The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge

Abstract

IL-12 plays a central role in both the induction and magnitude of a primary Th1 response. A critical question in designing vaccines for diseases requiring Th1 immunity such as Mycobacterium tuberculosis and Leishmania major is the requirements to sustain memory/effector Th1 cells in vivo. This report examines the role of IL-12 and antigen in sustaining Th1 responses sufficient for protective immunity to L. major after vaccination with LACK protein (LP) plus rIL-12 and LACK DNA. It shows that, after initial vaccination with LP plus rIL-12, supplemental boosting with either LP or rIL-12 is necessary but not sufficient to fully sustain long-term Th1 immunity. Moreover, endogenous IL-12 is also shown to be required for the induction, maintenance, and effector phase of the Th1 response after LACK DNA vaccination. Finally, IL-12 is required to sustain Th1 cells and control parasite growth in susceptible and resistant strains of mice during primary and secondary infection. Taken together, these data show that IL-12 is essential to sustain a sufficient number of memory/effector Th1 cells generated in vivo to mediate long-term protection to an intracellular pathogen.

Figure 1

The role of continuous IL-12 and LP in mediating long-term control of infection after primary immunization with LP plus rIL-12. BALB/c mice (n = 6–8 per group) were initially vaccinated in the footpad and were boosted 2 weeks later (primary immunization) with LACK DNA (100 μg), control DNA (100 μg), LP (50 μg) with IL-12 or control DNA (100 μg), or LP plus rIL-12 (1 μg). Mice were then challenged with 1 × 10⁵ L. major (WHOM/IR/-/173) metacyclic promastigotes in their hind footpads 2 weeks (A) or 12 weeks (B) later, and weekly foot pad measurements were recorded by using a metric caliper. In addition, some of the mice (n = 6–8 per group) that were initially vaccinated and boosted with LP plus rIL-12 (primary immunization) were given supplemental treatment every 2 weeks in the same foot pad with either LP (50 μg), rIL-12 (1 μg), or LP plus rIL-12 (1 μg) for a total of four supplemental treatments. Infectious challenge was done 12 weeks after the primary immunization (4 weeks after the last supplementary treatment). Weekly footpad measurements were recorded by using a metric caliper. These data are representative of three independent experiments.

Figure 2

The role of LP and rIL-12 in sustaining the frequency of CD4⁺ IFN-γ-producing T cells after vaccination and infection. In the same experiment as in Fig. 1, draining lymph node cells were pooled from groups of vaccinated mice (n = 6–8 per group) that were challenged with L. major either 2 weeks (A) or 12 weeks (B) after primary immunization. Lymph node cells were harvested 6 weeks after infection. The frequency of LACK-specific IFN-γ- and IL-4-producing cells was assessed by intracellular cytokine as described in Materials and Methods. (C) In a separate experiment, draining lymph node cells were pooled from groups of vaccinated mice (n = 6–8 per group) challenged with L. major 12 weeks after primary immunization. In this experiment, supplemental treatment with rIL-12 was done in a manner similar to that described in Fig. 1. (D) Groups of mice (n = 6–8 per group) were initially vaccinated and boosted with LP plus rIL-12 (primary immunization), and the frequencies of CD4⁺ IFN-γ-producing cells was determined 1 and 4 weeks after the primary immunization. In three groups of mice vaccinated with LP plus rIL-12, a single supplemental treatment with LP, rIL-12, or both was given 2 weeks after primary immunization, and the frequency of CD4⁺ IFN-γ-producing cells was determined 2 weeks later (4 weeks post-primary immunization).

Figure 3

The role of endogenous IL-12 in the induction and maintenance of CD4⁺ IFN-γ-producing cells after LACK DNA vaccination. In two independent experiments, the frequency of LACK-specific CD4⁺ IFN- or IL-4-producing cells was assessed from pooled (n = 6–8 per group) draining lymph node cells 1 week (A) and 2 weeks (B) after primary immunization with LACK DNA with or without treatment with a single dose of anti-IL-12 (1 mg) i.p. or isotype-matched control antibody. In a third experiment (C), the frequency of LACK-specific CD4⁺ IFN-γ-producing cells was determined from draining lymph node cells of LACK- and control DNA-vaccinated mice 2 or 3 weeks after primary immunization. In this experiment, a single injection of anti-IL-12 was given to LACK DNA-vaccinated mice 2 weeks after primary immunization. (D) In a separate experiment, the frequency of LACK-specific CD4⁺ IFN-γ- and IL-4-producing cells was determined from pooled lymph node cells from LACK DNA or control DNA (n = 6–8) vaccinated mice 6 weeks after infectious challenge. In this experiment, a group of LACK DNA-vaccinated mice were treated with a single injection of anti-IL-12 at the time of infection.

Figure 4

The role of IL-12 in sustaining IL-12Rβ2 expression and control of infection in wild-type and IL-12^−/− BALB/c mice after infection with L. major. Groups of wild-type BALB/c or IL-12^−/− mice (n = 6–8 per group) were infected with L. major as described above. In some groups of mice, treatment with rIL-12 (1 μg) i.p. was initiated at the time of infection and was continued for the following 4 days for a total treatment of 5 days. A group of IL-12^−/− mice initially treated with IL-12 for 5 days was given supplemental treatment weekly with rIL-12 (1 μg) i.p. for the next 5 weeks. Weekly footpad swelling measurements were recorded by using a metric caliper.