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The blood-brain barrier (BBB) continues to represent one of the most significant challenges for successful drug-based treatments of neurological disease. Mechanical modulation of the BBB using focused ultrasound (FUS) and microbubbles (MBs) has shown considerable promise in enhancing the delivery of therapeutics to the brain, but questions remain regarding possible long-term effects of such forced disruption. This review examines the evidence for inflammation associated with ultrasound-induced BBB disruption and potential strategies for managing such inflammatory effects to improve both the efficacy and safety of therapeutic ultrasound in neurological applications.

Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises.

T follicular helper (TFH) cells promote expansion of germinal center (GC) B cells and plasma cell differentiation. Whether cognate peptide-MHCII (pMHCII) density instructs selection and cell fate decisions in a quantitative manner remains unclear. Using αDEC205-OVA to differentially deliver OVA peptides to GC B cells on the basis of DEC205 allelic copy number, we find DEC205+/+ B cells take up 2-fold more antigen than DEC205+/− cells, leading to proportional TFH cell help and B cell expansion. To validate these results, we establish a caged OVA peptide, which is readily detected by OVA-specific TFH cells after photo-uncaging. In situ uncaging of peptides leads to multiple serial B-T contacts and cell activation. Differential CD40 signaling, is both necessary and sufficient to mediate 2-fold differences in B cell expansion. While plasmablast numbers are increased, pMHCII density does not directly control the output or quality of plasma cells. Thus, we distinguish the roles TFH cells play in expansion versus differentiation.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds., Al-Aghbar et al propose a TCR bending mechanosignal model that demonstrates how local mechanical membrane bending may influence T cell receptor binding events and thus T-cell activation.

Hematopoietic stem and progenitor cells (HSPCs) use specialized adhesive structures referred to as magnupodium to stay in hematopoietic niches. Bessey et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202005085) define new characteristics of the magnupodium, including centriole polarization and the necessary and sufficient role of CXCR4 signaling.

The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

The PI3K pathway plays a key role in B cell activation and is important for the differentiation of Ab producing plasma cells (PCs). Although much is known about the molecular mechanisms that modulate PI3K signaling in B cells, the transcriptional regulation of PI3K expression is poorly understood. In this study, we identify the zinc finger protein Zbtb18 as a transcriptional repressor that directly binds enhancer/promoter regions of genes encoding class I PI3K regulatory subunits, subsequently limiting their expression, dampening PI3K signaling and suppressing PC responses. Following activation, dividing B cells progressively downregulated Zbtb18, allowing gradual amplification of PI3K signals and enhanced development of PCs. Human Zbtb18 displayed similar expression patterns and function in human B cells, acting to inhibit development of PCs. Furthermore, a number of Zbtb18 mutants identified in cancer patients showed loss of suppressor activity, which was also accompanied by impaired regulation of PI3K genes. Taken together, our study identifies Zbtb18 as a repressor of PC differentiation and reveals its previously unappreciated function as a transcription modulator of the PI3K signaling pathway.

Although cognate peptide-MHCII (pMHCII) is critical for B cell selection in the germinal center (GC), it is unclear how cell intrinsic differences in peptide levels contribute to selection and cell fate decisions. Here, we applied the a-DEC205-OVA (dec) model system, to deliver different levels of OVA peptide to GC B cells in situ in order to interrogate how intermediate and high levels of pMHCII affect selection and cell fate on a per cell basis. We used Ly75+/− (DEC-het) B cells, which expressed ~50% of surface DEC205 protein compared to WT B cells, and presented proportional amount of OVA peptide after dec treatment. Using competitive co-transfers, we found that WT B cells expanded two-fold more than DEC-het B cells. This 2-fold difference in expansion was maintained at a wide range of dec administration. To study T cell recognition in situ, we developed a novel caged ovalbumin peptide, which is readily detected by OT-II T cells upon photo-uncaging, and revealed that initial T cell recognition in the GC leads to increased B-T serial contacts. Differential CD40 signaling, was both necessary and sufficient to mediate 2-fold differences in GC B cell expansion and also promoted GC-like B cell morphology, suggesting CD40 signaling is a rheostat of pMHCII dose. Surprisingly, we found that while plasmablast numbers were increased upon dec stimulation, both WT and DEC-het GC B cells were equally capable of differentiating into plasma cells, suggesting that pMHCII density does not directly control the output or quality of plasma cells generated. Thus, these results delineate distinct roles pMHCII play in expansion vs. differentiation in the GC. Supported by grants from NIH (R01 HL141491-05)

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be ‘dialled-in’ as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable., Summary: Immune cell–cell interactions are reconstituted in free-standing vesicles, allowing the spatiotemporal aspects of immune synapse formation to be investigated.

Regulatory T-cells (Treg) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T-cells, where protein kinase C-T (PKC-T) localizes to the contact-point between T-cells and antigen-presenting cells, in Treg, PKC-T localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-T phosphotarget and show that vimentin forms a DPC superstructure on which PKC-T accumulates. Treatment of Treg with either a clinically relevant PKC-T inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted Treg were significantly better than controls in suppressing alloreactive T-cell priming in graftversus-host disease, and graft-versus-host disease lethality. Interestingly, vimentin disruption augmented suppressor function of PKC-T-deficient Treg. This suggests that enhanced Treg activity after PKC-T inhibition is secondary to effects on vimentin, not just PKC-T kinase activity inhibition. Our data demonstrated that vimentin is a key metabolic and functional controller of Treg activity, and provide proof-of-principle that disrupting vimentin is a feasible, translationally relevant method to enhance Treg potency.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

The European Molecular Biology Organization (EMBO) meeting Visualizing Immune System Complexity, held in January 2009, covered multiple scales, from imaging single molecules to imaging whole animals. In addition to experimental details, there was an emphasis on modeling both for data analysis and as a predictive tool to support experimental design. Imaging technologies discussed included total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, two-photon laser scanning microscopy, and magnetic resonance imaging. The biological systems included basic aspects of adaptive and innate immunity. The type 1 diabetes model was used to illustrate how a human disease was dissected at all the scales, from single-molecule analysis of the interactions of T cell receptors with peptide-loaded major histocompatibility complexes to dynamics of immune cell infiltrates by intravital microscopy, as well as the application of imaging diagnostics in humans.

The T cell and antigen-presenting cell communicate to initiate an immune response through formation of an immunological synapse. This specialized cell-cell junction is compartmentalized into adhesion molecule and T cell receptor enriched regions or SMACs. Distinct signals seem to be generated in the T cell receptor and adhesion molecule-dominated regions. This review focuses on how these distinct signaling pathways may be integrated within the T cell to set thresholds for T cell activation, proliferation, and survival.

Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions.

The interaction of activated leukocytes with the rheumatoid synovial environment is a key process in arthritis. Understanding this process will play an important role in designing effective treatments. In vivo imaging approaches combined with molecular genetics in animal models provide important tools to address these issues. The present review will focus on approaches to in vivo imaging, with particular attention to approaches that are proving useful for, or have promise for, research on animal models of rheumatoid arthritis. These approaches will probably shed light on the specific local mechanisms involved in chronic inflammation and provide real time monitoring approaches to follow cellular and molecular events related to disease development.

The asymmetric distribution of antigen during B cell division affects the fate of B cells and their function.

Cell adhesion plays a fundamental role in cell motility and immune response. We investigated the adhesion between a Jurkat cell, chosen for its high expression of CD2, and a glass-supported planar membrane containing either the laterally mobile, lipid-anchored isoform (GPI LFA-3) or the immobile, transmembrane isoform (TM LFA-3) of the counter-receptor LFA-3 at the same concentration of 2,000 LFA-3 molecules/μm2 by using a novel micromanipulation method. In this technique, the pipette holding the cell is micromanipulated in the direction perpendicular to a glass-supported lipid bilayer reconstituted with a given type of surface adhesion molecules. In experiments using the planar membrane containing GPI LFA-3, the adhered Jurkat cell deformed extensively in response to the pipette force, as the cell detachment proceeded by peeling at the edges of the contact area. When the cell elongation in the direction of the pipette reached a maximum, the cell separated rapidly from the planar membrane. In experiments using the planar membrane containing TM LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape. Our experimental data showed that the aspiration pressure required to detach a Jurkat cell from a membrane containing mobile LFA-3 is about one order of magnitude greater than that for the immobile LFA-3.

