An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection

Abstract

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.

FIGURE 1

Survival of islet allografts with a combined treatment of CTLA4/Fc and IL-15 mutant/Fcγ2a. a, Islets from BALB/c (H-2^d) donors were transplanted under the renal capsule of C57BL/6 (H-2^b) recipients. Recipients were treated with CTLA4/Fc (0.1 mg), IL-15 mutant/Fcγ2a (1.5 µg), or a combination of both proteins as indicated in Materials and Methods. Untreated recipients (■) or recipients treated with a control IgG2a protein (1.5 µg) rejected allografts with a MST of 15 days and 14 days, respectively (□). Graft rejection was delayed by 30 days in recipients treated with CTLA4/Fc (◊), CTLA4/Fc plus IgG2a (♦), or IL-15 m/Fcγ2a (○), whereas in recipients receiving the combined treatment, the MST was of 120 days (●). b, Second islet transplantation. Nephrectomy of the left kidney was performed on five recipients 150 days after transplantation. Then four nephrectomized mice received a second BALB/c islet allograft under the right renal capsule (following the same procedure) without further immunosuppression. All of them accepted the second transplant for more than 50 days, demonstrating a state of tolerance (●). One mouse received a third part islet allograft (B10. BR, H-2^k; ○) which was rejected at day 12, confirming that the immune response is intact.

FIGURE 2

Histology of serial sections of islet allografts isolated 8 days after islet allograft transplantation. A dense tissue infiltration by various mononuclear cells with destruction of islets is observed in untreated recipients (a). Recipients receiving CTLA4/Fc or IL-15 mutant/Fcγ2a evidenced a decreased tissue infiltration by mononuclear leukocytes with some islets preserved (b and c, respectively). Graft sections from recipients treated with a combination of CTLA4/Fc and IL-15 mutant/Fcγ2a reveal almost normal histology with minimal mononuclear cell infiltration and intact islets (d). Cryostat sections, hematoxylin counterstain; original magnification, ×100. These are representative sections from two allograft from each treatment group.

FIGURE 3

Immunohistologic evaluation of islet allografts isolated 8 days after islet transplantation. Untreated recipients show dense tissue infiltration by mononuclear leukocytes surrounding and invading the islets with a predominance of CD4⁺ and CD8⁺ T cells (a and b). Recipients treated with CTLA4/Fc (c and d) or IL-15 mutant/Fcγ2a (e and f) show a decreased mononuclear cell infiltrate with preservation of the tissue structure. Recipient mice receiving a combined treatment present rare CD4⁺ and CD8⁺ T cell infiltrates (g and h). Cryostat sections, hematoxylin counterstain; original magnification, ×100. These are representative sections from two recipients from each treatment group.

FIGURE 4

Cytolytic gene transcripts were analyzed by RT-PCR. Tissues were isolated on day 8 after transplantation in untreated, CTLA4/Fc-treated, IL-15 mutant/Fcγ2a-treated, or CTLA4/Fc plus IL-15 mutant/Fcγ2a-treated recipients. Three allografts from each group were analyzed. Each lane represents an individual mouse, the first lane being the negative control. GAPDH primers were used as an internal control. Densitometric scanning quantification of gene expression for perforin, FasL, and granzyme B normalized to GAPDH was performed and the results are as follows: untreated mice: 0.733 ± 0.25, 0.362 ± 0.15, and 2.72 ± 0.57, respectively; CTLA4/Fc-treated mice: 0.98 ± 0.71, 0.56 ± 0.27, and 1.09 ± 0.24, respectively; IL-15 mutant/Fcγ2a-treated mice: 0.094 ± 0.09, 0.03 ± 0.03, and 0.13 ± 0.15, respectively; CTLA4/Fc plus IL-15 mutant/Fcγ2a-treated mice: 0.123 ± 0.15, 0.017 ± 0.02, and 0, respectively. Molecular weight markers are in the left part of the gel.

FIGURE 5

Analysis of alloreactive CD4⁺ and CD8⁺ T cell proliferation in vivo. CFSE-labeled CD8^−/− C57BL/6 (a) and CD4^−/− C57BL/6 (b) lymphocytes were injected through the lateral vein into irradiated BALB/c recipient mice. Recipient mice were then untreated or treated with CTLA4/Fc (0.1 mg), IL-15 mutant/Fcγ2a (1.5 µg) or a combination of both fusion proteins for 3 days. a, CFSE-labeled allogeneic CD4⁺ T cells from the host spleen. b, CFSE-labeled allogeneic CD8⁺ T cells from the host spleen. Data are represented as percentage of proliferation of CFSE-labeled CD4⁺ and CD8⁺ T cells. Data are representative of four individual experiments.