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Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81−/− mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection., Lactate is a by-product of glycolysis that can function via its G protein receptor GPR81. Here the authors show that LPS or Salmonella infection enhances glycolytic metabolism in bone marrow neutrophils, resulting in lactate production, which increases endothelial barrier permeability and mobilization of these neutrophils by targeting endothelial GPR81.

Enduring immunity against harmful pathogens depends on the generation of immunological memory. Serum immunoglobulins are constantly secreted by long-lived antibody-producing cells, which provide extended protection from recurrent exposures. These cells originate mainly from germinal center structures, wherein B cells introduce mutations to their immunoglobulin genes followed by affinity-based selection. Generation of high-affinity antibodies relies on physical contacts between T and B cells, a process that facilitates the delivery of fate decision signals. T-B cellular engagements are mediated through interactions between the T cell receptor and its cognate peptide presented on B cell major histocompatibility class II molecules. Here, we describe the cellular and molecular aspects of these cognate T-B interactions, and highlight exceptional cases, especially those arising at intestinal lymphoid organs, at which T cells provide help to B cells in an atypical manner, independent of T cell specificity.

Long-lasting immunity depends on generation of antibody forming cells in germinal centers (GCs). Conventional methods such as immunohistology and intravital live imaging have been used extensively to investigate the location of cellular assemblies within tissues as well as their dynamic motility and cellular interactions. Two photon laser scanning microscopy (TPLSM) intravital imaging allows scanning of large areas within tissues and reveals multiple immune cell niches. Nonetheless, this type of imaging is limited by the depth of penetration and cannot capture effectively all of the GC niches within lymphoid organs. Here we describe a method to visualize antigen-specific T and B cells in multiple microanatomical locations and niches at the level of a whole organ. This large-scale imaging approach can greatly increase our understanding of the spatial distribution of immune cells and help obtain detailed 3D maps of their locations and quantities.

Activation and differentiation of B cells depend on extensive rewiring of gene expression networks through changes in chromatin structure and accessibility. The chromatin remodeling complex BAF with its catalytic subunit Brg1 was previously identified as an essential regulator of early B cell development, however, how Brg1 orchestrates gene expression during mature B cell activation is less clear. Here, we find that Brg1 is required for B cell proliferation and germinal center formation through selective interactions with enhancers. Brg1 recruitment to enhancers following B cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle-associated genes. Accordingly, Brg1-deficient B cells were unable to mount germinal center reactions and support the formation of class-switched plasma cells. Our findings show that changes in B cell transcriptomes that support B cell proliferation and GC formation depend on enhancer activation by Brg1. Thus, the BAF complex plays a critical role during the onset of the humoral immune response.

Antibody-mediated long-lasting protection from harmful pathogens depends on collaboration of immune cells within immunological niches. Stoler-Barak et al. introduce an approach that enables the visualization of all the germinal center niches and activated B cells within intact lymph nodes., Germinal centers (GCs) are sites wherein B cells proliferate and mutate their immunoglobulins in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). Here, we mapped the location of single B cells in the context of intact lymph nodes (LNs) throughout the GC response, and examined the role of BCR affinity in dictating their position. Imaging of entire GC structures and proximal single cells by light-sheet fluorescence microscopy revealed that individual B cells that previously expressed AID are located within the LN cortex, in an area close to the GC LZ. Using in situ photoactivation, we demonstrated that B cells migrate from the LZ toward the GC outskirts, while DZ B cells are confined to the GC. B cells expressing very-low-affinity BCRs formed GCs but were unable to efficiently disperse within the follicles. Our findings reveal that BCR affinity regulates B cell positioning during the GC response.

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, sub-family 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). Importantly, 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.

