Production Of High-affinity Antibodies Research Articles

Isotype switching in human memory B cells sets intrinsic antigen-affinity thresholds that dictate antigen-driven fates

Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.

Marginal zone B cells produce 'natural' atheroprotective IgM antibodies in a T cell-dependent manner.

The adaptive immune response plays an important role in atherosclerosis. In response to a high-fat/high-cholesterol (HF/HC) diet, marginal zone B (MZB) cells activate an atheroprotective programme by regulating the differentiation and accumulation of 'poorly differentiated' T follicular helper (Tfh) cells. On the other hand, Tfh cells activate the germinal centre response, which promotes atherosclerosis through the production of class-switched high-affinity antibodies. We therefore investigated the direct role of Tfh cells and the role of IL18 in Tfh differentiation in atherosclerosis. We generated atherosclerotic mouse models with selective genetic deletion of Tfh cells, MZB cells, or IL18 signalling in Tfh cells. Surprisingly, mice lacking Tfh cells had increased atherosclerosis. Lack of Tfh not only reduced class-switched IgG antibodies against oxidation-specific epitopes (OSEs) but also reduced atheroprotective natural IgM-type anti-phosphorylcholine (PC) antibodies, despite no alteration of natural B1 cells. Moreover, the absence of Tfh cells was associated with an accumulation of MZB cells with substantially reduced ability to secrete antibodies. In the same manner, MZB cell deficiency in Ldlr-/- mice was associated with a significant decrease in atheroprotective IgM antibodies, including natural anti-PC IgM antibodies. In humans, we found a positive correlation between circulating MZB-like cells and anti-OSE IgM antibodies. Finally, we identified an important role for IL18 signalling in HF/HC diet-induced Tfh. Our findings reveal a previously unsuspected role of MZB cells in regulating atheroprotective 'natural' IgM antibody production in a Tfh-dependent manner, which could have important pathophysiological and therapeutic implications.

The lipid globotriaosylceramide promotes germinal center B cell responses and antiviral immunity.

Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes. In influenza infection, elevating Gb3, either endogenously or exogenously, promoted broadly reactive antibody responses and cross-protection. These data demonstrate that Gb3 determines the affinity and breadth of B cell immunity and has potential as a vaccine adjuvant.

Infliximab aggregates produced in severe and mild elevated temperature stress conditions induce an extended specific CD4 T-cell response

Aggregation has been widely described as a factor contributing to therapeutic antibody immunogenicity. Although production of high-affinity anti-drug antibodies depends on the activation of CD4 T lymphocytes, little is known about the T-cell response induced by antibody aggregates, especially for aggregates produced in mild conditions resulting from minor handling errors of vials. Large insoluble infliximab (IFX) aggregates produced in severe elevated temperature stress conditions have been previously shown to induce human monocyte-derived dendritic cell (moDC) maturation. We here showed that large IFX aggregates recruit in vitro a significantly higher number of CD4 T-cells compared to native IFX. Moreover, a larger array of T-cell epitopes encompassing the entire variable regions was evidenced compared to the native antibody. We then compared the responses of moDCs to different types of aggregates generated by submitting IFX to mild conditions of various times of incubation at an elevated temperature. Decreasing stress duration reduced aggregate size and quantity, and subsequently altered moDC activation. Of importance, IFX aggregates generated in mild conditions and not altering moDC phenotype generated an in vitro T-cell response with a higher frequency of CD4 T cells compared to native IFX. Moreover, cross-reactivity studies of aggregate-specific T cells showed that some T cells could recognize both native and aggregated IFX, while others responded only to IFX aggregates. Taken together, our results suggest that aggregation of antibodies in mild elevated temperature stress conditions is sufficient to alter moDC phenotype in a dose-dependent manner and to increase T-cell response.

