Adhesion to laminin-1 and collagen IV induces the formation of Ca2+ microdomains that sensitize mouse T cells for activation.

During an immune response, T cells migrate from blood vessel walls into inflamed tissues by migrating across the endothelium and through extracellular matrix (ECM). Integrins facilitate T cell binding to endothelial cells and ECM proteins. Here, we report that Ca2+ microdomains observed in the absence of T cell receptor (TCR)/CD3 stimulation are initial signaling events triggered by adhesion to ECM proteins that increase the sensitivity of primary murine T cells to activation. Adhesion to the ECM proteins collagen IV and laminin-1 increased the number of Ca2+ microdomains in a manner dependent on the kinase FAK, phospholipase C (PLC), and all three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes and promoted the nuclear translocation of the transcription factor NFAT-1. Mathematical modeling predicted that the formation of adhesion-dependent Ca2+ microdomains required the concerted activity of two to six IP3Rs and ORAI1 channels to achieve the increase in the Ca2+ concentration in the ER-plasma membrane junction that was observed experimentally and that required SOCE. Further, adhesion-dependent Ca2+ microdomains were important for the magnitude of the TCR-induced activation of T cells on collagen IV as assessed by the global Ca2+ response and NFAT-1 nuclear translocation. Thus, adhesion to collagen IV and laminin-1 sensitizes T cells through a mechanism involving the formation of Ca2+ microdomains, and blocking this low-level sensitization decreases T cell activation upon TCR engagement.

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens.

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Menstrual health and period poverty among young people who menstruate in the Barcelona metropolitan area (Spain): protocol of a mixed-methods study.

INTRODUCTION: The importance of menstrual health has been historically neglected, mostly due to taboos and misconceptions around menstruation and androcentrism within health knowledge and health systems around the world. There has also been a lack of attention on 'period poverty', which refers to the financial, social, cultural and political barriers to access menstrual products and education. The main aim of this research is to explore menstrual health and experiences of period poverty among young people who menstruate (YPM). METHODS AND ANALYSIS: This is a convergent mixed-methods study, which will combine a quantitative transversal study to identify the prevalence of period poverty among YPM (11-16 years old), and a qualitative study that will focus on exploring menstruation-related experiences of YPM and other groups (young people who do not menstruate (YNM); primary healthcare professionals; educators and policy-makers). The study will be conducted in the Barcelona metropolitan area between 2020 and 2021. Eighteen schools and 871 YPM will be recruited for the quantitative study. Sixty-five YPM will participate in the qualitative study. Forty-five YNM and 12 professionals will also be recruited to take part in the qualitative study. Socioeconomic and cultural diversity will be main vectors for recruitment, to ensure the findings are representative to the social and cultural context. Descriptive statistics will be performed for each variable to identify asymmetric distributions and differences among groups will be evaluated. Thematic analysis will be used for qualitative data analyses ETHICS AND DISSEMINATION: Several ethical issues have been considered, especially as this study includes the participation of underage participants. The study has received ethical approval by the IDIAPJGol Research Ethics Committee (19/178 P). Research findings will be disseminated to key audiences, such as YPM, YNM, parents/legal tutors, health professionals, educators, youth (and other relevant) organisations, general community members, stakeholders and policy-makers, and academia.

ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.