Mast cells help organize the Peyer's patch niche for induction of IgA responses.

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 TGFß-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.

Dynamic encounters with red blood cells trigger splenic marginal zone B cell retention and function.

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 Gα13 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.

Platelets and mast cells promote pathogenic eosinophil recruitment during invasive fungal infection via the 5-HIAA-GPR35 ligand-receptor system.

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.

B cell peripheral tolerance is promoted by cathepsin B protease.

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.

CD97 promotes spleen dendritic cell homeostasis through the mechanosensing of red blood cells.

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 Gα13 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.

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation.

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.

Abcc1 and Ggt5 support lymphocyte guidance through export and catabolism of S-geranylgeranyl-l-glutathione.

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.

First Report of Soft Rot Caused by Aspergillus niger sensu lato on Mother-in-law's Tongue in China.

"Mother-in-law's tongue" (MLT) [Dracaena trifasciata (Prain) Mabb. (syn. Sansevieria trifasciata Prain.)], also known as "Saint George's sword", "snake plant", "tiger's tail orchid", etc., is an evergreen perennial ornamental plant grown worldwide. In September 2016, severe soft rot occurred on the leaves of MLT in a flower market in Nanyang city (32º56´N, 112º32´E), Henan province, China with 25% disease incidence (n=100). Water-soaked spots initially appeared on the leaf margin, enlarged rapidly, and became soft rot under excessively watered conditions. A blight zone was visualized at the margin of a developing lesion in backlit conditions. Severely affected leaves folded down from the lesions. Lesion expansion stopped under dry conditions. Grey or dark brown mycelia were frequently seen on the lesions. Tissue pieces (4×4 mm2) at the margin of lesions were cut out, treated with 75% ethanol for 10 s, followed by 70 s in 0.1% HgCl2, rinsed eight times with sterile water, and plated on potato dextrose agar (PDA) medium. Pure Aspergillus cultures were obtained from the surface-disinfected lesions after 4 days of incubation at 26°C. Two single-spore-derived isolates (An-1 and An-2) were randomly selected and used for morphological and molecular identifications as well as pathogenicity tests. The isolates formed round dark brown colonies with a large number of conidia after 5 days of incubation on PDA at 28°C. Conidia were subsphaeroidal or oblate, unicellular, dark brown, 2.9-4.2(3.5) × 1.9-3.4(2.7) µm in size (n=100), developed from a two-series of strigmata born on a conidial head, with ridge or stab-shaped prominences. For pathogenicity tests, the two isolates were separately grown on oatmeal agar and incubated at 30°C for 6 days. Mycelial plugs (5 mm diam.) were inoculated on the scalpel incision X-shaped wounds of surface-disinfected leaves of MLT. The inoculated leaves were kept on a two-layer of wet napkin in a steel basin covered with a plastic film. Soft rot symptoms developed from the wounds 6 days after incubation, similar to those observed on naturally affected leaves. The An-1- and An-2-inoculated unwounded leaves remained symptomless during the pathogenicity tests. Fungal cultures with the same phenotypes as the inocula were consistently reisolated from the lesions of the leaves inoculated by each of the two isolates, verifying the isolates as the causal agent of the disease based on Koch's postulates. Both ß-tubulin gene and rDNA-ITS (internal transcribed spacer) sequences of the two isolates were separately amplified and sequenced. Sequences were submitted to GenBank with accession numbers MN259522 and MN259523 for the ß-tubulin gene sequences, and accession numbers MN227322 and MN227324 for the rDNA-ITS sequences of An-1 and An-2, respectively. Both An-1 and An-2 were clustered with members of Aspergillus niger van Tieghem in the phylogenetic tree of rDNA-ITS, clearly separated from other Aspergillus spp. In the phylogenetic tree of ß-tublin gene, both An-1 and An-2 formed a subclade inside a large clade consisting of members of A. niger in strict sense. Based on the molecular and morphological results, both An-1 and An-2 clearly separated from other Aspergillus spp. and can be considered as A. niger sensu lato. Foliar diseases of MLT are known to be caused by a few fungal species such as Chaetomella spp. (Li et al. 2014) and Colletotrichum sansevieriae (Nakamura et al. 2006). This is the first report of A. niger sensu lato causing soft rot on MLT in China.

