T cell-independent rescue of B lymphocytes from peripheral immune tolerance

Abstract

Autoimmunity arises when immune tolerance to specific self-antigens is broken. The mechanisms leading to such a failure remain poorly understood. One hypothesis proposes that infectious agents or antigens can break B or T lymphocyte self-tolerance by expressing epitopes that mimic self. Using a transgenic immunoglobulin model, we show that challenge with self-mimicking foreign antigen rescues B cells from peripheral tolerance independent of T cell help, resulting in the accumulation of self-reactive cells in the lymph nodes and secretion of immunoglobulins that bind to a liver-expressed self-antigen. Therefore, our studies reveal a potentially important mechanism by which B lymphocytes can escape self-tolerance.

Fig. 1

Bacteriophage P31 immunization induces a strong immune response in 3-83 nondeletor mice (ND) and breaks tolerance in 3-83 peripheral deletor (PD) mice. (A) In vivo deletion of B cells reactive to liver-expressed K^b antigen. Bone marrow, spleen, and lymph node lymphocytes of B10.D2 (WT), 3-83 nondeletor (3-83 ND), and (3-83 × pAlbumin-K^b) F₁ mice (3-83 PD) were analyzed by two-color flow cytometry. Staining was done with anti-IgM and rat 3-83 idiotype antibody (54.1). (B) ND and PD mice were immunized on day 0 with 300 μg of wild-type bacteriophage (Pwt) or a phage that binds to the 3-83 BCR (P31) in PBS, or with PBS alone. After the mice were bled on the indicated days, the sera were analyzed for 3-83 Ig levels by solid-phase enzyme-linked immunosorbent assay (ELISA). Data represent mean ± SD of six independent experiments. (C) 3-83 PD mice were challenged with PBS or the indicated phage immunogens; after 24 hours, mesenteric lymph node cells were stained and analyzed as in (A).

Fig. 2

Tolerance breakdown results in massive IgM accumulation in the liver. (A) Frozen sections of livers taken 4 days after immunization were stained with FITC-conjugated anti-IgM or control antibody (rat IgG2a, κ). Photos were taken at 200× magnification with 5-s exposure. (B) Similar staining as in (A), showing clusters of B cells; photos were taken at 1-s exposure. This exposure preferentially reveals antibody-forming cells. The two lower panels are images from different mice.

Fig. 3

B lymphopoiesis inhibition does not affect P31-induced tolerance breakdown. (A) Experimental design. Mice were injected every third day with 1.2 mg of anti–IL-7 ip as indicated (downward arrows). Phage injection was on day 9, and analyses of antibody secretion and cell surface markers were done on day 13. (B) Anti-IgM and anti-B220 staining and flow cytometry analysis of the bone marrow of nontransgenic B10.D2 (WT), untreated 3-83 PD, and anti–IL-7–treated 3-83 PD mice taken at day 13. Gate 1, IgM⁺B220⁺ recirculating B cells; gate 2, IgM⁺B220^low immature B cells; gate 3, IgM⁻B220^low pro- and pre-B cells. (C and D) 3-83 idiotype expression levels on splenic B220⁺IgM⁺ cells of the indicated PBS-injected mice at day 13. Relative to ND B cells, PD cells have reduced idiotype levels and anti–IL-7–treated PD cells have very low idiotype levels. Three-color analysis (D) shows IgM and idiotype densities on B220⁺ gated spleen cells. (E) Sera of anti–IL-7–treated and/or immunized 3-83 PD mice were analyzed by ELISA for 3-83 Ig levels. Each symbol represents a value obtained with an individual mouse.

Fig. 4

The absence of T cells does not alter B cell tolerance breakdown induced upon bacteriophage immunization. (A) 3-83 PD RAG-1^−/− mice were immunized three times on days −6, −3, and 0, then analyzed on day 4 for serum level of 3-83 IgM. Each symbol represents an individual mouse. Similar results were obtained with mice injected only on day 0 (not shown). (B) 3-83 PD mice were injected with 0.5 mg of anti-CD4 ip on days −2 and −1 and immunized with phage at day 0. Spleen cells were stained with anti-CD4 and anti-CD8 on days 0 or 4 (1 or 5 days after anti-CD4 injection) to reveal the extent of T cell depletion. (C) At days 0 and 4, sera of anti-CD4–treated and immunized 3-83 PD mice were analyzed for 3-83 Ig level by ELISA.