Checkpoint inhibitors: Better outcomes among advanced cutaneous head and neck melanoma patients.

OBJECTIVE:The aim of this study was to investigate if the treatment outcomes of checkpoint inhibitors (CPI) in patients with advanced-stage skin head and neck melanoma (HNM) differs from outcomes in patients with non-HNM. DESIGN:A retrospective cohort study of patients with unresectable AJCC stage III and stage IV, who received CPI between 2010 and 2017. PARTICIPANTS:Overall, 122 unresectable AJCC stage III and metastatic stage IV melanoma adult patients were treated with CPI during the study period (consecutive patients). The HNM group of patients was comparable with limbs and trunk melanoma group except different distant metastatic (M1a/b/c/d) pattern (p = 0.025). MAIN OUTCOMES:Comparison of overall survival and clinical response to CPI in patients with advanced-stage skin melanoma of the head and neck with non-HNM. RESULTS:We analyzed 38 patients with melanoma arising in the head and neck skin regions, 33 with melanoma of limbs and 51 with trunk melanoma. Most of the head and neck patients were men (89.5%), the average age of melanoma diagnosis was 61.4±16.7 years (range 16.4-85.6). More than a third of HNM group of patients (36.8%) were 70 years and older. Overall response rate (ORR) to CPI was 50% (CR 31.6% and PR 18.4%) in the head and neck study group of patients, compared to an ORR of 36.3% and 23.5% in melanoma of the limbs and of the trunk, respectively (p = 0.03). The median overall survival of HNM group of patients was 60.2±6.3 months, CI 95% [47.7-72.7], 63% were alive at 30 months, reaching a plateau. Whereas, the median survival time of limbs and trunk melanoma were 51.2 and 53.4 months, which did not reach significance. CONCLUSIONS AND RELEVANCE:Response rate to CPI is significantly improved in patients with melanoma of the head and neck and they have a trend towards improved, long standing, overall survival

Human T Cell Crosstalk Is Induced by Tumor Membrane Transfer

<div><p>Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8⁺ T cells to act as APCs (CD8⁺T-APCs). We demonstrate that, following trogocytosis, CD8⁺T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8⁺T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8⁺ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.</p></div

Exploring the potential of mucin 13 (MUC13) as a biomarker for carcinomas and other diseases

Abstract Background: Mucin 13 (MUC13) is a cell surface glycoprotein aberrantly expressed in a variety of epithelial carcinomas. Thus far, the role of MUC13 in various diseases remains elusive. To the best of our knowledge, this is the first study to examine the potential of MUC13 as a serum biomarker in a variety of carcinomas and other conditions. Methods: We developed a recombinant MUC13 protein, mouse monoclonal antibodies and enzyme immunoassay (ELISA) for MUC13. We used this assay to measure MUC13 levels in the supernatants of cancer cell lines and a large cohort of serum samples from healthy and diseased individuals. Results: MUC13 is secreted from cancer cell lines, with highest levels found in ovarian cancer cell lines. MUC13 levels in human sera were significantly increased in patients with renal failure and 20%–30% of patients with ovarian, liver, lung and other cancers. MUC13 was also elevated in 70% of patients with active cutaneous melanoma, but not uveal melanoma. Furthermore, we identified significant MUC13 elevations in the serum of patients with vasculitis (ANCA-positive) autoantibodies, but not in those with inflammatory bowel disease. Conclusions: Serum MUC13 is frequently elevated not only in a variety of malignant cases but also in some benign pathologies, thus appearing to be a non-specific disease biomarker. Nonetheless, serum MUC13 is clearly highly elevated in some carcinoma patients, and its relationship with tumor progression in this context warrant further research. Future studies that examine the correlation between serum MUC13 levels to stage of cancer could elucidate prognostic potential.</jats:sec

CD8⁺ T-APCs induce degranulation of effector CTLs with different antigen specificity.

<p>(<b>A, B</b>) CD8⁺ clones with different antigen specificity were used as CD8⁺T-APCs and effector CTLs. Biotinylated effector CTLs were co-cultured with CD8⁺T-APCs, stained with anti-CD107A mAb and streptavidin-allophycocyanin and analyzed by flow cytometry. (<b>A</b>) The gp100_154–162-specific clone 1G2 was used as CD8⁺T-APC for the MART-1_26–35-specific CTL clones (2E2 and 2D11). <b>(B)</b> The MART-1_26–35-specific clones 2E2 and 2D11 were used as CD8⁺T-APC for the gp100_154–162-specific clone (1G2). Numbers in upper right quadrants indicate the percentage of CD107A⁺streptavidin⁺ lymphocytes, gated on the CD8⁺ population (effector CTLs). Data are representative of three independent experiments. (<b>C</b>) Graphic presentation of intra- and inter-clonal T cell cross talk.</p

CD8⁺T-APCs activate anti-tumor CD8 T cells of the same antigen specificity.

