Haplosufficiency of MHCII in autoreactive germinal center B cells.

Utilizing an autoimmune bone marrow chimera model we determined that B cells depend critically on MHCII expression for participation in the germinal center, but cells displaying a 50% reduction in surface MHCII compete efficiently with their wild-type counterparts. This provides insights into the requirements for germinal center participation.

B cell MHC haplotype affects follicular inclusion, germinal center participation and plasma cell differentiation in a mouse model of lupus.

Introduction: MHC class II molecules are essential for appropriate immune responses against pathogens but are also implicated in pathological responses in autoimmune diseases and transplant rejection. Previous studies have shed light on the systemic contributions of MHC haplotypes to the development and severity of autoimmune diseases. In this study, we addressed the B cell intrinsic MHC haplotype impact on follicular inclusion, germinal center (GC) participation and plasma cell (PC) differentiation in the context of systemic lupus erythematosus (SLE). Methods: We leveraged the 564Igi mouse model which harbors a B cell receptor knock-in from an autoreactive B cell clone recognizing ribonuclear components, including double-stranded DNA (dsDNA). This model recapitulates the central hallmarks of the early stages of SLE. We compared 564Igi heterozygous offspring on either H2b/b, H2b/d, or H2d/d background. Results: This revealed significantly higher germinal center (GC) B cell levels in the spleens of H2b/b and H2b/d as compared to H2d/d (p<0.0001) mice. In agreement with this, anti-dsDNA-antibody levels were higher in H2b/b and H2b/d than in H2d/d (p<0.0001), with H2b/b also being higher compared to H2b/d (p<0.01). Specifically, these differences held true both for autoantibodies derived from the knock-in clone and from wild-type (WT) derived clones. In mixed chimeras where 564Igi H2b/b, H2b/d and H2d/d cells competed head-to-head in the same environment, we observed a significantly higher inclusion of H2b/b cells in GC and PC compartments relative to their representation in the B cell repertoire, compared to H2b/d and H2d/d cells. Furthermore, in mixed chimeras in which WT H2b/b and WT H2d/d cells competed for inclusion in GCs associated with an epitope spreading process, H2b/b cells participated to a greater extent and contributed more robustly to the PC compartment. Finally, immature WT H2b/b cells had a higher baseline of BCRs with an autoreactive idiotype and were subject to more stringent negative selection at the transitional stage. Discussion: Taken together, our findings demonstrate that B cell intrinsic MHC haplotype governs their capacity for participation in the autoreactive response at multiple levels: follicular inclusion, GC participation, and PC output. These findings pinpoint B cells as central contributors to precipitation of autoimmunity.

Antigen presentation by B cells enables epitope spreading across an MHC barrier.

Circumstantial evidence suggests that B cells may instruct T cells to break tolerance. Here, to test this hypothesis, we used a murine model in which a single B cell clone precipitates an autoreactive response resembling systemic lupus erythematosus (SLE). The initiating clone did not need to enter germinal centers to precipitate epitope spreading. Rather, it localized to extrafollicular splenic bridging channels early in the response. Autoantibody produced by the initiating clone was not sufficient to drive the autoreactive response. Subsequent epitope spreading depended on antigen presentation and was compartmentalized by major histocompatibility complex (MHC). B cells carrying two MHC haplotypes could bridge the MHC barrier between B cells that did not share MHC. Thus, B cells directly relay autoreactivity between two separate compartments of MHC-restricted T cells, leading to inclusion of distinct B cell populations in germinal centers. Our findings demonstrate that B cells initiate and propagate the autoimmune response.

Increased maternofoetal transfer of antibodies in a murine model of systemic lupus erythematosus, but no immune activation and neuroimmune sequelae in offspring.

