T_regs reduced MCP-1 production <i>in vitro</i> and <i>in vivo</i>.

<p>(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.</p

Cytokine production by human CD4 T cells from hu-NSG mice <i>in vitro</i>.

<p>Human CD4⁺ T cells were stimulated with CD3/CD28 beads for three days. Supernatants were analyzed by cytometric bead array for cytokine production (n = 3). Cytokines IL-4, IL-5, IL-6 and IL-12p70 were not detectable in the supernatants. Spl:CD4 denotes CD4 cells from the spleen of a hu-NSG mouse. AdPBMC:CD4 indicates CD4 cells from PBMC of a healthy donor. *: undetectable. **: mean ± SD.</p

Diabetic hu-NSG mice reject the grafted human islets.

<p>(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).</p

Adoptive transfer of <i>ex vivo</i> expanded T_regs protects human islet allograft from rejection.

<p>(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.</p

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

<p>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.</p

Reconstitution of NSG mice with human CD34⁺ stem cells.

<p>(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).</p

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

<p>(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.</p

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

<p>(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.</p

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

<p>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 (<b>a–b, e–f and i</b>) and antibodies to human antigens: insulin (<b>c</b> and <b>g</b>, brown), CD45 (<b>d and h</b>, green), CD4 (<b>j</b>, green), CD8 (<b>k</b>, red), CD11b (<b>l</b>, green), CD66b (<b>m</b>, red), and C3d (<b>n</b>, brown). Arrow indicates the kidney capsule or the area around islet grafts. Red square indicates the site of the infiltration. Scale bar: 100 µm.</p

DataSheet_1_Inhibition of thrombin on endothelium enhances recruitment of regulatory T cells during IRI and when combined with adoptive Treg transfer, significantly protects against acute tissue injury and prolongs allograft survival.pdf

Ischemia-reperfusion injury (IRI) amplifies T cell alloimmune responses after transplantation with thrombin playing a key pro-inflammatory role. To explore the influence of thrombin on regulatory T cell recruitment and efficacy we used a well-established model of IRI in the native murine kidney. Administration of the cytotopic thrombin inhibitor PTL060 inhibited IRI, and by skewing expression of chemokines (reducing CCL2 and CCL3 but increasing CCL17 and CCL22) increased the infiltration of M2 macrophages and Tregs. When PTL060 was combined with infusion of additional Tregs, these effects were further amplified. To test the benefits of thrombin inhibition in a transplant model, BALB/c hearts were transplanted into B6 mice with or without perfusion with PTL060 in combination with Tregs. Thrombin inhibition or Treg infusion alone led to small increments in allograft survival. However, the combined therapy led to modest graft prolongation by the same mechanisms as in renal IRI; graft survival was accompanied by increased numbers of Tregs and anti-inflammatory macrophages, and reduced expression of pro-inflammatory cytokines. While the grafts succumbed to rejection associated with the emergence of alloantibody, these data suggest that thrombin inhibition within the transplant vasculature enhances the efficacy of Treg infusion, a therapy that is currently entering the clinic to promote transplant tolerance.</p