<p><b>(A-C)</b> Cytokine production by effector CTLs in response to activation by T-APCs. <b>(A)</b> DiIC₁₈-labeled CD8⁺T-APCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118244#sec002" target="_blank">materials and methods</a>) were incubated for 6 hours with surface-biotinylated effector CTLs. Cytokine-producing effector CTLs were defined based on intracellular IFN-γ or TNF-α staining of CD8⁺streptavidin⁺ lymphocytes. Numbers in upper right quadrants indicate the percentage of IFN-γ ⁺ (upper panel) or TNF-α ⁺ (lower panel) effector CTLs. Labels indicate cells used as targets for CTLs: CTLs co-cultured with 624<i>mel</i> melanoma cells are designated T-APC; CTLs co-cultured with irrelevant M171 melanoma cells are designated non T-APC. <b>(B)</b> Confocal images of cytokine-producing effector CTLs. Calcein AM labeled CD8⁺T-APCs (<i>green</i>, upper panel) or non T-APCs (<i>green</i>, lower panel) were co-cultured for 6 hours with streptavidin-allophycocyanin-stained effector CTLs (<i>red</i>). Intracellular TNF-α production (<i>blue</i>) by effector CTLs is shown. Scale bars are 10 μm (upper panel) and 20 μm (lower panel). <b>(C)</b> Time period that CD8⁺T-APCs activate effector CTLs. Effector CTLs were co-cultured with CD8⁺T-APCs either immediately or 6, 24 and 48 hours after CD8⁺T-APC purification. Data are mean ± SE (n = 3 replicates/group) percentage of IFN-γ ⁺ effector CTLs, gated on CD8⁺ T cells. <b>(D)</b> CD8⁺T-APCs trigger degranulation of effector CTLs. CD8⁺T-APCs were generated as described above (1A) and co-cultured with effector CTLs. Cytolytic activity of T cells was measured by detection of surface CD107A on CD8⁺streptavidin⁺ effector CTLs. Number in upper right quadrants indicates the percentage of CD107A⁺ streptavidin⁺ effector CTLs. Data are representative of at least three independent experiments.</p

CD8⁺T-APC induce secondary trogocytosis by tumor specific CTL.

<p><b>(A, B)</b> CD8⁺T-APCs and non-T-APCs were generated by co-incubation with cognate or irrelevant melanoma, respectively. They were then sorted, labeled with DiIC18 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118244#sec002" target="_blank">Methods</a>) and co-cultured with surface biotinylated 2C7 or 2E2 clones (effector CTL). The culture was then stained with anti-CD8 antibodies and streptavidin- allophycocyanin, and subjected to flow cytometry. <b>(A)</b> Histograms indicate secondary trogocytosis by the presence of DiIC18 on 2C7 or 2E2 CTLs, gated on CD8⁺streptavidin⁺ populations, following co-culture with CD8⁺T-APC (<i>blue</i>) or non-T-APC (<i>grey</i>). <b>(B)</b> Secondary trogocytosis was measured by presence of DiIC18 on the CD8⁺streptavidin⁺ population (effector CTL) and streptavidin-allophycocyanin on the CD8⁺DiIC18⁺ population (<i>left column</i>, non-T-APC, <i>right column</i>, T-APC). Numbers in upper right quadrants indicate the percentage of DiIC18- stained effector CTL (performing secondary trogocytosis). <b>(C)</b> PKH67-labeled CD8⁺T-APCs (<i>red</i>) were co-cultured with PKH26-labeled effector CTLs (<i>blue</i>). The lymphocytes were co-incubated in a chambered cover-glass and subjected to confocal microscopy. A snapshot series of 8 min is presented. <i>Arrows</i>, transfer of membrane fragments (secondary trogocytosis) from CD8⁺T-APC to CTL. Scale bars, 15 μm. Data are representative of at least five independent experiments (A, B) or of three experiments (C).</p

The effect of CD8⁺T-APCs on effector CTLs is mediated by tumor-derived pMHC.

<p><b>(A)</b> Ova-expressing EG7 and parental EL4 target cell lines (<i>Left column</i>) and target-entrained CD8⁺T-APC (generated following co-culture of OT-I CD8⁺ T cells with designated targets, <i>right column</i>) were labeled with Ova_257–264/H-2Kb-specific mAb (<i>black histogram</i>). <i>Grey histogram</i>, background staining with isotype control antibody. <b>(B)</b> Proliferation of OT-I CD8⁺ T cells stimulated with CD8⁺T-APCs. CFSE-labeled OT-I T cells were left untreated (no target) or co-cultured with the following T-APCs: OT-I CD8⁺ pre-incubated with EL4 (EL4) or OT-I CD8⁺ pre-incubated with EG7 cells (EG7). ConA stimulation was used as positive control (right). <i>Scale bars</i>, proliferating lymphocytes that divided at least twice. <i>Numbers</i>, percentage of dividing CD8⁺ T cells. Data are representative of two independent experiments.</p

Assessment of CTL fratricide activity based on detection of cleaved caspase-3.

<p>MART-1- or MUC-1- reactive CTLs were co-cultured with DDAO-SE-tagged CD8⁺T-APCs and non T-APCs, generated by co-incubation of 2E2 cells with 624<i>mel</i> and M171 melanoma cells, respectively. T-APC damage was examined based on detection of intracellular cleaved caspase-3 in the DDAO-SE⁺CD8⁺ population. Numbers in upper right quadrants indicate the percentage of cleaved caspase 3-positive CD8⁺T-APC cells. Data are representative of three independent experiments.</p