CD8 Tumor-infiltrating Lymphocytes Research Articles

IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

Bispecific Tcell engagers (TCEs) show promising clinical efficacy in blood tumors, but their application to solid tumors remains challenging. Here, we show that Fc-fused IL-7 (rhIL-7-hyFc) changes the intratumoral CD8 Tcell landscape, enhancing the efficacy of TCE immunotherapy. rhIL-7-hyFc induces a dramatic increase in CD8 tumor-infiltrating lymphocytes (TILs) in various solid tumors, but the majority of these cells are PD-1-negative tumor non-responsive bystander Tcells. However, they are non-exhausted and central memory-phenotype CD8 Tcells with high Tcell receptor (TCR)-recall capacity that can be triggered by tumor antigen-specific TCEs to acquire tumoricidal activity. Single-cell transcriptome analysis reveals that rhIL-7-hyFc-induced bystander CD8 TILs transform into cycling transitional Tcells by TCE redirection with decreased memory markers and increased cytotoxic molecules. Notably, TCE treatment has no major effect on tumor-reactive CD8 TILs. Our results suggest that rhIL-7-hyFc treatment promotes the antitumor efficacy of TCE immunotherapy by increasing TCE-sensitive bystander CD8 TILs in solid tumors.

Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell-Mediated Cytotoxicity.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell-mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation invivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

PD1 and CD39 expression on tumor infiltrating lymphocytes in patients with gastric cancer.

377 Background: Activation and clonal expansion of tumor antigen-specific T cells in cancer patients are crucial for immunotherapies. Recent reports indicate that CD39 is a marker to distinguish tumor-reactive CD8 T cell populations from bystander populations. In gastric cancer (GC), anti-CD39 antibody, anti-PD-1 antibody, and FOLFOX combination therapy may be effective therapy at AACR 2022. Methods: We retrospectively reviewed the data of 62 GC patients who underwent gastrectomy at our hospital (2017 - 2018) and examined their rejected specimen’s immune-microenvironment using immunohistochemistry. Then we prospectively enrolled 16 GC patients. Their tumor tissue and tumor-infiltrating lymphocytes (TILs) were extracted from surgical specimens, these fractions were subjected to the RNA-seq analysis ( n = 3), CD107a degranulation assay ( n = 7) and 3H-Thymidine incorporation assay ( n = 7). Results: The densities of CD39+CD8 TILs positively correlated with the densities of CD4 T cells and B-cells in GC tissues ( P = 0.01, < 0.01, respectively). Furthermore, higher densities of CD39+CD8 TILs in GC patients were associated with better overall survival ( P < 0.01). We classified TILs into four groups based on expression levels of the PD-1 and CD39. The proportion of CD39+CD8 T cells was significantly higher in GC tissues than their PBMCs ( n = 13) and non-cancerous tissues ( n = 4). CD39+PD-1+CD8 (DP) TILs in primary tumors were elevated expression of multiple inhibitory receptors such as LAG3, TIGIT and reduced expression of IL-2 by RNA-seq analysis. T cells expanded in vitro from DP-TILs ( n = 5) efficiently recognized autologous tumor cells in CD107a assay compared with other populations, and considered to be tumor-reactive T cells fraction. However, since these DP TILs in primary tumors show exhausted phenotypes, capacities to kill tumor are expected to be reduced in the tumor-microenvironment (TME). By adding ATP to the DP-TILs in vitro at the concentration reported in the TME, we observed reduction of cell proliferation by 3H-thymidene incorporation assay and of degranulation by CD107a assay, and we observed recovery of these changes by adding anti-CD39 inhibitory antibody. Conclusions: DP-TILs may have superior T cell receptors recognizing tumor-antigens and contribute to tumor eradication, especially when administrated anti-CD39 antibody.

Metformin Alters Tumor Immune Microenvironment, Improving the Outcomes of Breast Cancer Patients With Type 2 Diabetes Mellitus.

This study investigated the clinical effect of metformin on breast cancer patients with preexisting type 2 diabetes mellitus (T2DM). We analyzed 177 patients with T2DM who underwent breast cancer surgery and assessed tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) in patients who underwent tumor resection with or without metformin treatment using multiplex immunohistochemistry (IHC). Patients who received metformin either pre- or postoperatively exhibited reduced distant organ recurrence and improved postoperative recurrence-free survival compared to those of patients who did not. Additionally, in a subgroup of 40 patients receiving preoperative systemic therapy, metformin treatment was associated with increased rates of pathological complete response. IHC analysis revealed significantly lower levels of cluster of differentiation (CD) 68(+) CD163(+) M2-type TAMs (p < 0.01) but higher CD3(+) and CD8(+) TIL densities in the metformin-treated group compared with the same parameters in those without metformin treatment, with a significant difference in the CD8(+)/CD3(+) TIL ratio (p < 0.01). Despite the constraints posed by our small sample size, our findings suggest a potential role for metformin in modulating the immunological microenvironment, which may contribute to improved outcomes in diabetes patients with breast cancer.