Maternally transferred autoantibodies can negatively impact the development and health of offspring, increasing the risk of neurodevelopmental disorders. We used embryo transfers to examine maternofoetal immune imprinting in the autoimmune BXSB/MpJ mouse model. Anti-double-stranded DNA antibodies and total immunoglobulins were measured, using allotypes of the IgG subclass to distinguish maternally transferred antibodies from those produced endogenously. Frequencies of germinal center and plasma cells were analysed by flow cytometry. Microglial morphology in offspring CNS was assessed using immunohistochemistry. In contrast to prior findings, our results indicate that BXSB/MpJ mothers display a mild autoimmune phenotype, which does not significantly impact the offspring.

Variants of beta-glucan polysaccharides downregulate autoimmune inflammation.

Common infections and polysaccharides, from bacteria and yeasts, could trigger psoriasis and psoriatic arthritis (PsA), and possibly rheumatoid arthritis (RA). The objective of this study was to investigate the effects of ß-glucan polysaccharides in the effector phase of arthritis and as regulators of psoriasis and PsA-like symptoms in mice. Collagen antibody induced arthritis was studied as a model of RA and mannan-induced psoriasis (MIP) was used as model for psoriasis and PsA, using mice with a mutation of Ncf1 on the B10.Q genetic background, making them highly disease susceptible. The mice were exposed to three common variants: 1,6-ß-glucan, 1,3-ß-glucan and 1,3-1,6-ß-glucan. These ß-glucans down-regulated disease in mice if administered simultaneously, before or after mannan. Interestingly, the protection was macrophage mannose receptor (MMR/CD206) dependent with a more pronounced protection long-term than short-term. The number of resident peritoneal macrophages decreased after in vivo challenge with ß-glucan and mannan compared to mannan alone, whereas the numbers of infiltrating cells correspondingly increased, further indicating macrophages as key for ß-glucan mediated regulation. At the doses tested, ß-glucans could not induce arthritis, psoriasis or PsA in wild-type mice. However, ß-glucans could ameliorate the PsA-like symptoms representing a new unforeseen possibility to explore for future clinical treatment.

Antibodies to Cartilage Oligomeric Matrix Protein Are Pathogenic in Mice and May Be Clinically Relevant in Rheumatoid Arthritis.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is an autoantigen in rheumatoid arthritis (RA) and experimental models of arthritis. This study was undertaken to investigate the structure, function, and relevance of anti-COMP antibodies. METHODS: We investigated the pathogenicity of monoclonal anti-COMP antibodies in mice using passive transfer experiments, and we explored the interaction of anti-COMP antibodies with cartilage using immunohistochemical staining. The interaction of the monoclonal antibody 15A11 in complex with its specific COMP epitope P6 was determined by x-ray crystallography. An enzyme-linked immunosorbent assay and a surface plasma resonance technique were used to study the modulation of calcium ion binding to 15A11. The clinical relevance and value of serum IgG specific to the COMP P6 epitope and its citrullinated variants were evaluated in a large Swedish cohort of RA patients. RESULTS: The murine monoclonal anti-COMP antibody 15A11 induced arthritis in naive mice. The crystal structure of the 15A11-P6 complex explained how the antibody could bind to COMP, which can be modulated by calcium ions. Moreover, serum IgG specific to the COMP P6 peptide and its citrullinated variants was detectable at significantly higher levels in RA patients compared to healthy controls and correlated with a higher disease activity score. CONCLUSION: Our findings provide the structural basis for binding a pathogenic anti-COMP antibody to cartilage. The recognized epitope can be citrullinated, and levels of antibodies to this epitope are elevated in RA patients and correlate with higher disease activity, implicating a pathogenic role of anti-COMP antibodies in a subset of RA patients.

B Cell Intrinsic STING Signaling Is Not Required for Autoreactive Germinal Center Participation.

