Upregulation Of CTLA-4 Research Articles

Abstract C029: Multiple non-redundant immune checkpoints direct therapeutic resistance to chemotherapy and anti-CSF1R in pancreatic ductal adenocarcinoma

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDA) is the formation of a myeloid rich immunosuppressive microenvironment which acts as a barrier to treatment. Therapeutic blockade/depletion of immunosuppressive myeloid cells in combination with chemotherapy or anti-PD-1 checkpoint blockade is under investigation in PDA but has yet to demonstrate broad clinical success. Here, we studied mechanisms of resistance to myeloid-targeted therapy to inform rational combination strategies. Spatial and molecular profiling of the tumor microenvironment in mouse and human PDA revealed vast intra-tumoral myeloid heterogeneity. Depletion of distinct myeloid cell populations triggered T cell infiltration into tumors but also provoked compensatory remodeling of the myeloid compartment. However, concurrent depletion of multiple myeloid subsets using drugs targeting CSF1R, CCR2/5, and CXCR2 was unexpectedly insufficient to overcome treatment resistance and failed to restore productive T cell surveillance in murine models of PDA. Therefore, we next investigated the role of the lymphoid compartment. High-dimensional single cell analyses of T cell infiltrates in PDA revealed an upregulation of multiple checkpoint molecules, including PD-1, LAG3 and CTLA-4, in both mice and humans, suggesting potential resistance mechanisms. In contrast to combinatory myeloid cell depletion, combined blockade of PD-1, LAG3, and CTLA-4 with gemcitabine and anti-CSF1R triggered peripheral CD4+ and CD8+ T cell activation and produced deep and durable tumor responses with a nearly 3-fold increase in median survival and in some cases, tumor cures. Taken together, these studies highlight the contribution of multiple immune checkpoints to cancer immune evasion and reveal a novel role for LAG3 in treatment resistance in PDA. Together, these findings suggest that a multipronged approach to disrupting negative regulatory signals controlling T cell function may be needed to realize the potential of immunotherapy in PDA. Citation Format: Meredith L. Stone, Veronica M. Herrera, Yan Li, Heather Coho, Yuqing Yue, Kathleen Graham, Devora Delman, Shaun O'Brien, Gregory L. Beatty. Multiple non-redundant immune checkpoints direct therapeutic resistance to chemotherapy and anti-CSF1R in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C029.

Identification of neutrophil-related genes and development of a prognostic model for cholangiocarcinoma.

Cholangiocarcinoma is a prevalent gastrointestinal tumor with limited effective early diagnostic methods. The role of neutrophils in the context of cholangiocarcinoma remains largely unexplored. A comprehensive analysis was performed on a cohort of cholangiocarcinoma samples (TCGA-CHOL) from the TCGA database to investigate the relationship between cholangiocarcinoma and neutrophils. Methodologies included single-sample gene set enrichment analysis (ssGSEA), differential expression analysis, weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA). The study identified a significant decrease of neutrophils in cholangiocarcinoma via ssGSEA. WGCNA and differential expression analysis led to the identification of a neutrophil-related gene module comprised of 1059 genes. Cluster 1, showing a higher proportion of neutrophils, was linked to better survival outcomes. GSEA disclosed downregulation of complement, inflammatory response and interferon response pathways in Cluster 2, hinting at possible cholangiocarcinoma development triggers. A notable upregulation of PD1, PD-L1 and CTLA4 was observed in Cluster 1, suggesting potential benefits from immunotherapy. A prognostic model was developed based on clinical data and expression levels of three prognostic genes (SOWAHD, TNFAIP8 and EBF3) showing satisfactory discrimination, calibration and clinical benefits. An overexpression of TNFAIP8 in cholangiocarcinoma cells was found, with its knockdown significantly inhibiting cell proliferation and migration. This study elucidates a neutrophil-related gene module and prognostic genes, offering insights into the role of neutrophils in cholangiocarcinoma development and progression. It also introduces a clinical prediction model for enhanced prognosis assessment. These findings may lay the groundwork for the development of innovative therapeutic strategies in cholangiocarcinoma treatment.

Circulating extracellular vesicles derived from tumor endothelial cells hijack the local and systemic anti-tumor immune response: Role of mTOR/G-CSF pathway

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model.4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.

