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BackgroundExpression 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 designWhole-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.ResultsNeoantigen-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.ConclusionCoexpression 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.

BackgroundHigh serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective.MethodsIL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1.control. Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6rafl/fl×LysMcre+ mice.ResultsOur therapeutic vaccination protocol elicits a strong tumor-specific CD8+ T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8+ T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6rafl/fl×LysMcre+ but not cre-negative control mice, while no differences in the vaccine-induced CD8+ T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages.ConclusionIL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections.

BackgroundCross-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.MethodsIn 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.ResultsHere, 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.ConclusionThese data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

BackgroundEpithelial ovarian cancer (EOC) is considered an immunogenic tumor, as illustrated by the clear correlation between T-cell infiltration and overall survival. This suggests that patients with EOC may be eligible for immunotherapy including adoptive cell therapy with autologous Tumor Infiltrating Lymphocytes (TIL). However, immunosuppressive cells including myeloid derived suppressor cells an regulatory T cells are also abundant in EOC and may need to be targeted simultaneously to achieve the full potential of the infused TIL. Carboplatin-paclitaxel chemotherapy (CPC) reduces the number of immunosuppressive cells in cervical cancer patients,1 creating a window-of-opportunity for TIL to exert their full effector function. Interferon-alpha further supports infused TIL. A phase I/II trial (NCT04072263) was initiated to study the feasibility and safety of TIL during CPC with or without additional interferon-alpha in patients with recurrent platinum-sensitive EOC.MethodsFifteen patients with recurrent platinum-sensitive EOC received 6 cycles of CPC intravenously every 3 weeks and TIL intravenously 2 weeks after the 2nd,3rd and 4th CPC cycle. Pegylated-interferon-alpha was added in the second cohort for 12 weeks, starting one week before the first TIL infusion. Patients who received 3 TIL infusions were evaluable. The primary endpoint was feasibility and safety of TIL administration during CPC with or without interferon-alpha. As secondary endpoints signs of activity, underlying mechanisms, immunomodulation, and T-cell reactivity were studied.ResultsThirteen patients were available for analysis. Median age 63 years (range, 29–77). TIL could be successfully expanded for all patients. Treatment with TIL during CPC was safe and did not add toxicity. Addition of IFNα resulted in grade 3 leucopenia and grade 3 trombocytopenia in the first 2 patients and was therefore omitted in subsequent patients. CPC alleviated the immunosuppressive status, reflected by reduced plasma IL-6 levels and circulating myeloid-cell numbers, while lymphocytes numbers are not affected. This was most prominently at 1–2 weeks after the 2nd CPC and is suggested to reflect improved conditions promoting intra-tumoral T-cell reactivity. Objective responses were observed in 10/13 (77%) patients and 3 patients had stable disease. Interestingly, in at least one patient the ongoing platinum-free interval of 25 months far exceeds the first platinum-free interval of 8 months after similar CPC. In depth studies on immune modulation by chemotherapy and by TIL/Interferon-alpha, and correlations between TIL phenotype and clinical outcome are ongoing and will be presented.ConclusionsCombined treatment with CP chemotherapy and properly timed TIL may result in clinical benefit for patients with EOC.AcknowledgementsThe unrestricted funding of the trial by Ovacure is greatly acknowledged.Trial RegistrationThe trial is registered at www.clinicaltrials.gov under number NCT04072263.ReferenceWelters MJ, van der Sluis TC, van Meir H, Loof NM, van Ham VJ, van Duikeren S, Santegoets SJ, Arens R, de Kam ML, Cohen AF, van Poelgeest MI, Kenter GG, Kroep JR, Burggraaf J, Melief CJ, van der Burg SH. Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses. Sci Transl Med 2016;8(334):334ra52. doi: 10.1126/scitranslmed.aad8307Ethics ApprovalThis study was approved by Leiden University Medical Center‘s Ethics Board; approval number L18-012 and the Central Committee on Research Involving Human Subjects; approval number NL63434.000.17.

Background Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed. Methods Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls. Results Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3+PD-1+), specifically helper T cells (CD3+CD8−Foxp3−), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4+PD-1++CD161−CD38+HLA-DR+ and CD8+CD103+CD161−NKG2A+/−PD1++CD38++HLA-DR+) effector memory T cells. Conclusion This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated. Electronic supplementary material The online version of this article (10.1186/s40425-019-0712-z) contains supplementary material, which is available to authorized users.

Background The capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds. Methods We generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8+ T cell response were determined. Mice were subsequently challenged with tumor cells, and the tumor protection was monitored. Results Immunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8+ T cell responses of > 0.3% of the total circulating CD8 T cell population fully protects mice against lethal tumor challenge. However, low dose inoculations via the IP or SC route or IN vaccination elicited vaccine-induced CD8+ T cell responses that did not reach protective thresholds for tumor protection. In addition, whereas weak pre-existing immunity did not alter the protective thresholds of the vaccine-specific T cell response following subsequent immunization with CMV-based vaccine vectors, strong pre-existing immunity inhibited the development of vaccine-induced T cells and their control on tumor progression. Conclusions This study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients. Electronic supplementary material The online version of this article (10.1186/s40425-019-0500-9) contains supplementary material, which is available to authorized users.

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. Given their profound effect on the outcome of immunotherapy trials, Treg frequency and function are being studied extensively. Unfortunately, no consensus has been reached about a) the (minimal number of) markers required to define human Tregs and b) which assay is the best to measure Treg suppressor function. We had organized a workshop on the detection and functional testing of (antigen-specific) Tregs. Based on the outcome of this workshop and subsequent discussions, we proposed the following essential marker set for flow cytometric Treg analysis: CD3, CD4, CD25, CD127, FoxP3, Ki67 and CD45RA. We have validated the use of this essential marker set in a series of PBMC from healthy donors and cancer patients, as well as in tumor draining lymph nodes and freshly isolated tumors. We here show that the CD3, CD4, CD25, CD127 and FoxP3 markers are the minimally required markers to define human Treg cells in peripheral blood, tumors and lymph nodes. Importantly, as Ki67 and CD45RA provide additional information on their activation status, we recommend to also include these markers in the essential Treg marker set. Similarly, the discussion on functional assays revealed that classical suppressor cell assays measuring the inhibition of responder cell proliferation or cytokine production in 3-7 day long assays required large numbers of Tregs and were technically challenging. Other assays, in particular an assay measuring Treg-mediated prevention of CD69 and CD154 up-regulation on CD4+ T cells was used as alternative and short (6-20h) assay for assessing human Treg suppressor function. We studied the ability of Tregs to inhibit CD25, CD69, CD154 and CD137 up-regulation on CD4+ and CD8+ T cells and compared this to the classical assay. Indeed Tregs were able to inhibit aCD3/CD28-bead-induced up-regulation of CD25, CD69 (on CD4 and CD8) and CD154 (on CD4), but not CD137 (on CD8) on responder T cells. Moreover, we showed that this excellently correlated with suppression of T cell proliferation. In conclusion, we have identified CD3, CD4, CD25, CD127, FoxP3, Ki67 and CD45RA as the minimally required marker to define Tregs and showed that the T cell activation marker assay is a simple, fast and robust assay to measure Treg suppressive activity. The proposed essential marker set will be used to launch proficiency panels and harmonize the phenotypic analysis of Tregs within the CIMT Immunoguiding Program (CIP) participating laboratories.