Cell Exhaustion Markers Research Articles

Abstract 1956: Stress-mediated reactive oxygen species from mitochondria dysregulates the tumor microenvironment in prostate cancer of African American men

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in men in United States. African American men (AAM) have higher rates of PCa incidence and mortality compared with European American men (EAM). While socioeconomics, access to optimal care and life-style factors contribute towards these disparities, underlying biologic differences in AAM remain important drivers of PCa disparities. In the current work, we aimed to explore the interplay between oxidative stress and inflammatory signaling in promoting progression of PCa in AAM. Oxidative stress results from excessive reactive oxygen species (ROS) generated mainly via membrane bound oxidases, peroxisomes or the mitochondrial electron transport chain (ETC). We performed a robust comparative genomics to determine whether there is a race dependent expression of genes related to ROS in publicly available PCa datasets. We then prospectively examined the differential gene expression of these genesets in PCa from clinically matched AAM and EAM in the VANDAAM prospective study. VANDAAM is a validation study of DecipherTM genomic testing in 243 men with localized PCa. We then combined computational and experimental approaches to explore the molecular mechanisms and consequences of exacerbated oxidative stress. We found that ETC complex I, III related genes are the most differentially expressed in AAM compared with EAM. To examine whether AAM vs EAM cell lines exhibits differential sensitivity to ETC modulation, we blocked key ETC enzymes using complex I inhibitor, rotenone, and complex III inhibitor, antimycin A. We used 2 types of mitochondria-specific labels that distinguish respiring and ROS-generating mitochondria. While the relative levels of respiring mitochondria remain constant, we found that antimycin A induced higher level of mitochondrial ROS in AAM PCa cell lines compared with EAM PCa cell lines. To understand the interplay between mitochondrial ETC complex I & III and immune microenvironment, we computed the correlational discordance of ETC complex I & III expression scores and expression of >1200 immune-related genes between AAM vs EAM in VANDAAM cohort. These analyses suggest that ETC complex I & III correlate with several immunosuppressive genes including the macrophage markers CD68 and CD163 and T cell exhaustion markers. In addition, intersecting discordant genes from several datasets suggests enrichment of inflammatory cytokines and type I interferon signature which we then validated using ex vivo culture of PCa tissues. In summary, we discovered a role of mitochondrial electron transport chain in regulating inflammatory circuits in PCa of AAM. Unraveling roles of mitochondria in promoting immune suppression will have significant impact on understanding the intersection between social pressures and biological stress responses. Citation Format: Asmaa El-Kenawi, Ryan Putney, Shivanshu Awasthi, Amparo Serna, Corrado Caslini, Jasreman Dhillon, Kosj Yamoah. Stress-mediated reactive oxygen species from mitochondria dysregulates the tumor microenvironment in prostate cancer of African American men [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 1956.

Abstract 2749: Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) model

Abstract Preclinical in vivo models are used to profile or refine CAR T therapies before advancing to human studies. Establishing long-term CAR T persistence and efficacy continues to challenge progress in the CAR T space, thus development of robust platforms that can provide longitudinal assessments of CAR T persistence and functionality is paramount. To this end, we used the NALM6-Luc ALL model to develop a flow cytometry platform that provides quantitative analysis of CAR T cells over time as well as surface markers that are documented to correlate with sustained T cell persistence and activation in vivo. Tumored NSG mice were enrolled into treatment groups based on tumor burden calculated from bioluminescence imaging (BLI) data. T cells transduced to express a CD19 CAR (or left untransduced (UTD)) were injected intravenously at 1.0E+07 cells/mouse. Tumor burden was monitored by BLI and flow cytometry was performed weekly on survival bleeds to measure CAR T persistence and examine phenotype. Treatment with CD19 CAR T cells delayed tumor growth resulting in an increase in time to progression of 107.1% compared to UTD controls. However, no regressions were observed. On day 1 post-transfer, CD3+ T cells were detectable in mice that received both CAR T and UTD cells (140+/-26 and 162+/-56 cells/uL blood respectively). T cells declined to near undetectable levels by Day 20, a point when all animals in the UTD treatment group had reached euthanasia criteria. After Day 20, T cells expanded in circulation in the CD19 CAR T treatment group reaching 695+/-327 cells/uL blood by day 40. T cell expansion coincided with exponential tumor outgrowth in the treatment group. To assess T cell functionality, flow cytometry was used to measure the expression of biomarkers for T cell activation (CD25, 4-1BB, and ICOS) and exhaustion markers (TIM-3, PD-1, and LAG-3). CD25, 4-1BB, and ICOS expression did not exceed positivity on more than 15% of CD8+ T cells and peaked by day 30 before downregulation was observed. Notably, PD-1 and LAG-3 expression levels continued to increase throughout the study, suggesting T cells were taking on an exhausted phenotype. Similar trends were observed on CD4+ T cells. To investigate whether the late phase T cell expansion was a graft vs. host response, CAR T cell measurements were compared to non-tumored animals. Expansion as well as PD-1/LAG-3 expression was only observed in tumor-bearing mice indicating the responses were tumor-specific. Taken together, these data demonstrate that CD19 CAR T cells can inhibit NALM6-Luc tumor growth in vivo and expand in circulation in an antigen-specific manner. Furthermore, CAR T cell failure to control tumor growth may be due to onset of an exhausted phenotype. Finally, we demonstrate that flow cytometry can be used to characterize T cell persistence and functionality in murine xenograft tumor models. Citation Format: David W. Draper, Derrik Germain, Stacey Roys, Olivia Nelson, Scott Wise. Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) 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 2749.