BACKGROUNDThe significant risks posed to mothers and fetuses by COVID-19 in pregnancy have sparked a worldwide debate surrounding the pros and cons of antenatal SARS-CoV-2 inoculation, as we lack sufficient evidence regarding vaccine effectiveness in pregnant women and their offspring. We aimed to provide substantial evidence for the effect of the BNT162b2 mRNA vaccine versus native infection on maternal humoral, as well as transplacentally acquired fetal immune response, potentially providing newborn protection.METHODSA multicenter study where parturients presenting for delivery were recruited at 8 medical centers across Israel and assigned to 3 study groups: vaccinated (n = 86); PCR-confirmed SARS-CoV-2 infected during pregnancy (n = 65), and unvaccinated noninfected controls (n = 62). Maternal and fetal blood samples were collected from parturients prior to delivery and from the umbilical cord following delivery, respectively. Sera IgG and IgM titers were measured using the Milliplex MAP SARS-CoV-2 Antigen Panel (for S1, S2, RBD, and N).RESULTSThe BNT162b2 mRNA vaccine elicits strong maternal humoral IgG response (anti-S and RBD) that crosses the placenta barrier and approaches maternal titers in the fetus within 15 days following the first dose. Maternal to neonatal anti-COVID-19 antibodies ratio did not differ when comparing sensitization (vaccine vs. infection). IgG transfer ratio at birth was significantly lower for third-trimester as compared with second trimester infection. Lastly, fetal IgM response was detected in 5 neonates, all in the infected group.CONCLUSIONAntenatal BNT162b2 mRNA vaccination induces a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.FUNDINGIsrael Science Foundation and the Weizmann Institute Fondazione Henry Krenter.

BackgroundThe significant risks posed to mothers and fetuses by COVID-19 in pregnancy have sparked a worldwide debate surrounding the pros and cons of antenatal SARS-CoV-2 inoculation, as we lack sufficient evidence regarding vaccine effectiveness in pregnant women and their offspring. We aimed to provide substantial evidence for the effect of BNT162b2 mRNA vaccine versus native infection on maternal humoral, as well as transplacentally acquired fetal immune response, potentially providing newborn protection.MethodsA multicenter study where parturients presenting for delivery were recruited at 8 medical centers across Israel and assigned to three study groups: vaccinated (n=86); PCR confirmed SARS-CoV-2 infected during pregnancy (n=65), and unvaccinated non-infected controls (n=62). Maternal and fetal blood samples were collected from parturients prior to delivery and from the umbilical cord following delivery, respectively. Sera IgG and IgM titers were measured using Milliplex MAP SARS-CoV-2 Antigen Panel (for S1, S2, RBD and N).ResultsBNT162b2 mRNA vaccine elicits strong maternal humoral IgG response (Anti-S and RBD) that crosses the placenta barrier and approaches maternal titers in the fetus within 15 days following the first dose. Maternal to neonatal anti-COVID-19 antibodies ratio did not differ when comparing sensitization (vaccine vs. infection). IgG transfer rate was significantly lower for third-trimester as compared to second trimester infection. Lastly, fetal IgM response was detected in 5 neonates, all in the infected group.ConclusionsAntenatal BNT162b2 mRNA vaccination induces a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.

Adrenergic signaling plays a central role in physiological regulation, including modulation of processes in the immune system. However, the effects of adrenergic activation on antibody-mediated immune response remain unknown. Here, we investigate the effects of stress - induced β-adrenergic receptor activation on the B cell response at molecular and the systemic levels. We find that β-adrenergic agonist treatment of B cells from three convalescent SARS coronavirus-2 donors, reduced both membrane IgG expression and clonal expansion when the cells were stimulated ex vivo with spike receptor binding domain (RBD). Interestingly, monoclonal anti-RBD antibodies cloned from B cells cultured in the presence of β-adrenergic agonist, exhibited higher affinity for RBD compared to antibodies cloned from control cultures, suggesting that clones under stress exhibit higher antigen affinity. As a corollary, following ovalbumin immunization in mice, physiological stress during germinal center reaction phase increased the levels of antigen specific serum IgG. At the same time, B cell clonal expansion and membrane IgG expression were reduced, along with an increase in MHC class II expression. These effects were abolished by treatment with the non-selective β-adrenergic antagonist, propranolol. Our study suggests that under stress conditions selection of high affinity variants comes in the expense of clonal expansion.

