Tim-3 Expression Research Articles

γδ T cells mediate robust anti-HIV functions during antiretroviral therapy regardless of immune checkpoint expression.

Although antiretroviral therapy (ART) efficiently suppresses HIV viral load, immune dysregulation and dysfunction persist in people living with HIV (PLWH). γδ T cells are functionally impaired during untreated HIV infection, but the extent to which they are reconstituted upon ART is currently unclear. Utilising a cohort of ART-treated PLWH, we assessed the frequency and phenotype, characterised in vitro functional responses and defined the impact of immune checkpoint marker expression on effector functions of both Vδ1 and Vδ2 T cells. We additionally explore the in vitro expansion of Vδ2 T cells from PLWH on ART and the mechanisms by which such expanded cells may sense and kill HIV-infected targets. A matured NK cell-like phenotype was observed for Vδ1 T cells among 25 ART-treated individuals (PLWH/ART) studied compared to 17 HIV-uninfected controls, with heightened expression of 2B4, CD160, TIGIT and Tim-3. Despite persistent phenotypic perturbations, Vδ1 T cells from PLWH/ART exhibited strong CD16-mediated activation and degranulation, which were suppressed upon Tim-3 and TIGIT crosslinking. Vδ2 T cell degranulation responses to the phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate at concentrations up to 2 ng mL-1 were significantly impaired in an immune checkpoint-independent manner among ART-treated participants. Nonetheless, expanded Vδ2 T cells from PLWH/ART retained potent anti-HIV effector functions, with the NKG2D receptor contributing substantially to the elimination of infected cells. Our findings highlight that although significant perturbations remain within the γδ T cell compartment throughout ART-treated HIV, both subsets retain the capacity for robust anti-HIV effector functions.

Effects of co-infection with Clonorchis sinensis on T cell exhaustion levels in patients with chronic hepatitis B.

To investigate the effects of co-infection with Clonorchis sinensis (C. sinensis) on T cell exhaustion levels in patients with chronic hepatitis B, we enrolled clinical cases in this study, including the patients with concomitant C. sinensis and HBV infection. In this study, we detected inhibitory receptors and cytokine expression in circulating CD4+ and CD8+ T cells by flow cytometry. PD-1 and TIM-3 expression levels were significantly higher on CD4+ T and CD8+ T cells from co-infected patients than on those from the HBV patients. In addition, CD4+ T cells and CD8+ T cells function were significantly inhibited by C. sinensis and HBV co-infection compared with HBV single infection, secreting lower levels of Interferon gamma (IFN-γ), Interleukin-2 (IL-2), and TNF-α. Our current results suggested that C. sinensis co-infection could exacerbate T cell exhaustion in patients with chronic hepatitis B. PD-1 and TIM-3 could be novel biomarkers for T cell exhaustion in patients with Clonorchis sinensis and chronic hepatitis B co-infection. Furthermore, it may be one possible reason for the weaker response to antiviral therapies and the chronicity of HBV infection in co-infected patients. We must realize the importance of C. sinensis treatment for HBV-infected patients. It might provide useful information for clinical doctors to choose the right treatment plans.

Relationship between TIM3 Expression on Peripheral T Lymphocytes and Post-Stroke Depression.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is a regulatory molecule expressed on a variety of cell types, including CD3+ T cells. Few studies have been conducted to look into the correlation between TIM3 expression on peripheral T lymphocytes and post-stroke depression (PSD). To investigate the relationship between TIM3 expressions on peripheral T lymphocytes in PSD patients. Acute stroke patients without depression (NPSD) (n=65), PSD patients (n=23), and body mass index (BMI), age, and education-matched healthy controls (HC) (n=59) were enrolled. Using flow cytometry, TIM3 expression was examined in the peripheral CD3+ CD4+ and CD3+ CD8+ T lymphocytes. Evaluation of the depressive severity in PSD patients was assessed using a 17-item Hamilton Depression Rating Scale (HAM-D-17). We used enzyme-linked immunosorbent assay (ELISA) to determine the serum concentrations of IL-1β, IL-6, IL-10, and IL-18. We further assessed the relationships between TIM3 expression, serum cytokine levels, and the HAM-D-17 scores. CD3+ CD4+ T cells reduced significantly in PSD patients compared with the NPSD patients and HC. Both NPSD patients and PSD patients had a significant increase in TIM3 expression in their peripheral CD3+ CD4+ T lymphocytes, compared with HC. In PSD patients, a higher frequency of peripheral CD3+ CD8+ T lymphocytes showed significant expression of TIM3 compared to NPSD patients and HC. High TIM3 level on peripheral CD3+ CD8+ T lymphocytes was positively associated with the HAM-D score. Patients with PSD exhibit immune dysfunction. TIM3 might contribute to the development and severity of PSD, making it a potential therapeutic target.

