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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

Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking. (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.
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fig4: Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking. (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.

Mentions: To test this, we incubated CD4+/Foxp3+ Treg for 72 hours in the presence of HMW-HA, plate-bound CD44 antibody, or IL-2, and examined Foxp3 expression. We observed that cross-linking CD44 receptor through either plate-bound CD44 antibody or HMW-HA promoted Foxp3 expression in CD4+ Tregs in a manner similar to IL-2 (Figure 4(a)).


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)

Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking. (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581548&req=5

fig4: Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking. (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.
Mentions: To test this, we incubated CD4+/Foxp3+ Treg for 72 hours in the presence of HMW-HA, plate-bound CD44 antibody, or IL-2, and examined Foxp3 expression. We observed that cross-linking CD44 receptor through either plate-bound CD44 antibody or HMW-HA promoted Foxp3 expression in CD4+ Tregs in a manner similar to IL-2 (Figure 4(a)).

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