Graft-vs-host disease (GvHD) is the major cause of non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT)[1]. We were the first to report that pharmacologic inhibition of Janus kinases 1 and 2 (JAK1/2) using ruxolitinib (RUX)[2-4] or baricitinib (BARI)[4, 5] prevents acute GvHD (aGvHD). While both BARI and RUX have a similar potency against JAK1/2, BARI is superior to RUX in preventing aGvHD in mice, which is associated with increases in GvHD-suppressive regulatory T cells (Tregs) and pSTAT5 in mice treated with BARI compared to those with RUX [5]. The survival and proliferation of Tregs require IL2R-dependent activation of JAK1/3-STAT5 signaling [6, 7]. Thus, we hypothesized that sparing JAK3 by BARI is critical for the increase in Tregs after allo-HSCT. We found that in vivo administration of JAK3-specific irreversible inhibitor (PF-06651600; iJAK3) along with BARI significantly reduced donor T cell-derived Tregs and overall survival compared to that of BARI alone (Fig. 1 and Supplementary Fig. 1a). In addition, consistent with our previous report [5], BARI elevated plasma levels of IL2 and pSTAT5 in CD4 T cells compared to vehicle control (Supplementary Fig. 1b, c). These increases in IL2 and pSTAT5 were partially reversed when BARI+ iJAK3 were administered (Supplementary Fig. 1b, c). Likewise, BARI+ iJAK3-significantly reduced Tregs and pSTAT5 in CD4 T cells in vitro compared to BARI alone (Supplementary Fig. 1d, e). JAK3 interacts with the common gamma chain in response to IL-2, IL-4, IL-7, IL-9, and IL-15 that are essential for T cell differentiation and proliferation. Thus, JAK3 inhibition significantly decreased Th1 differentiation compared to vehicle control in vitro (Supplementary Fig. 1f), which might be the reason that the overall survival of iJAK3 group was not worse than that of vehicle control (Fig. 1 B). Furthermore, BARI dramatically improved overall survival and clinical GvHD scores regardless of the number of donor T cells transplanted (Supplementary Fig. 2).

Since BARI significantly increases Tregs during the first two weeks of the treatment [5], we hypothesized that two weeks of BARI administration would be sufficient to prevent GvHD. To test our hypothesis, we treated allo-HSCT recipient mice with BARI for a period of one, two, three, or four weeks following allo-HSCT. Although all mice treated with BARI demonstrated significantly improved overall survival rates and clinical GvHD scores compared to vehicle control (Fig. 1 C), two weeks of BARI treatment had a similar preventative effect as that of three or four weeks. Thus, we conclude that two weeks of BARI treatment is sufficient to prevent GvHD. We also observed a significantly improved multilineage engraftment (especially platelets) as well as full donor chimerism in the BARI-treated groups compared to vehicle control (Supplementary Fig. 3a, b). In addition, all BARI-treated mice showed significantly decreased percentages of donor bone marrow (BM)-derived CD4/CD8 double-negative cells in the peripheral blood compared to vehicle control (Supplementary Fig. 3b), suggesting that BARI reduces thymic damage. Furthermore, higher percentages of donor BM-derived T/B lymphocytes were observed in BARI-treated groups compared to vehicle control, which is consistent with less clinical GvHD (Supplementary Fig. 3b). We previously reported that BARI treats established GvHD [5]. To determine the time at which BARI needs to be administered for a complete reversal of established GvHD, we administrated BARI starting on day 10 (D10), 17 (D17), or 24 (D24) after allo-HSCT. Despite this latency, all BARI-treated groups …