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Suboptimal activation of antigen-specific CD4+ effector cells enables persistence of M. tuberculosis in vivo.

Bold TD, Banaei N, Wolf AJ, Ernst JD - PLoS Pathog. (2011)

Bottom Line: Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice.Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity.These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, New York University School of Medicine, New York City, New York, United States of America.

ABSTRACT
Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

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Low frequency of IFN-γ-producing endogenous CD4+                            T cells in lungs of M. tuberculosis-infected                            mice.A. Frequency of IFN-γ expression by endogenous,                            polyclonal CD4+ T cells in the lungs of M.                                tuberculosis-infected mice throughout infection, assayed by                            intracellular cytokine staining without ex vivo                            restimulation. Flow cytometry dot plots show lung CD4+                            cells from a representative mouse at the indicated time point                            post-infection. Values indicate the proportion of cells expressing                            IFN-γ in the CD4+ population for each mouse.                                B. Mean frequency of IFN-γ+ cells                            among lung CD4+ T cells for each group of 4 mice at each                            time point post-infection, assayed by intracellular cytokine staining                            without ex vivo restimulation. Asterisks indicate                            statistical significance of differences in frequency of T cell                            activation observed between adjacent time points * p<0.05;                            ** p<0.005.
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ppat-1002063-g001: Low frequency of IFN-γ-producing endogenous CD4+ T cells in lungs of M. tuberculosis-infected mice.A. Frequency of IFN-γ expression by endogenous, polyclonal CD4+ T cells in the lungs of M. tuberculosis-infected mice throughout infection, assayed by intracellular cytokine staining without ex vivo restimulation. Flow cytometry dot plots show lung CD4+ cells from a representative mouse at the indicated time point post-infection. Values indicate the proportion of cells expressing IFN-γ in the CD4+ population for each mouse. B. Mean frequency of IFN-γ+ cells among lung CD4+ T cells for each group of 4 mice at each time point post-infection, assayed by intracellular cytokine staining without ex vivo restimulation. Asterisks indicate statistical significance of differences in frequency of T cell activation observed between adjacent time points * p<0.05; ** p<0.005.

Mentions: We hypothesized that M. tuberculosis evades adaptive immunity by modulating the activation of CD4+ effector T cells at the site of infection in the lungs. Since in vitro studies have revealed evidence that M. tuberculosis modulates MHC class II antigen presentation [10], [23], [24], [25], [26], we focused on in vivo activation of CD4+ T cells in the lungs. We reasoned that, if M. tuberculosis-infected cells do not present antigens efficiently to effector T cells in the lungs, then the frequency of activation of effector functions of CD4+ cells would also be low at the site of infection. To test this, we used direct intracellular cytokine staining of lung cells from infected mice for IFN-γ, without ex vivo restimulation. We found that that the frequency of IFN-γ expression by CD4+ T cells in the lungs varied with the time of infection (Figure 1B). IFN-γ+ CD4+ cells were undetectable in the lungs at day 14, increased in frequency beginning by day 21 to a peak at day 35 post-infection, and then markedly decreased afterward; no more than 7% of the bulk population of CD4+ T cells expressed IFN-γ at any time point after infection, and fewer than 1% expressed IFN-γ during the chronic phase. Other studies investigating IFN-γ production by CD8+ T cells in vivo have used treatment of mice with brefeldin A or inclusion of brefeldin A during cell isolation and staining [27], [28]. However, we determined that these methods did not improve detection of intracellular IFN-γ by CD4+ T cells during M. tuberculosis infection (Figure S1). These data indicate that a small minority of polyclonal CD4+ T cells recruited to the lungs of M. tuberculosis-infected mice are activated to produce IFN-γ at a given time, and are consistent with defective antigen presentation, costimulation, and/or inhibition of effector T cell activation at the site of infection.


Suboptimal activation of antigen-specific CD4+ effector cells enables persistence of M. tuberculosis in vivo.

Bold TD, Banaei N, Wolf AJ, Ernst JD - PLoS Pathog. (2011)

Low frequency of IFN-γ-producing endogenous CD4+                            T cells in lungs of M. tuberculosis-infected                            mice.A. Frequency of IFN-γ expression by endogenous,                            polyclonal CD4+ T cells in the lungs of M.                                tuberculosis-infected mice throughout infection, assayed by                            intracellular cytokine staining without ex vivo                            restimulation. Flow cytometry dot plots show lung CD4+                            cells from a representative mouse at the indicated time point                            post-infection. Values indicate the proportion of cells expressing                            IFN-γ in the CD4+ population for each mouse.                                B. Mean frequency of IFN-γ+ cells                            among lung CD4+ T cells for each group of 4 mice at each                            time point post-infection, assayed by intracellular cytokine staining                            without ex vivo restimulation. Asterisks indicate                            statistical significance of differences in frequency of T cell                            activation observed between adjacent time points * p<0.05;                            ** p<0.005.
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ppat-1002063-g001: Low frequency of IFN-γ-producing endogenous CD4+ T cells in lungs of M. tuberculosis-infected mice.A. Frequency of IFN-γ expression by endogenous, polyclonal CD4+ T cells in the lungs of M. tuberculosis-infected mice throughout infection, assayed by intracellular cytokine staining without ex vivo restimulation. Flow cytometry dot plots show lung CD4+ cells from a representative mouse at the indicated time point post-infection. Values indicate the proportion of cells expressing IFN-γ in the CD4+ population for each mouse. B. Mean frequency of IFN-γ+ cells among lung CD4+ T cells for each group of 4 mice at each time point post-infection, assayed by intracellular cytokine staining without ex vivo restimulation. Asterisks indicate statistical significance of differences in frequency of T cell activation observed between adjacent time points * p<0.05; ** p<0.005.
Mentions: We hypothesized that M. tuberculosis evades adaptive immunity by modulating the activation of CD4+ effector T cells at the site of infection in the lungs. Since in vitro studies have revealed evidence that M. tuberculosis modulates MHC class II antigen presentation [10], [23], [24], [25], [26], we focused on in vivo activation of CD4+ T cells in the lungs. We reasoned that, if M. tuberculosis-infected cells do not present antigens efficiently to effector T cells in the lungs, then the frequency of activation of effector functions of CD4+ cells would also be low at the site of infection. To test this, we used direct intracellular cytokine staining of lung cells from infected mice for IFN-γ, without ex vivo restimulation. We found that that the frequency of IFN-γ expression by CD4+ T cells in the lungs varied with the time of infection (Figure 1B). IFN-γ+ CD4+ cells were undetectable in the lungs at day 14, increased in frequency beginning by day 21 to a peak at day 35 post-infection, and then markedly decreased afterward; no more than 7% of the bulk population of CD4+ T cells expressed IFN-γ at any time point after infection, and fewer than 1% expressed IFN-γ during the chronic phase. Other studies investigating IFN-γ production by CD8+ T cells in vivo have used treatment of mice with brefeldin A or inclusion of brefeldin A during cell isolation and staining [27], [28]. However, we determined that these methods did not improve detection of intracellular IFN-γ by CD4+ T cells during M. tuberculosis infection (Figure S1). These data indicate that a small minority of polyclonal CD4+ T cells recruited to the lungs of M. tuberculosis-infected mice are activated to produce IFN-γ at a given time, and are consistent with defective antigen presentation, costimulation, and/or inhibition of effector T cell activation at the site of infection.

Bottom Line: Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice.Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity.These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, New York University School of Medicine, New York City, New York, United States of America.

ABSTRACT
Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

Show MeSH
Related in: MedlinePlus