scholarly journals 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 ◽  
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.

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

Blood ◽  
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.

scholarly journals 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

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.

A Failure to Start: Aborted Activation of CAR T Cells in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 681-681
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Meng Wang ◽  
Stefan M Lundh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
Low Levels

Introduction: Chronic Lymphocytic Leukemia (CLL) is a CD19+ B-cell malignancy that accounts for approximately 25% of adult leukemia diagnoses in the developed world. While conventional therapies have some efficacy, there are few curative therapeutic options and many patients ultimately progress to relapsed or refractory disease. CD19-targeting chimeric antigen receptor (CAR) T cell therapy has provided some hope, but induces complete remission in only 26% of patients. This suboptimal response rate is believed to be due to T cell dysfunction and immune-suppression by CLL cells, the mechanisms of which are poorly understood. Results: To understand the causes of CAR T cell dysfunction in CLL we investigated the defects that CLL cells induced in normal donor CD19-targeting CAR T cells. CAR T cells were repeatedly stimulated at 5-day intervals with either primary CLL cells from patients or a CD19-expressing control cell line (aAPC). Repeat stimulation of CAR T cells with aAPCs resulted in 5.36 ± .94 population doublings after three stimulations, whereas CLL cells only evoked 2.39 ± .92 population doublings. We performed phenotyping, proliferation analysis, and cytokine analysis of stimulated CAR T cells. CLL-stimulated T cells appeared un-activated, with low levels of PD-1, LAG3, and TIM3, low levels of cytokine production, and a high proportion of non-cycling cells as measured by Ki67 staining. We first hypothesized that CLL cells induce an altered epigenetic program that prevents effector function and is stabilized by successive stimulations. To test this, we stimulated CAR T cells with CLL cells or aAPCs as indicated in Fig. 1A. CLL-stimulated CAR T cells failed to proliferate or produce cytokines, but subsequent stimulation with aAPCs rescued these functions (Fig. 1B). Further, CLL-stimulated CAR T cells did not differentiate, suggesting that CLL cells do not induce stable defects but rather insufficiently activate CAR T cells (Fig. 1C). These cells also appeared un-activated as indicated by low levels of PD-1 and Ki67. We then used flow cytometry to assess expression of costimulatory and inhibitory molecules on the primary CLL samples. We found that the levels of co-stimulatory and adhesion molecules, namely CD80/CD86 and CD54/CD58 respectively were found at low frequencies, and where present were expressed at low levels. This suggested that one mechanism behind the lack of CAR T cell effector responses may be that a lack of co-stimulation prevents proper CAR T cell targeting of these cells. Towards this, we incubated CLL cells with a murine fibroblast line expressing CD40 ligand for 24 hours with IL-4 to activate the CLL cells. We found that this activation significantly increased expression of CD80, CD86, CD54, and CD58 on the CLL cells. We then used these cells to stimulate CAR T cells in our re-stimulation assay and found that CAR T cells were able to proliferate in response to these activated CLLs (Fig. 1D). In addition, CAR T cells stimulated with activated CLL cells formed more cell-to-cell conjugates than those stimulated with un-activated CLL cells. These data suggest not only that insufficient activation of CAR T cells may be responsible for the poor response rates to CAR T cell therapy in CLL, but also implicate a need for additional co-stimulation in this CAR T cell setting. Another contributing factor may be immune suppression by CLL cells. To determine if CLL cells are immune-suppressive, we used a co-culture assay to stimulate CAR T cells with aAPCs and CLL cells mixed at known ratios. Interestingly, all mixed cultures proliferated similarly, suggesting that CLL cells did not prevent T cell activation in the presence of a strong activation signal. We also found that CLL cells are responsive to IL-2, as addition of this cytokine to culture media prolongs survival of CLL cells out to 10 days depending on the dose. This suggests that CLL cells express a functional IL-2 receptor and may be taking up IL-2 from the culture media, further impairing T cell activation. In support of this, supplementing IL-2 into our CLL/CAR T cell co-cultures rescued T cell proliferative capacity. Taken together, these data suggest that T cell dysfunction in CLL is the result of insufficient activation rather than true functional defects. Disclosures June: Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; Incyte: Research Funding.

