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Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways.

Ruppert SM, Falk BA, Long SA, Bollyky PL - Int J Cell Biol (2015)

Bottom Line: We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment.This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation.Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Geographic Medicine, Department of Medicine, 300 Pasteur Drive, Stanford University School of Medicine, Stanford, CA 94305-5107, USA.

ABSTRACT
Cyclosporine A (CSA) is an immunosuppressive agent that specifically targets T cells and also increases the percentage of pro-tolerogenic CD4+Foxp3+ regulatory T cells (Treg) through unknown mechanisms. We previously reported that CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA), promotes Treg stability in IL-2-low environments. Here, we asked whether CD44 signaling also promotes Treg resistance to CSA. We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment. This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation. Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking. Together, these data support a model whereby CD44 cross-linking by HA promotes IL-2-independent Foxp3 expression and Treg survival in the face of CSA.

No MeSH data available.


Related in: MedlinePlus

CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included Figure 2(a). (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in Figure 2(a) and the other experiments are in Figure 2(b).
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fig2: CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included Figure 2(a). (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in Figure 2(a) and the other experiments are in Figure 2(b).

Mentions: We observed that Foxp3 and CD25 expression was abrogated by treatment with CSA but that both CD44 cross-linking and supplementation with 100 IU/mL of IL-2 maintained Foxp3 expression. Indeed, CD44 cross-linking and IL-2 supplementation were roughly equivalent in their ability to promote Treg homeostasis in the setting of CSA (Figures 2(a) and 2(b)). Of note, this beneficial effect of CD44 cross-linking on Foxp3 levels was predicated on the concomitant presence of TCR signals; CD44 cross-linking in the absence of aCD3/28 did not promote resistance to CSA (data not shown).


Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways.

Ruppert SM, Falk BA, Long SA, Bollyky PL - Int J Cell Biol (2015)

CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included Figure 2(a). (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in Figure 2(a) and the other experiments are in Figure 2(b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included Figure 2(a). (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in Figure 2(a) and the other experiments are in Figure 2(b).
Mentions: We observed that Foxp3 and CD25 expression was abrogated by treatment with CSA but that both CD44 cross-linking and supplementation with 100 IU/mL of IL-2 maintained Foxp3 expression. Indeed, CD44 cross-linking and IL-2 supplementation were roughly equivalent in their ability to promote Treg homeostasis in the setting of CSA (Figures 2(a) and 2(b)). Of note, this beneficial effect of CD44 cross-linking on Foxp3 levels was predicated on the concomitant presence of TCR signals; CD44 cross-linking in the absence of aCD3/28 did not promote resistance to CSA (data not shown).

Bottom Line: We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment.This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation.Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Geographic Medicine, Department of Medicine, 300 Pasteur Drive, Stanford University School of Medicine, Stanford, CA 94305-5107, USA.

ABSTRACT
Cyclosporine A (CSA) is an immunosuppressive agent that specifically targets T cells and also increases the percentage of pro-tolerogenic CD4+Foxp3+ regulatory T cells (Treg) through unknown mechanisms. We previously reported that CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA), promotes Treg stability in IL-2-low environments. Here, we asked whether CD44 signaling also promotes Treg resistance to CSA. We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment. This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation. Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking. Together, these data support a model whereby CD44 cross-linking by HA promotes IL-2-independent Foxp3 expression and Treg survival in the face of CSA.

No MeSH data available.


Related in: MedlinePlus