Purpose Repeated exposure to bacterial superantigens, such as staphylococcal enterotoxin A or B (SEA or SEB), is known to induce Vβ-specific anergic T cells which suppress the proliferative and cytokine responses of naïve T cells to SEA or SEB. We sought to determine if repeated exposure to another bacterial superantigen, Toxic Shock Syndrome Toxin-1 (TSST-1), could also generate regulatory T cells.
Method 6-8 week-old female C57B6 mice were injected subcutaneously with 4μg TSST-1 or saline for three doses at 4-day intervals. After the 3rd injection, mice were sacrificed, splenocytes were obtained, and CD4+ T cells were purified by magnetic separation. Splenocytes from TSST-1 or saline-treated mice were stimulated with either TSST-1 (22 ng/ml) or SEB (1 μg/ml) for 48h. The suppressive effect of CD4+ T cells from TSST-1 or saline-treated mice were evaluated by co-culture with naïve splenocytes and stimulation with TSST-1 or SEB in vitro. The proliferative response was measured by carboxy-fluorescein diacetate (CFSE)-staining and flow cytometry, and various cytokines in the culture supernatant were assayed by ELISA.
Results Splenocytes from TSST-1 treated mice produced lower levels of IL-2 and higher levels of IL-10 and IFN-γ, compared to saline-treated mice (p≤0.05), when re-stimulated with TSST-1 in vitro, consistent with a Tr1 phenotype. Stimulation with SEB in vitro induced a similar cytokine production profile as with TSST-1. Moreover, TSST-1 primed CD4+ T cells proliferated at a higher rate than saline-primed CD4+ T cells when re-stimulated with TSST-1 in vitro (74% vs. 16%; p≤0.01, paired t test), indicating that in vivo administration of TSST-1 did not result in anergy of TSST-1 responsive CD4+ T cells. Co-culture of TSST-1 primed CD4+ T cells with naïve splenocytes in the presence of TSST-1 led to decreased production of both IL-2 and IL-12, and increased production of IL-10 (p≤0.05). Furthermore, naïve CD4+ T cells proliferated in response to TSST-1 at a higher rate when co-cultured with TSST-1 primed CD4+ T cells, than with saline-primed CD4+ T cells (55% vs. 27%; p≤0.05). Co-culture of naïve splenocytes with TSST-1 primed CD4+ T cells in the presence of SEB showed a similar pattern of cytokine production as with TSST-1 stimulation.
Conclusion Repeated subcutaneous injection of TSST-1 induced CD4+ regulatory T cells that were non-anergic, and mediated both antigen-specific (TSST-1) and by-stander (SEB) inhibition of IL-2 and IL-12 production by naïve splenocytes. The therapeutic application of these regulatory T cells is currently under investigation.