T-cell Exhaustion Markers Research Articles

First-in-human Intravesical Delivery of Pembrolizumab Identifies Immune Activation in Bladder Cancer Unresponsive to Bacillus Calmette-Guérin

BackgroundIntravenous immune checkpoint inhibition is an effective anticancer strategy for bacillus Calmette-Guérin (BCG)-unresponsive non–muscle-invasive bladder cancer (NMIBC) but may be associated with greater systemic toxicity compared with localized therapies. ObjectiveWe assessed the safety and antitumor activity of intravesical pembrolizumab combined with BCG. Design, setting, and participantsA 3 + 3 phase 1 trial of pembrolizumab + BCG was conducted in patients with BCG-unresponsive NMIBC (NCT02808143). InterventionPembrolizumab was given intravesically (1–5 mg/kg for 2 h) beginning 2 weeks prior to BCG induction until recurrence. Urine profiling during treatment and spatial transcriptomic profiling of pre- and post-treatment tumors were conducted to identify biomarkers that correlated with response. Outcome measurements and statistical analysisSafety and tolerability of immune checkpoint inhibition were assessed, and Kaplan-Meier survival analysis was performed. Results and limitationsNine patients completed therapy. Median follow-up was 35 months for five patients still alive at the end of the trial. The trial was closed due to the COVID-19 pandemic. Grade 1–2 urinary symptoms were common. The maximum tolerated dose was not reached; however, one dose-limiting toxicity was reported (grade 2 diarrhea) in the only patient who reached 52 weeks without recurrence. One death occurred from myasthenia gravis that was deemed potentially related to treatment. The 6-mo and 1-yr recurrence-free rates were 67% (95% confidence interval [CI]: 42–100%) and 22% (95% CI: 6.5–75%), respectively. Pembrolizumab was detected in the urine and not in blood. CD4+ T cells were significantly increased in the urine after treatment, and a transcriptomic analysis identified decreased expression of T-cell exhaustion markers in late recurrences. ConclusionsWe demonstrate that intravesical pembrolizumab is safe, feasible, and capable of eliciting strong immune responses in a clinical setting and should be investigated further. Patient summaryDirect application of pembrolizumab to the bladder is a promising alternative for non–muscle-invasive bladder cancer unresponsive to Bacillus Calmette-Guérin and should be investigated further.

Persistent T-cell exhaustion in relation to prolonged pulmonary pathology and death after severe COVID-19: Results from two Norwegian cohort studies.

T-cell activation is associated with an adverse outcome in COVID-19, but whether T-cell activation and exhaustion relate to persistent respiratory dysfunction and death is unknown. To investigate whether T-cell activation and exhaustion persist and are associated with prolonged respiratory dysfunction and death after hospitalization for COVID-19. Plasma and serum from two Norwegian cohorts of hospitalized patients with COVID-19 (n = 414) were analyzed for soluble (s) markers of T-cell activation (sCD25) and exhaustion (sTim-3) during hospitalization and follow-up. Both markers were strongly associated with acute respiratory failure, but only sTim-3 was independently associated with 60-day mortality. Levels of sTim-3 remained elevated 3 and 12 months after hospitalization and were associated with pulmonary radiological pathology after 3 months. Our findings suggest prolonged T-cell exhaustion is an important immunological sequela, potentially related to long-term outcomes after severe COVID-19.

TIMM13 as a prognostic biomarker and associated with immune infiltration in skin cutaneous melanoma (SKCM)

ObjectiveProviding protection against aggregation and guiding hydrophobic precursors through the mitochondria’s intermembrane space, this protein functions as a chaperone-like protein. SLC25A12 is imported by TIMM8 as a result of its interaction with TIMM13. In spite of this, it is still unknown how TIMM13 interacts with skin cutaneous melanoma (SKCM) and tumor-infiltrating lymphocytes (TILs).MethodsAberrant expression of TIMM13 in SKCM and its clinical outcome was evaluated with the help of multiple databases, including the Xiantao tool (https://www.xiantao.love/), HPA, and UALCAN. TISIDB and Tumor Immune Estimation Resources (TIMER) databases were applied to explore the association between TIMM13 and tumor infiltration immune cells. OS nomogram was constructed, and model performance was examined. Finally, TIMM13 protein expression was validated by immunohistochemistry (IHC).ResultsTIMM13 expression was higher in SKCM samples than in peritumor samples. TIMM13 was strongly associated with sample type, subgroup, cancer stage, lymph node stage, and worse survival. Further, upregulation of TIMM13 was significantly associated with immunoregulators, and chemokines, as well as T cells, B cells, monocytes, neutrophils, macrophages, and T-cell regulators. An analysis of bioinformatic data uncovered that TIMM13 expression was strongly associated with PD1 (T-cell exhaustion marker). The nomogram showed good predictive performance based on calibration plot. TIMM13 was highly expressed in melanoma tissue samples than in normal samples.ConclusionIn brief, TIMM13 may be a prognostic biomarker for SKCM. It might modulate the tumor immune microenvironment and lead to a poorer prognosis. In addition, it is necessary to study the targeted therapy of TIMM13.

