Figure 7

Differentiation of CD45.1⁺Lin⁻ cells into CD115⁺ MDSCs in inflamed skin.

(a) Flow cytometric analysis of CD45.1⁺ cells in the inflamed skin and BM of recipients of CD45.1⁺Lin⁻ or Lin⁺ cells on day 7 post-transplantation. CD45.1⁺ cells were analyzed by flow cytometry and representative data are shown. Percentage values of CD115⁺ cells within the CD11b⁺Ly6G^intLy6C⁺ and CD11b⁺Ly6G^hiLy6C⁺ cell populations are plotted. (b,c) Immune suppression assays of CD45.1⁺ cells isolated from the inflamed skin or BM (b), or those from skin in the presence (+) or absence (−) of L-NMMA (1 mM). CFSE dilution by CFSE-labeled CD3⁺ T cells was analyzed via flow cytometry after stimulation in the presence sorted CD45.1⁺ cells. (d) qRT-PCR analysis of CD45.1⁺ cells isolated from the skin and BM. Relative levels of transcripts expressed by CD45.1⁺ cells in the BM and skin of mice transplanted with Lin⁻ cells and Lin⁺ cells were compared. Delta-delta Ct values were normalized to those obtained from amplification of β-actin and were expressed as fold changes compared with gene profiles of Lin⁻ cells in inflamed skin. (e) Flow cytometric analysis for expression of CD11b and NOS2 by CD45.1⁺ cells in the inflamed skin and BM of recipients of CD45.1⁺Lin⁻ cells on day 7. Representative flow cytometric data are shown and the percentage values of NOS2⁺ or arginase-1⁺ cells within the CD45.1⁺ cell populations are plotted. (f) qRT-PCR analysis using RNA extracted from the inflamed skin of Lin⁻ or Lin⁺ cell recipients, along with negative control mice that received PBS (no Tpl; no transplantation). Relative transcript levels are shown. Normalized values are expressed as fold changes compared with gene profiles of the no transplantation sample. (g) Flow cytometric analysis of skin-infiltrating cells after staining with anti-CD4-PE, anti-CD8-PE, anti-B220-APC, anti-CD45.1-eFlour 450 antibodies on day 7 post-transplantation. Representative flow cytometric data are shown after CD45.1⁻ cell gating. Data shown (a–g) are representative of two independent experiments (n = 3–5 mice/group/experiment). Data (a,d–g) are presented as means ±SEM. P values were determined using two-tailed unpaired Student’s t-tests; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.