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The peritoneal cavity (PerC) is an important site for immune responses to infection and cancer metastasis. Yet few ligand-receptor axes are known to preferentially govern immune cell accumulation in this compartment. GPR34 is a lysophosphatidylserine (lysoPS)-responsive receptor that frequently harbors gain-of-function mutations in mucosa-associated B cell lymphoma. Here, we set out to test the impact of a GPR34 knock-in (KI) allele in the B-lineage. We report that GPR34 KI promotes the PerC accumulation of plasma cells (PC) and memory B cells (MemB). These KI cells migrate robustly to lysoPS ex vivo, and the KI allele synergizes with a Bcl2 transgene to promote MemB but not PC accumulation. Gene expression and labeling studies reveal that GPR34 KI enhances PerC MemB proliferation. Both KI PC and MemB are specifically enriched at the omentum, a visceral adipose tissue containing fibroblasts that express the lysoPS-generating PLA1A enzyme. Adoptive transfer and chimera experiments revealed that KI PC and MemB maintenance in the PerC is dependent on stromal PLA1A. These findings provide in vivo evidence that PLA1A produces lysoPS that can regulate GPR34-mediated immune cell accumulation at the omentum., (© 2024 Tam et al.)

Pain, detected by nociceptors, is an integral part of injury, yet whether and how it can impact tissue physiology and recovery remain understudied. Here, we applied chemogenetics in mice to locally activate dermal TRPV1 innervations in naive skin and found that it triggered new regenerative cycling by dormant hair follicles (HFs). This was preceded by rapid apoptosis of dermal macrophages, mediated by the neuropeptide calcitonin gene-related peptide (CGRP). TRPV1 activation also triggered a macrophage-dependent induction of osteopontin (Spp1)-expressing dermal fibroblasts. The neuropeptide CGRP and the extracellular matrix protein Spp1 were required for the nociceptor-triggered hair growth. Finally, we showed that epidermal abrasion injury induced Spp1-expressing dermal fibroblasts and hair growth via a TRPV1 neuron and CGRP-dependent mechanism. Collectively, these data demonstrated a role for TRPV1 nociceptors in orchestrating a macrophage and fibroblast-supported mechanism to promote hair growth and enabling the efficient restoration of this mechano- and thermo-protective barrier after wounding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Intravital microscopy has enabled the study of immune dynamics in the pulmonary microvasculature, but many key events remain unseen because they occur in deeper lung regions. We therefore developed a technique for stabilized intravital imaging of bronchovascular cuffs and collecting lymphatics surrounding pulmonary veins in mice. Intravital imaging of pulmonary lymphatics revealed ventilation-dependence of steady-state lung lymph flow and ventilation-independent lymph flow during inflammation. We imaged the rapid exodus of migratory dendritic cells through lung lymphatics following inflammation and measured effects of pharmacologic and genetic interventions targeting chemokine signaling. Intravital imaging also captured lymphatic immune surveillance of lung-metastatic cancers and lymphatic metastasis of cancer cells. To our knowledge, this is the first imaging of lymph flow and leukocyte migration through intact pulmonary lymphatics. This approach will enable studies of protective and maladaptive processes unfolding within the lungs and in other previously inaccessible locations., Competing Interests: Declaration of interests N.S. is now employed by Arcus Biosciences and M.F.K. is a Founder & Managing Member of Foundery Therapeutics, working on projects not related to this manuscript. The authors declare no other competing interests.