TRLS-09. USE OF A PAN-PEPTIDE CHECKPOINT INHIBITOR IN THE TREATMENT OF CENTRAL NERVOUS SYSTEM TUMORS

Abstract Immunotherapy has revolutionized clinical management of a select group of malignancies by offering a long-term, durable treatment response. Unfortunately, only a small percentage of brain cancers respond. The combination of multiple checkpoint inhibitors may result in serious immune-related adverse events. We have developed a Pan-Immune Checkpoint Ligand that simultaneously controls multiple immune checkpoints. The CD200 immune checkpoint modulates the immune system through a single inhibitory receptor (CD200R1) and multiple activation receptors (CD200ARs). We developed a peptide ligand, CD200AR-L, that targets the CD200ARs, which results in activation of the immune system and suppression of the inhibitory effects of CD200 and other immune checkpoints. Treatment of high-grade glioma in companion dogs with autologous tumor lysate vaccinations and CD200AR-L resulted in a two-year progression-free survival rate of 20% with no significant adverse events. We believe this response is due to the ability of CD200AR-L to modulate multiple immune checkpoints through shared signaling molecules in both the CD200 and PD-1/PD-L1 checkpoint pathways. Our preliminary data demonstrate that CD200R1 and PD-1 mediated immune checkpoint signaling is through SHIP1. CD200AR-L overcomes the immunosuppressive effects of the tumor-derived CD200 and PD-L1 by downregulating these checkpoints on both antigen-presenting cells (APC) and T-cells. CD200AR-L also downregulates PD-1 on APCs and inhibits the upregulation of PD-1 and CTLA4 on T cells. This translational research has led to the initiation of a phase I dose-escalation clinical trial for recurrent glioblastoma in adults (NCT04642937) and the writing of an IND for a pediatric HGG/DIPG trial.

Abstract 5869: The concordant disruption of B7/CD28 immune regulators predicts the prognosis of oral carcinoma

Abstract Immune-escape is a critical factor determining the survival of malignancies including oral squamous cell carcinoma (OSCC). The immune escape of cancer cells may be driven by B7/CD28 family members and others on the tumor cells that form ligand-inhibitory receptor complexes with immune cells. Since the family members of B7/CD28 can functionally compensate, the concomitant disruption of multiple B7/CD28 family members in OSCC pathogenesis remains elusive. RNAseq performed on OSCC and paired oral mucosa retrieved the transcriptome of OSCC, and algorithms signified the general alterations of T cell population and the increase of macrophage population in tumors relative to controls. Upregulation of CD80, CD86, PD-L1, PD-L2, CD276 and CTLA4, and downregulation of L-ICOS in OSCC relative to normal mucosa was noted. In addition, there was a high concordance in the expression profile across the dysregulated regulators. The co-upregulation of CD276 and CTLA4 was associated with the decrease of T helper and NK cell population, and the increase of Treg and myeloid dendritic cell population. Interestingly, tumors harboring higher PD-L1 and/or PD-L2 expression accompany with lower ICOS or PD1 expression exhibited worse prognosis. In combination with these immunity features in primary tumors, the survival of patients with lymph node metastasis was further worsened. In the isografic models of murine OSCC cell lines, the decrease of CD4+T population in the lymph node and increase of myeloid dendritic cell population in the spleen were induced in the host mice. Many miRNAs are involved in the modulation of immune escape for malignancies. With the treatment of inhibitors against oncogenic miRNAs which enabled the OSCC suppression, the inhibitors of miR-146a and miR-211 also resulted in the down-regulation in the protein expression of multiple B7/CD28 members. This study concludes the eminent co-disruption of B7/CD28 members in OSCC tumors, which would confound the patient survival. The efficacy of miRNA inhibitors in abrogating oncogenicity and B7/CD28 members could be a therapeutic strategy. Citation Format: Sih-Rou Chang. The concordant disruption of B7/CD28 immune regulators predicts the prognosis of oral 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 5869.

The Concordant Disruption of B7/CD28 Immune Regulators Predicts the Prognosis of Oral Carcinomas.

Immune modulation is a critical factor in determining the survival of patients with malignancies, including those with oral squamous cell carcinoma (OSCC) and head and neck SCC (HNSCC). Immune escape or stimulation may be driven by the B7/CD28 family and other checkpoint molecules, forming ligand-receptor complexes with immune cells in the tumor microenvironment. Since the members of B7/CD28 can functionally compensate for or counteract each other, the concomitant disruption of multiple members of B7/CD28 in OSCC or HNSCC pathogenesis remains elusive. Transcriptome analysis was performed on 54 OSCC tumors and 28 paired normal oral tissue samples. Upregulation of CD80, CD86, PD-L1, PD-L2, CD276, VTCN1, and CTLA4 and downregulation of L-ICOS in OSCC relative to the control were noted. Concordance in the expression of CD80, CD86, PD-L1, PD-L2, and L-ICOS with CD28 members was observed across tumors. Lower ICOS expression indicated a worse prognosis in late-stage tumors. Moreover, tumors harboring higher PD-L1/ICOS, PD-L2/ICOS, or CD276/ICOS expression ratios had a worse prognosis. The survival of node-positive patients was further worsened in tumors exhibiting higher ratios between PD-L1, PD-L2, or CD276 and ICOS. Alterations in T cell, macrophage, myeloid dendritic cell, and mast cell populations in tumors relative to controls were found. Decreased memory B cells, CD8+ T cells, and Tregs, together with increased resting NK cells and M0 macrophages, occurred in tumors with a worse prognosis. This study confirmed frequent upregulation and eminent co-disruption of B7/CD28 members in OSCC tumors. The ratio between PD-L2 and ICOS is a promising survival predictor in node-positive HNSCC patients.

Characteristics of immunological events in Epstein-Barr virus infection in children with infectious mononucleosis.

