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Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection.

Prasad S, Hu S, Sheng WS, Singh A, Lokensgard JR - PLoS ONE (2015)

Bottom Line: Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells.These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion.Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Laboratory, Center for Infectious Diseases and Microbiology Translational Research, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, 55455, United States of America.

ABSTRACT
Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8+ and CD4+ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8+ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4+ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8+ and CD4+ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion. Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as TRM cells.

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Tregs limit IFN-γ production by T lymphocytes within MCMV-infected brains.Single cell suspension of brain tissue obtained from MCMV-infected DTx-treated and untreated mice (2–4 animals per time point) were banded on a 70% percoll cushion. Brain leukocytes at the 30–70% percoll interface were collected. For intracellular IFN-γ staining, brain leukocytes (2 x 106 cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with a MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots shows the ratio of CD8+ T lymphocytes producing IFN-γ among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8+ T-cells producing IFN-γ at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30 dpi.
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pone.0145457.g006: Tregs limit IFN-γ production by T lymphocytes within MCMV-infected brains.Single cell suspension of brain tissue obtained from MCMV-infected DTx-treated and untreated mice (2–4 animals per time point) were banded on a 70% percoll cushion. Brain leukocytes at the 30–70% percoll interface were collected. For intracellular IFN-γ staining, brain leukocytes (2 x 106 cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with a MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots shows the ratio of CD8+ T lymphocytes producing IFN-γ among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8+ T-cells producing IFN-γ at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30 dpi.

Mentions: Studies from our laboratory have previously demonstrated that MCMV brain infection results in long-term persistence of antigen-specific CD8+ T-cells which may contribute to chronic microglial cell activation [4]. To further investigate the role of Tregs and to assess their significance, we then studied the functional ability of CD8+ T-cells to respond to re-stimulation. This was done by performing intracellular cytokine staining to evaluate cytokine production by cells specific for the MCMV peptide M45, which has previously been identified as MCMV-specific major histocompatibility complex (MHC) class I restricted epitope [27, 28]. Brain mononuclear cells derived from infected brain at different time points were incubated with either CD3/CD28 antibodies for control, or MCMV M45 peptide for 5 h, then subjected to intracellular staining for IFN-γ and TNF-α after immunostaining for appropriate surface markers. In these studies, the frequency of CD8+ T-cells producing IFN-γ and TNF-α was found to be increased in Treg-ablated, MCMV-infected mice when compared to Treg sufficient animals (Figs 6A and 7A). These differences reached statistical significance at each time point for IFN-γ (P<0.001, Fig 6B), and at 14 and 30 dpi for TNF-α (P<0.001, Fig 7B). Thus, these data show elevated proinflammatory cytokine production following Treg depletion. Additionally, we also evaluated the number of CD8+ T-cells that were polyfunctional in terms of their ability to produce both IFN-γ and TNF-α. We found that in Treg-deficient mice, there were more CD8+ T-cells displaying dual cytokine production when compared to Treg-sufficient animals (Fig 8). The differences were found to be significant at each time point, indicating a more proinflammatory environment in Treg-deficient animals.


Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection.

Prasad S, Hu S, Sheng WS, Singh A, Lokensgard JR - PLoS ONE (2015)

Tregs limit IFN-γ production by T lymphocytes within MCMV-infected brains.Single cell suspension of brain tissue obtained from MCMV-infected DTx-treated and untreated mice (2–4 animals per time point) were banded on a 70% percoll cushion. Brain leukocytes at the 30–70% percoll interface were collected. For intracellular IFN-γ staining, brain leukocytes (2 x 106 cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with a MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots shows the ratio of CD8+ T lymphocytes producing IFN-γ among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8+ T-cells producing IFN-γ at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30 dpi.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697843&req=5

pone.0145457.g006: Tregs limit IFN-γ production by T lymphocytes within MCMV-infected brains.Single cell suspension of brain tissue obtained from MCMV-infected DTx-treated and untreated mice (2–4 animals per time point) were banded on a 70% percoll cushion. Brain leukocytes at the 30–70% percoll interface were collected. For intracellular IFN-γ staining, brain leukocytes (2 x 106 cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with a MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots shows the ratio of CD8+ T lymphocytes producing IFN-γ among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8+ T-cells producing IFN-γ at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30 dpi.
Mentions: Studies from our laboratory have previously demonstrated that MCMV brain infection results in long-term persistence of antigen-specific CD8+ T-cells which may contribute to chronic microglial cell activation [4]. To further investigate the role of Tregs and to assess their significance, we then studied the functional ability of CD8+ T-cells to respond to re-stimulation. This was done by performing intracellular cytokine staining to evaluate cytokine production by cells specific for the MCMV peptide M45, which has previously been identified as MCMV-specific major histocompatibility complex (MHC) class I restricted epitope [27, 28]. Brain mononuclear cells derived from infected brain at different time points were incubated with either CD3/CD28 antibodies for control, or MCMV M45 peptide for 5 h, then subjected to intracellular staining for IFN-γ and TNF-α after immunostaining for appropriate surface markers. In these studies, the frequency of CD8+ T-cells producing IFN-γ and TNF-α was found to be increased in Treg-ablated, MCMV-infected mice when compared to Treg sufficient animals (Figs 6A and 7A). These differences reached statistical significance at each time point for IFN-γ (P<0.001, Fig 6B), and at 14 and 30 dpi for TNF-α (P<0.001, Fig 7B). Thus, these data show elevated proinflammatory cytokine production following Treg depletion. Additionally, we also evaluated the number of CD8+ T-cells that were polyfunctional in terms of their ability to produce both IFN-γ and TNF-α. We found that in Treg-deficient mice, there were more CD8+ T-cells displaying dual cytokine production when compared to Treg-sufficient animals (Fig 8). The differences were found to be significant at each time point, indicating a more proinflammatory environment in Treg-deficient animals.

Bottom Line: Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells.These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion.Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Laboratory, Center for Infectious Diseases and Microbiology Translational Research, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, 55455, United States of America.

ABSTRACT
Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8+ and CD4+ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8+ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4+ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8+ and CD4+ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion. Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as TRM cells.

Show MeSH
Related in: MedlinePlus