Background: Statins lower circulating low-density lipoprotein cholesterol (LDLC) levels and reduce cardiovascular disease risk. Though highly efficacious in general, there is considerable inter-individual variation in statin efficacy that remains largely unexplained., Methods: To identify novel genes that may modulate statin-induced LDLC lowering, we used RNA-sequencing data from 426 control- and 2 µM simvastatin-treated lymphoblastoid cell lines (LCLs) derived from European and African American ancestry participants of the Cholesterol and Pharmacogenetics (CAP) 40 mg/day 6-week simvastatin clinical trial (ClinicalTrials.gov Identifier: NCT00451828). We correlated statin-induced changes in LCL gene expression with plasma LDLC statin response in the corresponding CAP participants. For the most correlated gene identified (ZNF335), we followed up in vivo by comparing plasma cholesterol levels, lipoprotein profiles, and lipid statin response between wild-type mice and carriers of a hypomorphic (partial loss of function) missense mutation in Zfp335 (the mouse homolog of ZNF335)., Results: The statin-induced expression changes of 147 human LCL genes were significantly correlated to the plasma LDLC statin responses of the corresponding CAP participants in vivo (FDR = 5%). The two genes with the strongest correlations were zinc finger protein 335 (ZNF335 aka NIF-1, rho = 0.237, FDR-adj p = 0.0085) and CCR4-NOT transcription complex subunit 3 (CNOT3, rho = 0.233, FDR-adj p = 0.0085). Chow-fed mice carrying a hypomorphic missense (R1092W; aka bloto) mutation in Zfp335 had significantly lower non-HDL cholesterol levels than wild-type C57BL/6J mice in a sex combined model (p = 0.04). Furthermore, male (but not female) mice carrying the Zfp335 R1092W allele had significantly lower total and HDL cholesterol levels than wild-type mice. In a separate experiment, wild-type mice fed a control diet for 4 weeks and a matched simvastatin diet for an additional 4 weeks had significant statin-induced reductions in non-HDLC (-43 ± 18% and -23 ± 19% for males and females, respectively). Wild-type male (but not female) mice experienced significant reductions in plasma LDL particle concentrations, while male mice carrying Zfp335 R1092W allele(s) exhibited a significantly blunted LDL statin response., Conclusions: Our in vitro and in vivo studies identified ZNF335 as a novel modulator of plasma cholesterol levels and statin response, suggesting that variation in ZNF335 activity could contribute to inter-individual differences in statin clinical efficacy., (© 2024. The Author(s).)

Antibodies are powerful modulators of ongoing and future B cell responses. While the concept of antibody feedback has been appreciated for over a century, the topic has seen a surge in interest due to the evidence that the broadening of antibody responses to SARS-CoV-2 after a third mRNA vaccination is a consequence of antibody feedback. Moreover, the discovery that slow antigen delivery can lead to more robust humoral immunity has put a spotlight on the capacity for early antibodies to augment B cell responses. Here, we review the mechanisms whereby antibody feedback shapes B cell responses, integrating findings in humans and in mouse models. We consider the major influence of epitope masking and the diverse actions of complement and Fc receptors and provide a framework for conceptualizing the ways antigen-specific antibodies may influence B cell responses to any form of antigen, in conditions as diverse as infectious disease, autoimmunity, and cancer., Competing Interests: Declaration of interests The authors make the following disclosures: J.G..C. is a member of the Scientific Advisory Board of BeBio Pharma and consults for Lycia Therapeutics and DrenBio Inc. P.C.W. is a member of the Scientific Advisory boards of Evozyne, Inc. and Invivyd, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ 4 exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ 4 -deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ 4 . In Gβ 4 knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

To surveil an organ for pathogens, lymphoid structures need to sample antigens locally. The full set of lymphoid structures involved in surveilling for brain-tropic pathogens has not been defined. Through comprehensive imaging of the mouse meninges, a new study by Fitzpatrick et al. describes dural-associated lymphoid tissue (DALT) and its contribution to humoral responses following intranasal viral infection., Competing Interests: Declaration of interests There are no interests to declare., (Copyright © 2024. Published by Elsevier Ltd.)

Peyer's patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFβ-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA + germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA + cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35 + cDC2s in the PP SED supports induction of intestinal IgA responses.

