T-cell Exhaustion Markers Research Articles

Evaluation of Immune Infiltrates in Ovarian Endometriosis and Endometriosis-Associated Ovarian Cancer: Relationship with Histological and Clinical Features.

the association between ovarian endometriosis (OE) and endometriosis-associated ovarian cancer (EAOC) is extensively documented, and misfunction of the immune system might be involved. The primary objective of this study was to identify and compare the spatial distribution of tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs) in OE and EAOC. Secondary objectives included the analysis of the relationship between immunosuppressive populations and T-cell exhaustion markers in both groups. TILs (CD3, CD4, and CD8) and macrophages (CD163) were assessed by immunochemistry. Exhaustion markers (PD-1, TIM3, CD39, and FOXP3) and their relationship with tumour-associated macrophages (CD163) were assessed by immunofluorescence on paraffin-embedded samples from n = 43 OE and n = 54 EAOC patients. we observed a predominantly intraepithelial CD3+ distribution in OE but both an intraepithelial and stromal pattern in EAOC (p < 0.001). TILs were more abundant in OE (p < 0.001), but higher TILs significantly correlated with a longer overall survival and disease-free survival in EAOC (p < 0.05). CD39 and FOXP3 significantly correlated with each other and CD163 (p < 0.05) at the epithelial level in moderate/intense CD4 EAOC, whereas in moderate/intense CD8+, PD-1+ and TIM3+ significantly correlated (p = 0.009). Finally, T-cell exhaustion markers FOXP3-CD39 were decreased and PD-1-TIM3 were significantly increased in EAOC (p < 0.05). the dysregulation of TILs, TAMs, and T-cell exhaustion might play a role in the malignization of OE to EAOC.

Intratumoral Abundance of M2-Macrophages is Associated With Unfavorable Prognosis and Markers of T-Cell Exhaustion in Small Cell Lung Cancer Patients

Small cell lung cancer (SCLC) accounts for about 10% to 15% of lung cancer cases. Unlike non-SCLC, therapy options for SCLC are limited, reflected by a 5-year survival rate of about 7%. At the same time, the rise of immunotherapeutic approaches in cancer therapy has rationalized to account for inflammatory phenotypes in tumors. However, the composition of the inflammatory microenvironment in human SCLC is poorly understood to date. In our study, we used in-depth image analysis of virtual whole-slide-images of 45 SCLC tumors and evaluated different markers of M2-macrophages (CD163 and CD204) together with global immunologic markers (CD4, CD8, CD68, CD38, FOXP3, and CD20) and characterized their abundance intratumorally using quantitative image analysis, combined with a deep-learning model for tumor segmentation. In addition, independent scoring, blinded to the results of the computational analysis, was performed by an expert pathologist (A.Q.) of both CD163/CD204 and PD-L1. To this end, we evaluated the prognostic relevance of the abundance of these cell types to overall survival. Given a 2-tier threshold of the median of the M2 marker CD163 within the study population, there was a 12-month overall survival rate of 22% (95% CI, 10%-47%) for patients with high CD163 abundance and 41% (95% CI, 25%-68%) for patients with low CD163 counts. Patients with increased CD163 had a median overall survival of 3 months compared to 8.34 months for patients with decreased CD163 counts (P = .039), which could be confirmed by an expert pathologist (A.Q., P = .018). By analyzing cases with increased CD163 cell infiltrates, a trend for higher FOXP3 counts and PD-L1 positive cells, together with increased CD8 T-cell infiltrates, was observed, which could be confirmed using an independent cohort at the transcriptional level. Together, we showed that markers of M2 were associated with unfavorable outcome in our study cohort.

Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with 89Zirconium oxine for PET imaging

BackgroundChimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products.MethodsTo determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89Zirconium-oxine (89Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells.ResultsWe observed that radiolabeling of CAR-T cells with 89Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells.ConclusionsImportantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89Zr-oxine retain critical product attributes and suggest 89Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

Biomarker analysis of phase II pembrolizumab maintenance treatment after chemotherapy response in patients with HER2-negative inflammatory breast cancer and triple-negative breast cancer.

