Page 195 - 2019_02-Haematologica-web

A TLR7 ligand prevents mouse GvHD
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Figure 6. R848 and anti-interleukin- 27p28 monoclonal antibody admin- istration to recipient mice prevents acute graft-versus-host disease in irradiated models. (A) BALB/c recipi- ent mice were irradiated with 8 Gy and treated or not with R848 (25 mg/mouse) 48 h and 0 h before transplantation of 2x106 B6 CD5+ splenocytes and 107 B6 T-cell deplet- ed bone marrow cells (TCD-BM). In addition, both recipient groups were treated or not (NT) with 0.5 mg anti- IL-27 (aIL-27) on days 0 and 6. Mice were monitored for survival. (B) Six days after B6-cell transfer, IFNγ and IL-27p28 were measured by enzyme- linked immunosorbent assay in plas- ma from irradiated-GvHD BALB/c mice. (C) B6D2F1 recipient mice were irradiated with 5 Gy and treated or not with R848 (25 mg/mouse) 48 and 24 h before transplantation of 40x106 splenocytes and 107 TCD-BM cells from B6 mice similarly treated with R848 or not. In addition, both recipient groups were treated or not with aIL-27 (0.5 mg) on days 0 and 6. Mice were monitored for survival. Data are representative of two to three experiments. (*P<0.05, **P<0.01, ***P<0.001 by the log rank test and Kruskal-Wallis test with Dunn multiple comparison test).
shown to contribute to GvHD pathology,10,11 was still pres- ent in the irradiated models, we tested the combination of R848 and anti-IL-27 and observed that this resulted in 100% survival of the 5 Gy-irradiated B6D2F1 recipients (Figure 6C).
The anti-IL-27-R848 combination also operated in B6→8Gy-BALB/c GvHD as it significantly decreased the concentration of plasma IFNγ, which was not the case when either agent was used separately (Figure 6B). However, it did not prevent T-cell engraftment since we observed the same numbers of CD4 and CD8 T cells (Figure 7A) and total B6 donor cell implantation (Figure 7B). Already after 6 days, the number of B6 CD4 T cells recovered from the BALB/c spleen was equivalent to the total number injected and for CD8 T cells the number of cells had increased ±10-fold. These numbers were not modified by R848, anti-IL-27 or the anti-IL-27-R848 com- bination (Figure 7A). Similarly, upregulation of the memo- ry marker CD44 and the activation marker CD69 in B6 CD4 and CD8 T cells recovered 6 days after transplanta- tion were not modified by R848. The only difference was observed for the CD62L naïve cell marker that was com- pletely lost in B6 CD4 and CD8 T cells recovered from control GvHD mice but remained significantly higher in the group treated with the anti-IL-27-R848 combination (Online Supplementary Figure S3A).
As we previously showed that Treg contribute to the R848 protection in B6→ncB6D2F1 GvHD, Treg popula-
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R848 cooperates with anti-interleukin-27p28 monoclonal antibody in regulatory T-cell upregulation and graft-versus-host disease prevention in conditioned models
B6 spleen cell transfer into ncB6D2F1 recipients is an opti- mal strategy for dissecting the mechanisms involved in immune-mediated hematopoietic cell destruction and can be used as a model of induced bone marrow failure23 but does not fully replicate allogeneic HCT. To test R848 in full allogeneic HCT, BALB/c mice were injected with R848 24 h before irradiation (8 Gy) and immediately after B6 spleen and bone marrow cell transfer (B6→8Gy-BALB/c). All untreated mice rapidly developed acute GvHD and started to die from day 7 whereas 90% of the mice injected with R848 survived more than 40 days (Figure 6A).
However, R848 did not completely prevent the GvHD reaction since IL-27p28 and IFNγ were still upregulated in R848-protected mice (Figure 6B). This incomplete suppres- sion of a GvHD reaction was even more striking in sub- lethally irradiated (5 Gy) B6D2F1 recipients of B6 spleen cells, in which R848 pre-treatment of host and donor before transplantation, as in the experiments described above in non-conditioned mice, solely delayed disease and no longer increased survival significantly (Figure 6C), contrary to the total protection seen in non-irradiated recipients. This also correlated with the persistence of pro-inflammatory cytokines IL-27p28 and IFNγ, which R848 treatment failed to decrease (data not shown). As IL-27, which has been
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