Abstract A004: Immune response to sub-ablative radiation before surgery in pleural mesothelioma – Results from the SMARTER trial

Abstract Introduction. We developed a new treatment paradigm in pleural mesothelioma using a short course of sub-ablative hemithoracic radiation followed by surgery and observed encouraging results in epithelioid subtypes. We therefore performed a phase I trial to determine the safety and immunological benefit of 3 fractions of radiation before surgery. Methods. The trial, Surgery for Mesothelioma After Radiation Therapy using Extensive pleural Resection (SMARTER), was designed as a 3+3 phase I trial with incremental hemithoracic radiation dose up to 18 Gy in 3 fractions (background dose) combined with a boost of at least 39 Gy to the gross disease (boost dose). Patients with histologically confirmed stage I-III pleural mesothelioma with tumor size of at least 2 cm in diameter on CT scan were eligible for the study. Surgery was performed 7-14 days after the last fraction of radiation. CyTOF and Luminex were performed on peripheral blood at baseline pre-radiation, post-radiation at the time of surgery, and during follow-up within 3 months from surgery. Tumor was collected at the time of surgery from the sites of low dose radiation (background, T1) and high dose radiation (boost, T2) and compared to pre-radiation (baseline, T0) using single cell RNA sequencing (scRNA seq). CD8 tumor infiltrating lymphocytes (TILs) was determined by immunostaining. The results were validated in a second cohort of patients treated off trial. Results. A total of 12 patients were included in the trial with all patients safely proceeding to surgery after a background radiation dose of 0 Gy, 6 Gy, 12 Gy and 18 Gy. Total radiation boost dose ranged from 39 Gy to 54 Gy. The results were validated in a second cohort of 12 patients. Circulating IL-15, FLT-3L, and CXCL10 significantly increased at the time of surgery compared to baseline. CXCL10 continued to rise during follow-up and was associated with better survival at 1 year (86% vs 17% in the absence of CXCL10 upregulation, p=0.005). The peak of circulating IL-15 correlated with CD8 TILs in the resected tumor specimen. scRNA seq on tumor samples demonstrated that monocytes expressed IL-15 and IL-15Ra, and increased after radiation. Pseudotime trajectory analysis showed an evolution from classical to non-classical monocytes with a shift from M2-like to M1-like phenotype. This was associated with reinvigoration of cytotoxic CD8 TILs and expansion of GITR negative NK cells. CyTOF analysis on peripheral blood confirmed the upregulation of circulating monocytes followed by upregulation of CD8 effector memory T cells, which progressively decreased before tumor recurrence. Tumors with larger populations of IFN-TAM and CD8 ISG were characterized by high CXCL10 expression at baseline and a lack of CXCL10 upregulation after radiation associated with an absence of CD8 T cell reinvigoration and poor survival. Conclusions. These results demonstrate the immunogenic benefit and clinical safety of sub-ablative radiation before surgery in pleural mesothelioma. The lack of benefit was observed in tumors expressing high CXCL10 at baseline. Citation Format: Marc de Perrot, Amin Zia, Licun Wu, Giselle Boukhaled, Ramy Gadalla, Ben Wang, David Brooks, Jeffrey Man, Trevor Pugh, Prodipto Pal, Fatemeh Zaeimi, Hebe Chu, Ming-Sound Tsao, Penelope Bradbury, John Cho. Immune response to sub-ablative radiation before surgery in pleural mesothelioma – Results from the SMARTER trial [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 A004.