Epstein-Barr virus (EBV) infection occurs commonly in children and may cause acute infectious mononucleosis (AIM) and various malignant diseases. Host immune responses are key players in the resistance to EBV infection. We here assessed the immunological events and laboratory indicators of EBV infection, as well as determined the clinical usefulness of evaluating the severity and efficacy of antiviral therapy in AIM patients. We enrolled 88 children with EBV infection. The immune environment was defined by immunological events such as frequencies of lymphocyte subsets, phenotypes of T cells, and their ability to secrete cytokines, and so on. This environment was analyzed in EBV-infected children with different viral loads and in children in different phases of infectious mononucleosis (IM) from disease onset to convalescence. Children with AIM had higher frequencies of CD3+ T and CD8+ T cells, but lower frequencies of CD4+ T cells and CD19+ B cells. In these children, the expression of CD62L was lower and that of CTLA-4 and PD-1 was higher on T cells. EBV exposure induced granzyme B expression, but reduced IFN-γ secretion, by CD8+ T cells, whereas NK cells exhibited reduced granzyme B expression and increased IFN-γ secretion. The frequency of CD8+ T cells was positively correlated with the EBV DNA load, whereas the frequencies of CD4+ T cells and B cells were negatively correlated. During the convalescent phase of IM, CD8+ T cell frequency and CD62L expression on T cells were restored. Moreover, patient serum levels of IL-4, IL-6, IL-10, and IFN-γ were considerably lower throughout the convalescent phase than throughout the acute phase. Robust expansion of CD8+ T cells, accompanied by CD62L downregulation, PD-1 and CTLA-4 upregulation on T cells, enhanced granzyme B production, and impaired IFN-γ secretion, is a typical characteristic of immunological events in children with AIM. Noncytolytic and cytolytic effector functions of CD8+ T cells are regulated in an oscillatory manner. Furthermore, the AST level, number of CD8+ T cells, and CD62L expression on T cells may act as markers related to IM severity and the effectiveness of antiviral treatment.

Chemokine receptors on human regulatory T cells during cutaneous leishmaniasis.

The aim of this work was to define the population of regulatory T cells (Tregs) which are circulating in the blood of Leishmania infected individuals clinically displaying a lesion (active disease-AD) and sub-clinical (SC) ones. We have individually collected blood samples, processed the PBMC and stained with fluorochrome-conjugated antibodies against CD3, CD4, Foxp3, CD25, CTLA-4, Ki-67, CCR4, CCR5, and CCR7. Cells were analyzed by flow cytometry. Our results suggest that CD25 and CTLA-4 are upregulated in Tregs of AD patients when compared to SC and uninfected (UN) controls. Moreover, Tregs proliferate upon infection based on Ki-67 nuclear antigen staining. Finally, we have observed that these Tregs of SC and AD patients upregulate CCR4, but not CCR5 and CCR7. There is an increase in the number of circulating Tregs in the blood of Leishmania infected individuals. These cells are potentially more suppressive based on the increased upregulation of CD25 and CTLA-4 during clinical infection (AD) when compared to SC infection. Tregs of both SC and AD cohorts are proliferating and express CCR4, which potentially guide them to the skin, but do not upregulate CCR5 and CCR7.

Augmenting the Persistence and Efficacy of Engineered Regulatory T Cells to Suppress Anti-Drug Antibody Formation

Background Adoptive cell therapy (ACT) using engineered antigen specific regulatory T cells (Tregs) is a powerful tool for delivering immune tolerance in autoimmune disease, transplantation and in suppressing undesirable anti-drug antibody (ADA) responses against therapeutic proteins. An important example of the latter is hemophilia A (HA), a genetic disorder characterized by deficiency of the blood clotting protein, factor VIII (FVIII). Replacement therapy with recombinant FVIII can be neutralized by ADA development in up to 30% of severe HA patients. We previously showed that ACT with Tregs expressing a chimeric CD3ε based T cell receptor fusion construct (TRuC), encoding extracellular antibody fragments with specificity to human FVIII, suppressed ADA formation to FVIII in a preclinical HA model [Mol Ther. 2021 29(9):2660-2676]. Ensuring adequate in vivo persistence and a suppressive phenotype without risk of plasticity are major concerns that impede translation of ACT with engineered Tregs. Here we evaluate a synthetic single chain antibody fusion IL2 cytokine product (IL2-IC) to selectively expand adoptively transferred FVIII-TRuC Tregs in vivo. We then combine IL2-IC delivery with FVIII-TRuC Tregs expressing the co-inhibitory receptor PD-L1 to test improved suppression to ADA formation. Methods IL2-IC was generated by fusing IL-2 cytokine to the anti-IL-2 monoclonal antibody F5111 via a flexible linker. This complex specifically delivers IL2 to cells that express high levels of IL2 receptor α (IL2Rα) (i.e., Tregs) while avoiding interaction with cells that express low levels (i.e., Tconv, NK and CD8+ T cells). To assess whether IL2-IC could expand TRuC Tregs in vivo, 2x106 FVIII TRuC Tregs were injected intravenously (IV) into HAmice followed 5 hrs later with low dose IL2-IC or IL2 fused to an irrelevant Ab (FITC-IC) as control. 24 hrs later, mice received 2 international units (IU) of FVIII, and spleens were assayed on days 3 and 6. Murine PD-L1 was co-expressed in FVIII TRuC Tregs and in vitro assessed by stimulating TRuC PDL1 Tregs with 2IU FVIII alone or in combination with 2 ug/ml of PD1-Fc to understand the impact of PDL1 signaling on the Treg phenotype. In vivo, 0.5x106 FVIII TRuC PD-L1 Tregs were IV injected in HAmice followed 5 hrs later by mock, IL2-IC or FITC-IC IV injections. 24 hrs later, mice received weekly IV injections of 1IU FVIII. Results IL2-IC treated FVIII TRuC Tregs were preferentially expanded over endogenous Tregs (fold expansion 7.47 vs 4.56 respectively) in spleens on day 3 post ACT. Whereas both IL2-IC and FITC-IC treated FVIII TRuC Tregs had higher division indices over mock treatment (4.047 ± 0.03 and 5.070 ± 0.07 over 2.00 ± 0.00), only IL2-IC treated FVIII TRuC Tregs showed increased frequencies (1.72% ± 0.376% of CD4+ T cells) as compared to FITC-IC (0.28% ± 0.005% of CD4+ T cells) or mock treatment (0.23% ± 0.023% of CD4+ T cells). FITC-IC treatment nonspecifically expanded CD8+ Tconv cells as well as NK cells, whereas IL2-IC selectively expanded Tregs. PD1-Fc triggered PD-L1 signals in FVIII stimulated TRuC Tregs resulted in significant upregulation of Treg associated activation markers FoxP3, CD69, LAP, CTLA4, PD1, ICOS and BCL2 in vitro. In vivo, IL2-IC administration combined with FVIII TRuC PD-L1 ACT led to complete suppression of ADA formation in HA mice. At 4 weeks, mice that only received FVIII infusions developed high functional ADA titers (29.53 ± 19.58 BU/mL), while IL2-IC, TRuC PDL1 Treg and TRuC PDL1 Treg + IL2-IC groups had significantly lower titers of 1.117 ± 0.952 BU/mL, 1.805 ± 0.702 BU/mL and 0.423 ± 0.243 BU/mL, respectively. FITC-IC combined with PDL1 TRuC Tregs resulted in high ADAs of average 109 ± 21.31 BU/mL, confirming the requirement for the IL2Rα targeting antibody to selectively expand Tregs. Conclusion We show that the PD-L1/PD1 axis is an important pathway that can be exploited to improve the suppressive capacity of engineered Tregs. We also demonstrate that exogenous administration of the novel IL2-IC can robustly expand FVIII TRuC Tregs. These 2 strategies can be readily combined to selectively expand and enhance the suppressive capacity of engineered Tregs. The concepts defined here can be extended for improving the persistence and suppressive potential of engineered Tregs for various autoimmune disorders and to prevent transplant rejection. We are currently evaluating long term suppression of ADA responses using this approach.

Construction, bioinformatics analysis, and validation of competitive endogenous RNA networks in ulcerative colitis.

Background: Ulcerative colitis (UC) is a common chronic disease of the digestive system. Recently, competitive endogenous RNAs (ceRNAs) have been increasingly used to reveal key mechanisms for the pathogenesis and treatment of UC. However, the role of ceRNA in UC pathogenesis has not been fully clarified. This study aimed to explore the mechanism of the lncRNA-miRNA-mRNA ceRNA network in UC and identify potential biomarkers and therapeutic targets. Materials and Methods: An integrative analysis of mRNA, microRNA (miRNA), and long non-coding RNA (lncRNA) files downloaded from the Gene Expression Omnibus (GEO) was performed. Differentially expressed mRNA (DE-mRNAs), miRNA (DE-miRNAs), and lncRNA (DE-lncRNAs) were investigated between the normal and UC groups by the limma package. A weighted correlation network analysis (WGCNA) was used to identify the relative model for constructing the ceRNA network, and, concurrently, miRWalk and DIANA-LncBase databases were used for target prediction. Consecutively, the Gene Ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway, and Reactome pathway enrichment analyses, protein-protein interaction (PPI) network, Cytohubba, and ClueGO were performed to identify hub genes. Additionally, we examined the immune infiltration characteristics of UC and the correlation between hub genes and immune cells using the immuCellAI database. Finally, the expression of potential biomarkers of ceRNA was validated via qRT-PCR in an experimental UC model induced by dextran sulfate sodium (DSS). Result: The ceRNA network was constructed by combining four mRNAs, two miRNAs, and two lncRNAs, and the receiver operating characteristic (ROC) analysis showed that two mRNAs (CTLA4 and STAT1) had high diagnostic accuracy (area under the curve [AUC] > 0.9). Furthermore, CTLA4 up-regulation was positively correlated with the infiltration of immune cells. Finally, as a result of this DSS-induced experimental UC model, CTLA4, MIAT, and several associate genes expression were consistent with the results of previous bioinformatics analysis, which proved our hypothesis. Conclusion: The investigation of the ceRNA network in this study could provide insight into UC pathogenesis. CTLA4, which has immune-related properties, can be a potential biomarker in UC, and MIAT/miR-422a/CTLA4 ceRNA networks may play important roles in UC.