1105 Background: Accumulating toxicities make indefinite chemotherapy unfeasible for many patients with metastatic/recurrent HER2-negative inflammatory breast cancer (IBC) or triple-negative breast cancer (TNBC). We conducted a single-arm phase II trial of pembrolizumab monotherapy to determine the efficacy of maintenance immunotherapy in patients with metastatic HER2-negative aggressive breast cancer and to identify biomarkers correlated with disease control. Methods: The study enrolled patients who had a complete response, partial response, or stable disease after a minimum of 3 cycles of chemotherapy for metastatic TNBC/IBC regardless of PD-L1 expression between 2015 and 2022. Pembrolizumab was administered at 200 mg every 3 weeks until disease progression, intolerable toxicity, or 2 years of exposure to the drug. The primary endpoint was the 4-month disease control rate (DCR), and the secondary endpoint was progression-free survival (PFS). Correlative studies were performed to determine associations between clinical response, and immune profiling consisting of T-cell clonality, immunophenotyping, and dendritic cell (DC) function with blood collected at baseline and after 2 cycles of pembrolizumab treatment. Results: Of 43 treated patients, 11 had metastatic IBC and 32 had TNBC. The median number of prior lines of chemotherapy for metastatic disease was 1. During a median follow-up of 11.4 months, 33 patients had progressive disease and 25 were deceased. The 4-month DCR was 58.1% (95% CI: 43.4%-72.9%) and the median PFS was 4.8 months (95% CI: 3.0-7.1 months). The median PFS of the metastatic IBC group (2.2 months) and TNBC group (4.8 months) did not differ significantly ( p = .12). Thirty-one patients discontinued treatment due to progressive disease rather than toxicity. Observed toxicities were consistent with the known profile of pembrolizumab. Correlative blood studies showed availability of PD-1 on CD8 T cells decreased on therapy. T-cell exhaustion markers 2B4, CTLA4, and TIM3 decreased in patients who had therapeutic benefit, while LAG3 increased in patients without benefit. During therapy, ex vivo cytokine synthesis (IL-6/IL-12/TNFα) by DCs was superior in patients who benefited from therapy and increased from pretreatment samples. High T-cell clonality at baseline predicted response to immunotherapy (10.4 vs 3.6 months, p = .04). Additionally, patients exhibiting the greatest clinical benefit also had a significant increase in T-cell clonality over the course of therapy (20% vs 5.9% mean increase, p = .04). Conclusions: Increased T-cell clonality and DC activity, along with reductions in T-cell exhaustion, were associated with prolonged disease control following pembrolizumab maintenance therapy, achieving acceptable disease control with manageable toxicity. Clinical trial information: NCT02411656 .

Non-Canonical Activin A Signaling Stimulates Context-Dependent and Cellular-Specific Outcomes in CRC to Promote Tumor Cell Migration and Immune Tolerance.

We have shown that activin A (activin), a TGF-β superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4-/-) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4-/- mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-β-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.

Abstract 4060: Selective Neoantigens Peptide TILs (snapTILs): A next generation tumor infiltrating lymphocytes (TIL) therapy platform

Abstract Background: Immunotherapies using autologous tumor infiltrating lymphocytes (TILs) have become effective treatment modalities for several patients with solid tumors, yielding a response rate of around 49% in patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TILs from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of these administered TILs recognize tumor antigens and most are non-mutated public antigens. Moreover, expression of these antigens on normal cells can trigger central and peripheral tolerance mechanisms. Against this background, cancer neoantigens derived from private mutations represent an ideal class of cancer antigens to target because they are highly tumor specific by nature, therefore reducing the potential induction of central and peripheral tolerance. We recently developed a new technology to enrich TILs with predefined neoantigen specificities, which we termed Selective Neo Antigens Peptides TILs (snapTILs). Here we describe the efficacy studies of a melanoma and pancreatic cancer patient’s snapTILs done in autologous settings both ex vivo and in vivo. Methods: Our workflow consisted of a multistep process performed on a patient-specific basis. Briefly, mutations were called by whole-exome sequencing of tumor vs. normal DNA to generate a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay. Selected peptides were then incubated with TILs isolated from the patient’s tumor biopsy, resulting in TIL population enriched of T cells that recognize the neopeptides (snapTILs). After rapid expansion, snapTIL efficacy was tested by checking their ability to infiltrate in patient-derived tumor organoids, their activation status (IFNγ), cytotoxicity (Granzyme B), and T-cell exhaustion markers. Lastly, snapTILs in vivo efficacy was tested in human melanoma and pancreatic cancer xenograft models using hIL-2 transgenic mice. Results: Our initial data showed that the levels of infiltration of snapTILs into tumor organoids was significantly higher compared to IL2-stimulated TILs in both cancer models. Also, snapTILs recognized tumors with great specificity as they showed significantly less infiltration in organoids generated from generally uninvolved tissues from the same patients. Moreover, snapTILs were found to be more cytotoxic in nature than the IL2-stimulated TILs in terms of IFNγ and Granzyme B secretion after coculture with autologous tumor cells. Our in vivo data confirmed these observations by showing 70% tumor growth inhibition in highly immunogenic melanoma and 50% tumor growth restriction in a poorly immunogenic pancreatic tumor model. In conclusion, our approach proves the potential for personally tailored immunotherapies to yield more effective and safer cancer treatments using snapTILs. Citation Format: Tithi Ghosh Halder, Shelby Rheinschmidt, Sydney Adamson, Anna Larson, Taylor Bargenquast, Ryan Rodriguez del Villar, Serina Ng, Kate Gutowsky, Alexis Weston, Trason Thode, Mohan Kaadige, Erin Kelley, Jorge Soria-Bustos, Jorge Giron, John Altin, Raffaella Soldi, Sunil Sharma. Selective Neoantigens Peptide TILs (snapTILs): A next generation tumor infiltrating lymphocytes (TIL) therapy platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4060.

