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

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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Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

Journal of Experimental Medicine ◽

10.1084/jem.20100637 ◽

2010 ◽

Vol 207 (10) ◽

pp. 2175-2186 ◽

Cited By ~ 725

Author(s):

Julien Fourcade ◽

Zhaojun Sun ◽

Mourad Benallaoua ◽

Philippe Guillaume ◽

Immanuel F. Luescher ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Tumor Antigen ◽

Cytokine Production ◽

Cd8 T Cell ◽

Advanced Melanoma ◽

Antigen Stimulation ◽

Cell Dysfunction ◽

T Cell Dysfunction

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8+ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8+ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1+ NY-ESO-1–specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1–specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

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PD-1 does not mark tumor-infiltrating CD8+ T cell dysfunction in human gastric cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000422 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000422

Author(s):

Yang Shen ◽

Yongsheng Teng ◽

Yipin Lv ◽

Yongliang Zhao ◽

Yuan Qiu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Human Gastric Cancer ◽

Cell Dysfunction ◽

T Cell Dysfunction ◽

Tgf Β1

BackgroundOverexpression of programmed cell death protein 1 (PD-1) is linked to CD8+ T cell dysfunction and contributes to tumor immune escape. However, the prevalence and functional regulations of PD-1 expression on CD8+ T cells in human gastric cancer (GC) remain largely unknown.MethodsFlow cytometry was performed to analyze the level, phenotype, functional and clinical relevance of PD-1+CD8+ T cells in GC patients. Peripheral blood CD8+ T cells were purified and subsequently exposed to culture supernatants from digested primary GC tumor tissues (TSN) in vitro for PD-1 expression and functional assays. Tumor responses to adoptively transferred TSN-stimulated CD8+ T cells or to the TSN-stimulated CD8+ T cell transfer combined with an anti-PD-1 antibody injection were measured in an in vivo xenograft mouse model.ResultsGC patients’ tumors showed a significantly increased PD-1+CD8+ T cell infiltration. However, these GC-infiltrating PD-1+CD8+ T cells showed equivalent function to their PD-1−CD8+ counterparts and they did not predict tumor progression. High level of transforming growth factor-β1 (TGF-β1) in tumors was positively correlated with PD-1+CD8+ T cell infiltration, and in vitro GC-derived TGF-β1 induced PD-1 expression on CD8+ T cells via Smad3 signaling, whereas Smad2 signaling was involved in GC-derived TGF-β1-mediated CD8+ T cell dysfunction. Furthermore, GC-derived TGF-β1-mediated CD8+ T cell dysfunction contributed to tumor growth in vivo that could not be attenuated by PD-1 blockade.ConclusionsOur data highlight that GC-derived TGF-β1 promotes PD-1 independent CD8+ T cell dysfunction. Therefore, restoring CD8+ T cell function by a combinational PD-1 and TGF-β1 blockade might benefit future GC immunotherapy.

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Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Journal of Experimental Medicine ◽

10.1084/jem.20081398 ◽

2008 ◽

Vol 205 (12) ◽

pp. 2763-2779 ◽

Cited By ~ 479

Author(s):

R. Brad Jones ◽

Lishomwa C. Ndhlovu ◽

Jason D. Barbour ◽

Prameet M. Sheth ◽

Aashish R. Jha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Surface Expression ◽

Cell Dysfunction ◽

Cd38 Expression ◽

Immunodeficiency Virus ◽

T Cell Dysfunction ◽

Novel Target ◽

Hiv 1

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

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Transforming growth factor beta orchestrates PD-L1 enrichment in tumor-derived exosomes and mediates CD8 T-cell dysfunction regulating early phosphorylation of TCR signalome in breast cancer

Carcinogenesis ◽

10.1093/carcin/bgaa092 ◽

2020 ◽

Author(s):

Soumya Chatterjee ◽

Annesha Chatterjee ◽

Samir Jana ◽

Subhasis Dey ◽

Himansu Roy ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Evasion ◽

Cd8 T Cells ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Cd8 T Cell ◽

Cell Dysfunction ◽

T Cell Dysfunction ◽

Growth Factor Beta

Abstract Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-β) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-β level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-β1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-β dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-β synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-β as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.