Abstract B005: Molecular, metabolic and functional CD4 T cell paralysis in lymph node impedes tumor control

Abstract Introduction: CD4 T cells are important in anti-tumor immunity, yet the regulation of CD4 tumor-specific T (TTS) cells during cancer development is still unclear. So far, studies have primarily focused on studying bulk CD4 tumor-infiltrating lymphocytes (TILs) with unknown antigen specificities, or transferring in vitro activated CD4 T cells as adoptive cell therapy (ACT), which fail to directly address how naïve CD4 TTS cells are activated and then differentiate in vivo in response to tumor progression. Therefore, studies addressing these questions will be critical to understand the state of anti-tumor CD4 T cells available for restorative therapies. Methods: To address these key questions, we developed two murine tumor cell lines, PyMG (an orthotopic breast tumor) and MC38GP (an adenocarcinoma tumor) expressing the LCMV glycoprotein 1-100 sequence (LCMV-GP1-100). The sequence contains an MHCII-epitope GP61-80 and an MHCI-epitope GP33-43, that can be recognized by TCR transgenic CD4 T cells (SMARTAs) and CD8 T cells (P14s), enabling direct in vivo identification of CD4 TTS cells. Results: We demonstrate that following tumor initiation, CD4 TTS cells are initially primed in the tumor draining lymph node (dLN), but rapidly frozen into a “paralyzed” state. The paralyzed state freezes CD4 TTS cell clonal expansion, impairs differentiation, limits IFNγ production, and redirects metabolic circuits, which together impede tumor control. Transcriptional programming of CD4 TTS cell paralysis is unique to the tumor setting and distinct from well-defined effector and exhaustion programming. In comparison, CD8 TTS cells do not manifest the proliferation defect. Paralysis is actively maintained throughout cancer progression by a functional interplay of regulatory T cells (Tregs) and CTLA4. Depleting Tregs induces robust proliferation of CD4 TTS cells, yet concurrently pushes the proliferated cells to become highly suppressive tumor-specific induced Tregs (iTregs). The differentiation of iTregs is dependent on the CTLA4 expression on CD4 TTS cells. Alleviating suppressive signals from both Tregs and CTLA4 is required to restore CD4 TTS cells proliferation, reduce iTreg differentiation and promote T helper cell differentiation. Overcoming CD4 TTS cell paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 TTS cells to favor tumor progression. Conclusion: Our study demonstrates that during early stages of cancer, the CD4 TTS cell development is quickly paralyzed at multiple levels of functional development. Overcoming their paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 TTS cells to favor tumor progression. Citation Format: Mengdi Guo, Diala Abd-Rabbo, Bruna Bertol, Madeleine Carew, David G Brooks. Molecular, metabolic and functional CD4 T cell paralysis in lymph node impedes tumor control [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 B005.

Characteristics and anatomic location of PD-1+TCF1+ stem-like CD8 T cells in chronic viral infection and cancer

CD8 T cells play an essential role in antitumor immunity and chronic viral infections. Recent findings have delineated the differentiation pathway of CD8 T cells in accordance with the progenitor-progeny relationship of TCF1+ stem-like and Tim-3+TCF1- more differentiated T cells. Here, we investigated the characteristics of stem-like and differentiated CD8 T cells isolated from several murine tumor models and human lung cancer samples in terms of phenotypic and transcriptional features as well as their location compared to virus-specific CD8 T cells in the chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. We found that CD8 tumor-infiltrating lymphocytes (TILs) in both murine and human tumors exhibited overall similar phenotypic and transcriptional characteristics compared to corresponding subsets in the spleen of chronically infected mice. Moreover, stem-like CD8 TILs exclusively responded and produced effector-like progeny CD8 T cells in vivo after antigenic restimulation, confirming their lineage relationship and the proliferative potential of stem-like CD8 TILs. Most importantly, similar to the preferential localization of PD-1+ stem-like CD8 T cells in T cell zones of the spleen during chronic LCMV infection, we found that the PD-1+ stem-like CD8 TILs in lung cancer samples are preferentially located not in the tumor parenchyma but in tertiary lymphoid structures (TLSs). The stem-like CD8 T cells are present in TLSs located within and at the periphery of the tumor, as well as in TLSs closely adjacent to the tumor parenchyma. These findings suggest that TLSs provide a protective niche to support the quiescence and maintenance of stem-like CD8 T cells in the tumor.

Intermittent fasting against cancer development and progression: Highlighting potential anticancer molecular mechanisms.

