Ceramide synthase 6 impacts T-cell allogeneic response and graft-versus-host disease through regulating N-RAS/ERK pathway

Abstract

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for various hematologic malignancies, predominantly through potent graft-versus-leukemia (GVL) effect. However, the mortality after allo-HCT is because of relapse of primary malignancy and followed by graft-vs-host-disease (GVHD) as a major cause of transplant-related mortality. Hence, strategies to limit GVHD while preserving the GVL effect are highly desirable. Ceramide, which serves a central role in sphingolipid metabolism, is generated by ceramide synthases (CerS1-6). In this study, we found that genetic or pharmacologic targeting of CerS6 prevented and reversed chronic GVHD (cGVHD). Furthermore, specific inhibition of CerS6 with ST1072 significantly ameliorated acute GVHD (aGVHD) while preserving the GVL effect, which differed from FTY720 that attenuated aGVHD but impaired GVL activity. At the cellular level, blockade of CerS6 restrained donor T cells from migrating into GVHD target organs and preferentially reduced activation of donor CD4 T cells. At the molecular level, CerS6 was required for optimal TCR signaling, CD3/PKCθ co-localization, and subsequent N-RAS activation and ERK signaling, especially on CD4⁺ T cells. The current study provides rationale and means for targeting CerS6 to control GVHD and leukemia relapse, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.

Figure 1.. Absence of CerS6 on donor T cells or inhibition of CerS6 ameliorates cGVHD development.

(A) The splenocytes from WT mice on B6 background were intravenously (i.v.) injected at 6×10⁶/mouse into lethally irradiated BALB/c mice. The recipient mice were injected with vehicle or ST1072 at 1mg/kg intraperitoneally (i.p.) from day −1 to day 3. Four days after cell transfer, serum was collected and subjected to mass spectrometry HPLC-MS analysis for different ceramide species. Data shown is pooled from two independent experiments (n=10 per group). BALB/c mice were lethally irradiated and transplanted with 5×10⁶/mouse T-cell depleted-bone marrow (TCD-BM) alone or plus splenocytes (0.5×10⁶/mouse) either from WT or CerS6KO B6 mice. One group of WT recipient mice was injected i.p. with ST1072 at 2 mg/kg from day-1 to day 28. The recipients were monitored for body weight and clinical score (B) until 60 days post-BMT. Data shown is from two combined experiments (N=10–15 per group). Recipient clinical scores and body weight loss were compared using a nonparametric Mann-Whitney U test. Asterisks indicate statistical significance *p< 0.05, ***p < 0.001.

Figure 2.. ST1072 modulates B- and T-cell functions during cGVHD development.

BALB/c mice were lethally irradiated and 5×10⁶/mouse TCD-BM or plus 5×10⁶/mouse splenocytes from B10.D2 donor mice were transplanted. One group of the recipient mice was injected i.p. with ST1072 at 2 mg/kg from day-1 to day 28 or day 28 to day 59. The recipients were monitored for body weight and clinical score until 60 days post-BMT. 60 days post cell transfer, spleens and peripheral lymph nodes (pLNs) were collected from the recipient mice and subjected to cell counting and FACS staining. Percentage of CD4⁺ or CD8⁺ among donor cells and percentage of IFN-γ among CD4⁺ or CD8⁺ donor cells are shown in recipient spleens (A) or periphery lymph nodes (pLNs) (B). Percentages of B220⁺ or B220^lowCD138⁺ and FAS⁺GL⁺ (germinal center cell, GC cell) are shown among donor B cells in recipient spleens (C). Pooled data is summarized with statistical analyses where either treatment (day 0 or 28) was compared with vehicle control. Data shown is one of the two replicate experiments. Significance is determined by one-way ANOVA (using multiple comparison test). Asterisks indicate statistical significance *p< 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. The effects of ST1072 or FTY720 on GVH and GVL responses.

BALB/c mice were lethally irradiated and transplanted with 5 × 10⁶/mouse TCD-BM (Ly5.1⁺) or plus purified T cells (1×10⁶) from B6 mice and mixed-lineage leukemia (MLL) as well. The recipient mice were injected i.p. with ST1072 or FTY720 at 1mg/kg per mouse/day from day −1 to day14. The mice were monitored for clinical score (A) survival (B) and tumor mortality (C). Data shown here is from two independent experiments (n=10 per group). For comparison of recipient survival and tumor mortality among groups, the log-rank test was used to determine statistical significance. Clinical scores were compared using a nonparametric Mann-Whitney U test. Asterisks indicate statistical significance *p< 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. ST1072 treatment modulates T-cell expansion and migration after allo-BMT.

