Monotherapy With Anti-CD70 Antibody Causes Long-Term Mouse Cardiac Allograft Acceptance With Induction of Tolerogenic Dendritic Cells

Abstract

Allograft rejection has been an obstacle for the long-term survival of patients. CD70, a tumor necrosis factor (TNF) family member critically expressed on antigen-presenting cells and strongly but transiently up-regulated during lymphocyte activation, represents an important co-stimulatory molecule that induces effective T cell responses. We used a mouse heterotopic cardiac transplantation model to evaluate the effects of monotherapy with the antibody targeting mouse CD70 (FR70) on transplantation tolerance and its immunoregulatory activity. FR70-treated C3H recipient mice permanently accepted B6 fully mismatched cardiac allografts. Consistent with the graft survival, the infiltration of CD8⁺ T cells in the graft was reduced, dendritic cells were differentiated into a tolerogenic status, and the number of regulatory T cells was elevated both in the graft and the recipient's spleen. In addition, naïve C3H given an adoptive transfer of spleen cells from the primary recipients with FR70 treatment accepted a heart graft from a matching B6 donor but not third-party BALB/c mice. Our findings show that treatment with FR70 induced regulatory cells and inhibited cytotoxic T cell proliferation, which led to long-term acceptance of mouse cardiac allografts. These findings highlight the potential role of anti-CD70 antibodies as a clinically effective treatment for allograft rejection.

Keywords: CD70; allograft; dendritic cell; regulatory T cell; rejection; tolerance.

Figure 1

Anti-CD70 antibody (FR70) treatment induced long-term acceptance of mouse cardiac grafts. (A) FR70 (500 µg) was intraperitoneally (i.p.) injected into C3H recipient mice immediately after heterotopic cardiac transplantation, and in the following 5 days, three additional half-dose (250 µg, i.p.) FR70 injections were performed. (B) The graft survival data are presented in detail. C3H recipients that underwent transplantation of a heart from B6 mice were either not treated or treated with FR70 (n = 18 mice for not treated group, n = 25 mice for treated group, mean ± SEM, pooled from five independent experiments). The mean survival time (MST) is shown in Table 1 . A statistical evaluation of the graft survival was performed using Kaplan–Meier curves and compared using log-rank tests, ****p < 0.0001. (C) Histologic studies of harvested cardiac grafts were performed with hematoxylin–eosin (HE) staining. Syngeneic B6 heart (Syngeneic), isotype control-treated B6 cardiac allograft (Allo) and FR70-treated B6 cardiac allograft (Allo + FR70) on POD7 are shown (n = 3 mice/group, scale bars: 200 µm).

Figure 2

The number of CD8⁺ CTLs and the related mRNA expression were decreased in the GILs and in the SPCs of the FR70-treated recipients. GILs and spleens from isotype control-treated (Allo) and FR70-treated (FR70) groups were collected on POD7 [Control (POD7), Treatment (POD7)]. In addition, spleens from the FR70-treated groups were collected at POD60 and 100 [Treatment (POD60), Treatment (POD100)]. GILs and SPCs were separated and stained with anti-CD4 and CD8 mAb. Samples were subjected to FCM. (A) A representative FCM analysis of the CD4 and CD8 in GILs and SPCs is shown (left panel). Gating strategy is shown in Supplementary Figures 2A , B . Quantification of the CD4 and CD8 in GILs and SPCs by FCM is shown (right panel) (Grafts: n = 4 mice/control group; n = 6 mice/FR70-treated group; Spleens: n = 5 mice/allo group; n = 6 mice/FR70-treated POD7 group; n = 8 mice/FR70-treated POD100 group, mean ± SEM, pooled from three independent experiments). Statistical analysis was determined by Student’s t-test, one-way ANOVA and Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) FR70-treated and control allografts were harvested on POD7 and stained with anti-CD8 (blue), collagen IV (yellowish-brown), and BrdU (red) by triple immunostaining (scale bars: 200 µm). (C) Quantitative RT-PCR of mRNA in GILs collected on POD7 from the FR70-treated and control groups (n = 4–5 mice/group) and SPCs collected on POD7 from the control group and on POD7, 60 and 100 from the FR70-treated group (n = 5 mice/group). The mean ± SEM are presented, pooled from three independent experiments with three biological replicates. Statistical analysis was determined by Student’s t-test, one-way ANOVA, and Tukey’s test. *p < 0.05, **p < 0.01.

