Multiple T cell subsets control Francisella tularensis LVS intracellular growth without stimulation through macrophage interferon gamma receptors

Abstract

A variety of data suggest that in vivo production of interferon (IFN)-gamma is necessary, but not sufficient, for expression of secondary protective immunity against intracellular pathogens. To discover specific IFN-gamma-independent T cell mediated mechanisms, we took advantage of an in vitro culture system that models in vivo immune responses to the intracellular bacterium Francisella tularensis live vaccine strain (LVS). LVS-immune lymphocytes specifically controlled 99% of the growth of LVS in wild-type murine bone marrow-derived macrophages. Surprisingly, LVS-immune lymphocytes also inhibited LVS intracellular growth by as much as 95% in macrophages derived from IFN-gamma receptor knockout (IFNgammaR KO) mice. CD8+ T cells, and to a lesser degree CD4+ T cells, controlled LVS intracellular growth in both wild-type and IFNgammaR KO macrophages. Further, a unique population of Thy1+alphabeta+CD4-CD8- cells that was previously suggested to operate during secondary immunity to LVS in vivo strongly controlled LVS intracellular growth in vitro. A large proportion of the inhibition of LVS intracellular growth in IFNgammaR KO macrophages by all three T cell subsets could be attributed to tumor necrosis factor (TNF) alpha. Thus, T cell mechanisms exist that control LVS intracellular growth without acting through the IFN-gamma receptor; such control is due in large part to TNF-alpha, and is partially mediated by a unique double negative T cell subpopulation.

Figure 1.

Control of LVS growth by wild-type LVS-immune splenocytes in (A) wild-type BMMØ and (B) BMMØ that lack the IFNγR. BMMØ from wild-type and IFNγR KO mice were infected with LVS at an MOI of 1:40 (bacterium-to-macrophage ratio). Infected BMMØs were cocultured with splenocytes from either uninfected mice (naive spleen), mice infected intradermally with LVS 4 wk previously (primed spleen), or no spleen cells (macrophages + LVS). Immediately after LVS infection of the BMMØs, splenocytes were added to the indicated wells at a ratio of 1:2 (splenocyte:BMMØ). 72 h after infection, the BMMØ were washed, lysed, and plated to determine the levels of intracellular bacteria. Values shown are the mean numbers of CFU/ml ± the SEM of viable bacteria (triplicate samples). Asterisks (*) indicate P values <0.01 as compared with cocultures containing naive spleen cells. These data are representative of five experiments of similar design.

Figure 2.

Specificity of control of LVS growth in (A) wild-type BMMØ and (B) IFNγR KO BMMØs. Infected BMMØs were cocultured with wild-type splenocytes from either uninfected mice (naive spleen), mice infected intradermally 4 wk previously with LVS (LVS primed spleen), or with L. monocytogenes (Listeria primed spleen). Immediately following LVS infection of the BMMØs, splenocytes were added to the indicated wells at a ratio of 1:2 (splenocyte:BMMØ). 72 h after infection, the BMMØ were assessed for levels of intracellular bacteria as described in the legend to Fig. 1. Values shown are the mean numbers of CFU/ml ± the SEM of viable bacteria (triplicate samples). Asterisks (*) indicate P values <0.01 as compared with cocultures containing naive spleen cells. These results are representative of three experiments of similar design.

Figure 3.

Effects of TNF-α neutralization and iNOS inhibition on control of LVS growth in (A) wild-type BMMØ and (B) IFNγR KO BMMØs. Infected BMMØs were cocultured with splenocytes from either uninfected mice (naive spleen), or wild-type mice infected intradermally with LVS 4 wk previously (primed spleen). Immediately following LVS infection of the BMMØs, splenocytes were added to the indicated wells at a ratio of 1:2 (splenocyte:BMMØ). In the indicated cultures, either anti–TNF-α Abs or control IgG Ab (20 μg/ml), or 1 mM NMMA, were added to the cultures at the time of addition of the splenocytes. 72 h after infection, the BMMØ were assessed for levels of intracellular bacteria as described in the legend to Fig. 1. Values shown are the mean numbers of CFU/ml ± the SEM of viable bacteria (triplicate samples). Asterisks (*) indicate P values <0.01 as compared with cocultures containing naive spleen cells. These results are representative of three experiments of similar design.

