Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice

Abstract

Bruton's Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Ibrutinib improves survival and reduces glomerulonephritis induced proteinuria.

Lethally irradiated BALB/c recipients were transplanted with TCD-BM (5 x 106 per mouse) from DBA/2 donors with (n = 26) or without (n = 16) CD25- splenocytes at 40 x 106 per mouse. Groups were either given no treatment, daily oral gavage of vehicle alone, or Ibrutinib 10 mg/kg beginning 2–4 h before BMT and continued for 4 weeks. Mice were monitored for 60 d for survival (A) and proteinuria twice/week (B). Data are shown from two replicate experiments combined. In separate experiments under the same conditions as in A and B, daily oral gavage of vehicle (n = 13) and Ibrutinib treatment (n = 26) was delayed by either 1 (n = 13) or 2 (n = 13) weeks post-BMT and continued for 4 weeks. Mice were monitored for 60 d for survival (C) proteinuria twice/week (D). Data are shown from three replicate experiments combined. Asterisk indicates statistical significance between vehicle treatment and Ibrutinib treatment groups: *p<0.05, **p<0.01, ***p<0.001.

Fig 2. Ibrutinib reduces B-cell proliferation, costimulatory molecules, and dsDNA autoantibodies in cGVHD.

Lethally irradiated BALB/c recipients were transplanted with or without CFSE labeled CD25- splenocytes from DBA/2 donors at a dose of 40 x 106 per mouse. Groups were either given no treatment (n = 6), daily oral gavage of vehicle alone (n = 12), or Ibrutinib 10 mg/kg (n = 12) beginning 2–4 hours before BMT and continued for 4 days. Recipient mice were euthanized 4 days after transplant and spleen was taken for FACS analysis. The percentage of CFSE dilution (A, B) represents the amount of proliferated donor B cells. The expression of CD40, CD80, and CD86 costimulatory molecules on B cells were analyzed by gating on B220+ cells and shown as mean florescence intensity (MFI) (C). D panel represents a separate experimental design where BMTs and the number of subjects were as described previously except recipients were sacrificed 28 days after BMT and the Ibrutinib treatment duration was 4 weeks. Serum from whole blood was taken on day 28 post-BMT for ELISA measuring autoantibodies IgG and IgG2a (D). Data are pooled from three replicate experiments. Asterisk indicates statistical significance: *p<0.05.

Fig 3. Ibrutinib reduces glomerulonephritis and dsDNA autoantibodies.

Unconditioned B6D2F1 recipients were transplanted with 80–95 x 106 splenocytes without BM from DBA/2 donors. Groups were either given no treatment (n = 6), daily oral gavage of vehicle (n = 15), or two different dosages of Ibrutinib, 10 mg/kg (n = 5) or 20mg/kg (n = 10) 2–4 hours before transplant and continued for 4 weeks. Animals were monitored for proteinuria (A). Serum from whole blood was taken bi-weekly to monitor levels of IgG (B) and IgG2a (C) autoantibodies. The data are pooled from three replicate experiments. Asterisk indicates statistical significance between vehicle treatment and Ibrutinib treatment groups: *p<0.05.

Fig 4. Ibrutinib delays onset and reduces severity of scleroderma development.

Lethally irradiated BALB/c recipients were transplanted with TCD-BM (5 x 106 per mouse) from B10.D2 donors with or without whole splenocytes at a dose of 5 x 106 per mouse. Groups were either given no treatment (n = 4), daily oral gavage of vehicle alone (n = 9), or Ibrutinib at the dose of 10 mg/kg (n = 9) beginning 2–4 hours before BMT and continued for 4 weeks. Animals were monitored for survival and clinical score using a scoring system (A) and visual representations (C). At day 60, recipients were sacrificed and skin biopsies were taken for H&E staining (B) and scoring (D). Spleens were excised for FACS analysis (E-H). Percentage of B220loCD138+ plasma cells were reported for BM, vehicle, and Ibrutinib treated groups (E). Representative flow plot (F) as well as the combined data (G) for the percentage of Tfh cells were shown. Absolute numbers of B220+ cells were calculated and presented (H). Data shown is pooled from two replicate experiments. Asterisk indicates statistical significance between vehicle treatment and Ibrutinib treatment groups: *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Ibrutinib improves survival and clinical score.

Lethally irradiated BALB/c recipients were transplanted with TCD-BM (5 x 106 per mouse) from B6 donors with or without whole splenocytes at a dose of 1–2 x 106 per mouse. Groups either received no treatment (n = 4), daily oral gavage of vehicle (n = 10), or Ibrutinib 10 mg/kg (n = 11) 2–4 hours before BMT and continued for 4 weeks. Survival rate (A) and cGVHD clinical scores (B) were shown from two replicate experiments combined. Asterisk indicates statistical significance between vehicle treatment and Ibrutinib treatment groups: *p<0.05. A one-tailed student t test was used in (B).

Fig 6. Ibrutinib improves survival and clinical manifestations of cGVHD independent of B cells.

Lethally irradiated BALB/c recipients were transplanted with TCD-BM (5 x 106 per mouse) plus purified T cells (0.5 x 106 per mouse) from B6.129S2-Ighmtm1Cgn/J (B-cell deficient; BKO) donors, and received daily oral gavage of vehicle (n = 10), or Ibrutinib at 10 mg/kg (n = 10), lasting 4 weeks. Bone marrow alone controls received TCD-BM (5 x 106 per mouse) from WT B6 mice without treatment (n = 4). Animals were monitored for survival (A) and aGVHD clinical score (B) for 80 days. Data are shown from two replicate experiments combined. Asterisk indicates statistical significance between vehicle treatment and Ibrutinib treatment groups: **p<0.01.