Preventing Allograft Rejection by Targeting Immune Metabolism

Abstract

Upon antigen recognition and co-stimulation, T lymphocytes upregulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic reprogramming in T cells regulates T cell activation and differentiation but is not just a consequence of antigen recognition. Although such metabolic reprogramming promotes the differentiation and function of T effector cells, the differentiation of regulatory T cells employs different metabolic reprogramming. Therefore, we hypothesized that inhibition of glycolysis and glutamine metabolism might prevent graft rejection by inhibiting effector generation and function and promoting regulatory T cell generation. We devised an anti-rejection regimen involving the glycolytic inhibitor 2-deoxyglucose (2-DG), the anti-type II diabetes drug metformin, and the inhibitor of glutamine metabolism 6-diazo-5-oxo-L-norleucine (DON). Using this triple-drug regimen, we were able to prevent or delay graft rejection in fully mismatched skin and heart allograft transplantation models.

Figure 1. 2-DG combined with Metformin inhibits T cell response through suppression of glycolysis

(A and B) ECAR and OCR of resting CD4⁺ cells measured in real time under basal conditions and in response to anti-CD3/CD28/cross-linking IgG1 (anti-CD3, 2 μg/ml; anti-CD28, 2 μg/ml; cross-linking IgG1, 1 μg/ml) with or without the presence of individual or combination of drugs (2-DG, 10 mM; Metformin, 50 mM). Bar graphs display data of ECAR and OCR measured at the endpoint of the experiment (205 min). Data are shown as mean ± SEM of 5 measurements. (C and D) Naïve splenocytes labeled with cell proliferation dye eFluor 670 were stimulated with anti-CD3 in in the presence of media control, 2-DG alone, metformin alone or 2-DG + metformin (2-DG, 0.6mM; Metformin, 1mM). (C) 24-hour IFN-γ secretion to supernatants was interrogated by enzyme-linked immunosorbent assay (ELISA). Data are shown as mean ± SEM of three independent samples. (D) 72-hour eFluor dilution of CD4⁺ and CD8⁺ T cells. n.s., not significant, *p<0.05, **p < 0.01, ****p < 0.0001 (Student’s t test). Data are representative of at least two independent experiments.

Figure 2. Combined inhibition of glycolysis and glutaminolysis profoundly suppresses T cell responses

(A) Glutaminase activity of CD4⁺ T cells cultured for 24 hrs in different conditions (anti-CD3, 2μg/ml; anti-CD28, 2μg/ml; DON, 5 μM; 2-DG, 0.6 mM; Metformin, 1 mM). Data are shown as mean ± SEM of three independent experiments. (B and C) Naïve WT C57BL/6 splenocytes labeled with eFluor 670 and stimulated with anti-CD3 in medium containing indicated metabolic inhibitors (DON, 5μM; 2-DG, 0.6mM; Metformin, 1mM). (B) 24-hour IFN-γ secretion to supernatants as interrogated by ELISA. Data are shown as mean ± SEM of three independent samples. (C) Proliferation of CD4⁺ and CD8⁺ T cells at 72h measured by dilution of eFluor 670. n.s., not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). Data are representative of at least two independent experiments. (D) ³H-acetate incorporation into lipids in preactivated and stimulated CD4+ T cells (anti-CD3, 1μg/ml; anti-CD28, 2μg/ml; cross-linking 0.75 μg/ml) with the presence of 2DG, Metformin, DON or in combination (2DG, 5mM; Metformin, 30mM; DON, 60μM). Data are shown as mean ± SEM of three independent samples. ***p < 0.001 (ANOVA). Data are representative of two independent experiments. (E) The phosphorylation state of the S6 ribosomal protein was measured in CD4⁺ T cells after 30 minutes stimulation (anti-CD3, 1μg/ml; anti-CD28, 2μg/ml; cross-linking 0.75 μg/ml) with the presence of Rapamycin, PP242 and metabolic inhibitors (Rapamycin, 1μM; pp242, 1μM; 2DG, 5mM, Metformin, 30mM; DON, 60μM). Data are representative of two independent experiments.

Figure 3. Metabolic Inhibitors suppress the proliferation and function of antigen-specific T cells while increase the relative frequency of Tregs in vivo

(A to D) OT-II (Thy1.1⁺) CD4⁺ T cells were adoptively transferred into WT (Thy1.2⁺) recipient mice. The recipients were infected with OVA-expressing vaccinia virus and treated with vehicle, 2-DG+Metformin or 2-DG+metformin+DON (2-DG, 500 mg/kg once daily; metformin, 150 mg/kg once daily; DON, 1.6 mg/kg once every other day) for 3 days. Splenocytes from the recipients were harvested at day 4 to interrogate the frequency of antigen-specific T cells and regulatory T cells. (A) Percentage of Thy1.1⁺ cells relative to CD4⁺ cells were analyzed by flow cytometry (left) and plotted as cumulative data (right). (B) OT-II cells were rechallenged with OVA peptide class II (10 μg/ml) ex vivo for 24hrs. The supernatants were interrogated for IFN-γ production by ELISA. Data are mean ± SEM (n=6). (C) Percentage of Foxp3⁺ cells relative to CD4⁺ cells (left) and plotted as cumulative data (right). (D) The ratio of OVA-specific Foxp3+ T cells:Effector cells. (E to G) OT-I (Thy1.1⁺) CD8⁺ T cells were adoptively transferred into WT (Thy1.2⁺) recipient mice. The hosts were infected with vaccinia-OVA and treated with vehicle, 2-DG+metformin or triple therapy for 5 days. Host splenocytes were harvested at day 6 to interrogate the proliferation and function of antigen-specific CD8⁺ T cells. (E) Percent Thy1.1⁺ cells relative to CD8⁺ cells were analyzed by flow cytometry (left) and plotted as cumulative data (right) (F) Percent IFN-γ-producing cells relative to CD8+ cells (left) and plotted as cumulative data (right). (G) OT-I cells were rechallenged with OVA peptide class I (10μg/ml) ex vivo for 24hrs. The supernatants were interrogated for IFN-γ production by ELISA. Data are mean ± SEM (n=5 ~6) (H) The ability of metabolic inhibitors to suppress endogenous effector CD8⁺T cells development was assessed with in vivo CTL assay. Percent of specific killing was determined at 10 hrs after transferring target cells. Data are mean ± SEM (n=3mice/group). Each symbol represents an individual mouse. Horizontal lines indicate mean± SEM. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). Data are representative of more than three independent experiments.

Figure 4. Metabolic inhibitors promote allograft survival

(A) Balb/c to C57BL/6 skin graft survival, as monitored daily by assessment of macroscopic signs of rejection. (B) Representative photomicrographs of skin graft histology (hematoxylin and eosion (H&E) staining) on post-transplant days 7 and 40 under an optical microscope (outlet, X100; inlet: X200). (C) Balb/c to C57BL/6 heart graft survival, as monitored daily by palpation of heart beating. (D) Representative photomicrographs of cardiac graft histology (H&E staining) on indicated post-transplant day under an optical microscope (outlet, X200; inlet, X400). The anti-metabolic treatment was started from the day of graft (day 0) until rejection in both models. The dosages of all drugs were the same as described in Figure 3. **p < 0.01, ****p < 0.001 (Log-rank analysis). Data are representative of two independent experiments.