In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia

Abstract

Regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving a beneficial graft-versus-leukemia (GVL) effect. Thus, their use in allogeneic stem cell transplantation (SCT) provides a promising strategy to treat GVHD. However, 3 obstacles prevent their routine use in human clinical trials: (1) low circulating number of Tregs in peripheral blood, (2) loss of suppressor function after in vitro expansion, and (3) lack of Treg-specific surface markers necessary for efficient purification. FOXP3 is exclusively expressed in Tregs and forced expression in CD4(+)CD25(-) T cells can convert these non-Tregs into Tregs with functional suppressor function. Here, we show that the FDA-approved hypomethylating agents, decitabine (Dec) and azacitidine (AzaC), induce FOXP3 expression in CD4(+)CD25(-) T cells both in vitro and in vivo. Their suppressor function is dependent on direct contact, partially dependent on perforin 1 (Prf1), but independent of granzyme B (GzmB), and surprisingly, Foxp3. Independence of Foxp3 suggests that genes responsible for the suppressor function are also regulated by DNA methylation. We have identified 48 candidate genes for future studies. Finally, AzaC treatment of mice that received a transplant of major histocompatibility complex mismatched allogeneic bone marrow and T cells mitigates GVHD while preserving GVL by peripheral conversion of alloreactive effector T cells into FOXP3(+) Tregs and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Tregs.

Figures

Figure 1

Effect of Dec on FOXP3 expression in anti-CD3/CD28 antibody coated bead-activated CD4+CD25− T cells. (A) Both human and mouse CD4+CD25− T cells express FOXP3 after Dec treatment in the presence of anti-CD3/CD28 antibody coated beads. (B) Real-time RT-PCR for human FOXP3 mRNA was performed in triplicate at various times after Dec treatment of activated T cells. Levels of mRNA for FOXP3 increase each day and are comparable to that seen in bead-activated Tregs (brown) by day 4. (C) CD4+CD25− T cells obtained from Foxp3-ires-GFP KI mice confirm induction of FOXP3 expression after Dec and AzaC treatment (indirectly measured as GFP expression). (D) DcT FOXP3 expression (blue) persists for at least 7 days, suggesting prolonged and stable expression of FOXP3 in vitro. Treg (red) is shown for comparison. A pool of 2 independent experiments.

Figure 2

DcTs suppress the proliferation of Teffs. CFSE-based proliferation assays and MLR were performed with each population 2.5 × 104 per well in 96-well round-bottom plates. For transwell plate experiments, each population (1 × 105 per well) was incubated in 96-well flat-bottom transwell plates in the presence of beads as stimulators. The proliferation of CFSE-labeled CD4+CD25− T cells was analyzed after 3 days (cells with beads) or 6 days (cells with γ-irradiated APCs) on a FACScan cytometer (BD Biosciences). All cultures were evaluated in triplicate. Hypomethylating agent–treated cells suppress the proliferation of anti-CD3/CD28 antibody coated bead-activated Teffs (A-B) and allogeneic APC-activated Teffs (C-D). Treg (1×), (2×), and (4×) indicate the ratios of Treg:Teff = 1:1, 2:1, and 4:1, respectively. azacT (a) and (b) indicate that these azacTs were generated in the presence of AzaC 1μM and 2μM, respectivley. dcTs, azacTs, and pbsTs (right) were generated in the presence of hIL-2 (500 μ/mL; panel A). Only FACS purified GFP+ (thus, FOXP3+) and not GFP− CD4+ cells obtained from Foxp3-ires-GFP KI mice are suppressive (panel B). CD45.1 was used to gate on CFSE-labeled Teffs. dcT: Dec-treated T cells, pbsT: PBS-treated T cells, azacT: AzaC-treated T cells. Neg: negative control, CFSE-labeled Teffs alone; pos: CFSE-labeled Teffs with stimulators, anti-CD3/CD28 antibody coated beads or allogeneic APC; all others contain both CFSE-labeled Teffs and stimulators plus indicated cells such as nTregs, dcTs, pbsTs, or azacTs. GFP+ azacT: MoFlo sorted GFP+ cells after treatment of AzaC; GFP− azacT: MoFlo sorted GFP− cells after treatment of AzaC.

Figure 3

DcTs mitigate GVHD. (A) Schema of the experiments. 9 Gy total-body irradiation was used to condition recipient mice (Balb/c). B6 mice (CD45.2) TCD BM (5 × 106 cells) were used as a stem cell source. To induce GVHD, 5 × 105 B6 Tconv (CD45.1) was infused along with donor TCD BM. To test suppressor function of dcTs, 5 × 105 B6 CD4+CD25+ nTregs (for control), pbsTs or dcTs (all CD45.2) generated from CD4+CD25− T cells were injected with TCD BM and Tconv. (B-D) Mice that underwent a transplantation with dcTs show significantly higher survival rate (B) and less weight loss (C) than mice infused with pbsTs and a trend toward increase of B cells (D) analyzed 1 month after transplantation. A pool of 2 independent experiments. (E) Schema of the experiments. B6 mice (CD45.1) TCD BM (5 × 106 cells) were used as a stem cell source. A total of 10 × 106 pbsTs/dcTs generated from Tconv (B6, CD45.2) were given. (F-G) Mice that underwent a transplantation with dcTs show significantly higher survival rate (F) and more B cells (G) than mice that received pbsTs. A pool of 3 independent experiments analyzed 1 month after transplantation.

Figure 4

AzaC treatment of mice that underwent a transplantation with delayed allogeneic T cells mitigates GVHD. (A) Schema of the experiments. B6 mice (CD45.2) TCD BM (5 × 106 cells) were used as a stem cell source. To induce GVHD, 2 × 106 Tconv (B6, CD45.1) were given on day 11 after SCT followed by the treatment with AzaC/Dec/PBS (every other day; 4 doses) starting on day 15 after SCT. (B-F) Mice treated with AzaC (2 mg/kg) show significantly higher survival rate (B), less weight loss (C), and more B cells (E) and T cells (F) with donor engraftment (D) than mice infused with pbsTs. (G) AzaC group also show increased Treg population in peripheral blood. T rec: T cells from recipient; T BM: T cells from donor BM; T donT: T cells from donor T cells; B BM: B cells from donor BM. (D-G) Analyzed 1 month after transplantation. A pool of 4 independent experiments.

Figure 5

AzaC increase FOXP3+ Tregs that may inhibit the proliferation of allogeneic Tconv in vivo. In vivo AzaC treatment (blue) attenuates the proliferation of allogeneic Tconv compared with the PBS control (red, left panel; peripheral blood on day 19 after SCT, gated on CD45.1+ donor T cells). This inhibited proliferation of Tconv is likely to be mediated by FOXP3+ Tregs induced by AzaC (middle and right; splenocytes on day 19 after SCT, gated on CD45.1+ donor T cells). One representative from each group with identical results is shown. AzaC (n = 4), PBS (n = 2).

Figure 6

AzaC treatment of mice that underwent a transplantation with delayed allogeneic T cells mitigates GVHD while preserving GVL. (A) Schema of the experiments. B6 mice (CD45.2) TCD BM (5 × 106 cells) were used as a stem cell source. To induce GVHD, 10 × 106 Tconv (B6, CD45.1) were given on day 11 after SCT followed by the treatment with AzaC or PBS (every other day; 4 doses) starting on day 15 after SCT. For examination of GVL effect, 1 × 104 A20-luc/egfp leukemic cells were given along with TCD BM. (B-C) Mice treated with AzaC show significantly higher survival rate (B) and lower leukemic burden (C). Y axis in top panels indicates photon flux (photons/sec) in log scale measured from the dorsal and the ventral view with a region of interest drawn over the entire body of each mouse. Actual images of 1 representative mouse from each group are shown in bottom panels (scale: photons/sec/cm2/sr). A pool of 3 independent experiments.

Figure 7

Mechanism of hypomethylating agent-induced generation of Tregs. (A) Perforin (Prf1) is partially required for the suppressor function of dcT. DcTs from both WT and granzyme B (GzmB)–deficient mice were equally suppressive while those from Prf1-deficient mice were significantly less suppressive. A pool of 2 independent experiments. (B-C) The impact of dcTs (B) and azacTs (C) from WT, Foxp3 heterozygote (Foxp3+/Y) and Foxp3 deficient (Foxp3−/Y) mice on Teffs were compared in proliferation assays. They are equally suppressive regardless of their origins. Neg: negative control, CFSE-labeled Teffs alone; pos: CFSE-labeled Teffs with stimulators, anti-CD3/CD28 antibody coated beads or allogeneic APC; all others contain both CFSE-labeled Teffs and stimulators plus indicated cells. (D) GA-Mantel analysis was run 100 times. Forty-nine probes appeared in at least 30 of 100 solutions (Mantel correlation = 0.97). Foxp3 is the most frequently selected probe. Heat map shows these 49 probes that separate the 3 Treg groups from the pbsT group. nnTreg: naive nTregs (n = 2), anTreg: activated nTregs (n = 3), dcT (n = 3), pbsT (n = 3). (E) AzaC treatment prolongs survival of Foxp3 deficient mice. A treatment of 2 mg/kg AzaC was given every other day (4 doses) starting at 16 to 26 days of age. A pool of 4 independent experiments. (F) Shown are 41-day-old Foxp3 KO mice treated with PBS (left) and AzaC (right); 25% (n = 8) had WT-looking ears.