Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival

Abstract

Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

Figure 1

(a) Schematic presentation of the plasmid pGCL-GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site (MCS) for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP fluorescent protein (marker gene) cassette flanked by loxp sites. (b) The stem-loop-encoded shRNA has sequence identity to a 21-nt region of CD80 mRNA.

Figure 2

Transduction efficiency was estimated 4 days after transduction at indicated MOIs. (a) GFP expression was observed under light microscopy (top) or fluorescence microscopy (bottom). (A) MOI of 5 (×100). (B) MOI of 20 (×100). DCs saw an increase in expression of GFP that peaked when MOI = 20. (b) The efficiency of infection was determined by assaying GFP expression by flow cytometry (green line) and comparing with uninfected control cells (grey peak).

Figure 3

DCs were transduced with nothing (No treated), CD80 lenti, CD86 lenti, NC lenti, or both CD80 lenti and CD86 lenti. (a) After 6 days of culture, the cells were then incubated with LPS (1 μg/mL) for 18 hours. Expression of CD80 and CD86 was determined by flow cytometry. The data show the effect of recombinant lentivirus on inhibition of target molecule expression. Values expressed as mean ± 1 SD from three independent experiments; *P < .05 compared to NC lenti/No treated; **P < .05 compared to CD80 lenti; ***P < .05 compared to CD86 lenti. (b) C3H BM-derived DCs were cultured over 6 days. DCs were stimulated or unstimulated with LPS (1 μg/mL) for 18 hours. Upregulation of CD80 and CD86 in response to LPS was also suppressed by CD80 lenti and CD86 lenti. The mean fluorescence intensity (MFI) of GFP+ cells expressing the marker of interest is indicated.

Figure 3

DCs were transduced with nothing (No treated), CD80 lenti, CD86 lenti, NC lenti, or both CD80 lenti and CD86 lenti. (a) After 6 days of culture, the cells were then incubated with LPS (1 μg/mL) for 18 hours. Expression of CD80 and CD86 was determined by flow cytometry. The data show the effect of recombinant lentivirus on inhibition of target molecule expression. Values expressed as mean ± 1 SD from three independent experiments; *P < .05 compared to NC lenti/No treated; **P < .05 compared to CD80 lenti; ***P < .05 compared to CD86 lenti. (b) C3H BM-derived DCs were cultured over 6 days. DCs were stimulated or unstimulated with LPS (1 μg/mL) for 18 hours. Upregulation of CD80 and CD86 in response to LPS was also suppressed by CD80 lenti and CD86 lenti. The mean fluorescence intensity (MFI) of GFP+ cells expressing the marker of interest is indicated.

Figure 4

(a) B6 alloAg-pulsed, CD80 lenti- and CD86 lenti-transduced DCs were inferior stimulators of naïve syngeneic C3H T cells, compared to alloAg-pulsed NC lenti-transduced DCs or no treated DCs. *P < .05 compared to NC lenti/No treated. (b) DCs loaded with debris B6 spleen cells were injected into syngeneic C3H mice (3 × 10⁶/mouse). Seven days later, recipients were sacrificed and splenic T cells challenged ex vivo with γ-irradiated C3H DCs pulsed with Ag (BALB/c, C3H, or B6 splenocyte lysates). Data are expressed as cpm ±1SD from triplicate cultures and are representative of four separate experiments.

Figure 5

AlloAg-pulsed DCs in NC lenti group, or CD80 lenti and CD86 lenti group, and third party Ag-pulsed DCs in CD80 lenti and CD86 lenti group were injected into C3H mice, 7 days before the transplantation with B6 heart grafts. *P < .05 compared to other groups.

Figure 6

Lymphocytes were isolated from recipient spleens, mesenteric lymph nodes, and grafts on day 5 after transplantation treated with recombinant lentivirus transduced DCs; these cells were double stained with FITC-conjugated anti-CD3 and PE-conjugated Annexin V, analyzed by flow cytometry. Numbers indicate the percentage of Annexin V positive cells in CD3+ cell population.

Figure 7

T cells isolated on day 5 after translation from the spleens of C3H recipients bearing B6 heart allografts treated with PBS or recombinant lentivirus transduced DCs were used for analysis of T cell apoptosis signaling pathway molecules by RT-PCR. All data are representative of three separate experiments.