αvβ5 integrin expression on PDAC-infiltrating Tregs in human samples.

The effect of peptides and a TGFβR1 kinase inhibitor on iTregs.

KPC78 PDAC cells activate LAP-TGF-β in a manner independent of NRP-1.

CRGDC does not inhibit Smad2 phosphorylation in PDAC mice.

Flow cytometry analysis of αvβ5 integrin + mouse iTregs.

Human nTregs express the αvβ5 integrin in response to TCR stimulation.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is notorious for its aggressive, therapy-resistant nature that is in part driven by the desmoplastic, hypoperfused, and immunosuppressive tumor microenvironment (TME). In this study, we demonstrated that the αv integrin and neuropilin-1 (NRP-1) dual targeting iRGD peptide reverses some of these TME features by inhibiting transforming growth factor β (TGFβ) activation in the tumor, a process mediated by the αvβ5 integrin. In addition to PDAC epithelial cells and fibroblasts, regulatory T cells (Treg) in PDAC tumors also expressed the αvβ5 integrin and NRP-1. The αvβ5+ Tregs potently inhibited T-cell proliferation, and systemic iRGD therapy not only depleted αvβ5+ Tregs from PDAC tumors but also reduced their αvβ5− counterparts. Mechanistically, iRGD inhibited the activation of TGFβ mediated by the αvβ5-rich TME, thereby depriving Tregs of the cytokine essential for their development and maintenance. NRP-1–dependent tumor penetration was required for this effect because a traditional RGD peptide without an NRP-1–binding motif failed to inhibit TGFβ signaling or deplete Tregs in vivo. Treatment with iRGD induced a series of additional TME changes, such as improved vascular patency and perfusion, reduced stromal fibers, and increased CD8+ T-cell entry into the core of the tumors. Combining iRGD with immune checkpoint blockade led to an enhanced antitumor effect. Together, these findings support targeting the αvβ5 integrin with affinity ligands such as iRGD as a potential approach to enhance immunotherapy efficacy against PDAC and other desmoplastic tumors with high TGFβ and αvβ5 expression. Significance: The iRGD tumor penetrating peptide inhibits αvβ5 integrin-dependent TGF-β activation in pancreatic cancer to normalize the desmoplastic and immunosuppressive tumor microenvironment.

Flow cytometry analysis of αvβ5 integrin + mouse iTregs.

Tumor-infiltrating T cells have been the primary focus of cancer immunotherapy; however, accumulating evidence points to a critical role for B cells and plasma cells in shaping responses to immune checkpoint blockade. In this study, we investigated the humoral immune response in 38 patients with hepatocellular carcinoma treated with neoadjuvant anti-programmed cell death protein 1 (PD-1) therapy. In responders, defined by more than 50% tumor necrosis, we observed on-treatment enrichment of clonally expanded IgG1+ plasma cells within the tumor. Clonal tracking revealed that anti-PD-1 treatment expanded preexisting B cell clones associated with favorable clinical outcomes. Moreover, serum from responders contained IgG1 antibodies specific to cancer/testis antigens, including NY-ESO-1, and these humoral responses were linked to tumor-reactive T cell activity. We independently validated these findings across seven additional cohorts, encompassing single-cell and bulk sequencing data from 500 patients, spatial transcriptomics from seven patients and survival analyses from 1,582 patients. Our findings apply to recently approved treatments, such as PD-1 and vascular endothelial growth factor A (VEGF-A) blockade, but not to chemotherapy alone, suggesting broad relevance to individuals treated with immunotherapy. Collectively, our results demonstrate that PD-1 blockade induces tumor-specific IgG1+ plasma cell responses that complement cellular immunity and contribute to clinical benefit, underscoring a coordinated humoral-cellular axis in effective antitumor immunity.

The antitumor effect of an anti–PD-L1 mAb alone and in combination with iRGD + Gem therapy on KPC78 tumors with a PD-L1high and GATA6Low background.

In vitro binding of FAM-CRGDC to Tregs and non-Tregs isolated from mouse PDAC and spleen.

M21L human melanoma cells as a detector of active TGFβ.

Abstract Regulatory T cells (Tregs) contribute to the immunosuppressive nature of pancreatic ductal adenocarcinoma (PDAC). Systemic depletion of Tregs can enhance anti-tumor T cell immunity, but can also cause unwanted autoimmune complications. A tumor-specific Treg-targeted therapy that effectively and safely enhances anti-tumor immunity for PDAC treatment remains an unmet need. We have recently found that αvβ5 integrin is expressed on Tregs that reside in the PDAC, but not those in the spleen, suggesting that the integrin can serve as a targetable marker for tumor-resident Tregs. Indeed, long-term treatment with the iRGD tumor-penetrating peptide, which effectively targets αvβ5+ cells in PDAC tissue, depleted Tregs in a tumor-specific manner leading to a significant expansion of CD8+ T cells in transgenic and syngeneic PDAC mouse models. αvβ5+ Tregs were induced from both naïve CD4+ T cells and naturally occurring Tregs. T cell receptor stimulation was found to be the common inducer of αvβ5+ Tregs. αvβ5+ Tregs formed a subpopulation of CCR8+ Tregs, which are preferentially found in the tumor tissue of various cancer types. Our most recent data show that the αvβ5 integrin marks a highly immunosuppressive fraction of the CCR8+ Tregs, suggesting that targeting αvβ5+ Tregs can lead to effective depletion of immunosuppressive Tregs in a tumor-specific manner. Citation Format: Yuki Kunisada, Kodai Suzuki, Norio Miyamura, Shingo Eikawa, Tatiana Hurtado de Mendoza, Evangeline S. Mose, Caisheng Lu, Yukihito Kuroda, Erkki Ruoslahti, Andrew M. Lowy, Kazuki N. Sugahara. αvβ5 Integrin serves as a tumor-specific marker for immunosuppressive regulatory T cells in pancreatic cancer [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 B026.

Cisplatin-based chemotherapy has been associated with durable disease control in a small subset of patients with metastatic urothelial cancer. However, the mechanistic basis for this phenomenon has remained elusive. Antitumor immunity may underlie these exceptional responders. In a phase II trial evaluating a phased schedule of gemcitabine and cisplatin followed by gemcitabine and cisplatin with ipilimumab for metastatic urothelial cancer, 4 of 36 patients achieved durable disease-free treatment-free survival (DDFTFS) and remain in remission over 5 years after enrolment on the study. We sought to identify the genomic and immunological mechanisms associated with functional cures of such patients. Whole exome sequencing was performed on pretreatment archival tumor tissue. Neoantigen prediction and ranking were performed using a novel pipeline. For a subset of patients with available biospecimens, selected peptides were tested for neoantigen-specific T cell reactivity in peripheral blood CD4+ and CD8+ T cells cultured with autologous antigen-presenting cells at baseline, postchemotherapy, and postchemotherapy and ipilimumab timepoints. Multiplex assays of serum protein analytes were also assessed at each time point. Serum proteomic analysis revealed that pretreatment, patients achieving DDFTFS demonstrated an immune activated phenotype with elevations in TH1 adaptive immunity, costimulatory molecules, and immune checkpoint markers. After combination cisplatin-based chemotherapy and ipilimumab treatment, DDFTFS patients again displayed enrichment for markers of adaptive immunity, as well as T cell cytotoxicity. CD27 was uniquely enriched in DDFTFS patients at all timepoints. Neoantigen reactivity was not detected in any patient at baseline or post two cycles of chemotherapy. Both CD4+ and CD8+ neoantigen-specific T cell reactivity was detected in two of two DDFTFS patients in comparison to zero of five non-DDFTFS patients after combination cisplatin-based chemotherapy and ipilimumab treatment. Antitumor immunity may underlie functional cures achieved in patients with metastatic urothelial cancer treated with cisplatin-based chemotherapy and immune checkpoint blockade. Probing the mechanistic basis for DDFTFS may facilitate the identification of biomarkers, therapeutic components, and optimal treatment sequences necessary to extend this ultimate goal to a larger subset of patients.

Pancreatic ductal adenocarcinoma (PDAC) has abundant immunosuppressive regulatory T cells (Tregs), which contribute to a microenvironment resistant to immunotherapy. Here, we report that Tregs in the PDAC tissue, but not those in the spleen, express the αvβ5 integrin in addition to neuropilin-1 (NRP-1), which makes them susceptible to the iRGD tumor-penetrating peptide, which targets cells positive for αv integrin- and NRP-1. As a result, long-term treatment of PDAC mice with iRGD leads to tumor-specific depletion of Tregs and improved efficacy of immune checkpoint blockade. αvβ5 integrin + Tregs are induced from both naïve CD4 + T cells and natural Tregs upon T cell receptor stimulation, and represent a highly immunosuppressive subpopulation of CCR8 + Tregs. This study identifies the αvβ5 integrin as a marker for activated tumor-resident Tregs, which can be targeted to achieve tumor-specific Treg depletion and thereby augment anti-tumor immunity for PDAC therapy.

