Impact of peptide:HLA complex stability for the identification of SARS-CoV-2-specific CD8+T cells.

Induction of a lasting protective immune response is dependent on presentation of epitopes to patrolling T cells through the HLA complex. While peptide:HLA (pHLA) complex affinity alone is widely exploited for epitope selection, we demonstrate that including the pHLA complex stability as a selection parameter can significantly reduce the high false discovery rate observed with predicted affinity. In this study, pHLA complex stability was measured on three common class I alleles and 1286 overlapping 9-mer peptides derived from the SARS-CoV-2 Spike protein. Peptides were pooled based on measured stability and predicted affinity. Strikingly, stability of the pHLA complex was shown to strongly select for immunogenic epitopes able to activate functional CD8+T cells. This result was observed across the three studied alleles and in both vaccinated and convalescent COVID-19 donors. Deconvolution of peptide pools showed that specific CD8+T cells recognized one or two dominant epitopes. Moreover, SARS-CoV-2 specific CD8+T cells were detected by tetramer-staining across multiple donors. In conclusion, we show that stability analysis of pHLA is a key factor for identifying immunogenic epitopes.

Arginase-2-specific cytotoxic T cells specifically recognize functional regulatory T cells.

BACKGROUND: High expression of the metabolic enzyme arginase-2 (ARG2) by cancer cells, regulatory immune cells, or cells of the tumor stroma can reduce the availability of arginine (L-Arg) in the tumor microenvironment (TME). Depletion of L-Arg has detrimental consequences for T cells and leads to T-cell dysfunction and suppression of anticancer immune responses. Previous work from our group has demonstrated the presence of proinflammatory ARG2-specific CD4 T cells that inhibited tumor growth in murine models on activation with ARG2-derived peptides. In this study, we investigated the natural occurrence of ARG2-specific CD8 T cells in both healthy donors (HDs) and patients with cancer, along with their immunomodulatory capabilities in the context of the TME. MATERIALS AND METHODS: A library of 15 major histocompatibility complex (MHC) class I-restricted ARG2-derived peptides were screened in HD peripheral blood mononuclear cells using interferon gamma (IFN-γ) ELISPOT. ARG2-specific CD8 T-cell responses were identified using intracellular cytokine staining and ARG2-specific CD8 T-cell cultures were established by enrichment and rapid expansion following in vitro peptide stimulation. The reactivity of the cultures toward ARG2-expressing cells, including cancer cell lines and activated regulatory T cells (Tregs), was assessed using IFN-γ ELISPOT and a chromium release assay. The Treg signature was validated based on proliferation suppression assays, flow cytometry and quantitative reverse transcription PCR (RT-qPCR). In addition, vaccinations with ARG2-derived epitopes were performed in the murine Pan02 tumor model, and induction of ARG2-specific T-cell responses was evaluated with IFN-γ ELISPOT. RNAseq and subsequent GO-term and ImmuCC analysis was performed on the tumor tissue. RESULTS: We describe the existence of ARG2-specific CD8+ T cells and demonstrate these CD8+ T-cell responses in both HDs and patients with cancer. ARG2-specific T cells recognize and react to an ARG2-derived peptide presented in the context of HLA-B8 and exert their cytotoxic function against cancer cells with endogenous ARG2 expression. We demonstrate that ARG2-specific T cells can specifically recognize and react to activated Tregs with high ARG2 expression. Finally, we observe tumor growth suppression and antitumorigenic immunomodulation following ARG2 vaccination in an in vivo setting. CONCLUSION: These findings highlight the ability of ARG2-specific T cells to modulate the immunosuppressive TME and suggest that ARG2-based immunomodulatory vaccines may be an interesting option for cancer immunotherapy.

Peptide vaccination activating Galectin-3-specific T cells offers a novel means to target Galectin-3-expressing cells in the tumor microenvironment.

Galectin-3 (Gal3) can be expressed by many cells in the tumor microenvironment (TME), including cancer cells, cancer-associated fibroblasts, tumor-associated macrophages, and regulatory T cells (Tregs). In addition to immunosuppression, Gal3 expression has been connected to malignant cell transformation, tumor progression, and metastasis. In the present study, we found spontaneous T-cell responses against Gal3-derived peptides in PBMCs from both healthy donors and cancer patients. We isolated and expanded these Gal3-specific T cells in vitro and showed that they could directly recognize target cells that expressed Gal3. Finally, therapeutic vaccination with a long Gal3-derived peptide epitope, which induced the expansion of Gal3-specific CD8+ T cells in vivo, showed a significant tumor-growth delay in mice inoculated with EO771.LMB metastatic mammary tumor cells. This was associated with a significantly lower percentage of both Tregs and tumor-infiltrating Gal3+ cells in the non-myeloid CD45+CD11b- compartment and with an alteration of the T-cell memory populations in the spleens of Gal3-vaccinated mice. These results suggest that by activating Gal3-specific T cells by an immune-modulatory vaccination, we can target Gal3-producing cells in the TME, and thereby induce a more immune permissive TME. This indicates that Gal3 could be a novel target for therapeutic cancer vaccines.