Pan-cancer analysis of TIM-3 transcriptomic expression reveals high levels in pancreatic cancer and interpatient heterogeneity.

T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), an immune checkpoint receptor, dampens immune function. TIM-3 antagonists have entered the clinic. We analyzed TIM-3 transcriptomic expression in 514 diverse cancers. Transcript abundance was normalized to internal housekeeping genes and ranked (0-100 percentile) to a reference population (735 tumors; 35 histologies [high≥75 percentile rank]). Ninety tumors (17.5%) demonstrated high TIM-3 expression. TIM-3 expression varied between and within tumor types. However, high TIM-3 expression was more common in pancreatic cancer (20/55 tumors, 36.4%; odds ratio, 95% confidence interval (pancreatic vs. other tumors) = 3.176 (1.733-5.818; p < 0.001, multivariate]). High TIM-3 also significantly and independently correlated with high PD-L1 (p = 0.014) and high CTLA-4 (p < 0.001) transcriptomic expression (multivariate). These observations indicate that TIM-3 RNA expression is heterogeneous, but more common in pancreatic cancer and in tumors exploiting PD-L1 and CTLA-4 checkpoints. Clinical trials with patient selection for matched immune-targeted combinations may be warranted.

TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis in the process of endometriosis

BackgroundT cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) initially discovered on the surface of Th1 cells, negatively regulates immune responses and mediates apoptosis of Th1 cells. An increasing number of studies have since shown that TIM-3 is crucial in the genesis and development of immune diseases, cancers, and chronic infectious illnesses. However, the effect of TIM-3 on endometriosis is still unknown.MethodsQuantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry were used to measure TIM-3 levels in endometriosis. Cell Counting Kit-8, 5-ethynyl-2’-deoxyuridine, colony-forming, Transwell® migration, Matrigel® invasion, and flow cytometry assays were used to explore the function of TIM-3 in vitro, and xenograft experiments in nude mice were used to assess its role in vivo. According to the RNA seq, brain-derived neurotrophic factor (BDNF) was screened. The involvement of specific proliferation-related signaling molecules was determined by transfecting a plasmid and adding an inhibitor in vivo and in vitro.ResultsTIM-3 mRNA and protein expression levels were significantly higher in eutopic and ectopic endometrial tissues than in normal endometrial tissues. By examining the effects of TIM-3 overexpression and knockdown on cell proliferation, migration, and invasion in vitro, and lesions formation in vivo, we found that the expression of TIM-3 was positively correlated with cell proliferation and clone formation in vitro, as well as lesions growth in nude mice. By adding the phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT) pathway inhibitor LY294002 and knocking down PI3K, we further verified that TIM-3 promotes proliferation in vivo and in vitro via the PI3K pathway. By transfecting the plasmid into ESC cells and gave inhibitors to endometriotic rats models, we tested that TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis.ConclusionTIM-3 can promote the proliferation of endometriosis by BDNF-mediated PI3K/AKT axis in vivo and in vitro, which may provide a new therapeutic target for the treatment of endometriosis.

Immunohistochemical Expression of Immune Checkpoints; CTLA-4, LAG3, and TIM-3 in Cancer Cells and Tumor-infiltrating Lymphocytes (TILs) in Colorectal Carcinoma.