457 CD39 Expression in the Glioblastoma Microenvironment does not Affect T Cell Exhaustion or Survival

INTRODUCTION: Glioblastoma (GBM), the most common malignant brain tumor, is notable for its dysfunctional T-cells. The initial optimism of Immune checkpoint inhibitors has been tempered as these agents have not been successful in treating GBM. CD39 is the first enzyme in the breakdown of ATP to adenosine and is highly upregulated in GBM. CD39 is also thought to contribute to T-cell exhaustion. However, inhibiting the CD39 ATP breakdown pathway did not improve survival in mice, even with PD-1 inhibition, leading to uncertainty about whether non-enzymatic functions of CD39 give it a role in T-cell exhaustion in GBM. METHODS: We performed intracranial injections of SB28 and GL261 GBM cell lines into CD39 global nulls (KO) and C57BL/6J (WT) mice and measured survival. Another cohort of mice were similarly injected and sacrificed after 2 weeks. These tumors were analyzed for T-cell exhaustion markers using flow cytometry. RESULTS: There was no survival benefit seen in either cell line when compared to wild type mice. The immune cell profile was not found to be statistically different between KO and WT mice. Next, expression of known exhaustion markers such as PD-1, TIM3, and LAG3 on tumor infiltrating lymphocytes (TILs) were analyzed. Expression of TIM3 and 2B4 were found to be increased in CD39KO TILs compared to WT (p &lt; 0.01). Additionally, numerous exhaustion markers such as LAG3, TIGIT, and CTLA4 were decreased on TILs compared to peripheral blood T-cells in tumor-bearing mice (p &lt; 0.01). CONCLUSIONS: These results suggest that CD39 lacks a direct impact on GBM outcomes but may affect the expression of other exhaustion markers. Further studies will continue to investigate the interaction between immune checkpoint molecules in GBM.

Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the QUAZAR AML-001 trial.

Oral azacitidine (Oral-AZA) maintenance therapy improved relapse-free (RFS) and overall survival (OS) significantly versus placebo for AML patients in remission after intensive chemotherapy (IC) in the phase 3 QUAZAR AML-001 study. Immune profiling was performed on the bone marrow (BM) at remission and on-treatment in a subset of patients with the aim of identifying prognostic immune features and evaluating associations of on-treatment immune effects by Oral-AZA with clinical outcomes. Post-IC, increased levels of lymphocytes, monocytes, T cells and CD34 + CD117+ BM cells were prognostically favourable for RFS. CD3+ T-cell counts were significantly prognostic for RFS in both treatment arms. At baseline, high expression of the PD-L1 checkpoint marker was identified on a subset of CD34 + CD117+ BM cells; many of which were PD-L2+. High co-expression of T-cell exhaustion markers PD-1 and TIM-3 was associated with inferior outcomes. Oral-AZA augmented T-cell numbers during early treatment, increased CD4+:CD8+ ratios and reversed T-cell exhaustion. Unsupervised clustering analysis identified two patient subsets defined by T-cell content and expression of T-cell exhaustion markers that were enriched for MRD negativity. These results indicate that Oral-AZA modulates T-cell activity in the maintenance setting of AML, and these immune-mediated responses are associated with clinical outcomes.

Exhausted intratumoral Vδ2− γδ T cells in human kidney cancer retain effector function

Gamma delta (γδ) T cells reside within human tissues including tumors, but their function in mediating antitumor responses to immune checkpoint inhibition is unknown. Here we show that kidney cancers are infiltrated by Vδ2− γδ T cells, with equivalent representation of Vδ1+ and Vδ1− cells, that are distinct from γδ T cells found in normal human tissues. These tumor-resident Vδ2− T cells can express the transcriptional program of exhausted αβ CD8+ T cells as well as canonical markers of terminal T-cell exhaustion including PD-1, TIGIT and TIM-3. Although Vδ2− γδ T cells have reduced IL-2 production, they retain expression of cytolytic effector molecules and co-stimulatory receptors such as 4-1BB. Exhausted Vδ2− γδ T cells are composed of three distinct populations that lack TCF7, are clonally expanded and express cytotoxic molecules and multiple Vδ2− T-cell receptors. Human tumor-derived Vδ2− γδ T cells maintain cytotoxic function and pro-inflammatory cytokine secretion in vitro. The transcriptional program of Vδ2− T cells in pretreatment tumor biopsies was used to predict subsequent clinical responses to PD-1 blockade in patients with cancer. Thus, Vδ2− γδ T cells within the tumor microenvironment can contribute to antitumor efficacy.

Inflammation burden score in multidrug-resistant HIV-1 infection

Four-class drug-resistant (4DR) people living with HIV (PLWH) are a fragile population with a high burden of disease. No data on their inflammation and T-cell exhaustion markers are currently available. Inflammation, immune activation and microbial translocation biomarkers were measured through ELISA in 30 4DR-PLWH with HIV-1 RNA ≥50 copies/mL, 30 non-viremic 4DR-PLWH and 20 non-viremic non-4DR-PLWH. Groups were matched by age, gender and smoking habit. T-cell activation and exhaustion markers were assessed by flow cytometry in 4DR-PLWH. An inflammation burden score (IBS) was calculated from soluble marker levels and associated factors were estimated through multivariate regression. The highest plasma biomarker concentrations were observed in viremic 4DR-PLWH, the lowest ones in non-4DR-PLWH. Endotoxin core immunoglobulin G showed an opposite trend. Among 4DR-PLWH, CD38/HLA-DR and PD-1 were more expressed on CD4+ (p=0.019 and 0.034, respectively) and CD8+ (p=0.002 and 0.032, respectively) cells of viremic compared to non-viremic subjects. An increased IBS was significantly associated with 4DR condition, higher values of viral load and a previous cancer diagnosis. Multidrug-resistant HIV infection is associated with a higher IBS, even when viremia is undetectable. Therapeutic approaches aimed to reduce inflammation and T-cell exhaustion in 4DR-PLWH need to be investigated.

CAR-tropic extracellular vesicles carry tumor-associated antigens and modulate CAR T cell functionality

Tumor-derived extracellular vesicles (EVs) are active contributors in metastasis and immunosuppression in tumor microenvironment. At least some of the EVs carry tumor surface molecules such as tumor-associated antigens (TAAs) and/or checkpoint inhibitors, and potentially could interact with T cells or CAR T cells. Upon contact with T cells, EVs could alter their phenotype and functions by triggering signaling through TCR or CAR reprogramming them to escape immune response. We hypothesize that EVs that possess TAA on the surface will probably interact with CAR T cells which can recognize and bind corresponding TAA. This interaction between EVs and CAR T cells may change the outcome of CAR T-based cancer immunotherapy since it should affect CAR T cells. Also, EVs could serve as adjuvants and antigenic components of antitumor vaccines. Herein, we isolated EVs from B cell precursor leukemia cell line (pre-B ALL) Nalm-6 and demonstrated that recognition and binding of CD19+EVs with CD19-CAR T cells strongly depends on the presence of CD19 antigen. CD19+EVs induce secretion of pro-inflammatory cytokines (IL-2 and IFN-y) and upregulated transcription of activation-related genes (IFNG, IFNGR1, FASLG, IL2) in CD19-CAR T cells. Tumor necrosis factor receptor superfamily (TNFRSF4 and TNFRSF9) and T-cell exhaustion markers (CTLA4, LAG3, TIM3 and PDCD1LG2) were also upregulated in CD19-CAR T cells after incubation with CD19+EVs. Long-term cultivation of CD19+ or PD-L1+EVs with CD19-CAR T cells led to increased terminal differentiation and functional exhaustion according to elevated expression of PD-1, TIGIT, CD57. In summary, our results suggest that chronic exposure of CD19-CAR T cells to CD19+EVs mediates activation and systemic exhaustion in antigen-specific manner, and this negative effect is accompanied by the impaired cytotoxic activity in vitro.