The spillover of animal coronaviruses (aCoVs) to humans has caused SARS, MERS, and COVID-19. While antibody responses displaying cross-reactivity between SARS-CoV-2 and seasonal/common cold human coronaviruses (hCoVs) have been reported, potential cross-reactivity with aCoVs and the diagnostic implications are incompletely understood. Here, we probed for antibody binding against all seven hCoVs and 49 aCoVs represented as 12,924 peptides within a phage-displayed antigen library. Antibody repertoires of 269 recovered COVID-19 patients showed distinct changes compared to 260 unexposed pre-pandemic controls, not limited to binding of SARS-CoV-2 antigens but including binding to antigens from hCoVs and aCoVs with shared motifs to SARS-CoV-2. We isolated broadly reactive monoclonal antibodies from recovered COVID-19 patients that bind a shared motif of SARS-CoV-2, hCoV-OC43, hCoV-HKU1, and several aCoVs, demonstrating that interspecies cross-reactivity can be mediated by a single immunoglobulin. Employing antibody binding data against the entire CoV antigen library allowed accurate discrimination of recovered COVID-19 patients from unexposed individuals by machine learning. Leaving out SARS-CoV-2 antigens and relying solely on antibody binding to other hCoVs and aCoVs achieved equally accurate detection of SARS-CoV-2 infection. The ability to detect SARS-CoV-2 infection without knowledge of its unique antigens solely from cross-reactive antibody responses against other hCoVs and aCoVs suggests a potential diagnostic strategy for the early stage of future pandemics. Creating regularly updated antigen libraries representing the animal coronavirome can provide the basis for a serological assay already poised to identify infected individuals following a future zoonotic transmission event.

Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer’s patch germinal center structures. Recently, we have shown that B cells responding to gut-derived antigens colonize the subepithelial dome (SED) in Peyer’s patches and rapidly proliferate independently of their relative BCR affinity. To evaluate B cell proliferation within different niches in Peyer’s patches, we applied in vivo EdU incorporation assay as described in this protocol.

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1’s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular andin vivotarget engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells andin vivo. Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

PROteolysis Targeting Chimeras (PROTACs) represent an exciting inhibitory modality with many advantages, including sub-stoichiometric degradation of targets. Their scope, though, is still limited to-date by the requirement for a sufficiently potent target binder. A solution that proved useful in tackling challenging targets is the use of electrophiles to allow irreversible binding to the target. However, such binding will negate the catalytic nature of PROTACs. Reversible covalent PROTACs potentially offer the best of both worlds. They possess the potency and selectivity associated with the formation of the covalent bond, while being able to dissociate and regenerate once the protein target is degraded. Using Bruton’s tyrosine kinase (BTK) as a clinically relevant model system, we show efficient covalent degradation by non-covalent, irreversible covalent and reversible covalent PROTACs, with 85% degradation. Our data suggests that part of the degradation by our irreversible covalent PROTACs is driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drive degradation primarily by covalent engagement. The PROTACs showed enhanced inhibition of B cell activation compared to Ibrutinib, and exhibit potent degradation of BTK in patients-derived primary chronic lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited enhanced selectivity towards BTK compared to non-covalent and irreversible covalent PROTACs. These compounds may pave the way for the design of covalent PROTACs for a wide variety of challenging targets.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1’s active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

T follicular helper (Tfh) cells regulate B cell selection for entry into the germinal center (GC) reaction or for differentiation into antibody forming cells. This process takes place at the border between the T and B zones in lymphoid organs and involves physical contacts between T and B cells. During these interactions, T cells endow the B cells with selection signals that promote GC seeding or plasmablast differentiation based on their B cell receptor affinity. In Peyer's patches (PPs), T cells promote B cell colonization of the subepithelial dome (SED) without effective affinity-based clonal selection. To specifically characterize the T cell population that resides within the SED niche, we performed ex vivo photoactivation of the SED compartment followed by flow cytometry analysis of the labeled cells, as described in this protocol. This technique integrates both spatial and cellular information in studies of immunological niches and can be adapted to various experimental systems.

In germinal centers, B cells interact with antigen in the light zone and clonally expand in the dark zone. Davidzohn et al. show that BCR-induced Syk degradation in the light zone attenuates signal transduction, impedes plasma cell formation, and promotes B cell transition to the dark zone., Germinal centers (GCs) are sites at which B cells proliferate and mutate their antibody-encoding genes in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). B cell antigen receptor (BCR) signals induce Syk activation followed by rapid phosphatase-mediated desensitization; however, how degradation events regulate BCR functions in GCs is unclear. Here, we found that Syk degradation restrains plasma cell (PC) formation in GCs and promotes B cell LZ to DZ transition. Using a mouse model defective in Cbl-mediated Syk degradation, we demonstrate that this machinery attenuates BCR signaling intensity by mitigating the Kras/Erk and PI3K/Foxo1 pathways, and restricting the expression of PC transcription factors in GC B cells. Inhibition of Syk degradation perturbed gene expression, specifically in the LZ, and enhanced the generation of PCs without affecting B cell proliferation. These findings reveal how long-lasting attenuation of signal transduction by degradation events regulates cell fate within specialized microanatomical sites., Graphical Abstract