Macrophage-derived CCL23 upregulates expression of T-cell exhaustion markers in ovarian cancer.

Macrophages are an important component of the tumour immune microenvironment (TME) and can promote tumour growth and metastasis. Macrophage-secreted chemokine-ligand-23 (CCL23) induces ovarian cancer cell migration via chemokine-receptor 1 (CCR1). However, the effect of CCL23 on other immune cells in the TME is unknown. CCL23 levels were measured by ELISA. The expression of surface markers in exhaustion assays was quantified by flow cytometry. Signalling pathways were identified by phosphokinase array and validated by western blot. Ascites from patients with high-grade serous ovarian cancer (HGSC) contain high levels of CCL23. Similarly, significantly higher CCL23 levels were found in plasma from HGSC patients compared to healthy individuals. RNA-seq analysis of ovarian cancer tissues from TCGA showed that expression of CCL23 correlated with the presence of macrophages. In tissues with high levels of CCL23 and macrophage content, the fraction of CD8 + T cells expressing exhaustion markers CTLA-4 and PD-1 were significantly higher compared to low-level CCL23 tissues. In vitro, CCL23 induced upregulation of immune checkpoint proteins on CD8 + T cells, including CTLA-4, TIGIT, TIM-3 and LAG-3 via phosphorylation of GSK3β in CD8 + T cells. Our data suggest that CCL23 produced by macrophages contributes to the immune-suppressive TME in ovarian cancer by inducing an exhausted T-cell phenotype.

P498: CLINICAL AND BIOLOGICAL MARKERS ASSOCIATED WITH LONG-TERM SURVIVAL FOR PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) IN REMISSION AFTER CHEMOTHERAPY IN THE QUAZAR AML-001 TRIAL OF ORAL AZACITIDINE

Background: In the randomized, phase 3 QUAZAR AML-001 trial, oral azacitidine (Oral-AZA) significantly prolonged overall survival (OS) compared with placebo (PBO) (median OS 24.7 vs 14.8 months [mo], respectively) in older patients (pts) with AML in first remission after intensive chemotherapy (IC). At an updated data cutoff performed in Sep 2020, 34.9% of pts in the Oral-AZA arm and 24.4% of pts in the PBO arm remained alive at ≥3 y from randomization. Aims: Assess clinical and biological variables associated with long-term survival (LTS) in QUAZAR AML-001. Methods: In all, 472 pts were randomized 1:1 to receive Oral-AZA 300 mg or PBO QD ×14d/28d within 4 mo of achieving first complete remission (CR) or CR with incomplete blood count recovery (CRi) after IC. The primary endpoint was OS, time from randomization until death, withdrawal of consent, or loss to follow-up. The LTS cohort comprised pts who were alive ≥3 y from randomization as of Sep 2020 and the non-LTS cohort included pts who died or were censored before 3 y. Variables assessed for association with LTS were diagnostic (Dx [pre-IC]) features (AML subtype, cytogenetic risk, NPM1 and FLT3 mutations [mut]); pre-study treatment (Tx) variables (response to IC [CR/CRi], receipt of consolidation, number of consolidation cycles); baseline (BL) demographic and disease characteristics, hematologic parameters (red blood cells [RBCs], hemoglobin, platelets, and leukocyte subsets), and measurable residual disease (MRD) status; and post-BL variables (MRD response [conversion from MRD+ at BL to MRD– on study], timing of MRD– [MRD– response on-study vs BL MRD–], and receipt of transplant after Tx discontinuation [D/C]). Associations of LTS with bone marrow immune parameters (CD3, CD4, and CD8 T-cell counts, and expression of PD-1/TIM-3 T-cell exhaustion markers) were investigated in a subset of pts (n=108). Variables were compared within Tx arms (LTS vs non-LTS) in univariate analyses with P values corrected for multiple testing. A logistic multivariable regression analysis of the effects of prognostic BL covariates on LTS was performed. Results: The LTS cohort included 83/238 pts (34.9%) in the Oral-AZA arm and 57/234 pts (24.4%) in the PBO arm. Within both arms, factors significantly associated with LTS were intermediate (Int)-risk cytogenetics and NPM1mut at Dx, and MRD response (MRD+ to MRD–) on study (Figure). MRD response rate was 2-fold higher with Oral-AZA vs PBO (37% vs 19%, respectively), and while early attrition was more common in the PBO arm, most MRD responses occurred within 6 mo. Factors significantly associated with LTS only in the PBO arm were BL (post-IC) MRD– status and receipt of transplant after Tx D/C. No significant associations were observed between BL hematological or immune parameters and LTS. The multivariable analysis (MVA) confirmed Oral-AZA Tx as independently significantly predictive of LTS vs PBO. Other covariates significantly associated with LTS in MVA were Int-risk cytogenetics and NPM1mut at Dx, and MRD– status at BL. Image:Summary/Conclusion: Oral-AZA Tx was significantly associated with LTS vs PBO. In univariate analysis, Int-risk cytogenetics and NPM1mut at Dx, and MRD response on-study, were significantly prognostic of LTS in both arms, whereas MRD– status at BL (post-IC) was associated with LTS only in the PBO arm.