Comprehensive analysis of prognostic value, immune implication and biological function of CPNE1 in clear cell renal cell carcinoma.

Background: Elevated expression of Copine-1 (CPNE1) has been proved in various cancers; however, the underlying mechanisms by which it affects clear cell renal cell carcinoma (ccRCC) are unclear. Methods: In this study, we applied multiple bioinformatic databases to analyze the expression and clinical significance of CPNE1 in ccRCC. Co-expression analysis and functional enrichment analysis were investigated by LinkedOmics, cBioPortal and Metascape. The relationships between CPNE1 and tumor immunology were explored using ESTIMATE and CIBERSORT method. In vitro experiments, CCK-8, wound healing, transwell assays and western blotting were conducted to investigate the effects of gain- or loss-of-function of CPNE1 in ccRCC cells. Results: The expression of CPNE1 was notably elevated in ccRCC tissues and cells, and significantly correlated with grade, invasion range, stage and distant metastasis. Kaplan-Meier and Cox regression analysis displayed that CPNE1 expression was an independent prognostic factor for ccRCC patients. Functional enrichment analysis revealed that CPNE1 and its co-expressed genes mainly regulated cancer-related and immune-related pathways. Immune correlation analysis showed that CPNE1 expression was significantly related to immune and estimate scores. CPNE1 expression was positively related to higher infiltrations of immune cells, such as CD8+ T cells, plasma cells and regulatory T cells, exhibited lower infiltrations of neutrophils. Meanwhile, elevated expression of CPNE1 was characterized by high immune infiltration levels, increased expression levels of CD8+ T cell exhaustion markers (CTLA4, PDCD1 and LAG3) and worse response to immunotherapy. In vitro functional studies demonstrated that CPNE1 promoted proliferation, migration and invasion of ccRCC cells through EGFR/STAT3 pathway. Conclusion: CPNE1 is a reliable clinical predictor for the prognosis of ccRCC and promotes proliferation and migration by activating EGFR/STAT3 signaling. Moreover, CPNE1 significantly correlates with immune infiltration in ccRCC.

PD-1+CD8+ T Cells Proximal to PD-L1+CD68+ Macrophages Are Associated with Poor Prognosis in Pancreatic Ductal Adenocarcinoma Patients

Immune complexity status in the TME has been linked to clinical outcomes in pancreatic ductal adenocarcinoma (PDAC) patients. TME assessments with current cell marker and cell density-based analyses do not identify the original phenotypes of single cells with multilineage selectivity, the functional status of the cells, or cellular spatial information in the tissues. Here, we describe a method that circumvents these problems. The combined strategy of multiplexed IHC with computational image cytometry and multiparameter cytometric quantification allows us to assess multiple lineage-selective and functional phenotypic biomarkers in the TME. Our study revealed that the percentage of CD8+ T lymphoid cells expressing the T cell exhaustion marker PD-1 and the high expression of the checkpoint PD-L1 in CD68+ cells are associated with a poor prognosis. The prognostic value of this combined approach is more significant than that of lymphoid and myeloid cell density analyses. In addition, a spatial analysis revealed a correlation between the abundance of PD-L1+CD68+ tumor-associated macrophages and PD-1+CD8+T cell infiltration, indicating pro-tumor immunity associated with a poor prognosis. These data highlight the implications of practical monitoring for understanding the complexity of immune cells in situ. Digital imaging and multiparameter cytometric processing of cell phenotypes in the TME and tissue architecture can reveal biomarkers and assessment parameters for patient stratification.

Development of Prognostic Features of Hepatocellular Carcinoma Based on Metabolic Gene Classification and Immune and Oxidative Stress Characteristic Analysis.

