Ablation of BATF Alleviates Transplant Rejection via Abrogating the Effector Differentiation and Memory Responses of CD8+ T Cells

Abstract

Allogeneic CD8⁺ T cells are prominently involved in allograft rejection, but how their effector differentiation and function are regulated at a transcriptional level is not fully understood. Herein, we identified the basic leucine zipper ATF-like transcription factor (BATF) as a key transcription factor that drives the effector program of allogeneic CD8⁺ T cells. We found that BATF is highly expressed in graft-infiltrating CD8⁺ T cells, and its ablation in CD8⁺ T cells significantly prolonged skin allograft survival in a fully MHC-mismatched transplantation model. To investigate how BATF dictates allogeneic CD8⁺ T cell response, BATF^-/- and wild-type (WT) CD8⁺ T cells were mixed in a 1:1 ratio and adoptively transferred into B6.Rag1^-/- mice 1 day prior to skin transplantation. Compared with WT CD8⁺ T cells at the peak of rejection response, BATF^-/- CD8⁺ T cells displayed a dysfunctional phenotype, evident by their failure to differentiate into CD127^-KLRG1⁺ terminal effectors, impaired proliferative capacity and production of pro-inflammatory cytokines/cytotoxic molecules, and diminished capacity to infiltrate allografts. In association with the failure of effector differentiation, BATF^-/- CD8⁺ T cells largely retained TCF1 expression and expressed significantly low levels of T-bet, TOX, and Ki67. At the memory phase, BATF-deficient CD8⁺ T cells displayed impaired effector differentiation upon allogeneic antigen re-stimulation. Therefore, BATF is a critical transcriptional determinant that governs the terminal differentiation and memory responses of allogeneic CD8⁺ T cells in the transplantation setting. Targeting BATF in CD8⁺ T cells may be an attractive therapeutic approach to promote transplant acceptance.

Keywords: BATF; CD8+ T cells; allograft rejection; effector differentiation; memory; transplantation.

Figure 1

Graft-infiltrating CD8⁺ T cells highly express BATF, and BATF deficiency in CD8⁺ T cells prolongs skin allograft survival. Wild-type B6 recipients were transplanted with Balb/c skin allografts or left untransplanted on day 0. The expression levels of BATF in CD8⁺ T cells in these recipients were detected by flow cytometry on day 8 post skin grafting. All plots were gated on live CD8⁺ T cells. (A) Schematic of the experimental design. (B, C) Representative plots and bar graphs display the BATF expression levels in CD8⁺ T cells from spleens and skin allografts. Data represent the mean ± SD (n = 3-4). **p < 0.01, ***p < 0.001; (unpaired Student’s t-test). B6.Rag1 ^–/– hosts were adoptive transferred with either 1 x 10⁶ WT CD8⁺ T cells or 1 x 10⁶ BATF^–/– CD8⁺ T cells, followed by Balb/c skin transplantation. (D) Schematic of the experimental design. (E) % skin allograft survival (n = 5). **p < 0.01; (Mann-Whitney test). (F) Representative images of the skin allografts on B6.Rag1 ^–/– recipients at indicated days post skin grafting.

Figure 2

BATF-sufficient CD8⁺ T cells outcompete their BATF-deficient counterparts in infiltration into skin allografts. B6.Rag1 ^–/– recipients were adoptively transferred with a mixture of 0.5 x 10⁶ CD45.1⁺ WT CD8⁺ T cells plus 0.5 x 10⁶ CD45.2⁺ BATF^–/– CD8⁺ T cells, followed by Balb/c skin transplantation. The frequencies of the transferred CD8⁺ T cells were determined on day 11 post skin grafting. (A) Schematic of the experimental design. (B) Gating strategy for detecting live WT and BATF^–/– CD8⁺ T cells. (C, D) Representative plots and bar graphs show the percentages of the CD45.1⁺ WT and CD45.2⁺ BATF^–/– cells among live CD8⁺ T cells. (E–G) Bar graphs display the absolute cell numbers of WT and BATF^–/– CD8⁺ T cells. Data represent the mean ± SD (n = 4). **p < 0.01, ***p < 0.001, ****p < 0.0001; (unpaired Student’s t-test).

Figure 3

BATF-deficient CD8⁺ T cells fail to differentiate into terminal effector cells in recipients. The effector phenotypes of the transferred CD8⁺ T cells in B6.Rag1 ^–/– recipients were analyzed on day 11 post skin transplantation. (A–C) Representative plots and bar graphs show % CD127^–KLRG1⁺ terminal effector cells and % CD127⁺KLRG1^– cells. (D, E) Representative plots and bar graphs show % CD62L^–CX3CR1⁺ effector cells. Data represent the mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; (unpaired Student’s t-test).

Figure 4

BATF deficiency in CD8⁺ T cells impairs their expression of proinflammatory cytokines and cytotoxic molecules. The production of the effector cytokines and cytotoxic molecules of the adoptively transferred CD8⁺ T cells in recipients were analyzed on day 11 post skin grafting. All plots were gated on live CD8⁺ T cells. (A, B) Representative plots and bar graph display % IFN-γ⁺ cells in spleens and dLNs. (C–E) Representative plots and bar graphs display % Granzyme A⁺ cells and % Granzyme B⁺ cells. Data represent the mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; (unpaired Student’s t-test).

Figure 5

BATF deficiency may perturb the transcriptional networks that control the effector programs of CD8⁺ T cells. B6.Rag1 ^–/– mice were reconstituted with a mixture of WT and BATF^–/– CD8⁺ T cells, and were transplanted with Balb/c skins 1 day later. The expression levels of several transcription factors were determined on day 11 after skin transplantation. All plots were gated on live splenic CD8⁺ T cells. (A–C) Representative plots and bar graphs show the expression levels of TCF1 and PD-1 of the transferred WT and BATF^–/– CD8⁺ T cells. (D–G) Representative histograms and bar graphs display the expression levels of T-bet, TOX and Ki67 in the transferred CD8⁺ T cells. Data represent the mean ± SD (n = 4). *p < 0.05, ****p < 0.0001; (unpaired Student’s t-test).

Figure 6

BATF-deficient CD8⁺ T cells display impaired memory responses upon allogeneic antigen re-stimulation. B6.Rag1 ^–/– mice were reconstituted with a mixture of WT and BATF CD8⁺ T cells^–/– (in a 1:1 ratio) on day -1, transplanted with Balb/c skins on day 0, and re-transplanted with Balb/c allografts on day 60 post-initial transplantation. On day 7 post-secondary transplantation, the cell states of the transferred CD8⁺ T cells were determined by flow cytometry. All the representative plots are gated on live CD8⁺ T cells. (A) Schematic of the experimental design. (B–D) Representative plots and bar graphs show % CD44⁺CD62L^– cells. (E–I) Representative plots and bar graphs display the expression of Granzyme A, Granzyme B, IL-2, and KLRG1. Data represent the mean ± SD (n = 3). ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; (unpaired Student’s t-test).