The synergism of B and T lymphocyte attenuator (BTLA) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) attenuated acute T-cell mediated rejection and prolonged renal graft survival

Abstract

Background: Acute T-cell mediated rejection (TCMR) continues to be a major problem in the area of kidney transplantation. The B and T lymphocyte attenuator (BTLA) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) were recently found costimulatory molecules. The research aims to explore the inhibitory synergism of BTLA and CTLA-4 in TCMR.

Methods: We investigated the suppressive role of overexpressed BTLA and CTLA-4 in vitro. The rat kidney transplantation model was established to explore the effect of combined overexpressed BTLA and CTLA-4 in recipients of kidney transplantation. The grafts and peripheral blood were harvested for renal function, histology, immunohistochemical and flow cytometry analysis.

Results: Combination therapy decreased the secretion of interleukin-2 (IL-2) and proliferation of T cells compared to the single therapy and the control group. Decrease of interstitium monocyte infiltration and especially intimal arteritis in the graft was observed with the combination therapy, with remarkable reduction of numbers and proliferation response of T cells in peripheral blood and grafts. Combined overexpressed BTLA and CTLA-4 attenuated the acute TCMR after kidney transplantation and improved the graft function and prolonged the graft survival. The inhibiting role against TCMR in the combination therapy group was more effective than single therapy.

Conclusions: The synergism of BTLA and CTLA-4 attenuated acute TCMR after kidney transplantation by suppressing T cell activation and proliferation.

Keywords: Kidney transplantation; animal model; graft rejection; graft survival; immunosuppression.

Figure 1

Overexpressed BTLA and CTLA-4 inhibit T cell activation and proliferation. (A) The BTLA protein and relative mRNA expression in splenic T lymphocytes among different transfected groups by western blot analysis and qRT-PCR. (B) The CTLA-4 protein and relative mRNA expression in splenic T lymphocytes from different treated groups by western blot analysis and qRT-PCR. (C) The cytokines IL-2 of supernatant on 48h after MLR were assayed by ELISA among the different MLR groups. (D) BrdU-positive cell percentage among different MLR groups by flow cytometry analysis. The results are presented as the means ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.

Figure 2

Establishment of the rat acute rejection of kidney transplantation model. (A) HE analysis of the histological features of graft tissue between the Syn and the Allo groups at Day 0, 3 and 7 following transplantation (magnification: 200×). (B) Semi-quantitative assessment of graft sections among the Syn and the Allo group based on the Banff 2017 classification at day 3 and 7 after kidney transplantation (n=5 for each time point and each group). Data are expressed as the means ± SD. **, P<0.01; ***, P<0.001.

Figure 3

Combined overexpressed BTLA and CTLA-4 attenuated the TCMR after kidney transplantation. (A) SCr among the six groups at Day 0, 1, 3, 5, 7 and 14 after transplantation. Data are contrast to the Allo + BTLA + CTLA-4 group. (B) Rat graft survival time among the six groups after kidney transplantation. (C) HE analysis of the graft sections among four groups on 7 days following transplantation (magnification: 200× and 400×). Upper: reflect the interstitium monocyte infiltration, Lower: the performance of endarterium. (D) Semi-quantitative assessment of graft tissues based on the Banff classification at day 7 after kidney transplantation (n=5 for each time point). Data are presented as the means ± SD. NS, not significant, *, P<0.05; ***, P<0.001.

Figure 4

Combined overexpressed BTLA and CTLA-4 reduced the numbers of T cell in peripheral blood and graft and down-regulated serum IL-2 level. (A) Flow cytometry analysis of CD3+ T cells in peripheral blood cells of SD recipients at day 7 after transplantation among the four groups. (B) The percentage of CD3+ cells in total lymphocyte in peripheral blood. (C) Postoperative CD3 expression of kidney grafts by IHC among the four groups (magnification: 200×). (D) Integrated optical density (IOD) value used to express the relative quantity of CD3. (E) Secretion of cytokines IL-2 in the serum of SD recipients was measured by ELISA. Data are presented as the means ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.