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493 Tired and hungry: a potential role for CD47 in T cell exhaustion

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0493 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A529-A529

Author(s):

Levi Mangarin ◽

Cailian Liu ◽

Roberta Zappasodi ◽

Pamela Holland ◽

Jedd Wolchok ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Effector Function ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Cell Dysfunction ◽

Melanoma Model ◽

T Cell Dysfunction ◽

Melanoma Patients

BackgroundMultiple suppressive mechanisms within the tumor microenvironment are capable of blunting anti-tumor T cell responses, including the engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8+ T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).1 2 While immune checkpoint blockade therapies aimed at reinvigorating T cell effector function have demonstrated their clinical effectiveness,3 4 not all patients demonstrate long-term disease control.5 The refractory nature of terminally differentiated, exhausted CD8+ T cells to be reinvigorated by PD-1 blockade is one potential cause.6–8 This limitation warrants the need to explore modulatory pathways that potentially program T cells toward exhaustion.MethodsSingle cell-RNA sequencing (scRNA-seq) data derived from the tumor-infiltrating lymphocytes (TILs) of melanoma patients9 were used for transcriptomic analysis and flow cytometry results were used to quantify protein levels in TILs. Murine B16-F10 (B16) melanoma model was used for both in vitro and in vivo studies. TCR-transgenic Pmel-1 and OT-1 transgenic mice, as well as CD47-/- (knockout, KO) mice were purchased from the Jackson Laboratory to generate CD47+/+ (wild-type, WT), CD47± (heterozygote, HET) mice with Pmel-1 or OT-1 background. For T cell co-transfer studies, Rag-deficient mice or C57BL/6j mice with sub-lethal irradiation (600cGy) were used as recipients. Naïve TCR-transgenic CD47-WT and CD47-HET CD8+ T cells were labelled, mixed in a 1:1 ratio for co-transfer experiments.ResultsFlow cytometry analysis of human melanoma TILs found a strong upregulation of CD47 expression in tumor-associated, exhausted CD8+ T cells. We confirmed that CD47 transcription is significantly elevated among CD8+ T cells with a phenotype consistent with exhaustion using scRNA-seq results of TILs derived from melanoma patients.9 Our study in murine B16 melanoma model confirms our finding in melanoma patients. To specifically address the role of CD47 in anti-tumor CD8 effector function, we conducted T cell co-transfer studies and found that CD8+ T cells with lower copy number of CD47 (CD47-HET) significantly outnumber the co-transferred CD47-WT CD8+ T cells within the tumor, exhibiting an enhanced effector function and less exhausted phenotype. Our study demonstrates a potentially novel role for CD47 in mediating CD8+ T cell exhaustion.ConclusionsCD47 expression in CD8+ T cells programs T cells toward exhaustion.Ethics ApprovalAll mice were maintained in microisolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the MSKCC Institutional Animal Care and Use Committee (IACUC).ReferencesWherry EJ and M Kurachi. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15(8): p. 486–99.Thommen DS and Schumacher TN. T Cell Dysfunction in Cancer. Cancer Cell 2018;33(4): p. 547–562.Ribas A and Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 2018. 359(6382): p. 1350–1355.Sharma P and Allison JP. The future of immune checkpoint therapy. Science 2015; 48(6230): p. 56–61.Sharma P, et al. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 2017. 168(4): p. 707–723.Schietinger, A., et al., Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity 2016;45(2): p. 389–401.Pauken KE, et al., Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354(6316): p. 1160–1165.Philip M, et al., Chromatin states define tumour-specific T cell dysfunction and reprogramming. Nature 2017;545(7655): p. 452–456.Sade-Feldman M, et al., Defining T Cell States associated with response to checkpoint immunotherapy in melanoma. Cell 2018;175(4): p. 998–1013e20.