Colorectal cancer is considered the third most prevalent cancer in both sexes. Immune checkpoint receptors that regulate T-cell response, stimulation, and development include lymphocyte activating gene 3 (LAG-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and T-cell immunoglobulin and mucin domain 3 (Tim-3). In addition, they are crucial for the advancement of cancer and tumor immune escape. This work's aim was to assess the immunohistochemistry expression of Tim-3, CTLA-4, and LAG-3 in cancer cells and tumor-infiltrating lymphocytes (TILs) in colorectal cancer (CRC) and the correlation between these markers and clinicopathological variables and survival data. This study involved 206 CRC specimens processed for CTLA-4, LAG3, and TIM-3 immunohistochemistry and correlated with the clinicopathological and survival parameters of the patients. High CTLA-4 epithelial expression was highly related to the old age group, large tumor size, low tumor-stroma ratio (TSR), high grade, advanced stage, the presence of distant metastasis (DM), perineural invasion (PNI), necrosis, lymphovascular invasion (LVI), relapse, mortality, overall survival (OS), and disease-free survival (DFS), while negative CTLA-4 TILs expression was highly linked with the presence of gross perforation, low TSR, high tumor budding (TB) score, high grade, advanced stage, the existence of lymph node (LN) metastasis, DM, necrosis, LVI, PNI, DFS, mortality, and OS. Positive LAG-3 TILs expression was highly correlated with large tumor size, gross perforation, low TSR, high TB score, high grade, advanced phase, the presence of LN, necrosis, LVI, PNI, relapse DFS, mortality, and OS. High Tim-3 epithelial expression was extremely linked with low TSR, advanced phase, the presence of LN, LVI, PNI, relapse, DFS, mortality, and OS, while positive Tim-3 TILs expression was related to gross perforation, low TSR, high TB score, advanced stage, the presence of LN, DM, necrosis, relapse, DFS, mortality, and OS. The patients' poor prognosis may be related to the immunohistochemistry expression of LAG-3, Tim-3, and CTLA-4 in CRC cancer tissue and TILs. Poor patient consequences can result from the CTLA-4, Tim-3, and LAG-3 co-expression, but CTLA-4 TILs' expression of these proteins may inhibit the growth of tumors.

Activated Tim-3/Galectin-9 participated in the development of multiple myeloma by negatively regulating CD4 T cells

ABSTRACT The interaction between Tim-3 on T cells and its ligand Galectin-9 negatively regulates the cellular immune response. However, the regulation of Tim-3/Galectin-9 on CD4 T cell subsets in multiple myeloma (MM) remains unclear. The aim of this study was to investigate the relationship between the regulation of CD4 T cell subsets by the Tim-3/Galectin-9 pathway and clinical prognostic indicators in MM. Tim-3/Galectin-9 were detected by flow cytometry, PCR and ELISA in 60 MM patients and 40 healthy controls, and its correlation with clinical prognostic parameters was analyzed. The expressions of Tim-3 on CD4 T cells, Galectin-9 mRNA in PBMC and level of Galectin-9 protein in serum were significantly elevated in MM patients, especially those with poor prognostic indicators. In MM patients, Tim-3 was highly expressed on the surfaces of Th1, Th2, and Th17 cells, but lowly expressed on Treg. Moreover, level of cytokine IFN-γ in serum was negatively correlated with Tim-3+Th1 cell and Galectin-9mRNA, Galectin-9 protein level. In addition, cell culture experiments showed that the anti-tumor effect and the ability to secrete IFN-γ were restored by blocking the Tim-3/Galectin-9 pathway. In MM patients, Tim-3/Galectin-9 is elevated and associated with disease progression, by inhibiting the cytotoxic function of Th1, and also promoting Th2 and Th17 to be involved in immune escape of MM. Therefore, Tim-3/Galectin-9 may serve as a new immunotherapeutic target for MM patients.

Combined TIM-3 and PD-1 blockade restrains hepatocellular carcinoma development by facilitating CD4+ and CD8+ T cell-mediated antitumor immune responses.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies. However, ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma (HCC) patients due to the complex pathological mechanisms of HCC. To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model, aiming to identify more effective immunotherapies and provide more treatment options for HCC patients. The levels of PD-1 and TIM-3 on CD4+ and CD8+ T cells from tumor tissues, ascites, and matched adjacent tissues from HCC patients were determined with flow cytometry. An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody (mAb) and/or anti-PD-1 mAb. Tumor growth in each group was measured. Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors. The percentage of CD4+ and CD8+ T cells in tissue samples from mice was tested with flow cytometry. The percentages of PD-1+CD8+, TIM-3+CD8+, and PD-1+TIM-3+ CD8+ T cells was accessed by flow cytometry. The levels of the cytokines including tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6, and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits. We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+ and CD8+ T cells isolated from tumor tissues and ascites of HCC patients. TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight, while combined blockade had more substantial anti-tumor effects than individual treatment. Then we showed that combined therapy increased T cell infiltration into tumor tissues, and downregulated PD-1 and TIM-3 expression on CD8+ T cells in tumor tissues. Moreover, combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ, and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues. Thus, we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model. Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+ T cell-mediated antitumor immune responses.

