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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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Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation.Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8+ T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8+ T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4+ T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8+ T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4+ T-cells with and without DTx treatment is shown. **p < 0.001 DTx- versus DTx+ animals
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pone.0145457.g004: Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation.Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8+ T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8+ T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4+ T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8+ T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4+ T-cells with and without DTx treatment is shown. **p < 0.001 DTx- versus DTx+ animals

Mentions: Discrimination between brain tissue-localized lymphocytes and those within the vasculature is important not only to evaluate immune cells within the brain parenchyma, but also to understand the role of these cells within the vessels. Because we first observed the kinetics of lymphocyte proliferation in whole brain tissue obtained from DTx-treated and untreated animals, we went on to characterized lymphocyte proliferation status within the parenchyma. For this we adapted a method of intravascular staining previously developed by other investigators [29, 31]. Using this approach, we employed, MCMV-infected, DTx treated and untreated FoxP3-DTR mice animals at 14 dpi when infiltration of leukocytes into the brain is negligible. The vascular population of CD8+ T-cells were identified by dual staining with anti–CD8a-PE (clone 53–6.7), administered by intravenous injection, and anti-CD8a-AF647 (clone YTS156.7.7) stained ex-vivo. Similarly, CD4+ T -cells were distinguished by the co-stained population using anti-CD4-AF700 (clone RM4-5) and anti–CD4-FITC (clone RM4-4). Leukocytes residing within the brain were identified by ex-vivo single staining (Fig 4A). Next, the distinguished populations of vascular and tissue lymphocytes were further characterized for their proliferation by Ki67 staining. In these studies, we observed that 35.5% of CD8+ T-cells and 10.6% of CD4+ T- cells in the brain tissue; and 32.6% of CD8+ T cells and 40.5% of CD4+ T- cells in the vasculature were positive for Ki67 in DTx-untreated animals. The lymphocytes in DTX-treated animals showed higher levels of proliferation with 59.5% of CD8+ T- cells and 39.6% of CD4+ T-cells in the brain tissue, and 42.6% of CD8+ T cells and 62.5% of CD4+ T- cells in the vasculature expressing Ki67 (Fig 4B and 4C). Thus, these data indicate that both CD8+ and CD4+ T lymphocytes proliferated within the brain parenchyma upon viral infection, with CD8+ T-cells showing higher levels. We also examined the number of parenchyma-localized CD8+ and CD4+ T lymphocyte in Treg-deficient and Treg-sufficient animals. Analysis of pooled data (n = 6 animals per group, two different experiments) revealed elevated levels of both types of T-cells in DTx treated animals when compared to untreated mice (Fig 4D and 4E).


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)

Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation.Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8+ T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8+ T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4+ T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8+ T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4+ T-cells with and without DTx treatment is shown. **p < 0.001 DTx- versus DTx+ animals
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4697843&req=5

pone.0145457.g004: Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation.Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained ex-vivo for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8+ T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8+ T-cells both within the tissue and in the vasculature. C. Contour plots represent proliferation of CD4+ T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8+ T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4+ T-cells with and without DTx treatment is shown. **p < 0.001 DTx- versus DTx+ animals
Mentions: Discrimination between brain tissue-localized lymphocytes and those within the vasculature is important not only to evaluate immune cells within the brain parenchyma, but also to understand the role of these cells within the vessels. Because we first observed the kinetics of lymphocyte proliferation in whole brain tissue obtained from DTx-treated and untreated animals, we went on to characterized lymphocyte proliferation status within the parenchyma. For this we adapted a method of intravascular staining previously developed by other investigators [29, 31]. Using this approach, we employed, MCMV-infected, DTx treated and untreated FoxP3-DTR mice animals at 14 dpi when infiltration of leukocytes into the brain is negligible. The vascular population of CD8+ T-cells were identified by dual staining with anti–CD8a-PE (clone 53–6.7), administered by intravenous injection, and anti-CD8a-AF647 (clone YTS156.7.7) stained ex-vivo. Similarly, CD4+ T -cells were distinguished by the co-stained population using anti-CD4-AF700 (clone RM4-5) and anti–CD4-FITC (clone RM4-4). Leukocytes residing within the brain were identified by ex-vivo single staining (Fig 4A). Next, the distinguished populations of vascular and tissue lymphocytes were further characterized for their proliferation by Ki67 staining. In these studies, we observed that 35.5% of CD8+ T-cells and 10.6% of CD4+ T- cells in the brain tissue; and 32.6% of CD8+ T cells and 40.5% of CD4+ T- cells in the vasculature were positive for Ki67 in DTx-untreated animals. The lymphocytes in DTX-treated animals showed higher levels of proliferation with 59.5% of CD8+ T- cells and 39.6% of CD4+ T-cells in the brain tissue, and 42.6% of CD8+ T cells and 62.5% of CD4+ T- cells in the vasculature expressing Ki67 (Fig 4B and 4C). Thus, these data indicate that both CD8+ and CD4+ T lymphocytes proliferated within the brain parenchyma upon viral infection, with CD8+ T-cells showing higher levels. We also examined the number of parenchyma-localized CD8+ and CD4+ T lymphocyte in Treg-deficient and Treg-sufficient animals. Analysis of pooled data (n = 6 animals per group, two different experiments) revealed elevated levels of both types of T-cells in DTx treated animals when compared to untreated mice (Fig 4D and 4E).

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