Spleen marginal zone (MZ) B cells are important for antibody responses against blood-borne antigens. The signals they use to detect exposure to blood are not well defined. Here, using intravital two-photon microscopy in mice, we observe transient contacts between MZ B cells and red blood cells that are in flow. We show that MZ B cells use adhesion G-protein-coupled receptor ADGRE5 (CD97) for retention in the spleen. CD97 function in MZ B cells depends on its ability to undergo autoproteolytic cleavage and signaling via Gα 13 and ARHGEF1. Red blood cell expression of the CD97 ligand CD55 is required for MZ B cell homeostasis. Applying a pulling force on CD97-transfected cells using an optical C-trap and CD55 + beads leads to accumulation of active RhoA and membrane retraction. Finally, we show that CD97 deficiency leads to a reduced T cell-independent IgM response. Thus, our studies provide evidence that MZ B cells use mechanosensing to position in a manner that enhances antibody responses against blood-borne antigens., (© 2023. The Author(s).)

Many orphan G protein-coupled receptors (GPCRs) remain understudied because their endogenous ligands are unknown. Here, we show that a group of class A/rhodopsin-like orphan GPCRs including GPR61, GPR161 and GPR174 increase the cAMP level similarly to fully activated D1 dopamine receptor (D1R). We report cryo-electron microscopy structures of the GPR61‒G s , GPR161‒G s and GPR174‒G s complexes without any exogenous ligands. The GPR174 structure reveals that endogenous lysophosphatidylserine (lysoPS) is copurified. While GPR174 fails to respond to exogenous lysoPS, likely owing to its maximal activation by the endogenous ligand, GPR174 mutants with lower ligand binding affinities can be specifically activated by lysoPS but not other lipids, in a dose-dependent manner. Moreover, GPR174 adopts a non-canonical G s coupling mode. The structures of GPR161 and GPR61 reveal that the second extracellular loop (ECL2) penetrates into the orthosteric pocket, possibly contributing to constitutive activity. Our work definitively confirms lysoPS as an endogenous GPR174 ligand and suggests that high constitutive activity of some orphan GPCRs could be accounted for by their having naturally abundant ligands., (© 2023. Springer Nature Limited.)

Neutrophil recruitment from circulation to sites of inflammation is guided by multiple chemoattractant cues emanating from tissue cells, immune cells, and platelets. Here, we focus on the function of one G-protein coupled receptor, GPR35, in neutrophil recruitment. GPR35 has been challenging to study due the description of multiple ligands and G-protein couplings. Recently, we found that GPR35-expressing hematopoietic cells respond to the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). We discuss distinct response profiles of GPR35 to 5-HIAA compared to other ligands. To place the functions of 5-HIAA in context, we summarize the actions of serotonin in vascular biology and leukocyte recruitment. Important sources of serotonin and 5-HIAA are platelets and mast cells. We discuss the dynamics of cell migration into inflamed tissues and how multiple platelet and mast cell-derived mediators, including 5-HIAA, cooperate to promote neutrophil recruitment. Additional actions of GPR35 in tissue physiology are reviewed. Finally, we discuss how clinically approved drugs that modulate serotonin uptake and metabolism may influence 5-HIAA-GPR35 function, and we speculate about broader influences of the GPR35 ligand-receptor system in immunity and disease., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cryptococcus neoformans is the leading cause of fungal meningitis and is characterized by pathogenic eosinophil accumulation in the context of type-2 inflammation. The chemoattractant receptor GPR35 is expressed by granulocytes and promotes their migration to the inflammatory mediator 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite. Given the inflammatory nature of cryptococcal infection, we examined the role of GPR35 in the circuitry underlying cell recruitment to the lung. GPR35 deficiency dampened eosinophil recruitment and fungal growth, whereas overexpression promoted eosinophil homing to airways and fungal replication. Activated platelets and mast cells were the sources of GPR35 ligand activity and pharmacological inhibition of serotonin conversion to 5-HIAA, or genetic deficiency in 5-HIAA production by platelets and mast cells resulted in more efficient clearance of Cryptococcus. Thus, the 5-HIAA-GPR35 axis is an eosinophil chemoattractant receptor system that modulates the clearance of a lethal fungal pathogen, with implications for the use of serotonin metabolism inhibitors in the treatment of fungal infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

