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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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Enhanced proliferation of CD8+ T-lymphocytes in MCMV-infected brains following Treg depletion.(A) Single cell suspensions of brain tissue obtained from infected Foxp3-DTR transgenic mice (2–4 animals per time point) were collected and stained for flow cytometry with PE-Cy5-conjugated Abs specific for CD45, PE-Cy7-labeled for CD8, and Ki67 FITC–conjugated Abs. An isotype Ab for Ki67 FITC was used as gating control. Contour plots showing the proliferation frequency of CD8+ T-cells from infected, untreated (-DTx), and DTx-treated (+DTx) animals at the indicated time points are representative of 2 independent experiments. (B) Pooled data show percentage proliferation of CD8+ T-cells (mean ±SD) in infected brains with and without treatment at the indicated time points. ***p < 0.0001 DTx- versus DTx+ MCMV-infected at 14 dpi. (C) The number of proliferating CD8+ T-cells in infected brains of animals with and without DTx treatment at the indicated time points is shown. **p < 0.001 DTx- versus DTx+ MCMV-infected animals
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pone.0145457.g002: Enhanced proliferation of CD8+ T-lymphocytes in MCMV-infected brains following Treg depletion.(A) Single cell suspensions of brain tissue obtained from infected Foxp3-DTR transgenic mice (2–4 animals per time point) were collected and stained for flow cytometry with PE-Cy5-conjugated Abs specific for CD45, PE-Cy7-labeled for CD8, and Ki67 FITC–conjugated Abs. An isotype Ab for Ki67 FITC was used as gating control. Contour plots showing the proliferation frequency of CD8+ T-cells from infected, untreated (-DTx), and DTx-treated (+DTx) animals at the indicated time points are representative of 2 independent experiments. (B) Pooled data show percentage proliferation of CD8+ T-cells (mean ±SD) in infected brains with and without treatment at the indicated time points. ***p < 0.0001 DTx- versus DTx+ MCMV-infected at 14 dpi. (C) The number of proliferating CD8+ T-cells in infected brains of animals with and without DTx treatment at the indicated time points is shown. **p < 0.001 DTx- versus DTx+ MCMV-infected animals

Mentions: Given the differences in T-lymphocyte number within MCMV-infected brains following Treg depletion, it was important to first evaluate if loss of Tregs altered the proliferation of infiltrating T-lymphocytes in response to MCMV brain infection. We examined single cell suspensions of infected brain tissue with and without the DTx treatment using multi-color flow cytometry. We observed increased proliferation (i.e., Ki67 staining) of CD8+ and CD4+ T-cells within brains of Treg-deficient, MCMV-infected mice when compared to Treg-sufficient animals (Figs 2A and 3A, respectively). Analysis of pooled data showed differences in proliferation were significant for both CD8+ and CD4+ T-cell populations at 14 dpi (P<0.0001 for both CD8+ and CD4+ T-cells), whereas Treg ablation generated no significant differences in either CD8+ and CD4+ T lymphocyte proliferation at 7 dpi. At 30 dpi, differences in CD8+ T-cell proliferation were not detected, but CD4+ T-cell proliferation was significantly elevated (P<0.001) within brains of DTx-treated animals (Figs 2B and 3B, respectively). Evaluation of the absolute numbers of proliferating CD8+ and CD4+ T-cells revealed robust differences between the two groups at each time point tested (Figs 2C and 3C, respectively).


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)

Enhanced proliferation of CD8+ T-lymphocytes in MCMV-infected brains following Treg depletion.(A) Single cell suspensions of brain tissue obtained from infected Foxp3-DTR transgenic mice (2–4 animals per time point) were collected and stained for flow cytometry with PE-Cy5-conjugated Abs specific for CD45, PE-Cy7-labeled for CD8, and Ki67 FITC–conjugated Abs. An isotype Ab for Ki67 FITC was used as gating control. Contour plots showing the proliferation frequency of CD8+ T-cells from infected, untreated (-DTx), and DTx-treated (+DTx) animals at the indicated time points are representative of 2 independent experiments. (B) Pooled data show percentage proliferation of CD8+ T-cells (mean ±SD) in infected brains with and without treatment at the indicated time points. ***p < 0.0001 DTx- versus DTx+ MCMV-infected at 14 dpi. (C) The number of proliferating CD8+ T-cells in infected brains of animals with and without DTx treatment at the indicated time points is shown. **p < 0.001 DTx- versus DTx+ MCMV-infected animals
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pone.0145457.g002: Enhanced proliferation of CD8+ T-lymphocytes in MCMV-infected brains following Treg depletion.(A) Single cell suspensions of brain tissue obtained from infected Foxp3-DTR transgenic mice (2–4 animals per time point) were collected and stained for flow cytometry with PE-Cy5-conjugated Abs specific for CD45, PE-Cy7-labeled for CD8, and Ki67 FITC–conjugated Abs. An isotype Ab for Ki67 FITC was used as gating control. Contour plots showing the proliferation frequency of CD8+ T-cells from infected, untreated (-DTx), and DTx-treated (+DTx) animals at the indicated time points are representative of 2 independent experiments. (B) Pooled data show percentage proliferation of CD8+ T-cells (mean ±SD) in infected brains with and without treatment at the indicated time points. ***p < 0.0001 DTx- versus DTx+ MCMV-infected at 14 dpi. (C) The number of proliferating CD8+ T-cells in infected brains of animals with and without DTx treatment at the indicated time points is shown. **p < 0.001 DTx- versus DTx+ MCMV-infected animals
Mentions: Given the differences in T-lymphocyte number within MCMV-infected brains following Treg depletion, it was important to first evaluate if loss of Tregs altered the proliferation of infiltrating T-lymphocytes in response to MCMV brain infection. We examined single cell suspensions of infected brain tissue with and without the DTx treatment using multi-color flow cytometry. We observed increased proliferation (i.e., Ki67 staining) of CD8+ and CD4+ T-cells within brains of Treg-deficient, MCMV-infected mice when compared to Treg-sufficient animals (Figs 2A and 3A, respectively). Analysis of pooled data showed differences in proliferation were significant for both CD8+ and CD4+ T-cell populations at 14 dpi (P<0.0001 for both CD8+ and CD4+ T-cells), whereas Treg ablation generated no significant differences in either CD8+ and CD4+ T lymphocyte proliferation at 7 dpi. At 30 dpi, differences in CD8+ T-cell proliferation were not detected, but CD4+ T-cell proliferation was significantly elevated (P<0.001) within brains of DTx-treated animals (Figs 2B and 3B, respectively). Evaluation of the absolute numbers of proliferating CD8+ and CD4+ T-cells revealed robust differences between the two groups at each time point tested (Figs 2C and 3C, respectively).

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