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Abstract 16744: Antibody Responses to Myocardial Antigens Released in the Context of a Myocardial Infarction

Our study aims to investigate antigen-specific B cell responses after myocardial infarction. Recent research has indicated that B cells and myocardial antibodies may contribute to the development of ischemic heart failure and dampen post-myocardial infarction repair. However, the antigen specificity of these myocardial antibodies remains unknown. To produce high-affinity antibodies, B cells typically undergo multiple rounds of somatic hypermutation and selection processes within the germinal center (GC) reactions of secondary lymphoid organs. Therefore, we sought to characterize the germinal center B cell responses and to examine the repertoire of B cell receptors (BCR) in C57BL/6J mice subjected to experimental myocardial infarction. Flow cytometry analyses showed increased frequencies of GC B cells in infarcted mice at 5 and 14 days post-surgery. However, sham-operated mice also showed similar increase in GC frequencies on day 14 post-surgery. Single-cell RNA/BCR sequencing analyses enabled the identification of GC B cell and plasma cell (antibody secreting) populations expressing unique B cell receptors in infarcted mice that were not found in sham-operated mice. Over time, the number of GC B cells increased accompanied by a progressive clonal expansion, indicating the development of antigen-driven B cell responses. By examining immunoglobulin variable gene rearrangements within single B cells, we identified distinct B cell lineages expressing mutated antigen receptors suggestive of affinity maturation. Selected BCRs of interested were then cloned into immunoglobulin expression vectors and expressed in immortalized human embryonic kidney cells. When incubated with histological myocardial slices, one of these unique BCR expressed showed binding to myocardial antigens. These findings support the notion that myocardial infarction can trigger germinal center B cell responses, which then lead to the production of high-affinity heart-specific antibodies. Moreover, the ‘track-and-clone’ approach herein described could help unraveling the antigen specificity and functional significance of these myocardial antibodies.

Suppressive might of a few: T follicular regulatory cells impede auto-reactivity despite being outnumbered in the germinal centres.

The selection of high-affinity B cells and the production of high-affinity antibodies are mediated by T follicular helper cells (Tfhs) within germinal centres (GCs). Therein, somatic hypermutation and selection enhance B cell affinity but risk the emergence of self-reactive B cell clones. Despite being outnumbered compared to their helper counterpart, the ablation of T follicular regulatory cells (Tfrs) results in enhanced dissemination of self-reactive antibody-secreting cells (ASCs). The specific mechanisms by which Tfrs exert their regulatory action on self-reactive B cells are largely unknown. We developed computer simulations to investigate how Tfrs regulate either selection or differentiation of B cells to prevent auto-reactivity. We observed that Tfr-induced apoptosis of self-reactive B cells during the selection phase impedes self-reactivity with physiological Tfr numbers, especially when Tfrs can access centrocyte-enriched GC areas. While this aided in selecting non-self-reactive B cells by restraining competition, higher Tfr numbers distracted non-self-reactive B cells from receiving survival signals from Tfhs. Thus, the location and number of Tfrs must be regulated to circumvent such Tfr distraction and avoid disrupting GC evolution. In contrast, when Tfrs regulate differentiation of selected centrocytes by promoting recycling to the dark zone phenotype of self-reactive GC resident pre-plasma cells (GCPCs), higher Tfr numbers were required to impede the circulation of self-reactive ASCs (s-ASCs). On the other hand, Tfr-engagement with GCPCs and subsequent apoptosis of s-ASCs can control self-reactivity with low Tfr numbers, but does not confer selection advantage to non-self-reactive B cells. The simulations predict that to restrict auto-reactivity, natural redemption of self-reactive B cells is insufficient and that Tfrs should increase the mutation probability of self-reactive B cells.

The TRIM37 variants in Mulibrey nanism patients paralyze follicular helper T cell differentiation

The Mulibrey (Muscle–liver–brain–eye) nanism caused by loss-of-function variants in TRIM37 gene is an autosomal recessive disorder characterized by severe growth failure and constrictive pericarditis. These patients also suffer from severe respiratory infections, co-incident with an increased mortality rate. Here, we revealed that TRIM37 variants were associated with recurrent infection. Trim37 FINmajor (a representative variant of Mulibrey nanism patients) and Trim37 knockout mice were susceptible to influenza virus infection. These mice showed defects in follicular helper T (TFH) cell development and antibody production. The effects of Trim37 on TFH cell differentiation relied on its E3 ligase activity catalyzing the K27/29-linked polyubiquitination of Bcl6 and its MATH domain-mediated interactions with Bcl6, thereby protecting Bcl6 from proteasome-mediated degradation. Collectively, these findings highlight the importance of the Trim37-Bcl6 axis in controlling the development of TFH cells and the production of high-affinity antibodies, and further unveil the immunologic mechanism underlying recurrent respiratory infection in Mulibrey nanism.

B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics.

Successful B cell activation, critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodelling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through Akt and MAPK axes.