Abstract Dysfunctional T cell states resulting from continuous exposure to self shared tumor antigens or neoantigens are key to understanding immune mechanisms that prevent proper antitumor immunity. We previously showed that repeated administration of recombinant tumor antigen vaccines without immunological adjuvant emulates a tolerized-like immune response that cannot be rescued even after in vivo re-challenge with adjuvant. We used bulk and single-cell RNA-seq of purified antigen-specific CD4+ T-cells (i.e., the orchestrator of immunity) from 12 ovarian and non-small cell lung cancer patients receiving NY-ESO-1 or MAGE-A3 vaccines with or without adjuvant, over a period of 13 weeks. First, using bulk RNA-seq data, we derived novel temporal, tolerization CD4+ T cell expression and clonotype evolution signatures using differential expression and VDJ-deconvolution analyses, highlighting significant association with cell cycle and immune activation. Then, we projected these signatures onto several recently published single-cell RNA-seq data profiling exhausted tumor-infiltrating and peripheral blood lymphocytes, finding reasonable predictive power (AUC~0.8) to classify dysfunctional T-cell states that could distinguish exhaustion from tolerance. Next, we characterized in depth the transcriptional profiles and heterogeneity of our antigen-specific CD4+ T-cells pre- and post-treatment at the single-cell level using combined scRNAseq and scTCRseq. Finally, we computationally inferred the regulatory axis of tolerized T-cells by deriving transcription factor signaling networks upstream of gene expression. In conclusion, we characterized transcriptional signatures of tumor antigen-specific CD4+ T-cells and derived a specific tolerization profile, distinguishable from exhaustion, and conserved across datasets. Through comprehensive identification of genes and pathways in a human experimental setting for induction of tolerization and exhaustion, these findings provide relevant and promising avenues to reverse dysfunctional CD4+ T cell states. Citation Format: Edgar Kozlova, Shingo Eikawa, Sacha Gnjatic, Bojan Losic. Identifying tumor antigen-specific CD4+ T cell dysfunctional states by single cell transcriptomics in immunotherapy-treated cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3381.

The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

The promise of checkpoint blockade treatment has yet to be fully realized in part due to a lack of knowledge of immune exhaustion and its efficient characterization, especially in the context of immunosuppressive tumor microenvironments which effectively tolerize the immune system.We hypothesized that mapping expression signatures defining regulatory T-cell states to previously studied and characterized exhaustion phenotypes of infiltrating T cells in NSCLC would reveal points of contact between T cell tolerization and exhaustion. Concretely, we profiled the gene expression and directly inferred CDR3/1 clonality between tolerized and immunized states in purified CD4+ T-Cells from 10 patients with ovarian or peritoneal and non-small cell lung cancer. The samples were taken before and during immunization therapy for the antigens MAGE-A3 or NY-ESO-1 over a period of 13 weeks, in the presence and absence of adjuvant to emulate the tolerized and immunized states.Further, by modeling the regulatory T-cell states as an interaction between the tolerized and immunized with time and type of antigen allowed us to identify significant differentially expressed gene clusters and their correlation to clonality. We then projected these signatures onto single cell RNAseq data from NSCLC-TCGA to compare the identified clusters to previously identified exhaustion phenotypes. We show that antigen vaccination is immunostimulating and induces lymphocyte activation, leading to a proinflammatory environment. During an immunized stated an increase in cytokine activity, including IL2, 4, 9, 10, 13 and 17, whereas tolerized T Cells, we observe an increase in clonality, BIRC6 and IL20 apart from alterations in chromosome organization pathways and cell cycle including senescence.In conclusion, we detail how the differences between tolerized and immunized states help unravel exhaustion mechanisms, which will ultimately identify molecular markers for exhaustion to assist during treatment and prognosis in clinical settings.Citation Format: Edgar Kozlova, Shingo Eikawa, Sacha Gnjatic, Bojan Losic. Mapping tolerized T cell states to exhaustion phenotypes in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4558.