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Legends for supplementary Figure 1 through 5

Figure S1. Kaplan-meier curves of the ACT-treated patients. Figure S2. Immunofluorescent staining of metastatic melanoma tissue with antibodies against CD3, CD8, FoxP3, CD14, CD163, galectin-1, galectin-3 and galectin-9. Figure S3. Macrophage and Tbet staining Figure S4. Interaction analyses of immune markers that are prognostic for survival Figure S5. Interaction analysis and immune signature of survival cohorts Table S1A. Immunoreactive score (IRS): scoring of staining intensity of markers expressed by tumor cells. S1B. Patient characteristics Table S2A. Number of infiltrating immune cells and expression of galectin-1 and galectin-3. Table S2B. Correlation between all analyzed immune markers Table S3. Immune signatures of the patients showing clinical benefit after ACT.

The presence of tumor-infiltrating immune cells is associated with longer survival and a better response to immunotherapy in early-stage melanoma, but a comprehensive study of the in situ immune microenvironment in stage IV melanoma has not been performed. We investigated the combined influence of a series of immune factors on survival and response to adoptive cell transfer (ACT) in stage IV melanoma patients. Metastases of 73 stage IV melanoma patients, 17 of which were treated with ACT, were studied with respect to the number and functional phenotype of lymphocytes and myeloid cells as well as for expression of galectins-1, -3, and -9. Single factors associated with better survival were identified using Kaplan–Meier curves and multivariate Cox regression analyses, and those factors were used for interaction analyses. The results were validated using The Cancer Genome Atlas database. We identified four parameters that were associated with a better survival: CD8+ T cells, galectin-9+ dendritic cells (DC)/DC-like macrophages, a high M1/M2 macrophage ratio, and the expression of galectin-3 by tumor cells. The presence of at least three of these parameters formed an independent positive prognostic factor for long-term survival. Patients displaying this four-parameter signature were found exclusively among patients responding to ACT and were the ones with sustained clinical benefit. Cancer Immunol Res; 5(2); 170–9. ©2017 AACR.

Immune-checkpoint inhibitors (ICIs) activate the immune system to assault cancer cells in a manner that is not antigen specific. We hypothesized that tolerance may also be broken to autoantigens, resulting in autoantibody formation, which could be associated with immune-related adverse events (irAEs) and antitumor efficacy. Twenty-three common clinical autoantibodies in pre- and posttreatment sera from 133 ipilimumab-treated melanoma patients were determined, and their development linked to the occurrence of irAEs, best overall response, and survival. Autoantibodies developed in 19.2% (19/99) of patients who were autoantibody-negative pretreatment. A nonsignificant association was observed between development of any autoantibodies and any irAEs [OR, 2.92; 95% confidence interval (CI) 0.85–10.01]. Patients with antithyroid antibodies after ipilimumab had significantly more thyroid dysfunction under subsequent anti–PD-1 therapy: 7/11 (54.6%) patients with antithyroid antibodies after ipilimumab developed thyroid dysfunction under anti–PD1 versus 7/49 (14.3%) patients without antibodies (OR, 9.96; 95% CI, 1.94–51.1). Patients who developed autoantibodies showed a trend for better survival (HR for all-cause death: 0.66; 95% CI, 0.34–1.26) and therapy response (OR, 2.64; 95% CI, 0.85–8.16). We conclude that autoantibodies develop under ipilimumab treatment and could be a potential marker of ICI toxicity and efficacy.

Supplementary Figure S1 Supplementary Figure S2 Supplementary Table S1 Supplementary Table S2

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.

