The Lack of WIP Binding to Actin Results in Impaired B Cell Migration and Altered Humoral Immune Responses

Wiskott-Aldrich syndrome protein (WASp) is a main cytoskeletal regulator in B cells. WASp-interacting protein (WIP) binds to and stabilizes WASp but also interacts with actin. Using mice with a mutated actin binding domain of WIP (WIPΔABD), we here investigated the role of WIP binding to actin during B cell activation. We found an altered differentiation of WIPΔABD B cells and diminished antibody affinity maturation after immunization. Mechanistically, WIPΔABD B cells showed impaired B cell receptor (BCR)-induced PI3K signaling and actin reorganization, likely caused by diminished CD81 expression and altered CD19 dynamics on the B cell surface. WIPΔABD B cells displayed reduced in vivo motility, concomitantly with impaired chemotaxis and defective F-actin polarization, HS1 phosphorylation, and polarization of HS1 to F-actin-rich structures after CXCL12 stimulation in vitro. We thus concluded that WIP binding to actin, independent of its binding to WASp, is critical for actin cytoskeleton plasticity in B cells

Protein Kinase C-beta Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis

PKCβ-null (Prkcb−/−) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCβ failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb−/−B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCβ as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate. Lymphocyte activation is associated with major changes in metabolism. Tsui and colleagues demonstrate that PKCβ promotes metabolic reprogramming to drive effector fate decision in B cells

Complete analysis of the B-cell response to a protein antigen, from in vivo germinal centre formation to 3-D modelling of affinity maturation

Somatic hypermutation of immunoglobulin variable region genes occurs within germinal centres (GCs) and is the process responsible for affinity maturation of antibodies during an immune response. Previous studies have focused almost exclusively on the immune response to haptens, which may be unrepresentative of epitopes on protein antigens. In this study, we have exploited a model system that uses transgenic B and CD4<sup>+</sup> T cells specific for hen egg lysozyme (HEL) and a chicken ovalbumin peptide, respectively, to investigate a tightly synchronized immune response to protein antigens of widely differing affinities, thus allowing us to track many facets of the development of an antibody response at the antigen-specific B cell level in an integrated system <i>in</i> <i>vivo</i>. Somatic hypermutation of immunoglobulin variable genes was analysed in clones of transgenic B cells proliferating in individual GCs in response to HEL or the cross-reactive low-affinity antigen, duck egg lysozyme (DEL). Molecular modelling of the antibody–antigen interface demonstrates that recurring mutations in the antigen-binding site, selected in GCs, enhance interactions of the antibody with DEL. The effects of these mutations on affinity maturation are demonstrated by a shift of transgenic serum antibodies towards higher affinity for DEL in DEL-cOVA immunized mice. The results show that B cells with high affinity antigen receptors can revise their specificity by somatic hypermutation and antigen selection in response to a low-affinity, cross-reactive antigen. These observations shed further light on the nature of the immune response to pathogens and autoimmunity and demonstrate the utility of this novel model for studies of the mechanisms of somatic hypermutation

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates

Optimality of mutation and selection in germinal centers

The population dynamics theory of B cells in a typical germinal center could play an important role in revealing how affinity maturation is achieved. However, the existing models encountered some conflicts with experiments. To resolve these conflicts, we present a coarse-grained model to calculate the B cell population development in affinity maturation, which allows a comprehensive analysis of its parameter space to look for optimal values of mutation rate, selection strength, and initial antibody-antigen binding level that maximize the affinity improvement. With these optimized parameters, the model is compatible with the experimental observations such as the ~100-fold affinity improvements, the number of mutations, the hypermutation rate, and the "all or none" phenomenon. Moreover, we study the reasons behind the optimal parameters. The optimal mutation rate, in agreement with the hypermutation rate in vivo, results from a tradeoff between accumulating enough beneficial mutations and avoiding too many deleterious or lethal mutations. The optimal selection strength evolves as a balance between the need for affinity improvement and the requirement to pass the population bottleneck. These findings point to the conclusion that germinal centers have been optimized by evolution to generate strong affinity antibodies effectively and rapidly. In addition, we study the enhancement of affinity improvement due to B cell migration between germinal centers. These results could enhance our understandings to the functions of germinal centers.Comment: 5 figures in main text, and 4 figures in Supplementary Informatio

Study of avidity of antigen-specific antibody as a means of understanding development of long-term immunological memory after Vibrio cholerae O1 infection