Requirements for cDC2 positioning in blood-exposed regions of the neonatal and adult spleen.

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 α4ß1 and αLß2 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.

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in 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.

Critical role of integrin CD11c in splenic dendritic cell capture of missing-self CD47 cells to induce adaptive immunity.

CD11c, also known as integrin alpha X, is the most widely used defining marker for dendritic cells (DCs). CD11c can bind complement iC3b and mediate phagocytosis in vitro, for which it is also referred to as complement receptor 4. However, the functions of this prominent marker protein in DCs, especially in vivo, remain poorly defined. Here, in the process of studying DC activation and immune responses induced by cells lacking self-CD47, we found that DC capture of CD47-deficient cells and DC activation was dependent on the integrin-signaling adaptor Talin1. Specifically, CD11c and its partner Itgb2 were required for DC capture of CD47-deficient cells. CD11b was not necessary for this process but could partially compensate in the absence of CD11c. Mice with DCs lacking Talin1, Itgb2, or CD11c were defective in supporting T-cell proliferation and differentiation induced by CD47-deficient cell associated antigen. These findings establish a critical role for CD11c in DC antigen uptake and activation in vivo. They may also contribute to understanding the functional mechanism of CD47-blockade therapies.

Splenic Dendritic Cells Survey Red Blood Cells for Missing Self-CD47 to Trigger Adaptive Immune Responses.

Sheep red blood cells (SRBCs) have long been used as a model antigen for eliciting systemic immune responses, yet the basis for their adjuvant activity has been unknown. Here, we show that SRBCs failed to engage the inhibitory mouse SIRPα receptor on splenic CD4(+) dendritic cells (DCs), and this failure led to DC activation. Removal of the SIRPα ligand, CD47, from self-RBCs was sufficient to convert them into an adjuvant for adaptive immune responses. DC capture of Cd47(-/-) RBCs and DC activation occurred within minutes in a Src-family-kinase- and CD18-integrin-dependent manner. These findings provide an explanation for the adjuvant mechanism of SRBCs and reveal that splenic DCs survey blood cells for missing self-CD47, a process that might contribute to detecting and mounting immune responses against pathogen-infected RBCs.

The lysophosphatidylserine receptor GPR174 constrains regulatory T cell development and function.

Regulatory T cell (T reg cell) numbers and activities are tightly calibrated to maintain immune homeostasis, but the mechanisms involved are incompletely defined. Here, we report that the lysophosphatidylserine (LysoPS) receptor GPR174 is abundantly expressed in developing and mature T reg cells. In mice that lacked this X-linked gene, T reg cell generation in the thymus was intrinsically favored, and a higher fraction of peripheral T reg cells expressed CD103. LysoPS could act in vitro via GPR174 to suppress T cell proliferation and T reg cell generation. In vivo, LysoPS was detected in lymphoid organ and spinal cord tissues and was abundant in the colon. Gpr174(-/Y) mice were less susceptible to experimental autoimmune encephalomyelitis than wild-type mice, and GPR174 deficiency in T reg cells contributed to this phenotype. This study provides evidence that a bioactive lipid, LysoPS, negatively influences T reg cell accumulation and activity through GPR174. As such, GPR174 antagonists might have therapeutic potential for promoting immune regulation in the context of autoimmune disease.

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Germinal center centroblasts transition to a centrocyte phenotype according to a timed program and depend on the dark zone for effective selection.

Germinal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73(+) memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions.

Oxysterol gradient generation by lymphoid stromal cells guides activated B cell movement during humoral responses.

7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G protein-coupled receptor EBI2; however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We showed that all three enzymes control EBI2 ligand concentration in lymphoid tissues. Lymphoid stromal cells were the main CH25H- and CYP7B1-expressing cells required for positioning of B cells, and they also mediated 7α,25-OHC inactivation. CH25H and CYP7B1 were abundant at the follicle perimeter, whereas CH25H expression by follicular dendritic cells was repressed. CYP7B1, CH25H, and HSD3B7 deficiencies each resulted in defective T cell-dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues, and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses.

Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers.

Follicular dendritic cells (FDCs) retain and display opsonized antigens in primary follicles and germinal centers (GCs). However, their roles beyond antigen presentation have been incompletely defined. In this study, we tested the impact of selective FDC ablation on short-term follicle and GC function. Within 2 d of FDC ablation, primary follicles lost their homogeneity and became disorganized bands of cells around T zones. These B cell areas retained CXCL13-expressing stromal cells but often exhibited inappropriate ER-TR7 and CCL21 expression. Ablation of GC FDCs led to the disappearance of GCs. When B cell death was prevented using a Bcl2 transgene, FDC ablation led to splenic GC B cell dispersal. Mesenteric lymph node GCs were more resistant but became dispersed when sphingosine-1-phosphate receptor-2 was also removed. These experiments indicate that FDCs help maintain primary follicles as a B cell exclusive niche and define a critical role for FDCs in cell retention within GCs.

GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood.

Lymphocytes egress from lymphoid organs in response to sphingosine-1-phosphate (S1P); minutes later they migrate from blood into tissue against the S1P gradient. The mechanisms facilitating cell movement against the gradient have not been defined. Here, we show that heterotrimeric guanine nucleotide-binding protein-coupled receptor kinase-2 (GRK2) functions in down-regulation of S1P receptor-1 (S1PR1) on blood-exposed lymphocytes. T and B cell movement from blood into lymph nodes is reduced in the absence of GRK2 but is restored in S1P-deficient mice. In the spleen, B cell movement between the blood-rich marginal zone and follicles is disrupted by GRK2 deficiency and by mutation of an S1PR1 desensitization motif. Moreover, delivery of systemic antigen into follicles is impaired. Thus, GRK2-dependent S1PR1 desensitization allows lymphocytes to escape circulatory fluids and migrate into lymphoid tissues.

Cannabinoid receptor 2 mediates the retention of immature B cells in bone marrow sinusoids.

Immature B cells developing in the bone marrow are found in the parenchyma and sinusoids. The mechanisms that control the positioning of B cells in the sinusoids are not understood. Here we show that the integrin alpha(4)beta(1) (VLA-4) and its ligand VCAM-1 were required, whereas the chemokine receptor CXCR4 was dispensable, for sinusoidal retention of B cells. Instead, cannabinoid receptor 2 (CB2), a Galpha(i) protein-coupled receptor upregulated in immature B cells, was required for sinusoidal retention. Using two-photon microscopy, we found immature B cells entering and crawling in sinusoids; these immature B cells were displaced by CB2 antagonism. Moreover, CB2-deficient mice had a lower frequency of immunoglobulin lambda-chain-positive B cells in the peripheral blood and spleen. Our findings identify unique requirements for the retention of B cells in the bone marrow sinusoidal niche and suggest involvement of CB2 in the generation of the B cell repertoire.

The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse strain and in a patient with severe combined immunodeficiency.

Mice carrying the recessive locus for peripheral T cell deficiency (Ptcd) have a block in thymic egress, but the mechanism responsible is undefined. Here we found that Ptcd T cells had an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus showed a point substitution of lysine for glutamic acid at position 26 in the actin regulator coronin 1A that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. The discovery of another coronin 1A mutant during an N-ethyl-N-nitrosourea-mutagenesis screen for T cell-lymphopenic mice prompted us to evaluate a T cell-deficient, B cell-sufficient and natural killer cell-sufficient patient with severe combined immunodeficiency, whom we found had mutations in both CORO1A alleles. Our findings establish a function for coronin 1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation and demonstrate that actin regulation is a biological process defective in human and mouse severe combined immunodeficiency.