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.

HIF1A Knockout by Biallelic and Selection-Free CRISPR Gene Editing in Human Primary Endothelial Cells with Ribonucleoprotein Complexes.

Primary endothelial cells (ECs), especially human umbilical vein endothelial cells (HUVECs), are broadly used in vascular biology. Gene editing of primary endothelial cells is known to be challenging, due to the low DNA transfection efficiency and the limited proliferation capacity of ECs. We report the establishment of a highly efficient and selection-free CRISPR gene editing approach for primary endothelial cells (HUVECs) with ribonucleoprotein (RNP) complex. We first optimized an efficient and cost-effective protocol for messenger RNA (mRNA) delivery into primary HUVECs by nucleofection. Nearly 100% transfection efficiency of HUVECs was achieved with EGFP mRNA. Using this optimized DNA-free approach, we tested RNP-mediated CRISPR gene editing of primary HUVECs with three different gRNAs targeting the HIF1A gene. We achieved highly efficient (98%) and biallelic HIF1A knockout in HUVECs without selection. The effects of HIF1A knockout on ECs' angiogenic characteristics and response to hypoxia were validated by functional assays. Our work provides a simple method for highly efficient gene editing of primary endothelial cells (HUVECs) in studies and manipulations of ECs functions.

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.