Combinatorial Effects of BH3 Mimetics and CAR T Cells Targeting T-Cell Lymphomas

T-cell lymphomas (TCL) represent a significant unmet need for improved therapeutic strategies. Recent advances in disease modeling and molecular characterization have greatly increased the potential for advancement. We previously showed that a subset of TCL cell lines, patient-derived xenografts (PDXs), and primary samples express high and uniform levels of the tetraspanin CD37 (PMID: 30089630). Selective expression on malignant T cells distinguishes CD37 from other CAR T cell targets under development against TCL and led TCL inclusion in our phase 1 clinical trial of autologous CD37-directed CAR T cells (CAR-37; NCT04136275). CAR-37 T cells specifically lysed CD37+ TCL cell lines in vitro. In an aggressive T cell prolymphocytic leukemia PDX model, we observed >100-fold reduction in disease burden in vivo with CAR-37 T cells, but all mice eventually relapsed. We transplanted the relapsed tumor cells and re-treated with CAR-37 T cells. All recipients experienced a second response, with relapse in only a subset. Thus, a third dose of CAR-37 T cells was administered, but only a subset of mice underwent a third response. At the time of relapse/progression, CD37 expression was detected on tumor cells from each mouse. In addition, a small percentage of CAR-37 T cells were present in the tumor. Based on these results, we sought to evaluate combinatorial strategies that potentiate the tumoricidal activity of CAR-37 T cells and increase the rate of complete disease eradication. CAR T cells kill primarily through driving the intrinsic pathway of apoptosis. We previously showed that BH3 mimetics of BCL2, BCL-xL, and MCL1 can selectively induce apoptosis in TCLs based on predicted BH3 protein dependencies (PMID: 30498064). Thus, to define novel therapeutic combinations with CAR-37 T cells, we performed BH3 profiling on CD37-expressing TCL cell lines. Corresponding to the predicted dependencies via BH3 profiling, FEPD and SMZ1 were most sensitive to the MCL1 inhibitor AZD5991, whereas HUT78 was most sensitive to the BCL-2/BCL-xL inhibitor navitoclax. We observed concordant sensitivity patterns to the BH3 mimetics by viability/apoptosis assay and decreased active mitochondrial membrane potential. Importantly, CD37 antigen density was unaffected by exposure to BH3 mimetics. To evaluate the feasibility of combining BH3 mimetics and CAR T cells, we determined the impact of BH3 mimetics on CAR-37 T cell phenotype. First, we did not observe a reduction in CAR-37 T cell viability when cultured in vitro in the presence of TCL target cells and either venetoclax 0.5μM, navitoclax 0.5μM, or AZD5991 0.1μM for 48 hours. The same culture conditions also did not affect the differentiation or subset distribution of CAR-37 T cells. Nearly 60% of the CAR-37 T cells had an effector memory phenotype, whereas approximately 20% had a central memory phenotype and the remaining were either effector memory T cells re-expressing CD45RA or naïve. In addition, cytokine production (GMCSF and IFN-γ) and expression of T-cell exhaustion markers (PD-1, TIM3, and LAG3) was not affected by co-culture with BH3 mimetics. Since BH3 mimetics did not alter CAR-37 T cell activity, we evaluated the cytotoxicity effect of their combination against target TCL cell lines. Using a luminescence based killing assay and xCELLigence RTCA (ACEA), we found that all 3 BH3 mimetics in combination with CAR-37 T cells resulted in greater specific lysis of FEPD tumor cells. The most significant increase occurred in combination with AZD5991, which corresponds to the predicted MCL1 dependence of FEPD cells. Next, we evaluated the combination of CAR-37 T cells and AZD5991 in NSG mice engrafted with FEPD. CAR-37 T cells were administered upon a total flux (photons/s) ≥106 and the combination cohort subsequently received AZD5991 (100 mg/kg) on days 1 and 2, 8 and 9, and 15 and 16 post-CAR T cells. We observed significantly reduced tumor burden and prolonged survival in the combination cohort (median 42 days) compared to CAR-37 T cell (median 18 days) or AZD5991 (median 31 days) alone. Together, our findings demonstrate that BH3 mimetics with predicted activity against individual TCLs can enhance the cytotoxic activity of CAR-37 T cells. These results could be used to nominate and/or deprioritize future combination strategies both for TCL and for other patients being treated with effector T cells for a range of hematologic malignancies. AZD5991 was kindly provided by AstraZeneca.

