Low-dose rapamycin treatment increases the ability of human regulatory T cells to inhibit transplant arteriosclerosis in vivo

Abstract

Regulatory T cells (T(reg)) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote T(reg) expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human T(reg) to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic T(reg) numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2(-/-) Il2rg(-/-) mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4(+) but not CD8(+) T lymphocytes were sensitive to T(reg) and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of T(reg)-based immunosuppressive protocols in clinical practice. By inhibiting TA, T(reg) and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival.

Figure 1. Low-dose rapamycin potentiates the inhibitory effects of T_reg on TA development

(A) Experiment set-up: Side branches of human internal mammary artery were transplanted into immunodeficient BALB/c Rag2^−/−Il2rg^−/− mice. The recipients were injected the following day with 10 × 10⁶ allogeneic human PMBCs administered i.p. Some of the mice additionally received 1 × 10⁶ ex vivo expanded CD127^lo T_reg-injected i.p. at the same time as the PMBCs or 300 μg/kg rapamycin injected i.p. on days 7, 8 and 10 after the transplant. A fourth group of mice received a combination of PBMCs, T_reg and rapamycin. The arterial grafts, blood and spleen were collected 30 days after the surgery. (B) Representative photomicrographs showing development of TA in the PBMC (n = 6); PBMC and rapamycin (n = 6); PBMC and T_reg (n = 4); PBMC and T_reg and rapamycin (n = 5) groups. The grafts have been stained with Elastin/van Gieson. The elastic lamina in the media stain purple and the cellular cytoplasm pink. The newly formed neointima is delineated by the internal elastic lamina (IEL) and the vascular lumen. (C) Quantification of TA expressed as luminal occlusion, percentage of the area inside the IEL occupied by the neointima. The box plots show median, 25th and 75th percentiles as well as the highest and lowest values. *p < 0.05, ***p < 0.001.

Figure 2. T_reg and rapamycin inhibit lymphocyte proliferation in vivo

(A) Experiment setup: We injected immunodeficient BALB/c Rag2^−/−Il2rg^−/- mice with 10 × 10⁶ human PMBCs i.p. and allowed 14 days for cellular reconstitution. On day 14, 10 × 10⁶ CFSE-labeled human PMBCs isolated from the same blood donor were administered i.v. alone or together with 1 × 10⁶ ex vivo expanded CD127^lo T_reg. Some of the mice also received 300 μg/kg rapamycin i.p. on days 14, 15 and 17. Spleens were recovered on day 19 and the different cellular populations were analyzed by flow cytometry. (B) Representative flow cytometry plots demonstrating the gating strategy for CFSE high (CFSE^hi), CFSE intermediate (CFSE^int) and CFSE negative (CFSE⁰) CD4 and CD8 T lymphocytes in the spleen. (C) Percentage of CFSE^hi cells of the total CFSE⁺ CD4 and CD8 T lymphocytes in the spleen at the time of recovery in the PBMC (n = 6); PBMC and rapamycin (n = 6); PBMC and T_reg (n = 3); PBMC and T_reg and rapamycin (n = 4) groups. The error bars represent standard deviation. (D) Numbers of human CD4⁺ and CD8⁺ T lymphocytes expressed as total number of cells per spleen in the four groups. *p < 0.05.

Figure 3. Combined therapy with T_reg and rapamycin inhibits CD4⁺ and CD8⁺ T-cell proliferation and potentiates apoptosis of CD4+ lymphocytes in vitro

CFSE-labeled PBMC (10⁵ per well) have been incubated with anti-CD3/anti-CD28 beads (cells:bead ratio 5:1) in the presence or absence of 10 nM rapamycin and/or 10⁴ ex vivo expanded CD127^lo T_reg cells per well. (A) Representative plots depicting CFSE dilution in CD4⁺ and CD8⁺ lymphocytes after 5 day of culture. The numbers represent percentage of undivided CFSE^hi cells in the gated populations. (B) Percentage of undivided CFSE^hi lymphocytes of the total lymphocyte population, as shown in A. (C) Representative plots demonstrating the binding of the apoptosis marker Annexin V to CD4⁺ and CD8⁺ T cells. The numbers represent percentage of AnnexinV⁺ cells of the total lymphocyte population. (D) Absolute numbers and percentage of AnnexinV⁺ apoptotic cells within the CD4⁺ and CD8⁺ gates as demonstrated in panel C.