Prognostic value of T cell immunoglobulin and mucin-domain containing-3 expression in upper gastrointestinal tract tumors: A meta-analysis.

There is a lack of robust prognostic markers for upper gastrointestinal (GI) tract cancers, including esophageal, gastric, and esophagogastric junction cancers. T cell immunoglobulin and mucin-domain containing-3 (TIM3) plays a key immunomodulatory role and is linked to the prognosis of various cancers. However, the significance of TIM3 in upper GI tract tumors is still uncertain. To investigate the prognostic value of TIM3 expression in upper GI tract tumors. A literature search was conducted on the PubMed, Embase, and Web of Science databases for relevant studies published until June 2023. After screening and quality assessment, studies that met the criteria were included in the meta-analysis. Statistical methods were used for the pooled analysis to assess the association of TIM3 expression in upper GI tract tumors with the prognosis and clinicopathological parameters. The results were reported with the hazard ratio (HR) and 95% confidence interval (CI). Nine studies involving 2556 patients with upper GI tract cancer were included. High TIM3 expression was associated with a worse prognosis in upper GI tract cancer (HR: 1.17, 95%CI: 1.01-1.36). Positive expression of TIM3 in gastric cancer was correlated with the T and N stage, but the difference was not statistically significant. However, TIM3 overexpression was significantly correlated with the TNM stage (odds ratio: 1.21, 95%CI: 0.63-2.33; P < 0.05). TIM3 expression showed no association with the other clinicopathological parameters. High expression of TIM3 in the upper GI tract cancer is associated with a worse prognosis and advanced T or N stages, indicating its potential value as a prognostic biomarker. These findings may provide a basis for the personalized treatment of upper GI tract cancers.

Itaconate promotes hepatocellular carcinoma progression by epigenetic induction of CD8+ T-cell exhaustion

Itaconate is a well-known immunomodulatory metabolite; however, its role in hepatocellular carcinoma (HCC) remains unclear. Here, we find that macrophage-derived itaconate promotes HCC by epigenetic induction of Eomesodermin (EOMES)-mediated CD8+ T-cell exhaustion. Our results show that the knockout of immune-responsive gene 1 (IRG1), responsible for itaconate production, suppresses HCC progression. Irg1 knockout leads to a decreased proportion of PD-1+ and TIM-3+ CD8+ T cells. Deletion or adoptive transfer of CD8+ T cells shows that IRG1-promoted tumorigenesis depends on CD8+ T-cell exhaustion. Mechanistically, itaconate upregulates PD-1 and TIM-3 expression levels by promoting succinate-dependent H3K4me3 of the Eomes promoter. Finally, ibuprofen is found to inhibit HCC progression by targeting IRG1/itaconate-dependent tumor immunoevasion, and high IRG1 expression in macrophages predicts poor prognosis in HCC patients. Taken together, our results uncover an epigenetic link between itaconate and HCC and suggest that targeting IRG1 or itaconate might be a promising strategy for HCC treatment.

T-cell Immunoglobulin and Mucin Domain 3 in Circulating Monocytes as a Novel Biomarker for Coronary Artery Disease.

The diagnostic role of T-cell immunoglobulin and mucin domain 3 (Tim-3) expression levels in circulating monocytes in coronary artery disease (CAD) remains to be determined. Here, we enrolled of 265 patients and isolated circulating monocytes from the blood of all participants. We found that the Tim-3 expression levels in monocytes were lower in CAD patients than in the control group. Spearman correlation analysis verified that the Tim-3 levels in monocytes were negatively correlated with the Gensini score and the number of coronary vessels. Multivariate logistic regression analysis showed that the Tim-3 levels in circulating monocytes were negatively correlated with CAD, severe CAD, and three-vessel CAD. The ROC curve showed that Tim-3 possessed high diagnostic value for CAD, severe CAD, and three-vessel CAD, with CAD prediction being the most significant of these values. In conclusion, Tim-3 in circulating monocytes is a novel biomarker for CAD.

PD-1 Expression on Intratumoral Regulatory T Cells Is Associated with Lack of Benefit from Anti-PD-1 Therapy in Metastatic Clear-Cell Renal Cell Carcinoma Patients.

