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.
Fig1A
This data file is associated with Fig1A of the accompanied publication. Survival data of bone marrow only, vehicle treated, and Ibrutinib treated recipients. Column A is an arbitrary identification number given to each mouse. Column B indicates the time-point at which a survival (marked as "0') or non-survival event (marked as "1") occurred. Column C indicates the bone marrow alone group. Column D indicates mice treated with vehicle. Column E indicates mice treated with Ibrutinib 10mg/kg. Any other relevant information, including experimental conditions and setup is detailed in the accompanied publication.
Fig1B
This data file is associated with Fig1B of the accompanied publication. Urine protein measurement data of bone marrow only, vehicle treated, and Ibrutinib treated recipients. Column A is an arbitrary identification number given to each mouse. Column B indicates the time-point at which a non-proteinuria event (marked as "0') or a proteinuria event (marked as "1") occurred. Proteinuria event "1" is defined as a mouse showing a >2,000mg/dL score using Albustix urine test strips.Column C indicates the vehicle group, Column D indicates mice treated with Ibrutinib 10mg/kg. Column E indicates mice treated with Ibrutinib 15mg/kg, Column F indicates bone marrow alone mice treated with phosphate buffered saline, Column G indicates bone marrow alone mice treated with vehicle, Column H indicates bone marrow alone mice treated with 10mg/kg Ibrutinib. Any other relevant information, including experimental conditions and setup is detailed in the accompanied publication.
Fig1C
This data file is associated with Fig1C of the accompanied publication. Survival data of bone marrow only, vehicle treated, and Ibrutinib treated recipients. Column A is an arbitrary identification number given to each mouse. Column B indicates the time-point at which a survival (marked as "0') or non-survival event (marked as "1") occurred. Column C indicates the bone marrow alone group. Column D indicates mice treated with vehicle. Column E indicates mice treated with Ibrutinib 10mg/kg. Column F indicates mice treated with 10mg/kg Ibrutinib beginning 14 days post transplantation. Any other relevant information, including experimental conditions and setup is detailed in the accompanied publication.
Fig1D
This data file is associated with Fig1D of the accompanied publication. Urine protein measurement data of bone marrow only, vehicle treated, and Ibrutinib treated recipients. Column A is an arbitrary identification number given to each mouse. Column B indicates the time-point at which a non-proteinuria event (marked as "0') or a proteinuria event (marked as "1") occurred. Proteinuria event "1" is defined as a mouse showing a >2,000mg/dL score using Albustix urine test strips.Column C indicates the BM only group, Column D indicates mice treated with vehicle. Column E indicates mice treated with Ibrutinib 10mg/kg beginning 7 days post-transplant, Column F indicates mice treated with 10mg/kg Ibrutinib beginning 14 days post-transplant. Any other relevant information, including experimental conditions and setup is detailed in the accompanied publication.
Fig2A
This data file is associated with Fig2A of the accompanied publication. Each panel is a representative histogram of one mouse from each group. Mice treated with either vehicle or 10mg/kg Ibrutinib were injected with 40 x 10^6 CD25 depleted CFSE labeled whole splenocytes. Flow cytometry readings from recipients were taken 4 days post-transplant. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig2B
This data file is associated with Fig2B of the accompanied publication. Mice treated with either vehicle or 10mg/kg Ibrutinib were injected with 40 x 10^6 CD25 depleted CFSE labeled whole splenocytes. Flow cytometry readings from recipients were taken 4 days post-transplant. Using the percentage of CFSE diluted or CFSE low, the amount of proliferated cells can be determined. Column and row labels are self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig2C
This data file is associated with Fig2C of the accompanied publication. Mice treated with either vehicle or 10mg/kg Ibrutinib were injected with 40 x 10^6 CD25 depleted CFSE labeled whole splenocytes. Flow cytometry readings from recipients were taken 4 days post-transplant. The amount of CD40,80, and 86 on donor B cells was analyzed. Column and row labels are self-explanatory. Value from Column "E" row "2" was excluded as an extreme outlier compared to other values within the same group. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig2D
This data file is associated with Fig2D of the accompanied publication. Mice treated with either vehicle or 10mg/kg Ibrutinib were injected with 40 x 10^6 CD25 depleted whole splenocytes. Peripheral blood was collected from each recipient and serum was taken for ELISA measurement of total IgG and IgG2a autoantibodies. Each value represents the reported optical density (OD) output of an ELISA plate-reader. Column and row labels are self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig3A
This data file is associated with Fig3A of the accompanied publication. Urine protein measurement data of no injection, vehicle treated, and Ibrutinib treated recipients. Column A indicates the time-point at which urine protein measurements were taken post-transplant using Albustix urine test strips. Column and row labels are self-explanatory. Any other relevant information, including experimental conditions and setup is detailed in the accompanied publication.
Fig3B
This data file is associated with Fig3B of the accompanied publication. Mice treated with no injection, vehicle, or 10mg/kg Ibrutinib were injected with 80-100 x 10^6 whole splenocytes. Peripheral blood was collected from each recipient and serum was taken for ELISA measurement of total IgG and IgG2a autoantibodies. Each value represents the reported optical density (OD) output of an ELISA plate-reader. Column A indicates the number of weeks post-transplant serum samples was taken. Additional Column and row labels are self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig3C
This data file is associated with Fig3C of the accompanied publication. Mice treated with no injection, vehicle, or 10mg/kg Ibrutinib were injected with 80-100 x 10^6 whole splenocytes. Peripheral blood was collected from each recipient and serum was taken for ELISA measurement of total IgG and IgG2a autoantibodies. Each value represents the reported optical density (OD) output of an ELISA plate-reader. Column A indicates the number of weeks post-transplant serum samples was taken. Additional Column and row labels are self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig4A
This data file is associated with Fig4A of the accompanied publication. Mice treated with BM alone, vehicle, or 10mg/kg Ibrutinib were injected with 5 x 10^6 whole splenocytes. Clinical scores representing 6 visible factors were recorded and tabulated once per week following Day 14 post-transplant. Column A represents the day post-transplant the scoring was conducted on. Additional column and row titles are self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig4B images
This data file is associated with Fig4B of the accompanied publication. Mice treated with BM alone, vehicle, 5mg/kg, or 10mg/kg Ibrutinib were injected with 5 x 10^6 whole splenocytes. Skin lesions from each recipient were excised after euthanasia and subjected to H&E staining for scoring by a professional pathologist. Representative microscope images were taken from one mouse from each group. #199 is from BM alone group, #202 is from vehicle treated group, #205 is from 10mg/kg Ibrutinib treated group, #211 is from 5mg/kg Ibrutinib treated group. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig4C images
This data file is associated with Fig4C of the accompanied publication. Mice treated with BM alone, vehicle, or 10mg/kg Ibrutinib were injected with 5 x 10^6 whole splenocytes. Representative images were taken of one mouse from each group using a typical smartphone on Day 41 post-transplant. The file name of each representative image indicates the group that the mouse belongs in and should be self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig4D
This data file is associated with Fig4D of the accompanied publication. Mice treated with BM alone, vehicle, or 10mg/kg Ibrutinib were injected with 5 x 10^6 whole splenocytes. Skin lesions from each recipient were excised after euthanasia and subjected to H&E staining for scoring by a professional pathologist. Skin sections were scored based on several criteria as described in the accompanied manuscript. Columns and row titles are self-explanatory. Any other relevant information including experimental conditions and setup are included in the accompanied publication.
Fig4E
This data file is associated with Fig4E of the accompanied publication. Flow cytometry readings of B220 and CD138 were analyzed. B220low and CD138+ cell populations are indicative of autoantibody producing plasma cells. Columns and row titles are self-explanatory. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig4F
This data file is associated with Fig4F of the accompanied publication. Flow cytometry of splenic CD4+PD-1+CXCR5+ cell populations was analyzed. CD4+PD-1+CXCR5+ cell populations are indicative of T-follicular helper (Tfh) cells. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig4G
This data file is associated with Fig4G of the accompanied publication. Flow cytometry of B220+ B cells was analyzed. High B cell counts following transplantation are indicative of improved immune reconstitution and reduced GVHD pathogenesis. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig4H
This data file is associated with Fig4H of the accompanied publication. Column and row labels are self-explanatory. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig5A
This data file is associated with Fig5A of the accompanied publication. Survival of mice given 1-2 x 10^6 whole splenocytes. Column A is the mouse identification number. Column B is the time-point at which either a survival ("0") or non-survival ("1") event occurred. Other column and row titles should be self-explanatory. Mouse #482 was excluded from the results due to a non-GVHD related death confirmed by a DLAR veterinarian. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig5B
This data file is associated with Fig5B of the accompanied publication. Clinical score of recipients given 1-2 x 10^6 whole splenocytes was monitored once per week following transplantation. Column A indicates time-point at which the clinical score was measured. Additional column and row titles are self-explanatory. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig6A
This data file is associated with Fig6A of the accompanied publication. Survival of mice receiving 0.5-0.75 x 10^6 purified T cells. Column A indicates mouse identification number, Column B indicates time at which a non-survival ("1") or survival ("0") even occurred. Other columns and rows are self-explanatory. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Fig6B
This data file is associated with Fig6B of the accompanied publication. Column A indicates time-point at which the clinical score was measured. Additional column and row titles are self-explanatory. Any relevant information for this figure, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.
Supplementary Information
This data file is associated with all supplementary figures and information of the accompanied publication. This archive includes data files for S1_Fig, S2_Fig, S3_Fig, S4_Fig, S5_Fig. Individual data files of graphs or data within each supplementary figure (e.g., A in S1_Fig) are also included. Any relevant information for these figures, including experimental conditions and setup are included in the methods, results, or discussion section of the accompanied publication.