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M. Gaignage et al.
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Figure 7. The combination of anti-interleukin-27p28 monoclonal antibody and R848 induces strong regulatory T-cell activation in acute graft-versus-host disease in irradiated recipients. BALB/c recipient mice were irradiated with 8 Gy and treated or not with R848 (25 mg/mouse) 48 and 0 h before transplantation of 2x106 CD5+ splenocytes from B6 mice. In addition, recipients were treated or not with anti-interleukin-27p28 (aIL-27) on days 0 and 6. After (A) 6 and (B) 40 days of GvHD induction, spleen cells were recovered and stained with anti-H-2Dd, -H-2Db, -CD4 and -CD8 for FACS spleen cell subset analysis. (C-D) H-2Dd, H-2Db, LIVE/DEAD®, CD4, CD8, LAP and Foxp3 staining was added on 6-day-GvHD spleen cells to evaluate Treg populations. Data are from two to three experiments (**P<0.01, ***P<0.001 by the Kruskal-Wallis test with Dunn multiple comparison test).
tions were also analyzed 6 days after B6→8 Gy-BALB/c GvHD induction. The numbers of Foxp3+ CD4 T cells were significantly higher in the R848-treated group than in the control group and adding anti-IL-27 to the R848 treatment further increased the Treg population 2-fold (Figure 7C). Sixty-five percent of these Foxp3+ CD4 cells were positive for latent TGF-β1-associated protein (LAP), attesting their state of activation (Figure 7D). The stimu- lating effect of the anti-IL-27-R848 combination on Foxp3 expression was even more striking for CD8 Foxp3+ T cells, which were barely detectable in the donor B6 spleen but reached levels equivalent to their CD4 counterparts in the anti-IL-27-R848 group. This was not seen in mice treated with either R848 or anti-IL-27 (Figure 7C). Interestingly, following anti-IL-27 treatment, B6 Foxp3+ CD4 T cells increased 3-fold more than in the control GvHD group and 75% of these cells were activated. In contrast, R848 administration did not significantly ampli- fy these cells, which correlated with the failure of the TLR7 ligand to increase survival in B6→5Gy-B6D2F1 mice. The anti-IL-27-R848 combination induced a strong increase of Foxp3+ CD4 T cells, which were 5-fold more numerous than in control GvHD mice and 85% were LAP+. In this irradiated model, the Foxp3+ CD8 popula- tion, although present, was substantially less abundant (Online Supplementary Figure S3B).
Thus, R848 treatment failed to provide complete protec- tion against conditioned GvHD but full protection was restored when anti-IL-27 was added. The combination was necessary to abrogate IFNγ production and to induce maxi- mal Foxp3+ CD4 and CD8 cell responses.
Discussion
Innate pattern recognition receptors, including TLR, are implicated in the control of GvHD.37 Most often TLR stim- ulation aggravates disease, as reported for TLR438 and TLR9,39 but the consequences may differ depending on the time of TLR stimulation with respect to allogeneic HCT as observed for the TLR7/8 agonist 3M-001.40,41
The present study demonstrates the prevention of murine GvHD by the TLR7 ligand resiquimod/R848 and further improvement of protection by inhibition of IL-27.10,11 Our results indicate that R848 administration to recipients 48 h before and at the time of allogeneic HCT promoted survival in lethally irradiated recipients of fully allogeneic hematopoietic cells and in the lethal form of GvHD induced in ncB6D2F1 recipients of B6 parental spleen cells. In this model, R848 inhibited IFNγ, IL-27 and TNFα production while upregulating TGF-β1, thus altering the cytokine bal- ance. However, this suppression of Th1 cytokine produc-
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haematologica | 2019; 104(2)