B cells that bind soluble autoantigens receive chronic signaling via the B cell receptor (signal-1) in the absence of strong costimulatory signals (signal-2), and this leads to their elimination in peripheral tissues. The factors determining the extent of soluble autoantigen-binding B cell elimination are not fully understood. Here we demonstrate that the elimination of B cells chronically exposed to signal-1 is promoted by cathepsin B (Ctsb). Using hen egg lysozyme-specific (HEL-specific) immunoglobulin transgenic (MD4) B cells and mice harboring circulating HEL, we found improved survival and increased proliferation of HEL-binding B cells in Ctsb-deficient mice. Bone marrow chimera experiments established that both hematopoietic and nonhematopoietic sources of Ctsb were sufficient to promote peripheral B cell deletion. The depletion of CD4 + T cells overcame the survival and growth advantage provided by Ctsb deficiency, as did blocking CD40L or removing CD40 from the chronically antigen-engaged B cells. Thus, we suggest that Ctsb acts extracellularly to reduce soluble autoantigen-binding B cell survival and that its actions restrain CD40L-dependent pro-survival effects. These findings identify a role for cell-extrinsic protease activity in establishing a peripheral self-tolerance checkpoint.

The activation of Sphingosine-1-phosphate receptor 1 (S1PR1) by S1P promotes lymphocyte egress from lymphoid organs, a process critical for immune surveillance and T cell effector activity. Multiple drugs that inhibit S1PR1 function are in use clinically for the treatment of autoimmune diseases. Cluster of Differentiation 69 (CD69) is an endogenous negative regulator of lymphocyte egress that interacts with S1PR1 in cis to facilitate internalization and degradation of the receptor. The mechanism by which CD69 causes S1PR1 internalization has been unclear. Moreover, although there are numerous class A GPCR structures determined with different small molecule agonists bound, it remains unknown whether a transmembrane protein per se can act as a class A GPCR agonist. Here, we present the cryo-EM structure of CD69-bound S1PR1 coupled to the heterotrimeric G i complex. The transmembrane helix (TM) of one protomer of CD69 homodimer contacts the S1PR1-TM4. This interaction allosterically induces the movement of S1PR1-TMs 5-6, directly activating the receptor to engage the heterotrimeric G i . Mutations in key residues at the interface affect the interactions between CD69 and S1PR1, as well as reduce the receptor internalization. Thus, our structural findings along with functional analyses demonstrate that CD69 acts in cis as a protein agonist of S1PR1, thereby promoting G i -dependent S1PR1 internalization, loss of S1P gradient sensing, and inhibition of lymphocyte egress., Competing Interests: HC, YQ, MC, JC, XL No competing interests declared, (© 2023, Chen et al.)

