Targeting JAK2 By Gene Knockout or Pacritinib Treatment Reduces Gvhd and Xenograft Rejection By Promoting Induced Treg Differentiation

Janus kinase 2 (JAK2) signal transduction is a critical mediator of immune response.  JAK2 activation promotes T-cell allosensitization, as well as Th1 and Th17 differentiation.  JAK2-mediated STAT3 phosphorylation limits the generation of induced regulatory T cells (iTregs) by disrupting interactions between STAT5 and Foxp3.  JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT).  Using murine models of allo-HCT we show here that transfer of donor JAK2^-/- T cells is associated with significantly less GVHD, compared with wild-type or JAK2 replete donors (Figure 1, P=.003 and 0.01, respectively).  Th1 differentiation among JAK2^-/- T-cells is dramatically decreased, compared with controls.  Conversely, iTreg polarization and stability are significantly increased among the JAK2 deficient T cells.  To investigate the effect of pharmacologic JAK2 inhibition on T-cell alloresponses, pacritinib (supplied by CTI BioPharma), was chosen as it does not induce myelosuppression or increase risk for opportunistic infections in myelofibrosis patients – unlike JAK1/JAK2 inhibitors.  Pacritinib potently inhibits JAK2, but also has suppressive activity toward JAK3, CSF1R, and IRAK1.  Pacritinib was administered at 100mg/kg twice a day by oral gavage for 4 weeks beginning on the day of MHC-disparate allo-HCT, significantly reducing GVHD in recipient mice compared with methylcellulose vehicle control.  In allogeneic mixed leukocyte reactions using human cells, pacritinib (2.5μM) significantly reduces T-cell proliferation after 5 days of culture (P<.0001).  In vitro studies verified pacritinib eliminates STAT3 activity by IL-6 in human CD4+ T-cells, while permitting IL-2 induced STAT5 phosphorylation despite its effects on JAK3.  This offers a platform to reduce Th17 development, while promoting iTreg populations.  As such, pacritinib dramatically suppresses Th17 differentiation reflected by RORgammaT expression among naïve CD4+ T cells stimulated by allogeneic dendritic cells (DC), compared with DMSO (P<.0001).  iTregs were generated with allogeneic DCs in the presence of pacritinib or DMSO for 5 days, and then cultured with self T-cell responders and fresh DCs without additional drug exposure.  The suppressive potency of either pacritinib- or DMSO-treated iTregs was similar, suggesting JAK2 is not required for iTreg function.  We then evaluated whether JAK2 inhibition could prevent human skin graft rejection in an NSG mouse xenograft model.  A 1x1 cm split-thickness human skin graft was transplanted onto the animal dorsally.  The mice rested for 30 days after surgery to permit engraftment, then received 5x10⁶ allogeneic peripheral blood mononuclear cells (PBMC) by intraperitoneal injection.  Pacritinib was administered at the same dose and schedule for 2 weeks beginning at time of PBMC injection as tolerated by the NSG mice. The treatment significantly delayed allograft rejection by the human donor PBMCs, compared with vehicle control (Figure 2: Median graft survival 32.5 v 51 days, Day +60 survival 0% v 33.3%, pooled data from 2 experiments, n=5-6 mice per arm, P=.0011).  Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after HCT or solid organ transplantation. 

*BCB and DB contributed equally to this work.

Figure 1:  Decreased murine GVHD with JAK2 deficient T-cells

Figure 2:  Decreased human skin graft rejection with JAK2 inhibitor, pacritinib