B lymphocytes are key players in host defence, but also autoimmune diseases. Their survival depends upon tonic signals transduced by surface immunoglobulin (BCR) and the process leading to antibody secretion is initiated by interaction of BCR with a cognate antigen. CD22 limits signalling of the BCR to strike a balance between tonic signalling, reactivity to pathogens and prevention of autoimmunity. In this issue, Gasparrini et al (2016) combined super‐resolution imaging approaches with single‐particle tracking and simulations to show how CD22 controls the signalling state of the BCR. They demonstrated that small CD22 nanoclusters run rings around the BCR in confined steady state to maintain low tonic signals, but releasing BCR from these corrals allows BCR cluster growth, which overcomes the harrying inhibition from highly mobile CD22.

The dream of stimulating the body's immune response to fight cancer has become a reality in the past decade. Several potent drugs have been clinically approved that block inhibitory checkpoints in the immune system, boosting the ability of the system's T cells to mount responses against a range of cancers1. Furthermore, patients' own T cells have been successfully genetically engineered to attack blood-cancer cells2. Although these studies have established the immune system as a powerful ally in cancer therapy, there are still many challenges to overcome, and further advances would increase the number of people who stand to benefit from immunotherapy. In this issue, Yang and colleagues3 (page 651) propose a way to boost the function of antitumour T cells, using a metabolic trick to increase the level of cholesterol in the cells' membranes.

The accumulation of antigen specific T lymphocytes at sites of antigenic challenge is an important potential example of regulated leukocyte migration. A key engine for lymphocyte migration in tissues is based on engagement of integrin receptors and coordinated cytoskeletal responses that translate integrin engagement into cell movement. We hypothesized that antigen receptor engagement would regulate lymphocyte migration by changing the outcome of integrin engagement from migration to firm in situ adhesion. To test this hypothesis we have developed an in vitro system where we can simultaneously view cell migration, integrin engagement and antigen receptor engagement. We found that interaction of the integrin LFA1 with its ligand ICAM-I promotes fast, random migration of effector lymphocytes. Strikingly, antigen receptor engagement stops migration. Thus, antigen receptor engagement provides a dominant stop signal for lymphocyte migration that allows prolonged interaction with antigen presenting cells. The stop signal was accompanied by a change in the organization of the T cell cytoskeleton and an increase in LFA-1 engagement Time-lapse observations show that stopped T cell remain highly dynamic, but focus on a central site of antigen receptor engagement. In contrast, chemokines also signal increased integrin engagement, but in a manner that leads to greater locomotion, rather than a stop signal. Thus, signals that activate adhesion mechanisms can have distinct outcomes-stop or go-depending upon simultaneous effects on cytoskeletal organization. Simple loss or gain of adhesion receptors cannot fully explain migratory decision making.

Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. To determine the breadth and underpinning of changes in immunocyte gene expression due to genetic variation in mice, we performed, as part of the Immunological Genome Project, gene expression profiling for CD4+T cells and neutrophils purified from 39 inbred strains of the Mouse Phenome Database. Considering both cell types, a large number of transcripts showed significant variation across the inbred strains, with 22% of the transcriptome varying by 2-fold or more. These included 119 loci with apparent complete loss of function, where the corresponding transcript was not expressed in some of the strains, representing a useful resource of "natural knockouts." We identified 1222 cis-expression quantitative trait loci (cis-eQTL) that control some of this variation. Most (60%) cis-eQTLs were shared between T cells and neutrophils, but a significant portion uniquely impacted one of the cell types, suggesting cell type-specific regulatory mechanisms. Using a conditional regression algorithm, we predicted regulatory interactions between transcription factors and potential targets, and we demonstrated that these predictions overlap with regulatory interactions inferred from transcriptional changes during immunocyte differentiation. Finally, comparison of these and parallel data from CD4+T cells of healthy humans demonstrated intriguing similarities in variability of a gene's expression: the most variable genes tended to be the same in both species, and there was an overlap in genes subject to strong cis-acting genetic variants. We speculate that this "conservation of variation" reflects a differential constraint on intraspecies variation in expression levels of different genes, either through lower pressure for some genes, or by favoring variability for others.

The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4+CD8+stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4+or CD8+lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation. © 2013 Nature America, Inc. All rights reserved.

After infection, many factors coordinate the population expansion and differentiation of CD8+effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+effector cells. Long-lived memory CD8+cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+T cell immunity. © 2013 Nature America, Inc. All rights reserved.