Intermittent fasting (IF) diets have been popular since the last few decades because of their provable clinical efficiency on weight control of the subjects. These diet types are generally safe, resulting in health promoting effects against several human diseases like cardiovascular diseases, diabetes mellitus, neurogenerative disorders and cancer. To review whether IF can act against cancer development and progression, highlighting potential anticancer molecular mechanisms in clinical studies. Applied summarization of the available clinical studies investigating the effectiveness of IF against cancer development and progression and cancer-induced indicators. Scientific databases, e.g., PubMed, and Scopus, were comprehensively searched using relative words to identify in vivo and in vitro data, as well as clinical studies. IF seems to exert health-promoting effects in cancer patients through induction of autophagy, which enhances the in vivo suppression of tumor development, by chemotherapy. IF provokes tumors to chemotherapy and defends the normal cells from its adverse side effects, increasing the immune response. In addition, it enhances the cytotoxic CD8(+) tumor-infiltrating lymphocytes and the bone marrow lymphoid progenitor cells, delaying the cancer progression. IF reduces oxidative stress via repression of translation and induces cellular apoptosis. Fasting exerts anti-aging properties modulating the secretion of IGF-1, IGFBP-1, glucose, and insulin while, at the same time, it integrates cell adaptive responses and activates cell signaling pathways which stimulates antioxidant defenses, DNA repairment, control of protein quality, mitochondrial synthesis while decreasing inflammation. IF appears to exert health promoting effects against cancer development and progression, suppressing several kinds of cancer. There are well-recognized and not well-recognized molecular processes accentuating its anticancer outcomes; however, well-designed clinical trials and further molecular studies are strongly recommended.

Concomitant Expression of CD39, CD69, and CD103 Identifies Antitumor CD8+ T cells in Breast Cancer Implications for Adoptive Cell Therapy.

In cancer, an effective immune response involves the action of several different cell types, among which CD8 T cells play a major role as they can specifically recognize and kill cancer cells via the release of cytotoxic molecules and cytokines, being of major importance for adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). The inflammation resulting from the tumor growth attracts both activated and bystander T cells. For an effective antitumor response, the T cell must express a specific group of chemokine receptors and integrins which include CD103, CD39, CD69, and CD25. These markers had already been analyzed in various cancers, not including breast cancer and their subsequent subtypes, until now. To analyze, the key receptors on ex vivo expanded tumor-infiltrating lymphocytes in luminal A and luminal B breast cancer (BC) subtypes. We were successful in expanding TILs ex vivo using a standard TIL culture condition from a cohort study of 15 primary luminal A and luminal B breast cancer patients. Furthermore, we examined the expression of CD103, CD39, CD69, and CD25 biomarkers after the expansion by flow cytometry. We found that the information about the percentage of TILs obtainable after the ex vivo expansion is not associated to nor it is dependent on the heterogeneity of the TIL population before the expansion and does not differ by the molecular subtype (p>0.05). We also found that there is a major population of memory-resident antitumor CD8+CD103+CD39+ and CD8+CD103+CD69+ TILs present in the stroma after the expansion when compared to CD4 immunosubtypes (p<0.0001). Only the CD8+CD103+CD39+ subpopulation was related to BC subtype (0.0009). Evidence from our study suggests that CD8 TILs present in the stroma of luminal A and luminal B breast cancer patients can be quantified and phenotyped by flow cytometry and be further expanded ex vivo. The immuno-phenotyping of these markers may be targeted to improve the success of immunotherapeutic approaches, such as adoptive cellular therapy (ACT) in patients with BC.

Prognostic and predictive value of tumor infiltrating lymphocytes in combination with systemic inflammatory markers in colon cancer

Objective. Systemic inflammatory indices and CD8(+) tumor infiltrating lymphocytes (TILs) in the tumor microenvironment are highly prognostic in colon cancer (CC) but combined assessment is less well studied. The purpose of this study was to investigate the prognostic and predictive value of CD8(+) TILs in combination with systemic inflammatory indices in patients with resected stage II-III colon cancer.Patients and methods. Patients with stage II-III CC (n = 304) diagnosed between 2008 and 2016 were included. Pan-immune inflammation value (PIV) was used as a comprehensive inflammatory index and was calculated as: [neutrophil count × platelet count × monocyte count]/lymphocyte count. The mean density of CD8+ TILs in the periphery and center of the tumor was assessed and dichotomized at the 75th percentile. Combined inflammation score (CIS) was classified as “high” in patients with high PIV (>median) plus low mean CD8(+) TILs density, and CIS “low” in the remaining patients.Results. 5-year DFS was 71% (78% in stage II, 63.4% in stage III). PIV was higher in right colon tumors, T4 tumors and in patients with obstruction / perforation. CD8(+) TIL density was lower in node positive tumors. High PIV and low CD8(+) TILs were associated with shorter disease-free survival (DFS). In multivariate analysis; age > 65 years, stage III disease and high CIS (PIVhigh / CD8low) were associated with shorter DFS. Among patients with stage II disease, patients with high CIS (PIVhigh / CD8low) derived significant benefit from adjuvant chemotherapy while those with low CIS derived no benefit.Conclusion. Combined inflammation score may represent a new prognostic factor for localized colon cancer and predictor of chemotherapy response in patients with stage II disease.

