Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo

Abstract

TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.

Fig. 1

Anti-CD3 Ab induces severe apoptosis in thymuses of IFN-γR KO mice. Groups of 3 IFN-γR KO and 3 wild-type mice were challenged with 0, 20 or 40 µg anti-CD3 Ab. Forty hours post anti-CD3 challenge, thymocytes were analysed by flow cytometry for (a) their DNA content and (b) their staining pattern for CD4 and CD8. (a) PI fluorescence intensity: a representative histogram from one mouse out of three is shown. Value represents the percentage of apoptotic cells. (b) Thymocyte subpopulations in IFN-γR KO and wild-type mice. Each plot represents analysis of one mouse. The absolute number of each subpopulation as well as the total number of thymocytes (in parenthesis) are shown (×10⁻⁶). Data are representative of three separate experiments.

Fig. 2

Anti-CD3 induction of IL-15 in spleens of wild-type (h), but not of IFN-γR KO (▪) mice. IFN-γR KO and wild-type mice were challenged with anti-CD3 Ab at the indicated dose. Six hours later, total RNA was extracted from spleens (a) or thymuses (b), and real-time PCR was performed for IL-15. (a) IL-15 mRNA expression in spleens of mice. Data of one mouse at each dose are shown. The inset shows a separate experiment performed with 20 µg anti-CD3 Ab. Each bar represents the mean ± S.E. of 3 mice. *P < 0·02 in comparison with wild-type mice (Mann–Whitney U-test). (b) IL-15 mRNA expression in thymuses of three IFN-γR KO and wild-type mice that had received 40 µg anti-CD3 Ab or saline.

Fig. 3

Anti-IL-15 Ab increases anti-CD3-induced apoptosis in (a) wild-type mice and (b) IL-15 protects IFN-γR KO mice from anti-CD3-induced apoptosis. (a) Two groups of wild-type mice (n = 4) were treated with anti-IL-15 Ab or control IgG. Five hours later, these mice and an additional group of 4 IFN-γR KO mice, were challenged with 20 µg anti-CD3 Ab. Forty hours post anti-CD3-challenge, apoptosis in the thymuses was measured with the use of the DNA dye PI. Each histogram represents analysis of a single thymus. Values indicate the average percentages of apoptotic cells (mean ± S.E.) for 4 mice in each group. *P < 0·05 in comparison with control IgG-treated wild-type mice (Mann–Whitney U-test). (b) Two groups of IFN-γR KO mice (n = 3) were injected with 20 µg anti-CD3-Ab and two injections of either 15 µg IL-15 in 100 µl PBS, or PBS as control, at 6 h and 18 h post anti-CD3 challenge. Wild-type mice that similarly received anti-CD3 and PBS were also included. Thymocyte apoptosis was analysed with PI. Each histogram represents analysis of a single thymus. Values indicate the average percentages of apoptotic cells (mean ± S.E.) for 3 mice in each group. *P < 0·05 in comparison with PBS-treated IFN-γR KO mice (Mann–Whitney U-test).