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Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-214. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity3-5. Although presentation of B:12-20 is evident in the islets3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in β-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

The role of aberrantly expressed proteins in tumors in driving immune-mediated control of cancer has been well documented for more than five decades. Today, we know that both aberrantly expressed normal proteins as well as mutant proteins (neoantigens) can function as tumor antigens in both humans and mice. Next-generation sequencing (NGS) and high-resolution mass spectrometry (MS) technologies have made significant advances since the early 2010s, enabling detection of rare but clinically relevant neoantigens recognized by T cells. MS profiling of tumor-specific immunopeptidomes remains the most direct method to identify mutant peptides bound to cellular MHC. However, the need for use of large numbers of cells or significant amounts of tumor tissue to achieve neoantigen detection has historically limited the application of MS. Newer, more sensitive MS technologies have recently demonstrated the capacities to detect neoantigens from fewer cells. Here, we highlight recent advancements in immunopeptidomics-based characterization of tumor-specific neoantigens. Various tumor antigen categories and neoantigen identification approaches are also discussed. Furthermore, we summarize recent reports that achieved successful tumor neoantigen detection by MS using a variety of starting materials, MS acquisition modes, and novel ion mobility devices. [ABSTRACT FROM AUTHOR]

A study was conducted to evaluate whether DNMT3A is recurrently mutated in acute myeloid leukemia samples and whether DNMT3A mutations are associated with poor survival rates. Results indicated that sNMT3A mutations are highly recurrent in such patients and are independently associated with poor outcomes.

Background: The tumor microenvironment plays an important role in the progression of cancer by mediating stromal--epithelial paracrine signaling, which can aberrantly modulate cellular proliferation and tumorigenesis. Exposure to environmental toxicants, such as inorganic arsenic (iAs), has also been implicated in the progression of prostate cancer. OBJECTIVE: The role of iAs exposure in stromal signaling in the tumor microenvironment has been largely unexplored. Our objective was to elucidate molecular mechanisms of iAs-induced changes to stromal signaling by an enriched prostate tumor microenvironment cell population, adipose-derived mesenchymal stem/stromal cells (ASCs). RESULTS: ASC-conditioned media (CM) collected after 1 week of iAs exposure increased prostate cancer cell viability, whereas CM from ASCs that received no iAs exposure decreased cell viability. Cytokine array analysis suggested changes to cytokine signaling associated with iAs exposure. Subsequent proteomic analysis suggested a concentration-dependent alteration to the HMOX1/THBS1/TGFβ signaling pathway by iAs. These results were validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, confirming a concentration-dependent increase in HMOX1 and a decrease in THBS1 expression in ASC following iAs exposure. Subsequently, we used a TGFβ pathway reporter construct to confirm a decrease in stromal TGFβ signaling in ASC following iAs exposure. CONCLUSIONS: Our results suggest a concentration-dependent alteration of stromal signaling: specifically, attenuation of stromal-mediated TGFβ signaling following exposure to iAs. Our results indicate iAs may enhance prostate cancer cell viability through a previously unreported stromal-based mechanism. These findings indicate that the stroma may mediate the effects of iAs in tumor progression, which may have future therapeutic implications. http://dx.doi.org/10.1289/ehp.1510090, Introduction Inorganic arsenic (iAs) is a ubiquitously distributed environmental toxicant that is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) (IARC 2012). The [...]

Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3β (GSK3β) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3β and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3β and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3β prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3β with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3β. A GSK3β-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3β regulation of Nav1.6 as a biosignature ofMSNs maladaptive plasticity. [ABSTRACT FROM AUTHOR]