Nivolumab-Based Salvage Therapy to Restore T Cell Fitness in Penta-Refractory Multiple Myeloma with Relapse to Anti-BCMA CAR T Cell Therapy

Background: Idecabtagene Vicleucel (ide-cel) is the first FDA-approved BCMA-directed CAR T cell therapy for the treatment of relapsed/refractory multiple myeloma (RRMM). Despite remarkable activity of 85% objective response rate and a progression-free survival (PFS) of 11.8 months reported in the CRB-401 trial (NCT02658929), most responses to ide-cel are not durable, and nearly all patients (pts) eventually relapse, even those achieving MRD-negative complete remission (CR). Factors associated with clinical relapse to ide-cel include BCMA loss, lack of CAR T persistence, T-cell exhaustion and PD-1/ PD-L1 impaired T-cell fitness. Here, we report clinical data and correlative findings for 4 pts who were salvaged with a nivolumab (anti-PD1)- based triplet therapy at the time of clinical relapse to first or second ide-cel infusion. Methods: Four pts with penta-refractory RRMM were treated as part of the CRB-401 phase 1 study as previously reported (Raje et al.N Engl J Med.2019;380:1726-37). All pts received nivolumab along with lenalidomide-dexamethasone (Nivo-Rd) or pomalidomide-dexamethasone (Nivo-Pd) at the time of relapse to ide-cel. CD3+ T cells (n=1,047) were isolated from the peripheral blood of 2 of these pts and full-length single-cell RNA sequencing (scRNA-seq) was conducted before and upon treatment with Nivo-Rd/-Pd. Response rates were assessed as per IMWG criteria. Results: The median age of pts was 48.5 years (range 37-56). Two out of 4 pts had cytogenetic high-risk MM and all pts were penta-refractory at the time of CRB-401 study inclusion. The median PFS and OS after first ide-cel infusion were 10.4 (range 3.0-13.8) months and 21.5 (range 16.0-34.6) months, respectively. Three pts received a second ide-cel infusion, whereof only 1 pt achieved a partial response (PR) at a PFS of 5.2 months. Two pts each were treated with Nivo-Rd and Nivo-Pd respectively at the time of progression to ide-cel. Responses to anti-PD1- based therapy varied markedly with 1 PR, 1 stable disease (SD) and 2 progressive disease (PD) as per IMWG criteria. The median PFS to nivolumab-based salvage therapy was 3.6 (range 0.4-8.1) months. Interestingly, pts MM1 and MM2 both had a CR to first ide-cel infusion (PFS 13.8 and 11.9 months, respectively), but while both pts did not respond to second CAR T cell infusion, the benefit from anti-PD1 treatment differed markedly in both pts. MM1 reached a PR and PFS of 105 days. In contrast, MM2 experienced immediate PD and succumbed to his disease 3.0 months later. Profiling of CD3+ T cells from MM1 and MM2 by scRNA-seq revealed rapid cellular state changes in response to PD1 inhibition. PAGODA2 clustering and t-Stochastic Neighbor Embedding (t-SNE) visualization revealed 3 distinct clusters, segregating to an individual pre-anti-PD1 cluster for MM1 and MM2 each and a shared post-anti-PD1 cluster comprising CD3+ T cells from both pts. The pre-anti-PD1 cluster for MM1 contained CD3+ T-cells with a memory T cell- like phenotype (IL7Rhigh, CD62Lhigh) and decreased expression of markers correlating with T-cell fitness (GNLY, PRF1, NKG7, GZMH,LY6C, KLRG1). This phenotype could be rescued, and T-cell fitness could be restored by Nivo-Pd treatment, potentially contributing to the clinical response observed in MM1. Opposingly, the pre-anti-PD1 cluster for MM2 showed intact T-cell fitness with low expression of T-cell exhaustion markers. No clinical benefit to Nivo-Rd was noted in this pt. Conclusions: Checkpoint inhibition and IMiD therapy can induce clinical responses at the time of relapse to first or second CAR T infusion. Efficacy however seems to depend on impaired T-cell fitness as premise for PD1 inhibition to re-induce clinical responses in selected pts.

Immuno(T)herapy for age-related diseases.

During the last decade, the stimulation of T-cell function by the blockage of immunosuppressive checkpoints has experienced an outstanding impact in the treatment of cancer. Development of the chimeric antigen receptor T-cell technology has also emerged as a powerful alternative for patients suffering from oncological processes, especially those affected by hematological neoplasms. Recent evidence suggest that the use of immunotherapy could be extended to non-oncological diseases and could be especially relevant for age-associated disorders, opening exciting therapeutic options for a wide range of diseases of the elderly. Here we comment on the emergence of T-cell-based immunotherapies as feasible approaches that could revolutionize the future of GeroScience.

T-cell dysfunction as a potential contributing factor in post-COVID-19 mucormycosis.

