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. 2007 Jul;121(3):349-58.
doi: 10.1111/j.1365-2567.2007.02581.x. Epub 2007 Mar 22.

Blockade of the 4-1BB (CD137)/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival in mice

Tatsuhiko Asai  1 , Beom K Choi, Patrick M Kwon, Won Y Kim, Jung D Kim, Dass S Vinay, Bryan M Gebhardt, Byoung S Kwon
Affiliations

Blockade of the 4-1BB (CD137)/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival in mice

Tatsuhiko Asai et al. Immunology. 2007 Jul.

Abstract

To explore the roles of 4-1BB (CD137) and CD28 in corneal transplantation, we examined the effect of 4-1BB/4-1BB ligand (4-1BBL) and/or CD28/CD80/CD86 blockade on corneal allograft survival in mice. Allogeneic corneal transplantation was performed between two strains of wild-type (WT) mice, BALB/c and C57BL/6 (B6), and between BALB/c and B6 WT donors and various gene knockout (KO) recipients. Some of the WT graft recipients were treated intraperitoneally with agonistic anti-4-1BB or blocking anti-4-1BBL monoclonal antibody (mAb) on days 0, 2, 4 and 6 after transplantation. Transplanted eyes were observed over a 13-week period. Allogeneic grafts in control WT B6 and BALB/c mice treated with rat immunoglobulin G showed median survival times (MST) of 12 and 14 days, respectively. Allogeneic grafts in B6 WT recipients treated with anti-4-1BB mAb showed accelerated rejection, with an MST of 8 days. In contrast, allogeneic grafts in BALB/c 4-1BB/CD28 KO and B6 CD80/CD86 KO recipients had significantly prolonged graft survival times (MST, 52.5 days and 36 days, respectively). Treatment of WT recipients with anti-4-1BB mAb resulted in enhanced cellular proliferation in the mixed lymphocyte reaction and increased the numbers of CD4(+) CD8(+) T cells, and macrophages in the grafts, which correlated with decreased graft survival time, whereas transplant recipients with costimulatory receptor deletion showed longer graft survival times. These results suggest that the absence of receptors for the 4-1BB/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival, whereas triggering 4-1BB with an agonistic mAb enhances the rejection of corneal allografts.