Increased expression of ST2 on regulatory T cells is associated with cancer associated fibroblast-derived IL-33 in laryngeal cancer

Interleukin (IL)− 33 plays an essential role in regulatory T cell (Treg)-mediated immunosuppression in cancers and underlies the crosstalk between Tregs and the tumor microenvironment. However, the phenotypic characteristics of subset Tregs modulated by IL-33 and its association with the tumor microenvironment are not fully understood. This study aimed to examine the expression of ST2, the receptor of IL-33, on Tregs in tumors and to evaluate their association with cancer associated fibroblasts (CAFs) and reciprocal influences on the prognosis of laryngeal cancer. Our results showed that increased numbers of Tregs were found in laryngeal tumor tissues. Tregs in stromal IL-33-positive tumor tissues demonstrated significantly higher expression of ST2 than those in IL-33- or adjacent nontumor tissues. ST2-expressing Tregs exhibited upregulation of Ki67 and CTLA4 compared with their ST2- negative counterparts. Furthermore, IL-33 in the tumor microenvironment was mainly derived from fibroblasts. ST2 expression on Tregs was correlated with the number of IL-33-positive CAFs. High ST2 expression on Tregs, combined high ST2 on Tregs and the presence of IL-33 expressing CAFs was associated with worse survival outcomes in laryngeal cancer. This study indicated that increased expression of ST2 on Tregs is associated with microenvironmental IL-33 signaling derived from CAFs in laryngeal cancer, unraveling the special role of Tregs and fibroblasts in modulating IL-33/ST2 involved immune-evasive tumor microenvironment.

Abstract 1361: Crosstalk between peripherally expanded T cells and Tfh-like cells in shaping effector functions of CD8 T cells in ovarian cancer

Abstract Background: Immune checkpoint blockade (ICB) of PD-1 and/or CTLA-4 can induce long-lasting clinical responses in several neoplasias. However, despite the successful response in some cancer, it is still ineffective for other malignant lesions such as ovarian cancer. Studies on T cell dynamics demonstrated that antitumor immune response from ICB is mediated by clonal replacement of CD8 T cells. Moreover, the presence of clones expanded in both tumor and peripheral tissue, also called dual-expanded, can predict response to ICB. To understand T cell dynamics in ovarian cancer, we characterized intratumoral and peripheral immune cells by single-cell transcriptome and TCR-sequencing. Methods and results: Using the 5’ Chromium assay (10X Genomics) on CD45+ cells isolated from 5 naïve-treatment patients undergoing cytoreductive surgery, we identified T cell states and classified clones by location (blood or tumor) and expansion pattern. In addition, the data revealed that clones expanded within the tumor were terminally exhausted (upregulation of PDCD1, HAVCR2, CTLA4, ENTPD1, and CXCL13 genes), while dual-expanded clones (expanded in blood and tumor) upregulated genes associated with the cytotoxic function of CD8 T cells, such as granzymes (GZMA and GZMB) and perforin (PRF1). Considering the importance of dual-expanded clones in antitumor immune response, we interrogated the cellular interactions they encountered in the tumor microenvironment. Using the tool NicheNet we identified IL21 as one of the top ligands mediating the interaction between dual-expanded clones and T follicular helper-like CD4+ cells (Tfh-like cells). Interestingly, the gene signature of Tfh-like cells showed that besides dual-expanded clones, these cells might attract other immune cells through CXCL13 and oxysterol metabolites. We identified B cells and plasmacytoid dendritic cells (pDCs) as the major cell types recruited by the CXCL13-CXCR5 axis and oxysterol-GPR183, respectively. Additionally, memory-like CD8 T cells were found to express GPR183, suggesting that a gradient of oxysterol might recruit them to a region defined by Tfh-like cells. Conclusions: Taken together, our study provides insights into additional functions of Tfh-like cells in the recruitment of immune cells through oxysterol gradient. A better understanding of the T cell dynamics and antitumor immune response can provide potential development of novel immunotherapy for the treatment of ovarian cancer. Citation Format: Mayra Carneiro, Cheng Zhao, Paméla Thébault, Yacine Bareche, Kurosh Rahimi, Jean-François Cailhier, Anne-Marie Mes-Masson, John Stagg, Sophie Petropoulos, Réjean Lapointe. Crosstalk between peripherally expanded T cells and Tfh-like cells in shaping effector functions of CD8 T cells in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1361.