The second wave of COVID-19 in India was followed by large number of mucormycosis cases. Indiscriminate use of immunosuppressive drugs, underlying diseases such as diabetes, cancers, or autoimmune diseases was thought to be the cause. However, the mortality was not as high as that seen in non-COVID mucormycosis. To study the detailed characteristics of T-cells for evaluating the underlying differences in the T-cell immune dysfunction in post-COVID and non-COVID mucor patients. The study included histopathologically confirmed cases of mucor (13 post-COVID, 13 non-COVID) and 15 healthy individuals (HI). Expression of T-cell activation (CD44, HLADR, CD69, CD38) and exhaustion (CTLA, PD-1, LAG-3 and TIM-3) markers was evaluated by flow cytometry. All cases showed significant depletion of T-cells compared to HI. Both post-COVID and non-COVID groups showed increased activation and exhaustion as compared to HI. Non-COVID mucor group showed significant activation of CD4+ T cells for HLADR and CD38 (p=.025, p=.054) and marked T-cell exhaustion in form of expression of LAG-3 on both CD4+ T and CD8+ T cells in comparison with post-COVID patients (p =.011, p=.036). Additionally, co-expression of PD-1 & LAG-3 and LAG-3 & TIM-3 on CD8+ T cells was statistically significant in non-COVID mucor patients (p=.016, p=.027). Immunosuppression in non-COVID mucor showed pronounced exhaustion of T-cells in comparison to post-COVID mucor cases implicating T-cell immune dysfunction is much more severe in non-COVID mucor which are in a state of continuous activation followed by extreme exhaustion leading to poorer outcome.

Microfibrillar-associated protein 2 is a prognostic marker that correlates with the immune microenvironment in glioma.

Aims: microfibrillar-associated protein 2 (MFAP2), a component of the extracellular matrix, plays key roles in regulating growth factor signal transduction and various malignant tumors. However, the clinicopathological features of microfibrillar-associated protein 2 in gliomas have not been elucidated to date. Methods: TCGA and CGGA databases were used to study the expression of microfibrillar-associated protein 2 in glioma and its relationship with clinicopathological features of patients with glioma. Western blotting was performed to detect the expression of microfibrillar-associated protein 2 protein in tissue samples from glioma patients. Gene set enrichment analysis (GSEA) was applied to detect biological processes and signal pathways related to microfibrillar-associated protein 2. Single-sample gene set enrichment analysis, TIMER 2.0, and TISIDB databases were used to evaluate the role of microfibrillar-associated protein 2 in tumor immune characteristics. The prognostic role of microfibrillar-associated protein 2 in glioma was analyzed using the Kaplan-Meier method and Cox regression. Survival data were used to establish a nomogram prediction model. Results: microfibrillar-associated protein 2 expression was significantly elevated in gliomas. receiver operating characteristic analysis revealed good discrimination of microfibrillar-associated protein 2 between glioma and normal tissues. High expression of microfibrillar-associated protein 2 was associated with malignant phenotypes, such as histological type. Based on gene set enrichment analysis, we identified pathways associated with high microfibrillar-associated protein 2 expression. High microfibrillar-associated protein 2 expression was related to the infiltration of tumor immune cells, including Th2 cells and macrophages, and correlated with key markers of T-cell exhaustion. Based on the TISIDB database, microfibrillar-associated protein 2 was observed to be associated with chemokines, chemokine receptors, and multiple immunoinhibitors in glioma. Kaplan–Meier survival analyses revealed that high microfibrillar-associated protein 2 expression predicted poor overall survival, DSS, and PFS in patients with glioma. By combining microfibrillar-associated protein 2 and other prognostic factors, a nomogram prognostic prediction model was constructed, which demonstrated an ideal prediction effect. Conclusion: microfibrillar-associated protein 2 is a potential prognostic marker that plays a key role in glioma development given its association with malignant phenotypes, cancer-related pathways and tumor immunity.

Integrated TCR repertoire analysis and single-cell transcriptomic profiling of tumor-infiltrating T cells in renal cell carcinoma identifies shared and tumor-restricted expanded clones with unique phenotypes.

