Hypomethylating therapy mitigates acute allograft rejection in a murine lung transplant model

Abstract

Introduction: Acute cellular rejection of transplanted lung allografts involves activated cytotoxic T cells and reduced Regulatory T (Treg) cell function. Calcineurin inhibitors, the cornerstone of immunosuppressive regimens, suppress T cell cytotoxicity but inhibit Treg proliferation. The DNA hypomethylating agent decitabine (DAC) can abrogate T cell cytotoxicity while stimulating Treg proliferation.

Methods: We sought to determine the effects of DAC treatment in a murine MHC-mismatched orthotopic lung transplant model.

Results: Rescue treatment with DAC maintains lung allograft gross and histologic integrity with a reduction in cytotoxic T cell responses. CD4+FoxP3+ T cell depletion in Foxp3DTR mice exacerbated rejection lung injury compared to CD4+FoxP3+ T cell sufficient mice and failed to abolish the protective effect of DAC in this model. The protective effect of DAC was associated with a reduction in cytokine production from host T-cells.

Discussion: Decitabine could offer a new line of treatment for acute lung allograft rejection, in part via its effects on Tregs.

Keywords: T regulatory cells; acute rejection; decitabine; immune tolerance; lung transplantation.

Figure 1

Decitabine attenuates lung allograft rejection. Gross morphology [(A) & (C)] and histologic hematoxylin and eosin (H&E) staining [(B) & (D), 1× and 20× magnifications] of BALB/c lung allografts harvested 10 days post-transplantation into wild-type C57BL/6 hosts. Mice were treated with either vehicle (DMSO, intraperitoneally) or decitabine (DAC, 1 mg/kg, intraperitoneally) on post-transplant days 3, 4, 5, and 8. DAC treatment preserved lung architecture and reduced inflammatory cell infiltration compared to DMSO-treated controls. TPX: Left Lung Allograft, L: Left, and R: Right.

Figure 2

(A) Histograms depicting the total single-cell suspension counts (gray histogram) and absolute number of live cells (white histogram). (B) Box and whiskers showing CD4⁺/CD8⁺ ratios, and (C) the percentages of live CD4⁺, CD8⁺, and CD4⁺FoxP3⁺ T cells as a function of host treatment regimen. Data are presented as mean ± SEM (N = 6–9 per group). P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****); ns = not significant.

Figure 3

DAC treatment restricts T-cell infiltration to the perivascular region and decreases airway inflammation. Representative immunofluorescence images of allografts harvested 10 days post-transplant from DMSO- and DAC-treated CD4⁺FoxP3⁺ Treg-sufficient hosts. Sections were stained for CK-19 (green), CD3 (yellow), CD4 (red), and CD8 (blue) to assess T-cell distribution across different lung regions. DAPI (cyan) marks nuclear staining. Scale bars = 200 um.

Figure 4

Histograms quantifying the effect of host DAC- (vs. DMSO-) treatment on T cell distribution and airway thickness in allografts. Histograms depict cell counts in (A) perivascular, (B) interstitial, and (C) peribronchial regions, as well as CD4:CD8 ratios (D, E, & F), and (G) alveolar wall thickness and (H) bronchial epithelial height/cross-sectional area. Four random sites per slide were analyzed, each covering an average total area of 2,400 cm². Perivascular T cell density was measured as CD4⁺ and CD8⁺ T cells per cm² of perivascular area using ImageJ's manual cell count plugin at 20× magnification, with counts normalized to the perivascular area measured, which varied between vessels. Interstitial and peribronchial T cell counts were obtained at 20× magnification and averaged across sites. Alveolar wall thickness was measured in micrometers (µm) at 40× magnification using an Inter-edge Distance Measurement Macro in ImageJ, averaging at least 10 distances per alveolar wall, with five alveoli measured per site. Bronchial epithelial height was also measured in µm at 40× magnification using the same macro, averaging at least 10 distances per bronchial wall, while bronchial cross-sectional area was manually outlined and measured using ImageJ, with values averaged across sites. Data are presented as mean ± SEM. P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****); ns = not significant.

