Memory B cells (MBCs) are essential for long-lived humoral immunity. However, the transcription factors involved in MBC differentiation are poorly defined. Here, by single-cell RNA-seq analysis, we identified a population of germinal center (GC) B cells in the process of differentiating into MBCs. Using an inducible Crispr/Cas9 screening approach we identified the hematopoietically expressed homeobox gene Hhex as a transcription factor regulating MBC differentiation. The co-repressor Tle3 was also identified in the screen and was found to interact with Hhex to promote MBC development. Bcl-6 directly repressed Hhex in GC B cells. Reciprocally, Hhex-deficient MBCs exhibited derepressed Bcl6 and reduced expression of Bcl-6-repressed Bcl2 . Overexpression of Bcl-2 was able to rescue MBC differentiation in Hhex-deficient cells. We also identified Ski as an Hhex-induced transcription factor involved in MBC differentiation. These findings establish an important role for Hhex-Tle3 in regulating the transcriptional circuitry governing MBC differentiation.

Highlights d PDGFRb cells function as initial sensors of systemic inflammation in the brain d PDGFRb cells relay the infection signal to neurons by secreting chemokine CCL2 d Col1a1 and Rgs5 subgroups of PDGFRb cells are sources of Ccl2 during early infection d PDGFRb-specific Ccl2 knockout blocked LPS-induced increase in synaptic transmission

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα 13 and adhesion G protein–coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

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A salient feature of neurons is their intrinsic ability to grow and extend neurites, even in the absence of external cues. Compared to the later stages of neuronal development, such as neuronal polarization and dendrite morphogenesis, the early steps of neuritogenesis remain relatively unexplored. Here we showed that redistribution of cortical actin into large aggregates preceded neuritogenesis and determined the site of neurite initiation. Enhancing actin polymerization by jasplakinolide treatment effectively blocked actin redistribution and neurite initiation, while treatment with the actin depolymerizing agents latrunculin A or cytochalasin D accelerated neurite formation. Together, these results demonstrate a critical role of actin dynamics and reorganization in neurite initiation. Further experiments showed that microtubule dynamics and protein synthesis are not required for neurite initiation, but are required for later neurite stabilization. The redistribution of actin during early neuronal development was also observed in the cerebral cortex and hippocampus in vivo.

Neurite initiation is critical for neuronal morphogenesis and early neural circuit development. Recent studies showed that local actin aggregation underneath the cell membrane determined the site of neurite initiation. An immediately arising question is what signaling mechanism initiated actin aggregation. Here we demonstrate that local clustering of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P), a phospholipid with relatively few known signaling functions, is necessary and sufficient for aggregating actin and promoting neuritogenesis. In contrast, the related and more extensively studied phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol (3,4,5)-trisphosphate (PIP) molecules did not have such functions. Specifically, we showed that beads coated with PI(3,4)P promoted actin aggregation and neurite initiation, while pharmacological interference with PI(3,4)P synthesis inhibited both processes. PI(3,4)P clustering occurred even when actin aggregation was pharmacologically blocked, demonstrating that PI(3,4)P functioned as the upstream signaling molecule. Two enzymes critical for PI(3,4)P generation, namely, SH2 domain-containing inositol 5-phosphatase and class II phosphoinositide 3-kinase α, were complementarily and non-redundantly required for actin aggregation and neuritogenesis, as well as for subsequent dendritogenesis. Finally, we demonstrate that neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex functioned downstream of PI(3,4)P to mediate neuritogenesis and dendritogenesis. Together, our results identify PI(3,4)P as an important signaling molecule during early development and demonstrate its critical role in regulating actin aggregation and neuritogenesis.

SignificanceModeling immune responses in vitro is critical for studying many facets of the B cell response. We show that during culture without stimulation, mouse B cells undergo massive changes in gene expression. Many of these changes are promoted by GPR174 signaling via Gαs. GPR174 and Gαs also contribute to reduced B cell viability during culture. We suggest that GPR174 antagonists may be useful to reduce the shift in gene expression and to augment B cell survival during culture. We also provide evidence that ligand engagement of GPR174 can activate this pathway in vivo. Variants in the GPR174 locus have been associated with autoimmune diseases. Our findings provide knowledge for understanding how alterations in GPR174 expression may contribute to disease.

Spleen dendritic cells (DC) are critical for initiation of adaptive immune responses against blood‐borne invaders. Key to DC function is their positioning at sites of pathogen entry, and their abilities to selectively capture foreign antigens and promptly engage T cells. Focusing on conventional DC2 (cDC2), we discuss the contribution of chemoattractant receptors (EBI2 or GPR183, S1PR1, and CCR7) and integrins to cDC2 positioning and function. We give particular attention to a newly identified role in cDC2 for adhesion G‐protein coupled receptor E5 (Adgre5 or CD97) and its ligand CD55, detailing how this mechanosensing system contributes to splenic cDC2 positioning and homeostasis. Additional roles of CD97 in the immune system are reviewed. The ability of cDC2 to be activated by circulating missing self‐CD47 cells and to integrate multiple red blood cell (RBC)‐derived inputs is discussed. Finally, we describe the process of activated cDC2 migration to engage and prime helper T cells. Throughout the review, we consider the insights into cDC function in the spleen that have emerged from imaging studies.