Pharmacologic inhibition of PKCα and PKCθ prevents GVHD while preserving GVL activity in mice

Abstract

Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.

Figure 1

Absence of PKCα and θ in donor T cells more comprehensively abrogates GVHD lethality and pathology than deficiency of PKCθ alone. Recipient BALB/c mice were lethally irradiated (800 cGy) and transplanted with 5 × 10⁶ genotype-matched TCD-BM cells alone (n = 2 for each genotype) or in addition to 1 × 10⁶ T cells (CD4⁺, CD8⁺, and CD25^–) from littermate WT C57BL/6 or PKCα^−/−, PKCθ^−/−, or PKCα^−/−/θ^−/− mice (n = 11, 10, 15, and 15 recipients, respectively). Recipient mice were monitored throughout the experimental period for survival (A) and weight change (B), and pooled data from 3 separate experiments are represented. In separate experiments, recipients (n = 4 recipients per group per experiment) were euthanized 2 weeks posttransplant, and samples of skin, liver, lung, small intestine, and large intestine were collected in formalin for routine hematoxylin and eosin and scored for microscopic GVHD severity by a pathologist blinded to the treatment groups. Photomicrographs depicting the average disease score morphology from 1 representative experiment out of 3 separate experiments (C) and average scores for GVHD target organs across 3 separate experiments (D) are depicted. Recipients surviving to 120 days post-BMT in experiments depicted in panels A-B were euthanized, and their spleens were subjected to total splenocyte count (E) and flow cytometric staining and analysis for H2kb, CD4, CD8, and B220 surface expression (F) and plated with 1 ug/mL anti-CD3 or 5 ug/mL LPS for 72 hours followed by overnight tritium thymidine incorporation to observe activation of T cells or B cells, respectively (G). T- and B-cell activities are depicted as per cell activity normalized to the original spleen count. *P < .05; **P < .01; ***P < .001 (compared with WT [brackets indicate statistical significance for comparisons between PKCα^−/−/θ^−/− and PKCθ^−/− treatment groups]). All error bars indicate standard error of the mean (SEM).

Figure 2

Donor T cells deficient for PKCα and θ produce reduced inflammatory cytokines in vivo. Lethally irradiated (800 cGy) BALB/c mice were transplanted with 5 × 10⁶ genotype-matched TCD-BM cells alone (n = 2 for each genotype) or in addition to 1 × 10⁶ T cells (CD4⁺, CD8⁺, and CD25^–) from littermate WT C57BL/6 or PKCα^−/−, PKCθ^−/−, or PKCα^−/−/θ^−/− mice (n = 4 recipients per group per experiment). Two weeks posttransplant, recipient spleens (A) and livers (B) were harvested, and organ cell counts determined and stained for H2K^b, CD4, CD8, IFNγ, TNFα, and IL-4/5 analysis by flow cytometry. Absolute cell numbers depicted were calculated from whole spleen and liver counts at necropsy with flow cytometric percentages. Serum collected at necropsy was subjected to cytokine bead analysis (C) to quantify serum cytokine concentrations of IFN, TNF, IL-2, IL-6, IL-10, and IL-17. Cytokines that are not graphically represented were undetectable in serum. Averages for all values are represented with error bars indicating SEM from 1 representative experiment out of 3 separate experiments. *P < .05; **P < .01; ***P < .001 (compared with WT [brackets indicate statistical significance for comparisons between PKCα^−/−/θ^−/− and PKCθ^−/− treatment groups]).

Figure 3

Inhibition of PKCα and θ significantly ameliorates acute GVHD after myeloablative allogeneic BMT. Recipient BALB/c mice were lethally irradiated (800 cGy) and transplanted with 5 × 10⁶ C57BL/6 TCD-BM cells alone (n = 6) or in addition to 1 × 10⁶ total T cells from WT C57BL/6 donors and treated twice daily by gavage with vehicle (n = 15) or 40 mg/kg R524 (n = 12) beginning on day 0 (the day of BMT) and continuing daily for 6 weeks. Recipient mice were monitored throughout the experimental period for survival (A) and weight change (B), and pooled data from 3 separate experiments are represented. In separate experiments, recipients (n = 4 recipients per group per experiment) were euthanized 2 weeks posttransplant, and samples of skin, liver, lung, small intestine, and large intestine were collected in formalin for routine hematoxylin and eosin and scored for microscopic GVHD severity by a pathologist blinded to the treatment groups. Average scores for GVHD target organs across 3 separate experiments (C) are depicted. *P < .05; **P < .01; ***P < .001 (compared with vehicle-treated recipients). All error bars indicate SEM.

