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MyD88/IRAK4 in donor T cells potentiates GVHD
through TLR stimulation towards production of inflam- matory cytokines such as tumor necrosis factor (TNF)-α and IL-1 family cytokines.16-19
In this study, we evaluated the role of MyD88 signaling in donor T cells by using a well-established mouse model of allogeneic bone marrow transplantation (allo-BMT), in which lethally irradiated recipient mice were transplanted with MyD88-/- T cells and T cell-depleted bone marrow cells (TCD-BM) from WT mice. We found that lack of MyD88 signaling in donor T cells directly modulated adaptive T-cell responses and reduced severity of GvHD in association with profoundly impaired donor Th1, Tc1, and Th17 responses. Administration of a pharmacological IRAK4 inhibitor, PF-06650833, significantly ameliorated GvHD. MyD88 in donor T cells was not essential for graft-versus-leukemia (GvL) effects, suggesting that MyD88 in T cells is a potential therapeutic target of GvHD, while sparing GvL effects.
Methods
Mice
Female C57BL/6 (B6, H-2b) and B6D2F1 (H-2b/d) mice were purchased from Charles River Japan (Yokohama, Japan). TLR2-/- and TLR7-/- mice with a B6 genetic back- ground were purchased from Oriental Bioservice (Chiba, Japan). B6-MyD88-/- mice were produced and maintained as previously described.20 Age of the mice was 8-10 weeks. All animal experiments were performed under the auspices of the Institutional Animal Care and Research Advisory Committee (approval n: 12-0106).
Bone marrow transplantation
Mice were transplanted as previously described.21 In brief, recipient B6D2F1 mice were intravenously (i.v.) injected with 5x106 TCD-BM cells form WT B6 donors plus 1x106 T cells purified from either wild-type (WT) or MyD88-/- B6 donors on day 0 following lethal total body irradiation (TBI, 12Gy) delivered in two doses at 3-hour intervals. BALB/c recipients were transplanted with 5x106 TCD-BM cells from WT B6 donors plus 1x106 T cells puri- fied from either WT or MyD88-/- B6 donors on day 0 fol- lowing 6 Gy TBI. Isolation of T cells and TCD were per- formed using a Pan T cell Isolation kit II and anti-CD90- MicroBeads, respectively, and the autoMACS Pro system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Mice were housed in sterilized microisolator cages and received autoclaved hyperchlorinated drinking water for the first three weeks after BMT, and filtered water thereafter.
Assessment of graft-versus-host disease and graft-versus-leukemia effect
Clinical GvHD scores were assessed as previously described.22 GvL was assessed by postmortem examina- tion or on in vivo bioluminescent imaging.23,24 Detailed pro- tocols are described in the Online Supplementary Methods.
Quantitative-polymerase chain reaction
RNA extraction and quantitative-polymerase chain reaction (Q-PCR) were performed as described in the Online Supplementary Methods. Specific primers and probes used for Q-PCR are listed in Online Supplementary Table S1.
Flow cytometric analysis
Flow cytometric analysis was performed as previously described.21 The cells isolated from the thymus or spleen were incubated with antibodies (Abs) (listed in Online Supplementary Table S2) at 4°C for 30 minutes (min). Detailed protocols are described in the Online Supplementary Methods.
Cell cultures
All culture media and incubation conditions have been previously described.21 TCR on purified T cells (5x104 T cells/well) were stimulated with 5x104 /well of Dynabeads Mouse T-Activator CD3/CD28 for T-cell expansion and activation (ThermoFisher Scientific, Waltham, MA, USA) in the presence or absence of TLR ligands at concentra- tions listed in Online Supplementary Table S3 or PF- 06650833 (20 mM) for up to 96 hours.
T-cell proliferation
To assess T-cell proliferation, purified T cells were labeled using a CellTrace Violet Cell Proliferation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. To measure cellular uptake of BrdU, recipi- ents were intraperitoneally (i.p.) injected with 1 mg of BrdU 2 hours before analyses.
Statistical analysis
Mann-Whitney U tests were used to analyze cell counts, the cytokine data, and the clinical scores. We used the Kaplan-Meier product limit method to obtain the survival probability. and the log-rank test was applied to compare the survival curves. P<0.05 was considered statistically significant.
Results
Donor T cells lacking MyD88 pathway induce attenuated graft-versus-host disease
We investigated whether ablation of MyD88 signaling in donor cells influenced GvHD in a well-established mouse model of haploidentical BMT. Lethally irradiated B6D2F1 mice were i.v. injected with 5x106 BM plus 5x106 splenocytes from either WT or MyD88-/- B6 donors. Frequencies and absolute numbers of CD4+ T cells, CD8+ T cells, memory T cells, and Foxp3+ Tregs in the spleen were equivalent in donor WT and MyD88-/- B6 mice (Online Supplementary Figure S1). While GvHD was severe in allogeneic controls with 80% mortality by day 50, 67% of recipients of MyD88-/- donors survived this period (Figure 1A). Clinical GvHD scores were also significantly lower in recipients of MyD88-/- graft compared to those of WT graft (Figure 1B).
Next, we evaluated if effects of MyD88 signaling in donor cells on GvHD could reside in the T-cell compart- ment of the donor graft. Lethally irradiated B6D2F1 mice were injected with 5x106 TCD-BM from WT B6 mice plus 1x106 T cells from either WT or MyD88-/- B6 mice. MyD88 deficiency in donor T cell alone significantly ameliorated mortality and morbidity of GvHD (Figure 1C and D). Histopathological examination of the small intestine and colon performed 6-8 weeks after BMT confirmed attenu- ated GvHD pathology in recipients of MyD88-/- T cells. GvHD pathology in the small intestine, including villous blunting, epithelial apoptosis, and Paneth-cell loss accom-
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