Multiple Inhibitory Receptors Are Expressed on Central Memory and Memory Stem T Cells Infiltrating the Bone Marrow of AML Patients Relapsing after Allo-HSCT

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4564-4564 ◽  
Author(s):  
Maddalena Noviello ◽  
Francesco Manfredi ◽  
Tommaso Perini ◽  
Giacomo Oliveira ◽  
Filippo Cortesi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Central Memory ◽  
Cd8 Cells ◽  
Effector Functions ◽  
Cell Dysfunction ◽  
T Cell Dysfunction ◽  
Cell Effector

Abstract Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p<0.01) and a preferential late differentiation profile (p<0.05) when compared to HD. A higher proportion of BM Tregs was documented at relapse (p<0.01), independently from the donor source. We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p<0.05) and HD (p<0.01). We then investigated the profile of each T-cell subset in our cohort. In the BM of HD the IR expression was confined to effector memory and effectors. While a similar IR distribution was observed in CR, at relapse, PD-1, 2B4 and Tim-3 were also upregulated in BM infiltrating central memory (p<0.01) and memory stem T cells (p<0.05). Interestingly, at relapse, leukemia expressed PD-L1 (9/9 cases) and Galectin-9 (6/9). The levels of Tim-3 on BM CD8 cells associates with that of Galectin-9 on autologous blasts (p<0.05), suggesting a preferential role for this immunomodulatory axis after HSCT. Based on phenotype similarities, the BH-SNE algorithm positioned HD samples separately from transplanted pts in bi-dimensional maps. 93 significant clusters were identified. Clusters associated with relapse after HLA-id (5) and after haplo (15) were composed of T cells expressing multiple IRs, while CR-specific clusters were diminished in IR fluorescence. To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p<0.05). In two patients, selected based on samples availability, we isolated and expanded by rapid expansion protocol (REP) T cells expressing one or more IRs (IR+) or no IR (IR-). Expansion rates were high and similar in IR+ and IR- T cells (mean fold increase 624 and 781, respectively at day 21). The degranulation ability measured ex-vivo in those patients (mean 4.4% on CD8 cells) was dramatically increased upon REP expansion (95% and 88.9% for IR+ and IR-, respectively). Similarly, the frequency of IFN-g producing CD8 cells increased in IR+ and IR- cells upon REP, indicating that the T-cell dysfunction observed at relapse can be efficiently reversed. We next challenged IR+ and IR- T cells against autologous blasts. Preliminary results suggest that IR+ T cells are enriched in leukemia specificity (elimination index of 66% and 44% in IR+ and IR- cells respectively at an E/T ratio of 100:1). Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

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

2008 ◽  
Vol 205 (12) ◽  
pp. 2763-2779 ◽  
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.

scholarly journals Acute Myeloid Leukemia (AML) Blasts Influence the Gene Expression Signature and Co-Signaling Receptor Expression of CD8+ T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1700-1700
Author(s):  
Hanna A. Knaus ◽  
Sofia Berglund ◽  
Hubert Hackl ◽  
Raúl Montiel-Esparza ◽  
Mark J. Levis ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Induction Chemotherapy ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Receptor Expression ◽  
Inhibitory Receptor ◽  
Canonical Pathways