The G allele of SNP rs3922 reduces the binding affinity between IGF2BP3 and CXCR5 correlating with a lower antibody production.

Effective vaccines that function through humoral immunity seek to produce high-affinity antibodies. Our previous research identified the single-nucleotide polymorphism rs3922G in the 3'UTR of CXCR5 as being associated with nonresponsiveness to the hepatitis B vaccine. The differential expression of CXCR5 between the dark zone (DZ) and light zone (LZ) is critical for organizing the functional structure of the germinal center (GC). In this study, we report that the RNA-binding protein IGF2BP3 can bind to CXCR5 mRNA containing the rs3922 variant to promote its degradation via the nonsense-mediated mRNA decay pathway. Deficiency of IGF2BP3 leads to increased CXCR5 expression, which results in the disappearance of CXCR5 differential expression between DZ and LZ, disorganized GCs, aberrant somatic hypermutations, and reduced production of high-affinity antibodies. Furthermore, the affinity of IGF2BP3 for the rs3922G-containing sequence is lower than that for the rs3922A counterpart, which may explain the nonresponsiveness to the hepatitis B vaccination. Together, our findings suggest that IGF2BP3 plays a crucial role in the production of high-affinity antibodies in the GC by binding to the rs3922-containing sequence to regulate CXCR5 expression.

We are memory: B-cell responses in allergy and tolerance.

The significance of B-cell memory in sustaining IgE-mediated allergies but also ensuring the development of long-term allergen tolerance has remained enigmatic. However, well-thought murine and human studies have begun to shed more light on this highly disputed subject. The present mini review highlights important aspects, like the involvement of IgG1 memory B cells, the meaning of low- or high-affinity IgE antibody production, the impact of allergen immunotherapy, or the relevance of local memory established by ectopic lymphoid structures. Based on recent findings, future investigations should lead to deeper knowledge and the development of improved therapies treating allergic individuals.

T follicular helper cells and T follicular regulatory cells in autoimmune diseases.

T follicular helper (Tfh) cells are heterogeneous and mainly characterized by expressing surface markers CXCR5, ICOS, and PD-1; cytokine IL-21; and transcription factor Bcl6. They are crucial for B-cell differentiation into long-lived plasma cells and high-affinity antibody production. T follicular regulatory (Tfr) cells were described to express markers of conventional T regulatory (Treg) cells and Tfh cells and were able to suppress Tfh-cell and B-cell responses. Evidence has revealed that the dysregulation of Tfh and Tfr cells is positively associated with the pathogenic processes of autoimmune diseases. Herein, we briefly introduce the phenotype, differentiation, and function of Tfh and Tfr cells, and review their potential roles in autoimmune diseases. In addition, we discuss perspectives to develop novel therapies targeting Tfh/Tfr balance.

Protein phosphatase 2A propels follicular T helper cell development in lupus

Follicular helper T (Tfh) cells are important for generating humoral immune responses by helping B cells form germinal centers (GCs) and the production of high-affinity antibodies. However, aberrant Tfh cell expansion also contributes to the generation of self-reactive autoantibodies and promotes autoantibody-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Protein phosphatase 2A catalytic subunit alpha isoform (PP2A Cα) expression levels are elevated in peripheral T cells of SLE patients and positively correlate with autoantibody titers and disease activity. Here, we demonstrate a critical role of PP2A in Tfh differentiation by using T cell restricted PP2A Cα deficient mice. We observed impaired Tfh differentiation and GC response in two different classical Tfh induction models. Mechanistic studies revealed that downregulation of protein translation of the Tfh lineage transcription factor BCL6 in PP2A deficient T cells. Importantly, we found that PP2A deficiency by either gene knockout or chemical inhibition alleviated lupus severity in mice. Lastly, we confirmed a positive correlation between PP2A Cα and BCL6 protein levels in human CD4+ T cells from patients with SLE. In summary, our study revealed a critical role of PP2A in regulating Tfh cells and suggests it is a potential therapeutic target for lupus.