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A Signature of T Cell Exhaustion Is Enriched in the Bone Marrow (BM) of AML Patients and Shared with Immune Exhaustion Signatures of Solid Tumors

Blood ◽

10.1182/blood-2019-128742 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2711-2711

Author(s):

Hanna A. Knaus ◽

Hubert Hackl ◽

Amanda Blackford ◽

Sofia Berglund ◽

Raul Montiel-Esparza ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Induction Chemotherapy ◽

Cd8 T Cells ◽

Research Funding ◽

Post Treatment ◽

Cell Dysfunction ◽

Immune Exhaustion ◽

T Cell Dysfunction

Background: T cells are key players in immunotherapy but our understanding of their role and function in AML is limited. We have previously shown that following induction chemotherapy, the phenotypic and transcriptional profiles of peripheral blood (PB) CD8+ T cells in AML diverge between complete responders (CR) and non-responders (NR) to treatment (Knaus et al, JCI insight 2018). Further defining AML-induced T cell dysfunction is critical for immune monitoring and developing novel immunotherapeutic strategies. Aims: 1. To characterize transcriptional differences between PB vs BM T cells of AML patients, similar to concept of circulating vs tumor infiltrating lymphocytes (TILs), and compare them to established immunologic signatures of solid tumors. 2. To examine the cumulative expression of multiple inhibitory receptors (IRs) on AML CD8+ T cells and their dynamics in PB vs BM at diagnosis (PRE-treatment) and POST-induction chemotherapy. Methods: To study transcriptional signatures, we FACS-purified CD8+ T cells from BM of 6 AML patients (3 CR and 3 NR) PRE- and POST-induction chemotherapy whose PB T cells we analyzed and published previously (Knaus et al, JCI insight 2018). Samples were hybridized to the Human Prime View Gene Expression Array (Affymetrix). Expression fold change (FC), p-values and FDR were calculated. We compared our data set to those of dysfunctional T-cell signatures from non-small cell lung cancer (NSCLC: Guo et al, Nature 2018), colorectal cancer (CRC; Zhang et al, Nature 2018) and hepatocellular carcinoma (HCC; Zheng et al, Cell 2017) using gene set enrichment analyses (GSEA) and considered a normalized enrichment score NES>2 and FDR<0.1 as enriched. Flow cytometry data on paired PB and BM samples from AML patients (n=32) PRE- and POST-treatment and healthy controls (HC; n=21) were used to calculate the IR-score (Thommen et al, Cancer Immunol Res 2015). The IR-score summarizes the cumulative relative amount of expression of 7 different IRs on CD8+ T cells. To determine the predictive ability of the IR score to differentiate CR from NR, Receiver Operating Characteristic (ROC) analysis was conducted using logistic regression, and the area under the ROC curve (AUC) was calculated. Results: Immune signatures of AML CD8+ T cells at diagnosis, particularly in the BM, overlapped with transcriptomic exhaustion signatures of TILs from other malignancies (Figure 1-Overlap CRC, HCC, NSCLC), in particular that of CRC. Further, AML BM but not PB T cells showed a high enrichment for a common immune exhaustion signature shared by all 3 solid tumor entities and included IRs like HAVCR2, PDCD1, CTLA4, LAG3 and TIGIT, and other genes (e.g. TNFRSF9, CD27, IFNG, FASLG, CD39/ENTPD1) (Figure 1). Interestingly, this signature seemed to discriminate CR from NR patients only in the BM compartment, and at both PRE- and POST-treatment, thus providing a rationale for future exploration as a predictive biomarker of response. We found that the flow-cytometry-based IR-score strongly discriminated between CR and NR in both the PB and the BM compartment POST-treatment with an AUC of 0.70 and 0.84, respectively. While IR-score including all 7 markers provided the best discriminatory value in the PB, the combination of only two senescence markers, CD57 and KLRG1, discriminated CR from NR patients in both PB and BM with an AUC of 0.69 and 0.79, respectively. The combination of 5 exhaustion markers (2B4, BTLA, Tim3, PD-1, and CD160) discriminated CR from NR patients only in the BM (AUC 0.79). Conclusion: We found that in AML, T cells display a signature of immune exhaustion which is more prominent in the tumor milieu (e.g BM), similar to exhaustion signatures of solid cancers, can discriminate CR from NR, and persists in CR. Multiple genes, such as TNFRSF9, CD27, IFNG, FASLG, and CD39/ENTPD1, were higher expressed in CR patients both PRE and POST-treatment, suggesting that this signature also includes genes that could be useful for discriminating less dysfunctional T cell states. The phenotypic IR score was increased only in NR POST-treatment, indicating that assessment of a limited number of phenotypic markers may not be sufficient to address overall changes within T cells. Further exploration of AML-imposed T cell dysfunction is ongoing to inform better integration of immune-based therapies to augment anti-leukemia immunity. Disclosures Zeidner: Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding; Pfizer: Honoraria; AsystBio Laboratories: Consultancy; Merck: Research Funding; Takeda: Research Funding; AbbVie: Honoraria; Agios: Honoraria. Gojo:Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Merck: Research Funding; Juno: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Amphivena: Research Funding. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder.