Abstract Background: T cell dysfunction in AML remains poorly understood. Our previous studies of AML-associated T cell dysfunction (Knaus, ASH 2015) have focused on expression of multiple inhibitory receptors by T cells in AML patients. Transcriptional signatures, however, remain relatively unexplored, as does the role of Blast/T cell interactions on T cell function. Deciphering those could be crucial for integration of future immunotherapies into clinical practice. Therefore, we aimed to characterize CD8+ T cell gene expression signatures in newly diagnosed AML patients before and after treatment, and to decipher the effects of AML blasts on the expression of co-signaling molecules by CD8+ T cells in co-culture experiments. Methods: Serial peripheral blood (PB) samples (at diagnosis and at the recovery after induction chemotherapy) were collected. To study transcriptional signatures, RNA isolated from FACS-purified PB CD8+ T cells from 6 patients [3 responders (R) and 3 non-responders (NR)] and 4 healthy controls (HC) was analyzed with the Human Prime View Gene Expression Array (Affymetrix). The data were normalized and log transformed. Expression fold change (FC), p values and false discovery rate were determined. Enrichment of canonical pathways was determined using Ingenuity Pathway Analysis (IPA, QIAGEN). To study AML blast-T cell interactions, we FACS-purified T cells and primary AML blasts at diagnosis (n=13) and T cells from HC (n=12). T cells were cultured in vitro for 3 days in the presence or absence of blasts (T cell:blast ratio 1:10) and analyzed by flow cytometry. Results: The transcriptional profile of CD8+ T cells at AML diagnosis significantly differed from that of HC. Genes were selected based on >2 FC between patient and HC, and p< 0.01. We identified a total of 453 dysregulated genes, of which 237 were up- and 216 down-regulated. Upregulated genes included immune inhibitory receptors LILRB1, 2B4, KLRG1, CD160, the transcription factors EOMES, TBET, TIGIT and cytokines (granzyme-A/B/K). In contrast, co-stimulatory receptor genes were downregulated, including CD40LG, CD28, CD30LG and CD28H. Canonical pathways analysis with IPA revealed that the NFAT pathway (involved in T cell differentiation and self-tolerance) was highly upregulated, while co-stimulatory CD28, ICOS and OX40 signaling pathways were downregulated in CD8+ T cells at AML diagnosis. Next, we compared R to NR after induction chemotherapy. There were a total of 351 dysregulated genes; 108/243 genes were up-/down-regulated, respectively. R patients upregulated immune stimulatory receptor genes like ICOS, whereas the top expressed genes for NR patients included the co-inhibitory receptor TIM3; several members of the inhibitory LIR receptor family; LST1 (involved in inhibition of lymphocyte proliferation); TWEAK-APRIL (associated with T cell apoptosis); and CD39 (terminally exhausted CD8+ T cells). In line with these findings, IPA showed that the co-stimulatory ICOS and OX40 signaling pathways were enriched in R patients. In contrast, the NFAT pathway, which had been highly upregulated at diagnosis, remained enriched in NR, but not in R patients. Results were confirmed by qPCR. The culture assay showed that the presence of primary AML blasts significantly reduced the viability of both AML and HC T cells (p <0.005 in both cases). The presence of AML blasts also significantly decreased the frequency of primary AML T cells expressing co-stimulatory receptors 41BB, ICOS and OX40, while it increased the frequency of HC T cells expressing co-inhibitory receptor 2B4 and the senescence/exhaustion marker CD57 compared to their counterparts cultured without blasts. Conclusions: Our study provides insight into the genomic CD8+ T cell signatures of AML patients at diagnosis and following chemotherapy. At diagnosis, T cells overexpressed genes that negatively regulate T cell immune responses, while genes that positively regulate immune responses were downregulated. Interestingly, after induction chemotherapy these changes persisted in NR only. Additionally, a pattern of decreased viability and co-stimulatory receptor expression was seen after in vitro co-culture of T cells with AML blasts, whereas immune inhibitory receptor expression was increased. Our data suggests that the blasts themselves influence the T cell phenotype and genotype in AML patients and that remission is associated with reversion to HC pattern. Disclosures Levis: Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Millennium: Consultancy, Research Funding.

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

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.

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

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.

scholarly journals Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3714-3714
Author(s):  
Jaco A. C. Van Bruggen ◽  
Fleur Peters ◽  
Gaspard Cretenet ◽  
J. Joseph Melenhorst ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Cell Function ◽  
Cell Dysfunction ◽  
T Cell Function ◽  
Car T ◽  
T Cell Dysfunction

Abstract Introduction Success rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction. Results Dynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms. Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ). Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C). Conclusion These results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. Figure 1 Figure 1. Disclosures Kater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

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

Blood ◽  
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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