MyD88 signaling in donor T cells accelerates graft-versus-host disease

Myeloid differentiation factor 88 (MyD88) signaling is vital in the activation of both innate and adaptive immunity. MyD88 transduces signal through Toll-like receptors (TLRs), and interleukin-1 receptors (IL-1R) superfamily to the NFκB pathway, and inflammasome by establishing a molecular complex with interleukin-1 receptor-associated kinase 4 (IRAK4).

While the role of MyD88 for immunity has been well studied its role in the development of graft-versus-host disease (GvHD) pathophysiology remains unknown.

Satomi Matsuoka, from the Department of Hematology, Faculty of Medicine, Hokkaido University, Sapporo, Japan and colleagues evaluated the role of MyD88 signaling in donor T cells by using a well-established mouse model of allogeneic bone marrow transplantation (allo-BMT), where lethally irradiated recipient mice were transplanted with MyD88- deficient T cells (MyD88-/- T cells), and T cell depleted bone marrow cells (TCD-BM) from wild-type (WT) mice.¹

Methods

Assessment of GVHD and GVL

• The clinical GvHD scores were assessed. The graft-versus-leukemia (GVL) was assessed using post-mortem examination or in vivo bioluminescent imaging.
• GVL: 1 X 10³ P815 cells (H-2d) were intravenously injected to BMT recipients on day 0
• Survival after BMT was monitored daily, and the clinical GvHD scores and pathological GvHD scores were assessed
• The cause of each death after BMT was determined by a post-mortem examination to be either GvHD or tumor death:
• Leukemia death: the occurrence of either hepatosplenomegaly, macroscopic tumor nodules in the liver and/or spleen, or hind-leg paralysis
• GvHD death: The absence of leukemia and by the presence of clinical signs of GvHD 

Flow cytometric analysis

• Cells isolated from the thymus or spleen were incubated with antibodies (Abs)
• Sections of the liver, small intestine, and colon were stained with hematoxylin and eosin (H&E).
• Pathological GVHD was assessed using a semi-quantitative scoring system

Table 1: List of primary antibodies used in Flow cytometry

 Cell cultures

• TCR on purified T cells (5 × 10⁴ T cells/well) were stimulated with 5 × 10⁴ /well of Dynabeads Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation in the presence or absence of TLR ligands at various concentrations or PF-06650833 (20 μM) for up to 96 hours 

Table 2: List of synthetic TLR ligands used in cell culture 

T cell proliferation

• Purified T cells were labeled
• To measure cellular uptake of BrdU, recipients were intraperitoneal injected(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

• The Kaplan-Meier product limit method was used to obtain the survival probability and the log-rank test was applied to compare the survival curves. The team defined P < 0.05 as statistically significant 

Results 

The team investigated whether ablation of MyD88 signaling in donor cells influenced GVHD in a well-established mouse model of haploidentical BMT: 

• GvHD was severe in allogeneic controls with 80% mortality by day 50, 67% of recipients of MyD88-/- donors survived this period
• Clinical GvHD scores were also significantly lower in recipients of MyD88-/- graft compared to those of WT graft
• MyD88 deficiency in donor T cell significantly ameliorated mortality and morbidity of GvHD

Next, the effects of MyD88 signaling in donor cells on GvHD that may reside in the T-cell compartment of the donor graft were evaluated:

• Histopathologic examination of the small intestine and colon performed 6-8 weeks after BMT confirmed attenuated GvHD pathology in recipients of MyD88-/- T cells
• GvHD pathology in the small intestine, including villous blunting, epithelial apoptosis, and Paneth-cell loss accompanied by inflammatory-cell infiltration was significantly less severe in recipients of MyD88-/- T cells
• Thymic GvHD characterized by the loss of CD4+ CD8+ double positive (DP) thymocytes was also less severe in MyD88-/- T-cell recipients than in controls
• Donor T-cell MyD88 signaling in GvHD was not strain dependent, as MyD88 deficiency in donor T cells ameliorate GvHD in the B6 into BALB/c mouse model