The molecular classification of HCC premised on metabolic genes might give assistance for diagnosis, therapy, prognosis prediction, immune infiltration, and oxidative stress in addition to supplementing the limitations of the clinical staging system. This would help to better represent the deeper features of HCC. TCGA datasets combined with GSE14520 and HCCDB18 datasets were used to determine the metabolic subtype (MC) using ConsensusClusterPlus. ssGSEA method was used to calculate the IFNγ score, the oxidative stress pathway scores, and the score distribution of 22 distinct immune cells, and their differential expressions were assessed with the use of CIBERSORT. To generate a subtype classification feature index, LDA was utilized. Screening of the metabolic gene coexpression modules was done with the help of WGCNA. Three MCs (MC1, MC2, and MC3) were identified and showed different prognoses (MC2-poor and MC1-better). Although MC2 had a high immune microenvironment infiltration, T cell exhaustion markers were expressed at a high level in MC2 in contrast with MC1. Most oxidative stress-related pathways are inhibited in the MC2 subtype and activated in the MC1 subtype. The immunophenotyping of pan-cancer showed that the C1 and C2 subtypes with poor prognosis accounted for significantly higher proportions of MC2 and MC3 subtypes than MC1, while the better prognostic C3 subtype accounted for significantly lower proportions of MC2 than MC1. As per the findings of the TIDE analysis, MC1 had a greater likelihood of benefiting from immunotherapeutic regimens. MC2 was found to have a greater sensitivity to traditional chemotherapy drugs. Finally, 7 potential gene markers indicate HCC prognosis. The difference (variation) in tumor microenvironment and oxidative stress among metabolic subtypes of HCC was compared from multiple angles and levels. A complete and thorough clarification of the molecular pathological properties of HCC, the exploration of reliable markers for diagnosis, the improvement of the cancer staging system, and the guiding of individualized treatment of HCC all gain benefit greatly from molecular classification associated with metabolism.

MUCOSAL IMMUNE SIGNATURES OF PERIANAL FISTULIZING CROHN’S DISEASE BY MASS CYTOMETRY

Abstract BACKGROUNDS Perianal fistulas occur in ~30-40% of patients associated with high morbidity and an impaired quality of life. The management of perianal fistulizing CD is a major clinical challenge, where its underlying etiology of is poorly understood. METHODS We recruited patients with a history of: 1) CD with perianal fistulas; 2) CD without perianal involvement; 3) idiopathic perianal fistulas. Biopsies were taken from the fistula tracts, external opening of the fistulas, and rectal mucosa during examination under anesthesia or colonoscopy. Isolated mucosal immune cells were analyzed using mass cytometry. Data was analyzed using viSNE for dimensionality reduction. RESULTS We have revealed previously unknown mucosal immune landscapes in perianal CD using mass cytometry. Key cell populations with significant difference in frequencies are summarize in Table 1 (data of external fistula opening not shown due to limited space): 1) CD45RO+ T cells including Tregs are elevated in CD fistulas compared to those from idiopathic fistulas, which is consistent with a prior histological study. The expression of co-inhibitory receptor TIGIT and co-stimulatory receptor CD226 are increased in Tregs in CD fistulas, although their function in IBD and especially perianal CD remains unclear. 2) Perianal CD is associated with increased Th17 cells in the fistula tracts and elevated IL-17-producing CD8 T cells (Tc17) in the rectum, which highlight the importance of IL-17 signaling in perianal CD. 3) CD fistulas show increased CD39 and decreased CD127 (IL-7Ra) in both CD4 and CD8 T cells. CD39 and CD127 are markers of T cell exhaustion, which may control the pathogenesis of perianal CD by regulating cytokine production. 4) Total B cells are elevated in CD fistulas compared to idiopathic fistulas. In contrast, regulatory B cells (Bregs), with immunomodulatory function in the gut, are dramatically diminished in CD fistulas compared to idiopathic fistulas. 5) Besides adaptive cells, CD3-CD19- innate immune cells including innate lymphoid cells (ILCs), macrophages, and NK cells are also skewed in perianal CD. Fistula tracts, external fistula opening (data not shown), and rectum of perianal CD all show dramatically elevated CD172+TREM1+ macrophages, which were previously found to promote chronic inflammation in luminal CD. CONCLUSIONS The immune responses in perianal CD demonstrate unique features that are not present in luminal CD (summarized in Figure 1). This indicates that perianal CD represents an immune identity distinct from luminal CD in the intestine. Importantly, altered T cell exhaustion markers (CD39 and CD127) and increased CD172+TREM1+ pathogenic macrophages are present in all three locations (fistula tracts, external opening, and rectum) of perianal CD, suggesting their roles in the pathogenesis of perianal CD and may become novel therapeutic targets.

Aging augments obesity-induced thymic involution and peripheral T cell exhaustion altering the "obesity paradox".