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CD3xCD19 Dart Treatment Is Efficient in Venetoclax Resistant CLL and Reverses T Cell Dysfunction

Blood ◽

10.1182/blood-2019-123350 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3043-3043

Author(s):

Anne W. J. Martens ◽

Susanne R. Janssen ◽

Ingrid A.M. Derks ◽

Sanne Tonino ◽

Eric Eldering ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Killing ◽

Advisory Committees ◽

Cell Dysfunction ◽

T Cell Dysfunction

Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been described. T cell mediated lysis is another tool currently exploited in hematologic malignancies. In contrast to acute lymphoblastic leukemia (ALL) however, efficacy of autologous based T cell therapy, such as CAR T cells, in CLL has been low. This is linked to a CLL mediated acquired T cell dysfunction. Bispecific T cell engagers targeting CD19 are successfully applied in ALL, but whether it overcomes the acquired T cell dysfunction in CLL is unknown. We therefore tested efficacy of a CD3xCD19 Dual Affinity Re-Targeting molecule (DART) in CLL. Since it has been observed that bispecific antibodies can overcome deficient synapse formation in CLL (Robinson et al, 2018) and based on our assumption that T cell mediated lysis differs from venetoclax-mediated killing, we hypothesized that usage of a CD3xCD19 DART in CLL overcomes T cell dysfunction and will be effective against venetoclax resistant CLL. Methods - Co-culture of CLL derived or aged-matched healthy donor (HD) CD4+ and/or CD8+ T cells with (CD40 activated) primary CLL or CD19+ cell lines JeKo-1 or Ramos in presence of CD3xCD19 (JNJ-64052781), CD3xFITC, anti-CD3/28 antibodies was performed. R esults - JeKo-1 cells were highly sensitive to CD3xCD19 mediated HD T cell killing with close to 70% of lysis in a concentration of 10ng/mL using an E:T ratio of 4:1. In the same conditions, primary CLL cells proved sensitive for CD3xCD19 mediated HD T cell killing with 50% of lysis. Killing was observed irrespective of IGHV mutation or chemorefractory status. We next compared HD with CLL-derived T cells by measuring activation levels between direct TCR (anti-CD3/CD28) and CD3xCD19 stimulation. As described, TCR stimulation resulted in impaired CLL CD4+ and CD8+ T cell activation and proliferation when compared to HD. In contrast, treatment of CLL derived PBMCs with CD3xCD19 did not resulted in dysfunctional CLL-derived T cell responses (Fig 1A-C). Consistently, co-culture of CLL derived CD4+, CD8+ or a combination with either JeKo-1 or allogeneic CLL cells in the presence of CD3xCD19 resulted in significant cytotoxicity (Fig. 1D). In the allogeneic setting, cytotoxic capacity of CD4+ T cells was similar to their CD8+ counterparts. When targeting autologous CLL, a benefit was observed when both CD4+ and CD8+ T cells were present (Fig. 1D). We then studied whether venetoclax resistant CLL cells could be targeted by CD3xCD19 mediated T cell killing. Bcl-2 overexpressing Ramos were equally lysed in presence of the CD3xCD19 DART as their wildtype counterpart, indicating that Bcl-2 expression does not inhibit CD3xCD19 mediated cell death. Following CLL cell stimulation by CD40 ligation, anti-apoptotic Bcl-XL, Bfl-1 and Mcl-1 are highly induced (Thijssen et al., 2015) resulting in venetoclax resistance (Fig 1E). Nevertheless, CD40L stimulated CLL cells were as efficiently lysed upon CD3xCD19 treatment as unstimulated CLL. (Fig 1F). This indicates that an augmented apoptotic threshold does not impact efficacy of CD3xCD19. Further examination of the mechanism of CD3xCD19 mediated killing showed that lysis depended on granzymes, as blocking granule exocytosis prevented cell death. Independence of the mitochondrial apoptotic pathway was shown by equal sensitivity to CD3xCD19 mediated T cell lysis comparing BAX/BAK knockout Jeko-1 cells to the parental cell line. Also, caspase blockage did not inhibit cell death, pointing to apoptosis independent killing. In concordance, PARP cleavage could only be detected when caspase activity was not blocked. Conclusion - This is the first report describing reversal of CLL mediated T cell dysfunction by applying a CD3xCD19 DART. Furthermore, it shows that venetoclax resistant CLL can still be efficiently targeted by T cells, in a non-apoptotic fashion. These results imply that T cell mediated therapy could be used alongside venetoclax. Figure 1 Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding. van der Windt:Genmab: Employment. Kater:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

