Igh-6(-/-) (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar typhimurium and show impaired Th1 T-cell responses to Salmonella antigens

Abstract

In the present study we evaluated the role of B cells in acquired immunity to Salmonella infection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6(-/-)). Igh-6(-/-) mice immunized with a live, attenuated aroA Salmonella enterica serovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5. Igh-6(-/-) mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However, Igh-6(-/-) mice, unlike Igh-6(+/+) C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance in Igh-6(-/-) mice. Total splenocytes and purified CD4(+) T cells obtained from Igh-6(-/-) mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained from Igh-6(+/+) C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer of Salmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization.

FIG. 1

Igh-6^−/− mice (closed diamonds) and C57BL/6 mice (open squares) were infected i.v. with 5 × 10⁵ CFU of serovar Typhimurium strain SL3261. Spleen and liver counts of viable bacteria were obtained thereafter. Results are means ± standard deviations from groups of four mice.

FIG. 2

Igh-6^−/− mice (closed diamonds) and C57BL/6 mice (open squares) were immunized with serovar Typhimurium strain SL3261 as for Fig. 1. Age-matched naive Igh-6^−/− mice (open triangles) and naive C57BL/6 mice (open circles) were used as unimmunized controls. Four months after vaccination, all mice were challenged orally with ca. 2.5 × 10⁹ CFU of virulent serovar Typhimurium strain C5.

FIG. 3

Igh-6^−/− mice (closed diamonds) and C57BL/6 mice (open squares) were immunized with serovar Typhimurium strain SL3261 as for Fig. 1. Age-matched naive Igh-6^−/− mice (open triangles) and naive C57BL/6 mice (open circles) were used as unimmunized controls. One additional group of Igh-6^−/− mice in each experiment (squares with bars) received immune serum from similarly immunized and orally reinfected C57BL/6 donors 2 h before challenge and at various times thereafter (days 1, 3, and 6 in experiment A and days 1, 3, 4, 6, and 12 in experiment B). All mice were challenged orally with serovar Typhimurium strain C5 (5 × 10⁹ CFU [experiment A] or 1 × 10⁹ CFU [experiment B]).

FIG. 4

Splenocytes were prepared from naive C57BL/6 mice and Igh-6^−/− mice as well as from mice of either strain which had been vaccinated 4 months earlier with 5 × 10⁵ CFU of serovar Typhimurium strain SL3261. The cells were restimulated in vitro with a crude (C5SE at 1.2 μg/ml) or detoxified (C5SENaOH at 20 μg/ml) Salmonella soluble extract. IL-2 and IFN-γ production was measured by ELISA. Values are means ± standard deviations of triplicate cultures.