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Immunoglobulin A (IgA) secretion by plasma cells, terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. Exposure to diet-derived and commensal-derived signals provides immune cells with organizing cues that instruct their effector function and dynamically shape intestinal immune responses at the mucosal barrier. Recent data have described metabolic and microbial inputs controlling T cell and innate lymphoid cell activation in the gut; however, whether IgA-secreting lamina propria plasma cells are tuned by local stimuli is completely unknown. Although antibody secretion is considered to be imprinted during B cell differentiation and therefore largely unaffected by environmental changes, a rapid modulation of IgA levels in response to intestinal fluctuations might be beneficial to the host. In the present study, we showed that dietary cholesterol absorption and commensal recognition by duodenal intestinal epithelial cells lead to the production of oxysterols, evolutionarily conserved lipids with immunomodulatory functions. Using conditional cholesterol 25-hydroxylase deleter mouse line we demonstrated that 7α,25-dihydroxycholesterol from epithelial cells is critical to restrain IgA secretion against commensal- and pathogen-derived antigens in the gut. Intestinal plasma cells sense oxysterols via the chemoattractant receptor GPR183 and couple their tissue positioning with IgA secretion. Our findings revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around plasma cell localization for efficient mucosal protection., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Multiple myeloma (MM), a malignancy of plasma cells (PCs), has diverse genetic underpinnings and in rare cases these include amplification of the lymphotoxin b receptor (Ltbr) locus. LTβR has well defined roles in supporting lymphoid tissue development and function through actions in stromal and myeloid cells, but whether it is functional in PCs is unknown. Here we showed that Ltbr mRNA was upregulated in mouse PCs compared to follicular B cells, but deficiency in the receptor did not cause a reduction in PC responses to a T-dependent or T-independent immunogen. However, LTβR overexpression (OE) enhanced PC formation in vitro after LPS or anti-CD40 stimulation. In vivo, LTβR OE led to increased antigen-specific splenic and bone marrow (BM) plasma cells responses. LTβR OE PCs had increased expression of Nfkb2 and of the NF-kB target genes Bcl2 and Mcl1, factors involved in the formation of long-lived BM PCs. Our findings suggest a pathway by which Ltbr gene amplifications may contribute to MM development through increased NF-kB activity and induction of an anti-apoptotic transcriptional program., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JGC is a scientific advisory board member of BeBio Pharma.

GPR174 is abundantly expressed in B and T lymphocytes and has a role in restraining T cell responses, but the function of GPR174 in B cells is less clear. Here we report that upon in vitro culture B cells undergo a spontaneous GPR174-dependent activation process that is associated with marked changes in gene expression, including up-regulation of Cd86, Nr4a1, Ccr7, and phosphodiesterases. B cells lacking Gαs show a block in induction of the GPR174-dependent program. Spontaneous up-regulation of CD86 in cultured B cells is dependent on protein kinase A. Both GPR174- and Gαs-deficient B cells show enhanced survival in culture. In vivo, GPR174 contributes to NUR77 expression in follicular B cells and is needed for establishing a marginal zone compartment of normal size. Treatment of mice with lysophosphatidylserine (lysoPS), a GPR174 ligand, is sufficient to promote CD86 up-regulation by follicular B cells. These findings demonstrate that GPR174 can signal via Gαs to modulate B cell gene expression and show this can occur in vivo in response to lysoPS. Additionally, the findings illuminate a pathway that might be targeted to improve systems for the in vitro study of B cell responses.

Sphingosine-1-phosphate (S1P) regulates immune cell trafficking, angiogenesis, and vascular function via its five receptors. Inherited mutations in S1P receptor 2 (S1PR2) occur in individuals with hearing loss, and acquired mutations in S1PR2 and G α13 occur in a malignant lymphoma. Here, we present the cryo-electron microscopy structure of S1P-bound S1PR2 coupled to the heterotrimeric G 13 . Interaction between S1PR2 intracellular loop 2 (ICL2) and transmembrane helix 4 confines ICL2 to engage the α5 helix of G α13 . Transforming growth factor-α shedding assays and cell migration assays support the key roles of the residues in S1PR2-G α13 complex assembly. The structure illuminates the mechanism of receptor disruption by disease-associated mutations. Unexpectedly, we showed that FTY720-P, an agonist of the other four S1PRs, can trigger G 13 activation via S1PR2. S1PR2 F274I variant can increase the activity of G 13 considerably with FTY720-P and S1P, thus revealing a basis for S1PR drug selectivity.

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis., Competing Interests: Disclosures:  J.G. Cyster reported "other" from Be BioPharma and MiroBio outside the submitted work. No other disclosures were reported., (© 2021 He et al.)

