Exhaustion Tim-3 Research Articles

Abstract 2789: Highly multiplexed imaging mass cytometry reveals immune cell composition and spatial heterogeneity in diffuse large B cell lymphoma associated with treatment outcome

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogenous entity characterized by its variable clinical and biological behaviour, and approximately 30% of patients experience relapsed or refractory disease after first-line therapy. We hypothesize that a better characterization of the tumor microenvironment (TME) might help identify patients who may benefit from individualized immunotherapies. Similar studies in this area have been limited by technical challenges - conventional highly multiplexed techniques require tissue disruptions that lose spatial information, while those that retain tissue architecture can only examine 6-8 markers simultaneously. We and others have previously reported that PD-L1 expression is correlated with decreased survival in a cohort of 85 DLBCL patients. In the present study, we characterized TME components, including their types, frequency and spatial interaction, in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution. Using a panel of 32 antibodies, IMC was performed on a subset of our previously studied cohort. We examined 41 cores from 33 DLBCL cases, 17 GCB and 16 non-GCB, by Hans criteria. Clinical outcome data were available for 29 patients, 22 of whom had complete response (CR) to R-CHOP therapy while 7 had primary refractory disease. Using both supervised gating and unsupervised clustering algorithm, IMC data were analyzed for relevant immunophenotypes and compared across clinical outcome groups. The TME was mainly composed of 13.1% ± 1.9% (mean ± SE) CD4+ T-helper cells, 10.8% ± 1.1% CD8+ cytotoxic T cells, 6.3% ± 0.9% CD68+ macrophages, 2.7% ± 0.5% FoxP3+ regulatory T cells, and 58.1% ± 3.4% tumor cells. In non-GCB group, higher ratio of regulatory T cells was associated with refractory disease. In contrast, activated granzyme-B+/CD8+ cytotoxic T cells were more frequent in CR group, while markers of exhaustion (Tim3, Lag3) were found in patients with refractory disease. To gain functional insight into the various immune subsets, we performed spatial analysis of the immune cells and their relation to blood vessels and tumor cells. Nearest distance analysis showed that CD4+ cells were most tightly clustered around blood vessels in patients with CR, while in those with refractory diseases, CD4+ cells were further away from the vessels (p=0.03). On the contrary, distances between cytotoxic T cells and regulatory T cells showed no difference between CR and refractory patients. Together, these results show variable composition of the different immune cells and their spatial heterogeneity to be associated with the clinical outcome of DLBCL patients and that spatial analysis of immune cells should be explored as a potential biomarker for patients treated with immunotherapies. Citation Format: Monirath Hav, Erik Gerdtsson, Mohan Singh, Anthony Colombo, James Hicks, Peter Kuhn, Imran Siddiqi, Akil Merchant. Highly multiplexed imaging mass cytometry reveals immune cell composition and spatial heterogeneity in diffuse large B cell lymphoma associated with treatment outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2789.

CD8+CXCR5+ T cells in tumor-draining lymph nodes are highly activated and predict better prognosis in colorectal cancer

Tumor-draining lymph nodes (TDLNs) are the primary sites of tumor antigen presentation, as well as the origin of metastasis in most cases. Hence, the type and function of immune cells in TDLNs are critical to the microenvironment and potentially affect the clinical outcome of the malignancy. CD8+CXCR5+ T cells are recently described to present high effector functions in infectious diseases, but their role in colorectal cancer (CRC) remains unclear. In forty-four Stage III CRC patients, we examined the CD8+CXCR5+ T cells in blood, tumor, and TDLN. CD8+CXCR5+ T cells represented lass than 2% of CD3+ T cells in blood, but a much larger population in tumor. In TDLN, the CD8+CXCR5+ T cells represented the vast majority of CD8+ T cells and between 9.3% and 32.9% of CD3+ T cells. The prevalence of CD8+CXCR5+ T cells in tumor was not associated with their frequency in peripheral blood, but was positively correlated with their frequency in TDLN. The transcription of effector genes, including IFNG, TNF, IL2, PRF1, and GZMB, and exhaustion markers, including PD1, TIM3, 2B4, and LAG3, were examined in CD8+CXCR5+ T cells and CD8+CXCR5− T cells. With a few exceptions, CD8+CXCR5+ T cell presented significantly higher effector gene expression, and significantly lower exhaustion marker expression than their CXCR5− counterparts. In addition, the prognosis of CRC patients was positively associated with the frequency of TDLN CD8+CXCR5+ T cells, and with the expression of IFNG, PRF1, and GZMB expression by tumor and TDLN CD8+CXCR5+ T cells. Together, these results demonstrated that CD8+CXCR5+ T cells were significant participants of CRC-associated immunity and could potentially serve as therapeutic options.

Abstract LB-235: Delta-24-RGD oncolytic adenovirus treatment downmodulates the key regulator of T-cell exhaustion TIM3 in malignant gliomas

Abstract T cell exhaustion is a key mechanism of tumor immune suppression and is commonly observed in several cancers. The exhausted T cells in the tumor microenvironment overexpress inhibitory receptors, show decreased effector cytokine production, and exhibit diminished cytolytic capabilities, resulting in the failure to eliminate cancer cells. Restoring the activity of exhausted T cells is a desirable strategy to improve immune-based anti-cancer therapies, and has yielded encouraging results. Exhausted T cells are characterized by the expression of high levels of PD-1 and TIM3, which cooperate to induce T-cell hyporesponsiveness. In this study, we sought to determine whether the infection of human gliomas with oncolytic viruses modifies the expression of T cell markers. As part of a phase 1, dose-escalation, biological-endpoint clinical study (NCT00805376) of Delta-24-RGD, a Rb-targeted, tumor-selective, replication-competent oncolytic adenovirus, conducted in 37 patients with recurrent malignant glioma, 12 patients received an intratumoral injection through an implanted catheter, followed 14-days later by en bloc resection and Delta-24-RGD injections into the post-resection tumor bed. 10 surgical post-treatment specimens and 5 pre-treated tumors were examined for markers of immune response. From all selected cases, formalin fixed, paraffin embedded tissue blocks were examined. IHC was performed using a Leica Bond Max automated stainer as previously described. The slides were scanned in an Aperio AT2 scanner (Leica Biosystems). The IHC scores for CD3, CD4, CD8, CD45Ro, FOXP3, TIM3, LAG-3, VISTA, CD20, CD57, CD68, and OX40 were expressed as cell density in the inflammatory cell population (number of positive cells per mm square of tumor area), except PD-L1, PD-L2, B7-H3, B7-H4, IDO-1, which were expressed using percentage of positive tumor cells, and also as H-Score of the tumor cells (which integrates percentage of tumor cells and intensity of staining, with a final H-score ranging from 0 to 300). Correlations between immunostaining marker values and survival and dose level were assessed using Spearman rank correlation analysis. Differences in immunostaining marker values before and after treatment were assessed using the Wilcoxon rank sum test. These analyses demonstrated that all the target proteins are expressed in gliomas in different patterns and tumor regions. Although, we did not observe significant treatment-related changes in the expression of PD-1 in lymphocytes and PD-L1 in glioma cells, TIM3, a marker for T cell exhaustion, was downregulated after Delta-24-RGD treatment. In addition, expression of B7-H3, a marker of poor prognosis in other tumors, correlated with poorer survival. Because PD-1 and TIM3 cooperate to maintain T-cell exhaustion our data provide a rationale for the combination of Delta-24-RGD and anti-PD-1 antibodies for the treatment of malignant gliomas. Importantly, a multicenter clinical trial to test this strategy has already been open (NCT02798406). Citation Format: Juan Fueyo, Candelaria Gomez-Manzano, Pamela Villalobos, Jaime Rodriguez-Canales, Barbara Mino, Ignacio Wistuba, Kenneth Hess, Marta Alonso, Sonia Tejada, Ricardo Diez-Valle, Brett Ewald, Frank Tufaro, Frederick Lang. Delta-24-RGD oncolytic adenovirus treatment downmodulates the key regulator of T-cell exhaustion TIM3 in malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-235. doi:10.1158/1538-7445.AM2017-LB-235

Bat3 promotes T cell responses and autoimmunity by repressing Tim-3–mediated cell death and exhaustion

T cell immunoglobulin and mucin domain–containing 3 (Tim-3) is an inhibitory receptor that is expressed on exhausted T cells during infection with HIV-1 and hepatitis C virus. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms modulating Tim-3 function are not well understood. Here we show that human leukocyte antigen B (HLA-B)-associated transcript 3 (Bat3) binds to, and represses the function of, Tim-3. Bat3 protects T helper type 1 (TH1) cells from galectin-9–mediated cell death and promotes both proliferation and proinflammatory cytokine production. Bat3-deficient T cells have elevated expression of exhaustion-associated molecules such as Tim-3, Lag3, Prdm1 and Pbx3, and Bat3 knockdown in myelin-antigen–specific CD4+ T cells markedly inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3hi, interferon-γ (IFN-γ)loCD4+ cell population. Furthermore, expression of Bat3 is reduced in exhausted Tim-3+ T cells from mouse tumors and HIV-1–infected individuals. These data indicate that Bat3 acts as an inhibitor of Tim-3–dependent exhaustion and cell death. Bat3 may thus represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers.

HIV-1 Infection Abrogates CD8+T Cell Mitogen-Activated Protein Kinase Signaling Responses

Mitogen-activated protein kinase (MAPK) signaling pathways are dynamic and sensitive regulators of T cell function and differentiation. Altered MAPK signaling has been associated with the inflammatory and autoimmune diseases lupus and arthritis and with some pathogenic viral infections. HIV-1 infection is characterized by chronic immune inflammation, aberrantly heightened CD8(+) T cell activation levels, and altered T cell function. The relationship between MAPK pathway function, HIV-1-induced activation (CD38 and HLA-DR), and exhaustion (Tim-3) markers in circulating CD8(+) T cells remains unknown. Phosphorylation of the MAPK effector proteins ERK and p38 was examined by "phosflow" flow cytometry in 79 recently HIV-1-infected, antiretroviral-treatment-naïve adults and 21 risk-matched HIV-1-negative controls. We identified a subset of CD8(+) T cells refractory to phorbol 12-myristate 13-acetate plus ionomycin-induced ERK1/2 phosphorylation (referred to as p-ERK1/2-refractory cells) that was greatly expanded in HIV-1-infected adults. The CD8(+) p-ERK1/2-refractory cells were highly activated (CD38(+) HLA-DR(+)) but not exhausted (Tim-3 negative), tended to have low CD8 expression, and were enriched in intermediate and late transitional memory states of differentiation (CD45RA(-) CD28(-) CD27(+/-)). Targeting MAPK pathways to restore ERK1/2 signaling may normalize immune inflammation levels and restore CD8(+) T cell function during HIV-1 infection.