Limits...
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.

Show MeSH

Related in: MedlinePlus

Treg proliferation and activation during MCMV infection.(A) Brain tissue from MCMV-infected, DTx-treated and untreated Foxp3-DTR transgenic mice were collected at 30 dpi. Brain-infiltrating leukocytes were isolated and stained for flow cytometry. CD4+ T-cells were gated on from the CD45+CD11blow leukocyte population for analysis of Foxp3 expression. Isotype control Abs for Foxp3 was used as a gating control. Plots show Ki67-positive and ICOS-expressing Tregs, and are representative of two separate experiments using four animals/time point. (B) Data show the MFI of Ki67-expressing Tregs from the DTx-treated and untreated groups at the indicated time points from 2 independent experiments. **p = 0.0047 DTx- versus DTx+ MCMV-infected at 30 dpi. (C) Pooled data present the MFI of ICOS expression on Tregs between groups at the indicated time points. *p = 0.0165 DTx- versus DTx+ MCMV-infected at 30 dpi. (D) The number of ICOS+ Treg cells in infected brains with and without DTx treatment at the indicated time points is shown. E. The number of Ki67+Treg cells in infected brains with and without DTx treatment at 7, 14, & 30 dpi is shown. **p < 0.001 DTx- versus DTx+
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697843&req=5

pone.0145457.g009: Treg proliferation and activation during MCMV infection.(A) Brain tissue from MCMV-infected, DTx-treated and untreated Foxp3-DTR transgenic mice were collected at 30 dpi. Brain-infiltrating leukocytes were isolated and stained for flow cytometry. CD4+ T-cells were gated on from the CD45+CD11blow leukocyte population for analysis of Foxp3 expression. Isotype control Abs for Foxp3 was used as a gating control. Plots show Ki67-positive and ICOS-expressing Tregs, and are representative of two separate experiments using four animals/time point. (B) Data show the MFI of Ki67-expressing Tregs from the DTx-treated and untreated groups at the indicated time points from 2 independent experiments. **p = 0.0047 DTx- versus DTx+ MCMV-infected at 30 dpi. (C) Pooled data present the MFI of ICOS expression on Tregs between groups at the indicated time points. *p = 0.0165 DTx- versus DTx+ MCMV-infected at 30 dpi. (D) The number of ICOS+ Treg cells in infected brains with and without DTx treatment at the indicated time points is shown. E. The number of Ki67+Treg cells in infected brains with and without DTx treatment at 7, 14, & 30 dpi is shown. **p < 0.001 DTx- versus DTx+

Mentions: Previous findings have demonstrated that Tregs infiltrate the CNS between 4 and 7 d following MCMV brain infection, indicating that accumulation of these cells may play a regulatory role in controlling immunopathogenesis. We next investigated the expansion and activation of the Treg cells themselves in response to MCMV brain infection. Data obtained show that Tregs within the brains of infected animals without DTx-treatment expanded gradually from the acute phase through chronic phase of infection. However, Treg cell proliferation was found to be more rapid during the acute phase infection when the percentage of Tregs was low. Proliferation of Treg cells as indicated by Ki67 staining was found to decrease by 30 dpi in DTx-untreated animals. In contrast, when Tregs were ablated during acute infection, their numbers rebounded by 14–20 dpi, with significantly increased proliferation at d 30 p.i. (P = 0.0047), (Fig 9A and 9B). Similarly, marked differences in the expression level of ICOS were observed between DTx-treated and untreated groups, indicating that Tregs which repopulate the brain were highly activated (Fig 9A and 9C). In contrast, in untreated groups ICOS expression on Tregs progressively decreased by 30 dpi (P = 0.0165). In addition, we evaluated the number of Treg cells that were activated and proliferative. We observed that in DTx-untreated animals, ICOS expressing Treg cells were present within the brain during the acute phase of infection, the number of which decreased by 30 dpi. Similar observations were made in proliferating Treg cells. In contrast, the number of activating and proliferating Treg cells which rebounded by 30 dpi was significant higher in DTx-treated animals (Fig 9D and 9E).