Abstract 5887: Association of ovarian tumor immune profiles with overall survival

Abstract Despite being immunogenic, high-grade serous ovarian carcinomas (HGSC), the most common histotype, generally are not responsive to immune checkpoint inhibitors. To understand potential reasons for this, we examined associations of tumor-infiltrating B cells and T cells, including sub-populations defined by expression of immune checkpoints and T cell exhaustion markers, with survival in three epidemiologic studies. Analyses included participants in the Nurses’ Health Study (NHS), NHSII, and New England Case-control study who were diagnosed with stage 1-3 HGSC for whom tumor tissue was obtained. B cells (CD19, CD20, CD138), T cells (CD3), and T cell subpopulations (CD3 co-localized with CD8, CD4, PD1, PDL1, LAG3, TIM3) were measured using multiplex immunofluorescence assays performed on tissue microarray slides containing 3 cores per case. For each participant, we summed counts of immune cells infiltrating the tumor and total tumor cells across cores. Counts of immune cell subpopulations were adjusted for the parent cell count using residuals from beta-binomial models. Hazard ratios (HR) and 95% confidence intervals (CI) for overall mortality according to presence/absence or quantiles of immune cells were calculated using Cox proportional hazards regression models adjusting for age at diagnosis, diagnosis year, stage, study, and total tumor cell count. Among 389 women with HGSC, presence vs. absence of CD19+ B cells was suggestively associated with lower mortality (HR 0.79, 95%CI:0.61-1.01); similar associations were observed for CD19-adjusted CD20+ B cells and CD138+ plasma cells (HRs 0.75-0.78, quintile 5 vs. 1).Higher total T cells was associated with improved survival (HR 0.67, 95%CI:0.45-0.99, quintile 5vs. 1). Similar reduced risks were observed with high levels of both CD19+ B cells and CD3+ T cells (HR 0.66) or high levels of one cell type (HRs 0.65-0.75) compared with low levels of both. For the two primary T cell subfractions, lower risk was observed with higher CD3-adjustedCD3+CD8+ cytotoxic T cells (HRs 0.68, 0.73, 0.69, 0.59 for quintiles 2-5 vs. 1), but notCD3+CD4+ helper T cells (HR 0.97, 95%CI: 0.72-1.31, tertile 3 vs. 1). T cells with immune checkpoint markers (PD1, PDL1) and/or markers of T-cell exhaustion (LAG3, TIM3) generally were not associated with mortality or tended to be associated with lower mortality risk, with a significant inverse association observed for CD3-adjusted CD3+PD1+TIM3- cells (HR 0.61,95%CI:0.41-0.92, quintile 5 vs. 1).Overall, higher levels of B cells and CD3+CD8+ T cells in tumor were associated with improved outcomes, consistent with other studies. Congruent with the apparent ineffectiveness of immune checkpoint inhibitors in ovarian cancer, our results do not support that T cells expressing immune checkpoint and/or T cell exhaustion markers are related to worse outcome among women with HGSC. Future analyses will consider multiple cell types simultaneously and the stroma compartment. Citation Format: Shelley S. Tworoger, Mary K. Townsend, Jose Conejo-Garcia, Brooke L. Fridley, Carlos Moran Segura, Daryoush Saeed-Vafa, Naoko Sasamoto, Kathryn L. Terry. Association of ovarian tumor immune profiles with overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5887.

