Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II

Abstract

Injection of AKR/N mice with fibroblasts co-expressing MHC class II and TPO in the absence of adjuvant induces IgG-class TPO antibodies that resemble spontaneously arising human thyroid autoantibodies. We have used this model to examine the effect of iodide on TPO antibody induction as well as to analyse the interaction between T and B cells. Despite its importance as a major environmental factor in thyroid autoimmunity, variable iodide intake had no detectable effects on TPO antibody levels, lymphocytic infiltration of the thyroid or thyroid hormone levels. In terms of T cell responsiveness, splenocytes from TPO fibroblast-injected mice, but not from control mice, proliferated in response to TPO. Intriguingly, B cell-depleted splenocytes (mainly T cells without reduction of macrophages) proliferated in response to TPO only when co-cultured with irradiated autologous splenocytes from TPO fibroblast-injected mice but not from control mice. These data suggest that TPO-specific B cells are involved in antigen presentation to sensitized T cells and are supported by the ability of spleen cells from TPO cell-injected (but not control) mice to secrete TPO antibodies spontaneously in culture. In conclusion, we provide the first evidence for the presence of thyroid autoantigen-specific B cells and their ability to present their autoantigen to sensitized T cells in mice induced to develop TPO antibodies resembling autoantibodies in humans.

Fig. 1

Protocol to investigate the effect of variable iodide intake on AKR/N mice that were untreated or injected on six occasions at 2-weekly intervals with viable, mitomycin C-treated TPO⁺ class II⁺ RT4.15HP fibroblasts. Lo I, Low iodide chow (see Materials and Methods) for 10 days plus 0.1% methimazole in drinking water; low iodide chow plus normal water for 2 days; Hi I, normal chow plus 0.05% potassium iodide in the drinking water for 2 days.

Fig. 2

Iodide intake has no effect on TPO antibody titres in AKR/N mice injected with TPO⁺ class II⁺ RT4.15HP fibroblasts. Serial dilutions (1:20–1:5000) of TPO antibody binding in mice on a normal iodide diet (a) and on a variable iodide diet (b). Binding values are corrected for background binding by normal mouse serum (approx. 3%, see Materials and Methods).

Fig. 3

Splenocytes from AKR/N mice injected with TPO⁺ class II⁺ fibroblasts, unlike splenocytes from uninjected mice or most mice on variable iodide intake, proliferate in response to TPO in vitro. (a,b) ³H-TdR incorporation by splenocytes incubated for 5 days with increasing concentrations of TPO in conventional cultures (a) and co-cultures containing irradiated autologous splenocytes (b). Data are shown as ct/min (mean + s.e.m.) for splenocytes from individual mice (indicated by ‘a’, ‘b’, etc.) previously injected with TPO⁺ cells (n = 4, (a); n = 5, (b)), mice exposed to variable iodide intake (n = 5, (b)) or untreated mice on normal iodide intake (n = 4, (b)). The dashed line in (b) indicates the mean + 2 s.d. of thymidine incorporation by irradiated splenocytes used in co-cultures. Values significantly different from the response in medium without TPO: *P < 0.05; **P < 0.01 (Student's t-test). (c) Stimulation indices (SI; mean ± s.e.m.) for the response to TPO by splenocytes from mice injected with TPO⁺ class II⁺ cells (n = 9) and from uninjected mice (n = 9). Values for spleen cells from injected mice significantly higher than those for spleen cells from uninjected mice cultured with the same concentrations of TPO: *P < 0.019; Mann–Whitney rank sum test).

Fig. 4

Removal of B220⁺ cells from spleen cells depletes B cells and enriches for T cells and macrophages. Spleen cells were analysed by flow cytometry before and after removal of cells labelled with biotinylated anti-B220 using streptavidin-coated beads. B cells, T cells and macrophages were labelled with biotinylated antibodies (anti-B220, anti-CD3ε and anti-Mac-1α, respectively) and detected with streptavidin–FITC (Materials and Methods).

Fig. 5

Splenic T cell proliferation in response to TPO requires irradiated ‘feeder’ splenocytes from TPO⁺ class II⁺ fibroblast-injected mice. (a) Protocol for studying proliferation to TPO by B-depleted spleen cells containing T cells and macrophages (‘Responders’). These cells were co-cultured with irradiated unseparated spleen cells from TPO fibroblast-injected mice or from control mice. (b,c) ³H-TdR uptake by B-depleted responder splenocytes. Responder cells from three mice were pooled (mice t and u, (b); mice h, t and u, (c)) and cultured with irradiated spleen cells from TPO fibroblast-injected mice or control mice with or without TPO. Data are the mean ± s.e.m. ct/min. Values significantly different from cultures without TPO: *P < 0.03 (t = 2.59, (b)); *P < 0.01 (t = 3.12, (c)).

Fig. 6

Evidence for TPO antibody synthesis by cultures of splenocytes from TPO⁺ class II⁺ fibroblast-injected mice. Supernatants were collected from splenocytes cultured for 10 days in the presence or absence of pokeweed mitogen (PWM). Cultures of splenocytes from TPO cell-injected (▪, n = 4) or uninjected (□, n = 6) mice were analysed by ELISA using plates coated with recombinant TPO or bovine serum albumin (BSA). Data are expressed as optical density (OD) 492 (mean + s.e.m.) after subtraction of background levels determined in parallel cultures for each donor mouse by freeze–thaw (× 3) of splenocyte suspensions.