Expression Of Immune Checkpoint Receptors Research Articles

T-Cell Immunoglobulin and Mucin Domain 3 (TIM-3) Gene Expression as a Negative Biomarker of B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) accounts for 85% of all childhood ALL. Malignancies exhaust T and B cells, resulting in an increased expression of immune checkpoint receptors (ICRs), such as T-cell immunoglobulin and mucin domain 3 (TIM-3). TIM-3 has been found to be dysregulated in different types of cancer. However, there is a lack of rigorous studies on the TIM-3 expression in B-ALL. The current study aimed to measure the expression of TIM-3 at the gene and protein levels and evaluate the potential of TIM-3 as a biomarker in B-ALL. A total of 28 subjects were recruited between 2021 and 2023, comprising 18 subjects diagnosed with B-ALL and 10 non-malignant healthy controls. The B-ALL patients were divided into three groups: newly diagnosed (four patients), in remission (nine patients), and relapse/refractory (five patients). The expression levels of TIM-3 were evaluated using the real-time qPCR and ELISA techniques. The results revealed that the TIM-3 expression was significantly downregulated in the malignant B-ALL patients compared to the non-malignant healthy controls in the mRNA (FC = -1.058 ± 0.3548, p = 0.0061) and protein blood serum (p = 0.0498) levels. A significant TIM-3 gene reduction was observed in the relapse/refractory cases (FC = -1.355 ± 0.4686, p = 0.0327). TIM-3 gene expression allowed for significant differentiation between patients with malignant B-ALL and non-malignant healthy controls, with an area under the curve (AUC) of 0.706. The current study addressed the potential of reduced levels of TIM-3 as a negative biomarker for B-ALL patients.

Plasma Gelsolin Inhibits Natural Killer Cell Function and Confers Chemoresistance in Epithelial Ovarian Cancer.

Plasma gelsolin (pGSN) overexpression in ovarian cancer (OVCA) disarms immune function, contributing to chemoresistance. The aim of this study was to investigate the immunoregulatory effects of pGSN expression on natural killer (NK) cell function in OVCA. OVCA tissues from primary surgeries underwent immunofluorescent staining of pGSN and the activated NK cell marker natural cytotoxicity triggering receptor 1 to analyze the prognostic impact of pGSN expression and activated NK cell infiltration. The immunoregulatory effects of pGSN on NK cells were assessed using apoptosis assay, cytokine secretion, immune checkpoint-receptor expression, and phosphorylation of STAT3. In OVCA tissue analyses, activated NK cell infiltration provided survival advantages to patients. However, high pGSN expression attenuated the survival benefits of activated NK cell infiltration. In the in vitro experiment, pGSN in OVCA cells induced NK cell death through cell-to-cell contact. pGSN increased T-cell immunoglobulin and mucin-domain-containing-3 expression (TIM-3) on activated NK cells. Further, it decreased interferon-γ production in activated TIM-3+ NK cells, attenuating their anti-tumor effects. Thus, increased pGSN expression suppresses the anti-tumor functions of NK cells. The study provides insights into why immunotherapy is rarely effective in patients with OVCA and suggests novel treatment strategies.

Peripheral-central network analysis of cancer cachexia status accompanied by the polarization of hypothalamic microglia with low expression of inhibitory immune checkpoint receptors

