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

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 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

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

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

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

data_sheet_1_Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice.PDF

<p>Fibrocytes are myeloid lineage cells implicated in wound healing, repair, and fibrosis. We previously showed that fibrocytes are mobilized into the circulation after vascular injury, including the immune-mediated injury that occurs after allogeneic transplantation. A common response to inflammatory vascular injury is intimal hyperplasia (IH), which, alongside vascular remodeling, results in progressive loss of blood flow, downstream ischemia, and end-organ fibrosis. This forms the pathological basis of transplant arteriosclerosis and other diseases including post-angioplasty re-stenosis. In investigating whether fibrocytes contribute to IH, we previously showed that subpopulations expressing smooth muscle actin and CD31 are recruited to the site of injury and accumulate in the neointima. Expression of tissue factor (TF) by these “CD31+ myofibrocytes” is needed for progressive neointimal expansion, such that TF inhibition limits the neointima to a single layer of cells by day 28 post-injury. The aim of this study was to determine pathophysiological mediators downstream of TF that contribute to myofibrocyte-orchestrated IH. We first show that myofibrocytes make up a significant component of the neointima 28 days following injury. Using a previously defined adoptive transfer model, we then show that CD31+ myofibrocytes get recruited early to the site of injury; this model allows manipulations of the adoptively transferred cells to study how IH develops. Having confirmed that inhibition of TF on adoptively transferred cells prevents IH, we then show that TF, primarily through the generation of thrombin, induces secretion of angiopoietin-2 by myofibrocytes and this directly stimulates proliferation, inhibits apoptosis, and induces CXCL-12 production by neointimal cells, including non-fibrocytes, all of which promote progressive IH in vivo. Prior incubation to inhibit angiopoietin-2 secretion by or block TIE-2 signaling on adoptively transferred fibrocytes inhibits IH. These novel data indicate that angiopoietin-2 production by early recruited myofibrocytes critically influences the development of IH after vascular injury and suggest new therapeutic avenues for exploration.</p

A multicenter randomized controlled trial indicates that paclitaxel-coated balloons provide no benefit during angioplasty of arteriovenous fistulas.

The role of paclitaxel-coated balloons has been established in the coronary and peripheral arterial circulations with recent interest in the use of paclitaxel-coated balloons to improve patency rates following angioplasty of arteriovenous fistulas. To assess the efficacy of paclitaxel-coated angioplasty balloons to prolong the survival time of target lesion primary patency in arteriovenous fistulas, we designed an investigator-led multi-center randomized controlled trial with follow up time variable for a minimum of one year. Patients with an arteriovenous fistula who were undergoing an angioplasty for a clinical indication were included but patients with one or more lesions outside the treatment segment were excluded. Following successful treatment with a high-pressure balloon, 212 patients were randomized. In the intervention arm, the second component was insertion of a paclitaxel-coated balloon. In the control arm, an identical procedure was followed, but using a standard balloon. The primary endpoint was time to loss of clinically driven target lesion primary patency. Primary analysis showed no significant evidence for a difference in time to end of target lesion primary patency between groups: hazard ratio 1.18 with a 95% confidence interval of 0.78 to 1.79. There were no significant differences for any secondary outcomes, including patency outcomes and adverse events. Thus, our study demonstrated no evidence that paclitaxel-coated balloons provide benefit, following standard care high-pressure balloon angioplasty, in the treatment of arteriovenous fistulas. Hence, in view of the benefit suggested by other trials, the role of paclitaxel-coated angioplasty balloons remains uncertain