Summary Protective immune responses depend on the formation of immune synapses between T cells and antigen-presenting cells (APCs). The two main LFA-1 ligands, ICAM-1 and ICAM-2, are co-expressed on many cell types, including APCs and blood vessels. Although these molecules were suggested to be key players in immune synapses studied in vitro , their contribution to helper T cell priming in vivo is unclear. Here, we used transgenic mice and intravital imaging to examine the role of dendritic cell (DC) ICAM-1 and ICAM-2 in naive CD4 T cell priming and differentiation in skin-draining lymph nodes. Surprisingly, ICAM deficiency on endogenous CD40-stimulated lymph node DCs did not impair their ability to arrest and prime CD4 lymphocyte activation and differentiation into Th1 and Tfh effectors. Thus, functional T cell receptor (TCR)-specific helper T cell synapses with antigen-presenting DCs and subsequent proliferation and early differentiation into T effectors do not require LFA-1-mediated T cell adhesiveness to DC ICAMs.

The eradication of pathogens and establishment of immunological memory depend on the generation of both effector and long-lived memory cells within specialized immune niches. Whole-organ imaging demonstrates that, during viral infection, the fate of CD8+ T cells in lymph nodes is coupled to chemotactic signals controlling their distribution within different microanatomical sites.

The molecular mechanism governing affinity-based B cell selection for germinal center colonization is unclear. Zaretsky et al. show that B cell ICAMs promote efficient B cell selection for clonal expansion by supporting sustained interactions with T follicular helper cells., The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T–B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh–B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells.

Somatic hypermutation (SHM) and class-switch recombination (CSR) increase the affinity and diversify the effector functions of antibodies during immune responses. Although SHM and CSR are fundamentally different, their independent roles in regulating B cell fate have been difficult to uncouple because a single enzyme, activation-induced cytidine deaminase (encoded by Aicda), initiates both reactions. Here, we used a combination of Aicda and antibody mutant alleles that separate the effects of CSR and SHM on polyclonal immune responses. We found that class-switching to IgG1 biased the fate choice made by B cells, favoring the plasma cell over memory cell fate without significantly affecting clonal expansion in the germinal center (GC). In contrast, SHM reduced the longevity of memory B cells by creating polyreactive specificities that were selected against over time. Our data define the independent contributions of SHM and CSR to the generation and persistence of memory in the antibody system.

B cells have a need for speed High-affinity antibodies provide long-lasting protective immunity against many infections. Generating such antibodies requires help, in the form of T cells, which interact with antibody-producing B cells. As B cells proliferate and mutate their antibody genes, T cells select the cells producing high-affinity antibodies. Gitlin et al. show in mice that B cells that receive T cell help transit through the cell cycle more quickly by increasing the speed at which replication forks progress. Such a rapid cell cycle transition gives high-affinity B cells a selective advantage. Science , this issue p. 643

Spatial information from NICHE-seq Immune functions depend on the interactions of heterogeneous cells in a range of microenvironments in the body. Although information regarding immune cell function has been collected using single-cell RNA-sequencing methods, these techniques have traditionally lacked spatial information. Medaglia et al. describe NICHE-seq, a technique that allows the sorting and analysis of cells from within visually selected territories in transgenic mice that express photoactivatable green fluorescent protein. The method successfully identified T and B cell-specific niches in mouse lymph nodes and spleens after virus infection. The approach will allow us to bridge the gap between cellular and spatial information in studies of organs. Science , this issue p. 1622

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Help Shared Germinal centers are specialized structures within lymph nodes, where B cells undergo the changes required to produce high-affinity antibodies. This process relies on T follicular helper (Tfh) cells. The dynamic properties of Tfh cells and how they affect the selection of B cells, however, are not well understood. Using two-photon laser scanning microscopy of mouse lymph nodes, Shulman et al. (p. 673 , published online 25 July) find that Tfh cells are not restricted to a single germinal center, but instead emigrate into neighboring germinal centers within the same lymph nodes. Furthermore, newly activated T cells can enter already established germinal centers and presumably influence ongoing B cell selection and differentiation. Such active movement may ensure maximal diversification of the B cell response and promote the production of high-affinity antibodies.