Figure 3

The number of TolDC and the related mRNA expression were significantly changed in graft and spleen of the FR70-treated recipients. (A) GILs were collected from control (Allo) and FR70-treated groups on POD7, and spleens were harvested from the control group (Allo) on POD7 and the FR70-treated group on POD7 and 100 for FCM. Gating strategy is shown in Supplementary Figure 3A (n = 3–5 mice/group, mean ± SEM, pooled from two independent experiments). Statistical analysis was determined by Student’s t-test, one-way ANOVA, and Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001. (B) The median fluorescence intensity (MFI) in CD11c and CD11b double-positive cells in graft and spleen was estimated for cells stained with anti-CD40, CD80, CD86, IA/IE, or PD-L1 antibodies and detected by FCM (Grafts: n = 2 mice/control group; n = 3 mice/FR70-treated group; Spleens: n = 4 mice for isotype control group; n = 5 mice/FR70-treated group POD7 and POD100, mean ± SEM, pooled from two independent experiments). Statistical analysis was determined by Student’s t-test, one-way ANOVA, and Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001. (C, D) Grafts and spleens were collected from the control and FR70-treated groups on POD7. In addition, GILs and SPCs from the FR70-treated groups were collected at POD60 and 100. The mRNA expression was quantified by quantitative RT-PCR (Grafts: n = 4 mice/control group; n = 4 mice/FR70-treated POD7 group; n = 5 mice/FR70-treated POD60 group; n = 5 mice/FR70-treated POD100 group; Spleens: n = 4 mice/isotype control group; n = 5 mice/FR70-treated POD7, 60 and 100 groups, mean ± SEM, pooled from two independent experiments with three biological replicates). Statistical analysis was determined by one-way ANOVA and Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

The Treg cell number and the related mRNA expression were significantly increased in graft and spleen of the FR70-treated recipients. (A) Grafts and spleens were collected on POD7, 60 and 100. The representative data from Foxp3 and CD25 staining with specific antibodies are presented (left panel). The percentage of CD25⁺ Foxp3⁺ cells among the CD4⁺ GILs and SPCs was determined by FCM (right panel, n = 4–8 mice for each group, mean ± SEM, pooled from two independent experiments). Gating strategy is presented in Supplementary Figure 3B . Statistical analysis was determined by one-way ANOVA and Tukey’s test. *p < 0.05. (B) Quantitative RT-PCR of the Foxp3 and IL-10 mRNA levels in GILs and SPCs collected at POD7, 60 and 100 (n = 4–5 mice for each group, mean ± SEM, pooled from three independent experiments with three biological replicates). Statistical analysis was determined by one-way ANOVA and Tukey’s test. *p < 0.05, **p < 0.01.

Figure 5

Tolerance induced by FR70 resulted in the acceptance of secondary donor-type cardiac grafts after adoptive splenocyte transfer. The splenocytes harvested at POD60 and 100 from the C3H recipients bearing B6 grafts were adoptively transferred into naïve C3H mice, which were then given B6 (donor type) or BALB/c (third party) cardiac allografts. The graft survival after adoptive transfer of splenocytes is shown (n = 7 mice for Naïve AT control group, n = 3 mice for 60 days-AT-D_type group, n = 8 mice for 100 days-AT-D_type group, n = 3 mice for 60 days-AT-Third party group, n =7 mice for 100 days-AT-Third party group, pooled from two independent experiments). Table 2 shows the detailed graft survival data. A statistical evaluation of the graft survival was performed using Kaplan–Meier curves with a comparison using log-rank tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.