Species restrictions in immune cell interactions have been demonstrated both in Ag-specific responses of T lymphocytes and the phenomenon of natural attachment. To determine the possible contribution of adhesion receptors to these restrictions, we have studied binding between the murine and human homologues of LFA-1 (CD11a/CD18) and ICAM employing purified human LFA-1 and ICAM-1 (CD54) bound to solid substrates. Murine cell lines bind to purified human LFA-1 through ICAM-1 and at least one other counter-receptor. This provides evidence for multiple counter-receptors for LFA-1 in the mouse as well as in the human. In contrast to binding of murine ICAM-1 to human LFA-1, murine LFA-1 does not bind to human ICAM-1. The species specificity maps to the LFA-1 alpha subunit, because mouse x human hybrid cells expressing the human alpha subunit associated with a mouse beta subunit bind to human ICAM-1, whereas those with a human beta subunit associated with a murine alpha subunit do not. Increased adhesiveness for ICAM-1 stimulated by phorbol esters could be demonstrated for hybrid LFA-1 molecules with human alpha and murine beta subunits.

Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton.

T11 target structure (T11TS) and lymphocyte function-associated antigen (LFA) 3 are the cell-surface glycoproteins on sheep and human erythrocytes (E) binding to cluster differentiation 2 (the E-receptor) on T cells in E rosette formation. Here we show that this functional cross-reactivity is most likely due to a structural homology of these molecules. A rabbit antiserum to sheep T11TS is shown to cross-react with LFA-3 in several independent assays: (a) rabbit anti-T11TS antiserum blocks the formation of E rosettes by human T cells with both autologous and xenogeneic (sheep) E by binding to the respective E; (b) the antiserum blocks the binding of anti-LFA-3 monoclonal antibody to human E; and (c) it reacts with purified LFA-3 in Western blotting. Together, these findings demonstrate that T11TS on sheep E and LFA-3 on human E are serologically related, providing further support for the notion that T11TS and LFA-3 are the sheep and human forms of the same cell interaction molecule.

Ligation of T cell receptor (TCR) to peptide–MHC (pMHC) complexes initiates signaling leading to T cell activation and TCR ubiquitination. Ubiquitinated TCR is then either internalized by the T cell or released toward the antigen-presenting cell (APC) in extracellular vesicles. How these distinct fates are orchestrated is unknown. Here, we show that clathrin is first recruited to TCR microclusters by HRS and STAM2 to initiate release of TCR in extracellular vesicles through clathrin- and ESCRT-mediated ectocytosis directly from the plasma membrane. Subsequently, EPN1 recruits clathrin to remaining TCR microclusters to enable trans-endocytosis of pMHC–TCR conjugates from the APC. With these results, we demonstrate how clathrin governs bidirectional membrane exchange at the immunological synapse through two topologically opposite processes coordinated by the sequential recruitment of ecto- and endocytic adaptors. This provides a scaffold for direct two-way communication between T cells and APCs.

The germinal centre (GC) response is an essential component of adaptive immunity, crucial for the generation of high-affinity antibodies, long-lived memory and plasma cells and is the basis for most current vaccines. While the dynamics of cognate B and T lymphocytes during the GC response are extensively studied, the roles of resident tissue macrophages in this process are less well understood, particularly in the spleen. A unique compartment to the spleen, called the marginal (MZ), is known to be important for adaptive immunity and harbours at least two distinct MZ-resident macrophage subsets. Both MZ macrophage populations are essential for capturing and preventing systemic dissemination of pathogens. However, their individual functions and potential role in adaptive immunity are not established. Here we used pharmacologic and genetic approaches to deplete SIGN-R1+ MZ macrophages and showed that these cells were specifically required for the optimal development of germinal centre responses in the spleen. SIGN-R1+ macrophages were dispensable for the initial GC establishment but were required for optimal maintenance of the response. The GC defect could be partially corrected by boosting T follicular helper (Tfh) cell responses or by inducing Tfh help prior to macrophage ablation. Furthermore, in the absence of SIGN-R1+ macrophages, 33D1+ DCs, a key population involved in Tfh priming, was displaced from the interfollicular regions to the MZ. Reconstituting the SIGN-R1+ subset restored DC positioning and rescued the GC phenotype. This work advances our understanding of how the splenic marginal zone regulates adaptive immunity, highlights the functional diversification of MZ macrophages, and provides the first genetic mouse model allowing for the specific depletion and modification of SIGN-R1+ cells.