Limits...
ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis.

Moguche AO, Shafiani S, Clemons C, Larson RP, Dinh C, Higdon LE, Cambier CJ, Sissons JR, Gallegos AM, Fink PJ, Urdahl KB - J. Exp. Med. (2015)

Bottom Line: When transferred into uninfected animals, these cells persist, mount a robust recall response, and provide superior protection to Mtb rechallenge when compared to terminally differentiated Th1 cells that reside preferentially in the lung-associated vasculature.Thus, the molecular pathways required to maintain Mtb-specific CD4 T cells during ongoing infection are similar to those that maintain memory CD4 T cells in scenarios of antigen deprivation.These results suggest that vaccination strategies targeting the ICOS and Bcl6 pathways in CD4 T cells may provide new avenues to prevent TB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Seattle Biomedical Research Institute (renamed Center for Infectious Disease Research), Seattle, WA 98109 Department of Immunology, University of Washington School of Medicine, Seattle, WA 98104.

Show MeSH

Related in: MedlinePlus

ESAT-6–specific CD4 T cells cluster into distinct functional subsets. Mice were infected as described in Figure 1 and lung CD4 T cells were analyzed by flow cytometry. (A) Representative flow cytometry plot depicts KLRG1 and PD-1 expression by ESAT-6 tetramer-binding CD4 T cells in the lung 90 d after infection. (B) Single-cell suspensions from the lungs of Mtb-infected mice (150 d after infection) were stimulated with ESAT-64-17 in vitro and intracellular cytokine staining was performed. A representative flow plot depicts KLRG1 and IL-2 expression within CD4 T cells coproducing IFN-γ and TNF (gate not depicted), and numbers in parentheses represent the percentage of KLRG1− (blue) and KLRG1+ (red) cells within this population that express IL-2. The graph shows this value for each mouse and the mean ± SEM for each group. (C) Flow cytometry plot shows direct ex vivo intracellular IFN-γ versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 34 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells, and the graph below shows the mean ± SEM of these values for each group at various time points after infection. (D) Flow cytometry histograms show T-bet expression by naive CD44low (gray), PD-1+KLRG1− (blue), and PD-1−KLRG1+ (red) ESAT-6 tetramer-binding CD4 T cells 90 d after infection. Graph below shows the T-bet mean fluorescent intensity (MFI) for each T cell subset in individual mice, and the mean ± SEM for each group. (E) Flow cytometry plot shows Ki67 versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 70 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells that express Ki67 and the graph below shows the mean ± SEM of these values for each group at various time points after infection. Significance was determined by two-tailed Student’s t test (***, P < 0.001; ****, P < 0.0001). Data are representative of three independent experiments with three to five mice per group at each time point.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4419347&req=5

fig3: ESAT-6–specific CD4 T cells cluster into distinct functional subsets. Mice were infected as described in Figure 1 and lung CD4 T cells were analyzed by flow cytometry. (A) Representative flow cytometry plot depicts KLRG1 and PD-1 expression by ESAT-6 tetramer-binding CD4 T cells in the lung 90 d after infection. (B) Single-cell suspensions from the lungs of Mtb-infected mice (150 d after infection) were stimulated with ESAT-64-17 in vitro and intracellular cytokine staining was performed. A representative flow plot depicts KLRG1 and IL-2 expression within CD4 T cells coproducing IFN-γ and TNF (gate not depicted), and numbers in parentheses represent the percentage of KLRG1− (blue) and KLRG1+ (red) cells within this population that express IL-2. The graph shows this value for each mouse and the mean ± SEM for each group. (C) Flow cytometry plot shows direct ex vivo intracellular IFN-γ versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 34 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells, and the graph below shows the mean ± SEM of these values for each group at various time points after infection. (D) Flow cytometry histograms show T-bet expression by naive CD44low (gray), PD-1+KLRG1− (blue), and PD-1−KLRG1+ (red) ESAT-6 tetramer-binding CD4 T cells 90 d after infection. Graph below shows the T-bet mean fluorescent intensity (MFI) for each T cell subset in individual mice, and the mean ± SEM for each group. (E) Flow cytometry plot shows Ki67 versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 70 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells that express Ki67 and the graph below shows the mean ± SEM of these values for each group at various time points after infection. Significance was determined by two-tailed Student’s t test (***, P < 0.001; ****, P < 0.0001). Data are representative of three independent experiments with three to five mice per group at each time point.

