Page 192 - 2019_02-Haematologica-web

M. Gaignage et al.
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Figure 3. In vivo treatment of mice with R848 affects responder and presenting cells in mixed lymphocyte culture: role of IFNAR-1. (A) B6D2F1 and B6 mice were treated or not with R848 (25 mg) 48 and 18 h before mixed lymphocyte culture of B6 responder cells and irradiated B6D2F1 APC. After 48 h, (left) proliferation and (right) IFNγ production were determined by 3H-thymidine incorporation and enzyme-linked immunosorbent assay, respectively. (B) Spleen cells from 129/Sv and 129/Sv IFNAR-1-/- mice were collected 48 h after in vivo R848 treatment and incubated with B6D2F1 APC. Proliferations and IFNγ were measured. (C) 129/Sv spleen cells cells were stained for CD4, LIVE/DEAD® and Foxp3 to determine the percentage and absolute numbers of Treg. (D) Treg were depleted with PC61 antibody in B6 mice 4 days before R848 treatment. B6 spleen cells were collected 48 h after in vivo R848 treatment and incubated with B6D2F1 APC and IFNγ was measured after 72 h. (E) FVB (H2q) splenocytes were incubated without APC or with CD11b+ cDC, CD8a+ cDC or pDC purified by MACS beads and FACS sorting from normal and R848-treated 129/Sv mice. After 48 h, proliferation was recorded. (F) Spleen cells from 129/Sv and 129/Sv IFNAR-1-/- mice were collected 48 h after R848 treatment and co-cultured with FVB responder splenocytes. Proliferation and IFNγ were measured. Data are from two to four experiments in all panels (*P<0.05, ***P<0.001 by the Kruskal-Wallis test with Dunn multiple comparison test).
addition, an increase was observed for CD69+ CD4 T cells in R848-protected mice as compared to control B6D2F1 mice (Online Supplementary Figure S1B).
We next examined the influence of R848 treatment on Treg in the same experiments. First, we observed a 10-fold decrease in the proportion of donor and recipient Foxp3+ CD4 T cells 14 days after B6 spleen cell transplantation. In contrast, in R848-treated GvHD mice, recipient Foxp3+ CD4 T cells increased 4-fold while donor Treg returned to nor- mal B6 levels (Figure 4E).
Together, these data indicate that during ncGvHD R848 affected donor CD4 and CD8 T-cell implantation and acti- vation but the inhibition was only partial and did not pre- vent the establishment of permanent chimerism. On the other hand, R848 prevented GvHD-induced loss of donor and recipient Treg and even increased the latter above nor- mal levels.
Regulatory T cells contribute to R848-mediated prevention of non-conditioned graft-versus-host disease
The contribution of Treg to the protective effect of R848 was tested by depletion of Treg using anti-CD25 PC61 anti- body treatment of B6 donors and B6D2F1 recipients. PC61 antibody was injected 4 days before R848 treatment and a second injection was administered 1 day after B6 spleen cell transplantation. As compared to donor CD4 T cells from R848-treated mice with GvHD, B6 CD4 T cells from the PC61-R848 GvHD group engrafted host spleen faster and their numbers were significantly higher at 14 and 20 days after B6 cell transfer and continued to expand up to day 50. PC61 antibody completely depleted their Foxp3+ Treg pop- ulation which remained totally absent during the course of GvHD in contrast to that in the R848-treated mice, in which they expanded more than 6-fold from day 14 to day 50 (Figure 5A). Regarding host Treg, in PC61-R848-treated
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