IL-15 promotes self-renewal of progenitor exhausted CD8 T cells during persistent antigenic stimulation.

In chronic infections and cancer, exhausted CD8 T cells exhibit heterogeneous subpopulations. TCF1+PD-1+ progenitor exhausted CD8 T cells (Tpex) can self-renew and give rise to Tim-3+PD-1+ terminally differentiated CD8 T cells that retain their effector functions. Tpex cells are thus essential to maintaining a pool of antigen-specific CD8 T cells during persistent antigenic stimulation, and only they respond to PD-1-targeted therapy. Despite their potential as a crucial therapeutic target for immune interventions, the mechanisms controlling the maintenance of virus-specific Tpex cells remain to be determined. We observed approximately 10-fold fewer Tpex cells in the spleens of mice chronically infected with lymphocytic choriomeningitis virus (LCMV) one-year post-infection (p.i.) than at three months p.i. Similar to memory CD8 T cells, Tpex cells have been found to undergo self-renewal in the lymphoid organs, prominently the bone marrow, during chronic LCMV infection. Furthermore, ex vivo treatment with IL-15 preferentially induced the proliferation of Tpex cells rather than the terminally differentiated subsets. Interestingly, single-cell RNA sequencing analysis of LCMV-specific exhausted CD8 T cells after ex vivo IL-15 treatment compared with those before treatment revealed increased expression of ribosome-related genes and decreased expression of genes associated with the TCR signaling pathway and apoptosis in both Tpex and Ttex subsets. The exogenous administration of IL-15 to chronically LCMV-infected mice also significantly increased self-renewal of Tpex cells in the spleen and bone marrow. In addition, we assessed the responsiveness of CD8 tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma patients to IL-15. Similar to the data we obtained from chronic viral infection in mice, the expansion of the Tpex subset of PD-1+ CD8 TILs upon ex vivo IL-15 treatment was significantly higher than that of the terminally differentiated subset. These results show that IL-15 could promote self-renewal of Tpex cells, which has important therapeutic implications.

Abstract 5112: Immune biomarkers of clinical response after NSAID treatment in combination with vaccination targeting EGFR-COX2-CDC25B in the APC(Min/+) mouse model

Abstract Studies from our group have shown that administration of NSAIDs in mouse models of Familial Adenomatous Polyposis (FAP) results in a decrease in the development in small intestinal polyps with a significant increase in CD8 tumor infiltrating lymphocytes (TIL) and downregulation of PD-L1 on tumors. Furthermore, we have demonstrated that NSAIDs could be used in combination with vaccination to even further reduce the development of intestinal polyps. We questioned what tissue specific biomarkers could define the efficacy of response with the combination treatment. APC(Min/+) male and female mice (4-6 weeks old) were immunized with a multi-antigen (EGFR, COX2, CDC25B) peptide vaccine with CFA/IFA as an adjuvant. Control mice received immune adjuvant alone. Regular chow or chow containing 400 ppm naproxen was provided intermittently (3 weeks on, 3 weeks off for 3 cycles) or continuously (18 weeks on) at the initiation of the vaccine regimen. Animals were euthanized at 22 weeks and tumors quantified. TIL were evaluated by flow cytometry. Since intermittent naproxen treatment was just as effective at inhibiting tumor development as continuous naproxen treatment, with or without vaccine, we did not differentiate between NSAID regimens. We observed an increase in CD8+ (83%; p&amp;lt;0.0001), Tbet+CD4+ (94%; p&amp;lt;0.001), effector memory (EM) CD4+ (76%; p&amp;lt;0.0001) and EM CD8+ (94%; p&amp;lt;0.0001) TIL in the tumors that did develop in the mice treated with the combination NSAID and vaccination as compared to control. There was increased CD8+ (Pearson correlation coefficient: −0.506, p&amp;lt;0001), Tbet+CD4+ (Pearson correlation coefficient: -0.6222, p&amp;lt;0.001), EM CD4+ (Pearson correlation coefficient: 0.-6074, p&amp;lt;0.001) and EM CD8+ (Pearson correlation coefficient: -0.7155, p&amp;lt;0.001) in mice with greater tumor inhibition than those with less protection. Receiver operator curves revealed that CD8+ (AUC: 0.7623; p=0.002), Tbet+CD4+ (AUC 0.7273; p=0.008), EM CD4+ (AUC 0.6782; p=0.04) and EM CD8 (AUC 0.7897; p&amp;lt;0.001) TIL can differentiate mice receiving the combination of NSAID and vaccination as compared to either monotherapy alone. These data define potential biomarkers of response that can guide human clinical trials of NSAIDs +/- vaccination in patients with FAP. Citation Format: Denise L. Cecil, Ying Liu, Lauren Corulli, Mary L. Disis. Immune biomarkers of clinical response after NSAID treatment in combination with vaccination targeting EGFR-COX2-CDC25B in the APC(Min/+) mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5112.

