Interleukin-12 primes CD4+ T cells for interferon-gamma production and protective immunity during Mycobacterium avium infection

Abstract

Interleukin-12 (IL-12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL-12-deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL-12 was similar to that of interferon-gamma (IFN-gamma)-deficient mice. These data indicate a crucial role of IL-12 in the development of a T-cell population able to produce IFN-gamma and to mediate protection against M. avium infection. Treatment of M. avium-infected mice with IL-12 induced CD4+ T cells with enhanced capacity to produce IFN-gamma as well as to confer increased protection against M. avium.

Figure 1

Failure of IL-12 p40^–/– mice to control M. avium infection and of their T cells to respond to specific antigens. IL-12 p40^–/– mice and their IL-12 p40^+/– littermates (a) and (c) or wild-type C57BL/6, IL-12 p40^–/– and IFN-γ^–/– mice (b) were infected i.v. with 10⁶ CFU of M. avium 2447. (a) Number of viable bacteria present in the organs of IL-12 p40^–/– (filled symbols) and IL-12 p40^+/– (open symbols) mice. Each time-point represents the geometric mean of the CFU from four or five animals. (b) Number of viable bacteria present in the organs of C57BL/6 (open bars), IL-12 p40^–/– (solid bars), and IFN-γ^–/– (hatched bars) mice at 90 days of infection. Each value represents the geometric mean of the CFU from four or five animals. (c) In vitro production of IFN-γ by spleen cells from uninfected (stippled bars) or infected mice at 60 days of infection (IL12 p40^+/–, open bars; IL12 p40^–/–, solid bars) after stimulation with ConA or specific antigens of M. avium (MEP and CFP) for 72 hr. Values from non-stimulated spleen cells were below the detection limit of the assay and were not represented. Data are presented as the means ± SD of triplicates from individual mice for each group (four mice per group). Statistically significant differences between the two groups of infected mice are labelled as **P < 0·01.

Figure 2

Effect of rmIL-12 administration on the ability of spleen cells or purified CD4⁺ T cells from spleens of M. avium infected mice to produce IFN-γ after stimulation in vitro with M. avium-specific antigens. BALB/c mice were infected i.v. with M. avium and treated i.p. with rmIL-12 (hatched bars) or PBS (open bars) every other day during the 15 days of the experimental infection. At 15 days of infection, total spleen cells (a) or purified CD4⁺ T spleen cells (b) obtained from uninfected mice or infected mice were cultured in the presence of ConA (5 µg/ml), CFP (4 µg/ml), or medium alone. Supernatants from stimulated and unstimulated cell cultures were harvested at 72 hr and the amount of IFN-γ was measured by ELISA. IFN-γ levels in culture supernatants of unstimulated cells were below the detection limit of the assay and were not represented. Values are reported as means ± SD of triplicates from four individual mice for each group or pool of CD4⁺ T spleen cells from mice for each group (n = 4). The statistical significance of the differences between rmIL-12 versus PBS treatments is shown as **P < 0·01.

Figure 3

The administration of rmIL-12 enhances the generation of a protective CD4⁺ T-cell population. The log₁₀ protection afforded by M. avium-immune whole T cells (open bars) or purified CD4⁺ T cells (stippled bars) from rmIL-12-treated and PBS-treated mice is shown. Five mice were used per group of donors and four were used per group of recipient animals. The total numbers of CD4⁺ T cells adoptively transferred to the irradiated recipient mice were 6·9 × 10⁶ from uninfected, 6·5 × 10⁶ from PBS-treated infected and 6·9 × 10⁶ from IL-12-treated and infected group. Statistically significant protection (*P < 0·05 and **P < 0·01) was found with all immune cells. No statistically significant differences were found when comparing protection afforded by purified CD4⁺ T cells versus whole T cells.