BALB/c mice were lethally irradiated and transplanted with 5×10⁶/mouse TCD-BM (Ly5.1⁺) or plus purified T cells (1×10⁶) from β-actin luciferase transgenic B6 mice. The recipient mice were injected i.p. with vehicle alone or ST1072 at 1 mg/kg daily from day −1 to day 14. Donor T-cell expansion and migration were monitored using bioluminescent imaging (BLI) at the time points indicated. Macro-photos are shown for BLI of total body (A) and individual organs (B) with a region of interest (ROI) summary (C). Data shown here is pooled from two independent experiments (n=10 per group). Significance is determined by Student’s t test. Asterisks indicate statistical significance *p < 0.05.

Figure 5.. The role of CerS6 in proximal TCR signaling.

(A) Freshly isolated T cells from WT or CerS6KO mice were either left unstimulated or stimulated with plate bound anti-CD3 at the time points indicated. Some WT T cells were also stimulated in the presence of ST1072 at 50ug/ml. These T cells were subjected to immunoprecipitation using GST-RAF RBD. Protein lysates were incubated with GST-RAF RBD conjugated glutathione sepharose beads. The proteins bound to the Glutathione beads (top 2 panels) were analyzed by Western blot using anti-NRAS or anti-GST antibodies. Cell lysates were also measured for total or phosphor-ERK antibodies. (B) Freshly isolated T cells from WT mice were stimulated in presence and absence of ST1072 at the time points indicated. The membrane and cytosolic fraction were isolated from these cells and were subjected to western blot analysis for NRAS, H-RAS, K-RAS and β-actin. Data shown here is from one of two independent experiments. The control or oncogenic NRAS (Q61K) plasmid DNA were transfected into naïve T cells using Amaxa Nucleofector kit V from Lonza per manufacturer’s instructions. Six hours after, the cells collected and co-cultured with allogeneic antigen presenting cells for 5 days in presence or absence of ST1072. Cultured cells were re-stimulated with PMA/Ionomycin for 4hrs and analyzed for the percentage of IFN-γ production on gated CD4⁺ or CD8⁺ cells (C). Data shown is one of the two replicate experiments. Significance is determined by Student’s t test. Asterisks indicate statistical significance **p < 0.01.

Figure 6.. The effect of CerS6 on CD3 and PKCθ capping in CD4 T cells after TCR stimulation.

Primary CD4 T cells (1×10⁶ cells) were engaged with TCR for the indicated time points followed by their incubation with anti-CD3 antibody for 30 minutes at 4°C. After PBS wash and incubation with goat anti-hamster IgG at 37°C, the reaction was stopped immediately, and the cells were washed and incubated with Alexa Fluor 488-conjugated antibody for 30 minutes at 4°C. The cells were fixed and permeabilized followed by incubation overnight at 4°C with PKCθ antibody, and then stained with Alexa Fluor 594 secondary antibody followed by analysis with confocal microscopy (original magnification x63). At least 6–7 fields in each condition per experiment were examined. The data shown here is one representative of two independent experiments.

Figure 7.. Preventative treatment with ST1072 reduces GVHD in NSG xenograft mouse model.

NSG-A2⁺ mice were irradiated (250 cGy) and were transplanted with HLA-A2^– human PBMCs (10×10⁶). The recipient mice were injected i.p. with vehicle or with ST1072 at 1mg/kg daily from day −1 to day 14. The recipients were monitored for body weight and survival (A) until 60 days post transplantation. Data shown here are pooled from two combined experiments (n=13–14 per group). In a separate experiment, Raji B cell lymphoma (1×10⁶) were infused together with or without HLA-A2^– human PBMCs (10×10⁶) into NSG-A2⁺ mice, which were treated with vehicle alone or with ST1072. The mice were monitored for clinical score, survival and tumor burden (B) until 50 days post transplantation. Data shown here are from two combined experiments (n=13–14/group). For comparison of recipient survival among groups, the log-rank test was used to determine statistical significance. Clinical scores were compared using a nonparametric Mann-Whitney U test. Asterisks indicate statistical significance *p < 0.05, *p < 0.01.