Abstract 4615: Differential immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes according to ProMisE classifier in endometrial cancer

Abstract Endometrial cancer (EC), one of the most common gynecological malignancies, has been gradually increasing worldwide. The Cancer Genome Atlas endometrial collaborative project discovered 4 distinct prognostic genomic subtypes, and the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) was developed: POLE-mutated, mismatch repair deficiency (MMR-D), non-specific molecular profile (NSMP), and p53-abnormal (p53abn). In the era of immunotherapy, immune checkpoint inhibitors (ICIs) become a definite management of EC, both as single agent or in combination with other targeted agents. However, the characteristics of the tumor immune microenvironment according to molecular classification in EC are not well known. This study was conducted to identify distinct immune properties of tumor infiltrating lymphocytes (TILs) according to molecular classification in EC and find optimal therapeutic targets in each molecular subtypes. We isolated peripheral blood mononuclear cells (PBMCs) and TILs from 81 patients with EC. The expression of immune checkpoint receptors and T-cell transcription factors of TILs were examined using flow cytometry. Sixty-six patients were evaluated according to the ProMisE algorithm based on sequencing for the presence of POLE mutations, immunohistochemistry for MMR proteins and p53 (POLE-mut, n=6; MMR-D, n=17; p53abn, n=20; NSMP, n=23). Tumors were categorized into one of the four molecular subtypes. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1, and their proliferation was assessed to predict ICI-induced reinvigoration capacity of CD8 TILs. Comparing T-cell transcription factors (TCF-1 and TOX), CD8 TILs showed higher expression of TOX, and lower expression of TCF-1. When comparing the immune characteristics of TILs according to molecular subtypes, Tregs of POLE-mut showed significantly lower expression of CTLA-4 and Ki-67 than that of MMR-D. In addition, CD8 TILs of MMR-D showed higher expression of immune checkpoint receptors, including PD-1, CTLA-4, and TIM-3, and lower expression of TCF-1 than those of p53abn. CD8+TILs of p53abn showed significantly higher expression of CD226 than those of MMR-D. In particular, the frequency of antigen reactive CD39+CD103+CD8 T cells significantly lower in p53abn than in MMR-D. CD8 TILs of NSMP showed higher expression of TOX than those of MMR-D. In ex vivo T cell proliferation assay (n=49), 80.0% POLE-mut (4 of 5), 46.2% MMR-D (6 of 13), 35.7% p53abn (5 of 14) and 35.36% NSMP (6 of 17) showed enhanced proliferation of CD8 TILs upon anti-PD-1 treatment. In endometrial cancer, TILs showed distinct immune properties according to ProMisE classification. Further study is needed to develop optimal treatment targets for the p53abn and NSMP subtype, which is non-immunogenic and hardly reinvigorated by ICIs. Citation Format: Jung Chul Kim, Junsik Park, Yong Jae Lee, Sunghoon Kim, Sang Wun Kim, Jung-Yun Lee. Differential immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes according to ProMisE classifier in endometrial cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4615.

Abstract 6424: Immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes in ovarian clear cell carcinoma relative to high-grade serous ovarian carcinoma

Abstract Recent clinical trials have demonstrated the potential efficacy of immune checkpoint inhibitors (ICIs) for ovarian clear cell carcinoma (OCCC). However, little is known about the immune characteristics of OCCC. In this study, we aimed to investigate the distinct immune characteristics of tumor-infiltrating lymphocytes (TILs) in OCCC to high-grade serous ovarian carcinoma (HGSOC). We isolated peripheral blood mononuclear cells (PBMCs) and TILs from patients with epithelial ovarian cancer. The expression of immune checkpoint receptors and T-cell transcription factors of TILs in OCCC (n=26) and HGSOC patients (n=44) were accessed using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-CTLA-4, and their proliferation was assessed to predict response to ICIs. CD8 TILs and tumor-infiltrating regulatory T cells (Tregs) exhibited higher expression of immune checkpoint receptors, including PD-1, CTLA-4, and 4-1BB, compared to PBMCs. Tregs were more infiltrated in advanced/recurrent stage than early stage OCCC. However, immune characteristics of CD8 TILs and Tregs were not significantly different regardless of stage. When we compared the characteristics of TILs in OCCC (n=26) with those in HGSOC (n=44), the expression of PD-1 on CD8 TILs was significantly lower in OCCC than in HGSOC. The frequencies of PD-1highCD8 TILs and PD-1+TOX+CD8 TILs were also lower in OCCC than HGSOC. In ex vivo assay (n=19), 9 cases responded to anti-PD-1 in terms of enhanced proliferation capacity of CD8 TILs. In addition, when anti-CTLA-4 was added to anti-PD-1, an additional increase in proliferation of CD8 TILs was observed in patients who responded to anti-PD-1. Responders to anti-PD-1 had significantly lower frequencies of effector Tregs in the TILs than non-responders. Overall, CD8 TIL in OCCC was found to be less exhausted than HGSOC in terms of PD-1 and TOX expressions. Tumor-infiltrating Tregs affected on reinvigoration ability of CD8 TILs upon ICIs treatment in OCCC. This study provides the rationale and evidence for establishing the optimal strategies for ICIs treatment in patients with OCCC. Citation Format: Junsik Park, Yong Jae Lee, Jung Chul Kim, Jung-Yun Lee. Immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes in ovarian clear cell carcinoma relative to high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6424.

Abstract 1268: The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus

Abstract Background: Although metformin (Met) is well known to suppress tumor growth, the detailed mechanisms of its anti-tumor effect remain unknown. In recent years, it has been pointed out that tumor-associated neutrophils (TAN) can produce neutrophil extracellular traps (NETs) in the tumor microenvironment (TME) which can tumor invasion and metastasis. Here, in this study, we investigated the effect of Met on tumor-associated NETs and its effect on the prognosis of cancer patients. Method: We retrospectively examined the outcome of colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM) who received curative surgery in Jichi Medical University Hospital and asked the impact of Met intake on their outcome. In addition, we performed multicolor immunohistochemistry to examine the phenotypes of tumor-infiltrating lymphocytes (TIL), TAN and NETs in surgically removed tissues specimens in 40 (colorectal cancer) CRC patients who had taken Met before surgery and propensity score-matched 40 patients who had not. Results: A total of 1858 patients underwent curative colectomy from April 2010 to March 2021. Among them, 283 patients had T2DM at the time of surgery, and 62 patients had been treated with drugs including Met. The postoperative disease-free survival rate (DFS) was significantly improved in the Met intake group compared with non-intake group (HR=0.21, p&amp;lt;0.01). The number of CD66b(+) TANs significantly decreased in Met-intake group (Median(M)=169 (36-553)/mm2 vs 99 (34-322)/mm2, p&amp;lt;0.01). The number of tumor-associated NETs defined as CD66b(+) cit-H3(+) cells significantly decreased in Met-intake group (M=36 (8-272)/mm2 vs 20 (0-104)/mm2, p&amp;lt;0.01), and tumor-associated NETs/TANs ratio significantly decreased in Met-intake group (29.9% vs 19.8%, p&amp;lt;0.01). The number of CD3(+) TILs significantly increased in Met intake group (M=122 (28-257)/mm2 vs 142 (48-386)/mm2, p&amp;lt;0.05). The number of CD8(+) TILs significantly increased in Met-intake group (M=60 (14-180)/mm2 vs 99 (52-318)/mm2, p&amp;lt;0.01), and CD8/CD3 ratio significantly increased in Met-intake group (51.2% vs 72.1%, p&amp;lt;0.01). In addition, there was a negative correlation between TANs or tumor-associated NETs and CD8 positive TILs in all 80 patients (r=-0.26, p&amp;lt;0.05). Conclusion: Met can inhibit neutrophil infiltration and NETs formation while promote the infiltration of cytotoxic T cells in TME, which may lead to the improvement of the outcome of the patients with CRC. Citation Format: Akira Saito, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Rie Kawashima, Hideyo Miyato, Joji Kitayama, Naohiro Sata. The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1268.

Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, the responses to ICI treatment are highly variable in different individuals and the underlying mechanisms remain poorly understood. Here, we employed a mouse squamous cell carcinoma (SCC) model where tumor-bearing recipients diverged into responders (R) versus non-responders (NR) upon anti-PD-L1 treatment. We performed in-depth TCRβ sequencing with immunoSEQ platform to delineate the differences in CD8 tumor-infiltrating lymphocytes (TILs). We found that R and NR CD8 TILs both exhibited evidence of clonal expansion, suggesting activation regardless of response status. We detected no differences in clonal expansion or clonal diversity indexes between R vs. NR. However, the top expanded (>1%) TCRβ clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, showing a preferential expansion of distinct TCRβ clonotypes in response to the same SCC tumor in R vs. NR. Notably, the mutual exclusivity of TCR clonotypes in R vs. NR was only observed when top TCRβ clonotypes were counted, because such top-expanded clonotypes are present in the opposite outcome group at a much lower frequency. Many TCRβ sequences were detected in only one recipient at a high frequency, implicating highly individualized anti-tumor immune responses. We conclude that differences in the clonal frequency of top TCR clonotypes between R and NR CD8 TILs may be one of the factors underlying differential anti-PD-L1 responses. This notion may offer a novel explanation for variable ICI responses in different individuals, which may substantially impact the development of new strategies for personalized cancer immunotherapy.

Targeting tumor vasculature to improve antitumor activity of T cells armed ex vivo with T cell engaging bispecific antibody

BackgroundSuccess of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of...

Discordance of PD-L1 expression in primary and metastatic ovarian high-grade serous carcinoma and its correlation with CD8 + tumor-infiltrating lymphocytes and patient prognosis.

Differential expression of programmed death-1 ligand (PD-L1) and its clinical significance in primary and metastatic ovarian high-grade serous carcinoma (HGSC) have not been defined. Thus, we investigated the PD-L1 expression of paired ovarian primary and omental metastatic HGSC and its correlation with CD8 + tumor-infiltrating lymphocyte (TILs) and patient survival. A total of 212 cases of ovarian HGSCs with matched primary ovarian and metastatic omental tumors accessioned between 2003 and 2018 were selected for further analysis. Using immunohistochemistry, we evaluated the density of CD8 + TILs and expression of PD-L1 on whole tissue sections. Applying tumor proportion score (TPS, cutoff 1%) and combined positive score (CPS, cutoff 1), the prevalence of PD-L1 expression was similar but with significant discordance in ovarian and omental tumor. Using TPS, patients with PD-L1-positive tumors demonstrated significantly worse recurrence free survival (RFS) and overall survival (OS) than patients with PD-L1-negative tumors. Using CPS, patients with PD-L1-positive ovarian tumors demonstrated significantly worse OS while no significant difference in RFS was found. Patients with PD-L1-positive omental tumors demonstrated significantly worse RFS and OS. Patients with omental PD-L1-positive tumors (TPS) were associated with poorer RFS and OS, while patients with ovarian PD-L1-positive tumors (TPS) were associated with OS not RFS, in COX multivariant analysis. Nonetheless, ovarian and omental high CD8 TILs density was not associated with worse OS in univariant and COX multivariant analysis. PD-L1 expression in ovarian and omental tumor associated with an increased CD8 + TILs density. PD-L1 expression by TPS was better correlated with survival than by CPS, and PD-L1 expression in omental tumors was a stronger prognostic indicator than that in ovarian tumors.

GLIS1 intervention enhances anti-PD1 therapy for hepatocellular carcinoma by targeting SGK1-STAT3-PD1 pathway

BackgroundGLI-similar 1 (GLIS1) is one of of Krüppel-like zinc finger proteins, which are either stimulators or inhibitors of genetic transcription. Nevertheless, its effects on T cell were elusive.MethodsIn this study,...

PD-L1, CD4+, and CD8+ Tumor-Infiltrating Lymphocytes (TILs) Expression Profiles in Melanoma Tumor Microenvironment Cells.

(1) Background: Because melanoma is an aggressive tumor with an unfavorable prognosis, we aimed to characterize the PD-L1 expression in melanomas in association with T cell infiltrates because PD-1/PD-L1 blockade represents the target in treating melanoma strategy. (2) Methods: The immunohistochemical manual quantitative methods of PD-L1, CD4, and CD8 TILs were performed in melanoma tumor microenvironment cells. (3) Results: Most of the PD-L1 positive, expressing tumors, have a moderate score of CD4+ TILs and CD8+TILs (5-50% of tumor area) in tumoral melanoma environment cells. The PD-L1 expression in TILs was correlated with different degrees of lymphocytic infiltration described by the Clark system (X2 = 8.383, p = 0.020). PD-L1 expression was observed often in melanoma cases, with more than 2-4 mm of Breslow tumor thickness being the associated parameters (X2 = 9.933, p = 0.014). (4) Conclusions: PD-L1 expression represents a predictive biomarker with very good accuracy for discriminating the presence or absence of malign tumoral melanoma cells. PD-L1 expression was an independent predictor of good prognosis in patients with melanomas.