GvHD Prophylaxis,   aGvHD,  cGvHD

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

Mice

  • Female C57BL/6 (B6, H-2b ) and B6D2F1 (H-2b/d) were used
  • Age range: 8-10 weeks
  • Mice received transplants between the ages of 10 and 14 weeks
  • Prior to transplantation, host mice received 11 Gy of total body irradiation (I3'Cs source) delivered in two fractions separated by 3 hours to reduce gastrointestinal toxicity2

BMT

  • Recipient mice were intravenously injected with 5 × 106 TCD-BM cells from WT B6 donors with 1 × 106 T cells purified from either 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 5 × 106 TCD-BM cells from WT B6 donors plus 1 × 106 T cells purified from either WT or MyD88-/- B6 donors on day 0 following 6 Gy TBI 

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.[iii]
  • 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

Target Ag

Clone

Fluorochrome

TCRβ

H57-597

FITC

TCRβ

H57-597

APC

H-2Kd

SF1-1.1

FITC

H-2Kd

SF1-1.1

Biotin

CD4

GK1.5

PE-Cy7

CD8a

53-6.7

BV510

CD8a

53-6.7

BV510

CD11b

M1/70

PerCP-Cy5.5

CD44

PE

IM7

CD62L

FITC

MEL-14

TLR1

eBioTR23

PE

LR2

T2.5

PE

TLR6

418601

PE

TLR7

polyclonal

PE

IFN-ϒ

XMG1.2

APC

IL-4

11B11

Alexa Fluor 647

IL-17A

TC11-18H10

PE

BrdU

Bu20a

APC

Foxp3

FJK-16s

PE

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 

TLR ligands

Corresponding TLR

Final concentration

Pam3CSK4

TLR1/2

1 µg/ml

LPS

TLR4

1 µg/ml

Flagellin

TLR5

1 µg/ml

ssRNA40

TLR7

5 µg/ml

R848

TLR7/8

2 µg/ml

ODN1826

TLR9

5 µM

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.

References
  1. Matsuoka S. et al. The myeloid differentiation factor 88 signaling in donor T cells accelerates graft-versus-host disease. Haematologica. 2019 May 2: haematol-2018. DOI: 10.3324/haematol.2018.203380.
  2. Cooke KR, et al.  An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood. 1996 Oct 15;88(8):3230-9. DOI: 88:3230-3239
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