Kaplan-Meier cardiac graft survival curve.

<p>Heterotopic abdominal heart transplantation was performed by using standard procedure. All transplanted mice were monitored every day until graft rejection, defined as the cessation of palpable cardiac activity. Although only three symbols of “triangle” are observed for group (WT B6B/c), two grafts survived for 7 days and two grafts survived for 8 days. It implies that each “triangle” on day 7 (60% remained to survive on day 7) and day 8 (20% remained to survive on day 8) represents two grafts. Graft survival of the allogeneic control group (C57BL/6BALB/c, n=5) is equivalent to IL-6 deficiency donor graft (IL-6KOBALB/c) treated with anti-CD25 mAb (n=5) (Mantel-Cox Test, p=0.17; Gehan-Breslow-Wilcoxon Test, p=0.28), whereas IL-6 deficiency donor graft survival (IL-6KOBALB/c) treated with iso-IgG (n=5) was significantly prolonged (Mantel-Cox Test, p=0.012; Gehan-Breslow-Wilcoxon Test, p=0.0031) in comparison to allogeneic control group.</p

(A) The frequency of MDSCs, CD4-CD8-NK1.1+, F4/80+ cells within cardiac graft of the transplant recipient. Graft-infiltrating these innate immune cells were collected for fluorochrome-labeling and subject to FACS analysis on day 3 after transplant. (B) Statistical analysis was performed for the groups G5 (n=3) and G6 (n=4). *p<0.05, **p<0.01.

<p>(A) The frequency of MDSCs, CD4-CD8-NK1.1+, F4/80+ cells within cardiac graft of the transplant recipient. Graft-infiltrating these innate immune cells were collected for fluorochrome-labeling and subject to FACS analysis on day 3 after transplant. (B) Statistical analysis was performed for the groups G5 (n=3) and G6 (n=4). *p<0.05, **p<0.01.</p

Graft histology and immunohistochemical analysis of grafts.

<p>(A) The heart transplanted recipients were sacrificed at day 9 after surgery. Allogeneic grafts were harvested, stained with H&E to assess inflammation and lymphocytes infiltration between different groups under microscope, as described in the Methods section. The figure shows that a mild leukocytes infiltration was observed in the IL-6 deficient donors (G1) (n=2), while a pronouncedly infiltration of leukocytes and preserved cardiac architecture were observed in the subepicardial area of the IL-6KO grafts treated by anti-CD25 mAb (PC61) (G2) (n=2) and wild-type allografts (G3) (n=2) without any treatment. (B) Grafts specimens harvested at day 9 post-transplant were snap-frozen into Tissue Tec and stained for Gr-1 protein as described in the Materials and Methods section. Immunohistochemical sections showed that depletion of Treg cells using anti-CD25 mAb (PC61) remarkably caused an infiltration of Gr-1+ cells in the IL-6 deficient grafts, which is similar to acutely rejected wild-type hearts. A mild subepicardial Gr-1+ cells infiltration was found within IL-6KO grafts without any treatment. Original magnification ╳ 40.</p

Administration of anti-CD25 mAb (PC61) depleted CD4+CD25+Foxp3+ cells (n=2).

<p>Peripheral blood CD4+CD25+Foxp3+ T cells was analyzed through flow cytometry at day -3 (pre-administration of anti-CD25 monoclonal antibody), day 0 (transplant surgery), day 3, and day 9 post-transplant surgery. Administration of anti-CD25mAb 3 days before transplant surgery resulted in a continuous decrease of frequency of peripheral CD4+CD25+Foxp3+ T cells (12.4% on day -3, 2.78% on day 0, 0.64% on day 3). On day 9, the frequency of peripheral CD4+CD25+Foxp3+ T cells reactively increased owing to acute rejection event.</p