The incidence of obesity, a condition characterized by systemic chronic inflammation, has reached pandemic proportions and is a poor prognostic factor in many pathologic states. However, its role on immune parameters has been diverse and at times contradictory. We have previously demonstrated that obesity can result in what has been called the "obesity paradox" which results in increased T cell exhaustion, but also greater efficacy of immune checkpoint blockade in cancer treatment. The role of obesity, particularly in the context of aging, has not been robustly explored using preclinical models. We therefore evaluated how age impacts the immune environment on T cell development and function using diet-induced obese (DIO) mice. We observed that DIO mice initially displayed greater thymopoiesis but then developed greater thymic involution over time compared to their lean counterparts. Both aging and obesity resulted in increased T cell memory conversion combined with increased expression of T cell exhaustion markers and Treg expansion. This increased T cell immunosuppression with age then resulted in a loss of anti-tumor efficacy by immune checkpoint inhibitors (ICIs) in older DIO mice compared to the younger DIO counterparts. These results suggest that both aging and obesity contribute to T cell dysfunction resulting in increased thymic involution. This combined with increased T cell exhaustion and immunosuppressive parameters affects immunotherapy efficacy reducing the advantage of obesity in cancer immunotherapy responses.

A Novel Lymphangiogenesis-Related Gene Signature can Predict Prognosis and Immunosuppressive Microenvironment in Patients with Clear Cell Renal Cell Carcinoma.

Background: Lymphangiogenesis represents a key event in the progression and metastasis of patients with clear cell renal cell carcinoma (ccRCC). Nevertheless, the prognostic value of lymphangiogenesis-related genes (LRGs) in ccRCC patients remains unknown. Method: Differential analyses were performed to identify differentially expressed LRGs between normal and tumor tissues. A univariate Cox analysis was performed to identify differently expressed LRGs associated with overall survival (OS). LASSO and multivariate Cox analyses were performed to construct and optimize the LRG signature. To further explore the molecular characterization of the LRG signature, a functional enrichment analysis, immune signature, somatic mutations, and drug sensitivity were assessed. Immunohistochemistry (IHC) and immunofluorescence staining were performed to validate the relationship between lymphangiogenesis and immunity using our ccRCC samples. Results: Four candidate genes (IL4, CSF2, PROX1, and TEK) were eventually available to construct the LRG signature in the training set. Patients in the high-risk group had a shorter survival than those in the low-risk group. The LRG signature was an independent prognostic factor of OS. These results were confirmed in the validation group. The LRG signature was correlated with immunosuppressive cell infiltration, T cell exhaustion markers, somatic mutations, and drug sensitivity. The IHC and immunofluorescence staining results confirmed the correlation between lymphangiogenesis and CD163+ macrophages, exhausted CD8+PD-1+, and CD8+ LAG3+ T cells. Conclusion: A novel prognostic signature based on LRGs could provide insight into the prognostic evaluation and treatment of ccRCC patients.

Interleukin-15 and chemokine ligand 19 enhance cytotoxic effects of chimeric antigen receptor T cells using zebrafish xenograft model of gastric cancer.

Chimeric antigen receptor (CAR) T cells have been proven effective for the treatment of B-cell-mediated malignancies. Currently, the development of efficient tools that supply CAR T cells for the treatment of other malignancies would have great impact. In this study, interleukin (IL)-15 and C-C motif chemokine ligand 19 (CCL19) were introduced into natural killer group 2D (NKG2D)-based CARs to generate 15×19 CAR T cells, which remarkably increased T-cell expansion and promoted the production of central memory T (Tcm) cells. 15×19 CAR T cells showed greater cytotoxicity to gastric cell lines than conventional CAR T cells and produced higher levels of IL-15 and CCL-19, which resulted in increased responder T cell chemotaxis and reduced expression of T cell exhaustion markers. A live zebrafish model was used for single-cell visualization of local cytotoxicity and metastatic cancers. Administration of 15×19 CAR T cells resulted in significant shrinking of gastric cancer xenograft tumors and expansion of 15×19 CAR T cells in zebrafish models. Taken together, these findings demonstrate that 15×19 CAR T cells are highly efficient in killing gastric cancer cells, are effective to avoid off-target effects, and migrate to local and metastatic sites for long-term surveillance of cancers.

Multimodal In Vivo Tracking of Chimeric Antigen Receptor T Cells in Preclinical Glioblastoma Models.

