In situ induction of dendritic cell-based T cell tolerance in humanized mice and nonhuman primates

Abstract

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.

Figure 1.

Development of an anti–human ICAM-1 antibody. (A) HEK293 cells were transfected with vector alone or vector encoding domains 1, 2, or 3–5 of human (h) or mouse (m) ICAM-1. MD-3 binding was assessed by flow cytometry. (B) PBMCs or neutrophils were incubated with human umbilical vein endothelial monolayers in the absence (negative control [NC]) or presence of monoclonal antibodies specific for CD18 (IB4) or ICAM-1 (R6.5) with MD-3, all at 20 µg/ml. Adhesion was measured as described in Materials and methods. Data are expressed as the mean adhesion relative to control ± SE of three experiments (except IB4 in the monocyte adhesion assay, where n = 2), with six replicates per experiment. n.s., not significant; *, P < 0.05; ***, P < 0.001.

Figure 2.

Human hematopoietic cell engraftment and immune cell development in humanized mice. (A) Data represent the percentage of human HLA-ABC⁺ or CD3⁺ cells in the peripheral blood of consecutively analyzed mice at the indicated weeks after transplantation of human CD34⁺ cells. Cumulative data (n = 111) were obtained from >10 independent experiments. Horizontal bars indicate the mean. (B) Flow cytometry data show CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells, B cells (CD19⁺), monocytes (CD14⁺), and conventional (CD11c⁺) or plasmacytoid (CD123⁺) DCs in the spleen. Data shown are representative of more than five independent experiments. (C) Flow cytometry data show the individual population of CD14⁺CD15⁻ monocytes and CD14⁻CD15⁺ neutrophils in the peripheral blood. Shown are representative data from one experiment of two mice.

Figure 3.

Assessment of graft survival in humanized mice. Porcine islets were transplanted under the renal capsule of humanized mice that had been rendered diabetic by STZ or were nondiabetic and received an injection of isotype-matched irrelevant control or MD-3 antibody (Ab). (A) Experimental protocol. (B) Fasting blood glucose (open circles; right y axis) and porcine C-peptide (closed squares; left y axis) levels were monitored weekly. (C) Based on serum level of porcine C-peptide, functional survival of islet xenografts was plotted over time. The dotted line indicates the day when a portion of the mice were sacrificed for ELISPOT and histopathological analyses. (D) Serial kidney sections of a representative mouse in the control or MD-3–treated groups were stained with H&E or antibodies specific for insulin, human CD3, or human CD68. Bar, 100 µm.

Figure 4.

Induction of antigen-specific T cell tolerance in humanized mice. To assess antigen-specific T tolerance in humanized mice that received the islet graft, some recipient mice were challenged with KLH at 4 wk after transplant. Splenocytes were isolated at 6 wk after transplant and tested for recall IL-2 and IFN-γ responses against donor islets, human allogeneic blood mononuclear cells (MLR), and KLH by ELISPOT assay. (A) Representative results. (B) Summarized data from 4–11 mice are presented as total numbers of cytokine-producing cells per 3 × 10⁵ splenocytes. As a negative control (NC) for anti-islet response, splenocytes from humanized mice that did not undergo transplantation (ungrafted) were stimulated with porcine islets. In contrast, splenocytes from engrafted mice cultured in the absence of stimulating antigen (responder only) were used as a negative control for MLR and anti-KLH responses. Horizontal bars represent mean values. Ab, antibody.

Figure 5.

Arrest of DC maturation at the semimature stage. Immature monocyte-derived DCs were generated from human CD14⁺ monocytes by incubation with GM-CSF and IL-4 in the presence of MD-3 or isotype-matched control antibody (control Ab) from the beginning of culture. After 6 d, DCs were stimulated or not with LPS. (A) Expression levels of MHC class I and II, CD80, CD86, CD40, PD-L1, PD-L2, and CTLA-4 on their surface were compared by flow cytometry. Cumulative data showing mean fluorescent intensity (MFI) ± SE of MHC class I and II, CD80, CD86, and CD40 were obtained from four independent experiments. Data showing mean fluorescent intensity ± SE of PD-L1, PD-L2, and CTLA-4 are representative of two independent experiments in triplicate. (B) Representative cytokine levels in the culture supernatants of immature and LPS-treated monocyte-derived DCs in the presence of MD-3 or control antibody. Results are the mean ± SE of triplicate cultures, and data are representative of three independent experiments. (C) Humanized mice received MD-3 or control antibody three times before LPS (100 µg/mouse) administration. Splenocytes were isolated 1 d after LPS injection and stained with HLA-ABC, CD11c, CD80, and CD86 antibodies. Representative dot plots of CD80 and CD86 expression on gated CD11c⁺ DCs are shown at the left. Numbers indicate the percentage of cells in each quadrant. Cumulative data (n = 3) showing mean fluorescent intensity were obtained from three independent experiments (middle). The percentages of CD11c⁺ cells among HLA-ABC⁺ cells in spleens were also calculated (right). Error bars indicate SE.

