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M. Gaignage et al.
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Figure 5. Foxp3+ regulatory T cells are involved in graft-versus-host disease prevention by R848. Treg were depleted with PC61 antibody in donor and recipient mice 4 days before R848 treatment and 1 day after B6 cell transfer in the non-conditioned GvHD model. (A) After 14, 20 and 50 days of B6 cell implantation, H-2Dd, H- 2Db, LIVE/DEAD®, CD4, TCRβ and Foxp3 staining was used to evaluate Treg populations by flow cytometry. Mice were monitored for (B) weight loss and (C) mortality. (D) Anti-CD44 and -CD69 antibodies were used to analyze T-cell activation after 14 and 20 days. (E) IFNγ, IL-27p28 and active TGFβ-1 were measured by enzyme- linked immunosorbent assay in plasma prepared from mice bled on days 6, 14 and 20. Data are from two to three experiments in all panels (*P<0.05, ** P<0.01, ***P<0.001 by the Kruskal-Wallis test with Dunn multiple comparison test, Mann–Whitney unpaired t-test and ANOVA–Bonferroni post-test).
mice, half of the initial Treg population recovered 14 days after GvHD induction. In contrast, in R848-treated GvHD mice, B6D2F1 Treg numbers almost doubled compared to normal B6D2F1 mice and increased four times versus PC61- R848 ncGVHD mice and their levels remained unchanged up to day 50 after transplantation (Online Supplementary Figure S2A,B).
PC61 treatment of the R848 GvHD mice resulted in weight loss starting from day 17, a few days later than in control GvHD mice. The percentage weight loss was finally the same in both groups, suggesting a significant contribu- tion of Treg in the prevention of ncGVHD morbidity by R848 (Figure 5B). However, PC61 treatment only partially decreased the survival of R848-treated mice (70% versus 90%) (Figure 5C). This trend was observed in two addition- al experiments.
In order to test whether Treg depletion affected the level of donor T-cell activation, we evaluated CD44 and CD69 expression levels 14 and 20 days after ncGVHD induction. When Treg were depleted in R848-treated mice, CD44+ and CD69+ B6 CD4 and CD8 T cells were significantly
increased and CD69 levels even exceeded those of the control ncGVHD group. Compared to day 14 levels, the B6 CD69+ T-cell population tripled at day 20, indicating that an absence of Treg increased expansion of memory and activated donor T cells (Figure 5D). However, Treg depletion by PC61 did not seem to influence early cytokine production since no significant differences in IFNγ, IL-27p28 and active TGF-β1 plasma concentrations were observed between R848- and PC61-R848-treated mice (Figure 5E).
Together, the data suggest that Treg from donors and recipients contributed to R848-mediated GvHD preven- tion. However, despite the depleting treatment, a small population of host Treg remained present, which could explain why R848 protection was not completely abrogat- ed and resulted in death of only 30% of PC61-R848-treat- ed mice. As shown previously, R848 GvHD protection correlates with a strong drop in pro-inflammatory cytokines and this was still observed after Treg depletion, which could also explain why the protective effect of R848 was not completely suppressed by Treg depletion.
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haematologica | 2019; 104(2)