Graft-Matched Pregnancy Imparts Profound Epigenetic Changes onto Alloreactive Memory T Cells to Enforce a Phenotypic and Transcriptional State of Tolerance

Abstract Pregnancy is an immunological paradox, where the maternal immune system develops tolerance to a semi-allogeneic fetus, yet pregnancy is a major sensitizing event in clinical transplantation. We previously reported that pregnancy robustly tolerizes fetus-specific T cells, allowing for the spontaneous acceptance of fetus-matched allografts when B cells and alloantibodies are absent at the time of transplantation(1). In this study, we report that mice sensitized to 2WOVA.B/c allogeneic skin grafts (Memory) underwent successful pregnancies comparable to Naïve mice when mated with 2WOVA.B/c males. We utilized a high-dimensional multi-omics approach to characterize post-partum (PP) fetus-specific T cells from Naïve or Memory mice after semi-allogeneic pregnancies. Strikingly, pregnancy induced fetus-specific naïve and memory T cells to achieve a nearly identical phenotype, characterized by upregulation of co-inhibitory molecules FR4, CD73, PD1, and CTLA4 in CD4+FoxP3- Tconvs and upregulated PD-1, FR4 and Lag3 in CD8+ T cells. We confirmed that naïve and memory PP CD8+ T cells acquired similar transcriptional profiles via RNA-Seq; unexpectedly, pregnancy induced profound epigenetic modifications in memory, but not naïve, CD8+ T cells. In vitro studies indicated reduced TNF aproduction by memory PP CD4+ and CD8+ T cells, and experiments examining their response to fetus-matched allografts are ongoing. Our studies suggest that significant epigenetic imprinting is necessary to constrain memory T cells to preserve fetal viability during semi-allogeneic pregnancy. These findings may provide insights into controlling antigen-specific memory T cells, a major barrier to clinical transplant success

Increased CDCA2 Level Was Related to Poor Prognosis in Hepatocellular Carcinoma and Associated With Up-Regulation of Immune Checkpoints.

BackgroundCell division cycle-associated protein 2 (CDCA2) is a member of cell cycle-related proteins. CDCA2 plays a role in the regulation of protein phosphatase 1(PP1) γ-dependent DNA damage response (DDR) and H3 phosphorylation. CDCA2 promotes the tumorigenesis and development of several types of cancers by promoting the proliferation of tumor cells. However, the relationship between CDCA2 expression and the clinicopathological characteristics of hepatocellular carcinoma (HCC) is unknown.MethodsGene expression information and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. The expression of CDCA2 and its correlation to clinical characteristics in HCC were analyzed. The expression level of CDCA2 was validated in HCC cell lines. The relationship between CDCA2 expression and the survival of patients with HCC was analyzed by using Kaplan–Meier method. The prognostic value of CDCA2 in HCC was estimated by Cox regression analysis. The expression difference of CDCA2 between HCC and normal tissues and its correlation to survival were verified in independent datasets. Gene set enrichment analysis (GSEA) was used to screen the CDCA2-related signaling pathways.ResultsCell division cycle-associated protein 2 expression was upregulated in HCC tissues (p < 0.001) and increased CDCA2 was correlated to increased T stage, pathologic stage, histologic grade, and alpha-fetoprotein (AFP) level (p < 0.001). In addition, CDCA2 was overexpressed in HCC cell lines HepG2 and LM3. High CDCA2 expression level was associated with poor overall survival [hazard ratio (HR) = 1.69; 95% CI, 1.20–1.40, p = 0.003], disease specific survival (HR = 1.73; 95% CI, 1.11–2.71, p = 0.016), and progress free interval (HR = 1.74; 95% CI, 1.30–2.34, p < 0.001). Overexpression of CDCA2 and its correlation to poor survival in HCC were verified in Gene Expression Omnibus (GEO) datasets and Kaplan–Meier plotter database. Increased CDCA2 expression was associated with upregulation of PD-L1 (Spearman's coefficient = 0.207, p < 0.001), PD-L2 (Spearman coefficient's = 0.118, p < 0.05), and CTLA4 (Spearman's coefficient = 0.355, p < 0.001). GSEA showed that homologous recombination pathway, insulin signaling pathway, mitogen-activated protein kinase (MAPK) pathway, mismatch repair pathway, mechanistic target of rapamycin (mTOR) pathway, Notch pathway, T cell receptor pathway, toll like receptor pathway, and WNT pathway were enriched in CDCA2 high expression phenotype.ConclusionCell division cycle-associated protein 2 may serve as an independent biomarker for poor prognosis in HCC and increased CDCA2 expression was associated with upregulation of immune checkpoints.

Oncolytic adenoviruses synergistically enhance anti-PD-L1 and anti-CTLA-4 immunotherapy by modulating the tumour microenvironment in a 4T1 orthotopic mouse model

Effective therapeutic strategies for triple-negative breast cancer (TNBC) are still lacking. Clinical data suggest that a large number of TNBC patients cannot benefit from single immune checkpoint inhibitor (ICI) treatment due to the immunosuppressive tumour microenvironment (TME). Therefore, combination immunotherapy is an alternative approach to overcome this limitation. In this article, we combined two kinds of oncolytic adenoviruses with ICIs to treat TNBC in an orthotopic mouse model. Histopathological analysis and immunohistochemistry as well as multiplex immunofluorescence were used to analyse the TME. The immunophenotype of the peripheral blood and spleen was detected by using flow cytometry. Oncolytic adenovirus-mediated immune activity in a coculture system of lytic supernatant and splenocytes supported the study of the mechanism of combination therapy in vitro. Our results showed that the combination of oncolytic adenoviruses with anti-programmed cell death-ligand 1 (anti-PD-L1) and anti-cytotoxic T lymphocyte-associated antigen-4 (anti-CTLA-4) (aPC) can significantly inhibit tumour growth and prolong survival in a TNBC model. The combination therapy synergistically enhanced the antitumour effect by recruiting CD8+ T and T memory cells, reducing the number of regulatory T cells and tumour-associated macrophages, and promoting the polarization of macrophages from the M2 to the M1 phenotype to regulate the TME. The rAd.GM regimen performed better than the rAd.Null treatment. Furthermore, aPC efficiently blocked oncolytic virus-induced upregulation of PD-L1 and CTLA-4. These findings indicate that oncolytic adenoviruses can reprogramme the immunosuppressive TME, while ICIs can prevent immune escape after oncolytic virus therapy by reducing the expression of immune checkpoint molecules. Our results provide a mutually reinforcing strategy for clinical combination immunotherapy.

A Prognostic Autophagy-Related Gene Pair Signature and Small-Molecule Drugs for Hepatocellular Carcinoma.

Dysregulation of autophagy-related genes (ARGs) is related to the prognosis of cancers. However, the aberrant expression of ARGs signature in the prognosis of hepatocellular carcinoma (HCC) remain unclear. Using The Cancer Genome Atlas and the International Cancer Genome Consortium database, 188 common autophagy-related gene pairs (ARGPs) were identified. Through univariate, least absolute shrinkage and selection operator analysis, and multivariate Cox regression analysis, a prognostic signature of the training set was constructed on the basis of 6 ARGPs. Further analysis revealed that the ARGP based signature performed more accurately in overall survival (OS) prediction compared to other published gene signatures. In addition, a high risk of HCC was closely related to CTLA4 upregulation, LC3 downregulation, low-response to axitinib, rapamycin, temsirolimus, docetaxel, metformin, and high-response to bleomycin. Univariate Cox and multivariate Cox analysis revealed that the risk score was an independent prognostic factor for HCC. These results were internally validated in the test and TCGA sets and externally validated in the ICGC set. A nomogram, consisting of the risk score and the TNM stage, performed well when compared to an ideal nomogram. In conclusion, a 6-ARGP-based prognostic signature was identified and validated as an effective predictor of OS of patients with HCC. Furthermore, we recognized six small-molecule drugs, which may be potentially effective in treating HCC.

Single-Cell RNA-Seq of T Cells in B-ALL Patients Reveals an Exhausted Subset with Remarkable Heterogeneity.

Characterization of functional T cell clusters is key to developing strategies for immunotherapy and predicting clinical responses in leukemia. Here, single‐cell RNA sequencing is performed with T cells sorted from the peripheral blood of healthy individuals and patients with B cell‐acute lymphoblastic leukemia (B‐ALL). Unbiased bioinformatics analysis enabled the authors to identify 13 T cell clusters in the patients based on their molecular properties. All 11 major T cell subsets in healthy individuals are found in the patients with B‐ALL, with the counterparts in the patients universally showing more activated characteristics. Two exhausted T cell populations, characterized by up‐regulation of TIGIT, PDCD1, HLADRA, LAG3, and CTLA4 are specifically discovered in B‐ALL patients. Of note, these exhausted T cells possess remarkable heterogeneity, and ten sub‐clusters are further identified, which are characterized by different cell cycle phases, naïve states, and GNLY (coding granulysin) expression. Coupled with single‐cell T cell receptor repertoire profiling, diverse originations of the exhausted T cells in B‐ALL are suggested, and clonally expanded exhausted T cells are likely to originate from CD8+ effector memory/terminal effector cells. Together, these data provide for the first‐time valuable insights for understanding exhausted T cell populations in leukemia.

Small cell lung cancer stem cells display mesenchymal properties and exploit immune checkpoint pathways in activated cytotoxic T lymphocytes

Small cell lung cancer (SCLC) is an aggressive tumor type with early dissemination and distant metastasis capacity. Even though optimal chemotherapy responses are observed initially in many patients, therapy resistance is almost inevitable. Accordingly, SCLC has been regarded as an archetype for cancer stem cell (CSC) dynamics. To determine the immune-modulatory influence of CSC in SCLC, this study focused on the characterization of CD44+CD90+ CSC-like subpopulations in SCLC. These cells displayed mesenchymal properties, differentiated into different lineages and further contributed to CD8+ cytotoxic T lymphocytes (CTL) responses. The interaction between CD44+CD90+ CSC-like cells and T cells led to the upregulation of checkpoint molecules PD-1, CTLA-4, TIM-3, and LAG3. In the patient-derived lymph nodes, CD44+ SCLC metastases were also observed with T cells expressing PD-1, TIM-3, or LAG3. Proliferation and IFN-γ expression capacity of TIM-3 and LAG3 co-expressing CTLs are adversely affected over long-time co-culture with CD44+CD90+ CSC-like cells. Moreover, especially through IFN-γ secreted by the T cells, the CSC-like SCLC cells highly expressed PD-L1 and PD-L2. Upon a second encounter with immune-experienced, IFN-γ-stimulated CSC-like SCLC cells, both cytotoxic and proliferation capacities of T cells were hampered. In conclusion, our data provide evidence for the superior potential of the SCLC cells with stem-like and mesenchymal properties to gain immune regulatory capacities and cope with cytotoxic T cell responses. With their high metastatic and immune-modulatory assets, the CSC subpopulation in SCLC may serve as a preferential target for checkpoint blockade immunotherapy.

Nivolumab (Nivo) with Azacytidine (AZA) in Patients (pts) with Relapsed Acute Myeloid Leukemia (AML) or Frontline Elderly AML

Nivolumab (Nivo) with Azacytidine (AZA) in Patients (pts) with Relapsed Acute Myeloid Leukemia (AML) or Frontline Elderly AML

Pilot Study of Pembrolizumab for Immunoprevention in Smoldering Multiple Myeloma

Background: The current management of patients with smoldering multiple myeloma (SMM) is watchful waiting. However, a fraction of these patients are at significant risk of progression to symptomatic multiple myeloma (MM). In a recent study, expression of PD-L1 on tumor cells correlated with increased risk of progression from SMM to MM (Dhodapkar et al Blood. 2015; 126(22): 2475-2478) suggesting a role for immune checkpoint blockade for immunoprevention of MM. Pembrolizumab is a humanized monoclonal antibody against programmed death receptor-1 (PD-1) and blocks the interaction between PD-1 and PD-L1. Here, we report the results of a pilot study evaluating the safety and efficacy of pembrolizumab in intermediate- and high-risk SMM patients (I-HR-SMM). Methods: Patients with I-HR-SMM by PETHEMA, Mayo or SWOG criteria were eligible. Permbrolizumab was administered at a dose of 200 mg IV every 21 days for up to 8 cycles. Patients were eligible to continue treatment for up to 24 cycles if they achieved ≥ minor response after 8 cycles. The primary objective was the overall response rate (ORR) after 8 cycles. The target ORR was 25%. The trial was conducted by the Simon's Minimax design and the ORR was estimated accordingly (Simon, 1989). Twelve patients were planned for the first stage. If at least one patient had ≥ partial response, 4 more patients could enroll for a total of 16 patients. Serial peripheral blood mononuclear cells and bone marrow samples were collected for comprehensive immunophenotyping using multiparametric flow cytometry, and molecular profiling with gene expression profiling (GEP) and exome sequencing. Results: Twelve patients with I-HR-SMM were dosed on the first stage between 08/2016 and 06/2017 (Table 1a/b). Median age was 67.5 years (range 49-74). Median number of cycles received was 8 (range 3-8) and all 12 were evaluable for safety and efficacy. Median follow up was 8.54 months (4.7 to 9.6 months). One patient achieved a stringent complete remission (sCR) (8%), ten patients had stable disease (83%), and one patient had progressive disease (8%). The patient with sCR had HR-SMM by GEP70 and FISH (deletion 17p and amplification of CKS1B) and 50% plasma cells infiltration in the bone marrow at study entry. The sCR with negative flow cytometry on bone marrow was noted in this patient after only 3 cycles of therapy and response is ongoing 8 months after the end of treatment. Five patients discontinued therapy due to related adverse events: grade 2 elevation in LFT (n=2), grade 3 myalgia (n=1), and grade 3 acute kidney injury (AKI) due to tubulo-interstitial nephritis (n=2) [all resolved with discontinuation of treatment or prednisone administration]. There were no grade 4/5 adverse events. Enrollment on second stage is ongoing. Imunophenotyping of paired baseline and end of treatment bone marrow samples showed that the patient who achieved sCR had high levels of PD-1 expression on CD4+ and CD8+ T cells at baseline and showed evidence of CD8+ T-cell activation after therapy with greater upregulation of CD69, CTLA-4, PD-1, TIM-3, 4-1BB, and ICOS relative to non-responders. In contrast, expression of other immune checkpoint molecules such as TIM-3, LAG-3, and CD244 were greater on T cells in non-responders at baseline relative to the responder suggesting greater exhaustion of T cells in non-responders. Molecular profiling including GEP, exome sequencing, and neoantigens analysis is ongoing and will be presented at the meeting. Conclusions: This pilot study of pembrolizumab monotherapy showed that it is tolerable and can induce sCR in patients with SMM. More importantly, it suggested that immunoprevention to prevent progression of SMM to MM is possible. Additional biomarker studies are needed to identify patients that are most likely to respond to this approach. Our correlative studies also provide insights into potential future combination approaches to further improve efficacy of immunoprevention strategies in SMM. [Display omitted] DisclosuresManasanch:takeda: Consultancy; celgene: Consultancy; sanofi: Research Funding; quest diagnostics: Research Funding; merck: Research Funding; adaptive biotechnologies: Consultancy. Lee:Eutropics Pharmaceuticals: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; Pimera Inc: Consultancy; Celgene: Consultancy; Daiichi Sankyo: Research Funding; Takeda: Consultancy. Patel:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Consultancy; Celgene: Consultancy. Thomas:Celgene: Research Funding; Bristol Myers Squibb: Research Funding. Neelapu:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karus: Research Funding; Cellectis Inc.: Research Funding; Poseida Therapeutics, Inc: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees.