Conditions that diminish myeloid-derived suppressor cell activities stimulate cross-protective immunity

Abstract

Immunity conferred by conventional vaccines is restricted to a narrow range of closely related strains, highlighting the unmet medical need for the development of vaccines that elicit protection against multiple pathogenic serotypes. Here we show that a Salmonella bivalent vaccine comprised of strains that lack and overproduce DNA adenine methylase (Dam) conferred cross-protective immunity to salmonella clinical isolates of human and animal origin. Protective immunity directly correlated with increased levels of cross-reactive opsonizing antibodies and memory T cells and a diminished expansion of myeloid-derived suppressor cells (MDSCs) that are responsible for the immune suppression linked to several conditions of host stress, including chronic microbial infections, traumatic insults, and many forms of cancer. Further, aged mice contained increased numbers of MDSCs and were more susceptible to Salmonella infection than young mice, suggesting a role for these cells in the immune declines associated with the natural aging process. These data suggest that interventions capable of reducing MDSC presence and activities may allow corresponding increases in B- and T-cell stimulation and benefit the ability of immunologically diverse populations to be effectively vaccinated as well as reducing the risk of susceptible individuals to infectious disease.

FIG. 1.

dam mutant immunization results in increased levels of cross-reactive opsonizing antibodies, blocking antibodies capable of inhibiting the infection of nonphagocytic cells, and cross-reactive memory CD4⁺ and CD8⁺ cells. BALB/c mice were orally immunized with aroA, dam, or dam/Dam^OP serovar Typhimurium (14028). After 11 weeks, pooled serum samples collected from immunized mice (heat inactivated, 1:50 dilution) were incubated with salmonellae (10⁹ CFU) for 1 h at 4°C. (A) Opsonized salmonellae were cocultured with RAW 264.7 macrophages (MOI, 20:1) for 1 h at 37°C. (B) Immune serum (1:40) was added directly to HeLa cell cultures followed by the addition of bacteria (MOI, 50:1). After 1 h at 37°C, the numbers of internalized bacteria were determined by direct colony counting after gentamicin treatment (1 h at 37°C) prior to cell lysis with Triton X-100. Values represent enhancement of phagocytosis and percent inhibition of invasion into nonphagocytic cells, respectively, relative to naïve serum. (C and D) Immunized and naïve mice were i.v. challenged with salmonellae (10⁶ CFU); 3 h later, splenocytes were isolated and stained with monoclonal antibodies to quantitate CD4⁺ CD11a⁺ IFN-γ⁺ or CD8⁺ CD11a⁺ IFN-γ⁺ cells by FACS. Values represent the number of memory CD4⁺ or CD8⁺ T cells producing IFN-γ per 10³ splenocytes. Memory CD4⁺ or CD8⁺ T cells from naïve challenged mice producing IFN-γ were less than 1 per 10³ (data not shown). SB, serovar Bovismorbificans; SD, serovar Dublin; SE, serovar Enteritidis; ST, serovar Typhimurium. Data represent the means of triplicates ± the standard deviations. *, P < 0.05; **, P < 0.005.

FIG. 2.

dam mutant immunization does not result in a significant expansion of myeloid-derived suppressor cells. BALB/c mice were i.p. infected (10⁴ CFU) with aroA or dam mutant serovar Typhimurium (14028). Spleens were collected from animals at days 3, 7, 10, 14, 21, 28, and 35 postinfection and were evaluated for number of Gr1⁺ CD11b⁺ MDSCs per spleen analyzed by FACS analysis (A), NO production in 48-h culture supernatants, quantitated by Griess assay (B), or CFU of infecting bacteria, determined by direct colony counting (C). Data represent the means of triplicates ± the standard deviations. Differences between the numbers of Gr1⁺ CD11b⁺ MDSCs, the levels of NO produced by Gr1⁺ CD11b⁺ MDSCs, and the CFU numbers in spleens of mice infected with aroA or dam mutant serovar Typhimurium were statistically significant (P < 0.004) on days 3, 5, and 7.

FIG. 3.

Ovalbumin-specific CD4⁺ T-cell proliferation was inhibited by myeloid-derived suppressor cells isolated from aroA Salmonella-infected mice. Purified MDSCs of the Gr1⁺ CD11b⁺ phenotype isolated from the spleens of BALB/c mice i.p. infected with aroA or dam mutant serovar Typhimurium (10⁴ CFU) at the times of peak Gr1⁺ CD11b⁺ cell numbers postinfection (days 7 and 14, respectively) were cocultured with splenocytes from T-cell receptor transgenic (ovalbumin 323-339-specific) DO11.10 mice at different ratios (1:8, 1:16, and 1:32) in the presence of ovalbumin (100 μg/ml). After 4 days of coculture, CD4⁺ T-cell proliferation was assessed by the ability of proliferating cells to incorporate [³H]thymidine. Values given reflect the percent inhibition of CD4⁺ T-cell proliferation compared to cell cultures containing similar ratios of Gr1⁺ CD11b⁺ cells derived from uninfected mice. Data represent the means of triplicates ± standard deviations. **, P < 0.002.

FIG. 4.

Aged mice contained an increased percentage of myeloid-derived suppressor cells and were more susceptible to Salmonella infection than young mice. (A and B) Splenocytes isolated from uninfected young (3-month-old) and aged (18- to 20-month-old) BALB/c or C57BL/6 mice were analyzed by FACS for the presence and percentage of MDSCs of the Gr1⁺ CD11b⁺ phenotype. (C) Young and aged mice were i.p. infected with aroA or dam (10³ CFU) or wild-type (10² CFU) serovar Typhimurium. Three days postinfection, spleens of infected mice were evaluated for bacterial load by direct colony count. Data represent the means of triplicates ± standard deviations. **, P < 0.001.