Targeting Sirt-1 controls GVHD by inhibiting T-cell allo-response and promoting Treg stability in mice

Abstract

Graft-versus-host disease (GVHD) remains one of the major complications after allogeneic bone marrow transplantation (allo-BMT). Sirtuin-1 (Sirt-1) plays a crucial role in various biological processes including cellular senescence, metabolism, and inflammatory responses. Sirt-1 deacetylation regulates different transcription factors that are important for modulating immune responses. In the current study, we addressed the role of Sirt-1 in GVHD induction by employing Sirt-1 conditional knockout mice as well as a pharmacological Sirt-1 inhibitor. Using major histocompatibility complex (MHC)-mismatched and MHC-matched murine BMT models, we found that Sirt-1^-/- T cells had a reduced ability to induce acute GVHD (aGVHD) via enhanced p53 acetylation. Sirt-1-deficient T cells also promoted induced regulatory T cell (iTreg) differentiation and inhibited interferon-γ production after allo-BMT. Sirt-1 deletion in iTregs increased Foxp3 stability and restrained iTreg conversion into pathogenic T cells. Furthermore, we found that administration with a Sirt-1 inhibitor, Ex-527, significantly improved recipient survival and clinical scores, with no signs of tumor relapse. These results indicate that Sirt-1 inhibition can attenuate GVHD while preserving the graft-versus-leukemia effect. Consistently, Sirt-1-deficient T cells also displayed a remarkably reduced ability to induce chronic GVHD (cGVHD). Mechanistic studies revealed that Sirt-1 deficiency in T cells enhanced splenic B-cell reconstitution and reduced follicular T helper cell development. Sirt-1 deficiency in T cells modulated donor B-cell responses reducing both B-cell activation and plasma cell differentiation. In addition, therapeutic Sirt-1 inhibition could both prevent cGVHD and reduce established cGVHD. In conclusion, Sirt-1 is a promising therapeutic target for the control of aGVHD and cGVHD pathogenesis and possesses high potential for clinical application.

Graphical abstract

Figure 1.

Sirt-1 regulates T-cell proliferation, differentiation, and aGVHD pathogenicity. (A) Purified T cells from either WT or Sirt-1^−/− donors (H-2^b) were labeled with CFSE and transferred into lethally irradiated BALB/c (H-2^d) mice at 2 × 10⁶ cells per mouse. Three days after cells transfer, recipient spleens were harvested and analyzed by flow cytometry. Representative figures and percentages of CFSE and IFN-γ are shown on gated live cells followed by H-2^b+CD4⁺ or CD8⁺ cells. (B) Average percentages of CFSE-diluted and IFN-γ⁺ cells are shown on gated live donor CD4⁺ or CD8⁺ T cells in recipient spleen (n = 5 mice/group). (C-E) Lethally irradiated BALB/c (700 cGy) mice underwent transplantation with 5 × 10⁶ TCD-BM per mouse plus 0.7 × 10⁶ CD25-depleted T cells. (C) Survival, (D) bodyweight loss, and (E) clinical scores were monitored. (F-G) Tissues from BALB/c recipients were collected on day 14 after allo-BMT and analyzed for pathology. (G) Hematoxylin and eosin staining of representative pictures of livers and large intestines are shown (n = 10 mice/group). Original magnification ×200. Log-rank (Mantel-Cox) test was used to analyze the survival curve. Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 2.

Sirt-1 inhibits iTreg differentiation after allo-BMT. Two weeks after allo-BMT (B6 to BALB/c model), spleen and liver of the recipients were harvested and analyzed. (A-D) Representative dot plots and the average percentages of Foxp3, IFN-γ, IL-4, and IL-17 expressions on gated donor CD4⁺ or CD8⁺ T cells from spleen are shown. (E-H) Representative dot plots and the frequency of Foxp3 and IFN-γ⁺ cells gated on donor CD4⁺ or CD8⁺ T cells from liver are shown. (F) The absolute number of total cells recovered from liver and absolute numbers of donor CD4⁺ and CD8⁺ T cells are depicted. (I) Cytokine analyses of mice serum on day 14 are shown (n = 8 mice/group). Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001.