Figure 4

Inhibition of PKCα and θ restricts alloreactive donor T-cell expansion and migration to GVHD target organs. BALB/c mice were irradiated at 800 cGy and transplanted with 5 × 10⁶ WT C57CL/6 TCD-BM cells alone (n = 2 per experiment) or in addition to 1 × 10⁶ total T cells from β-actin luciferase transduced C57BL/6 mice and treated twice daily by gavage with vehicle or 40 mg/kg R524 (n = 4 per group per experiment) beginning on day 0 and continuing daily for 2 weeks. Fourteen days posttransplant, recipients were subjected to intraperitoneal luciferin injection and whole body BLI followed by a second luciferin injection, euthanization, organ removal, and BLI of GVHD target organs (A). Bioluminescence was quantified using whole body (B) or whole organ (C) region of interest gating and signal normalization with Living Image software. Average representative whole body images, ex vivo organ images, and average bioluminescence intensities ± SEM depicted are from 1 representative experiment of 2 separate experiments. *P < .05; **P < .01; ***P < .001 (compared with vehicle-treated controls).

Figure 5

Inhibition of PKCα and θ reduces alloreactive chemokine receptor expression and inflammatory cytokine production in vivo. Total-body irradiation (800 cGy) was dosed to BALB/c mice, which were then transplanted with 5 × 10⁶ WT C57BL/6 TCD-BM cells alone (n = 2 per experiment) or in addition to 1 × 10⁶ total T cells and treated twice daily by gavage with vehicle or 40 mg/kg R524 (n = 4 per group per experiment) beginning on day 0 and continuing daily for 2 weeks. Fourteen days posttransplant, recipient spleens, livers, and lungs were harvested; organ cell counts determined; and samples stained for H2Kb, CD4, CD8, CXCR3, CCR6, IFN-γ, and TNF-α. Average percentages ± SEM are based on live H2K^b+ (donor) cell gate (A), T-cell chemokine receptor expression (B), and intracellular T-cell cytokine expression (C). Serum collected at necropsy was subjected to cytokine bead analysis (D) to quantify serum cytokine concentrations of IFN, TNF, IL-2, IL-6, IL-10, and IL-17. Cytokines that are not graphically represented were undetectable in serum. Data are representative of 1 experiment out of 3 separate repeat experiments. *P < .05; **P < .01; ***P < .001 (compared with vehicle-treated controls).

Figure 6

GVL is preserved in myeloablated allogeneic BMT recipients treated with R524. BALB/c mice were lethally irradiated (800 cGy) and transplanted with 5 × 10⁶ C57BL/6 TCD-BM cells alone (n = 4) or in addition to 1 × 10⁶ total T cells from WT C57BL/6 and treated twice daily by gavage with vehicle (n = 10) or 40 mg/kg R524 (n = 10) beginning on day 0 (the day of BMT) and continuing daily for 6 weeks. Additionally, recipients were intravenously injected with 2 × 10³ A20 luciferase-transduced lymphoma cells at the time as BMT. Recipients were monitored throughout the experimental period for survival (A), weight change (B), and tumor expansion by luciferin intraperitoneal injection and whole body BLI (C). Average survival and weight changes across 2 separate repeat experiments and recipient BLI images from 1 of the 2 replicated experiments are shown. *P < .05; **P < .01; ***P < .001 (compared with vehicle-treated recipients). In a separate experiment in which recipient mice were treated with vehicle or R524 twice daily for 14 days post-BMT, splenocytes were assayed for preserved cytotoxic T lymphocyte (CTL) activity against control EL4 or mismatched P815 tumor target cells ex vivo (D). CTL assay was run in triplicate and normalized to % CD8⁺ cells in recipient spleens.

Figure 7

GVHD induction is impaired, whereas GVL effects are preserved in R524-treated recipients in an MiHA-mismatch BMT model. C57BL/6 recipients received total-body irradiation (1200 cGy) and were transplanted with donor C3SW.H TCD-BM alone (n = 6) or in combination with 3 × 10⁶ T cells (CD4⁺, CD8⁺, CD25^–, and CD44^–) and treated with vehicle (n = 6) or 40 mg/kg R524 (n = 6) twice daily for 6 weeks beginning on day 0. Additional recipients were intravenously injected with 5 × 10⁴ C1498 luciferase-transduced leukemia cells at the time as BMT (n = 10 vehicle treated and n = 10 R524 treated). Recipients without tumor (A-B) and with tumor (C-E) were monitored throughout the experimental period for survival (A,C) and weight change (B,D). In leukemia recipients, tumor expansion was tracked by luciferin intraperitoneal injection and whole body BLI (E). Leukemia growth was confirmed in recipients at necropsy. Average survival and weight changes across 3 separate repeat experiments and recipient BLI images from 1 of 2 repeating experiments are shown. *P < .05; **P < .01; ***P < .001 (compared with vehicle-treated recipients).