Figure 5

DAC treatment inhibits cytokine production in host CD4⁺ and CD8⁺ T cells. Histograms displaying percentage change in cytokine production from rested CD4⁺ FoxP3⁺ Treg-sufficient host right lung cells after exposure to BALB/c splenocytes. Production of IFN-γ, TNF-α, and IL-17 was assessed in (A–C) CD4⁺ T cells and (D–F) CD8⁺ T cells. Native right lungs from host wild-type C57BL/6, having received BALB/c orthotopic left lung transplants and being treated with DMSO or DAC, were harvested. Their cells were incubated overnight (in R10 medium) and cultured with BALB/c spleen cells (1:1 ratio, 37°C, 5% CO2 × 5 h with GogliStop^TM present the final 2 h). Cells were then harvested, stained for surface antibodies and intracellular cytokines, and analyzed with flow cytometry. Data are presented as mean ± SEM, with individual data points overlaid. P ≤ 0.05 (*), 0.05 < P < 0.09 (#). N = 4–5 per group.

Figure 6

Decitabine inhibition of lung allograft rejection requires CD4 ⁺ FoxP3⁺ treg-sufficient host for maximal effect. Gross morphology [(A) & (C)] and histologic hematoxylin and eosin (H&E) staining [(B) & (D), 1× and 20× magnifications] of BALB/c lung allografts harvested 10 days post-transplantation from FoxP3^DTR hosts. Mice were treated with either DMSO (vehicle, intraperitoneally) or decitabine (DAC, 1 mg/kg, intraperitoneally) on post-transplant days 3, 4, 5, and 8. Hosts also received diphtheria toxin (dT) on post-transplant days 3 (20 ng/kg), 5 (10 ng/kg), and 7 (10 ng/kg) for CD4⁺FoxP3⁺ Treg depletion. TPX: Left Lung Allograft, L: Left, and R: Right.

Figure 7

(A) Histograms depicting the total single-cell suspension counts (gray histogram) and absolute number of live cells (white histogram). (B) Box and whiskers showing CD4⁺/CD8⁺ ratios, and (C) the percentages total live cell population composed by CD4⁺, CD8⁺, and CD4⁺FoxP3⁺ T cells. CD4⁺FoxP3⁺ depletion with dT is very effective at removing this cell population. Data are presented as mean ± SEM (N = 6–9 per group). P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****); ns = not significant.

Figure 8

Effect of DAC on the phenotype of CD4 ⁺ FoxP3⁺ T cells in CD4 ⁺ FoxP3⁺ treg sufficient hosts. Volcano plot demonstrating the effect on the percentage of allograft live CD4⁺FoxP3⁺ T cells (A) and on live CD8⁺FoxP3⁺ T cells (B) of DAC vs. DMSO treatment of CD4⁺FoxP3⁺ Treg-sufficient hosts. Effect (x-axis) is presented as Fold Change (Log₂) in the percentage of live allograft cells expressing a given marker when the host is treated with DAC rather than DMSO. Markers expressed on a higher percentage of cells are depicted more to the right, and increasing statistical significance is depicted by ascending position on the Y axis. The green-shaded region denotes markers with statistically significant differences (p < 0.05, -log₁₀ > 1.301). Markers with increased expression in DAC-treated hosts are shown in blue, while those with reduced expression are in red.

Figure 9

Cd4 ⁺ FoxP3⁺ treg depletion alters the effect of DAC treatment on allograft CD4⁺ T cells. Volcano plot demonstrating the effect on percentage of allograft live CD4⁺ T cells expressing various markers following (A) DAC (vs. DMSO) treatment of CD4⁺FoxP3⁺ Treg-sufficient hosts; (B) DAC (vs. DMSO) treatment of CD4⁺FoxP3⁺ Treg-depleted hosts; (C) DAC treatment of CD4⁺FoxP3⁺ Teg-sufficient vs. –depleted hosts. Effect (x-axis) is presented as Fold Change (Log₂) in the percentage of live CD4⁺ allograft cells expressing a given marker when the (A) CD4⁺FoxP3⁺ Treg–sufficient host is treated with DAC (vs. DMSO); (B) CD4⁺FoxP3⁺ Treg-depleted host is treated with DAC (vs. DMSO); (C) CD4⁺FoxP3⁺ Treg-sufficient vs. –depleted host is treated with DAC. Markers expressed on a higher percentage of cells are depicted more to the right, and increasing statistical significance is depicted by ascending position on the Y axis (-Log₁₀). The green-shaded region denotes markers with statistically significant differences (p < 0.05, -log₁₀ > 1.301). Markers with increased expression in DAC-treated hosts are shown in blue, while those with reduced expression are in red.

Figure 10

Cd4 ⁺ FoxP3⁺ treg depletion alters the effect of DAC treatment on allograft CD8⁺ T cells. Volcano plot demonstrating the effect on the percentage of allograft live CD8⁺ T cells expressing various markers following (A) DAC (vs. DMSO) treatment of CD4⁺FoxP3⁺ Treg-sufficient hosts; (B) DAC (vs. DMSO) treatment of CD4⁺FoxP3⁺ Treg-depleted hosts; (C) DAC treatment of CD4⁺FoxP3⁺ Treg-sufficient vs. –depleted hosts. Effect (x-axis) is presented as Fold Change (Log₂) in the percentage of live CD8⁺FoxP3⁺ allograft cells expressing a given marker when the (A) CD4⁺FoxP3⁺ Treg–sufficient host is treated with DAC (vs. DMSO); (B) CD4⁺FoxP3⁺ Treg-depleted host is treated with DAC (vs. DMSO); (C) CD4⁺FoxP3⁺ Treg-sufficient vs. –depleted host is treated with DAC. Markers expressed on a higher percentage of cells are depicted more to the right, and increasing statistical significance is depicted by ascending position on the Y axis (-Log₁₀). The green-shaded region denotes markers with statistically significant differences (p < 0.05, -log₁₀ > 1.301). Markers with increased expression in DAC-treated hosts are shown in blue, while those with reduced expression are in red.

Figure 11

Cd4 ⁺ FoxP3⁺ treg depletion alters the effect of DAC treatment on allograft's CD8⁺FoxP3⁺ T cells. Volcano plot demonstrating the effect on the percentage of allograft live CD8⁺FoxP3⁺ T cells expressing various markers following (A) DAC (vs. DMSO) treatment of CD4⁺FoxP3⁺ Treg-sufficient hosts; (B) DAC (vs. DMSO) treatment of CD4⁺FoxP3⁺ Treg-depleted hosts; (C) DAC treatment of CD4⁺FoxP3⁺ Treg-sufficient vs. –depleted hosts. Effect (x-axis) is presented as Fold Change (Log₂) in the percentage of live CD8⁺FoxP3⁺ allograft cells expressing a given marker when the (A) CD4⁺FoxP3⁺ Treg–sufficient host is treated with DAC (vs. DMSO); (B) CD4⁺FoxP3⁺ Treg-depleted host is treated with DAC (vs. DMSO); (C) CD4⁺FoxP3⁺ Treg-sufficient vs. –depleted host is treated with DAC. Markers expressed on a higher percentage of cells are depicted more to the right, and increasing statistical significance is depicted by ascending position on the Y axis (-Log₁₀). The green-shaded region denotes markers with statistically significant differences (p < 0.05, -log₁₀ > 1.301). Markers with increased expression in DAC-treated hosts are shown in blue, while those with reduced expression are in red.

Figure 12

DAC treatment increases the percentage of CD8⁺ T cells expressing markers of immune tolerance in CD4 ⁺ FoxP3⁺ treg-sufficient and -depleted hosts. Violin plots display the percentage of allograft live (A) CD8 ⁺ CD44 ⁺ CD62l ⁺ CD103⁺, (B) CD8 ⁺ CD44 ⁺ CD62l⁺, and (C) CD8 ⁺ CD103⁺ T-cells in transplanted lungs 10 days post-transplant. CD4 ⁺ FoxP3⁺ Treg-sufficient hosts were wild-type C57BL/6 mice treated with either diluent (DMSO) or DAC. CD4 ⁺ FoxP3⁺ Treg-depleted hosts were diphtheria toxin (dT)-treated FoxP3^DTR mice receiving either diluent or DAC. DAC (1 mg/kg, i.p.) or DMSO was administered on post-transplant days 3, 4, 5, and 8, while dT was given on post-transplant days 3 (20 ng/kg), 5 (10 ng/kg), and 7 (10 ng/kg). Data are presented as median, minimum, and maximum with individual data points overlaid. P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****); ns = not significant.