of effector memory and high levels of PD-1 expression, which suggest T-cell dysfunction as a possible mechanism. Further studies of T-cell dysfunction in this model may identify targets for therapeutic blockade following adoptive immunotherapy with particular relevance to those clinical situations where GVHD does not enhance GVT. Disclosures: No relevant conflicts of interest to declare.

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Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion

Science Advances ◽

10.1126/sciadv.abd2710 ◽

2021 ◽

Vol 7 (18) ◽

pp. eabd2710

Author(s):

Chen Zhu ◽

Karen O. Dixon ◽

Kathleen Newcomer ◽

Guangxiang Gu ◽

Sheng Xiao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adaptor Protein ◽

Transcriptional Analysis ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Cell Dysfunction ◽

Autoreactive T Cell ◽

Autoimmune Conditions ◽

T Cell Dysfunction

T cell exhaustion has been associated with poor prognosis in persistent viral infection and cancer. Conversely, in the context of autoimmunity, T cell exhaustion has been favorably correlated with long-term clinical outcome. Understanding the development of exhaustion in autoimmune settings may provide underlying principles that can be exploited to quell autoreactive T cells. Here, we demonstrate that the adaptor molecule Bat3 acts as a molecular checkpoint of T cell exhaustion, with deficiency of Bat3 promoting a profound exhaustion phenotype, suppressing autoreactive T cell–mediated neuroinflammation. Mechanistically, Bat3 acts as a critical mTORC2 inhibitor to suppress Akt function. As a result, Bat3 deficiency leads to increased Akt activity and FoxO1 phosphorylation, indirectly promoting Prdm1 expression. Transcriptional analysis of Bat3−/− T cells revealed up-regulation of dysfunction-associated genes, concomitant with down-regulation of genes associated with T cell effector function, suggesting that absence of Bat3 can trigger T cell dysfunction even under highly proinflammatory autoimmune conditions.

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TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

10.21203/rs.3.rs-641407/v1 ◽

2021 ◽

Author(s):

qi shao ◽

Lei Wang ◽

maoling yuan ◽

Xiaohong Jin ◽

changping wu

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

T Cell ◽

Glucose Metabolism ◽

Tumor Growth ◽

Tumor Cells ◽

Peripheral Blood ◽

Cancer Tissue ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

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663 Media based on the metabolic composition of tumor interstitial fluid reveals persistent T cell dysfunction induced through arginine deprivation and exposure to the oncometabolite phosphoethanolamine

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.663 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A691-A691

Author(s):

Yupeng Wang ◽

Chufan Cai ◽

Dayana Rivadeneira ◽

Alexander Muir ◽

Greg Delgoffe

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Interstitial Fluid ◽

Cytokine Production ◽

Cell Dysfunction ◽

Kennedy Pathway ◽

T Cell Dysfunction ◽

Tumor Interstitial Fluid

BackgroundWhile CD8 T cells are crucial for anti-tumor immunity, tumor infiltrating CD8 T cells encounter stressors which deviate their differentiation to a dysfunctional, exhausted phenotype. T cell functions are closely regulated by T cell metabolism, and the dysfunctional vasculature in tumor tissues and the deregulated metabolism of tumor cells lead to depletion of nutrients and accumulation of metabolic wastes in the tumor microenvironment (TME). Thus, the unbalanced levels of the nutrients and the metabolic wastes might skew the metabolism of T cells thus contributing to T cell dysfunction.MethodsOvalbumin-specific OT-I cells were activated with SIINFEKL/IL2 and cultured with IL2. The tumor interstitial fluid media (TIFM) was formulated based on the concentrations of the metabolites measured in the tumor interstitial fluid of pancreatic ductal adenocarcinoma.1 Purified arginine and phosphoethanolamine (PEtn) were used to change their levels in TIFM/RPMI1640 culture. Expression level of cytokines and PD-1 was measured by flow cytometry.ResultsWe sought to determine how T cells would differentiate, in vitro, if they were exposed only to the metabolites present in the TME. Using media formulated to model the metabolic composition of tumor interstitial fluid (TIFM),1 we show that CD8 T cells develop features of exhausted T cells in the TIFM culture: reduced proliferation, increased expression of PD-1 and decreased cytokine production. Using 'dropout' and 'add-back' approaches, we found arginine levels as a major contributor to the proliferation defect observed in TIFM-cultured T cells. Arginine was sufficient to restore proliferative capacity to T cells cultured in TIFM, but had no effect on the inhibited cytokine production. We then asked which metabolites were enriched in the TIFM, finding that PEtn, an intermediate in the ethanolamine branch of the Kennedy pathway and an oncometabolite enriched in the interstitial of many solid tumors, up-regulates PD-1 expression and compromises the cytokine production of the cells in culture. Depletion of Pcyt2, the metabolizing enzyme of PEtn and the rate limiting enzyme in the Kennedy pathway, makes CD8 T cells resistant to the effects of PEtn.ConclusionsOur data shows that the metabolic environment in the TME can be recapitulated in vitro and is sufficient to drive T cell dysfunction. Arginine depletion acts as a major inhibitor of T cell proliferation in the TME, but the oncometabolite PEtn drives a hypofunctional effector fate of T cells. Targeting PEtn metabolism via Pcyt2 depletion or inhibition is a potential target to reinvigorate T cells and enhance anti-tumor immunity.ReferenceSullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. Elife 2019;;8:e44235. doi: 10.7554/eLife.44235. PMID: 30990168; PMCID: PMC6510537.