Abstract 1722: Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer (NSCLC)

Abstract The rapid clinical expansion of immune checkpoint blockers (ICB) has transformed the therapeutic arsenal for non-small cell lung cancer (NSCLC). However, only a relatively small fraction of patients benefits from these treatments and those who respond can develop acquired resistance. This underlines the need to identify factors or biomarkers associated with treatment sensitivity and resistance to guide clinical decisions. It has been proposed that elevated levels of tumor infiltrating lymphocytes (TIL) in the tumor microenvironment could be associated with favorable clinical outcomes after treatment with ICB. However, few studies have explored this relationship and they used semi-quantitative scoring methods and/or analyzed small tumor areas. Here, we used multiplexed quantitative immunofluorescence (QIF) panels to study major TIL subsets (DAPI, CK, CD4, CD8 and CD20) and T-cell exhaustion markers (DAPI, CD3, LAG-3, PD-1, TIM-3) in full-face baseline whole-tumor slides from a large multi-institutional patient cohort (n=179). The analysis of numerous fields of view per case allowed us to examine the spatial distribution of immune cells and spatial immune heterogeneity. Our results demonstrate that CD8+ effector T-cells are the largest TIL subpopulation and that they are preferentially located in the stromal compartment. We also show that tertiary lymphoid structures have limited association with survival and their detection depends on the size of the tissue area analyzed. Additionally, higher levels of baseline CD8+ T-cells in the stroma were significantly associated with better survival in PD-L1 positive patients but not in PD-L1 negative. To validate these results, we used whole-exome sequencing to analyze the TCR-burden in an independent cohort of ICI-treated NSCLC patients. Increased expression of the exhaustion markers LAG-3 and TIM-3 in CD3 positive T-cells was associated with reduced survival. Finally, we used a novel multiparametric heterogeneity metric termed Rao’s quadratic entropy that enabled us to measure the spatial immune heterogeneity and was associated with worse survival. In summary, we quantitatively measured the density, functional properties, and spatial distribution of major TIL subsets in a large cohort of NSCLC patients treated with ICB. Our results highlight the prominent role of TILs in NSCLC and their potential role as a biomarker. These results could be easily translated into the clinic and used to guide optimal treatment options. Citation Format: Miguel Lopez de Rodas Gregorio, Venkata Nagineni, Arvind Ravi, Ila J. Datar, Mari Mino-Kenudson, German Corredor, Cristian Barrera, Lindsey Behlman, David L. Rimm, Roy S. Herbst, Anant Madabhushi, Jonathan W. Riess, Vamsidhar Velcheti, Matthew Hellmann, Justin F. Gainor, Kurt A. Schalper. Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1722.

Abstract 236: Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer

Abstract Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3΄-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl2) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8+ and CD4+ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8+ and CD4+ T-cell exhaustion. Analysis of CD8+ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8+ and CD4+ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8+ (tumors and spleen) and CD4+ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes. Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 236.

Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer

IntroductionEpithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. MethodsWe evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). ResultsWe uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. ConclusionOur results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses.

Objective: The purpose of current research is to explore the function of retinoic acid-induced protein 14 (RAI14), being a reciprocal protein of carboxypeptidase N1 (CPN1), and as a biomarker for prognosis and immunoregulatory effects in breast cancers. Methods: Interacting proteins of CPN1 were characterized by co-immunoprecipitation (CO-IP) and mass spectrometry. We evaluated RAI14 expression and related clinical prognosis based on bioinformatics methods. The level of relevance between RAI14 and infiltrating immune cells biomarkers was investigated by using TIMER and certificated by immunohistochemical staining and cytology experiments. Results: RAI14 is an interacting protein of CPN1. Higher RAI14 expression in TNBC was significantly correlated with poor prognosis in TNBC, especially (RFS: HR = 1.32, p = 0.015; DFS: HR = 1.18, p = 0.035). The estrogen receptor (ER), P53 status, and histological types and triple-negative status were observed and correlated with RAI14 expression. Moreover, the level of RAI14 was positive in relation with the expression of CD163 (M2 macrophages marker, r = 0.393, p = 1.89e-06) and PD-1 (T-cell exhaustion marker, r = 0.626, p = 4.82e-03), indicating RAI14 levels were mainly related to M2 macrophages and T-cell exhaustion infiltration in TNBC. Furthermore, CPN1 overexpression was accompanied by RAI14 and PD-L1 upregulation, and a correlation was found among them. Conclusions: RAI14 is a potential downstream molecule of CPN1, which may be a potential prognostic biomarker and identification of an immunosuppressive tumor microenvironment in TNBC.

ITGAL as a Prognostic Biomarker Correlated With Immune Infiltrates in Gastric Cancer.