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)

Treg proliferation and activation during MCMV infection.(A) Brain tissue from MCMV-infected, DTx-treated and untreated Foxp3-DTR transgenic mice were collected at 30 dpi. Brain-infiltrating leukocytes were isolated and stained for flow cytometry. CD4+ T-cells were gated on from the CD45+CD11blow leukocyte population for analysis of Foxp3 expression. Isotype control Abs for Foxp3 was used as a gating control. Plots show Ki67-positive and ICOS-expressing Tregs, and are representative of two separate experiments using four animals/time point. (B) Data show the MFI of Ki67-expressing Tregs from the DTx-treated and untreated groups at the indicated time points from 2 independent experiments. **p = 0.0047 DTx- versus DTx+ MCMV-infected at 30 dpi. (C) Pooled data present the MFI of ICOS expression on Tregs between groups at the indicated time points. *p = 0.0165 DTx- versus DTx+ MCMV-infected at 30 dpi. (D) The number of ICOS+ Treg cells in infected brains with and without DTx treatment at the indicated time points is shown. E. The number of Ki67+Treg cells in infected brains with and without DTx treatment at 7, 14, & 30 dpi is shown. **p < 0.001 DTx- versus DTx+
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145457.g009: Treg proliferation and activation during MCMV infection.(A) Brain tissue from MCMV-infected, DTx-treated and untreated Foxp3-DTR transgenic mice were collected at 30 dpi. Brain-infiltrating leukocytes were isolated and stained for flow cytometry. CD4+ T-cells were gated on from the CD45+CD11blow leukocyte population for analysis of Foxp3 expression. Isotype control Abs for Foxp3 was used as a gating control. Plots show Ki67-positive and ICOS-expressing Tregs, and are representative of two separate experiments using four animals/time point. (B) Data show the MFI of Ki67-expressing Tregs from the DTx-treated and untreated groups at the indicated time points from 2 independent experiments. **p = 0.0047 DTx- versus DTx+ MCMV-infected at 30 dpi. (C) Pooled data present the MFI of ICOS expression on Tregs between groups at the indicated time points. *p = 0.0165 DTx- versus DTx+ MCMV-infected at 30 dpi. (D) The number of ICOS+ Treg cells in infected brains with and without DTx treatment at the indicated time points is shown. E. The number of Ki67+Treg cells in infected brains with and without DTx treatment at 7, 14, & 30 dpi is shown. **p < 0.001 DTx- versus DTx+
Mentions: Previous findings have demonstrated that Tregs infiltrate the CNS between 4 and 7 d following MCMV brain infection, indicating that accumulation of these cells may play a regulatory role in controlling immunopathogenesis. We next investigated the expansion and activation of the Treg cells themselves in response to MCMV brain infection. Data obtained show that Tregs within the brains of infected animals without DTx-treatment expanded gradually from the acute phase through chronic phase of infection. However, Treg cell proliferation was found to be more rapid during the acute phase infection when the percentage of Tregs was low. Proliferation of Treg cells as indicated by Ki67 staining was found to decrease by 30 dpi in DTx-untreated animals. In contrast, when Tregs were ablated during acute infection, their numbers rebounded by 14–20 dpi, with significantly increased proliferation at d 30 p.i. (P = 0.0047), (Fig 9A and 9B). Similarly, marked differences in the expression level of ICOS were observed between DTx-treated and untreated groups, indicating that Tregs which repopulate the brain were highly activated (Fig 9A and 9C). In contrast, in untreated groups ICOS expression on Tregs progressively decreased by 30 dpi (P = 0.0165). In addition, we evaluated the number of Treg cells that were activated and proliferative. We observed that in DTx-untreated animals, ICOS expressing Treg cells were present within the brain during the acute phase of infection, the number of which decreased by 30 dpi. Similar observations were made in proliferating Treg cells. In contrast, the number of activating and proliferating Treg cells which rebounded by 30 dpi was significant higher in DTx-treated animals (Fig 9D and 9E).

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