The avidity of antibodies to specific antigens and the relationship of avidity to memory B cell responses to these antigens have not been studied in patients with cholera or those receiving oral cholera vaccines. We measured the avidity of antibodies to cholera toxin B subunit (CTB) and Vibrio cholerae O1 lipopolysaccharide (LPS) in Bangladeshi adult cholera patients (n = 30), as well as vaccinees (n = 30) after administration of two doses of a killed oral cholera vaccine. We assessed antibody and memory B cell responses at the acute stage in patients or prior to vaccination in vaccinees and then in follow-up over a year. Both patients and vaccinees mounted CTB-specific IgG and IgA antibodies of high avidity. Patients showed longer persistence of these antibodies than vaccinees, with persistence lasting in patients up to day 270 to 360. The avidity of LPS-specific IgG and IgA antibodies in patients remained elevated up to 180 days of follow-up. Vaccinees mounted highly avid LPS-specific antibodies at day 17 (3 days after the second dose of vaccine), but the avidity waned rapidly to baseline by 30 days. We examined the correlation between antigen-specific memory B cell responses and avidity indices for both antigens. We found that numbers of CTB- and LPS-specific memory B cells significantly correlated with the avidity indices of the corresponding antibodies (P < 0.05; Spearman's ρ = 0.28 to 0.45). These findings suggest that antibody avidity after infection and immunization is a good correlate of the development and maintenance of memory B cell responses to Vibrio cholerae O1 antigens

Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human Natural Killer cells

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions

Structural and Thermodynamic Approach to Peptide Immunogenicity

In the conventional paradigm of humoral immunity, B cells recognize their cognate antigen target in its native form. However, it is well known that relatively unstable peptides bearing only partial structural resemblance to the native protein can trigger antibodies recognizing higher-order structures found in the native protein. On the basis of sound thermodynamic principles, this work reveals that stability of immunogenic proteinlike motifs is a critical parameter rationalizing the diverse humoral immune responses induced by different linear peptide epitopes. In this paradigm, peptides with a minimal amount of stability (ΔGX<0 kcal/mol) around a proteinlike motif (X) are capable of inducing antibodies with similar affinity for both peptide and native protein, more weakly stable peptides (ΔGX>0 kcal/mol) trigger antibodies recognizing full protein but not peptide, and unstable peptides (ΔGX>8 kcal/mol) fail to generate antibodies against either peptide or protein. Immunization experiments involving peptides derived from the autoantigen histidyl-tRNA synthetase verify that selected peptides with varying relative stabilities predicted by molecular dynamics simulations induce antibody responses consistent with this theory. Collectively, these studies provide insight pertinent to the structural basis of immunogenicity and, at the same time, validate this form of thermodynamic and molecular modeling as an approach to probe the development/evolution of humoral immune responses

Modeling of B cell Synapse Formation by Monte Carlo Simulation Shows That Directed Transport of Receptor Molecules Is a Potential Formation Mechanism

The formation of the protein segregation structure known as the “immunological synapse” in the contact region between B cells and antigen presenting cells appears to precede antigen (Ag) uptake by B cells. The mature B cell synapse consists of a central cluster of B cell receptor/Antigen (BCR/Ag) complexes surrounded by a ring of LFA-1/ICAM-1 complexes. In this study, we used an in silico model to investigate whether cytoskeletally driven transport of molecules toward the center of the contact zone is a potential mechanism of immunological synapse formation in B cells. We modeled directed transport by the cytoskeleton in an effective manner, by biasing the diffusion of molecules toward the center of the contact zone. Our results clearly show that biased diffusion of BCR/Ag complexes on the B cell surface is sufficient to produce patterns similar to experimentally observed immunological synapses. This is true even in the presence of significant membrane deformation as a result of receptor–ligand binding, which in previous work we showed had a detrimental effect on synapse formation at high antigen affinity values. Comparison of our model’s results to those of experiments shows that our model produces synapses for realistic length, time, and affinity scales. Our results also show that strong biased diffusion of free molecules has a negative effect on synapse formation by excluding BCR/Ag complexes from the center of the contact zone. However, synapses may still form provided the bias in diffusion of free molecules is an order-of-magnitude weaker than that of BCR/Ag complexes. We also show how diffusion trajectories obtained from single-molecule tracking experiments can generate insight into the mechanism of synapse formation

Dynamics of Macrophage Trogocytosis of Rituximab-Coated B Cells

Macrophages can remove antigen from the surface of antibody-coated cells by a process termed trogocytosis. Using live cell microscopy and flow cytometry, we investigated the dynamics of trogocytosis by RAW264.7 macrophages of Ramos B cells opsonized with the anti-CD20 monoclonal antibody rituximab. Spontaneous and reversible formation of uropods was observed on Ramos cells, and these showed a strong enrichment in rituximab binding. RAW-Ramos conjugate interfaces were highly enriched in rituximab, and transfer of rituximab to the RAW cells in submicron-sized puncta occurred shortly after cell contact. Membrane from the target cells was concomitantly transferred along with rituximab to a variable extent. We established a flow cytometry-based approach to follow the kinetics of transfer and internalization of rituximab. Disruption of actin polymerization nearly eliminated transfer, while blocking phosphatidylinositol 3-kinase activity only resulted in a delay in its acquisition. Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis. The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates. Selective pre-treatment of Ramos cells with phenylarsine oxide blocked uropod formation, reduced enrichment of rituximab at cell-cell interfaces, and reduced the efficiency of trogocytic transfer of rituximab. Our findings highlight that dynamic changes in target cell shape and surface distribution of antigen may significantly influence the progression and extent of trogocytosis. Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies