Kai-Michael Toellner

IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

Extrafollicular antibody responses

Summary:  In adaptive antibody responses, B cells are induced to grow either in follicles where they form germinal centers or in extrafollicular foci as plasmablasts. Extrafollicular growth typically occurs in the medullary cords of lymph nodes and in foci in the red pulp of the spleen. It is not a feature of secondary lymphoid tissue associated with the internal epithelia of the body. All types of naïve and memory B cells can be recruited into extrafollicular responses. These responses are associated with immunoglobulin class switching but, at the most, only low‐level hypermutation.

The changing preference of T and B cells for partners as T-dependent antibody responses develop

Summary: Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T‐cell priming starts when processed of interdigitating dendritic cells. T‐cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move of the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class swith‐recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones. B cells both B and T cells; much of this growth occurs outside the T zones. B cells migrate to follicles. Where they form germinal centres, and to extrafollicular sites of B‐cell growth, where they differentiate into mainly short‐lived plasma cells. T cells do not move to the extrafollicular foci but to the follicles; there they proliferate and are subsequently involved in the selection of B cell that have mutated their Ig variable‐region genes. During primary antibody responses T‐cell proliferation in follicles produces many times the peak number of T cell found in that site; a substantial proportion of the CD4+ memory T‐cell pool may originate from growth in follicles.

Low-level Hypermutation in T Cell–independent Germinal Centers Compared with High Mutation Rates Associated with T Cell–dependent Germinal Centers

Exceptionally germinal center formation can be induced without T cell help by polysaccharide-based antigens, but these germinal centers involute by massive B cell apoptosis at the time centrocyte selection starts. This study investigates whether B cells in germinal centers induced by the T cell–independent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to Ficoll undergo hypermutation in their immunoglobulin V region genes. Positive controls are provided by comparing germinal centers at the same stage of development in carrier-primed mice immunized with a T cell–dependent antigen: NP protein conjugate. False positive results from background germinal centers and false negatives from non-B cells in germinal centers were avoided by transferring B cells with a transgenic B cell receptor into congenic controls not carrying the transgene. By 4 d after immunization, hypermutation was well advanced in the T cell–dependent germinal centers. By contrast, the mutation rate for T cell–independent germinal centers was low, but significantly higher than in NP-specific B cells from nonimmunized transgenic mice. Interestingly, a similar rate of mutation was seen in extrafollicular plasma cells at this stage. It is concluded that efficient activation of hypermutation depends on interaction with T cells, but some hypermutation may be induced without such signals, even outside germinal centers.

Salmonella Induces a Switched Antibody Response without Germinal Centers That Impedes the Extracellular Spread of Infection

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.

Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes

During T cell‐dependent antibody responses lymph node B cells differentiate either to plasmablasts that grow in the medullary cords, or to blasts that proliferate in follicles forming germinal centers. Many plasmablasts differentiate to plasma cells locally, but some leave the medullary cords and migrate to downstream lymph nodes. To assess the basis for this migration, changes in the responsiveness of B cells to a range of chemokines have been studied as they differentiate. Naive B cells express high levels of CCR6, CCR7, CXCR4 and CXCR5. When activated B cells grow in follicles the expression of these chemokine receptors and the responsiveness to the respective chemokines is retained. During the extrafollicular response, plasmablast expression of CXCR5 and responsiveness to B‐lymphocyte chemoattractant (CXCR5) as well as to secondary lymphoid tissue chemokine (CCR7) and stromal cell‐derived factor (SDF)‐1 (CXCR4) are lost while a weak response towards the CCR6 chemokine LARC is maintained. Despite losing responsiveness to SDF‐1, extrafollicular plasmablasts still express high levels of CXCR4 on the cell surface. These results suggest that the combined loss of chemokine receptor expression and of chemokine responsiveness may be a necessary prerequisite for cells to migrate to the medullary cords and subsequently enter the efferent lymph.

Germinal center B cells govern their own fate via antibody feedback

High-affinity antibodies reenter germinal centers (GCs) and limit antigen access, thus causing sustained directional evolution in GCs toward higher-affinity antibody production.

Responses to the soluble flagellar protein FliC are Th2, while those to FliC on Salmonella are Th1

Features of the Th1 or Th2 phenotype start to develop during CD4 T cell priming. This study of the response to the bacterial flagellar protein FliC shows that either Th1 or Th2 responses can be induced in mice depending upon how FliC is presented. This is shown by assessing the cytokine mRNA and class of FliC‐specific plasma cells induced in situ. Soluble recombinant (r)FliC and polymerized FliC are strongly Th2 polarizing, inducing IL‐4, NIP45 and c‐Maf mRNA as well as ϵ and γ1 switch transcripts and switching to IgG1. CD28‐requirement for this switching shows its T cell dependence. rFliC was unable to induce markers of Th1 activity including IL‐12, T‐bet and IFN‐γ. Conversely, when FliC is presented in its native context surface‐bound on live, flagellated Salmonella, switching is predominantly to IgG2a (IgG2c in C57BL/6 mice), reflecting Th1 activity. The development of divergent FliC‐specific polarization to either Th1 or Th2 indicates that the context in which this antigen is encountered rather than its intrinsic immunostimulatory properties determines the direction of Th polarization.

T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci

During the primary splenic response to the T‐independent type 2 (TI‐2) antigen (4‐hydroxy‐3‐nitrophenyl) acetyl (NP)‐Ficoll, small numbers of antigen‐specific B cells have entered S phase of the cell cycle 24 h after intraperitoneal immunization. These are distributed in all splenic compartments  (T zones, marginal zones, follicles, and red pulp), indicating early proliferation induced by NP‐Ficoll does not require accessory signals delivered in a particular splenic microenvironment. Subsequently B blasts accumulate selectively in the outer T zone areas, but exponential growth leading to plasma cell production occurs only in extra‐follicular foci. This growth peaks after 5 days, but 20 % of peak numbers of antibody‐containing cells are still present 3 months after immunization and 9 % of these cells are proliferating. It is unclear if these late plasmablasts are sustained by self‐renewal or continued recruitment of virgin cells into the response. Unlike TD and TI‐1 responses NP‐specific memory cells do not accumulate in the splenic marginal zones. The level of Cγ3 switch transcripts increases during the first 24 h of the response, but does not increase further during exponential plasmablast growth.

Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis

Objectives In rheumatoid arthritis (RA), a complex cytokine network drives chronic inflammation and joint destruction. So far, few attempts have been made to identify the cellular sources of individual cytokines systematically. Therefore, the primary objective of this study was systematically to assess the cytokine messenger RNA expression profiles in the five largest cell populations in the synovial fluid and peripheral blood of RA patients. To reflect the in vivo situation as closely as possible, the cells were neither cultured nor stimulated ex vivo. Methods Inflammatory cells from 12 RA patients were sorted into CD4 and CD8 T cells, B cells, macrophages and neutrophils. mRNA expression for 41 cytokines was determined by real-time PCR using microfluidic cards. Receptor activator nuclear factor kappa B ligand (RANKL) (TNFSF11) expression by B cells was further confirmed by flow cytometry and by immunofluorescence staining of frozen sections of synovial tissue from patients with RA. Results The detection of cytokines characteristic for T cells and myeloid cells in the expected populations validated this methodology. Beyond the expected cytokine patterns, novel observations were made. Striking among these was the high expression of mRNA for RANKL in B cells from synovial fluid. This observation was validated at the protein level in synovial tissue and fluid. Conclusions RANKL, the key cytokine driving bone destruction by osteoclast activation, is produced by synovial B cells in RA. This observation is of importance for our understanding of the role of B cells in RA and their therapeutic targeting.