Figure 4.

Effects of T cell depletion on control of LVS growth in (A) wild-type BMMØ and (B) IFNγR KO BMMØs. Infected BMMØs were cocultured with splenocytes obtained from either uninfected mice (naive spleen), wild-type mice infected intradermally with LVS 4 wk previously (primed spleen), or LVS-primed wild-type mice that had been in vivo depleted of the indicated T cell subsets by intraperitoneal injection with Abs specific for CD4, CD8, CD4 and CD8 simultaneously, or Thy1.2 cell surface markers. Immediately after LVS infection of the BMMØs, splenocytes were added to the indicated wells at a ratio of 1:2 (splenocyte:BMMØ). 72 h after infection, the BMMØ were assessed for levels of intracellular bacteria as described in the legend to Fig. 1. Values shown are the mean numbers of CFU/ml ± the SEM of viable bacteria (triplicate samples). Asterisks (*) indicate P values <0.01 as compared with cocultures containing naive spleen cells. These results are representative of three experiments of similar design.

Figure 5.

Ability of various T cell subsets to control LVS growth in (A) wild-type BMMØ and (B) IFNγR KO BMMØs. Infected BMMØs were cocultured with splenocytes obtained from either uninfected mice (naive spleen), whole primed splenocytes from immune wild-type mice (primed spleen), or various T cell subsets enriched from immune wild-type mice (CD4⁺ cells, CD8⁺ cells, and Thy1⁺CD4⁻CD8⁻ cells). All splenocyte populations were added at a 1:2 ratio (splenocyte to BMMØ). 72 h after infection, the BMMØ were assessed for levels of intracellular bacteria as described in the legend to Fig. 1. Values shown are the mean numbers of CFU/ml ± the SEM of viable bacteria (triplicate samples). Asterisks (*) indicate P values <0.01 as compared with cocultures containing naive spleen cells. These results are representative of three experiments of similar design.

Figure 6.

Secretion of cytokines and NO into culture supernatants after coculture of LVS infected BMMØs and immune splenocytes. Wild-type (black bars) and IFNγR KO (gray bars) BMMØ were infected with LVS and cocultured with the indicated splenocyte populations. Splenocytes from either unprimed mice or mice infected 4 wk previously with an intradermal LVS infection were cocultured with LVS-infected macrophages at a 1:2 ratio (splenocytes to BMMØ). Similarly, CD4⁺ and CD8⁺ and Thy1⁺CD4⁻CD8⁻ splenocyte subpopulations were added to infected BMMØ cultures at a 1:2 ratio. Culture supernatants were collected 72 h later and tested for IFN-γ (A), TNF-α (B), IL-12 (C), and NO (D). Values shown are the mean ng/ml ± the SEM of the indicated cytokine (A–C) or mean μM/ml nitrite (D) (triplicate samples). These results are representative of three experiments of similar design.

Figure 7.

Effects of TNF-α neutralization on the ability of different T cell subsets to control LVS growth in (A) wild-type BMMØ and (B) IFNγR KO BMMØs. Infected BMMØs were cocultured with splenocytes obtained from either uninfected mice (naive spleen), whole primed splenocytes from immune wild-type mice (primed spleen), or various T cell subsets enriched from immune wild-type mice (CD4⁺ cells, CD8⁺ cells, and CD4⁻CD8⁻Thy1.2⁺ cells). All splenocyte populations were added at a 1:2 ratio (splenocyte to BMMØ). Anti–TNF-α Abs were added to the wells at the time of addition of the splenocytes to the infected BMMØs. 72 h after infection, the BMMØ were assessed for levels of intracellular bacteria as described in the legend to Fig. 1. Values shown are the mean numbers of CFU/ml ± the SEM of viable bacteria (triplicate samples). Asterisks (*) indicate P values <0.01 as compared with cocultures containing naive spleen cells. These results are representative of three experiments of similar design.