CTLA4-Ig in combination with FTY720 promotes allograft survival in sensitized recipients

Abstract

Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4+ and CD8+ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-γ-producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.

Keywords: Immunology; Transplantation.

Figure 1. Generation of allosensitized recipients.

(A) C57BL/6 mice were sensitized with 2W-OVA.B/c splenocytes (DST; s.c.) at ~14 months (n = 10–12/group). (B) Donor-specific IgG as mean fluorescence intensity (MFI). (C) Total number/mouse of naive (CD44^–CD62L⁺), central memory (TCM; CD44⁺CD62L⁺), and effector memory (TEM; CD44⁺CD62L^–) of 2W:I-A^b CD4⁺ and OVA:K^b CD8⁺ T cells in B/c-sensitized (n = 6) and naive mice (n = 8). Combined spleens and axial, brachial, and inguinal lymph nodes were stained, gated on Dump^–CD90⁺ T cells, separated into CD4⁺ and CD8⁺ gates, and examined for 2W:I-A^b or OVA:K^b multimer binding and CD44/CD62L expression. (C and D) Gating strategy is provided in Supplemental Figure 1A and symbols represent individual mice, pooled from 2 to 3 independent experiments. Data are presented as mean ± SEM, and statistical significance was determined by (B) unpaired t test or (C and D) ANOVA and Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 2. CLTA4-Ig delays graft rejection in allosensitized recipients.

(A) C57BL/6 mice sensitized with 2W-OVA.B/c or B/c splenocytes (DST; s.c.) for more than 14 months were transplanted with 2W-OVA.B/c or B/c hearts, respectively, and treated with 500 μg CTLA4-Ig/mouse on day –2, 0, and 2 (i.v.), and then at 250 μg/mouse (i.p.) twice per week until the end of the experiment. (B) Survival of the heart allografts pooled from more than 2 experiments/group. From recipients sensitized for 14 or more months, (C) donor-specific IgG, and (D) frequency of in vitro–stimulated IFN-γ–producing CD4⁺ or CD8⁺ T effector memory (TEM) or central memory (TCM) cells on day 30 after transplantation (n = 5–12/group). Donor-specific IFN-γ production was evaluated by incubating responder splenocytes with previously LPS-stimulated and T cell–deficient B/6 or T cell–depleted B/6xB/c.F1 cells in vitro. The percentage of donor-specific IFN-γ⁺ T cells was determined after subtracting syngeneic cell stimulation from allogeneic cell stimulation (n = 2–5/group). Symbols represent individual mice, pooled from 1 to 3 independent experiments. Data are presented as mean ± SEM, and statistical significance was determined by (B) log-rank test or (C and D) ANOVA and Holm-Sidak’s multiple comparison test. *P < 0.05; **P < 0.01; ****P < 0.001. MFI, mean fluorescence intensity; N, naive; S, sensitized; HTx, heart transplant.

Figure 3. Control of endogenous T cell expansion in sensitized recipients treated with CTLA4-Ig.

(A and B) Total numbers, (C and D) percentage of naive, central (TCM), or effector memory (TEM), and (E and F) total number of TEM of 2W:I-A^b or OVA:K^b T cells per sensitized mouse at day 30 after heart transplant. Data were pooled from 2 to 4 independent experiments (n = 6–8/group; same mice as analyzed for A–F), with the minimum number of cells set at 100. Data are presented as mean ± SEM and statistical analyses by ANOVA and Kruskal-Wallis or Holm-Sidak’s multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. N, nonsensitized; S, sensitized; HTx, heart transplant.

Figure 4. T cell infiltration into allografts of sensitized recipients treated with CTLA4-Ig.

(A) Histology of allografts from sensitized + CTLA4-Ig (n = 5) and naive + CTLA4-Ig (n = 6) at day 30 after transplantation. Original magnification, ×20. Scale bars: 200 μm. (B) Histological scores and (C) quantification of the total numbers of CD4⁺ and CD8⁺ T cells recovered from heart allografts (n = 3–4/group). Data were pooled from 2 independent experiments and presented as mean ± SEM, and statistical analyses were by (B) unpaired t test or (C) ANOVA and Kruskal-Wallis tests. *P < 0.05; ***P < 0.005. N, nonsensitized; S, sensitized; HTx, heart transplant.

Figure 5. CTLA4-Ig plus FTY720 combination therapy prevents rejection in sensitized recipients.

(A) Donor-specific antibody (DSA)-IgG and (B) IFN-γ T cell responses were assessed in ~4-month post-sensitization recipients (S) prior to transplantation, or on day 30 after transplantation (HTx) and treatment with CTLA4-Ig and/or FTY720. Nonsensitized tolerant recipients (N-Tol) received anti-CD154 (day 0, 7, and 14) plus B/c spleen cells (day 0) to induce tolerance to B/c heart grafts, and were analyzed on day 60 after transplantation. Data were pooled from 2 to 3 independent experiments (n = 6–8/group), with the minimum number of IFN-γ⁺ T cells set at 100. Data are presented as mean ± SEM, and statistical analyses were by ANOVA and Kruskal-Wallis tests. *P < 0.05; **P < 0.01, ***P < 0.005.

Figure 6. Efficacy of CTLA4-Ig plus FTY720 combination therapy in preventing T cell infiltration and rejection in sensitized mice.

(A) FTY720 in combination with CTLA4-Ig reduced the total number of CD4⁺ and CD8⁺ T cells and (B) TCR75 T cells infiltrating the B/c heart grafts transplanted into ~4-month-sensitized recipients on day 30 after transplantation. (C) Histology of the heart grafts on day 30 after transplant. Original magnification, ×20. Scale bars: 200 μm. (D) Histological scores are presented as mean ± SEM (n = 5/group). (E) Cumulative allograft survival of recipients sensitized for 4 or more months ± TCR75. Data are pooled from 2 to 4 independent experiments, and statistical analyses were by (A) ANOVA and Kruskal-Wallis tests, (B and D) unpaired t test, or (E) log-rank test. *P < 0.05; **P < 0.01; ***P < 0.005. HTx, heart transplant; S, sensitized; N-Tol, nonsensitized tolerant mice as described in Figure 5.