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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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Chronic Lymphocytic Leukemia (CLL) Cells Are Susceptible to γδ-T Cell Mediated Killing, Provided CLL-Derived γδ-T Cell Dysfunction Can be Reversed

Blood ◽

10.1182/blood.v126.23.2914.2914 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2914-2914 ◽

Cited By ~ 1

Author(s):

Iris de Weerdt ◽

Sanne Terpstra ◽

Tom Hofland ◽

Roeland Lameris ◽

Renee C.G. de Bruin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cytokine Production ◽

Ex Vivo ◽

Cell Dysfunction ◽

Cd107a Expression ◽

T Cell Dysfunction ◽

Vγ9vδ2 T Cells ◽

Daudi Cells

Abstract Background: Although T cell immunotherapy is considered a promising therapeutic approach in B cell malignancies, autologous T cell based therapy proved to be far less effective in CLL than in more aggressive B cell malignancies. This has been attributed to an acquired state of T cell dysfunction. Disturbances in conventional (αβ-)T cells include expansion of CD4+ and CD8+ T cells, increased expression of exhaustion markers and impaired cytotoxicity and cytokine production. Vγ9Vδ2-T cells are a conserved subset of cytotoxic T lymphocytes with potent antitumor activity, due to recognition of phosphoantigen-induced changes in CD277 in tumor cells. Aminobisphosphonate (ABP) treatment leads to intracellular accumulation of phosphoantigens and increased Vγ9Vδ2 antitumor responses. Vγ9Vδ2-T cells have been shown to effectively kill malignant B cell lines in vitro. Moreover, in clinical trials Vγ9Vδ2-T cells have been shown to recognize and kill B cell lymphomas. Whether Vγ9Vδ2-T cells could be exploited for CLL immunotherapy has not yet been explored. The aim of this study is to investigate the phenotype and function of Vγ9Vδ2-T cells in CLL patients, in order to determine whether Vγ9Vδ2-T cells can effectively kill CLL cells. Results: Frequencies of Vγ9Vδ2-T cells do not differ between untreated CLL patients (n=46) and age-matched healthy controls (HC) (n=20) as assessed by flow cytometry. Vγ9Vδ2-T cell subpopulations are skewed towards effector type (CD27- CD45RA-) in CLL patients, while numbers of naïve (CD27+ CD45RA+) Vγ9Vδ2-T cells are decreased. Expression of exhaustion markers PD-1 and BTLA is comparable between CLL and HC, as is expression of CD16, mediating antibody-dependent cellular cytotoxicity. Next, we compared the functionality of Vγ9Vδ2-T cells from CLL patients and HC. We first examined cytokine production and CD107a expression, a marker of degranulation. Production of TNFα and IFNγ upon PMA/ionomycin stimulation was significantly diminished in CLL Vγ9Vδ2-T cells as compared to HC Vγ9Vδ2-T cells. Similarly, CD107a expression was significantly reduced. Overnight coculture with primary CLL cells or the Vγ9Vδ2-T cell sensitive Daudi lymphoma cell line also induced expression of TNFα, IFNγ and CD107a. However, upon co-culture, HC Vγ9Vδ2-T cells expressed significantly more TNFα, IFNγ and CD107 than CLL Vγ9Vδ2-T cells. Subsequently, we compared cytotoxicity of Vγ9Vδ2-T cells towards Daudi cells. HC-derived Vγ9Vδ2-T cells killed Daudi cells 3-4 times more effectively at 1:5 and 1:2.5 effector:target ratios. Although ABP pretreatment of Daudi cells increased both CLL-derived and HC-derived Vγ9Vδ2-mediated killing, differences between CLL and HC could not be overcome. We then looked at Vγ9Vδ2-T cell cytotoxicity towards CLL cells. Vγ9Vδ2-T cells from HCs effectively recognized and killed primary CLL cells, irrespective of ABP pretreatment. CLL-derived Vγ9Vδ2-T cells killed allogeneic CLL cells significantly less efficiently. Finally, we investigated whether the Vγ9Vδ2-T cell dysfunction in CLL patients was reversible upon ex vivo activation without the presence of leukemic B cells. Purified Vγ9Vδ2-T cells were cocultured with mature monocytic-derived dendritic cells in the presence of ABP for 8 days. Following these culture conditions, no difference was observed in production of TNFα, IFNγ and IL-4 upon PMA/ionomycin stimulation between HC- and CLL-derived activated Vγ9Vδ2-T cells. Likewise, there was no difference in CD107a expression. The activated Vγ9Vδ2-T cells of HCs and CLL patients were equally effective at killing Daudi cells. Conclusion: Vγ9Vδ2-T cells are capable of recognizing and killing CLL cells. Yet, CLL-derived Vγ9Vδ2-T cells are functionally impaired in terms of cytokine production and cytotoxic capacity in comparison to age-matched HCs. Functional impairments of Vγ9Vδ2-T cells are reversible upon ex vivo activation. If dysfunction can be overcome effectively, the antileukemic properties of autologous Vγ9Vδ2-T cells can be efficiently employed. Disclosures No relevant conflicts of interest to declare.

