The ABCs of Antigen Presentation by Stromal Non-Professional Antigen-Presenting Cells

Professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, are known for their ability to present exogenous antigens to T cells. However, many other cell types, including endothelial cells, fibroblasts, and lymph node stromal cells, are also capable of presenting exogenous antigens to either CD8+ or CD4+ T cells via cross-presentation or major histocompatibility complex (MHC) class II-mediated presentation, respectively. Antigen presentation by these stromal nonprofessional APCs differentially affect T cell function, depending on the type of cells that present the antigen, as well as the local (inflammatory) micro-environment. It has been recently appreciated that nonprofessional APCs can, as such, orchestrate immunity against pathogens, tumor survival, or rejection, and aid in the progression of various auto-immune pathologies. Therefore, the interest for these nonprofessional APCs is growing as they might be an important target for enhancing various immunotherapies. In this review, the different nonprofessional APCs are discussed, as well as their functional consequences on the T cell response, with a focus on immuno-oncology.Experimental cancer immunology and therap

Cancer immunotherapy: broadening the scope of targetable tumours

MTG2 - Moleculaire genetica van gastrointestinale tumorenMolecular tumour pathology - and tumour genetic

Predictive biomarkers for outcomes of immune checkpoint inhibitors (ICIs) in melanoma: a systematic review

Simple Summary Immune checkpoint inhibitors (ICIs) have revolutionized treatment of advanced melanoma and survival of melanoma patients has radically improved since. However, as durable responses after ICIs are only observed in 30-50% of melanoma patients, there is an unmet need to identify predictive biomarkers for response. This systematic review demonstrates the substantial number of publications that have studied a wide variety of possible biomarkers. Covering 177 publications that investigated 128 unique biomarkers, we provide an overview of all studied biomarkers in correlation with response or survival. We highlight blood, tumor and fecal biomarkers that were associated with response to ICIs in multiple studies. Of these, only T-cell inflamed gene expression profiling was predictive for response in a large clinical trial and validated in other studies, thus representing a promising biomarker for clinical practice. Large validation studies are warranted to confirm the predictive utility of other biomarkers, thereby further personalizing immunotherapy treatment. Immune checkpoint inhibitors (ICIs) have strongly improved the survival of melanoma patients. However, as durable response to ICIs are only seen in a minority, there is an unmet need to identify biomarkers that predict response. Therefore, we provide a systematic review that evaluates all biomarkers studied in association with outcomes of melanoma patients receiving ICIs. We searched Pubmed, COCHRANE Library, Embase, Emcare, and Web of Science for relevant articles that were published before June 2020 and studied blood, tumor, or fecal biomarkers that predicted response or survival in melanoma patients treated with ICIs. Of the 2536 identified reports, 177 were included in our review. Risk of bias was high in 40%, moderate in 50% and low in 10% of all studies. Biomarkers that correlated with response were myeloid-derived suppressor cells (MDSCs), circulating tumor cells (CTCs), CD8+ memory T-cells, T-cell receptor (TCR) diversity, tumor-infiltrating lymphocytes (TILs), gene expression profiling (GEP), and a favorable gut microbiome. This review shows that biomarkers for ICIs in melanoma patients are widely studied, but heterogeneity between studies is high, average sample sizes are low, and validation is often lacking. Future studies are needed to further investigate the predictive utility of some promising candidate biomarkers.Surgical oncolog

CD103 and CD39 coexpression identifies neoantigen-specific cytotoxic T cells in colorectal cancers with low mutation burden

Background Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.Experimental design Whole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+ T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+ cytotoxic T cells in tumors.Results Neoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+ (double positive, DP) CD8+ T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+ T cells could be attributed to CD4+ T cells. CD8+ T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.Conclusion Coexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+ T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients.Experimental cancer immunology and therap

Neutralizing antibodies impair the oncolytic efficacy of reovirus but permit effective combination with T cell-based immunotherapies

Reovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell–deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell–based immunotherapeutic strategies. Cellular mechanisms in basic and clinical gastroenterology and hepatolog

PRAME and CTCFL-reactive TCRs for the treatment of ovarian cancer

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2 '-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Enhanced antigen cross-presentation in human colorectal cancer-associated fibroblasts through upregulation of the lysosomal protease cathepsin S

Background Cross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown. Methods In this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function. Results Here, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression. Conclusion These data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Blood-based kinase activity profiling: A potential predictor of response to immune checkpoint inhibition in metastatic cancer

Background Many cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome. Methods Here a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation. Results The kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-

Adoptive cell therapy: a highly successful individualized therapy for melanoma with great potential for other malignancies

Experimental cancer immunology and therap

Apoptin Enhances Radiation-Induced Cell Death in Poorly Responding Head and Neck Squamous Cell Carcinoma Cells

Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumours by combining irradiation with other therapeutics to induce apoptosis are widely investigated. We examined whether chicken anaemia virus-derived apoptin protein would have a beneficial effect on irradiation of radiosensitive SCC61 and radioresistant SQD9 human head and neck squamous carcinoma cell lines. In both cell lines, concurrent exposure to irradiation and apoptin resulted in analysed mitochondrial cytochrome c release and in cleavage of caspase-3, whereas irradiation alone of SQD9 cells under identical conditions did not. Moreover, in comparison with the irradiation, only the synchronized treatment of apoptin and irradiation resulted in increased cell death in especially the radioresistant SQD9 cells, as measured by means of a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumours responding poorly to radiotherapy.Experimental cancer immunology and therap