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How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4+ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca2+ buffering in NKT2 cells. In addition, the low Ca2+ extrusion rate may also contribute to the higher Ca2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8+ T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8+ CAR+ PD-1high TIM3high ("exhausted") tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8+ T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8+ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8+ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti-PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.

This document is a response to a study that questioned the results of a previous study on the effects of L-Type Ca2+ channel blockers on synthetic arterial myocytes. The authors of the response defend their conclusions and provide evidence to support their findings. They argue that the previous study did not replicate their key observations and that their conclusions are not incorrect due to the fluorescence of amlodipine. They also present evidence of the activation of CRAC currents by amlodipine and other Ca2+ channel blockers. The text also discusses a study by Bird et al. that challenges the findings of another study by Johnson et al. on the effects of L-type calcium channel blockers on store-operated calcium entry (SOCE) and heart failure. Johnson et al. argue that L-type calcium channel blockers can promote vascular remodeling through the activation of STIM proteins. Bird et al. dispute these findings and suggest that L-type calcium channel blockers are not effective in preventing heart failure. The text also addresses criticisms and responses from other researchers regarding the two studies. [Extracted from the article]

Background: Chronically hospitalized children are at risk for neurodevelopmental delay, compounded by restricted social interactions, movement and environmental stimulation. We measured patients' movements and interactions to characterize developmentally relevant aspects of our inpatient environment and identify opportunities for developmental enrichment. Methods: As part of a quality improvement initiative to inform neurodevelopmental programming for children with medical complexity at our paediatric post‐acute care specialty hospital, we conducted >232 hours of time‐motion observations. Trained observers followed 0‐ to 5‐year‐old inpatients from 7 am to 7 pm on weekdays, categorizing observations within five domains: Where, With, Position, State and Environment. Observations were collected continuously utilizing REDCap on iPads. A change in any domain initiated a new observation. Results: Patients were median 1 year and 8 months of age (range 2 months to 3 years 9 months) with a median length of hospitalization of 514 days (range 66–1298). In total, 2636 unique observations (or median 134 observations per patient‐day [range 95–210]) were collected. Patients left their rooms up to 4 times per day for median 1 h and 34 min (range 41 min to 4 h:30 min). Patients spent 4 h:6 min (2 h:57 min to 6 h:30 min) interacting with someone and 3 h:51 min (57 min to 6 h:36 min) out of bed each day. Patients were simultaneously out of their beds, interacting with someone and awake for 2 h:21 min (51 min to 4 h:19 min) each day. Conclusions: Despite a care model prioritizing time out of bed and social interaction, time‐motion observations indicate patients spent many of their waking hours in bed and alone. Quantifying our inpatients developmental opportunities will inform neurodevelopmental programming initiatives. [ABSTRACT FROM AUTHOR]

Skin and soft tissue infections (SSTIs) are frequent in student athletes and are often caused by community-associated methicillin-resistant "Staphylococcus aureus" (CA-MRSA). We evaluated the awareness of CA-MRSA among high school coaches and athletic directors in Missouri (n = 4,408) and evaluated hygiene practices affecting SSTI transmission. Of 1,642 (37%) respondents, 61% received MRSA educational information during the past year and 32% indicated their school had written guidelines for managing SSTIs in athletes. Coaches and athletic directors aware of written guidelines reported a lower incidence of SSTIs in student athletes (26%) compared to those without written policies (34%, p = 0.03). When confronted with SSTIs, 49% of respondents referred student athletes to the school nurse or a physician. A relationship exists between school policies for SSTI management and lower incidence of SSTIs. Educational initiatives by school nurses in conjunction with athletic staff may lead to practices that limit SSTIs in this at-risk population. (Contains 5 tables and 5 figures.)