Figure 4
Depletion of PDCA-1+Siglec-H+pDCs results in abrogation of plerixafor mediated prolongation of heart allograft survival. To investigate the contribution of pDCs to the prolongation of allograft survival and the increase of FoxP3+ Treg cell numbers, we performed pDCs depletion experiments in the allogeneic heart transplantation setting (outlined in A) and investigated the allografts for myocyte lesions (B), allograft fibrosis (C), allograft infiltrating immune cells (D), and Treg cells (E) 14 days after transplantation. Example data for the stainings can be found in Supplemental Fig. S4. The scatter plots in (B–E) depict data of 3 to 6 heart transplant specimens. In these experiments, pDCs were depleted in vivo by intraperitoneally injecting a rat anti-mouse PDCA-1 mAb two days before transplantation and following every other day until 14 days post transplantation along with the general treatment. Treatment groups (study arms in red, control arms in blue and green) and time points of drug or vehicle injections for post-transplant in vivo anti-PDCA-1 depletion experiments are depicted in (A). Allograft sections taken from P5R treated recipients that underwent pDC-depletion (P5R-aPDCA) showed significantly higher myocyte lesion scores (B), a tendency of increased fibrosis (C) and increased cellular allograft infiltrations (D) compared to animals with isotype injections (P5R). The scatter plots in (E) demonstrate that pDC-depletion resulted in a significant decrease of allograft infiltrating CD4+FoxP3+ Treg cells determined as ratio in between Foxp3+ cells among CD3+ T cell infiltrations. (F) HTX survival after pDC depletion: P5R treatment with concomitant anti-PDCA-1 depletion resulted in abrogation of the prolongation of allograft survival compared to P5R treated animals that received isotype injections (P5R + anti-PDCA, n = 9 mice, median survival 20 days; P5R + Isotype, n = 5 mice, median survival 63 days; p < 0.01). Examined groups of allogeneic heart transplanted animals included vehicles + mAb-isotype injections (NT-Iso, n = 6), plerixafor + mAb-isotype injections (P5-Iso, n = 5), rapamycin + mAb-isotype injections (R-Iso, n = 5), plerixafor + rapamycin + mAb-isotype injections (P5R-Iso, n = 5), plerixafor + anti-PDCA mAb (P5-aPDCA, n = 5), rapamycin + anti-PDCA mAb (R-aPDCA, n = 9), plerixafor + rapamycin + anti-PDCA mAb (P5R-aPDCA, n = 9). Horizontal lines of scatter plots in (B–E) show the mean ± SD, the p value was calculated using the One-way ANOVA with Bonferroni post-hoc test (n.s., p > 0.05; *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005). SC Syngeneic heart transplanted mice receiving only vehicles for plerixafor as well as rapamycin plus isotype mAb. #Since both control groups of P5 and P5-aPDCA (green filled and open triangles) were recovered 14 days after transplantation but did undergo full rejection several days ago and were thus not beating anymore after HTX recovery these animals were excluded from statistical analysis (indicated by the vertical black line in scatter plot graphs A–D).
