Thyrotrophin receptor-specific memory T cell responses require normal B cells in a murine model of Graves' disease

Abstract

The role of B cells as antigen-presenting cells is being recognized increasingly in immune responses to infections and autoimmunity. We compared T cell responses in wild-type and B cell-deficient mice immunized with the thyrotrophin receptor (TSHR), the major autoantigen in Graves' disease. Three B cell-deficient mouse strains were studied: JHD (no B cells), mIgM (membrane-bound monoclonal IgM+ B cells) and (m + s)IgM (membrane-bound and secreted monoclonal IgM). Wild-type and B cell-deficient mice (BALB/c background) were studied 8 weeks after three injections of TSHR or control adenovirus. Only wild-type mice developed IgG class TSHR antibodies and hyperthyroidism. After challenge with TSHR antigen, splenocyte cultures were tested for cytokine production. Splenocytes from TSHR adenovirus injected wild-type and mIgM-mice, but not from JHD- or (m + s)IgM- mice, produced interferon (IFN)-gamma in response to TSHR protein. Concanavalin A and pokeweed mitogen induced comparable IFN-gamma secretion in all groups of mice except in the JHD strain in which responses were reduced. The absence in (m + s)IgM mice and presence in mIgM mice of an anamnestic response to TSHR antigen was unrelated to lymphoid cell types. Surprisingly, although TSHR-specific antibodies were undetectable, low levels of serum IgG were present in mIgM- but not (m + s)IgM mice. Moreover, IFN-gamma production by antigen-stimulated splenocytes correlated with IgG levels. In conclusion, T cell responses to TSHR antigen developed only in mice with IgG-secreting B cells. Consequently, in the TSHR-adenovirus model of Graves' disease, some normal B cells appear to be required for the development of memory T cells.

Fig. 1

Serum IgM and IgG in wild-type and three strains of B cell-deficient mice, all on the BALB/c background. Mice were studied at euthanasia 8 weeks after three injections of TSHR– or control– adenovirus (Ad–TSHR or Ad–Con, respectively). Seva were analysed by ELISA for IgM (a) and IgG (b). The data are expressed as the mean + s.e.m. µg/ml IgG or IgM; the number of mice in each group is indicated in parentheses. *Values significantly higher in Ad–TSHR immunized mIgM mice than in Ad–TSHR immunized (m + s)IgM mice (P = 0·007, t-test).

Fig. 2

B cells, T cells and macrophages in B cell-deficient and wild-type BALB/c mice. Splenocytes from mice euthanized 8 weeks after three injections of TSHR– or control–adenovirus (Ad–TSHR or Ad–Con, respectively) were analysed by flow cytometry using anti-CD19, anti-IgM, anti-CD3, anti-CD4 and anti-CD11b. Positive cells (%) are shown as the mean + s.d. for two mice of each type, namely J_HD, mIgM (m + s)IgM and wild-type.

Fig. 3

TSHR antibodies in B-cell deficient and wild-type BALB/c mice determined by ELISA and by inhibition of TSH binding to the TSHR (TBI). (a) and (b) Binding to the TSHR A subunit in ELISA (sera diluted 1 : 100) for antibodies of IgM class (a) and IgG class (b). (c) TBI activity (25 µl undiluted serum) is reported as percentage inhibition. Data are shown as the mean + s.e.m. with the number of mice in each group indicated in parentheses. In (c), the vertical dotted line is the mean + 2 s.d. for wild-type mice immunized with control adenovirus.

Fig. 4

Splenocytes from mIgM- and wild-type mice, but not (m + s)IgM- or J_HD- mice, respond to in vitro challenge with TSHR–antigen. Spleen cells from mice euthanized 8 weeks after three injections of TSHR– (or control–) adenovirus were cultured for 6 days with TSHR protein and supernatants were analysed for IFN-γ. Data are reported as net IFN-γ production (pg/ml) after subtracting values for culture medium alone. Responses are shown for individual mice (open or solid circles) and as the mean + s.e.m. for each group with the number of animals in parentheses). *Values significantly greater in mIgM- than (m + s)IgM- mice injected with Ad–TSHR (P < 0·003, Mann–Whitney rank sum test).

Fig. 5

Normal mitogenic responses in mIgM and (m + s)IgM mice but not in J_HD mice. Splenocytes from mice euthanized 8 weeks after three injections of TSHR– (or control–) adenovirus were cultured with Concanavalin A (ConA) or pokeweed mitogen (PWM). Data are reported as IFN-γ production (pg/ml, mean + s.e.m., with the number of mice in each group in parentheses). **Values significantly different between J_HD mice and all other groups of mice (P < 0·05, anova on ranks, pairwise comparison by Bonferroni t-test; *values significantly different between J_HD mice and wild-type mice (P < 0·05, anova on ranks; pairwise comparison by Dunn's test).

Fig. 6

Splenocyte response to TSHR–antigen in culture is associated with ‘leaky’ production of serum IgG in mIgM- but not (m + s)IgM- mice. IFN-γ produced in response to TSHR–antigen (log₁₀ net pg/ml) correlates with serum IgM (µg/ml). The individual values for mIgM⁻ and (m + s)IgM⁻ mice are shown together with the regression line and 95% confidence limits.