DOI: 10.5061/dryad.qh53f
title: Data from: Inhibition of BTK and ITK with ibrutinib is effective in the prevention of chronic graft-versus-host disease in mice

description: 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.
description: Fig1AThis 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.Fig1BThis 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 &gt;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.Fig1CThis 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.Fig1DThis 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 &gt;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.Fig2AThis 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.Fig2BThis 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.Fig2CThis 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.Fig2DThis 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.Fig3AThis 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.Fig3BThis 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.Fig3CThis 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.Fig4AThis 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
 imagesThis 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
 imagesThis 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.Fig4DThis 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.Fig4EThis 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.Fig4FThis 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.Fig4GThis 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.Fig4HThis 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.Fig5AThis 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.Fig5BThis 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.Fig6AThis 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.Fig6BThis 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
 InformationThis 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.