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Figures

Figure 1
Figure 1
Role of 4-1BB, CD28 and/or CD80/86 in the survival of corneal allografts. (a) Corneal allografts in B6 WT recipients untreated or treated with anti-4-1BB mAb. Recipients treated with anti-4-1BB showed accelerated rejection in a dose-dependent manner. The decreased survival with the larger dose was significantly different from the survival of the IgG-treated control (P < 0·0007). Graft survival data are presented as Kaplan–Meier survival curves. (b) Corneal allografts in B6 WT untreated recipients or in recipients treated with anti-4-1BBL mAbs. Treatment with anti-4-1BBL did not significantly affect allogeneic graft survival, compared with control rat IgG treatment. (c) Corneal allograft in BALB/c mice deficient in 4-1BB and/or CD28. Survival in KO recipients was significantly longer than the survival of the control WT BALB/c recipients (P < 0·0085, 0·047, and 0·0001, respectively). The 4-1BB KO, CD28KO and 4-1BB/CD28 DKO recipients had no significant difference among them. (d) Corneal allograft in B6 mice deficient for 4-1BB, CD28 or CD80/86. All of the survival in KO recipients was significantly longer than the survival of the control WT B6 recipients (P < 0·001 for 4-1BB KO, CD28 KO and P < 0·0001 for CD80/86 KO).
Figure 2
Figure 2
Roles of 4-1BB, CD28 and/or CD80/86 in MLR. Effect of anti-4-1BB and anti-4-1BBL on MLR between WT BALB/c donor and WT B6 recipients on day 7 (a) and day 14 (b). C57BL/6 recipients were treated with either control IgG, anti-4-1BB, or anti-4-1BBL (200 μg/mouse, i.p.) on days 0, 2, 4 and 6 after transplantation. On days 7 and 14, mice were killed and DLN (cervical) cells were collected (n = 3 in each group). Enumeration of antigen-specific recall responses by in vitro MLR revealed greater proliferation in the DLN of anti-4-1BB-treated recipients on day 7, compared with anti-4-1BBL-treated or IgG-treated recipients, but no significant differences on day 14 in DLN. *P < 0·01, **P < 0·001, compared with IgG-treated WT controls. Results are shown as mean ± SD. Effect of costimulatory receptor gene deletion on MLR between WT B6 donor and BALB/c KO recipients (c), or MLR between WT BALB/c donor and B6 KO recipients (d). Both KO recipients showed reduced MLR responses on day 14 (**P < 0·001). Results are shown as means ± SD.
Figure 3
Figure 3
Profiles of infiltrating leucocytes in corneal allografts by flow cytometry. (a) Mice were killed on days 7, 14 and 21 after transplantation and the population of infiltrating leucocytes in the corneal allografts was measured by flow cytometry. By day 21, CD11c+ cells, CD4+ T cells, and CD8+ T cells were increased in the anti-4-1BB (3H3)-treated recipients. (n = 3 in each group). (b) BALB/c recipient mice were killed on day 14 after transplantation (n = 3 in each group). 4-1BB/CD28 KO recipients showed increased numbers of DX5+ cells. All of the KO recipients showed reduced numbers of CD11b+ and CD8+ cells, and tended to have a reduced population of Gr-1+ cells, which matched the observed longer graft survival times. (c) C57BL/6 KO recipients were killed on day 14 after transplantation (n = 3 in each group). Similarly to the BALB/c recipients in (b), increased numbers of DX5+ cells, as well as reduced populations of CD11c+, Gr-1+, CD4+ and CD8+ cells, were seen in the grafts with longer survival times. The CD80/86 KO mice showed an increased CD8+ T-cell population, similar to that seen in the WT control. Results shown in (b) and (c) are representative of triplicate experiments with similar results. Data are expressed as means ± SD.
Figure 4
Figure 4
4-1BB, 4-1BBL, cytokine and chemokine gene expression during orthotopic corneal allograft rejection in B6 recipients. (a) RT-PCR was performed on total RNA isolated from corneal allografts and contralateral normal corneas (negative controls) from B6 WT recipients on days 0, 3, 6 and 9 after transplantation. 4-1BB and 4-1BBL mRNA was expressed in allografted corneas on days 3, 6 and 9. The 4-1BBL was significantly elevated on day 6 after transplantation. IL-10 mRNA was expressed on days 6 and 9, and IFN-γ mRNA on day 9 (n = 9 in each group). (b) Chemokine mRNA levels were quantified by RPA analysis. Samples from corneal allografts, contralateral normal corneas (negative control), and DLN were evaluated on days 0, 3, 6 and 9 after transplantation. On the basis of the migration patterns of undigested probes, specific bands were identified for each chemokine. In comparison with the chemokine mRNA levels in the contralateral normal corneas, the allografts showed significant expression of MIP-2 mRNA that peaked on day 6 and weak expression of MIP-1α, MIP-1β, IP-10, MCP-1 and eotaxin mRNA from days 3 to 9. Expression of Ltn, RANTES and TCA-3 was undetectable in all the eyes studied. Declining expression of RANTES was detectable in DLN from day 0 through day 6 (n = 9 in each group).
Figure 5
Figure 5
Comparison of the number of CD4+ or CD8+ T cells infiltrating into corneal grafts. (a) C57BL/6 mice treated i.p. with anti-4-1BB, anti-4-1BBL, or control rat IgG (200 μg) on days 0, 2, 4 and 6 after transplantation, were killed on days 7, 14 and 21. Corneal sections were stained immunohistochemically, and CD4+ and CD8+ T cells were counted under × 100 magnification (two sections/eye and three eyes/time-point). Agonistic anti-4-1BB treatment significantly increased both CD4+ and CD8+ T-cell infiltration into the corneal grafts. *P < 0·05, **P < 0·01, compared with IgG-treated WT control. Data are expressed as means ± SD. (b) BALB/c and (c) B6 WT and KO recipient mice were killed 14 days after transplantation. Corneal sections were stained immunohistochemically and CD4+ and CD8+ T cells were counted under × 100 magnification (two sections/eye and three eyes/time-point). Cells were counted separately at the host–graft junction and in the central cornea. Results are shown as means ± SD. The absence of costimulatory ligands or receptors significantly decreased leucocyte infiltration into the corneal grafts. *P < 0·05, **P < 0·01, ***P < 0·001, compared with WT control.
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