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TOX defines the degree of CD8+ T cell dysfunction in distinct phases of chronic HBV infection

Gut ◽

10.1136/gutjnl-2020-322404 ◽

2020 ◽

pp. gutjnl-2020-322404

Author(s):

Kathrin Heim ◽

Benedikt Binder ◽

Sagar ◽

Dominik Wieland ◽

Nina Hensel ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Hbv Infection ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Cell Dysfunction ◽

Chronic Hepatitis B Virus ◽

T Cell Dysfunction

ObjectiveChronic hepatitis B virus (HBV) infection is characterised by HBV-specific CD8+ T cell dysfunction that has been linked to Tcell exhaustion, a distinct differentiation programme associated with persisting antigen recognition. Recently, Thymocyte Selection-Associated High Mobility Group Box (TOX) was identified as master regulator of CD8+ T cell exhaustion. Here, we addressed the role of TOX in HBV-specific CD8+ T cell dysfunction associated with different clinical phases of infection.DesignWe investigated TOX expression in HBV-specific CD8+ T cells from 53 HLA-A*01:01, HLA-A*11:01 and HLA-A*02:01 positive patients from different HBV infection phases and compared it to hepatitis C virus (HCV)-specific, cytomegalovirus (CMV)-specific, Epstein-Barr virus (EBV)-specific and influenza virus (FLU)-specific CD8+ T cells. Phenotypic and functional analyses of virus-specific CD8+ T cells were performed after peptide-loaded tetramer-enrichment and peptide-specific expansion.ResultsOur results show that TOX expression in HBV-specific CD8+ T cells is linked to chronic antigen stimulation, correlates with viral load and is associated with phenotypic and functional characteristics of T-cell exhaustion. In contrast, similar TOX expression in EBV-specific and CMV-specific CD8+ T cells is not linked to T-cell dysfunction suggesting different underlying programmes. TOX expression in HBV-specific CD8+ T cells is also affected by targeted antigens, for example, core versus polymerase. In HBV-specific CD8+ T cells, TOX expression is maintained after spontaneous or therapy-mediated viral control in chronic but not self-limiting acute HBV infection indicating a permanent molecular imprint after chronic but not temporary stimulation.ConclusionOur data highlight TOX as biomarker specific for dysfunctional virus-specific CD8+ T cells in the context of an actively persisting infection.

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