Type 2 T helper (T H 2) cells are protective against parasitic worm infections but also aggravate allergic inflammation. Although the role of dendritic cells (DCs) in T H 2 cell differentiation is well established, the underlying mechanisms are largely unknown. Here, we show that DC induction of T H 2 cells depends on membrane-associated RING-CH-1 (MARCH1) ubiquitin ligase. The pro-T H 2 effect of MARCH1 relied on lymph node (LN)–resident DCs, which triggered T cell receptor (TCR) signaling and induced GATA-3 expression from naïve CD4 + T cells independent of tissue-driven migratory DCs. Mice with mutations in the ubiquitin acceptor sites of MHCII and CD86, the two substrates of MARCH1, failed to develop T H 2 cells. These findings suggest that T H 2 cell development depends on ubiquitin-mediated clearance of antigen-presenting and costimulatory molecules by LN-resident DCs and consequent control of TCR signaling.

B cells within germinal centers (GCs) enter cycles of antibody affinity maturation or exit the GC as memory cells or plasma cells. Here, we examined the contribution of interleukin (IL)-4 on B cell fate decisions in the GC. Single-cell RNA-sequencing identified a subset of light zone GC B cells expressing high IL-4 receptor-a (IL4Ra) and CD23 and lacking a Myc-associated signature. These cells could differentiate into pre-memory cells. B cell-specific deletion of IL4Ra or STAT6 favored the pre-memory cell trajectory, and provision of exogenous IL-4 in a wild-type context reduced pre-memory cell frequencies. IL-4 acted during antigen-specific interactions but also influenced bystander cells. Deletion of IL4Ra from follicular dendritic cells (FDCs) increased the availability of IL-4 in the GC, impaired the selection of affinity-matured B cells, and reduced memory cell generation. We propose that GC FDCs establish a niche that limits bystander IL-4 activity, focusing IL-4 action on B cells undergoing selection and enhancing memory cell differentiation., Competing Interests: Declaration of interests J.G.C. is an Scientific Advisory Board member of Be Biopharma and MiroBio., (Copyright © 2021 Elsevier Inc. All rights reserved.)

P2RY8 promotes the confinement and growth regulation of germinal center (GC) B cells, and loss of human P2RY8 is associated with B cell lymphomagenesis. The metabolite S -geranylgeranyl-l-glutathione (GGG) is a P2RY8 ligand. The mechanisms controlling GGG distribution are poorly understood. Here, we show that gamma-glutamyltransferase-5 (Ggt5) expression in stromal cells was required for GGG catabolism and confinement of P2RY8-expressing cells to GCs. We identified the ATP-binding cassette subfamily C member 1 (Abcc1) as a GGG transporter and showed that Abcc1 expression by hematopoietic cells was necessary for P2RY8-mediated GC confinement. Furthermore, we discovered that P2RY8 and GGG negatively regulated trafficking of B and T cells to the bone marrow (BM). P2RY8 loss-of-function human T cells increased their BM homing. By defining how GGG distribution was determined and identifying sites of P2RY8 activity, this work helps establish how disruptions in P2RY8 function contribute to lymphomagenesis and other disease states., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The spleen contains a myriad of conventional dendritic cell (cDC) subsets that protect against systemic pathogen dissemination by bridging antigen detection to the induction of adaptive immunity. How cDC subsets differentiate in the splenic environment is poorly understood. Here, we report that LTα1β2-expressing Rorgt+ ILC3s, together with B cells, control the splenic cDC niche size and the terminal differentiation of Sirpα+CD4+Esam+ cDC2s, independently of the microbiota and of bone marrow pre-cDC output. Whereas the size of the splenic cDC niche depended on lymphotoxin signaling only during a restricted time frame, the homeostasis of Sirpα+CD4+Esam+ cDC2s required continuous lymphotoxin input. This latter property made Sirpα+CD4+Esam+ cDC2s uniquely susceptible to pharmacological interventions with LTβR agonists and antagonists and to ILC reconstitution strategies. Together, our findings demonstrate that LTα1β2-expressing Rorgt+ ILC3s drive splenic cDC differentiation and highlight the critical role of ILC3s as perpetual regulators of lymphoid tissue homeostasis., Competing Interests: Disclosures:   E. Vivier is an employee of Innate Pharma. C.F. Ware reported grants from Capella Biosciences, grants from Eli Lilly, and grants from Boehringer Ingelheim Pharmaceuticals outside the submitted work; in addition, C.F. Ware had a patent to USP 8,974,787 issued and a patent to USP 8,349,320 issued. No other disclosures were reported., (© 2021 Vanderkerken et al.)