Type 2 helper T cells convert into Interleukin-13-expressing follicular helper T cells after antigen repriming

Anaphylaxis caused by allergen sensitization and vaccination is a serious health concern. The severity of anaphylaxis is associated with the presence of immunoglobulin E (IgE) antibodies that bind to allergens with a high affinity. Here, we report the development of a unique Interleukin-13 (IL-13)-producing follicular helper T (TFH) cell subset, designated TFH2 cells, which is tightly associated with the production of high-affinity IgE antibodies. TFH2 cells had a transcriptionally hybrid phenotype between TH2 and TFH cells, which express GATA-binding protein 3 (Gata3) and B-cell/CLL lymphoma 6 (BCL-6), respectively. Adaptive transfer experiments demonstrated that type 2 helper T (TH2) cells were capable of differentiating into TFH2 cells upon secondary antigen stimulation. The type 2 TFH (TFH2) conversion process was entirely attenuated in T cell-specific deficient mice of Bcl6 (Bcl6f/f cd4-cre; Bcl6ΔT). Moreover, the transfer of IL-13 defective TH2 cells partially inhibited IgE responses and significantly decreased the high-affinity IgE, even though TFH development was intact. Therefore, IL-13 from TFH2 cells controlled the selective enrichment of IgE+ B-cells with a high affinity for allergens. A previously undefined unique TFH subset, TFH2, likely contributes to IgE responses. This suggests that the accumulation of antigen-specific TH2 cells in human patients with allergies is a high-risk factor for IgE-dependent allergic diseases.

Production of high-affinity monoclonal antibody and development of immunoassay for 3-methyl-quinoxaline-2-carboxylic acid detection in swine muscle and liver

Production of high-affinity and specific antibodies to small molecules with molecular weight (MW) lower than 200 Da is challenging. Here, we designed a novel hapten, named hapten H6, for the detection of 3-methyl-quinoxaline-2-carboxylic acid (MQCA, MW of 189 Da), a residual marker of olaquindox, one of important veterinary antibiotics. The hapten H6 maintained all structural features of MQCA, especially in mulliken atomic charge distribution. Then, a monoclonal antibody (mAb) named 8C9 was obtained with an IC50 value of 0.2 µg/L, yielding a 15.5- to 88.5-fold improvement compared to previously prepared specific antibodies against MQCA. In addition, mAb 8C9 exhibited ignorable cross-reactivity with other structural analogs. Finally, a highly sensitive and specific indirect competitive ELISA based on mAb 8C9 was developed for the detection of MQCA in swine muscle and liver samples with limit of detection values of 0.04 µg/kg and 0.09 µg/kg, respectively.

H3K36 methyltransferase NSD1 is essential for normal B1 and B2 cell development and germinal center formation.

B cells, which consist of two well-defined populations: B1 and B2 cells, which can produce antibodies that are essential for host protection against infections, through virus neutralization, opsonization and antibody-dependent cellular cytotoxicity. Epigenetic modifications, such as DNA methylation and histone modification could regulate immune cell differentiation and functions. In this study, we found a significant reduction of GC response in the B cell specific knockout of H3K36 methyltransferase NSD1 (Mb1-Cre+ NSD1fl/fl, NSD1B KO) mice compared with the wildtype control (Mb1-Cre+ NSD1+/+, NSD1B WT). We also demonstrated reduced production of high-affinity antibody, but increased production of low-affinity antibody in the NSD1B KO mice. Further analysis revealed that loss of NSD1 promoted the development of B1 cells by increasing the expression of Rap1b and Arid3a. In conclusion, our data suggest that NSD1 plays an important role in regulation the development of B1 and B2 cells, and the process of germinal center formation and high-affinity antibody production.

Advanced hybridoma technology for selective production of high-affinity monoclonal antibodies through B-cell receptors

In general, it is difficult to raise novel monoclonal antibodies against relatively low-molecular weight antigen, and particularly those with high homology for the mouse protein. The optimized B-cell targeting (BCT) technique can overcome this limitation. The point of this advanced technology is the selection of sensitized B lymphocytes by the antigen through B-cell receptors (BCRs). This strict selection by specific and strong interaction between antigen and antibody enables the efficient production of monoclonal antibodies with high specificity and affinity. It also offers the condensation of sensitized target B lymphocytes to selectively generate hybridoma cells secreting desired monoclonal antibodies. In this study, several kinds of biotinylated human myoglobin (hMyo) were prepared to select sensitized B lymphocytes via BCRs. Biotinylated hMyo prepared by a 3.75- and 7.5-fold molar excess of N-hydroxysuccinimide (NHS)-biotin provided high antigenicity of 68–88%. B lymphocytes selected by these biotinylated antigens had an ELISA-positive rate >17 times higher than that with usual biotinylated antigen. Monoclonal antibodies generated by the optimized BCT technology by preselecting sensitized B lymphocytes with the target antigen were identified to specifically recognize lower antigenic epitopes in hMyo with high affinity, while this would be impossible by the polyethylene glycol (PEG) method. Furthermore, combination of these high-affinity monoclonal antibodies gave the best binding rate in an epitope binning assay. These outcomes could be attributed to the unique characteristic that BCRs on sensitized B lymphocytes themselves can select the target epitopes in the antigen. The BCRs may act as a strict sensor of B lymphocytes to precisely select the target epitopes, even though the number of immunized B lymphocytes is low.

Characteristics and Roles of T Follicular Helper Cells in SARS-CoV-2 Vaccine Response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is critical to controlling the coronavirus disease 2019 (COVID-19) pandemic. However, a weak response to the vaccine and insufficient persistence of specific antibodies may threaten the global impact of mass vaccination campaigns. This study summarizes the internal factors of the body that affect the effectiveness of the SARS-CoV-2 vaccine. T follicular helper (Tfh) cells support germinal center B cells to produce vaccine-specific immunoglobulins. A reduction in the Tfh cell number and a shift in the subset phenotypes caused by multiple factors may impair the production and persistence of high-affinity antibodies. Besides efficacy differences caused by the different types of vaccines, the factors that affect vaccine effectiveness by intervening in the Tfh cell response also include age-related defects, the polarity of the body microenvironment, repeated immunization, immunodeficiency, and immunosuppressive treatments. Assessing the phenotypic distribution and activation levels of Tfh cell subsets after vaccination is helpful in predicting vaccine responses and may identify potential targets for improving vaccine effectiveness.

Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes.

Immune synapse formation is a key step for lymphocyte activation. In B lymphocytes, the immune synapse controls the production of high-affinity antibodies, thereby defining the efficiency of humoral immune responses. While the key roles played by both the actin and microtubule cytoskeletons in the formation and function of the immune synapse have become increasingly clear, how the different events involved in synapse formation are coordinated in space and time by actin-microtubule interactions is not understood. Using a microfluidic pairing device, we studied with unprecedented resolution the dynamics of the various events leading to immune synapse formation and maintenance in murine B cells. Our results identify two groups of events, local and global, dominated by actin and microtubules dynamics, respectively. They further highlight an unexpected role for microtubules and the GEF-H1-RhoA axis in restricting F-actin polymerization at the lymphocyte-antigen contact site, thereby allowing the formation and maintenance of a unique competent immune synapse.

Intracellular trafficking of antigen in B lymphocytes

B cells are an essential part of the adaptive immune system by providing the humoral arm of the response, and specific and long-lasting protection against a pathogens. Successful antigen internalization, intracellular trafficking, processing and presentation are critical for full B cell activation and high-affinity antibody production. Despite the fundamental importance of this pathway for the immune response, the mechanisms underlying antigen trafficking for successful processing and MHCII presentation remain unclear. By using confocal and super-resolution microscopy, we followed the traffic of internalized antigen along the endosomal pathway in B cells. To identify different vesicle populations, we took advantage of different Rab GTPases as well-known markers for early-, late-and recycling-endosomes. After internalization, antigen traffics from early-to late-endosomes and typically reaches a dense endosomal compartment located in the perinuclear region. This compartment is rich in lysosomal markers and Cathepsin-S, as well as MHCII, suggesting antigen loading compartment. Interestingly however, when we investigated the early timepoints after activation in detail, we found the antigen in peripheral endosomes with atypical features combining both early (EEA1+, Rab5+) and late (LAMP1+) markers. These endosomes also showed colocalization with Rab11, a marker of recycling-endosomes, and MHCII, suggesting that already these early peripheral vesicles could degrade antigen and load peptides on MHCII for presentation. In support of their role in antigen processing, these vesicles also contained Cathepsin-S and showed hydrolysis of antigen labeled with DQ, a dye that only fluoresces upon hydrolysis. We postulate that these early vesicles could play a role in fast antigen processing, or could promote presentation of different peptide repertoire as compared to the central, perhaps more mature, perinuclear compartment. Further studies are needed to unravel how these pathways are regulated and how their balance affects T cell help and thereby B cell fate and production of high-affinity antibodies.