Kindlin-3 is a key lymphocyte function–associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3–null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3–null T lymphocytes failed to trigger the robust LFA-1–mediated T-cell spreading on ICAM-1 and ICAM-1–expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3–null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, β I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1–driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3–null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.

Lymphocyte arrest and spreading on ICAM-1–expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca2+. In contrast to LFA-1–activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative beta(1) Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes. Israel Science Foundation US-Israel Binational Science Foundation Minerva Foundation, Germany

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer’s patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1–3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.

Rac GTPases are key regulators of leukocyte motility. In lymphocytes, chemokine-mediated Rac activation depends on the CDM adaptor DOCK2. The present studies addressed the role of DOCK2 in chemokine-triggered lymphocyte adhesion and motility. Rapid chemokine-triggered activation of both LFA-1 and VLA-4 integrins took place normally in DOCK2–/– T lymphocytes under various shear flow conditions. Consequently, DOCK2–/– T cells arrested normally on TNFα-activated endothelial cells in response to integrin stimulatory chemokine signals, and their resistance to detachment was similar to that of wild-type (wt) T lymphocytes. Nevertheless, DOCK2–/– T lymphocytes exhibited reduced microvillar collapse and lamellipodium extension in response to chemokine signals, ruling out a role for these events in integrin-mediated adhesion strengthening. Strikingly, arrested DOCK2–/– lymphocytes transmigrated through a CCL21-presenting endothelial barrier with similar efficiency and rate as wt lymphocytes but, unlike wt lymphocytes, could not locomote away from the transmigration site of the basal endothelial side. DOCK2–/– lymphocytes also failed to laterally migrate over multiple integrin ligands coimmobilized with chemokines. This is a first indication that T lymphocytes use 2 different chemokine-triggered actin remodeling programs: the first, DOCK2 dependent, to locomote laterally along apical and basal endothelial surfaces; the second, DOCK2 independent, to cross through a chemokine-bearing endothelial barrier.

SummaryEndothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.

The germinal center (GC) reaction in Peyer′s patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7− B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines., Gut lumen antigens must be continuously sampled by the immune system to maintain proper immune homeostasis. Here the authors show that activated CCR6+CCR1+GL7- gut B cells retrieve lumen antigens from specialized M cells and transfer them across the subepithelial dome in the Peyer’s patch to contribute to the maintenance of gut humoral immunity.

Summary Antibodies secreted within the intestinal tract provide protection from the invasion of microbes into the host tissues. Germinal center (GC) formation in lymph nodes and spleen strictly requires SLAM-associated protein (SAP)-mediated T cell functions; however, it is not known whether this mechanism plays a similar role in mucosal-associated lymphoid tissues. Here, we find that in Peyer’s patches (PPs), SAP-mediated T cell help is required for promoting B cell selection in GCs, but not for clonal diversification. PPs of SAP-deficient mice host chronic GCs that are absent in T cell-deficient mice. GC B cells in SAP-deficient mice express AID and Bcl6 and generate plasma cells in proportion to the GC size. Single-cell IgA sequencing analysis reveals that these mice host few diversified clones that were subjected to mild selection forces. These findings demonstrate that T cell-derived help to B cells in PPs includes SAP-dependent and SAP-independent functions., Graphical Abstract, Highlights • Chronic germinal centers in Peyer’s patches are formed in SAP-deficient mice • SAP-independent germinal centers arise in response to influenza infection • Few highly diversified clones dominate the SAP-independent germinal centers • Germinal center B cells in SAP-deficient mice are subjected to mild selection forces, SAP is required for proper T cell help in germinal centers (GCs). Biram et al. show that SAP-independent GCs are formed within Peyer’s patches. These GCs host highly diversified clones that are subjected to mild selection forces, demonstrating that clonal diversification can be uncoupled from clonal selection in chronic GCs.