Background Due to high melanoma immunogenicity, germline genetic variants in immune pathways have been studied for association with melanoma prognosis. However, limited candidate selection, inadequate power, or lack of independent validation have hampered the reproducibility of these prior findings, preventing personalised clinical applicability in melanoma prognostication. Our objective was to assess the prognostic utility of genetic variants in immunomodulatory pathways for prediction of melanoma clinical outcomes. Methods We genotyped 72 tag single nucleotide polymorphisms (SNPs) in 44 immunomodulatory genes in a population sample of 1022 melanoma patients and performed Cox regression analysis to test the association between SNPs and melanoma recurrence-free (RFS) and overall survival (OS). We have further investigated the most significant associations using a fine mapping strategy and followed with functional analyses in CD4+ T cells in a subset of 75 melanoma patients. Results The most significant associations were found with melanoma OS for rs3024493 in IL10 at chromosome 1q32.1 (heterozygous HR 0.58, 95% CI 0.39 to 0.86; p = 0.0006), a variant previously shown to be linked with autoimmune conditions. Multiple additional SNPs at 1q32.1 were also nominally associated with OS confirming at least two independent association signals in this locus. In addition, we found rs3024493 associated with the downregulation of interleukin 10 (IL10) secretion in CD4+ T cells. Conclusions We discovered novel associations of IL10 with melanoma survival at 1q32.1, suggesting this locus should be considered as a novel melanoma prognostic biomarker with potential for aiding melanoma patient management. Our findings also provide further support for an alternative role of IL10 in stimulation of anti-tumour immune response.

CD8+ T cells develop increased sensitivity following antigen experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that more than 50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with less than 20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM. Further, we observed superior cytotoxic effector function in TEM compared with TCM, and provide evidence that this results from a lower probability of TCM reaching threshold signaling due to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase (Csk), and use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM. Together, this work demonstrates a role for constitutive Lck activity in controlling antigen sensitivity, and suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.

Human embryonic stem cell (hESC) cultures are heterogeneous and constituting paracrine signals are required to maintain pluripotency. The cellular interplay and dynamic nature of this heterogeneity is not understood. Here, long-term hESC imaging and tracking revealed that hESC heterogeneity is dynamic and hESC self-renewal is dependent on colony-proximal distributions of paracrine signals. Tracking of hESCs forming colonies revealed that a biologically distinct cell type arises at the colony periphery in the absence of feeders. Higher rates of cell death occur in these hESC-derived cells, leading to clonal selection of colony reestablishing cells. hESC-derived feeders co-transferred during passaging promoted rapid colony recovery and expansion and reduced overall clonal selection of self-renewing hESCs. Our findings demonstrate that hESCderived feeders arise from a distinct subpopulation of hESCs that respond to paracrine cues at the colony periphery that are required to sustain and establish clonal hESC self-renewal. ' 2010 International Society for Advancement of Cytometry Key termslive cell imaging; cell tracking; lineage analysis; human embryonic stem cells; human embryonic stem cell niche STEM cell developmental potential is maintained by self-renewal, which is thought to be partially controlled in vivo through extrinsic signals that regulate stem cell survival, self-renewal, and differentiation. Similarly, recent evidence has demonstrated that human embryonic stem cell (hESCs) both create and are reliant on a supportive in vitro niche (1,2). Similar to in vivo stem cell niches, the hESC in vitro niche consists of supportive differentiated cells, including hESC-derived fibroblast-like cells (hdFs), paracrine signals, and interactions with extracellular matrix (1-3). Recently, multiple niche-independent hESC cultures have demonstrated a variety of features suggestive of early transformation events, including growth factor independence, increased proliferation, and dramatically reduced differentiation potential (4). These results illustrate the importance of the in vitro niche, and that niche components are defining factors regulating hESC fate within the culture. However, the cellular dynamics involved in hESC niche regulation are not well understood.Differentiation of hESCs to hdFs is observed in all hESC culture formats, but it is more prevalent as the culture becomes more defined and feeder layer-free (i.e. moving from mouse embryonic feeder (MEF) layers, to conditioned media, to defined media) (5). Repeated passaging for expansion of hESC cultures causes disruption of the hESC microenvironment and requires the reestablishment of the cellular and noncellular niche (such as ECM produced by hESCs) (6). To establish a cellular niche in the absence of MEFs, hESCs generate hdFs (1,7). This reestablishment period, in which niche signals for survival are suboptimal, appears to invoke significant cell death, based on observed cell debris following passage and the established disparity between hESC

BackgroundMatrix metalloproteinase-23 (MMP-23) can block the voltage-gated potassium channel Kv1.3, whose function is important for sustained Ca2+ signaling during T cell activation. MMP-23 may also alter T cell activity and phenotype through cleavage of proteins affecting cytokine and chemokine signaling. We therefore tested the hypothesis that MMP-23 can negatively regulate the anti-tumor T cell response in human melanoma.MethodsWe characterized MMP-23 expression in primary melanoma patients who received adjuvant immunotherapy. We examined the association of MMP-23 with the anti-tumor immune response - as assessed by the prevalence of tumor-infiltrating lymphocytes and Foxp3+ regulatory T cells. Further, we examined the association between MMP-23 expression and response to immunotherapy. Considering also an in trans mechanism, we examined the association of melanoma MMP-23 and melanoma Kv1.3 expression.ResultsOur data revealed an inverse association between primary melanoma MMP-23 expression and the anti-tumor T cell response, as demonstrated by decreased tumor-infiltrating lymphocytes (TIL) (P = 0.05), in particular brisk TILs (P = 0.04), and a trend towards an increased proportion of immunosuppressive Foxp3+ regulatory T cells (P = 0.07). High melanoma MMP-23 expression is also associated with recurrence in patients treated with immune biologics (P = 0.037) but not in those treated with vaccines (P = 0.64). Further, high melanoma MMP-23 expression is associated with shorter periods of progression-free survival for patients receiving immune biologics (P = 0.025). On the other hand, there is no relationship between melanoma MMP-23 and melanoma Kv1.3 expression (P = 0.27).ConclusionsOur data support a role for MMP-23 as a potential immunosuppressive target in melanoma, as well as a possible biomarker for informing melanoma immunotherapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0342-7) contains supplementary material, which is available to authorized users.

BackgroundThere has been a dramatic increase in T cell receptor (TCR) sequencing spurred, in part, by the widespread adoption of this technology across academic medical centers and by the rapid commercialization of TCR sequencing. While the raw TCR sequencing data has increased, there has been little in the way of approaches to parse the data in a biologically meaningful fashion. The ability to parse this new type of 'big data' quickly and efficiently to understand the T cell repertoire in a structurally relevant manner has the potential to open the way to new discoveries about how the immune system is able to respond to insults such as cancer and infectious diseases.

A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.

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