Iron oxide nanoparticles have been used to track the accumulation of chimeric antigen receptor (CAR) T cells with magnetic resonance imaging (MRI). However, the only nanoparticle available for clinical applications to date, ferumoxytol, has caused rare but severe anaphylactic reactions. MegaPro nanoparticles (MegaPro-NPs) provide an improved safety profile. We evaluated whether MegaPro-NPs can be applied for in vivo tracking of CAR T cells in a mouse model of glioblastoma multiforme. We labeled tumor-targeted CD70CAR (8R-70CAR) T cells and non-tumor-targeted controls with MegaPro-NPs, followed by inductively coupled plasma optical emission spectroscopy, Prussian blue staining, and cell viability assays. Next, we treated 42 NRG mice bearing U87-MG/eGFP-fLuc glioblastoma multiforme xenografts with MegaPro-NP-labeled/unlabeled CAR T cells or labeled untargeted T cells and performed serial MRI, magnetic particle imaging, and histology studies. The Kruskal-Wallis test was conducted to evaluate overall group differences, and the Mann-Whitney U test was applied to compare the pairs of groups. MegaPro-NP-labeled CAR T cells demonstrated significantly increased iron uptake compared with unlabeled controls ( P < 0.01). Cell viability, activation, and exhaustion markers were not significantly different between the 2 groups ( P > 0.05). In vivo, tumor T2* relaxation times were significantly lower after treatment with MegaPro-NP-labeled CAR T cells compared with untargeted T cells ( P < 0.01). There is no significant difference in tumor growth inhibition between mice injected with labeled and unlabeled CAR T cells. MegaPro-NPs can be used for in vivo tracking of CAR T cells. Because MegaPro-NPs recently completed phase II clinical trial investigation as an MRI contrast agent, MegaPro-NP is expected to be applied to track CAR T cells in cancer immunotherapy trials in the near future.

Pan-cancer analysis of DEPDC1 as a candidate prognostic biomarker and associated with immune infiltration.

DEP domain containing 1 (DEPDC1) gene is upregulated in several malignancies and contributes to tumorigenesis. Although the role of DEPDC1 in tumor is becoming increasingly popular, the function of DEPDC1 in pan-cancer still needs to be systematically elucidated. Data were downloaded from Genotype-Tissue Expression Data (GTEx), The Cancer Genome Atlas (TCGA) TIMER2.0, TISIDB, STRING, and CancerSEA databases and analyzed to determine the functionality of the DEPDC1. The results were visualized using tools provided by the databases and the R language. The results showed that DEPDC1 was significantly upregulated in 29 of the 33 human cancers analyzed. In addition, there were significant differences in DEPDC1 expression among cancer immune and molecular subtypes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPDC1 was mainly involved in the cell cycle, and CancerSEA analysis showed that DEPDC1 promoted cell cycle, DNA repair, DNA damage, and proliferation in pan-cancer. Receiver operating characteristic (ROC) curve analysis showed high predictive accuracy for pan-cancer. DEPDC1 expression was positively correlated with activated CD4+ T helper 2 cells and common lymphoid progenitor cells, and negatively correlated with natural killer (NK) T cells, CD4+ central memory T cells, and CD4+ effector memory T cells. Furthermore, DEPDC1 was significantly positively correlated with T cell exhaustion marker genes, such as CD274, transforming growth factor beta receptor 1 (TGFBR1), kinase insert domain receptor (KDR), programmed cell death 1 ligand 2 (PDCD1LG2), granzyme B (GZMB), and granulysin (LAG2). Additionally, DEPDC1 was associated with overall survival (OS), disease-specific survival (DSS), and progress-free interval (PFI) prognosis in multiple tumor types. The ROC analysis showed high predictive accuracy for pan-cancer. Collectively, DEPDC1 is aberrantly expressed and plays animmune-oncogenic role in pan-cancer, and DEPDC1 may serve as a biomarker for cancer diagnosis and therapy.

Abstract C013: Response to combination immunotherapy in a mouse model of PDAC is a function of baseline T cell content

