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. 2017 May 18;2(10):e91701.
doi: 10.1172/jci.insight.91701.

Ceramide synthesis regulates T cell activity and GVHD development

M Hanief Sofi  1 , Jessica Heinrichs  1 , Mohammed Dany  2 , Hung Nguyen  1 , Min Dai  1 , David Bastian  1 , Steven Schutt  1 , Yongxia Wu  1 , Anusara Daenthanasanmak  1 , Salih Gencer  2 , Aleksandra Zivkovic  3 , Zdzislaw Szulc  2 , Holger Stark  3 , Chen Liu  4 , Ying-Jun Chang  5 , Besim Ogretmen  2 , Xue-Zhong Yu  1
Affiliations

Ceramide synthesis regulates T cell activity and GVHD development

M Hanief Sofi et al. JCI Insight. .

Abstract

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for a variety of hematologic malignances, yet its efficacy is impeded by the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production, and migration of alloreactive donor T cells. Hence, strategies to limit GVHD are highly desirable. Ceramides are known to contribute to inflammation and autoimmunity. However, their involvement in T-cell responses to alloantigens is undefined. In the current study, we specifically characterized the role of ceramide synthase 6 (CerS6) after allo-HCT using genetic and pharmacologic approaches. We found that CerS6 was required for optimal T cell activation, proliferation, and cytokine production in response to alloantigen and for subsequent induction of GVHD. However, CerS6 was partially dispensable for the T cell-mediated antileukemia effect. At the molecular level, CerS6 was required for efficient TCR signal transduction, including tyrosine phosphorylation, ZAP-70 activation, and PKCθ/TCR colocalization. Impaired generation of C16-ceramide was responsible for diminished allogeneic T cell responses. Furthermore, _targeting CerS6 using a specific inhibitor significantly reduced T cell activation in mouse and human T cells in vitro. Our study provides a rationale for _targeting CerS6 to control GVHD, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.

Keywords: Immunology; Transplantation.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Effects of ceramide synthase 4 or ceramide synthase 6 on T cell phenotype.
Spleens were obtained from unmanipulated age- and sex-matched WT, ceramide synthase 4 (CerS4) KO, or ceramide synthase 6 (CerS6) KO mice on a B6 background. Splenocytes were individually processed, counted, and stained for the expression of CD4, CD8, Foxp3, CD44, and CD62L. (A and B) Percentages of CD4+ or CD8+ cells on total live splenocytes, percentages of CD25+Foxp3+ cells on gated CD4+ cells, and (C and D) CD44 and CD62L expression on gated CD4+ or CD8+ cells are shown. (A and C) Data shown are from 1 representative mouse. The data are from 1 representative experiment of 3 independent experiments (mean ± SD with 3 mice per group). Significance was determined by using ANOVA test. **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. Role of ceramide synthase 6 in T cell response to alloantigen in vivo.
Purified T cells from WT, ceramide synthase 4 (CerS4) KO, or ceramide synthase 6 (CerS6) KO mice on a B6 background were labeled with CFSE and i.v. injected into lethally irradiated BALB/c mice at 2 × 106 per mouse. Four days after cell transfer, spleens were collected from recipient mice and subjected to cell counting and FACS staining. (A) Percentages of donor-derived (H2Kb+) CD4+ and CD8+ cells among gated live cells and representative flow figures of CFSE dilution and IFN-γ+ cells on gated donor CD4+ or CD8+ cells. (B) Percentages of CFSE diluted and IFN-γ+ cells on gated donor CD4+ and CD8+ cells. Error bars represent one standard deviation in each group. Data shown are replicate of 2 independent experiments. Significance was determined by using ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3. Essential role of ceramide synthase 6 in T cell–induced graft-versus-host disease.
(A) BALB/c or (B) BD2F1 mice were lethally irradiated and transplanted with 5 × 106 T cell–depleted BM cells (Ly5.1+) per mouse or BM plus purified T cells (Ly5.2+) (0.5 × 106/mouse for BALB/c or 2 × 106/mouse for BD2F1) from WT, ceramide synthase 4 (CerS4) KO, or ceramide synthase 6 (CerS6) KO B6 mice. BALB/c and BDF1 recipients were monitored for body weight change, survival, and clinical score until 80 days after BMT (n = 5 per group). Data shown are from 1 of 3 independent experiments for BALB/c mice. For comparison of recipient survival among groups, the log-rank test was used to determine statistical significance. Clinical scores and body weight loss were compared using a nonparametric Mann-Whitney U test. *P < 0.05, **P < 0.01.
Figure 4
Figure 4. Effect of ceramide synthase 6 on T cell–mediated–GVL activity.
B cell lymphoma was infused with BM or BM plus 0.5 × 106/mouse T cells into lethally irradiated allogeneic recipients. Mice were monitored for (A) body weight, (B) survival, (C) clinical score, and (D) tumor signal. Data shown are from 1 of 2 independent experiments (n = 6–7 per group for WT and CerS6 KO). For comparison of recipient survival among groups, the log-rank test was used to determine statistical significance. Clinical scores and body weight loss were compared using a nonparametric Mann-Whitney U test. ***P < 0.001.
Figure 5
Figure 5. Effect of ceramide synthase 6 on donor T cell differentiation after allo-BMT.
BMT was carried out as outlined in Figure 3 using BALB/c mice as the recipients. Three weeks after BMT, livers and colons were collected from the recipients for H&E staining and were scored for microscopic GVHD severity by a pathologist blinded to the treatment groups. Data on small intestine, lung, and skin are not shown. (A) Pathological score of GVHD _target organs (mean ± SD). (B) Recipient spleen and liver cells were subjected to cell counting and FACS staining. The expression of IFN-γ, IL-4/5, or Foxp3 on gated H2Kb+ donor CD4+ or CD8+ cells from a representative mouse from each group. (C and D) The absolute numbers of (C) IFN-γ+, IL-4/5+, and Foxp3+ donor CD4+ cells and (D) CD8+ (H2Kb+Ly5.1–) cells in recipient spleens and (E and F) for livers, respectively. Data shown are from 2 independent experiments (n = 6). Significance was determined by ANOVA (A) and Student’s t tests (for C and D). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6. Role of ceramide synthase 6 in proximal TCR signaling.
(A) Western blot was performed using cell lysates prepared from freshly isolated splenocytes before and after TCR engagement at the indicated time points. The membrane was probed with an antibody detecting total phosphotyrosine, stripped, and reprobed with anti–β actin. (B) CD3ζ material was immunoprecipitated from WT or ceramide synthase 6 (CerS6) KO T cells before and after TCR engagement. The presence of coimmunoprecipitated protein was then evaluated by Western blot using total phosphotyrosine. (C and D) Splenocytes were subjected to TCR engagement for the indicated time points and stained for CD3 and PKCθ expression followed by analysis with confocal microscopy (original magnification, ×63). At least 4–5 fields in each condition per experiment were examined. Data shown are 1 representative of 3 independent experiments.
Figure 7
Figure 7. Effect of ceramide synthase 6 on lipid metabolism.
(A) T cells from WT or ceramide synthase 6 (CerS6) KO mice were stimulated with allogeneic APCs for 5 days, and unstimulated T cells were used as controls. These T cells were subjected to mass spectrometry HPLC-MS analysis for different ceramide species. Data shown are from 3 independent experiments (n = 9). Splenocytes from (B) WT or (C) CerS6 KO mice were incubated with C16-cer for 1 hour at 37°C and were either left unstimulated or subjected to TCR engagement for the indicated time points. Cells were then stained for CD3 and PKCθ expression, followed by analysis with confocal microscopy (original magnification, ×63). Data shown are from 1 representative experiment of 3 independent experiments. Significance was determined by ANOVA test. **P < 0.01, ***P < 0.001.
Figure 8
Figure 8. Effect of C16-ceramide on T cell proliferation and cytokine expression.
Purified T cells from WT or ceramide synthase 6 (CerS6) KO mice were labeled with CFSE and i.v. injected into lethally irradiated BALB/c recipients at 2 × 106/mouse. Recipients were also injected intraperitoneally at 15 mg/kg with C16-ceramide or vehicle from day 1 to day 3. Four days after cell transfer, recipient spleens were collected and subjected to cell counting and FACS staining. (A) Percentages of donor-derived (H2Kb+) CD4+ and CD8+ cells on gated live cells. (B–E) Representative flow figures of CFSE dilution and percentages of IFN-γ+ cells on gated donor CD4+ and CD8+ cells (mean ± SD of 3–4 mice per group). Data shown are from 1 representative experiment of 2 independent experiments. Significance was determined by ANOVA test for (C, E, and F). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 9
Figure 9. Effects of ceramide synthase 6 inhibition on T cell allogeneic responses.
CFSE-labeled T cells from WT or ceramide synthase 6 (CerS6) KO mice were stimulated with allogeneic APCs in the presence or absence of 50 μM ST1072 for 5 days. Cells were subjected to FACS staining and analyzed for T cell proliferation and cytokine expression. (A and B) CFSE dilution and percentage of IFN-γ+ on gated CD4+ or CD8+ cells. (C and D) CFSE-diluted or IFN-γ+ cells (mean ± SD). CFSE-labeled human T cells were stimulated with human DCs generated from a HLA-mismatched donor for 5 days in the presence or absence of 50 μM ST1072. Cells were subjected to FACS staining and analysis for proliferation and IFN-γ production. (E and F) representative flow figures of CFSE dilution and percentages of IFN-γ+ cells on gated donor CD4 cells T cells. (G) The mean ± SD for CFSE–diluted and IFN-γ+ cells, respectively. Data shown are 1 of 3 independent experiments. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.
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References

    1. Ferrara JL, Reddy P. Pathophysiology of graft-versus-host disease. Semin Hematol. 2006;43(1):3–10. doi: 10.1053/j.seminhematol.2005.09.001. - DOI - PubMed
    1. Socié G, Ritz J. Current issues in chronic graft-versus-host disease. Blood. 2014;124(3):374–384. doi: 10.1182/blood-2014-01-514752. - DOI - PMC - PubMed
    1. Shlomchik WD. Graft-versus-host disease. Nat Rev Immunol. 2007;7(5):340–352. doi: 10.1038/nri2000. - DOI - PubMed
    1. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet. 2009;373(9674):1550–1561. doi: 10.1016/S0140-6736(09)60237-3. - DOI - PMC - PubMed
    1. Welniak LA, Blazar BR, Murphy WJ. Immunobiology of allogeneic hematopoietic stem cell transplantation. Annu Rev Immunol. 2007;25:139–170. doi: 10.1146/annurev.immunol.25.022106.141606. - DOI - PubMed
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