Memory B cells (MBCs) are critical for the rapid development of protective immunity following re-infection. MBCs capable of neutralizing distinct subclasses of pathogens, such as influenza and HIV, have been identified in humans. However, efforts to develop vaccines that induce broadly protective MBCs to rapidly mutating pathogens have not yet been successful. Better understanding of the signals regulating MBC development and function are essential to overcome current challenges hindering successful vaccine development. Here, we discuss recent advancements regarding the signals and transcription factors regulating germinal centre-derived MBC development and function.

The marginal zone (MZ) of the spleen contains multiple cell types that are involved in mounting rapid immune responses against blood-borne pathogens, including conventional dendritic cells (cDCs) and MZ B cells. MZ B cells develop later than other B cell types and are sparse in neonatal mice. Here, we show that cDC2s are abundant in the MZ of neonatal compared with adult mice. We find that conditions associated with reduced MZ B cell numbers in adult mice cause increased cDC2 occupancy of the MZ. Treatment with the S1PR1-modulating drug, FTY720, causes cDC2 movement into the MZ through the indirect mechanism of displacing MZ B cells into follicles. Splenic cDC2s express high amounts of α4β1 and αLβ2 integrins and depend on these integrins and the adaptor Talin for their retention in blood-exposed regions of the spleen. Splenic CD4 T cell activation by particulate antigens is increased in mice with higher cDC2 density in the MZ, including in neonatal mice. Our work establishes requirements for homeostatic cDC2 positioning in the spleen and provides evidence that localization in blood-exposed regions around the white pulp augments cDC2 capture of particulate antigens. We suggest that MZ positioning of cDC2s partially compensates for the lack of MZ B cells during the neonatal period., Competing Interests: Disclosures: J. Cyster reported consulting for several biotech companies and serving on the scientific advisory board of ALX Oncology Inc. and MiroBio Ltd. No other disclosures were reported., (© 2020 Liu et al.)

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

Memory B cells (MBCs) are essential for long-lived humoral immunity. However, the transcription factors involved in MBC differentiation are poorly defined. Here, using single-cell RNA sequencing 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 protein Hhex as a transcription factor regulating MBC differentiation. The corepressor 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 increased Bcl6 expression and reduced expression of the Bcl-6 target gene 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.

Germinal centers (GCs) are confined anatomic regions where rapidly proliferating B cells undergo somatic mutation and selection and eventual differentiation into memory B cells or long-lived plasma cells. GCs are also the origin of malignancy, namely follicular lymphoma (FL), GC B cell-diffuse large B cell lymphoma (GCB-DLBCL), and Burkitt lymphoma (BL). GC B cell lymphomas maintain their GC transcriptional signatures and sustain many features of the GC microenvironment, including CD4 + T follicular helper (Tfh) cells. Tfh cells are essential for the formation and maintenance of GCs, providing critical helper signals such as CD40L. Large-scale sequencing efforts have led to new insights about the tightly regulated selection mechanisms that are commonly targeted during GC B cell lymphomagenesis. For instance, HVEM, a frequently mutated surface molecule in GC-derived lymphomas, engages the inhibitory receptor BTLA on Tfh cells and loss of HVEM leads to exaggerated T cell help. Here, we review current understanding of how Tfh cells contribute to the selection of GC B cells, with a particular emphasis on how Tfh cell signals may contribute to lymphomagenesis. The possibility of targeting Tfh cells for the treatment of GC-derived lymphomas is discussed., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The mechanisms that regulate germinal center (GC) B cell responses in the spleen are not fully understood. Here we use a combination of pharmacologic and genetic approaches to delete SIGN-R1 + marginal zone (MZ) macrophages and reveal their specific contribution to the regulation of humoral immunity in the spleen. We find that while SIGN-R1 + macrophages were not essential for initial activation of B cells, they were required for maturation of the response and development of GC B cells. These defects could be corrected when follicular helper T (Tfh) cells were induced before macrophage ablation or when Tfh responses were enhanced. Moreover, we show that in the absence of SIGN-R1 + macrophages, DCIR2 + dendritic cells (DCs), which play a key role in priming Tfh responses, were unable to cluster to the interfollicular regions of the spleen and were instead displaced to the MZ. Restoring SIGN-R1 + macrophages to the spleen corrected positioning of DCIR2 + DCs and rescued the GC B cell response. Our study reveals a previously unappreciated role for SIGN-R1 + macrophages in regulation of the GC reaction and highlights the functional specification of macrophage subsets in the MZ compartment., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Antigen receptor-dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein-coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.