Figure 6.

Abrogation of T cell tolerance induction after DC ablation. Humanized mice received anti-CD11c IT (α-CD11c; 5 µg/mouse) or PBS every other day from 3 d before porcine islet transplantation up to the fifth day after transplant (D+5). These mice were then immunized with KLH on the 12th day after transplant (D+12). (A) Experimental scheme. (B) Flow cytometric analysis on the indicated days after islet transplantation to assess depletion of CD11c⁺ DCs in the spleen of humanized mice. (C) Splenocytes were isolated 14 d after KLH immunization and tested for recall IL-2 and IFN-γ responses via ELISPOT assay against donor islets and KLH. The data from individual mice are presented as total numbers of cytokine producing cells per 3 × 10⁵ splenocytes or normalized anti-islet response (Islet/KLH) by dividing the anti-islet spot number by the anti-KLH spot number in each mouse. Horizontal bars represent mean values. (D) Splenocytes from each mouse were stained with anti–human CD11c and anti–HLA-ABC antibodies, and the total number of CD11c⁺ DCs was calculated after flow cytometric analysis. Error bars indicate SE.

Figure 7.

Induction of T cell tolerance in a nonhuman primate. (A) HEK293 cells were transfected with Rhesus ICAM1 gene or chimeric genes of Rhesus and mouse ICAM-1, and MD-3 binding was assessed by flow cytometry (solid line). As the negative control (dotted line), the cells were stained with only FITC-conjugated secondary antibody. (B) Adult porcine islets (50,000 IEQs/kg) were intraportally transplanted into three Rhesus monkeys (R043, R042, and R038) that received MD-3 antibody alone. PBMCs were isolated on the indicated days after transplantation, and the frequency of T cells secreting IL-2 or IFN-γ in response to donor islets was determined by ELISPOT assay. Results are presented as numbers of cytokine-producing cells per 2.5 × 10⁵ PBMCs in each triplicate culture. R, responder cells only; R+S, responder cells stimulated with porcine islet cells; (−), negative control responder cells from unsensitized monkeys stimulated with porcine islet cells; (+), positive control responder cells from sensitized monkeys stimulated with porcine islet cells. Error bars indicate SE. (C) Anti-Gal IgG levels were measured at the indicated time before and after porcine islet transplantation via ELISA.

Figure 8.

Achievement of long-term survival of a porcine islet xenograft in a nonhuman primate via combination therapy including MD-3. (A) After successfully inducing type 1 diabetes in Rhesus monkey via STZ administration, hyperglycemia was controlled by s.c. injecting human recombinant insulin (Exotic insulin). Adult porcine islets (100,000 IEQs/kg) were intraportally transplanted into Rhesus monkeys (R052 and R049) that received MD-3 combined with rapamycin and anti-CD154 antibody. Blood glucose level and serum porcine C-peptide concentration were measured at the indicated time after porcine islet transplantation. (B) PBMCs were isolated at 127 and 7 d after transplantation from R052 and R049, respectively, and the frequency of T cells secreting IL-2 or IFN-γ in response to donor islets (I) or allogeneic PBMCs (A) was determined by ELISPOT assay. Results are presented as numbers of cytokine-producing cells per 5 × 10⁵ PBMCs in each triplicate culture. R, responder cells only; R+I, responder cells stimulated with porcine islet cells; R+A, responder cells stimulated with allogeneic PBMCs; (−), unsensitized monkeys as a negative control; (+), sensitized monkeys as a positive control. Error bars indicate SE. (C) Anti-Gal IgG levels were measured at the indicated time before and after porcine islet transplantation via ELISA.