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Virus-specific regulatory T cells ameliorate encephalitis by repressing effector T cell functions from priming to effector stages.

Zhao J, Zhao J, Perlman S - PLoS Pathog. (2014)

Bottom Line: In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain.Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance.These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
Several studies have demonstrated the presence of pathogen-specific Foxp3+ CD4 regulatory T cells (Treg) in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg) and conventional CD4 T cells (M133 Tconv) in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN). Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.

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M133 Tregs inhibit M133 Tconv proliferation in, and egress from, the DCLN.M133 Tconv or M133 Tconv and M133 Tregs (1∶2 ratio) were labeled with Violet and transferred into Thy1 congenic mice prior to rJ2.2 infection as described in Figure 5A. Organs were harvested at the indicated time points and lymphocyte populations examined directly ex vivo. (A) Representative plots showing gating of transferred M133 Tconv (GFP−) in the absence (black) or presence of (red) co-transferred M133 Tregs, and of M133 Tregs (GFP+, blue). These gates and colors were applied in B–E. (B) Representative dot plots show CD69 and CD25 expression on M133 Tconv and M133 Tregs in DCLN at day 3 p.i. Boxed cells are positive for CD69 or CD25 expression. Summary data show expression levels of CD69 and CD25 on undivided and divided M133 Tconv. (C) Overlayed histograms showing proliferation of M133 Tconv in the absence or presence of co-transferred M133 Tregs in DCLN and spleen at days 3, 4 and 5 p.i. Expansion index (EI) of M133 Tconv was calculated using Flowjo software. (D) Apoptosis of M133 Tconv in the DCLN at day 4 p.i. was analyzed by Annexin V/7-AAD staining. (E) CXCR3 and Tbet levels on M133 Tconv and M133 Tregs in DCLN at day 4 p.i. The data in B-E are representative of 2–6 independent experiments with 3–5 mice/time point. *P<0.05, ***P<0.001.
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ppat-1004279-g006: M133 Tregs inhibit M133 Tconv proliferation in, and egress from, the DCLN.M133 Tconv or M133 Tconv and M133 Tregs (1∶2 ratio) were labeled with Violet and transferred into Thy1 congenic mice prior to rJ2.2 infection as described in Figure 5A. Organs were harvested at the indicated time points and lymphocyte populations examined directly ex vivo. (A) Representative plots showing gating of transferred M133 Tconv (GFP−) in the absence (black) or presence of (red) co-transferred M133 Tregs, and of M133 Tregs (GFP+, blue). These gates and colors were applied in B–E. (B) Representative dot plots show CD69 and CD25 expression on M133 Tconv and M133 Tregs in DCLN at day 3 p.i. Boxed cells are positive for CD69 or CD25 expression. Summary data show expression levels of CD69 and CD25 on undivided and divided M133 Tconv. (C) Overlayed histograms showing proliferation of M133 Tconv in the absence or presence of co-transferred M133 Tregs in DCLN and spleen at days 3, 4 and 5 p.i. Expansion index (EI) of M133 Tconv was calculated using Flowjo software. (D) Apoptosis of M133 Tconv in the DCLN at day 4 p.i. was analyzed by Annexin V/7-AAD staining. (E) CXCR3 and Tbet levels on M133 Tconv and M133 Tregs in DCLN at day 4 p.i. The data in B-E are representative of 2–6 independent experiments with 3–5 mice/time point. *P<0.05, ***P<0.001.

Mentions: To probe this in more detail, M133 Tconv activation and proliferation, based on Violet dilution, was analyzed with or without co-transferred M133 Tregs (Figure 6). M133 Tconv were gated as shown in Figure 6A. Activation was assessed by measuring levels of CD25 and CD69 on Tconv in the DCLN at day 3 p.i. (Figure 6B). Equivalent levels of CD25 and CD69 were detected on undivided M133 Tconv. However, even though expression of CD25 and CD69 followed similar kinetics in the presence or absence of Tregs, levels of both molecules were lower in the presence of Tregs after cells began to divide. These results suggest that the initial activation of M133 Tconv was not affected by the presence of Tregs, perhaps because of the lag in Treg relative to Tconv activation, shown in Figure 3. However, as M133 Tregs became activated and proliferated, they functioned to downregulate both molecules on Tconv. Of note, CD69 levels were lower on M133 Tregs than on M133 Tconv in mice that received both types of cells, while, as expected, CD25 levels were higher on M133 Tregs (Figure 6B, blue bars).


