Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation

Abstract

Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. 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). Transfer of JAK2-/- donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2-/- T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2-/- T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2-/- T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://ichgcp.net/clinical-trials-registry/NCT02891603).

Keywords: GVHD; GVL; JAK2; graft rejection.

Conflict of interest statement

Conflict of interest statement: B.C.B. has participated in advisory boards related to pacritinib (CTI BioPharma) and ruxolitinib (Incyte) in GVHD. J.S. is employed by CTI BioPharma with related equity ownership. A.O. is an employee at BioSeek. All other authors have no competing financial interests to declare.

Figures

Fig. 1.

JAK2 contributes to T cell-mediated GVHD, but is dispensable for GVL. Lethally irradiated BALB/c (A and B) or BALB/b (C and D) mice were transplanted with 5 × 106 TCD-BM alone or plus 1 × 106 (BALB/c) or 3 × 106 (BALB/b) purified T cells from WT B6 or JAK2 KO mice. Survival and body weight loss of BALB/c (A and B) and BALB/b recipients (C and D) are shown. Data shown are pooled from two to three replicate experiments with a total of 6–15 mice per group. Lethally irradiated BALB/c mice were transplanted with 5 × 106 TCD-BM alone or plus 2 × 103 luc-A20 cells and either 1 × 106 purified T cells from WT or JAK2 KO mice plus 2 × 103 luc-A20 cells. Recipient survival (E), body weight loss (F), and tumor burden (G) are shown. Percentage survival and tumor mortality data shown were pooled from three replicate experiments with a total of 6–15 mice per group. Representative BLI images were taken from one of three replicate experiments. **P < 0.01; ***P < 0.001.

Fig. 2.

Donor T cells deficient for JAK2 are prone to Th2 and Treg polarization in vivo. Lethally irradiated BALB/c mice were transplanted with 5 × 106 Ly5.1+ TCD-BM alone or plus 1 × 106 purified T cells (Ly5.2+) from WT B6 or JAK2 KO mice. Recipient splenic mononuclear cells were isolated for immunophenotyping on day +14 post-BMT. Data depict one representative mouse per group for IFNγ+, IL-4/5+, IL-17+, Foxp3+ (Tregs), or IL-10+ among gated H2Kb+Ly5.1-CD4+ or CD8+ cells (A). Average percentages +SD (B) or absolute numbers (C) of splenic T cell subsets are shown from one of three replicate experiments. Splenic cells from 9 to 11 mice per group were analyzed in total. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

JAK2 contributes to the migratory capacity of donor T cells. Lethally irradiated BALB/c mice were transplanted with 5 × 106 Ly5.1+ TCD-BM alone or plus 1 × 106 purified T cells (Ly5.2+) from WT B6 or JAK2 KO mice. Recipient splenic mononuclear cells were isolated for T cell migration surface markers on day +14 post-BMT. Flow plots show expression of CXCR3, CCR6, or α4β7 (A) among gated H2Kb+Ly5.1-CD4+ or CD8+ cells. (B) The mean frequency of T cell CXCR3, CCR6, or α4β7 expression ±SD for one of three replicate experiments is shown. A total of 9–11 mice per group were analyzed. (C) Quantified GVHD tissue damage scores ±SD from one representative experiment are shown (BMA: 6 mice; WT: 14 mice; JAK2 KO: 15 mice). (D) Representative H&E sections of GVHD target organs from each cohort showing vacuolar changes in the skin (↓); endothelialitis (#) and mononuclear infiltrates in the liver (*); crypt regeneration in the large intestine (LI, ^); and lamina propria inflammation in the small bowel (SI, +). *P < 0.05; **P < 0.01. (Magnification: 200×.)

Fig. 4.

Pharmacological inhibition of JAK2 with pacritinib reduces GVHD and spares GVL. Lethally irradiated BALB/c mice were transplanted with 5 × 106 TCD-BM alone or plus 1 × 106 T cells/mouse from WT B6 donors. Pacritinib 100 mg/kg or methylcellulose vehicle was given by oral gavage daily for 3 wk starting on day 0 of BMT. Recipient survival (A) and body weight loss (B) are shown. In separate experiments, lethally irradiated BALB/c mice were transplanted with carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells from either WT or JAK2 KO donors and treated with pacritinib or vehicle. Average percentages +SD of CFSE-diluted CD4+ and CD8+ T cells positive for IFNγ, IL-4/5, and IL-17 for are shown (C). A total of 12 mice/group were used across the three experiments (A–C). *P < 0.05; **P < 0.01; ***P < 0.001. Pac, pacritinib; Veh, vehicle.

Fig. 5.

Pacritinib polarizes a Th2 response by human T cells after allogeneic stimulation in vitro and permits the differentiation of suppressive iTreg. Data show the effect of pacritinib (2.5 μM) on (A) Th1 (IFNγ) versus Th2 (IL-4), (B) T-BET expression (replicate means ± SEM, n = 4 experiments), and (C) RORγT expression ±SD among CD4+ T cells after DC allostimulation (one of two experiments is shown, performed in triplicate). (D and E) iTregs were generated from Treg-depleted, DC-allostimulated CD4+ T cells in the presence of pacritinib (2.5 μM) or vehicle control. Contour plots show iTregs and corresponding Foxp3 expression. (F and G) Graphs show the mean frequency of iTreg and ratio of iTreg to alloreactive Tconv (CD4+, CD25+, CD127+) ±SD after DC allostimulation. n = 6 experiments. (H) iTreg-suppressive potency is demonstrated against self-T cell responders stimulated by allogeneic DCs without additional drugs. One of two experiments are shown, each performed in triplicate. *P < 0.05; **P < 0.01.

Fig. 6.

Pacritinib reduces xenograft rejection. NSG mice received a 1-cm2 split thickness human skin graft. After 30 d of rest to ensure engraftment, an inoculum of 5 × 106 human PBMCs (allogeneic to the skin) were administered by i.p. injection. Unique pairs of donor skin and allogeneic PBMCs were used for each set of experiments. Pacritinib 100 mg/kg or vehicle was given twice a day from days 0 to +14. (A) Graph shows human skin graft survival among pacritinib- or vehicle-treated NSG hosts (log-rank test). (B) Representative images show skin at time of suture removal (day −30) and at day +35. (C) Histologic representations of the skin grafts uniformly harvested on day +21 demonstrate that pacritinib reduces lymphocytic infiltration (^) and severe basal vacuolar changes of the graft, such as the subepidermal blister (#). Low power at 6×, high-power Inset at 20×. (Scale bar, 400 µm.) (D) Bar graph shows pathologic skin graft rejection scores at day +21 among vehicle- and pacritinib-treated mice. (E and F) Immunohistochemistry and accompanying bar graph shows skin-resident Th1 cells at day +21 by dual staining of CD4 (red) and T-BET (brown). n = 2 experiments, 5–6 mice per arm. (G) Graph depicts mean specific lysis ±SEM by human CD8+ CTL generated in vivo using NSG mice transplanted with human PBMCs (30 × 106) and vaccinated with irradiated U937cells (10 × 106) on days 0 and +7. Mice were treated with pacritinib (100 mg/kg twice a day), ruxolitinib (30 mg/kg twice a day), or vehicle from day 0 up to day +12. Human CD8+ T cells were harvested from euthanized mice between days +10 to +12. Results shown are from one of two independent experiments. U937 lysis was measured by colorimetric assay after 4 h. *P < 0.05; **P < 0.01; ****P < 0.0001. (Magnification: C and E, slides were scanned using a 20×/0.8 N.A. objective and viewed using a 6.5× digital zoom in ImageScope software.)