Journal: Scientific Reports

Article Title: Attenuation of cGVHD by C5a/C5aR blockade is associated with increased frequency of Treg

doi: 10.1038/s41598-017-03700-1

Figure Lengend Snippet: Expression of C5aR is increased and is associated with Treg reduction in cGVHD patients. ( A ) Representative flow cytometry plots showed C5aR expression in CD14+ monocytes from healthy controls, non-GVHD patients, or cGVHD patients. The statistical results revealed that C5aR expression was significantly up-regulated on the monocytes in cGVHD patients compared with healthy controls as well as non-GVHD patients who had undergone HSCT. ( B ) The lymphocytes were gated, and C5aR expression on these cells was also markedly increased in cGVHD groups. ( C ) Representative flow cytometry plots showed CD25 and Foxp3 expression in CD4+ cells from healthy controls, non-GVHD patients, or cGVHD patients. ( D ) Spearman’s correlation coefficient analysis revealed no significant correlation between C5aR expression on monocytes and the frequency of Treg, although the two parameters appeared to be negatively related. ( E ) There was a significant negative correlation between C5aR expression on lymphocytes and the frequency of Treg. * p < 0.05, ** p < 0.005, *** p < 0.001.

Article Snippet: Sections were incubated with rabbit anti-Foxp3 primary antibody (diluted 1:200 in blocking buffer, Abcam) overnight at 4 °C.

Techniques: Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Attenuation of cGVHD by C5a/C5aR blockade is associated with increased frequency of Treg

doi: 10.1038/s41598-017-03700-1

Figure Lengend Snippet: C5aR deficiency promoted the development of Treg, whereas C5a activation abolished the occurrence of Treg cell in vitro . ( A , upper panel) Splenocytes from the C5aR deficient mice and WT mice were isolated and analyzed directly for the frequency of Treg. Representative flow cytometry plots showed CD25 and Foxp3 expression in CD4+ cells. ( A , lower panel) CD4+ T cells from the C5aR deficient mice and WT mice were sorted and stimulated in the Treg polarization condition. Representative flow cytometry plots showed CD25 and Foxp3 expression in sorted CD4+ cells from WT and C5aR−/− mice. ( B , C ) The cultured splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days. The frequency of Treg was reduced in response to 100 ng/ml or 500 ng/ml mC5a stimulation. However, the splenocytes from the C5aR-deficient mice were activated and inclined to differentiate toward CD4 + CD25 + Foxp3 + Treg. ( D ) The supernatant protein level of TGF-β1 was decreased in the mC5a stimulation group, whereas it was significantly increased in the C5aR−/− splenocytes. At least five mice were used in flow cytometry experiment (N = 5) and there were at least three cell samples (N = 3) in ( B–D ); n.s. = no significance, * p < 0.05, ** p < 0.005, *** p < 0.001.

Article Snippet: Sections were incubated with rabbit anti-Foxp3 primary antibody (diluted 1:200 in blocking buffer, Abcam) overnight at 4 °C.

Techniques: Activation Assay, In Vitro, Isolation, Flow Cytometry, Expressing, Cell Culture

Journal: Scientific Reports

Article Title: Attenuation of cGVHD by C5a/C5aR blockade is associated with increased frequency of Treg

doi: 10.1038/s41598-017-03700-1

Figure Lengend Snippet: PMX53 treatment promoted the development of Treg and increased the accompanied expression of TGF-β1. ( A ) Representative flow cytometry plots showed C5aR expression in monocytes from cGVHD mice with or without PMX53 treatment. ( B ) ELISA result revealed that PMX53 treatment reduced the level of C5a in plasma from cGVHD mice. ( C ) Representative flow cytometry plots showed CD25 and Foxp3 expression in CD4+ cells from cGVHD mice with or without PMX53 treatment. ( D ) Expression of Foxp3, a key transcription factor in Treg, was increased in histological sections of spleen from cGVHD mice after PMX53 treatment. ( E ) PMX53 treatment also promoted the expression of TGF-β1 in the plasma in cGVHD mice compared with the control group. At least five mice were used in each group for flow cytometry and ELISA (N = 5) and three mice were used for histological analysis (N = 3), * p < 0.05, *** p < 0.001. Scale bar: 200 μm.

Article Snippet: Sections were incubated with rabbit anti-Foxp3 primary antibody (diluted 1:200 in blocking buffer, Abcam) overnight at 4 °C.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay