Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice

Abstract

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

Figure 1. Reconstitution of NSG mice with human CD34⁺ stem cells.

(A) Strategy for generation of humanized mouse model of islet transplantation. (B) A representative FACS profile of human CD45⁺, human CD19⁺ and human CD3⁺ cells in peripheral blood of hu-NSG mice, 12–16 weeks after transfer of CD34⁺ cells (n = 15). Human CD19⁺ B cells and CD3⁺ T cells were identified in the gate for human CD45⁺ cells. (C) Splenocytes from hu-NSG mice were analyzed by FACS for human CD45RA⁺ (naïve T cells), human CD45RO⁺ (memory T cells), human CD4⁺CD25⁺FoxP3⁺ (Tregs), human CD11c⁺ (dendritic cells), human CD14⁺ (macrophages) and human CD16⁺CD56⁺ (NK cells) expression. (D–E) Data are summarized. Plotted lines represent the mean (n = 15, D; n = 3, E).

Figure 2. Diabetic hu-NSG mice reject the grafted human islets.

(A) The function of grafted human islets was evaluated by measuring blood glucose levels. As a control, NSG mice without CD34⁺ cell reconstitution remained normoglycemic after islet transplantation. The insert shows a representative image of a kidney and a spleen from hu-NSG mice that received (bottom) or did not receive (top) islet transplants (n = 6). STZ: streptozotocin. (B) Serum samples from hu-NSG mice that were grafted with human islets were measured by ELISA for human insulin when blood glucose was 7, 20 and 28 mM (n = 3).

Figure 3. Human innate and adaptive immune responses mediate islet rejection in the hu-NSG mice.

(A) Sera were analyzed by ELISA for human C3 levels in hu-NSG mice with/without human islet transplants or NSG mice with/without transplants (n = 3). Control: no islet transplant. (B) Sera were collected at the time of graft rejection. Cytokines were measured by cytokine bead array (n = 3). Control: sera from hu-NSG mice without islet transplant. *: undetectable. **: mean ± SD.

Figure 4. Infiltration of immune cells and C3 deposit in the grafted human islets.

Tissue samples of islet allograft bearing kidney in hu-NSG mice were collected at the time of islet allograft rejection. Sections were stained with H & E (a–b, e–f and i) and antibodies to human antigens: insulin (c and g, brown), CD45 (d and h, green), CD4 (j, green), CD8 (k, red), CD11b (l, green), CD66b (m, red), and C3d (n, brown). Arrow indicates the kidney capsule or the area around islet grafts. Red square indicates the site of the infiltration. Scale bar: 100 µm.

Figure 5. Adoptive transfer of ex vivo expanded T_regs protects human islet allograft from rejection.

(A) Percentage graft survival in hu-NSG mice with or without T_reg-treatments (n = 15 for islets alone group; n = 10 for T_regs-treated group; Log-rank test, p = 0.0004). (B) Histological examination of islet grafts determined by immunostaining with antibodies for human antigens: insulin (brown), CD11b (green), CD66b (red), and CD4 (green). Nuclei were stained with DAPI (blue). Tissues were harvested at the time of islet allograft rejection in T_regs-untreated animals and at day 21 post-islet transfer in T_reg-treated animals. (C) Quantitative data analysis of islet graft immunostaining. Data represent results from three individual mice per group. (D) Identification of T_regs in the islet grafts. The harvested grafts were double-stained with FITC-conjugated CD4 (green) and TRITC-conjugated FoxP3 (red). CD4/FoxP3 double-positive cells are shown by yellow colour. Inset images show enlarged area indicated by a white arrow. Black arrow indicates islets grafts. **: p<0.01. Scale bar: 50 µm.

Figure 6. T_regs accumulate in the draining lymph nodes and inhibit CD4⁺ T cells in the draining lymph nodes and spleen.

(A) The absolute number of CD4⁺CD25⁺FoxP3⁺ cells in the draining lymph nodes and spleens, at the time of graft rejection (islets alone group) or at day 21 post-islet transfer (islets+Tregs group) was determined by flow cytometry. (B) Percentage of cells positive for human CD4 in draining lymph nodes and spleens from (A). (C) Data are representative dot plots from three independent experiments. Plotted lines represent the mean (n = 5). **: p<0.01. dLN: draining lymph nodes. Post-tx: post islet transfer.

Figure 7. T_regs reduced MCP-1 production in vitro and in vivo.

(A) MCP-1 expression in islet grafts at the time of rejection (islets alone group) or at day 21 post-islet transfer (islets+T_regs group) was measured by real-time RT-PCR (n = 5). β-actin was used as the endogenous control. (B) The expression of mRNA was analyzed by real-time RT-PCR in islet cells co-cultured with T_regs for three days. (C–D) Cumulative data (mean ± SD) for MCP-1 expression in cultured islet cells. MFI: mean fluorescence intensity. (E–F) Representative plots from 4 independent experiments are shown. *: p<0.05; **: p<0.01. Post-tx: post islet transfer.

Figure 8. T_regs regulate human cytokine production in islet-transplanted hu-NSG mice.

Sera were collected at the time of rejection (islets alone group) or at day 21 post-islet transfer (islets+T_regs group). Cytokines were measured by cytokine bead array (n = 3). Control: sera from hu-NSG mice without islet transplant. **: p<0.01.