Loss of Zbtb32 in NOD mice does not significantly alter T cell responses. [version 2; referees: 2 approved]

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2+ dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2+ DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32-/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32-/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype

Depression, anxiety, stress, and fear of COVID-19 among Bangladeshi medical students during the first wave of the pandemic: a mixed-methods study

AimThis study aims to investigate depression, anxiety, stress, and fear of the COVID-19 pandemic and the associated risk factors among Bangladeshi medical students. It also explored qualitative insights on mental health from medical students during the first wave of the pandemic.MethodsThis mixed-methods study was conducted online in Bangladesh from June 2020 to September 2020. Participants were Bangladeshi medical students from the first year to the final year. The quantitative part included a structured online survey. One focus group discussion (FGD) was organized using the Zoom platform to collect qualitative insights from the students. To determine levels of stress, anxiety, and depression, the Bangla-validated version of the Depression, Anxiety, and Stress Scale 21 (DASS-21) was used. A 7-item and Bangla-validated Fear of COVID-19 Scale, also known as FCV-19S, was used to explore the COVID-19-specific fear of the students. A semi-structured topic guide was used for exploring the qualitative insights of medical students' perceptions of fear of COVID-19, mental health impacts during COVID-19, overall recommendations to support students, and the impact of the pandemic on the future of the medical curriculum.ResultsThe study reported that 51.20%, 59.40%, and 64% of the 406 respondents had moderate to severe stress, anxiety, and depressive symptoms, respectively, according to the DASS-21. The mean fear score for the COVID-19 scale was 19.4 (SD 6.4). Respondents with family members aged 50 years or older (B = 2.1; CI: 0.3-3.9) and those who had infected family members (B = 1.9; 95% CI: 0.1-3.7) exhibited a higher level of fear of COVID-19. Moreover, depression was associated with a history of having cancer among family members (AOR = 2.9, CI: 1.1-7.5), anxiety was strongly associated with having symptoms of COVID-19 (AOR = 2, CI: 1.3-3.2), and stress was associated with having symptoms of COVID-19 infection among family members (AOR = 1.9, CI: 1.3-3). Altered sleep was a potential risk factor for developing stress, anxiety, and depression symptoms. Manual thematic analysis of qualitative data generated four major themes, including the perception of fear of COVID-19, the perception of mental health impacts during COVID-19, the change in the medical curriculum along with the pandemic, and recommendations from the medical students to support the mental health concerns of medical students during public health crises like this pandemic. Qualitative findings showed that the participants experienced fear of their parents becoming infected by COVID-19, and this fear was more prominent in those who had their loved ones hospitalized. They were also stressed and anxious, with thoughts of death. Their fear also extended to their thoughts on academic progress and the effectiveness of online classes.ConclusionA substantial proportion of medical students experienced mental health difficulties in Bangladesh. Appropriate interventions should be designed, and adequate support should be provided to the medical students to protect their mental health and wellbeing, considering their potential impact on the future health system in a low-resource setting like Bangladesh

Loss of Zbtb32 in NOD mice does not significantly alter T cell responses. [version 1; referees: 2 approved]

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2+ dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2+ DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32-/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32-/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype

Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation.

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions

Innate immune stimulation of whole blood reveals IFN-1 hyper-responsiveness in type 1 diabetes

International audienceSelf-antigen-specific T cell responses drive type 1 diabetes pathogenesis, but alterations in innate immune responses are also critical and not as well understood. Innate immunity in human type 1 diabetes has primarily been assessed via gene-expression analysis of unstimulated peripheral blood mononuclear cells, without the immune activation that could amplify disease-associated signals. Increased responsiveness in each of the two main innate immune pathways, driven by either type 1 IFN (IFN-1) or IL-1, have been detected in type 1 diabetes, but the dominant innate pathway is still unclear. This study aimed to determine the key innate pathway in type 1 diabetes and assess the whole blood immune stimulation assay as a tool to investigate this

Role of NFAT1 and NFAT1^S79 in NFAT2 and cytokine expression.

<p>(A) Interleukin (IL)-2 and tumor necrosis factor alpha (TNF-α) production in supernatants of wild-type (WT) or N1KO Jurkat clones stimulated with anti-CD3/CD28, phorbol myristate acetate (PMA)/ionomycin, or medium alone for 20 hours. The results represent the mean of 3 independent experiments ± SEM (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s004" target="_blank">S4 Data</a>). (B) WT or N1KO Jurkat clones were stimulated with anti-CD3/CD28, PMA/ionomycin, or medium alone for 48 hours, and NFAT2 expression was determined by immunoblotting. (C) The N1KO Jurkat clone was infected with retrovirus encoding HA-NFAT or HA-NFAT1^S79A, followed by single cell sorting of green fluorescent protein-positive (GFP⁺) cells. Quantitation of transduced gene product expression in 2 independent clones from each transduction was determined by immunoblotting with anti-HA. (D) Quantitation of IL-2 in the supernatants of HA-NFAT1 or HA-NFAT1^S79A Jurkat clones stimulated with anti-CD3/CD28, PMA/ionomycin, or medium alone. The results represent the mean of 3 independent experiments ± SEM (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s004" target="_blank">S4 Data</a>).</p

Phosphorylation of NFAT1^S79A is required for nuclear migration upon T-cell antigen receptor (TCR) stimulation.

<p>(A) Purified T cells from wild-type (WT) mice were infected with retrovirus encoding HA-NFAT1 or HA-NFAT1^S79A. The cells were stimulated with anti-CD3/CD28 for 1 hour and examined for NFAT1 (red) localization by confocal microscopy. DAPI was used to stain the nucleus. Scale bar = 10 μM. (B) Purified primary T cells from WT mice were stimulated and infected as in panel A, and NFAT1 levels in the cytosolic and nuclear fractions were assessed by immunoblotting. (C) Stable Jurkat cell lines expressing HA-NFAT1 or HA-NFAT1^S79A were stimulated with anti-CD3/CD28 for 1 hour, and the lysates were immunoprecipitated (IP) with anti-HA and immunoblotted (IB) for calcineurin A and HA. (D) Confocal images of in situ proximity ligation assay (PLA) of stable Jurkat cell clones expressing HA-NFAT or HA-NFAT1^S79A that had been stimulated with anti-CD3/CD28 for 15 minutes. Alexa Fluor 488 (green)-conjugated wheat germ agglutinin (WGA) was used to stain plasma membrane. Scale bar = 100 pixels (left panel). Quantification of the average dots and intensity per cell (WT-Uns [<i>n</i> = 166], anti-CD3/CD28 [<i>n</i> = 132]; S79A-Uns [<i>n</i> = 162], and anti-CD3/CD28 [<i>n</i> = 132]) (right panel) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004111#pbio.2004111.s005" target="_blank">S5 Data</a>). **<i>p</i> < 0.01, ****<i>p</i> < 0.0001. NS, not significant.</p