TGFß-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is associated with very poor survival, making it the third and fourth leading cause of all cancer-related deaths in the USA and European Union, respectively. The tumor microenvironment (TME) in PDAC is highly immunosuppressive and desmoplastic, which could explain the limited therapeutic effect of immunotherapy in PDAC. One of the key molecules that contributes to immunosuppression and fibrosis is transforming growth factor-ß (TGFß). The aim of this study was to target the immunosuppressive and fibrotic TME in PDAC using a novel immune modulatory vaccine with TGFß-derived peptides in a murine model of pancreatic cancer. METHODS: C57BL/6 mice were subcutaneously inoculated with Pan02 PDAC cells. Mice were treated with TGFß1-derived peptides (major histocompatibility complex (MHC)-I and MHC-II-restricted) adjuvanted with Montanide ISA 51VG. The presence of treatment-induced TGFß-specific T cells was assessed by ELISpot (enzyme-linked immunospot). Changes in the immune infiltration and gene expression profile in tumor samples were characterized by flow cytometry, reverse transcription-quantitative PCR (RT-qPCR), and bulk RNA sequencing. RESULTS: Treatment with immunogenic TGFß-derived peptides was safe and controlled tumor growth in Pan02 tumor-bearing mice. Enlargement of tumor-draining lymph nodes in vaccinated mice positively correlated to the control of tumor growth. Analysis of immune infiltration and gene expression in Pan02 tumors revealed that TGFß-derived peptide vaccine increased the infiltration of CD8+ T cells and the intratumoral M1/M2 macrophage ratio, it increased the expression of genes involved in immune activation and immune response to tumors, and it reduced the expression of myofibroblast-like cancer-associated fibroblast (CAF)-related genes and genes encoding fibroblast-derived collagens. Finally, we confirmed that TGFß-derived peptide vaccine actively modulated the TME, as the ability of T cells to proliferate was restored when exposed to tumor-conditioned media from vaccinated mice compared with media from untreated mice. CONCLUSION: This study demonstrates the antitumor activity of TGFß-derived multipeptide vaccination in a murine tumor model of PDAC. The data suggest that the vaccine targets immunosuppression and fibrosis in the TME by polarizing the cellular composition towards a more pro-inflammatory phenotype. Our findings support the feasibility and potential of TGFß-derived peptide vaccination as a novel immunotherapeutic approach to target immunosuppression in the TME.

Leveraging Endogenous Dendritic Cells to Enhance the Therapeutic Efficacy of Adoptive T-Cell Therapy and Checkpoint Blockade.

Adoptive cell therapy (ACT), based on treatment with autologous tumor infiltrating lymphocyte (TIL)-derived or genetically modified chimeric antigen receptor (CAR) T cells, has become a potentially curative therapy for subgroups of patients with melanoma and hematological malignancies. To further improve response rates, and to broaden the applicability of ACT to more types of solid malignancies, it is necessary to explore and define strategies that can be used as adjuvant treatments to ACT. Stimulation of endogenous dendritic cells (DCs) alongside ACT can be used to promote epitope spreading and thereby decrease the risk of tumor escape due to target antigen downregulation, which is a common cause of disease relapse in initially responsive ACT treated patients. Addition of checkpoint blockade to ACT and DC stimulation might further enhance response rates by counteracting an eventual inactivation of infused and endogenously primed tumor-reactive T cells. This review will outline and discuss therapeutic strategies that can be utilized to engage endogenous DCs alongside ACT and checkpoint blockade, to strengthen the anti-tumor immune response.

The metabolic enzyme arginase-2 is a potential target for novel immune modulatory vaccines.

One way that tumors evade immune destruction is through tumor and stromal cell expression of arginine-degrading enzyme arginase-2 (ARG2). Here we describe the existence of pro-inflammatory effector T-cells that recognize ARG2 and can directly target tumor and tumor-infiltrating cells. Using a library of 34 peptides covering the entire ARG2 sequence, we examined reactivity toward these peptides in peripheral blood mononuclear cells from cancer patients and healthy individuals. Interferon-γ ELISPOT revealed frequent immune responses against several of the peptides, indicating that ARG2-specific self-reactive T-cells are natural components of the human T-cell repertoire. Based on this, the most immunogenic ARG2 protein region was further characterized. By identifying conditions in the microenvironment that induce ARG2 expression in myeloid cells, we showed that ARG2-specific CD4T-cells isolated and expanded from a peripheral pool from a prostate cancer patient could recognize target cells in an ARG2-dependent manner. In the 'cold' in vivo tumor model Lewis lung carcinoma, we found that activation of ARG2-specific T-cells by vaccination significantly inhibited tumor growth. Immune-modulatory vaccines targeting ARG2 thus are a candidate strategy for cancer immunotherapy.