Programmed cell death protein 1 (PD-1) expression on CD8+TIM-3-LAG-3- tumor-infiltrating cells predicts positive response to PD-1 blockade in metastatic clear-cell renal cell carcinoma (mccRCC). Because inhibition of PD-1 signaling in regulatory T cells (Treg) augments their immunosuppressive function, we hypothesized that PD-1 expression on tumor-infiltrating Tregs would predict resistance to PD-1 inhibitors. PD-1+ Tregs were phenotyped using multiparametric immunofluorescence in ccRCC tissues from the CheckMate-025 trial (nivolumab: n = 91; everolimus: n = 90). Expression of CD8, PD-1, TIM-3, and LAG-3 was previously determined (Ficial and colleagues, 2021). Clinical endpoints included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). In the nivolumab (but not everolimus) arm, high percentage of PD-1+ Tregs was associated with shorter PFS (3.19 vs. 5.78 months; P = 0.021), shorter OS (18.1 vs. 27.7 months; P = 0.013) and marginally lower ORR (12.5% vs. 31.3%; P = 0.059). An integrated biomarker (PD-1 Treg/CD8 ratio) was developed by calculating the ratio between percentage of PD-1+Tregs (marker of resistance) and percentage of CD8+PD-1+TIM-3-LAG-3- cells (marker of response). In the nivolumab (but not everolimus) arm, patients with high PD-1 Treg/CD8 ratio experienced shorter PFS (3.48 vs. 9.23 months; P < 0.001), shorter OS (18.14 vs. 38.21 months; P < 0.001), and lower ORR (15.69% vs. 40.00%; P = 0.009). Compared with the individual biomarkers, the PD-1 Treg/CD8 ratio showed improved ability to predict outcomes to nivolumab versus everolimus. PD-1 expression on Tregs is associated with resistance to PD-1 blockade in mccRCC, suggesting that targeting Tregs may synergize with PD-1 inhibition. A model that integrates PD-1 expression on Tregs and CD8+TIM-3-LAG-3- cells has higher predictive value.

Significance of Tim-3 and Its Ligand Galectin-9 in Th1/Th2 Imbalance in Patients with Multiple Myeloma

To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM). 55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4+T cells, the proportion of Th1, Th2, Tim-3+Th1 and Tim-3+Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3+Th1/Tim-3+Th2 ratio and corresponding clinical features were analyzed. Compared with the control group, the expression of Tim-3 on CD4+T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3+Th1 cells, Tim-3+Th2 cells and Tim-3+Th1/Tim-3+Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3+CD4+T cells (r=0.663), Tim-3+Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3+Th1/Tim-3+Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632). These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3+Th1/Tim-3+Th2 is associated with poor clinical prognosis.

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

BackgroundAdoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long-term protection due to T cell exhaustion induced by chronic...

Abstract A034: VV-iVP55: A Superior Vaccinia Virus Platform with Enhanced Oncolytic Potency and Immunogenicity

Abstract This research introduces VV-iVP55, a conditionally replicating vaccinia virus platform, as a promising alternative to thymidine kinase (Tk) deleted oncolytic vaccinia viruses. The unique feature of VV-iVP55 is its growth control using doxycycline, a trait not found in conventional Tk-deleted viruses. Laboratory studies revealed that VV-iVP55 achieves higher virus titers and lower cell viability in B16-F10 and MC38 syngeneic models compared to Tk-deleted viruses. Notably, VV-iVP55 demonstrated lower toxicity in immunocompromised mice upon doxycycline withdrawal, unlike Tk-deleted viruses that showed significant toxicity. In vivo efficacy studies further highlighted the superiority of VV-iVP55 over Tk-deleted viruses in CT26 and B16 subcutaneous models, as well as MC38 subcutaneous and intraperitoneal models. An immune microenvironment analysis conducted six days post-intratumoral injection revealed beneficial alterations induced by VV-iVP55, such as increased T and B cell content in the tumor and reduced MDSCs in both the tumor and spleen. Moreover, VV-iVP55 was linked to a significant change in T cell differentiation. Unlike the PBS and Tk viruses, which showed almost complete differentiation towards effector T cells, VV-iVP55 maintained a substantial population of T cells in naive and transition phases, significantly in both CD4 and CD8 T cells. This likely led to lower expression of exhaustion markers PD1, TIM3, TIGIT, and LAG3, suggesting less exhaustion and more potential for immune response. In summary, VV-iVP55 emerges as a powerful alternative to Tk-deleted oncolytic vaccinia viruses, offering enhanced immunogenicity, controlled toxicity, and preserved oncolytic properties. Citation Format: Reza Rezaei, Taha Azad, Julia Petryk, John C. Bell. VV-iVP55: A Superior Vaccinia Virus Platform with Enhanced Oncolytic Potency and Immunogenicity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A034.

Immune checkpoint status and exhaustion-related phenotypes of CD8+ T cells from the tumor-draining regional lymph nodes in breast cancer.

Functional status of T cells determines the responsiveness of cancer patients to immunotherapeutic interventions. Even though T cell-mediated immunity is inaugurated in the tumor-adjacent lymph nodes, peripheral blood has been routinely sampled for testing the immunological assays. The purpose of this study is to determine the immune checkpoint molecule expression and the exhaustion-related phenotype of cytotoxic T cells in the regional lymph nodes from breast cancer patients. Multicolor immunophenotyping was used to determine the expression of PD-1, TIM-3, LAG3, CTLA-4, CCR7, CD45RO, CD127, CD25, CXCR5, and ICOS molecules on CD3+ CD4- CD56- CD8+ cytotoxic T cells freshly obtained from the lymph nodes and the peripheral blood samples of the breast cancer patients. The results were assessed together with the clinical data. A population of cytotoxic T cells was noted with high PD-1 and CXCR5 expression in the lymph nodes of the breast cancer patients. Co-expression of PD-1, CXCR5, TIM-3, and ICOS indicated a follicular helper T cell (Tfh)-like, exhaustion-related immunophenotype in these cytotoxic T cells. Only a minor population with CTLA-4 and LAG3 expression was noted. The PD-1+ CXCR5+ cytotoxic T cells largely displayed CD45RO+ CCR7+ central memory markers. The amount of CXCR5-expressing PD-1- cytotoxic T cells was elevated in the lymph nodes of the patients. The regional lymph nodes of breast cancer patients harbor Tfh-like exhausted cytotoxic T lymphocytes with high PD-1 and TIM-3 checkpoint molecule expression. The immunological conditions in the regional lymph nodes should be implicated for immune checkpoint immunotherapy (ICI) of cancer.

Upregulation of TIM3 and reduced expression of PD-1 on immune cell subsets in advanced prostate cancers

Abstract Introduction/Objective Although most prostate cancers behave in an indolent manner, a small proportion is highly aggressive. Both primary and advanced prostate cancer is widely known as a non-inflamed cancer that is characterized by a paucity of immune infiltration. Methods/Case Report To assess the spatial interplay of more than 30 TIM3, CTLA-4, PD-1/-L1 expressing leukocyte subpopulations in 453 prostate cancers, tissue microarrays were stained with 21 antibodies using our BLEACH&amp;STAIN multiplex fluorescence immunohistochemistry approach and analyzed using a deep learning-based image analysis framework. Results (if a Case Study enter NA) The immune cell density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, FOXP3+ regulatory T-cells, M1/ M2 macrophages, as well as CD11c+ dendritic cells increased consistently along with the Gleason grade in primary prostate cancer (p≤ 0.034 each). In recurrent prostate cancers under therapy, the density of FOXP3+regulatory T-cells and M1 macrophages further increased, while the density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, as well as CD11c+ dendritic cells decreased (p≤0.017 each). Although the immune checkpoint expression of TIM3 on T-cell subsets, macrophages and dendritic cells was upregulated in advanced/ recurrent tumors, the expression level of PD-1 was downregulated in all analyzed T-cell subsets. Conclusion Although prostate cancer is a generally considered a low inflamed tumor, the degree of tumor infiltration by various immune cell subtypes increases markedly along with tumor progression and in recurrent tumors under therapy. Taken together, these data suggest that the evaluation of the spatial distribution of immune cell types along with their immune checkpoint expression can provide relevant clinical information in prostate cancer.

Oncogenic RAS Signaling Promotes an Immunosuppressive Niche in Acute Lymphoblastic Leukemia

Emerging evidence suggests that genomic aberrations in cancer cells may impact the composition of the tumor microenvironment leading to a tumor-specific supporting niche. Previous genomic profiling studies have identified somatic alterations in RAS pathway genes that drive treatment resistance in B-cell acute lymphoblastic leukemia (B-ALL): yet it is unknown whether such genetic events may specifically hijack the leukemic niche. Here, we analyzed bulk RNA-sequencing data of bone marrow mononuclear cells from 185 pediatric B-ALL patients entering our center and performed mutation calling of RAS pathway genes, namely KRAS, NRAS, BRAF and PTPN11. Transcriptomic analysis revealed attenuated immune responses and decreased T cell activation in samples harboring RAS mutations (n= 62) compared with wild type cases (n= 123). To further explore this finding at single-cell resolution, we sorted bone marrow mononuclear cells from 8 primary B-ALL pediatric patient samples (4 RAS mutant and 4 wild type), enriching on the hematopoietic and non-hematopoietic compartments, followed by single-cell RNA-sequencing using the 10x Genomics platform. Twelve cell populations were identified by the expression of bona fide markers, mapping different immune and stromal cells in the ALL landscape. Specifically, the RAS-mutant microenvironment was defined by dysfunctional CD8+ T-cells, characterized by high expression of TIM-3, TIGIT and LAG-3, as well as regulatory T cells, displaying expression of CTLA-4, ICOS and FOXP3, suggesting a RAS-mediated immunosuppressive mechanism. In light of these changes, we examined flow cytometry data of bone marrow mononuclear cells collected at diagnosis from our cohort of 185 patient samples. Whereas the overall proportion of lymphoid subsets (T, B and NK cells) was not significantly changed among RAS mutant and wild type groups, we detected a decrease in myeloid subsets (neutrophils, eosinophils, and monocytes) in RAS mutant ALL, which was more prominent in monocytes. Cross comparison of the transcriptional state of monocytes in our single-cell data unravel that monocytes in the RAS mutant niche displayed a non-classical phenotype, characterized with high expression of the immune checkpoint TIM-3, which is a major driver of T cell exhaustion. Taken together, the bone marrow microenvironment of ALL patients harbouring oncogenic RAS lymphoblasts is defined by dysfunctional immune cell populations. Further studies on the cellular dynamics in the niche will provide the basis for the development of therapeutic opportunities to target the immune micro-environment in RAS mutant ALL.

Toll-like Receptor-9 Agonist Reverses T Cell Exhaustion in a Murine Model of Pediatric B-ALL

Despite significant treatment advances, the poor long-term prognosis for children with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) persists. The application of immune cell-based therapies, such as chimeric antigen receptor-expressing T cells and bispecific T-cell engagers, has improved progression-free survival for many B-ALL patients. However, the majority of these patients still experience disease relapse. An improved understanding of the mechanisms that impair T cell function in B-ALL patients could inform strategies to further improve outcomes. Recent studies have revealed T cell exhaustion, especially as indicated by PD-1 and Tim- 3 expression, to be predictive of disease relapse and overall survival in B-ALL. However, the precise contribution of T cell dysfunction to B-ALL development remains unclear. In this study, we explore the biology that leads to impaired T cell function and investigate potential approaches to restore it using both transgene-driven and syngeneic leukemia cell transplant models of B-ALL. First, we used the Emu-Ret transgenic mouse model of hyperdiploid B-ALL to investigate the induction of exhaustion-associated marker expression (PD-1, CTLA-4 and Tim-3) on CD4 and CD8 T cells. Longitudinal analysis of marker expression in various settings revealed that an upregulation of PD-1 and Tim-3, but not CTLA-4, on T cells occurred only in the presence of leukemia cells, not during the extended, nascent preleukemia phase. This suggests that T cell exhaustion occurs exclusively during the established malignancy phase, highlighting the dynamic nature of T cell responses as disease progresses. Next, B-ALL cells from moribund Emu-Ret mice were transplanted into 4-6-week-old wild type BALB/c mice and markers of T cell exhaustion were measured as disease progressed. Expression of PD-1 and Tim-3 on T cells correlated with disease progression. Notably, a significant increase in these markers was already detectable at low leukemia burden. These results indicate that T cell exhaustion is not simply a reflection of high disease burden and confirm the differing abilities of preleukemic and leukemic cells to affect T cell phenotype. Previously, we have demonstrated the ability of toll-like receptor (TLR) ligands to induce immune control over B-ALL cells. Thus, we investigated the impact of CpG oligodeoxynucleotides (CpG ODN), a ligand for TLR-9, on B-ALL depletion and T cell dysfunction. The treatment of leukemia bearing mice with CpG ODN led to a decrease in PD-1 and Tim-3 expression on T cells, which was also associated with a reduction in leukemia burden. We are currently investigating the functional implications of these expression changes using in vitro assays of T cell proliferation, cytokine production, and cytotoxicity. Additionally, we are also assessing if anti-tumour control by T cells can be revived in vivo, by comparing B-ALL progression and mouse survival after administration of antibodies against exhaustion markers, with or without CpG ODNs as adjuvant therapy. Overall, this study reveals that the functional impact of interactions between B-ALL cells and the immune system evolves during leukemia development. These insights could inform therapeutic strategies to overcome B-ALL immune evasion mechanisms and enhance patient outcomes.

Role of Cell Therapy Product Characteristics in Patient Response to TAK-940 (CD19(T2)28z1xx Chimeric Antigen Receptor (CAR) T): Biomarker Analyses from a Phase I Study in Adult Patients with Relapsed or Refractory B-Cell Malignancies

TAK-940 (CD19(T2)28z1XX Chimeric Antigen Receptor (CAR) T) is an autologous CD19 CAR T cell therapy utilizing an optimized 1XX signaling domain substituting for the natural CD3ζ immunoreceptor tyrosine-based activation motifs. This 1XX design extends the functional persistence of CAR T cells with increased potency in mice (Feucht J et al. Nat Med 2019). TAK-940 is being evaluated in a Ph1 trial in patients with Relapsed or Refractory B-cell malignancies (NCT04464200) with demonstrated early signals of clinical efficacy (Park J et al. ASH 2022). The present study aims to provide a better understanding of the clinical performance of CD19(T2)28z1XX CAR T cells by in depth characterization of the cellular products and patient samples from this Ph1 trial. A total of 28 subjects were enrolled and treated in the dose escalation and expansion cohorts of the Ph1 trial. Responses were observed across all dose levels (25x10 6 to 200x10 6 CAR T cells), achieving ORR 82%, CR 71%. Immunophenotyping by flow cytometry was conducted on patient apheresis material, un-transduced cells, transduced cellular drug product and peripheral blood samples from infused subjects. Circulating cytokine profiling was performed on patient serum samples collected pre and post treatment with TAK-940. In the transduced cellular drug products, CAR+ and CAR- CD8+ central memory and transitional memory T cells were more abundant than naïve, stem cell memory or terminal effector populations. This elevation in central and transitional memory T cells seen in the drug product was not observed in the patient apheresis material, indicating that this effect is likely driven by the production process. CD57 and TIM3 expression in both CD4+ and CD8+ CAR+ T cells in the drug product showed a positive association with clinical response, while CTLA4 or LAG3 expression independently was negatively associated with response. The drug product also contained low numbers of dysregulated/exhausted T cells, with no observable difference between responders and non-responders. Interestingly, in the starting apheresis material, increased expression of TIM3 as well as decreased expression of either LAG3 or KLRG1 in CD8 T cells, independently associated with responses. Current analysis is based on associations with complete response. Additional analyses to be reported will include associations with ORR and durability of responses. These findings suggest potential utility of these T cell markers in predicting patient response and warrant evaluation in a larger population. Although CD57 expression has been associated with a senescent phenotype, we hypothesize that the observed increase in CD57 expression in CD4+ and CD8+ CAR+ T cells in the product may indicate higher cytotoxic potential of those cells. Increase in CD57+ CAR+ T cell subsets has been previously associated with durable CRs in large B cell lymphoma patients treated with axi-cel (Good Z et al. Nat Med 2022). Additional analyses to further characterize the CD57+ cell populations and establish their role in clinical responses to CAR T cell therapy with the 1XX CAR design are in progress. The increased expression of CD57 and the activation marker TIM3, and decreased expression of inhibitory receptors CTLA4 and LAG3 in the drug product from responders, together with the robust clinical responses observed with doses as low as 25x10 6 CAR T cells, suggest that the product consists of highly potent T cells, in accordance with the preclinical results obtained with the 1XX CAR. Low levels of circulating cytokines were observed following treatment with TAK-940, including IFNg, IL-6, IL-10, indicating a safe and tolerable CAR T product consistent with the observed clinical safety profile of TAK-940. Correlative analysis of T cell immunophenotypes observed in the product to clinical parameters including durability of responses, metabolic tumor volume/tumor burden, and induction of cytokines and chemokines is ongoing and will be reported. This will further contribute to our understanding of CD19 CAR T cells endowed with a 1XX signaling domain and potentially support the identification of product and/or apheresis biomarkers that may predict patient response.