Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small-cell lung cancer.

BACKGROUND: Despite the importance of tumor-infiltrating T lymphocytes (TILs) in cancer biology, the relationship between TIL phenotypes and their prognostic relevance for localized non-small-cell lung cancer (NSCLC) has not been well established. PATIENTS AND METHODS: Fresh tumor and normal adjacent tissue was prospectively collected from 150 patients with localized NSCLC. Tissue was comprehensively characterized by high-dimensional flow cytometry of TILs integrated with immunogenomic data from multiplex immunofluorescence, T-cell receptor sequencing, exome sequencing, RNA sequencing, targeted proteomics, and clinicopathologic features. RESULTS: While neither the magnitude of TIL infiltration nor specific TIL subsets were significantly prognostic alone, the integration of high-dimensional flow cytometry data identified two major immunotypes (IM1 and IM2) that were predictive of recurrence-free survival independent of clinical characteristics. IM2 was associated with poor prognosis and characterized by the presence of proliferating TILs expressing cluster of differentiation 103, programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing protein 3, and inducible T-cell costimulator. Conversely, IM1 was associated with good prognosis and differentiated by an abundance of CD8+ T cells expressing cytolytic enzymes, CD4+ T cells lacking the expression of inhibitory receptors, and increased levels of B-cell infiltrates and tertiary lymphoid structures. While increased B-cell infiltration was associated with good prognosis, the best prognosis was observed in patients with tumors exhibiting high levels of both B cells and T cells. These findings were validated in patient tumors from The Cancer Genome Atlas. CONCLUSIONS: Our study suggests that although the number of infiltrating T cells is not associated with patient survival, the nature of the infiltrating T cells, resolved in distinct TIL immunotypes, is prognostically relevant in NSCLC and may inform therapeutic approaches to clinical care.

Genetic and molecular characterization of uveal melanoma cell lines.

The recent identification of frequent activating mutations in GNAQ or GNA11 in uveal melanoma provides an opportunity to better understand the pathogenesis of this melanoma subtype and to develop rational therapeutics to target the cellular effects mediated by these mutations. Cell lines from uveal melanoma tumors are an essential tool for these types of analyses. We report the mutation status of relevant melanoma genes, expression levels of proteins of interest, and DNA fingerprinting of a panel of uveal melanoma cell lines used in the research community.

Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells.

Our previous studies demonstrated that inhibitors of arachidonate-phospholipid remodeling [i.e. the enzyme CoA-independent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate-phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.

Residues involved in co-factor and substrate binding of the short-chain dehydrogenase/reductase PTR1 producing methotrexate resistance in Leishmania.

The Leishmania short-chain dehydrogenase/reductase gene ptr1 has been isolated while characterizing antifolate-resistant Leishmania mutants. PTR1 is active as a tetramer and can reduce pterins and folates. PTR1 has several of the hallmarks of the short chain dehydrogenase/reductase family, including a glycine rich co-factor binding site at its N-terminus, and the consensus catalytic site TyrXaa3Lys. To start probing the structure/function of PTR1, we have generated by site-directed mutagenesis five mutants, either in the co-factor-binding site, Y38D or in the catalytic site Y195F, Y195W, K199R or in a PTR1-specific region, Y175F. The mutated versions of PTR1 were studied in vivo in Leishmania and at the biochemical level using purified proteins. The Y175F mutant showed properties similar to wild-type PTR1 in every aspect tested, but all the other mutants were inactive even if they were purified as tetramers. To test the ability of the mutated PTR1 versions to bind its co-factor and substrates, trypsin digestion experiments were carried out under conditions upon which binding will prevent wild-type PTR1 of being digested by trypsin. The wild-type PTR1 as well as Y175F and Y195F mutants were protected against trypsin digestion whereas Y38D and K199R mutants were not. Mutations in regions involved in co-factor binding (Y38D) or in catalytic site (K199R) altered the binding of the ligands, explaining why those protein versions are inactive.

Functional analysis of Mycoplasma arthritidis-derived mitogen interactions with class II molecules.

The ability of superantigens (SAGs) to trigger various cellular events via major histocompatibility complex (MHC) class II molecules is largely mediated by their mode of interaction. Having two MHC class II binding sites, staphylococcal enterotoxin A (SEA) is able to dimerize MHC class II molecules on the cell surface and consequently induces cytokine gene expression in human monocytes. In contrast, cross-linking with specific monoclonal antibodies or T-cell receptor is required for staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1) to induce similar responses. In the present study, we report how Mycoplasma arthritidis-derived mitogen (MAM) may interact with MHC class II molecules to induce cytokine gene expression in human monocytes. The data presented indicate that MAM-induced cytokine gene expression in human monocytes is Zn2+ dependent. The MAM-induced response is completely abolished by pretreatment with SEA mutants that have lost their capacity to bind either the MHC class II alpha or beta chain, with wild-type SEB, or with wild-type TSST-1, suggesting that MAM induces cytokine gene expression most probably by inducing dimerization of class II molecules. In addition, it seems that SEA and MAM interact with the same or overlapping binding sites on the MHC class II beta chain and, on the other hand, that they bind to the alpha chain most probably through the regions that are involved in SEB and TSST-1 binding.