Integrin alpha L (ITGAL) is a member of the integrin family in which the abnormal expression is linked with carcinogenesis and immune regulation. However, the relation between ITGAL and the prognosis of gastric cancer (GC) and tumor-infiltrating lymphocytes (TILs) are not well understood. The differential expressions of ITGAL in human tumors and the clinical prognosis in GC were systematically analyzed via multiple databases including Gene Expression Profiling Interaction Analysis (GEPIA), UALCAN, Tumor Immune Estimation Resource (TIMER), and Kaplan–Meier (KM) plotter. TIMER, GEPIA, and TISIDB databases were used to comprehensively investigate the correlation between ITGAL and tumor infiltration immune cells. Also, further results were investigated by immunohistochemistry, qRT-PCR, and Western blot. We found that ITGAL expression in GC samples was considerably increased than in peritumor samples. Sample type, subgroup, cancer stage, lymphatic node stage, and worse survival were strongly related to high ITGAL expression. Moreover, upregulated ITGAL expression was strongly connected with immunomodulators, chemokines, and infiltrating levels of CD8+, CD4+ T cell, B cell, monocyte, neutrophil, macrophage, T-cell regulatory, NK cell, and myeloid dendritic cell in stomach adenocarcinoma (STAD). Specifically, immunohistochemistry and bioinformatic analysis showed that ITGAL expression was shown to have strong relationships with various immunological marker sets including PD1 (T-cell exhaustion marker). In conclusion, ITGAL is a prognostic biomarker for GC patients. It might regulate tumor immune microenvironment leading to poor prognosis. Furthermore, studies are essential to explore therapeutic targeting ITGAL.

Abstract P5-06-10: Tumor microenvironment modulates ganglioside expression leading to immunosuppression in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive human malignancies, characterized by a strikingly poor prognosis linked to high rates of distant metastases and disease relapse. Among several mechanisms that contribute to TNBC aggressiveness is the ability of TNBC cells to evade the anti-tumor immune response. Numerous efforts have been directed to identify and target molecules and signaling pathways that contribute to TNBC immune evasion. A considerable proportion of TNBCs are resistant to immune-checkpoint therapy, implying that additional molecules are involved in immune suppression in these malignancies. We have previously reported that ganglioside GD2 identifies breast cancer stem-like cells (BCSCs) and promotes tumorigenesis. GD3 synthase (GD3S), a key enzyme that regulates GD2 biosynthesis, is significantly upregulated in TNBCs. Gangliosides have been found to promote an immunosuppressive tumor microenvironment. In this study, we hypothesize that tumor-microenvironment influences expression of gangliosides downstream of GD3S, including GD2/GD3, suppressing the anti-tumor immune response while inhibiting GD3S expression enhances immune-mediated killing of TNBC cells. We performed immunohistochemistry (IHC) on primary TNBC patient samples (N=89) and measured GD2 expression using an automated imaging system (Vectra-Polaris). Immunohistochemistry analysis revealed that approximately 60% of samples had detectable GD2 expression of different intensity and was associated with poor overall survival of patients with TNBC (p=0.002). Our IHC data also demonstrated that GD2 is expressed not only in tumors but also in the tumor-associated stroma in ~60% of TNBC samples, suggesting the effect of crosstalk between TME and TNBC cells on GD2 expression. Additionally, to substantiate the role of TME on GD2 expression, we cultured bone marrow-derived mesenchymal stromal cell (BM-MSCs) with MDA-MB-231 cells and found a 2-fold increase in GD2 expression in BM-MSC and MDA-MB-231 cells (p<0.001), suggesting microenvironmental factors contribute to GD2 expression on both tumor and stromal cells. To examine the immunomodulatory role of gangliosides we used the TCGA dataset to analyze the co-expression of GD3S mRNA with T-cell exhaustion markers in all breast cancer patients. We found a significant (p<0.001) positive correlation between ST8SIA1 and CTLA-4 (pearson= 0.4), LAG-3 (pearson= 0.33), and PD1 (pearson=0.3) in breast cancer patients. Furthermore, we overexpressed GD3S in TNBC and non-TNBC cells and observed its effect on T-cell mediated killing of breast cancer (BC) cells by a live-cell imaging system (IncuCyte). Overexpression of GD3S caused upregulation of GD2 in BT549, HIM3 (TNBC), and MCF7 (non-TNBC) cells, and their co-culture with T-cells resulted in a significant reduction (p<0.001) in cell death compared to the controls. In conclusion, GD2 is a tumor-specific marker in patients with TNBC and that GD3S overexpression is associated with T-cell exhaustion and maintenance of an immunosuppressive microenvironment in breast cancer. This immunosuppressive microenvironment in TNBCs fostered by gangliosides downstream of GD3S is also a manifestation of the cross-talk between tumor and tumor-associated stromal cells. Citation Format: Vivek Anand, Fouad El-Dana, Stanley Ly, Michael Andreeff, Venkata Lokesh Battula. Tumor microenvironment modulates ganglioside expression leading to immunosuppression in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-10.

High CD169 Monocyte/Lymphocyte Ratio Reflects Immunophenotype Disruption and Oxygen Need in COVID-19 Patients.

Background: Sialoadhesin (CD169) has been found to be overexpressed in the blood of COVID-19 patients and identified as a biomarker in early disease. We analyzed CD169 in the blood cells of COVID-19 patients to assess its role as a predictive marker of disease progression and clinical outcomes. Methods: The ratio of the median fluorescence intensity of CD169 between monocytes and lymphocytes (CD169 RMFI) was analyzed by flow cytometry in blood samples of COVID-19 patients (COV) and healthy donors (HDs) and correlated with immunophenotyping, inflammatory markers, cytokine mRNA expression, pulmonary involvement, and disease progression. Results: CD169 RMFI was high in COV but not in HDs, and it correlated with CD8 T-cell senescence and exhaustion markers, as well as with B-cell maturation and differentiation in COV. CD169 RMFI correlated with blood cytokine mRNA levels, inflammatory markers, and pneumonia severity in patients who were untreated at sampling, and was associated with the respiratory outcome throughout hospitalization. Finally, we also report the first evidence of the specific ability of the spike protein of SARS-CoV-2 to trigger CD169 RMFI in a dose-dependent manner in parallel with IL-6 and IL-10 gene transcription in HD PBMCs stimulated in vitro. Conclusion: CD169 is induced by the spike protein and should be considered as an early biomarker for evaluating immune dysfunction and respiratory outcomes in COVID-19 patients.

188P TIM-3 expression and markers of immune escape on leukemia cells

TIM-3 is a known marker of T-cell exhaustion but it is also commonly found on leukemia blasts in acute myeloid leukemia (AML). TIM-3 positivity of AML blasts was associated with higher relapse rate and worse overall survival. Although TIM-3 function in AML cells is far from elucidated, TIM-3 was reported to mediate Galectin-9 secretion, and TIM-3/Galectin-9 release in the plasma might contribute to inhibition of T-cell effector function.

TCR-independent Activation in Presence of a Src-family Kinase Inhibitor Improves CAR-T Cell Product Attributes.

Chimeric antigen receptor expressing T cells (CAR-T cells) have shown remarkable efficacy against some blood cancers and have potential to treat many other human diseases. During CAR-T cell manufacturing, T cells are activated via engagement of the T-cell receptor (TCR); however, persistent TCR engagement can induce unchecked activation, differentiation, and exhaustion, which can negatively affect CAR-T cell product quality and in vivo potency. In addition, T cells may not uniformly respond to TCR-dependent activation (TCRD) contributing to lot-to-lot variability, poor expansion, and manufacturing failures. TCRD also presents challenges during manufacturing of allogeneic CAR-T cells when endogenous TCR is deleted to prevent graft-versus-host disease. Thus, novel strategies to activate T cells may help improve CAR-T cell product attributes and reduce manufacturing failures. In this study, we compared the effect of TCRD and TCR-independent activation (TCRI) on CAR-T cell product attributes. We found that TCRI in presence of a Src-kinase inhibitor significantly improved CAR-T cell expansion and yield without affecting viability and CD4/CD8 ratio. Markers of T-cell activation, exhaustion and differentiation were also reduced in these CAR-T cells compared with CAR-T cells manufactured by TCRD. TCRI did not affect CAR-T cell in vitro potency; however, following co-culture with target cells, CAR-T cells manufactured by TCRI released significantly less inflammatory cytokines compared with CAR-T cells manufactured by TCRD. Together, these data suggest that manufacturing CAR-T cells by TCRI activation in the presence of a Src-kinase inhibitor improves product quality attributes and may help reduce manufacturing failures and improve CAR-T cell safety and efficacy in vivo.

Single Cell RNA Sequencing Driven Characterization of Rare B/Myeloid and T/Myeloid Mixed Phenotype Acute Leukemia

Introduction: Pediatric mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia, contains features of both myeloid and lymphoid lineage blasts, which makes the disease more difficult to diagnose/treat. More information is needed to understand the origins of the major pediatric MPAL subtypes, B/Myeloid (B-MPAL) and T/Myeloid (T-MPAL), and how they relate to other leukemias. Single-cell RNA sequencing (scRNA-seq) analysis of bone marrow (BM) can provide in-depth information about the leukemia microenvironment and reveal differences/similarities between MPAL subtypes and other types of leukemia that could be exploited to develop novel diagnostics/therapies.Methods: We analyzed ~16,000 cells from five pediatric MPAL BM samples to generate a transcriptomic landscape of B-MPAL and T-MPAL blasts and associated microenvironment cells. Samples collected at the time of diagnosis (Dx) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy et al. JCI 2019, Tellechea et al. JID, 2020). After data normalization, cell clusters were identified using principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al. Nat. Biotech 2018). Meta-analysis was performed using single cell samples from ongoing studies in the Bhasin lab (Bhasin, et al. Blood 2020 (ASH), Thomas et al. Blood 2020 (ASH)) and publicly available single cell data from GEO biorepository. Unsupervised analysis using UMAP and PCA was performed to determine the overall relationship among B-MPAL, T-MPAL and other leukemias (acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell ALL (T-ALL)). Supervised differentially expressed gene (DEG) analysis was performed to identify B- and T- MPAL blast cell signatures (P value < 0.001 and log2 FC > 0.5). Transcriptomic profiles in MPAL samples and normal BM stem and immune cells were compared using data from the Human Cell Atlas Data Portal (humancellatlas.org). Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes (P value < 0.001).Results: PCA analysis showed transcriptome similarity between B-MPAL and both B-ALL and AML, while T-MPAL transcriptome correlated with T-ALL and AML (Fig. 1A). B- and T-MPAL subtype blasts clustered separately from each other in UMAP analyses, with T-MPAL blasts clustering with T-ALL blasts, and B-MPAL somewhat overlapping with B-ALL blasts. Subtype DEG analysis of leukemia blasts and healthy BM revealed distinct significantly upregulated gene signatures in B-MPAL (YBX3, SOCS2, BCL11A, and HIST1H1C) and T-MPAL (ITM2A, HPGD, PDLIM1, and TRDC) blasts (Fig. 1B). Pathway analysis showed upregulated gene activity related to TNFA signaling via NFKB, B-cell survival, and the AP1, FRA, and NGF transcription factors in B-MPAL blasts. In contrast, IL-17 and IL-12, T-cell apoptosis, and Stathmin pathways were upregulated in T-MPAL blasts (Fig. 1C). T-MPAL T-cells also expressed higher levels of T-cell exhaustion markers compared to T-cells in B-MPAL samples and healthy bone marrow.After filtering out genes that are significantly expressed in immune cells, we identified genes that are differentially expressed at diagnosis in MPAL blasts from patients that relapsed after treatment (Dx1) versus patients that achieved remission (Dx2). These genes are potential prognostic markers for B-MPAL and T-MPAL relapse/remission. These include MDM2 and NEIL1 from Dx1 and FOSL2 and CDKN1A in Dx2 B-MPAL blasts. In T-MPAL, expression of HES4 and SPINK2 is associated with Dx1 blasts and GNAQ and ITGA4 with Dx2 blasts. Pathway enrichment analysis on B-MPAL blasts revealed upregulation of interferon gamma and PD-1 signaling in Dx1 samples and increased HSP27 and Cell Cycle pathways in the Dx2 subset. T-MPAL Dx1 associated pathways included prostaglandin synthesis and IL-17, while cell-cell junction and extracellular matrix interactions were increased in T-MPAL Dx2 samples (Fig. 1D).Conclusion: Single-cell profiling was used to characterize the molecular landscapes of MPAL blasts and the bone marrow microenvironment and identified gene signatures and pathways that are specifically enriched in B- and T-MPAL subtypes. [Display omitted] DisclosuresDeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Inverted CD8 T-Cell Exhaustion and Co-Stimulation Marker Balance Differentiate Aviremic HIV-2-Infected From Seronegative Individuals.