Abstract Findings from the recently reported phase 2 PRINCE trial for patients with metastatic pancreatic cancer suggest potential clinical benefit with certain combinations of chemotherapy and immunotherapy. Overall survival following gemcitabine/nab-paclitaxel and either PD-1 blocking antibody or CD40 agonistic antibody (aCD40) correlated with the prevalence of distinct T cell subsets in circulation at baseline and with the extent of certain tumor infiltrating T cells (Padron et al., Nature Medicine, 2022). Here, we modeled the therapeutic effect of aCD40/PD-1/CTLA-4 therapy in the absence of chemotherapy in mice bearing tumors derived from the autochthonous pancreatic ductal adenocarcinoma (PDAC) KPC model and studied the dependency of tumor regression on the degree of tumor infiltrating T cells. We used a panel of KPC tumor cell clones that exhibit reproducible and transplantable levels of T cell infiltration ranging between high, intermediate, and low amounts. Mice were subcutaneously injected with clonal PDAC lines and treated 14-21 days later with either aCD40 alone or aCD40/PD-1/CTLA-4 (all i.p., n=5-8 mice per cohort of T cell high, intermediate and low clones). Systemic immune dynamics were surveyed by bleeding mice on day 0, 7, and 14 of treatment and analyzing for immune cell activation and exhaustion markers. In mice bearing T cell high and intermediate tumors, aCD40/PD-1/CTLA-4 induced higher rates of complete tumor regressions (71% for T cell high; 25% for T cell intermediate) and prolonged survival (P &amp;lt; 0.001 for T cell high; P = 0.013 for T cell intermediate) compared to untreated controls. Treatment with aCD40 alone was effective in mice with T cell intermediate tumors, leading to regressions in 29% of mice. In mice bearing T cell low tumors, however, tumor regressions after aCD40/PD-1/CTLA-4 were less frequent (12.5%), although survival was improved significantly following the treatment (P &amp;lt;0.001). aCD40 alone improved overall survival of T cell low bearing mice (P = 0.026) without inducing tumor regressions. Regardless of T cell high, intermediate, or low tumors, aCD40/PD-1/CTLA-4 increased the proportions of circulating activated and exhausted CD4+ and CD8+ T cells. aCD40/PD-1/CTLA-4 also increased T cell infiltration into T cell low tumors, but this enhanced infiltration did not correlate with complete regressions. Thus, using tumor clones derived from the same autochthonous genetic model, we show that response to combination immunotherapy is a function of baseline T cell content, with ‘hot’ tumors responding the best and ‘cold’ tumors responding the worst. Additional tumor cell-intrinsic factors such as those revealed in other mouse experiments and in the human samples from the PRINCE study likely restrict a robust anti-tumor immune response even when T cells have been recruited. Citation Format: Noah C. Cheng, Nune Markosyan, Robert H. Vonderheide. Response to combination immunotherapy in a mouse model of PDAC is a function of baseline T cell content [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C013.

MTAP deficiency contributes to immune landscape remodelling and tumour evasion.

Methylthioadenosine phosphorylase (MTAP) deficiency occurs in various malignancies and is associated with poor survival in cancer patients. However, the mechanisms underlying tumour progression due to MTAP loss are yet to be elucidated. Utilizing integrated analyses of the transcriptome, proteome and secretome, we demonstrated that MTAP deficiency alters tumour-intrinsic, immune-related pathways and reprograms cytokine profiles towards a tumour-favourable environment. Additionally, MTAP-knockout cells exhibited a marked increase in the immune checkpoint protein PD-L1. Upon co-culturing primary T cells with cancer cells, MTAP loss-mediated PD-L1 upregulation inhibited T cell-mediated killing activity and induced several T cell exhaustion markers. In two xenograft tumour models, we showed a modest increase in average volume of tumours derived from MTAP-deficient cells than that of MTAP-proficient tumours. Surprisingly, a remarkable increase in tumour size was observed in humanized mice bearing MTAP-deficient tumours, as compared to their MTAP-expressing counterparts. Following immunophenotypic characterization of tumour-infiltrating leukocytes by mass cytometry analysis, MTAP-deficient tumours were found to display decreased immune infiltrates with lower proportions of both T lymphocytes and natural killer cells and higher proportions of immunosuppressive cells as compared to MTAP-expressing tumour xenografts. Taken together, our results suggest that MTAP deficiency restructures the tumour immune microenvironment, promoting tumour progression and immune evasion.

Intratumor heterogeneity and T cell exhaustion in primary CNS lymphoma

BackgroundPrimary central nervous system lymphoma (PCNSL) is a rare lymphoma of the central nervous system, usually of diffuse large B cell phenotype. Stereotactic biopsy followed by histopathology is the diagnostic standard. However, limited material is available from CNS biopsies, thus impeding an in-depth characterization of PCNSL.MethodsWe performed flow cytometry, single-cell RNA sequencing, and B cell receptor sequencing of PCNSL cells released from biopsy material, blood, and cerebrospinal fluid (CSF), and spatial transcriptomics of biopsy samples.ResultsPCNSL-released cells were predominantly activated CD19+CD20+CD38+CD27+ B cells. In single-cell RNA sequencing, PCNSL cells were transcriptionally heterogeneous, forming multiple malignant B cell clusters. Hyperexpanded B cell clones were shared between biopsy- and CSF- but not blood-derived cells. T cells in the tumor microenvironment upregulated immune checkpoint molecules, thereby recognizing immune evasion signals from PCNSL cells. Spatial transcriptomics revealed heterogeneous spatial organization of malignant B cell clusters, mirroring their transcriptional heterogeneity across patients, and pronounced expression of T cell exhaustion markers, co-localizing with a highly malignant B cell cluster.ConclusionsMalignant B cells in PCNSL show transcriptional and spatial intratumor heterogeneity. T cell exhaustion is frequent in the PCNSL microenvironment, co-localizes with malignant cells, and highlights the potential of personalized treatments.