Objective responses of metastatic renal cell carcinoma (RCC) associated with systemic immunotherapies suggest the potential for T-cell-mediated tumor clearance. Recent analyses associate clonally expanded T cells present in the tumor at diagnosis with responses to immune checkpoint inhibitors (ICIs). To identify and further characterize tumor-associated, clonally expanded T cells, we characterized the density, spatial distribution, T-cell receptor (TCR) repertoire, and transcriptome of tumor-infiltrating T cells from 14 renal tumors at the time of resection and compared them with T cells in peripheral blood and normal adjacent kidney. Multiplex immunohistochemistry revealed that T-cell density was higher in clear cell RCC (ccRCC) than in other renal tumor histologies with spatially nonuniform T-cell hotspots and exclusion zones. TCR repertoire analysis also revealed increased clonal expansion in ccRCC tumors compared with non-clear cell histologies or normal tissues. Expanded T-cell clones were most frequently CD8+ with some detectable in peripheral blood or normal kidney and others found exclusively within the tumor. Divergent expression profiles for chemokine receptors and ligands and the Ki67 proliferation marker distinguished tumor-restricted T-cell clones from those also present in blood suggesting a distinct phenotype for subsets of clonally expanded T cells that also differed for upregulated markers of T-cell activation and exhaustion. Thus, our single-cell level stratification of clonally expanded tumor infiltrating T-cell subpopulations provides a framework for further analysis. Future studies will address the spatial orientation of these clonal subsets within tumors and their association with treatment outcomes for ICIs or other therapeutic modalities.

P1.11-01 Cobolimab with Dostarlimab and Docetaxel in Patients with Advanced Non-small Cell Lung Cancer (NSCLC): COSTAR Lung

TIM-3 and PD-1 are markers of T-cell exhaustion co-expressed on tumor-infiltrating T cells (CD4+, CD8+) and antigen presenting cells in lung cancer. TIM-3 expression has also been associated with poor overall survival (OS) outcomes in NSCLC.

Immune checkpoint receptor VISTA on immune cells is associated with expression of T-cell exhaustion marker TOX and worse prognosis in renal cell carcinoma with venous tumor thrombus

PurposeThe study aimed to determine the expression of VISTA and TOX within venous tumor thrombus and primary clear cell renal cell carcinoma (ccRCC) and to assess their prognostic value.MethodsThe study enrolled 82 patients with ccRCC and coexisting venous tumor thrombus treated radically from 2012 to 2019 in two tertiary centers. Tissue microarrays were prepared and stained with respective antibodies. The expression of markers was assessed separately on tumor cells (TCs) and/or tumor-associated immune cells (TAICs).ResultsTOX expression was positively correlated with the percentage of VISTA-positive TAICs in venous thrombus (p = 0.011), but not in the primary tumor (p = 0.674). High TOX expression was associated with a higher percentage of PD-L1-positive TAICs in both compartments (p = 0.001, p = 0.011, respectively). Positive expression of VISTA on TAICs was associated with PD-L1 expression on TCs (p = 0.005) and TAICs (p = 0.004) in the primary tumor, and only with PD-L1 on TAICs in thrombus (p = 0.006). The presence of VISTA-positive TAICs in venous thrombus was significantly more common in females (p = 0.034), and positively correlated with metastases (p = 0.028), and tumor necrosis (p = 0.013). The cases with VISTA-positive TAICs in venous tumor thrombi had significantly shorter OS than VISTA-negative cases (p = 0.041).ConclusionFor the first time, we demonstrated the expression of VISTA- and TOX-positive TAICs in the venous tumor thrombus. We found the association between immune checkpoint receptors and T cell exhaustion markers in both tumor mass and venous thrombus. Finally, we demonstrated that abundance of VISTA-positive TAICs in venous tumor thrombus correlates with worse outcomes in ccRCC.

Intratumoral PD-1+CD8+ T cells associate poor clinical outcomes and adjuvant chemotherapeutic benefit in gastric cancer

BackgroundAlthough PD-1 has been reported to be a marker of T-cell exhaustion in several malignancies, the biological role of PD-1+CD8+ T cells in gastric cancer (GC) remains unclear. Herein, we aimed to investigate the role of PD-1+CD8+ T cells in the tumour microenvironment and its clinical significance in GC.DesignsThis study included 441 tumour microarray specimens and 60 Flow cytometry specimens of GC patients from Zhongshan Hospital, and 250 GC patients from the Asian Cancer Research Group.ResultsHere, we demonstrated that PD-1+CD8+ T cells functioned as an independent adverse prognosticator in GC. In addition, an abundance of intratumoral PD-1+CD8+ T cells indicated worse chemotherapeutic responsiveness to fluorouracil in Stage III GC patients. Mechanistically, PD-1+CD8+ T cell high infiltration indicated an exhausted phenotype of global CD8+ T cells in GC tissues, which was characterised by elevated immune checkpoint expression including CTLA-4 and TIM-3, whereas decreased expression of perforin. Furthermore, PD-1+CD8+ T cell high-infiltration patients with Stage III GC held elevated activity of several therapeutic signal pathways.ConclusionsOur study highlighted that PD-1+CD8+ T cell abundance predicts inferior prognosis in GC, and may serve as a novel predictive biomarker to guide therapeutic option.