The effects of MyD88 signaling on donor T-cell expansion was then evaluated in the spleen early after BMT:

• 7 days after BMT The numbers of CD8+ T cells were significantly less in MyD88-/- T-cell recipients than those in controls
• Both groups showed complete donor T cell chimerism
• Analysis of cell division using showed equivalent cell division between WT and MyD88-/- T cells at both 72 and 96 hours after BMT
• T-cell proliferation was much more robust in vivo compared to in vitro probably due to the presence of potent inflammatory milieu induced by total body irradiation (TBI)
• On the other hand, annexin V+ apoptotic donor CD8+ T cells were significantly increased in the recipients of MyD88-/- T cells on day +7

 To evaluate the role of MyD88 in donor T-cell differentiation after allo-BMT, cytokine production was evaluated in donor T cells isolated on day +7 after BMT:

• MyD88-/- CD4+ T cells produced considerably fewer IFN-γ and IL-17,
• MyD88-/- donor CD8+ T cells produced fewer INF-γ than their WT counterpart
• IL-4 production from MyD88-/- donor CD4+ T cells was found to be greater than that from WT CD4+ T cells
• Impaired Th1/Tc1, not Th2, differentiation in MyD88-/- T cells was confirmed in vitro after CD3/CD28 stimulation
• Flow cytometry analysis on day +7 confirmed that absolute numbers of donor CD4+ Foxp3+ Tregs and proportions of Foxp3+ cells among donor CD4+ T cells were considerably greater in the spleen of recipients of MyD88-/- T cells than those in controls 

Considering the significant reduction of GvHD in the absence of MyD88 signaling in donor  T cells, it is of interest to evaluate the impact of MyD88 signaling in donor T cells on GVL effect after allogeneic BMT.

Lethally irradiated B6D2F1 mice were injected with 5 × 10⁶ TCD-BM from WT B6 plus 1 × 10⁶ T cells from either WT or MyD88-/- B6 mice, with the addition of 1 × 103 host type P815 leukemia cells to the donor inoculum. All allogeneic TCD-BM recipients died from leukemia within 2 weeks after BMT, whereas leukemia mortality was significantly suppressed in the recipients of both WT and MyD88-/- T cells. While leukemia mortality was not significantly different between the allogeneic recipients of WT and MyD88-/- T cells, overall survival time was significantly prolonged in recipients of MyD88-/- T cells compared to controls, suggesting that MyD88 signaling in donor T cells is dispensable for GVL effect and T-cell MyD88 is a therapeutic target of GVHD without GVL reduction.

To further assess GVL effects, the research team performed in vivo BLI to track luciferase-transfected P815 (P815-luc) cells after BMT. Survivals were again significantly prolonged in recipients of MyD88-/- T cells compared to those of WT T cells. Whole body BLI clearly demonstrated that growth of P815-luc cells were suppressed both in the recipients of MyD88-/- T cells and WT T cells, while P815-luc expanded vigorously in the recipients of TCD-BM alone. Altogether, the authors concluded that GVL effects were preserved without donor T-cell MyD88 signaling. 

Conclusion

Dr. Matsuoka and colleagues found that the deficiency of MyD88 signaling in the donor T cells 4 directly modulated the adaptive T cell response thereby reducing the severity of GvHD in relation with the impaired donor Th1, Tc2, and Th17 responses.  The authors found that by administrating a pharmacological IRAK4 inhibitor (PF-06650833) ameliorated the effects of GVHD. The authors highlighted that MyD88 in donor T cells were dispensable for graft versus leukemia (GVL) effects, thereby suggesting that MyD88 in T cells may be a potential therapeutic target for GvHD, while sparing GVL effects.

Understanding the critical role of IRAK4 in the activation of T cells and the development of GvHD should help to test IRAK4 inhibitors in clinical studies to explore their prophylactic and therapeutic potentials against GvHD.