Blood lymphocytes of a HLA-A2 positive breast cancer patient were stimulated with either MCF-7 or MDA-MB-231, i.e., HLA-A2-matched allogeneic breast carcinoma cell lines. Several CD8 + CTL clones with reactivity against the stimulator cells but not against K562 were generated. Reactivity could be blocked with monoclonal antibody (mAb) W6/32, MA2.1, and/or BB7.2, indicating that the clones are HLA-class I and HLA-A2 restricted. The CTL clones generated following stimulation with MCF-7, recognized various other allogeneic HLA-A2 + tumor cell lines, including breast carcinoma, renal cell carcinoma, and melanoma cell lines, but not HLA-A2 − tumor cell lines. The CTL clones did not recognize normal HLA-A2 + cells including breast epithelial cells, renal proximal tubular epithelial cells (PTEC), or EBV-transformed B cells including the autologous EBV cell line. In contrast to the CTL clones induced with MCF-7, the reactivity of the clones stimulated with MDA-MB-231, was limited to the stimulator cell MDA-MB-231. Cytotoxicity assays utilizing T2 cells loaded with peptides as target cells indicated that none of the examined CTL-epitopes derived from HER-2/neu, Muc-1, Ep-CAM-1, and p53 were recognized by the CTL clones generated. Our findings underscore that breast cancer is an immunogenic tumor and that HLA-class I-matched allogeneic tumor cells can be used as stimulator cells to generate tumor-specific CTL from peripheral blood of a breast cancer patient with specificity for an antigenic determinant that is broadly expressed on tumor cells from various origins or with specificity limited to the breast cancer stimulator cell.

Recognition of the neo-antigens that are formed as a consequence of DNA damage is likely to be a major driving force behind the clinical activity of T cell checkpoint blockade and adoptive T cell therapy for treatment of non-virally induced solid cancers. Consequently, strategies to selectively enhance T cell reactivity against genetically defined neo-antigens are currently under development. In mouse models, T cell pressure has been shown to sculpt the antigenicity of tumors through selection of tumor cell variants that do not express the relevant neo-antigens. However, it is unclear whether the T cell-recognized neo-antigen repertoire is constant over time in human cancers. To address this issue, we analyzed the stability of neo-antigen specific T cell responses and the antigens they recognize in stage IV melanoma patients treated by adoptive T cell transfer. Fresh tumor tissue resected before and after treatment was used to establish autologous melanoma cell lines, to culture tumor-infiltrating lymphocytes (TIL) and to generate T-cell infusion products by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Material from 2 patients with objective clinical responses after infusion of autologous tumor-specific CD4+ and CD8+ T cells was analyzed by whole exome and RNA sequencing. Subsequently, 31-mer peptides covering all the non-synonymous somatic mutations were used to test the reactivity of both the T-cell infusion products and TIL isolated from subsequent tumor lesions by ELISA and flow cytometry. The expression of genes encoding T cell-recognized neo-antigens was then analyzed within the tumor cell lines and within the corresponding paraffin-embedded tumor tissue, by real-time PCR analysis, a competitive allele-specific PCR assay, and by analysis of heterozygosity and next generation amplicon sequencing. Analysis of the infusion products showed that 50-80% of T cells reacted to autologous tumor cells, with only a very low percentage of these cells recognizing known shared tumor-associated epitopes. Instead, tumor reactivity was predominantly mediated by CD4+ and CD8+ T cells recognizing clonally expressed neo-antigens formed by private mutations. CD8+ T cells within the infusion products and within TIL populations generated from subsequent tumor lesions of these two patients recognized a total of 6 different neo-antigens, and analysis of these neo-antigens in both the index lesion and subsequent lesions revealed profound alterations in the T cell-recognized neo-antigen landscape. In particular, in 2 cases, the mutant allele was lost from a subsequent tumor, in one case, expression of the mutant gene was substantially reduced in a recurring lesion, and in another case expression of the mutant gene was increased approx. 40-fold in a subsequent tumor. Notably, in 4 out of 4 cases these changes in neo-antigen expression were paralleled by loss or gain of the respective neo-antigen specific T cell response. Collectively, our data demonstrate that under conditions in which a high frequency of tumor-specific T cells is present, tumor cell variants with reduced or lost neo-antigen expression can emerge, similar to what has been observed in mice. This, and the observation of concurrent acquisition of novel T cell reactivity, reveal the dynamics of T cell - neo-antigen interaction in human cancer, and indicate that immunotherapies that maximize the capacity to respond to an altering neo-antigen landscape may offer the highest chance to achieve long-term tumor control. Citation Format: Els M.E. Verdegaal, Noel de Miranda, Marten Visser, Tom Harryvan, Marit van Buuren, Rikke Andersen, Sine Hadrup, Caroline van der Minne, Remko Schotte, Hergen Spits, John Haanen, Ellen Kapiteijn, Ton Schumacher, Sjoerd H. Van Der Burg. Neo-antigen landscape dynamics during human melanoma-T cell interactions [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR11.