Identification of tumor microenvironment-related signature for predicting prognosis and immunotherapy response in patients with bladder cancer.

The tumor microenvironment (TME) not only provides fertile soil for tumor growth and development but also widely involves immune evasion as well as the resistance towards therapeutic response. Accumulating interest has been attracted from the biological function of TME to its effects on patient outcomes and treatment efficacy. However, the relationship between the TME-related gene expression profiles and the prognosis of bladder cancer (BLCA) remains unclear. The TME-related genes expression data of BLCA were collected from The Cancer Genome Atlas (TCGA) database. NFM algorithm was used to identify the distinct molecular pattern based on the significantly different TME-related genes. LASSO regression and Cox regression analyses were conducted to identify TME-related gene markers related to the prognosis of BLCA and to establish a prognostic model. The predictive efficacy of the risk model was verified through integrated bioinformatics analyses. Herein, 10 TME-related genes (PFKFB4, P4HB, OR2B6, OCIAD2, OAS1, KCNJ15, AHNAK, RAC3, EMP1, and PRKY) were identified to construct the prognostic model. The established risk scores were able to predict outcomes at 1, 3, and 5 years with greater accuracy than previously known models. Moreover, the risk score was closely associated with immune cell infiltration and the immunoregulatory genes including T cell exhaustion markers. Notably, the predictive power of the model in immunotherapy sensitivity was verified when it was applied to patients with metastatic urothelial carcinoma (mUC) undergoing immunotherapy. In conclusion, TME risk score can function as an independent prognostic biomarker and a predictor for evaluating immunotherapy response in BLCA patients, which provides recommendations for improving patients’ response to immunotherapy and promoting personalized tumor immunotherapy in the future.

Immune checkpoint receptor VISTA on immune cells is associated with expression of T-cell exhaustion marker TOX and worse prognosis in renal cell carcinoma with venous tumor thrombus

PurposeThe study aimed to determine the expression of VISTA and TOX within venous tumor thrombus and primary clear cell renal cell carcinoma (ccRCC) and to assess their prognostic value.MethodsThe study enrolled 82 patients with ccRCC and coexisting venous tumor thrombus treated radically from 2012 to 2019 in two tertiary centers. Tissue microarrays were prepared and stained with respective antibodies. The expression of markers was assessed separately on tumor cells (TCs) and/or tumor-associated immune cells (TAICs).ResultsTOX expression was positively correlated with the percentage of VISTA-positive TAICs in venous thrombus (p = 0.011), but not in the primary tumor (p = 0.674). High TOX expression was associated with a higher percentage of PD-L1-positive TAICs in both compartments (p = 0.001, p = 0.011, respectively). Positive expression of VISTA on TAICs was associated with PD-L1 expression on TCs (p = 0.005) and TAICs (p = 0.004) in the primary tumor, and only with PD-L1 on TAICs in thrombus (p = 0.006). The presence of VISTA-positive TAICs in venous thrombus was significantly more common in females (p = 0.034), and positively correlated with metastases (p = 0.028), and tumor necrosis (p = 0.013). The cases with VISTA-positive TAICs in venous tumor thrombi had significantly shorter OS than VISTA-negative cases (p = 0.041).ConclusionFor the first time, we demonstrated the expression of VISTA- and TOX-positive TAICs in the venous tumor thrombus. We found the association between immune checkpoint receptors and T cell exhaustion markers in both tumor mass and venous thrombus. Finally, we demonstrated that abundance of VISTA-positive TAICs in venous tumor thrombus correlates with worse outcomes in ccRCC.

Stem-like memory T cells are generated during hollow fiber perfusion-based expansion and enriched after cryopreservation in an automated modular cell therapy manufacturing process

Background aimsModular automation is a flexible and reliable option to build the foundation of a new or evolving process or to introduce automation to a process that is already established. Herein the authors demonstrate that modular automation provides both high-quality and high-yield T-cell products. MethodsCells from three individual donors collected on an automated continuous flow centrifugation system were successfully expanded in a functionally closed, automated, perfusion-based hollow fiber bioreactor. These cells were then prepared for cryopreservation in an automated closed-system device that maintains temperature and aliquots a mixed cell product and cryoprotectant into product bags. Cell product bags were thawed and expanded in flasks. Samples taken throughout this manufacturing process were analyzed for cell phenotype, exhaustion markers and functionality. The proportion of CD4+ and CD8+ T cells was maintained through each step, from pre-expansion and post-expansion to immediately after thaw and 24 h after thaw. ResultsInterestingly, phenotypic markers such as CD45RO, CD45RA and CCR7 evolved throughout the process and stem-like memory T cells emerged as the predominant phenotype in the clinically relevant 24-h post-thaw sample. ConclusionsModular automation supported the generation of stem-like memory T cells that were not terminally exhausted and were able to produce effector cytokines upon restimulation.

