MHC class-I-restricted CD8 T cells play a protective role during primary Salmonella infection

Abstract

Protective immunity against Salmonella infection is known to require CD4 Th1 cells and B cells, but the role of MHC class-I-restricted CD8 T cells is less clear. Previous studies have suggested that CD8 T cells participate in secondary, but not primary, bacterial clearance. However, these studies have used experimental models that are difficult to interpret and do not clearly isolate the role of MHC class-I-restricted CD8 T cells from other cell populations. Here, we examined the role of class-I-restricted T cells in protection against Salmonella infection using mice lacking all classical MHC class-Ia molecules, perforin, or granzyme B. Immunized K(b)D(b)-, perforin-, granzyme B-, or perforin/granzyme B-deficient mice were able to resolve secondary infection with virulent Salmonella, demonstrating that class-I-restricted CTLs are not required for acquired immunity. However, during primary infection with attenuated bacteria, bacterial clearance was delayed in each of these mouse strains when compared to wild-type mice. Taken together, these data demonstrate that CD8 T cells are not required for acquired immunity to Salmonella, but can play a protective role in resolving primary infection with attenuated bacteria.

Figure 1. CD8 T cells are not required for immunity to secondary Salmonella infection

C57BL6 (B6) Wild-type, K^bD^b-, perforin-, granzyme B- and perforin/granzyme B-deficient (PG) mice were intravenously infected with 5X10⁵ BRD509. On day 60 post-infection, all the mice that resolved the primary infection with BRD509 as well as naïve strains were rechallenged intravenously with 1000 virulent Salmonella SL1344. Infected mice were monitored daily for signs for disease and sacrificed after the development of a moribund state. Bacterial burden was tested at day 6 after SL1344 infection. Spleens and livers from infected mice were homogenized in PBS and serial dilutions plated on MacConkey agar plates to determine bacterial burden. Scatter plots represent the bacterial burden of individual mice in spleens (left) or livers (right) from naïve strains (top) or mice that resolved BRD509 infection (bottom). “+” Indicates a dead mouse in this group before day 6. Data with K^bD^b-, perforin-, and granzyme B-deficient mice are pooled from two different experiments. Bacterial burden was analyzed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test (***: p<0.001).

Figure 2. K^bD^b-deficient mice have slightly higher bacteria loads at the late stage of primary Salmonella infection

B6 Wild-type and K^bD^b-deficient mice were intravenously infected with 5X10⁵ attenuated Salmonella, BRD509. Bacterial burden was examined at day 7, 21, and 35 post-infection. Spleens and livers from infected mice were homogenized in PBS and serial dilutions plated onto MacConkey agar plates to determine bacterial numbers at each time points. Data show mean bacterial burden in spleen (top) and liver (bottom) with individual mice shown as scatter plots. Data are pooled from at least three separate experiments. Numbers above each group indicate statistical significance and show the p value of a comparison between groups.

Figure 3. Perforin-deficient mice have moderately higher bacterial burden at late stage of primary Salmonella infection

B6 Wild-type and perforin-deficient mice were intravenously infected with 5×10⁵ BRD509. Bacterial burden was examined at day 21 and 35 post-infection. Spleens and livers from infected mice were homogenized in PBS and serial dilutions plated on MacConkey agar plates to determine bacterial burden at each time points. Data show mean bacterial burden in spleen (top) and liver (bottom) with individual mice shown as scatter plots. Data are pooled from at least three separate experiments. Numbers above each group indicate statistical significance and show the p value of a comparison between groups.

Figure 4. Granzyme B-deficient mice are competent to resolve the primary bacteria infection

B6 Wild-type and granzyme B-deficient mice were intravenously infected with 5X10⁵ BRD509 Salmonella. Bacteria colonization in vivo was tested at day 21 and 35 post-infection. Spleens and livers from infected mice were homogenized in PBS and serial dilutions plated on MacConkey agar plates to specify bacterial burden at each time points. Data show mean bacterial burden in spleen (top) and liver (bottom) with individual mice shown as scatter plots. Data are combined from at least three different experiments. Numbers above each group indicate statistical significance and show the p value of a comparison between groups.

Figure 5. Perforin/granzyme B-deficient mice have the ability to resolve the primary Salmonella infection

B6 Wild-type and perforin/granzyme B-deficient (PG) mice were intravenously infected with 5×10⁵ BRD509. Bacterial burden was tested at day 21 and 35 post-infection. Spleens and livers from infected mice were homogenized in PBS and serial dilutions plated on MacConkey agar plates to determine bacterial burden at each time points. Data show mean bacterial burden in spleen (top) and liver (Bottom) with individual mice shown as scatter plots. Day 21 data are combined from two different experiments and day 35 data is from one experiment. Numbers above each group indicate statistical significance and show the p value of a comparison between groups.