HIV-2 is less pathogenic compared to HIV-1. Still, disease progression may develop in aviremic HIV-2 infection, but the driving forces and mechanisms behind such development are unclear. Here, we aimed to reveal the immunophenotypic pattern associated with CD8 T-cell pathology in HIV-2 infection, in relation to viremia and markers of disease progression. The relationships between pathological differences of the CD8 T-cell memory population and viremia were analyzed in blood samples obtained from an occupational cohort in Guinea-Bissau, including HIV-2 viremic and aviremic individuals. For comparison, samples from HIV-1- or dually HIV-1/2-infected and seronegative individuals were obtained from the same cohort. CD8 T-cell exhaustion was evaluated by the combined expression patterns of activation, stimulatory and inhibitory immune checkpoint markers analyzed using multicolor flow cytometry and advanced bioinformatics. Unsupervised multidimensional clustering analysis identified a cluster of late differentiated CD8 T-cells expressing activation (CD38+, HLA-DRint/high), co-stimulatory (CD226+/-), and immune inhibitory (2B4+, PD-1high, TIGIThigh) markers that distinguished aviremic from viremic HIV-2, and treated from untreated HIV-1-infected individuals. This CD8 T-cell population displayed close correlations to CD4%, viremia, and plasma levels of IP-10, sCD14 and beta-2 microglobulin in HIV-2 infection. Detailed analysis revealed that aviremic HIV-2-infected individuals had higher frequencies of exhausted TIGIT+ CD8 T-cell populations lacking CD226, while reduced percentage of stimulation-receptive TIGIT-CD226+ CD8 T-cells, compared to seronegative individuals. Our results suggest that HIV-2 infection, independent of viremia, skews CD8 T-cells towards exhaustion and reduced co-stimulation readiness. Further knowledge on CD8 T-cell phenotypes might provide help in therapy monitoring and identification of immunotherapy targets.

An autologous culture model of nodal B-cell lymphoma identifies ex vivo determinants of response to bispecific antibodies

AbstractBispecific antibodies (BsAbs) can induce long-term responses in patients with refractory and relapsed B-cell lymphoma. Nevertheless, response rates across patients are heterogeneous, and the factors determining quality and duration of responses are poorly understood. To identify key determinants of response to BsAbs, we established a primary, autologous culture model allowing us to mimic treatment with CD3xCD19 and CD3xCD20 BsAbs within the lymph node microenvironment ex vivo. T cell–mediated killing of lymphoma cells and proliferation of T cells varied significantly among patients but highly correlated between BsAbs targeting CD20 or CD19. Ex vivo response to BsAbs was significantly associated with expansion of T cells and secretion of effector molecules (eg, granzyme B, perforin) but not with expression of T-cell exhaustion (eg, PD1, TIM3) or activation markers (eg, CD25, CD69) or formation of intercellular contacts. In addition, we identified a distinct phenotype of regulatory T cells that was linked to ex vivo response independently from T-cell frequency at baseline. High expression levels of Aiolos (IKZF1), ICOS, and CXCR5 were positively associated with ex vivo response, whereas strong expression of Helios (IKZF2) had an unfavorable impact on ex vivo response to BsAbs. We further showed that lenalidomide, nivolumab, and atezolizumab improved ex vivo response to BsAbs by potentiating T-cell effector functions. In summary, our ex vivo study identified a distinct regulatory T-cell phenotype as a potential contributor to treatment failure of BsAbs and suggests drug combinations of high clinical relevance that could improve the efficacy of BsAbs.

A randomized phase 2 trial of idiotype vaccination and adoptive autologous T-cell transfer in patients with multiple myeloma

AbstractWe hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)–keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial, patients received either control (KLH only) or Id-KLH vaccine, autologous transplantation, vaccine-specific costimulated T cells expanded ex vivo, and 2 booster doses of assigned vaccine. In 36 patients (KLH, n = 20; Id-KLH, n = 16), no dose-limiting toxicity was seen. At last evaluation, 6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms, respectively (P = .22), and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with patients receiving KLH only, there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically, upregulation of genes associated with activation, effector function induction, and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly, in responding patients across both arms, upregulation of genes associated with T-cell activation was seen. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion, in this combination immunotherapy approach, we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828.

1359TiP RELATIVITY-104: First-line relatlimab (RELA) + nivolumab (NIVO) with chemotherapy vs nivo with chemotherapy in stage IV or recurrent non-small cell lung cancer (NSCLC): A phase II, randomized, double-blind study

Immune checkpoint inhibitors combined with platinum doublet chemotherapy (PDCT) have improved outcomes for NSCLC; however, some patients with metastatic NSCLC may not benefit from this strategy due to intrinsic or acquired resistance. Lymphocyte-activation gene 3 (LAG-3) is a inhibitory immune checkpoint that is a marker of T-cell exhaustion limiting their activity. RELA is a LAG-3-blocking monoclonal antibody that restores effector function in exhausted T cells. The combination of RELA + NIVO demonstrated superior progression-free survival (PFS) benefit in previously untreated melanoma and was well tolerated with no new safety signals.