While the excessive inflammation in cancer cachexia is well-known to be induced by the overproduction of inflammatory mediators in the periphery, microflora disruption and brain dysfunction are also considered to contribute to the induction of cancer cachexia. Hypothalamic microglia play a crucial role in brain inflammation and central-peripheral immune circuits via the production of inflammatory mediators. In the present study, we evaluated possible changes in excessive secretion of gut microbiota-derived endotoxin and the expression timeline of several inflammation-regulatory mediators and their inhibiting modulators in hypothalamic microglia of a mouse model of cancer cachexia following transplantation of pancreatic cancer cells. We demonstrated that the plasma level of lipopolysaccharide (LPS) was significantly increased with an increase in anaerobic bacteria, especially Firmicutes, in the gut at the late stage of tumor-bearing mice that exhibited dramatic appetite loss, sarcopenia and severe peripheral immune suppression. At the early stage, in which tumor-bearing mice had not yet displayed “cachexia symptoms”, the mRNA expression of pro-inflammatory cytokines, but not of the neurodegenerative and severe inflammatory modulator lipocalin-2 (LCN2), was significantly increased, whereas at the late “cachexia stage”, the level of LCN2 mRNA was significantly increased along with significant decreases in levels of inhibitory immune checkpoint receptors programmed death receptor-1 (PD-1) and CD112R in hypothalamic microglia. In addition, a high density of activated neurons in the paraventricular nucleus (PVN) of the hypothalamus region and a significant increase in corticosterone secretion were found in cachexia model mice. Related to the cachexia state, released corticosterone was clearly increased in normal mice with specific activation of PVN neurons. A marked decrease in the natural killer cell population was also observed in the spleen of mice with robust activation of PVN neurons as well as mice with cancer cachexia. On the other hand, in vivo administration of LPS in normal mice induced hypothalamic microglia with low expression of inhibitory immune checkpoint receptors. These findings suggest that the induction of cancer cachexia may parallel exacerbation of the hypothalamic inflammatory status with polarization to microglia expressed with low levels of inhibitory immune checkpoint receptors following LPS release from the gut microflora.

Abstract 5176: TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 12% 5-year survival rate (SEER) due to a lack of early detection biomarkers and resistance to standard therapeutic options (surgery, chemotherapy, radiation). Black African Americans (BAA) have 20% increased incidence of PDAC compared to those with European Ancestry (EA). TIGIT, an immune checkpoint receptor, is a marker of T cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. Recent studies have demonstrated that immune checkpoint receptor expression (PD-1 and TIGIT) on specific T cell populations correlates to worse overall survival (OS). TIGIT inhibitors are being explored in clinical trials in pancreatic cancer due to implications of its ligand (CD155) promoting immune evasion. We hypothesize that targeted anti-TIGIT therapy, in conjunction with other therapies targeting the tumor microenvironment, could reverse the immune suppression that is characteristic of PDAC. We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA (combined with a nuclear counterstain) on 79 tissue samples. The cohort of tissue samples included 8 biopsies (endoscopic-guided fine needle biopsies at time of diagnosis), 66 primary (from surgical resection), and 5 metastatic (liver core biopsies from patients with a primary PDAC diagnosis) formalin-fixed paraffin-embedded (FFPE) tissue sections from patients with histologically confirmed PDAC. After cells were segmented based on nuclear recognition, the presence of TIGIT probe within the cytoplasm of each cell was determined and quantified. Percent positive cell values were then exported for statistical analysis in R. De-identified clinical metadata was obtained from REDCap, a cloud-based HIPAA-compliant database used to compile patient data for research purposes. We tested for associations between %TIGIT present and clinical covariates, using linear regression for continuous outcomes, and logistic regression for binary outcomes. ScRNAseq revealed that TIGIT mRNA is enriched in, but not exclusive to the T/NK cellular compartments in PDAC. Staining analysis showed that TIGIT expression did not differ significantly between racial groups (comparing BAA to non-BAA). The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with clinical stage, where an increase in stage was associated with increasing %TIGIT (p < 0.05).The TIGIT biomarker assay can be conducted at time of diagnosis, time of surgical resection, and time of metastatic biopsy. If patients’ samples contain high levels of TIGIT expression, these patients may be candidates for anti-TIGIT drug therapy. Considering that TIGIT expression correlates with advancing clinical stage, patients with more advanced staging at diagnosis (stage IIB, III, IV) especially may benefit from anti-TIGIT therapy. Citation Format: Madison George, Julie Clark, Kendyll Gartrelle, Georges Nassif, Kailee Hartway, Daniel Long, Daniel Salas-Escabillas, Allison Wombwell, Thais Pichardo, Hui-Ju Wen, Simone Benitz, Samuel Zwernik, Rupen Shah, Hakmin Park, Philip, Gazala Khan, Howard Crawford, David Kwon, Brian Theisen, Nina Steele. TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5176.

Tumor-immune metaphenotypes orchestrate an evolutionary bottleneck that promotes metabolic transformation.

Metabolism plays a complex role in the evolution of cancerous tumors, including inducing a multifaceted effect on the immune system to aid immune escape. Immune escape is, by definition, a collective phenomenon by requiring the presence of two cell types interacting in close proximity: tumor and immune. The microenvironmental context of these interactions is influenced by the dynamic process of blood vessel growth and remodelling, creating heterogeneous patches of well-vascularized tumor or acidic niches. Here, we present a multiscale mathematical model that captures the phenotypic, vascular, microenvironmental, and spatial heterogeneity which shapes acid-mediated invasion and immune escape over a biologically-realistic time scale. The model explores several immune escape mechanisms such as i) acid inactivation of immune cells, ii) competition for glucose, and iii) inhibitory immune checkpoint receptor expression (PD-L1). We also explore the efficacy of anti-PD-L1 and sodium bicarbonate buffer agents for treatment. To aid in understanding immune escape as a collective cellular phenomenon, we define immune escape in the context of six collective phenotypes (termed "meta-phenotypes"): Self-Acidify, Mooch Acid, PD-L1 Attack, Mooch PD-L1, Proliferate Fast, and Starve Glucose. Fomenting a stronger immune response leads to initial benefits (additional cytotoxicity), but this advantage is offset by increased cell turnover that leads to accelerated evolution and the emergence of aggressive phenotypes. This creates a bimodal therapy landscape: either the immune system should be maximized for complete cure, or kept in check to avoid rapid evolution of invasive cells. These constraints are dependent on heterogeneity in vascular context, microenvironmental acidification, and the strength of immune response. This model helps to untangle the key constraints on evolutionary costs and benefits of three key phenotypic axes on tumor invasion and treatment: acid-resistance, glycolysis, and PD-L1 expression. The benefits of concomitant anti-PD-L1 and buffer treatments is a promising treatment strategy to limit the adverse effects of immune escape.

Tumor-infiltrating γδ T cells as targets of immune checkpoint blockade in melanoma.

Melanoma is one of the most sensitive tumors to immune modulation, and the major challenge for melanoma patients' survival is immune checkpoint inhibitor (ICI) therapy. γδ T lymphocytes play an antitumoral role in a broad variety of tumors including melanoma and they are optimal candidates for cellular immunotherapy. Thus, a comprehensive analysis of the correlation between γδ T cells and immune checkpoint receptors in the context of melanoma was conducted, with the aim of devising an innovative combined immunotherapeutic strategy. In this study, using the GEPIA2.0 database, a significant positive correlation was observed between the expression of γδ T cell-related genes (TRGC1, TRGC2, TCRD) and immune checkpoint genes (PDCD1, HAVCR2, LAG3), highlighting the potential role of γδ T cells in the immune response within melanoma. Moreover, flow cytometry analysis unveiled a significant augmentation in the population of γδ T cells within melanoma lesions, which exhibited the expression of immune checkpoint receptors including LAG3, TIM3, and PD1. Analysis of single-cell RNA sequencing data revealed a significant enrichment and functional reprogramming of γδ T cell clusters in response to ICIs. Interestingly, the effects of ICI therapy varied between Vδ1 and Vδ2 γδ T cell subsets, with distinct changes in gene expression patterns. Last, a correlation analysis between γδ T cell abundance, immune checkpoint gene expression, and clinical outcomes in melanoma patients showed that low expression of immune checkpoint genes, including LAG3, HAVCR2, and PDCD1, was associated with improved 1-year overall survival, emphasizing the significance of these genes in predicting patient outcomes, potentially outweighing the impact of γδ T cell abundance. This study offers critical insights into the dynamic interaction between γδ T cells, immune checkpoint receptors, and melanoma, providing valuable perspectives for potential therapeutic avenues and predictive markers in this intricate interplay.

