B Cell-Specific MHC Class II Deletion Reveals Multiple Nonredundant Roles for B Cell Antigen Presentation in Murine Lupus

Abstract

B cells have both Ab-dependent and Ab-independent functions in systemic autoimmune diseases, including systemic lupus erythematosus (SLE). Ab-independent functions are known to be important, because mice with B cells but no secreted Ig have severe disease. These functions could include roles in lymphoid development, cytokine secretion, and Ag presentation; however, these possibilities have not been directly tested in SLE models. In this study, we show by lineage-specific ablation of MHC class II (MHCII) that B cell Ag presentation plays a nonredundant role in CD4(+) T cell activation and effector differentiation in the MRL.Fas(lpr) mouse model of SLE. MHCII-mediated interactions between B and T cells further promote B cell proliferation and differentiation, and, in fact, inefficient MHCII deletion on B cells led to strong selection of escaped cells in activated and plasmablast compartments, further underscoring the central role of B cell Ag presentation. Despite the leakiness in the system, B cell-specific MHCII deletion resulted in substantially ameliorated clinical disease. Hence, B cell Ag presentation is critical for T and B cell activation and differentiation, as well as target organ damage.

Figure 1. Deletion of MHCII in B cells

(A) Representative histograms of MHCII staining of splenic B cells from control, MHCII^fl/fl, and CD19-Cre MHCII^fl/fl mice. Cells were first gated as EMA⁻ TCRβ⁻, then CD19^HiCD138⁻. (B) Frequency of MHCII⁺ splenic B cells, as identified as in (A), in the experimental cohorts. Data are pooled from 6 independent cohorts of 12 week old mice; control n=30 and CD19-Cre n=32. Data are represented as mean +/− SEM. Statistics were calculated by two-tailed Mann-Whitney test. ****p < 0.0001.

Figure 2. Selective differentiation of residual MHCII⁺ B cells in CD19-Cre mice

(A) FACS strategy to identify naive B cells, activated B cells, and plasmablasts. The cells were first gated as EMA⁻ TCRβ⁻ CD11c⁻ CD11b⁻. (B) Deletion efficiency of MHCII in the three sorted cell populations was determined by qRT-PCR. Deletion efficiency was calculated with the equation (1-residual MHCII) × 100. Residual MHCII was calculated as 2^−ΔΔCt. Data represent a total of 9 CD19-Cre mice from 2 independent experiments; each dot is an individual mouse. Horizontal bars mark the mean. (C) Representative staining plots of MHCII staining on total B cells (EMA⁻ TCRβ⁻CD22⁺) and GC B cells (EMA⁻ TCRβ⁻CD22⁺ CD38^Int PNA⁺) from one CD19-Cre mouse. (D) Summary data are represented as mean +/− SEM. Data are pooled from 2 independent cohorts of 12 week old mice; control n=16 and CD19-Cre n=16. Statistics were calculated by two-tailed Mann-Whitney test. **p < 0.01; ****p < 0.0001.

Figure 3. MHCII⁺ B cells have a significant proliferation advantage

One hour before sacrifice, mice were injected with 1mg of BrdU i.p. (A) Splenic B cells (EMA⁻ TCRβ⁻CD22⁺) were evaluated for BrdU incorporation by flow cytometry. Data are represented as mean +/− SEM. (B) Representative staining plot of MHCII expression and BrdU incorporation of B cells from a CD19-Cre mouse. The bar graph represents the percent of MHCII⁺ B cells within the BrdU⁻ and BrdU⁺ B cell populations as the mean +/− SEM. (C) Plasmablasts (EMA⁻ TCRβ⁻CD22^IntCD138⁺) were evaluated for BrdU incorporation by flow cytometry. Data are represented as mean +/− SEM. Data are pooled from 2 independent experiments; control n=21, CD19-Cre n=20. Statistics were calculated by two-tailed Mann-Whitney test. ****p < 0.0001.

Figure 4. Expression of MHCII is critical for plasmablast and GC B cell differentiation

(A) Representative staining plots of plasmablast and B cell gating. Cells were first gated as EMA⁻ TCRβ⁻. (B) Total number of B cells and (C) plasmablasts per spleen determined by flow cytometry. Data are represented as mean +/− SEM. (D) Isotype switched plasmablasts (IgM⁻) were normalized to the non-switched (IgM⁺). Data are represented as mean +/− SEM. Data are pooled from 6 independent cohorts of 12 week old mice; control n=30 and CD19-Cre n=32. (E) Representative staining plots of GC B cell gating from one control mouse and one CD19-Cre mouse. Cells were first gated on EMA⁻ TCRβ⁻CD22⁺. (F) Total number of GC B cells per spleen identified as in (E). Data are represented as mean +/− SEM. Data are pooled from 2 independent cohorts of 12 week old mice; control n=26 and CD19-Cre n=16. Statistics were calculated by two-tailed Mann-Whitney test. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5. Isotype switch in antibody-forming cells is dependent on MHCII

(A) Total numbers of AFCs per spleen were determined by ELISpot for IgM, IgG2a, and IgG1. Each dot is an individual mouse. Bars show geometric means. Data are pooled from 6 independent cohorts of 12 week old mice; control n=29 and CD19-Cre n=32. (B) Serum immunoglobulin concentrations determined by multiplex ELISAs. Data are represented as mean +/− SEM. Data are pooled from 7 independent cohorts of 12 week old mice; control n=35 and CD19-Cre n=32. Statistics were calculated by two-tailed Mann-Whitney test. *p < 0.05; ****p < 0.0001.

Figure 6. Specific autoantibodies differ in their dependence on B cell MHCII expression

(A) Serum concentration of anti-nucleosome IgM and IgG, (B) anti-RNA IgM and IgG, and (C) anti-Sm IgM and IgG were determined by ELISA. Bars show geometric means. Data are pooled from 7 independent cohorts of 12 week old mice; control n=35 and CD19-Cre n=39. Statistics were calculated by two-tailed Mann-Whitney test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7. B cell antigen presentation plays a non-redundant role in T cell activation and differentation

(A) Total numbers of CD4, CD8, DN, and T cells enumerated by flow cytometry. Frequency of naive (CD44⁻CD62L⁺) and activated (CD44⁺CD62L⁻) (B) CD4, and (C) CD8 T cells determined by flow cytometry. Representative intracellular IFN-γ staining histograms and numbers of (D) CD4 and (E) CD8 T cells stimulated with PMA and ionomycin. Control mice are shown in bold. Data are represented as mean +/− SEM. Data are pooled from 6 independent cohorts of 12 week old mice; control n=30 and CD19-Cre n=32. Statistics were calculated by two-tailed Mann-Whitney test. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 8. T extrafollicular helper cells are decreased in CD19-Cre mice

(A) Representative staining plots of T_EFH gating. Cells are first gated on EMA⁻ CD19⁻. Percentage is the mean of T_EFH of CD4 T cells in CD19-Cre mice. (B) The number of T_EFH cells per spleen as identified in (A). Data are pooled from 2 independent cohorts; control n=9, CD19-Cre n=12. (C) Representative intracellular IL-21 staining plots of CD4 T cells unstimulated (left) and stimulated with PMA and ionomycin (right). The bar graphs represent numbers. Data are pooled from 2 independent cohorts of 12 week old mice; control n=15 and CD19-Cre n=10. Statistics were calculated by two-tailed Mann-Whitney test. *p < 0.05.

Figure 9. Deletion of MHCII on B cells ameliorates clinical disease

(A) Glomerular and interstitial nephritis (GN and IN) were scored from 0 to 6. (B) Proteinuria was scored from 0 to 5. Each dot represents an individual mouse. Horizontal lines represent the medians. (C) Weights of combined axillary lymph nodes and (D) spleen were measured. Data are represented as median +/− SEM. Data are pooled from 6 independent cohorts of 12 week old mice; control n=30 and CD19-Cre n=32. Statistics were calculated by two-tailed Mann-Whitney test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 10. Activation and pathogenesis in systemic autoimmunity

(1) Autoreactive B cells, such as those specific for DNA, become activated through engagement of their BCR and TLRs in a MyD88-dependent manner. (2) Activated autoreactive B cells present antigen on MHCII and upregulate costimulatory molecules, resulting in the activation of cognate autoreactive CD4 T cells. (3) B cells and DCs promote subsequent differentation into T_H1 and T_EF cells. (4) Cognate activated T cells provide help such as CD40 ligation and cytokines, resulting in significant expansion and isotype switch of the plasmablasts. (5) Activated autoreactive B cells promote epitope spreading and expansion of the anti-self response. B cells have the potential to present anything that is endocytosed through their BCR, not only the protein sequences recognized by the CDR3 region. (6) Activated T cells migrate and infiltrate target tissues, such as the kidneys, in a DC-dependent manner. These T cells may activate resident or migratory DCs through CD40-CD40L ligation which induces ICOSL expression on the DCs. DC-expressed ICOSL promotes kidney damage.