The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintenance of a population of IL-17-committed γδ T cells in the dermis is important in promoting tissue immunity. However, the signals facilitating γδ T cell retention within the dermis remain poorly understood. Here, we find that sphingosine-1-phosphate receptor 2 (S1PR2) acts in a cell-intrinsic manner to oppose γδ T cell migration from the dermis to the skin draining lymph node (dLN). Migration of dermal γδ T cells to the dLN under steady-state conditions occurs in an S1PR1-dependent manner. S1PR1 and CD69 are reciprocally expressed on dermal γδ T cells, with loss of CD69 associated with increased S1PR1 expression and enhanced migration to the dLN. γδ T cells lacking both S1PR2 and CD69 are impaired in their maintenance within the dermis. These findings provide a mechanism for how IL-17 + γδ T cells establish residence within the dermis and identify a role for S1PR2 in restraining the egress of tissue-resident lymphocytes., (© 2019 Laidlaw et al.)

B-cell responses are dynamic processes that depend on multiple types of interactions. Rare antigen-specific B cells must encounter antigen and specialized systems are needed-unique to each lymphoid tissue type-to ensure this happens efficiently. Lymphoid tissue barrier cells act to ensure that pathogens, while being permitted entry for B-cell recognition, are blocked from replication or dissemination. T follicular helper (Tfh) cells often need to be primed by dendritic cells before supporting B-cell responses. For most responses, antigen-specific helper T cells and B cells need to interact, first to initiate clonal expansion and the plasmablast response, and later to support the germinal center (GC) response. Newly formed plasma cells need to travel to supportive niches. GC B cells must become confined to the follicle center, organize into dark and light zones, and interact with Tfh cells. Memory B cells need to be positioned for rapid responses following reinfection. Each of these events requires the actions of multiple G-protein coupled receptors (GPCRs) and their ligands, including chemokines and lipid mediators. This review will focus on the guidance cue code underlying B-cell immunity, with an emphasis on findings from our laboratory and on newer advances in related areas. We will discuss our recent identification of geranylgeranyl-glutathione as a ligand for P2RY8. Our goal is to provide the reader with a focused knowledge about the GPCRs guiding B-cell responses and how they might be therapeutic targets, while also providing examples of how multiple types of GPCRs can cooperate or act iteratively to control cell behavior., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

B cells and the antibodies they produce have a deeply penetrating influence on human physiology. Here, we review current understanding of how B cell responses are initiated; the different paths to generate short- and long-lived plasma cells, germinal center cells, and memory cells; and how each path impacts antibody diversity, selectivity, and affinity. We discuss how basic research is informing efforts to generate vaccines that induce broadly neutralizing antibodies against viral pathogens, revealing the special features associated with allergen-reactive IgE responses and uncovering the antibody-independent mechanisms by which B cells contribute to health and disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)