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Circulating CD8 T Lymphocytes in Human Immunodeficiency Virus-Infected Individuals Have Impaired Function and Downmodulate CD3ζ, the Signaling Chain of the T-Cell Receptor Complex

Blood ◽

10.1182/blood.v91.2.585.585_585_594 ◽

1998 ◽

Vol 91 (2) ◽

pp. 585-594 ◽

Cited By ~ 2

Author(s):

Linda A. Trimble ◽

Judy Lieberman

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

T Cell Receptor ◽

Cell Receptor ◽

Receptor Complex ◽

Cd8 T Lymphocytes ◽

Immunodeficiency Virus ◽

Specific Cytotoxicity

Although human immunodeficiency virus (HIV)-infected subjects without acquired immunodeficiency syndrome have a high frequency of HIV-specific CD8 T lymphocytes, freshly isolated lymphocytes frequently lack detectable HIV-specific cytotoxicity. However, this effector function becomes readily apparent after overnight culture. To investigate reasons for T-cell dysfunction, we analyzed T-cell expression of the cytolytic protease granzyme A and of CD3ζ, the signaling component of the T-cell receptor complex. An increased proportion of CD4 and CD8 T cells from HIV-infected donors contain granzyme A, consistent with the known increased frequency of activated T cells. In 28 HIV-infected donors with mild to advanced immunodeficiency, a substantial fraction of circulating T cells downmodulated CD3ζ (fraction of T cells expressing CD3ζ, 0.74 ± 0.16 v 1.01 ± 0.07 in healthy donors; P < .0000005). CD3ζ expression is downregulated more severely in CD8 than CD4 T cells, decreases early in infection, and correlates with declining CD4 counts and disease stage. CD3ζ expression increases over 6 to 16 hours of culture in an interleukin-2–dependent manner, coincident with restoration of viral-specific cytotoxicity. Impaired T-cell receptor signaling may help explain why HIV-specific cytotoxic T lymphocytes fail to control HIV replication.

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P03.23 Evolution of the immune landscape within partially controlled murine melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.61 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A31.2-A32

Author(s):

C Qing ◽

E Ghorani ◽

I Solomon ◽

F Gálvez-Cancino ◽

F Vargas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Partial Response ◽

Tumor Vaccine ◽

Combined Treatment ◽

Effector Memory ◽

Partial Control ◽

Treg Depletion ◽

Differentiated Cells ◽

Over Time

BackgroundRegulatory T cell (Treg) depletion with antibodies against CD25 is effective in tumor models but response rates are low in poorly infiltrated B16 melanomas. Combination with a tumor vaccine enhances efficacy, but relapse usually occurs following partial control, similar to what is seen clinically. How resistance develops is unknown.Materials and MethodsC57BL/6 mice were injected subcutaneously with B16 cells. Treatments included a depleting mouse IgG2a αCD25 antibody and/or a genetically modified, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting whole B16 tumor vaccine (Gvax). Changes in the immune landscape were assessed with high dimensional flow cytometry.ResultsCompared to monotherapies, combined Gvax/αCD25 significantly delayed tumour growth and prolonged survival, in association with enhanced infiltration of T cells with an activated phenotype. Approximately 50% of mice achieved partial response with relapse at day 35–45 post tumor injection. To characterize immune evolution prior to relapse, we analysed stable, partially responding tumors and paired draining lymph nodes (DLNs). Over time, activated PD-1+ICOS+TCF7- T cells with an effector memory (CD44+CD62L-) phenotype fell from 30% to 10% whilst resting, TCF7+ early differentiated cells rose in abundance towards levels seen in untreated tumors. Abundance of Ki67-, resting Tregs also recovered. Similar results were obtained in analysis of DLNs.ConclusionsCombined Treg depletion/whole tumor vaccination therapy is effective in a poorly infiltrated B16 melanoma model. Combined treatment promotes T cell infiltration and activation. In mice achieving a partial response, treatment effects on the immune landscape were observed to decay over time suggesting a return to immune equilibrium. Further studies to explore the mechanistic basis of this observation are underway.Disclosure InformationC. Qing: None. E. Ghorani: None. I. Solomon: None. F. Gálvez-Cancino: None. F. Vargas: None. K. Peggs: None. S. Quezada: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; F. HOFFMANN-LA ROCHE LTD.

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