Autoreactive thymic B cells are efficient antigen-presenting cells of cognate self-antigens for T cell negative selection

Abstract

The thymus contains a population of B cells that colocalize with dendritic cells and medullary thymic epithelial cells in the thymic medulla. The development and functional significance of these cells are largely unknown. Using recombination-activating gene 2 GFP reporter mice along with parabiosis experiments, we demonstrate that the vast majority of thymic B cells develop from progenitors within the thymus. Thymic B cells express unique phenotypic markers compared with peripheral B cells; particularly they express high levels of MHC class II, suggesting that they are poised to present self-antigens efficiently. Using Ig knock-in and T-cell receptor transgenic mice specific for the self-antigen glucose-6-phosphate isomerase, we show that autoreactive thymic B cells serve as efficient antigen-presenting cells for T cell negative selection even when they are present at low frequencies. Furthermore, the endogenous thymic B-cell repertoire also functions in this capacity. These results suggest that developing thymic B cells could efficiently capture a broad array of autoantigens through their B-cell receptors, presenting peptides derived from those autoantigens to developing thymocytes and eliminating cognate T cells.

Fig. 1.

B-cell development in the thymus. (A) Costaining of B220 and GFP in CD4/CD8 depleted thymi of Rag2-GFP⁻ (B6) and Rag2-GFP⁺ mice. (B) Characterization of three B-cell populations defined in A: B220^hiGFP⁻ (black line), B220^hiGFP^lo (blue line), B220^loGFP⁺ cells (red line), and B220⁻ thymocytes (gray-filled) for all panels except CD43 where B220⁺ splenocytes (gray-filled) are shown. (C) BrdU incorporation in thymic B cells with continuous labeling for the indicated duration. n = 3–5 mice for each time point.

Fig. 2.

Analysis of thymic B-cell recirculation by parabiosis. A CD45.1⁺ mouse was joined to a CD45.2⁺ partner for 6–8 wk. The percent of host-derived cells in each population, BM immature B cell (B220^loIgM⁻), thymus B-cell progenitor (CD19^loB220^lo), thymus mature B cell (CD19^hiB220^hi), and spleen mature B cell (CD19⁺B220⁺) was determined by CD45.1 and CD45.2 expression. Each symbol represents an individual mouse.

Fig. 3.

Reconstitution of thymic B cells by fetal liver and bone marrow cells in Rag^−/− host. (A) Representative FACS plots in the thymus (gated on lymphocytes) and peritoneal cavity (gated on CD19⁺B220⁺ cells). (B) Quantitation of B cells as a percentage of total cells in the thymus and spleen. n = 4 mice per group.

Fig. 4.

Comparison of B-cell markers and costimulatory molecules between thymic and splenic B cells. Representative flow plots of IgM and IgD (A), B220 and AA4.1 (B), CD21 and HSA (C), CD21 and CD23 (D), and MHC class II and costimulatory markers (E) on thymic B cells (black) relative to bulk splenic B cells (gray). Average median fluorescence intensities and SDs are listed for each marker, n = 3–5 mice per group, representative of three independent experiments. Gated on CD19⁺B220⁺ cells.

Fig. 5.

Development of autoreactive B cells in the thymus. (A) Expression of MHC class II, IgM, and CD69 in 121⁺ (black) and 121⁻ (gray) B cells. Gated on CD19⁺ B cells. Representative of five mice. (B) Quantification of the percentage of 121⁺ as detected in A in the spleen versus the thymus over time. Each symbol represents an individual mouse. Asterisks represent significant difference between thymus and spleen at the time point assayed.

Fig. 6.

Increased negative selection with enhanced thymic B-cell presentation of GPI in KRN TCR transgenic mice. (A) Thymic cellularity, DP/DN ratio, and number of CD4 single positive cells of KRN TCR⁺ mice expressing the A^g7 MHC, with either a WT B-cell repertoire (K/g7), the heavy chain of the 121 BCR (K/H/g7) or the heavy and light chain of the 121 BCR (K/HL/g7). (B) Representative FACS plot of Nur77-GFP induction in KRN-TCR DP thymocytes (maintained on B6 background) by thymic B cells of B6, K/g7, or K/HL/g7 mice. Gated on Thy1⁺ DP cells. (C) Quantification of GFP+ DP thymocytes after culturing with various APCs. n = 3–6 mice per group from five independent experiments.

Fig. 7.

Decreased negative selection of KRN TCR in mice lacking B cells. (A) Absence of mature thymic B cells in K/BxN mice on μMT^+/− or μMT^−/− background. (B) Thymic cellularity, DP/DN ratio, and number of CD4 SP thymocytes in μMT^+/− and μMT^−/− littermates analyzed at 4 wk of age (n = 11 and 14, respectively).