Personalizing Oncolytic Immunovirotherapy Approaches.

Development of successful cancer therapeutics requires exploration of the differences in genetics, metabolism, and interactions with the immune system among malignant and normal cells. The clinical observation of spontaneous tumor regression following natural infection with microorganismhas created the premise of their use as cancer therapeutics. Oncolytic viruses (OVs) originate from viruses with attenuated virulence in humans, well-characterized vaccine strains of known human pathogens, or engineered replication-deficient viral vectors. Their selectivity is based on receptor expression level and postentry restriction factors that favor replication in the tumor, while keeping the normal cells unharmed. Clinical trials have demonstrated a wide range of patient responses to virotherapy, with subgroups of patients significantly benefiting from OV administration. Tumor-specific gene signatures, including antiviral interferon-stimulated gene (ISG) expression profile, have demonstrated a strong correlation with tumor permissiveness to infection. Furthermore, the combination of OVs with immunotherapeutics, including anticancer vaccines and immune checkpoint inhibitors [ICIs, such as anti-PD-1/PD-L1 or anti-CTLA-4 and chimeric antigen receptor (CAR)-T or CAR-NK cells], could synergistically improve the therapeutic outcome. Creating response prediction algorithms represents an important step for the transition to individualized immunovirotherapy approaches in the clinic. Integrative predictors couldinclude tumor mutational burden (TMB), inflammatory gene signature, phenotype of tumor-infiltrating lymphocytes, tumor microenvironment (TME), and immune checkpoint receptor expression on both immune and target cells. Additionally, the gut microbiota has recently been recognized as a systemic immunomodulatory factor and could further be used in the optimization of individualized immunovirotherapy algorithms.

A transcriptional evaluation of the melanoma and squamous cell carcinoma TIL compartment reveals an unexpected spectrum of exhausted and functional T cells.

Significant heterogeneity exists within the tumor-infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and may retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted T cell subpopulations critical to the improvement of therapeutic approaches. To investigate mechanisms associated with terminally exhausted T cells, we sorted and performed transcriptional profiling of CD8+ tumor-infiltrating lymphocytes (TILs) co-expressing the exhaustion markers PD-1 and TIM-3 from large-volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single-cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TILs; these include bile acid and peroxisome pathway-related metabolism and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Based on bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma.

Ibrutinib-based therapy reinvigorates CD8+ T cells compared to chemoimmunotherapy: immune monitoring from the E1912 trial

AbstractBruton tyrosine kinase inhibitors (BTKis) that target B-cell receptor signaling have led to a paradigm shift in chronic lymphocytic leukemia (CLL) treatment. BTKis have been shown to reduce abnormally high CLL-associated T-cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T-cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pretreatment and on-treatment (6 and 12 months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab with fludarabine, cyclophosphamide, and rituximab (FCR). Intriguingly, we report that despite reduced overall T-cell counts; higher numbers of T cells, including effector CD8+ subsets at baseline and at the 6-month time point, associated with no infections; and favorable progression-free survival in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T-cell killing function during ibrutinib-rituximab treatment, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T-cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T-cell cytotoxicity during ibrutinib-rituximab treatment, using the bispecific antibody glofitamab, supporting combination immunotherapy approaches.

The immune microenvironment characterisation and dynamics in hormone receptor-positive breast cancer before and after neoadjuvant endocrine therapy.

Oestrogen receptor positive (ER+)/HER-2 negative breast cancer (BC) is considered to be an immunologically cold tumour compared to triple negative breast cancer. Therefore, the tumour microenvironment (TME) of ER+/HER-2 negative BC is understudied. The aim of this project is to investigate the TME and the immune response during neoadjuvant endocrine therapy (NET) and to correlate this with the treatment response in a real life setting. Expression of immune checkpoint receptors and immune cells was examined immunohistochemically, pre- and post-NET in a cohort of 56 ER+/HER-2 negative BC patients. They were treated with tamoxifen (n = 16), an aromatase inhibitor (n = 40) or a combination of an aromatase inhibitor with a PI3K inhibitor (n = 11) for a median duration of 6 months (range 1-32 months). Immunohistochemical staining with monoclonal antibodies for PDL-1, PD-1, TIM-3, LAG-3, CTLA-4, CD4, CD68 and FOXP3 were performed. All staining procedures were done according to validated protocols, and scoring was done by a pathologist specialized in breast cancer. Positivity was defined as staining >1% on TILs. Response to NET was evaluated according to tumour size change on imaging and Ki-67 change. The median age was 61.02 (37-90) years. Diameter of tumour size decreased with a mean of 8.1 mm (-16 mm to 45 mm) (p < 0.001) during NET and the value of Ki-67 value decreased with a median of 9 after NET (p < 0.001). An increase in PD-L1 expression after NET showed a trend towards significant (p = 0.088) and CD-4+ T cells significantly increased after NET (p = 0.03). A good response to NET defined as a decrease in tumour size and/or decrease of Ki-67 was found to be associated with a longer duration of NET, a change of CD4+ T-cells and a higher number of CD68+ tumour-associated macrophages before the start of NET. The immune microenvironment plays an important role in ER+/HER-2 negative BC. NET influences the composition and functional state of the infiltrating immune cells. Furthermore, changes in the immune microenvironment are also associated with treatment response.

Diverging prognostic effects of CD155 and CD73 expressions in locally advanced triple-negative breast cancer.

Immune checkpoint inhibition, combined with novel biomarkers, may provide alternative pathways for treating chemotherapy-resistant triple-negative breast cancer (TNBC). This study investigates the expression of new immune checkpoint receptors, including CD155 and CD73, which play a role in T and natural killer (NK) cell activities, in patients with residual TNBC after neoadjuvant chemotherapy (NAC). The expression of biomarkers was immunohistochemically examined by staining archival tissue from surgical specimens (n = 53) using specific monoclonal antibodies for PD-L1, CD155, and CD73. Of those, 59.2% (29/49) were found to be positive (>1%) for PD-L1 on the tumour and tumour-infiltrating lymphocytes (TILs), while CD155 (30/53, 56.6%) and CD73 (24/53, 45.3%) were detected on tumours. Tumour expressions of CD155 and CD73 significantly correlated with PD-L1 expression on the tumour (p = 0.004 for CD155, p = 0.001 for CD73). Patients with CD155 positivity ≥10% were more likely to have a poor chemotherapy response, as evidenced by higher MDACC Residual Cancer Burden Index scores and Class II/III than those without CD155 expression (100% vs 82.6%, p = 0.03). At a median follow-up time of 80 months (range, 24-239), patients with high CD73 expression showed improved 10-year disease-free survival (DFS) and disease-specific survival (DSS) rates compared to those with low CD73 expression. In contrast, patients with CD155 (≥10%) expression exhibited a decreasing trend in 10-year DFS and DSS compared to cases with lower expression, although statistical significance was not reached. However, patients with coexpression of CD155 (≥10%) and low CD73 were significantly more likely to have decreased 10-year DFS and DSS rates compared to others (p = 0.005). These results demonstrate high expression of CD73 and CD155 in patients with residual tumours following NAC. CD155 expression was associated with a poor response to NAC and poor prognosis in this chemotherapy-resistant TNBC cohort, supporting the use of additional immune checkpoint receptor inhibitor therapy. Interestingly, the interaction between CD155 and CD73 at lower levels resulted in a worse outcome than either marker alone, which calls for further investigation in future studies.

TIGIT Expression Delineates T-cell Populations with Distinct Functional and Prognostic Impact in Pancreatic Cancer.

Immunotherapy has led to a fundamental shift in the treatment of several cancers. However, its efficacy in pancreatic ductal adenocarcinoma (PDAC) is limited. Understanding the expression of inhibitory immune checkpoint receptors (ICR) by intratumoral T cells may help to unravel their involvement in insufficient T-cell-mediated antitumor immunity. Using multicolor flow cytometry, we analyzed circulating and intratumoral T cells from blood (n = 144) and matched tumor samples (n = 107) of patients with PDAC. We determined the expression of programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) by CD8+ T-cells, conventional CD4+ T-cells (Tconv) and regulatory T cells (Treg) and their association with T-cell differentiation, tumor reactivity, and cytokine expression. A comprehensive follow-up was used to determine their prognostic value. Intratumoral T cells were characterized by increased PD-1 and TIGIT expression. Both markers delineated distinct T-cell subpopulations. PD-1+TIGIT- T cells highly expressed proinflammatory cytokines and markers of tumor reactivity (CD39, CD103), whereas TIGIT expression was linked to antiinflammatory and exhausted phenotypes. In addition, the enhanced presence of intratumoral PD-1+TIGIT- Tconv was associated with improved clinical outcomes, while high ICR expression on blood T cells was a significant hazard for overall survival (OS). Our results uncover the association between ICR expression and T-cell functionality. PD-1 and TIGIT characterized intratumoral T cells with highly divergent phenotypes linked to clinical outcomes, further underscoring the relevance of TIGIT for immunotherapeutic approaches in PDAC. The prognostic value of ICR expression in patient blood may be a valuable tool for patient stratification.

CpG therapeutic efficacy in a murine model of metastatic Lymphangioleiomyomatosis (LAM) is mediated by plasmacytoid dendritic cell and T cell immune responses

Abstract Introduction: LAM is a slow growing, metastasizing neoplasm affecting women of reproductive age, marked by abnormal growth of smooth muscle-like cells leading to cystic lung destruction. LAM has hallmarks of cancer, like expression of immune checkpoint receptors. Effective cancer therapies promote robust T cell responses through activated dendritic cells (DCs). Anti-cancer strategies, like toll like receptor (TLR) activation and immune checkpoint inhibition could work as LAM therapy. In this study, we examine the effect of CpG, a TLR9 agonist, on the DC and T cell responses in murine LAM. Methods: We used a mouse model of metastatic LAM to determine survival after biweekly intranasal CpG therapy (10μg/ 5μg) with/ without systemic α-PD-1, rapamycin, or α-CD317 therapy. We used ELISA to measure the cytokine profile and flow cytometry to quantify cell populations between CpG-treated and untreated LAM lungs. Results: We found that CpG treatment enhances median survival from 32 to 60 days in murine LAM (p &amp;lt;0.0001). Efficacy of CpG treatment in LAM is facilitated by plasmacytoid DCs. pDC depletion in CpG-treated mice decreases survival from 60 to 52 days (p=0.028). CpG-treated LAM lungs also produce more IFN-α (p=0.031). CpG-treated LAM lungs have increased IFN-γ (p= 0.012) and IL-12 (p= 0.005), cytokines secreted by cytotoxic T cells. CpG-treatment is synergistic with α-PD-1 checkpoint inhibition (p=0.004) and can be administered with rapamycin without adverse effects on median survival. Conclusions: In LAM, CpG therapy is mediated by pDC dependent immune responses and cytotoxic T cells. This suggests that adjuvant immunotherapy, like CpG, may offer new treatment options for LAM compatible with the current standard of care, rapamycin. The LAM Foundation Research Grant Award

Restoring T cell function to promote resolution of chronic HBV infection

Abstract An estimated 296 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, with 1.5 million new infections occurring each year despite the availability of an effective vaccine. Chronic HBV infection persists in a dysfunctional immune environment, manifested by an ineffective T cell response to the virus. Patient HBV-specific CD8 +T cells have diminished proliferative capacity and cytokine production, and they exhibit increased and sustained expression of inhibitory immune checkpoint receptors. In mice with persistent HBV replication, reduction of viral antigens can be achieved by therapeutic immunization with alphavirus- and vesiculovirus-based vaccine platforms expressing HBV antigens. However, blocking PD-1 and CTLA-4 signaling may further enhance antiviral effector T cell function and therapeutic efficacy. Using a mouse model of HBV replication in which mice are transduced with adeno-associated virus encoding the HBV genome (AAV-HBV), we investigated the role of these inhibitory receptors in the T cell response to HBV. Antibody-mediated blockade of either PD-1 or CTLA-4 independently in AAV-HBV mice did not promote viral clearance. Likewise, PD-1 and CTLA-4 inhibition combined with therapeutic immunization with VSV expressing an HBV antigen did not improve anti-HBV T cell responses or virus clearance in high-antigen mice with persistent viral replication. However, in mice with lower initial antigen loads, blockade of these receptors along with VSV therapeutic immunization resulted in an enhanced decrease of circulating HBV antigen. These results suggest new methodologies for improving HBV-specific immune responses to promote a functional cure of the infection. Supported by the National Institute of Allergy and Infectious Diseases and the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award numbers R01AI148354 and R44DK113858. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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 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.

Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.

Tumor-Infiltrating T Cells in EBV-Associated Gastric Carcinomas Exhibit High Levels of Multiple Markers of Activation, Effector Gene Expression, and Exhaustion

Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with 10% of all gastric cancers (GCs) and 1.5% of all human cancers. EBV-associated GCs (EBVaGCs) are pathologically and clinically distinct entities from EBV-negative GCs (EBVnGCs), with EBVaGCs exhibiting differential molecular pathology, treatment response, and patient prognosis. However, the tumor immune landscape of EBVaGC has not been well explored. In this study, a systemic and comprehensive analysis of gene expression and immune landscape features was performed for both EBVaGC and EBVnGC. EBVaGCs exhibited many aspects of a T cell-inflamed phenotype, with greater T and NK cell infiltration, increased expression of immune checkpoint markers (BTLA, CD96, CTLA4, LAG3, PD1, TIGIT, and TIM3), and multiple T cell effector molecules in comparison with EBVnGCs. EBVaGCs also displayed a higher expression of anti-tumor immunity factors (PDL1, CD155, CEACAM1, galectin-9, and IDO1). Six EBV-encoded miRNAs (miR-BARTs 8-3p, 9-5p, 10-3p, 22, 5-5p, and 14-3p) were strongly negatively correlated with the expression of immune checkpoint receptors and multiple markers of anti-tumor immunity. These profound differences in the tumor immune landscape between EBVaGCs and EBVnGCs may help explain some of the observed differences in pathological and clinical outcomes, with an EBV-positive status possibly being a potential biomarker for the application of immunotherapy in GC.

Twenty-One Flavors of Type 1 Innate Lymphoid Cells with PD-1 (Programmed Cell Death-1 Receptor) Sprinkles.

Innate lymphoid cells (ILCs) are tissue-resident immune cells that have been recently implicated in initiating and driving anti-tumor responses. ILCs are classified into three main groups, namely type 1 ILCs (ILC1), type 2 ILCs, and type 3 ILCs. All three groups have been implicated in either eliciting pro or anti-tumor immune responses in different cancer subtypes with the consensus that ILCs cannot be overlooked within the field of anti-tumor immune responses. In this review, we will specifically expand on the knowledge on ILC1, their characterization, function, and plasticity in anti-cancer immune responses. Within this premise, we will discuss caveats of ILC1 characterization, and expand on the expression and function of immune checkpoint receptors within ILC1 subsets, specifically focusing on the role of programmed cell death-1 receptor in controlling specific ILC1 responses. We summarize that ILC1s are a vital component in initiating anti-tumor responses and can be boosted by checkpoint receptors.

CD69+ Bone Marrow-Resident CD8+ T Cells Represent a Functionally Impaired Tumor-Reactive Population and Can be Restored By Blocking PD-1 and Tigit in Multiple Myeloma

Recently, novel anti-myeloma therapies utilizing T cell-mediated immune responses, including chimeric antigen receptor (CAR) T cells or bispecific antibodies engaging T cells to target tumor cells, have been actively studied in clinical trials and shown significant clinical efficacy. To enhance the clinical activity of T cell-exploiting immunotherapies in MM further, a better understanding of immune evasion mechanisms by characterizing myeloma-specific T cells and the strategic reinvigoration of the impaired T cell functions are essential prerequisites. In the present study, we demonstrated that bone marrow (BM)-resident CD69-expressing CD8+ T cells showed more prominent pathologic features including tumor-antigen associated exhaustion and impaired function in MM patients. In addition, using direct ex vivo experimental techniques, we examined the strategy how to restore anti-myeloma responses of the tumor-specific T cells, based on the characteristics of BM-resident CD69+ CD8+ T cell population. We first tried to evaluate various immunophenotypes of CD8+ T cells in BM and peripheral blood (PB) compartments of MM patients and found that the frequency of CD69+ cells among CD8+ T cells was significantly higher in BM than in peripheral circulation. These CD69+CD8+ T cells in BM had an effector memory phenotype negative for CCR7. Also, CD8+ T cells obtained from BM of normal controls contained similar frequency of CD69+ cells to the CD8+ T cells from BM of MM patients, suggesting that the CD69+ population could represent a BM-resident memory population even in a physiologic condition. To this end, we isolated CD69+ and CD69- memory CD8+ T cells from BM of MM patients and analyzed their transcriptomic difference using RNA-sequencing analysis. CD69, EPAS1, EOMES, PDCD1, TIGIT, and TOX were significantly upregulated in CD69+ cells when compared to CD69- cells among memory CD8+ T cells. On the contrary, S1PR1, KLF2, KLF4, SELL, GZMB, and GNLY were downregulated in CD69+ cells when compared to CD69- cells. These results indicate that transcriptomic signatures of CD69+ cells were similar to previously known features of tissue-resident memory T cells and functionally exhausted CD8+ tumor-infiltrating lymphocytes. We then compared the surface expression level of immune checkpoint receptors between CD69+ and CD69- cells among BM CD8+ T cells. CD69+ cells showed significantly higher positive frequencies of PD-1, Lag-3 and TIGIT than CD69- cells, but expression of Tim-3 did not differ between two populations. Notably, TOX, the key transcription factor regulating T cell exhaustion, was higher in CD69+ cells than in CD69- cells. When we examined the expression of other transcription factors as indicators of T cell exhaustion, the EomeshiT-betlo cells were significantly enriched in CD69+ population, indicating that CD69+ cells have features of terminally exhausted CD8+ T cells. More importantly, the myeloma antigen (NY-ESO-1157-165 and HM1.2422-30)-specific CD8+ T cells in BM of MM patients, identified with the MHC-multimer technique, also enriched in CD69+ population. When we compared the expression of immune checkpoint receptors among myeloma antigen-specific CD8+ T cells, the frequencies of PD-1+, TIGIT+ and PD-1+TIGIT+ cells were higher in CD69+ cells than CD69- cells. In fact, CD69+CD8+ T cells from BM of MM patients showed reduced production of effector cytokines in response to direct ex vivo TCR stimulation, compared to CD69- counterpart. Next, we investigated whether blocking PD-1 and/or TIGIT could restore the function of BM-resident CD8+ T cells of MM patients. Although a single blockade using either anti-PD-1 or anti-TIGIT antibody was not effective, their combination resulted in significant increase of IFN-γ+ cells in total CD8+ T cells and CD69+CD8+ T cells but not in CD69-CD8+ T cells Taken together, we demonstrated that BM-resident CD69+CD8+ T cells represent a population of exhausted T cells which are functionally impaired but potentially restorable. Indeed, we provide an ex vivo evidence of dual blockade of immune checkpoint receptors to enhance anti-tumor T cell responses in MM. Detailed understanding of BM-resident CD8+ T cells could provide more fundamental information for improving the current T cell-based immunotherapeutic approaches against MM.