CD4 T-cell-mediated heterologous immunity between mycobacteria and poxviruses

Abstract

The bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis is used in many parts of the world as a vaccine against Mycobacterium tuberculosis. Some epidemiological evidence has suggested that BCG immunization may have unpredicted effects on resistance to other pathogens. We show here in a mouse model that BCG immunization followed by antibiotic treatment to clear the host of the pathogen rendered three strains of mice partially resistant to infection with vaccinia virus (VV) but not to lymphocytic choriomeningitis virus (LCMV). VV-challenged BCG-immune mice developed a striking splenomegaly and elevated CD4 and CD8 T-cell responses by 6 days postinfection (p.i.). However, resistance to VV infection could be seen as early as 1 to 2 days p.i. and was lost after antibody depletion of CD4 T-cell populations. BCG- but not LCMV-immune memory phenotype CD4 T cells preferentially produced gamma interferon (IFN-gamma) in vivo after VV challenge. In contrast, LCMV-immune CD8 T cells preferentially produced IFN-gamma in vivo in response to VV infection. In BCG-immune mice the resistance to VV infection and VV-induced CD4 T-cell IFN-gamma production were ablated by cyclosporine A, which inhibits signaling through the T-cell receptor. This study therefore demonstrates CD4 T-cell-mediated heterologous immunity between a bacterium and virus. Further, it poses the question of whether BCG immunization of humans alters resistance to unrelated pathogens.

FIG. 1.

Increased spleen size and spleen T-cell numbers 6 days after VV infection of BCG-immunized mice. (A) Sizes of spleens before and after VV challenge. (B and C) CD8 and CD4 cell numbers in spleens and PEC, respectively, as determined by flow cytometry. There were four mice per group in this representative experiment. Statistically significant differences between BCG- and sham-immunized mice: *, P < 0.05; **, P < 0.01.

FIG. 2.

Moderate loss of CFSE in adoptively transferred BCG-immune CD4 T-cell populations. A total of 3 × 10⁷ BCG- or sham-immune spleen leukocytes were transferred intravenously into naïve B6 female mice, which were challenged 4 h later with VV. At 6 days postinfection splenocytes were harvested and analyzed for CD4, CD8, and expression of CFSE. The cells were gated on CD4 or CD8 and plotted against IFN-γ (y axis) and CFSE (x axis). This was part of a larger experiment that included stimulation of intracellular IFN-γ production in vitro, but these plots are intended to show just cell number and are of the nonstimulated controls, so they all are IFN-γ-negative.

FIG. 3.

In vivo IFN-γ production by T cells infiltrating the visceral fat and the peritoneal cavity of day 2 (D2) VV-challenged BCG-or sham-immunized mice. As described in Materials and Methods, immunized mice were challenged with VV for 2 days and injected with BFA for 6 h, and isolated leukocytes were stained for antigenic phenotype and cytoplasmic IFN-γ. Some mice were treated with CsA on day 0 and day 1 before the day 2 harvest.

FIG. 4.

In vivo IFN-γ production by T cells infiltrating the visceral fat and the peritoneal cavity of day 2 (D2) VV-challenged LCMV-immune mice. Immune mice were challenged with VV and at 2 days postinfection their lymphocytes were tested for IFN-γ production, as described in Materials and Methods and in the legend to Fig. 2. The same three mice (two infected and one uninfected) were used for the CD4 and CD8 data.

FIG. 5.

Hierarchies of VV-encoded class II epitope-specific CD4 T cells in BCG- or sham-immunized mice 9 days after VV challenge, as determined by peptide-induced intracellular IFN-γ production in vitro.

FIG. 6.

Hierarchies of VV-encoded class I epitope-specific CD8 T cells in BCG- or sham-immunized mice 6 days after VV challenge, as determined by peptide-induced intracellular IFN-γ production in vitro.