Mentions: ESAT-6–specific T cells in the lung can be separated into two distinct populations based on their expression of the inhibitory receptors PD-1 and KLRG1 (Fig. 3 A). Consistent with the findings of a recent report (Reiley et al., 2010), we found that KLRG1+ cells produced more IFN-γ and TNF than their PD-1+ KLRG1− counterparts after in vitro stimulation with ESAT-6 peptide or anti-CD3 and anti-CD28 antibodies (unpublished data). In contrast, KLRG1+ cells produced very little IL-2, a cytokine almost exclusively produced by a population of polyfunctional PD-1+ KLRG1− cells that also produced IFN-γ and TNF (Fig. 3 B and not depicted). No TNF or IL-2 staining was detected in lung T cells using the direct ex vivo approach. However, consistent with the in vitro restimulation data, a higher percentage of KLRG1+ than PD-1+ KLRG1− CD4 T cells within the tetramer-binding population produced IFN-γ when assessed immediately after isolation from the lung (Fig. 3 C). Furthermore, KLRG1+ cells expressed high levels of the Th1-defining transcription factor T-bet, whereas PD-1+KLRG1− cells had intermediate T-bet levels (Fig. 3 D). We also found that PD-1+KLRG1− ESAT-6–specific CD4 T cells exhibited more proliferation (assessed by Ki67 expression) than KLRG1+ cells at time points beyond day 28 after infection (Fig. 3 E). Overall, these results indicate KLRG1+ cells comprise the primary ESAT-6–specific CD4 T cell population producing IFN-γ in vivo, but PD-1+ cells have a higher capacity to produce IL-2 and undergo more extensive proliferation.


ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis.

Moguche AO, Shafiani S, Clemons C, Larson RP, Dinh C, Higdon LE, Cambier CJ, Sissons JR, Gallegos AM, Fink PJ, Urdahl KB - J. Exp. Med. (2015)

ESAT-6–specific CD4 T cells cluster into distinct functional subsets. Mice were infected as described in Figure 1 and lung CD4 T cells were analyzed by flow cytometry. (A) Representative flow cytometry plot depicts KLRG1 and PD-1 expression by ESAT-6 tetramer-binding CD4 T cells in the lung 90 d after infection. (B) Single-cell suspensions from the lungs of Mtb-infected mice (150 d after infection) were stimulated with ESAT-64-17 in vitro and intracellular cytokine staining was performed. A representative flow plot depicts KLRG1 and IL-2 expression within CD4 T cells coproducing IFN-γ and TNF (gate not depicted), and numbers in parentheses represent the percentage of KLRG1− (blue) and KLRG1+ (red) cells within this population that express IL-2. The graph shows this value for each mouse and the mean ± SEM for each group. (C) Flow cytometry plot shows direct ex vivo intracellular IFN-γ versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 34 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells, and the graph below shows the mean ± SEM of these values for each group at various time points after infection. (D) Flow cytometry histograms show T-bet expression by naive CD44low (gray), PD-1+KLRG1− (blue), and PD-1−KLRG1+ (red) ESAT-6 tetramer-binding CD4 T cells 90 d after infection. Graph below shows the T-bet mean fluorescent intensity (MFI) for each T cell subset in individual mice, and the mean ± SEM for each group. (E) Flow cytometry plot shows Ki67 versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 70 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells that express Ki67 and the graph below shows the mean ± SEM of these values for each group at various time points after infection. Significance was determined by two-tailed Student’s t test (***, P < 0.001; ****, P < 0.0001). Data are representative of three independent experiments with three to five mice per group at each time point.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419347&req=5

fig3: ESAT-6–specific CD4 T cells cluster into distinct functional subsets. Mice were infected as described in Figure 1 and lung CD4 T cells were analyzed by flow cytometry. (A) Representative flow cytometry plot depicts KLRG1 and PD-1 expression by ESAT-6 tetramer-binding CD4 T cells in the lung 90 d after infection. (B) Single-cell suspensions from the lungs of Mtb-infected mice (150 d after infection) were stimulated with ESAT-64-17 in vitro and intracellular cytokine staining was performed. A representative flow plot depicts KLRG1 and IL-2 expression within CD4 T cells coproducing IFN-γ and TNF (gate not depicted), and numbers in parentheses represent the percentage of KLRG1− (blue) and KLRG1+ (red) cells within this population that express IL-2. The graph shows this value for each mouse and the mean ± SEM for each group. (C) Flow cytometry plot shows direct ex vivo intracellular IFN-γ versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 34 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells, and the graph below shows the mean ± SEM of these values for each group at various time points after infection. (D) Flow cytometry histograms show T-bet expression by naive CD44low (gray), PD-1+KLRG1− (blue), and PD-1−KLRG1+ (red) ESAT-6 tetramer-binding CD4 T cells 90 d after infection. Graph below shows the T-bet mean fluorescent intensity (MFI) for each T cell subset in individual mice, and the mean ± SEM for each group. (E) Flow cytometry plot shows Ki67 versus KLRG1 expression for ESAT-6 tetramer-binding CD4 T cells 70 d after infection. Numbers in parentheses denote the percentage of KLRG1− (blue) or KLRG1+ (red) cells that express Ki67 and the graph below shows the mean ± SEM of these values for each group at various time points after infection. Significance was determined by two-tailed Student’s t test (***, P < 0.001; ****, P < 0.0001). Data are representative of three independent experiments with three to five mice per group at each time point.
Mentions: ESAT-6–specific T cells in the lung can be separated into two distinct populations based on their expression of the inhibitory receptors PD-1 and KLRG1 (Fig. 3 A). Consistent with the findings of a recent report (Reiley et al., 2010), we found that KLRG1+ cells produced more IFN-γ and TNF than their PD-1+ KLRG1− counterparts after in vitro stimulation with ESAT-6 peptide or anti-CD3 and anti-CD28 antibodies (unpublished data). In contrast, KLRG1+ cells produced very little IL-2, a cytokine almost exclusively produced by a population of polyfunctional PD-1+ KLRG1− cells that also produced IFN-γ and TNF (Fig. 3 B and not depicted). No TNF or IL-2 staining was detected in lung T cells using the direct ex vivo approach. However, consistent with the in vitro restimulation data, a higher percentage of KLRG1+ than PD-1+ KLRG1− CD4 T cells within the tetramer-binding population produced IFN-γ when assessed immediately after isolation from the lung (Fig. 3 C). Furthermore, KLRG1+ cells expressed high levels of the Th1-defining transcription factor T-bet, whereas PD-1+KLRG1− cells had intermediate T-bet levels (Fig. 3 D). We also found that PD-1+KLRG1− ESAT-6–specific CD4 T cells exhibited more proliferation (assessed by Ki67 expression) than KLRG1+ cells at time points beyond day 28 after infection (Fig. 3 E). Overall, these results indicate KLRG1+ cells comprise the primary ESAT-6–specific CD4 T cell population producing IFN-γ in vivo, but PD-1+ cells have a higher capacity to produce IL-2 and undergo more extensive proliferation.

Bottom Line: When transferred into uninfected animals, these cells persist, mount a robust recall response, and provide superior protection to Mtb rechallenge when compared to terminally differentiated Th1 cells that reside preferentially in the lung-associated vasculature.Thus, the molecular pathways required to maintain Mtb-specific CD4 T cells during ongoing infection are similar to those that maintain memory CD4 T cells in scenarios of antigen deprivation.These results suggest that vaccination strategies targeting the ICOS and Bcl6 pathways in CD4 T cells may provide new avenues to prevent TB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Seattle Biomedical Research Institute (renamed Center for Infectious Disease Research), Seattle, WA 98109 Department of Immunology, University of Washington School of Medicine, Seattle, WA 98104.

Show MeSH
Related in: MedlinePlus