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Antibody affinity maturation depends on the formation of germinal centers (GCs) in lymph nodes. This process generates a massive number of apoptotic B cells, which are removed by a specialized subset of phagocytes, known as tingible body macrophages (TBMs). Although defects in these cells are associated with pathological conditions, the identity of their precursors and the dynamics of dying GC B cell disposal remained unknown. Here, we demonstrate that TBMs originate from pre-existing lymph node–resident precursors that enter the lymph node follicles in a GC-dependent manner. Intravital imaging shows that TBMs are stationary cells that selectively phagocytose GC B cells via highly dynamic protrusions and accommodate the final stages of B cell apoptosis. Cell-specific depletion and chimeric mouse models revealed that GC B cells drive TBM formation from bone marrow–derived precursors stationed within lymphoid organs prior to the immune challenge. Understanding TBM dynamics and function may explain the emergence of various antibody-mediated autoimmune conditions.

Protecting the upper airways and brain from viral invasion through the olfactory mucosa is critical. Wellford et al. describe a barrier that restricts the passage of circulating antibodies and prevents them from reaching the olfactory mucosa. Instead, plasma cells are recruited into this site and prevent viral infection of the airways and the brain through local antibody production.

Single cell data for"The cellular states and fates of shed intestinal cells" paper. Folder: Count_tables and Objects This folder contains BG subtracted UMI tables and metadata for both the fecal wash UMAP and the fecal/tissue enterocytes combined UMAP 1. BG_subtracted_count_table_all_Fecal_cells_before_filt.txt - BG subtracted UMI table of all cells before Seurat filtration and analysis 2. BG_subtracted_count_table_all_Fecal_cells_after_filt.txt - BG subtracted UMI table of all cells after Seurat filtration and analysis. 3. Metadata_all_Fecal_cells_after_filteration.txt - Metadata for "BG_subtracted_count_table_all_Fecal_cells_after_filt.txt" 4. Combine_all_Fecal_cells_Obj.rds - Seurat Object for BG_subtracted_count_table_all_Fecal_cells_after_filt.txt 5. BG_subtracted_count_table_all_Enterocytes_before_filt.txt - BG subtracted UMI table of all cells before Seurat filtration and analysis 6. BG_subtracted_count_table_all_Enterocytes_after_filt.txt - BG subtracted UMI table of all cells after Seurat filtration and analysis. 7. Metadata_all_Enterocytes_after_filteration.txt - Metadata for "BG_subtracted_count_table_all_Enterocytes_after_filt.txt" 8. Combine_Enterocytes_Obj.rds - Seurat Object for BG_subtracted_count_table_all_Enterocytes_after_filt.txt

Protection from viral infections depends on immunoglobulin isotype switching, which endows antibodies with effector functions. Here, we found that the protein kinase DYRK1A is essential for B cell-mediated protection from viral infection and effective vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells were impaired in CSR activity in vivo and in vitro. Phosphoproteomic screens and kinase-activity assays identified MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site impaired CSR. After CSR and germinal center seeding, DYRK1A was required for proper clonal expansion of antigen-specific B cells through attenuation of proliferation. These findings reveal DYRK1A-mediated biological mechanisms of B cell immune responses that may be used for manipulation in antibody-mediated autoimmunity.

Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition–elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition–elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.

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.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell–cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection.

Antibody-mediated immunity is initiated by B cell differentiation into multiple cell subsets, including plasmablast, memory, and germinal center (GC) cells. B cell differentiation trajectories are determined by transcription factors, yet very few mechanisms that specifically determine early B cell fates have been described. Here, we report a post-transcriptional mechanism that suppresses the plasmablast genetic program and promotes GC B cell fate commitment. Single-cell RNA-sequencing analysis reveals that antigen-specific B cell precursors at the pre-GC stage upregulate YTHDF2, which enhances the decay of methylated transcripts. Ythdf2-deficient B cells exhibit intact proliferation and activation, whereas differentiation into GC B cells is blocked. Mechanistically, B cells require YTHDF2 to attenuate the plasmablast genetic program during GC seeding, and transcripts of key plasmablast-regulating genes are methylated and bound by YTHDF2. Collectively, this study reveals how post-transcriptional suppression of gene expression directs appropriate B cell fate commitment during initiation of the adaptive immune 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.

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.

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation–related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.

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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.

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1

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.

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 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.

The c-Jun NH2-terminal kinase (JNK) signaling pathway is central to the cell response to stress, inflammatory signals, and toxins. While selective inhibitors are known for JNKs and for various upstream MAP3Ks, no selective inhibitor is reported for MKK7--one of two direct MAP2Ks that activate JNK. Here, using covalent virtual screening, we identify selective MKK7 covalent inhibitors. We optimized these compounds to low-micromolar inhibitors of JNK phosphorylation in cells. The crystal structure of a lead compound bound to MKK7 demonstrated that the binding mode was correctly predicted by docking. We asserted the selectivity of our inhibitors on a proteomic level and against a panel of 76 kinases, and validated an on-target effect using knockout cell lines. Lastly, we show that the inhibitors block activation of primary mouse B cells by lipopolysaccharide. These MKK7 tool compounds will enable better investigation of JNK signaling and may serve as starting points for therapeutics.

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

The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression, while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles.

T and B cells' intricate molecular dance Generating high-affinity antibodies to fight infection is no easy task. To do so requires multiple steps, including T cells interacting with antibody-producing B cells in lymph nodes. These interactions select B cells expressing high-affinity antibodies for further proliferation, ensuring that the immune response generates high-affinity antibodies in large quantities. Shulman et al. use fluorescent live-cell imaging in mice to determine the molecular details of these interactions. They find that T cells engage B cells in short-lived mobile contacts during selection. These contacts cause T cells to flux calcium and produce proteins called cytokines, which probably drive B cells to proliferate and produce high-affinity antibodies. Science , this issue p. 1058

During immune responses, B lymphocytes clonally expand and undergo secondary diversification of their immunoglobulin (Ig) genes in germinal centers (GCs)1–4. High affinity B cells are expanded through iterative interzonal cycles of division and hypermutation in the GC dark zone (DZ) followed by migration to the GC light zone (LZ) where they are selected based on affinity to return to the DZ5–10. Here we combine a transgenic strategy to measure cell division and a photoactivatable fluorescent reporter to examine whether the extent of clonal expansion and hypermutation are regulated during interzonal GC cycles. We find that both cell division and hypermutation are directly proportional to the amount of antigen captured and presented by GC B cells to T follicular helper (TFH) cells in the LZ. Our data explain how GC B cells with the highest affinity for antigen are selectively expanded and diversified.

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.