Long-term caloric restriction ameliorates T cell immunosenescence in mice

Aging is associated with a decrease in the function of the immune system, a phenomenon known as immunosenescence, which results in reduced resistance to infection. Caloric restriction (CR) is known to prolong lifespan and to regulate immune function. However, whether and how CR affects immunosenescence remains unclear. Here, we evaluated the effect of long- and short-term CR on immunosenescence by subjecting wild-type mice to CR between 6 and 18 months of age or between 17 and 18 months of age, respectively. Compared with a normal diet or short-term CR, long-term CR induced marked or complete attenuation of age-related decreases in the frequency of spleen NK cells and NKT cells; naïve CD4+ and CD8+ T cells; and cytokine- and granzyme B-secreting T cells. In contrast, both long- and short-term CR significantly suppressed age-related upregulation of the T cell exhaustion markers PD-1, Tim-3, and KLRG1, as well as the transcription factors NR4A1 and TOX, which regulate the expression of genes associated with the T cell exhaustion phenotype. These results suggest that CR might suppress age-associated immunosenescence by regulating the expression of transcription factors and target genes that control T cell exhaustion.

Circulating T Cell Activation and Exhaustion Markers Are Associated With Radiation Pneumonitis and Poor Survival in Non-Small-Cell Lung Cancer.

IntroductionPersistent inflammation and immune activation in the lungs are associated with adverse outcomes such as radiation pneumonitis (RP) and poor survival in non-small-cell lung cancer (NSCLC) patients. However, it is unknown how this is reflected by leukocyte activation markers in serum.ObjectiveThe aim was to evaluate the serum levels of activation of different leukocyte subsets and to examine those in relation to the pathogenesis of RP and survival in NSCLC.MethodsWe analyzed the serum levels of MPO, sCD25, sTIM-3, sPD-L1, sCD14, sCD163, CCL19 and CCL21 in 66 inoperable NSCLC patients with stage IA-IIIA disease. The patients were treated with stereotactic body radiation therapy (SBRT) or concurrent chemoradiation therapy (CCRT), followed by regular blood sampling for 12 months after treatment and for 5 years for survival.ResultsNineteen (29%) patients developed RP, which occurred more frequently and earlier in patients receiving CCRT than in those receiving SBRT. Increases in sCD25, sTIM-3 and CCL21 levels were observed at the last 6 months of follow-up in patients who had RP after SBRT. Patients who had RP after CCRT had higher sTIM-3 levels during the first 3 months of follow-up. Baseline sCD25 was independently associated with both 2- and 5-year mortality outcomes, while baseline sTIM-3 was independently associated with 2-year mortality.ConclusionWe showed that T cell activation and exhaustion markers such as sCD25 and sTIM-3 are enhanced in patients developing RP and are associated with poor survival in NSCLC.

CDK4/6 blockade provides an alternative approach for treatment of mismatch-repair deficient tumors

ABSTRACT Mismatch repair-deficient (dMMR) tumors show a good response toward immune checkpoint inhibitors (ICI), but developing resistance impairs patients’ outcomes. Here, we compared the therapeutic potential of an α-PD-L1 antibody with the CDK4/6 inhibitor abemaciclib in two preclinical mouse models of dMMR cancer, focusing on immune-modulatory effects of either treatment. Abemaciclib monotherapy significantly prolonged overall survival of Mlh1−/− and Msh2loxP/loxP;TgTg(Vil1- cre) mice (Mlh1−/−: 14.5 wks vs. 9.0 wks (α-PD-L1), and 3.5 wks (control); Msh2loxP/loxP;TgTg(Vil1- cre): 11.7 wks vs. 9.6 wks (α-PD-L1), and 2.0 wks (control)). The combination was not superior to either monotherapy. PET/CT imaging revealed individual response profiles, with best clinical responses seen with abemaciclib mono- and combination therapy. Therapeutic effects were accompanied by increasing numbers of tumor-infiltrating CD4+/CD8+ T-cells and lower numbers of M2-macrophages. Levels of T cell exhaustion markers and regulatory T cell counts declined. Expression analysis identified higher numbers of dendritic cells and neutrophils within tumors together with high expression of DNA damage repair genes as part of the global stress response. In Mlh1−/− tumors, abemaciclib suppressed the PI3K/Akt pathway and led to induction of Mxd4/Myc. The immune-modulatory potential of abemaciclib renders this compound ideal for dMMR patients not eligible for ICI treatment.