Virus-specific regulatory T cells ameliorate encephalitis by repressing effector T cell functions from priming to effector stages.

Zhao J, Zhao J, Perlman S - PLoS Pathog. (2014)

M133 Tregs inhibit M133 Tconv proliferation in, and egress from, the DCLN.M133 Tconv or M133 Tconv and M133 Tregs (1∶2 ratio) were labeled with Violet and transferred into Thy1 congenic mice prior to rJ2.2 infection as described in Figure 5A. Organs were harvested at the indicated time points and lymphocyte populations examined directly ex vivo. (A) Representative plots showing gating of transferred M133 Tconv (GFP−) in the absence (black) or presence of (red) co-transferred M133 Tregs, and of M133 Tregs (GFP+, blue). These gates and colors were applied in B–E. (B) Representative dot plots show CD69 and CD25 expression on M133 Tconv and M133 Tregs in DCLN at day 3 p.i. Boxed cells are positive for CD69 or CD25 expression. Summary data show expression levels of CD69 and CD25 on undivided and divided M133 Tconv. (C) Overlayed histograms showing proliferation of M133 Tconv in the absence or presence of co-transferred M133 Tregs in DCLN and spleen at days 3, 4 and 5 p.i. Expansion index (EI) of M133 Tconv was calculated using Flowjo software. (D) Apoptosis of M133 Tconv in the DCLN at day 4 p.i. was analyzed by Annexin V/7-AAD staining. (E) CXCR3 and Tbet levels on M133 Tconv and M133 Tregs in DCLN at day 4 p.i. The data in B-E are representative of 2–6 independent experiments with 3–5 mice/time point. *P<0.05, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125232&req=5

ppat-1004279-g006: M133 Tregs inhibit M133 Tconv proliferation in, and egress from, the DCLN.M133 Tconv or M133 Tconv and M133 Tregs (1∶2 ratio) were labeled with Violet and transferred into Thy1 congenic mice prior to rJ2.2 infection as described in Figure 5A. Organs were harvested at the indicated time points and lymphocyte populations examined directly ex vivo. (A) Representative plots showing gating of transferred M133 Tconv (GFP−) in the absence (black) or presence of (red) co-transferred M133 Tregs, and of M133 Tregs (GFP+, blue). These gates and colors were applied in B–E. (B) Representative dot plots show CD69 and CD25 expression on M133 Tconv and M133 Tregs in DCLN at day 3 p.i. Boxed cells are positive for CD69 or CD25 expression. Summary data show expression levels of CD69 and CD25 on undivided and divided M133 Tconv. (C) Overlayed histograms showing proliferation of M133 Tconv in the absence or presence of co-transferred M133 Tregs in DCLN and spleen at days 3, 4 and 5 p.i. Expansion index (EI) of M133 Tconv was calculated using Flowjo software. (D) Apoptosis of M133 Tconv in the DCLN at day 4 p.i. was analyzed by Annexin V/7-AAD staining. (E) CXCR3 and Tbet levels on M133 Tconv and M133 Tregs in DCLN at day 4 p.i. The data in B-E are representative of 2–6 independent experiments with 3–5 mice/time point. *P<0.05, ***P<0.001.
Mentions: To probe this in more detail, M133 Tconv activation and proliferation, based on Violet dilution, was analyzed with or without co-transferred M133 Tregs (Figure 6). M133 Tconv were gated as shown in Figure 6A. Activation was assessed by measuring levels of CD25 and CD69 on Tconv in the DCLN at day 3 p.i. (Figure 6B). Equivalent levels of CD25 and CD69 were detected on undivided M133 Tconv. However, even though expression of CD25 and CD69 followed similar kinetics in the presence or absence of Tregs, levels of both molecules were lower in the presence of Tregs after cells began to divide. These results suggest that the initial activation of M133 Tconv was not affected by the presence of Tregs, perhaps because of the lag in Treg relative to Tconv activation, shown in Figure 3. However, as M133 Tregs became activated and proliferated, they functioned to downregulate both molecules on Tconv. Of note, CD69 levels were lower on M133 Tregs than on M133 Tconv in mice that received both types of cells, while, as expected, CD25 levels were higher on M133 Tregs (Figure 6B, blue bars).

Bottom Line: In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain.Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance.These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
Several studies have demonstrated the presence of pathogen-specific Foxp3+ CD4 regulatory T cells (Treg) in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg) and conventional CD4 T cells (M133 Tconv) in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN). Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.

Show MeSH
Related in: MedlinePlus