Figure 3.

Sirt-1 enhances T-cell alloreactivity through p53 deacetylation. (A-B) Purified WT or Sirt-1^−/− T cells were labeled with CFSE and cocultured with T-cell-depleted splenocytes as APCs from BALB/c mice for 5 days in the presence of either dimethyl sulfoxide or Ex-527 (10 µg/mL). Cells were restimulated with phorbol 12-myristate 13-acetate and ionomycin for cytokine measurement. Average percentages of CFSE-diluted and IFN-γ⁺ gated on donor CD4 or CD8 T cells are shown. (C) CD4⁺ T cells isolated from WT or Sirt-1^−/− or WT T cells treated with Ex-527 were polarized into Th1 cells in the presence of syngeneic APCs with 1 μg/mL anti-mouse CD3ε (clone 145-2C11); 10 ng/mL mIL-12, and 1 ng/mL mIFN-γ were used for Th1 polarization. For western blot analysis, polarized cells were pretreated with 2 μM Trichostatin A (Sigma Aldrich) for 45 minutes to induce basal protein acetylation for detection of global acetylation. (D) Purified CD4 T cells from WT or Sirt-1^−/− or Ex-527-treated cells were polarized under Th1 condition for 3 days and analyzed by western blot analysis for α-acetylated p53 and total p53. (E-F) T cells isolated from WT were labeled with CFSE and cocultured with allogeneic APCs for 5 days in the presence of Ex-527 or Pifithrin-μ, p53 inhibitor (10 μg/mL each) or in combinations. The frequency of CFSE diluted and IFN-γ gated on donor CD4 or CD8 T cells were analyzed. Data represent mean ± standard error of the mean. Ordinary 1-way analysis of variance with Sidak multiple comparisons test was used for statistical analysis (n = 6 per group). *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 4.

Sirt-1 deacetylates Foxp3 and decreases iTregs stability. (A) Allogeneic CD4 iTregs were generated in vitro by coculturing naive CD4 T cells with allogeneic dendritic cells in the presence of IL-2, TGF-β, and retinoic acid for 5 days. CD4 iTregs were enriched for CD25^hi cells. (B) Enriched CD25⁺ CD4iTregs were analyzed by western blot analysis for detection of global acetylation. (C) Five-day in vitro-generated CD4 iTregs were restimulated with IL-2 and analyzed for pSTAT5 expression. (D) Enriched CD4 iTregs were cocultured with allogeneic APCs in the presence of IL-2 or IL-2+IL-12 for 3 days. Percentage Foxp3 retention was analyzed on day 3 (n = 4). (E) Experimental scheme: lethally irradiated BALB/c mice were adoptively transferred with 5 × 10⁶ Rag1^−/− BM and 0.5-1 × 10⁶ CD4 iTregs (Ly5.2). On day 3, 0.5-2 × 10⁶ CD25-depleted T-cells from B6Ly5.1 congenic mice were injected to induce GVHD in recipients. On day 7 or 14 after allo-BMT, spleen was harvested and analyzed. (F) Percentages of Foxp3 and CD25 expressions on CD4iTregs analyzed on day 7 (gated on Ly5.2⁺CD4⁺) are shown. (G) Percentages of Foxp3 and IFN-γ expressions of transferred CD4 iTregs are shown (n = 4-5 mice per group). Data represent the mean ± standard error of the mean. Student t-test was used for statistical analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 5.

Pharmacological inhibition of Sirt-1 with Ex-527 alleviates aGVHD while preserving the GVL activity. (A-B) GVHD experiments: Lethally irradiated (700 cGy) BALB/c mice underwent transplantation with 5 × 10⁶ TCD-BM alone or plus 0.7 × 10⁶ CD25⁻ T cells per mouse isolated from WT and administered with either phosphate-buffered saline or 2 mg/kg Ex-527/mouse/day daily for 3 weeks. (A) GVHD clinical score. (B) Survival (n = 10 mice/group). (C-G) GVL experiments: (C) Bar graph shows quantified percentages of MLL-AF9-GFP in the recipients’ peripheral blood on days 20, 30, 45, and 60 (n = 10 mice/group). (D) Survival of recipient mice was monitored. (E-G) Lethally irradiated (1100 cGy, split dose) BDF1 recipients were transplanted with 5 × 10⁶ TCD-BM and 5 × 10³ P815 mastocytoma plus 3 × 10⁶ CD25⁻ T cells. Recipients were monitored for tumor burden. (E) Representative images of tumor growth measured by bioluminescent imaging. (F) Tumor mortality and (G) mouse survival were monitored. Data were combined from 2 independent experiments (n = 10 mice/group). Log-rank (Mantel-Cox) test was used to compare the tumor mortality and survival. *P < .05; **P < .01; ***P < .001.

Figure 6.

Sirt-1 modulates T- and B-cell activation in cGVHD. Lethally irradiated (700 cGy) BALB/c mice underwent transplantation with 5 × 10⁶ TCD-BM alone or plus 5 × 10⁵ CD25⁻ splenocytes per mouse isolated from WT or Sirt-1^−/− donors. (A) Body weight loss. (B) cGVHD clinical and (C) pathology scores. (D) Representative pictures of skin and large intestine biopsies stained with hematoxylin and eosin (original magnification ×200). (E) Foxp3 expression on gated donor CD4 in spleen and frequency of follicular Treg cells (Foxp3⁺CXCR5⁺PD-1⁺) and Tfh (Foxp3⁻CXCR5⁺PD-1⁺) were determined on day 60. (F) IFN-γ expression was shown on gated donor CD4 and CD8 T cells. (G) Splenic B cells were analyzed for B220⁺, B220⁻CD138⁺, and (H) activation markers, CD86 and MHCII. Data were combined from 2 independent experiments (n = 6-10 mice/group). Log-rank (Mantel-Cox) test was used to analyze body weight, and Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 6.

Sirt-1 modulates T- and B-cell activation in cGVHD. Lethally irradiated (700 cGy) BALB/c mice underwent transplantation with 5 × 10⁶ TCD-BM alone or plus 5 × 10⁵ CD25⁻ splenocytes per mouse isolated from WT or Sirt-1^−/− donors. (A) Body weight loss. (B) cGVHD clinical and (C) pathology scores. (D) Representative pictures of skin and large intestine biopsies stained with hematoxylin and eosin (original magnification ×200). (E) Foxp3 expression on gated donor CD4 in spleen and frequency of follicular Treg cells (Foxp3⁺CXCR5⁺PD-1⁺) and Tfh (Foxp3⁻CXCR5⁺PD-1⁺) were determined on day 60. (F) IFN-γ expression was shown on gated donor CD4 and CD8 T cells. (G) Splenic B cells were analyzed for B220⁺, B220⁻CD138⁺, and (H) activation markers, CD86 and MHCII. Data were combined from 2 independent experiments (n = 6-10 mice/group). Log-rank (Mantel-Cox) test was used to analyze body weight, and Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 7.

Treatment with Ex-527 attenuates cGVHD. Lethally irradiated (700 cGy) BALB/c mice underwent transplantation with 5 × 10⁶ TCD-BM of B10.D2 BM plus 5 × 10⁶ whole splenocytes containing CD25 per mouse isolated from WT donor. The recipients were treated either prophylactically on day 0 or day 28 posttransplant for 21 days with 2 mg/kg/mouse/day Ex-527. (A) Survival (B) cGVHD clinical scores. (C-D) Analysis of IFN-γ and IL-17 expressions on donor CD4 in spleen. (E-F) BCL6 expression and IL-21 secretion by donor CD4 were determined on day 50. (G-H) Analysis of Foxp3 expression and frequency of Tfh (Foxp3⁻CXCR5⁺PD-1⁺) and PD-1⁺CD8⁺ were measured on donor T cells. Splenic B cells were analyzed for B220⁺, B220^-CD138⁺, and MHCII. Data were combined from 2 independent experiments (n = 5-7 mice/group). Student t test was used for statistical analysis. *P < .05; **P < .01.