Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4β1 integrin) and LFA-1 (αLβ2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin β2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin β1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin β2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.
© 2021. Published by The Company of Biologists Ltd.

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.
Copyright © 2020 Elsevier Inc. All rights reserved.

CD18 is the common beta subunit of β2 integrins, which are expressed on hematopoietic cells. β2 integrins are essential for cell adhesion and leukocyte trafficking.
Here we have analyzed the expression of CD18 in different subsets of human hematopoietic stem and progenitor cells (HSPCs) from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB) samples. CD34+ cells were classified into CD18high and CD18low/neg, and each of these populations was analyzed for the expression of HSPC markers, as well as for their clonogenity, quiescence state, and repopulating ability in immunodeficient mice.
A downregulated membrane expression of CD18 was associated with a primitive hematopoietic stem cells (HSC) phenotype, as well as with a higher content of quiescent cells and multipotent colony-forming cells (CFCs). Although no differences in the short-term repopulating potential of CD18low/neg CD34+ and CD18high CD34+ cells were observed, CD18low/neg CD34+ cells were characterized by an enhanced long-term repopulating ability in NSG mice.
Overall, our results indicate that the downregulated membrane expression of CD18 characterizes a primitive population of human hematopoietic repopulating cells.

This chapter describes a two-dimensional "monolayer" system for differentiating human pluripotent stem cells (PSCs) into "primitive" hematopoietic progenitor cells (HPCs) resembling those produced in vivo by the early embryonic yolk sac. This experimental system utilizes defined conditions without serum or feeder cells. Cytokines are added sequentially to stimulate the formation of mesoderm and its subsequent patterning to hematopoietic progenitors. The HPCs produced by this protocol have multi-lineage potential (erythroid, megakaryocyte, and myeloid) and can be isolated as a homogeneous population for use in standard hematopoietic studies including liquid expansion to mature lineages and colony assays. In addition, the HPCs can be cryopreserved for distribution or analysis at later times. The HPCs generated by this protocol have been used successfully to better define intrinsic variation in hematopoietic potential between different PSC lines and to model human hematopoietic diseases using patient-derived induced pluripotent stem cells.