Germinal centers (GCs) are induced microanatomical structures wherein B cells undergo affinity maturation to improve the quality of the antibody response. Although GCs are crucial to appropriate humoral responses to infectious challenges and vaccines, many questions remain about the molecular signals driving B cell participation in GC responses. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an important mediator of type I interferon and proinflammatory cytokine responses during infection and cellular stress. Recent studies have reported important roles for STING in B cell responses, including an impact on GC B cells and downstream antibody responses, which could have great consequences for vaccine design and understanding STING-associated interferonopathies. GCs are also involved in untoward reactions to autoantigens in a plethora of autoimmune disorders, and it is generally thought that these responses coopt the mechanisms used in foreign antigen-directed GCs. Here, we set out to investigate the importance of the cGAS-STING pathway in autoreactive B cell responses. In a direct competition scenario in a murine mixed bone marrow chimera model of autoreactive GCs, we find that B cell intrinsic deficiency of cGAS, STING, or the type I interferon receptor IFNAR, does not impair GC participation, whereas Toll-like receptor (TLR)-7 deficiency mediates a near-complete block. Our findings suggest that physiological B cell responses are strictly sustained by signals linked to BCR-mediated endocytosis. This wiring of B cell signals may enable appropriate antibody responses, while at the same time restricting aberrant antibody responses during infections and in autoimmune or autoinflammatory settings.

Comparison of gamma and x-ray irradiation for myeloablation and establishment of normal and autoimmune syngeneic bone marrow chimeras.

Murine bone marrow (BM) chimeras are a versatile and valuable research tool in stem cell and immunology research. Engraftment of donor BM requires myeloablative conditioning of recipients. The most common method used for mice is ionizing radiation, and Cesium-137 gamma irradiators have been preferred. However, radioactive sources are being out-phased worldwide due to safety concerns, and are most commonly replaced by X-ray sources, creating a need to compare these sources regarding efficiency and potential side effects. Prior research has proven both methods capable of efficiently ablating BM cells and splenocytes in mice, but with moderate differences in resultant donor chimerism across tissues. Here, we compared Cesium-137 to 350 keV X-ray irradiation with respect to immune reconstitution, assaying complete, syngeneic BM chimeras and a mixed chimera model of autoimmune disease. Based on dose titration, we find that both gamma and X-ray irradiation can facilitate a near-complete donor chimerism. Mice subjected to 13 Gy Cesium-137 irradiation and reconstituted with syngeneic donor marrow were viable and displayed high donor chimerism, whereas X-ray irradiated mice all succumbed at 13 Gy. However, a similar degree of chimerism as that obtained following 13 Gy gamma irradiation could be achieved by 11 Gy X-ray irradiation, about 85% relative to the gamma dose. In the mixed chimera model of autoimmune disease, we found that a similar autoimmune phenotype could be achieved irrespective of irradiation source used. It is thus possible to compare data generated, regardless of the irradiation source, but every setup and application likely needs individual optimization.

T follicular regulatory cells: Guardians of the germinal centre?

It is a central tenet of the clonal selection theory, that lymphocyte repertoires are tolerized to self-antigens during their ontogeny. Germinal centres are the sites in secondary lymphoid tissues where B cells undergo affinity maturation and class-switching to produce high-affinity antibodies. This process is crucial, both in our ability to mount protective humoral responses to infections and to vaccinations, but it is also involved in untoward reactions to self-antigens, which underlie autoimmunity. The process of affinity maturation poses a significant challenge to tolerance, as the random nature of somatic hypermutation can introduce novel reactivities. Therefore, it has been a long-standing idea that mechanisms must exist which limit the emergence of autoreactivity at the germinal centre level. One of these mechanisms is the requirement for linked recognition, which imposes on B cells a dependence on centrally tolerant T follicular helper cells. However, as linked recognition can be bypassed by adduct formation of autoantigenic complexes, it has been an appealing notion that there should be an additional layer of dominant mechanisms regulating emergence of autoreactive specificities. About a decade ago, this notion was addressed by the discovery of a novel subset of T regulatory cells localizing to the germinal centre and regulating germinal centre B-cell responses. Here, we detail the progress that has been made towards characterizing this T follicular regulatory cell subset and understanding the functions of these 'guardians of the germinal centre'.