Shounan Yi

T Cell-Activated Macrophages Are Capable of Both Recognition and Rejection of Pancreatic Islet Xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4+ T cells from BALB/c mice. Twelve days after CD4+ T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4+ T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4+ T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4+ T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4+ T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.

Pancreatic islet xenograft tolerance after short-term costimulation blockade is associated with increased CD4+ T cell apoptosis but not immune deviation.

BACKGROUND Our purpose was to determine if short-term inhibition of the CD40/CD40L and CD28/B7 costimulatory pathways was capable of inducing specific unresponsiveness to pancreatic islet xenografts and to ascertain the mechanism of tolerance induction. METHODS Diabetic B6AF1 mice were transplanted with Wistar or DA rat islets and were treated short term with CTLA4-Fc and anti-CD40L mAb (MR1). RESULTS Coadministration of CTLA4-Fc with MR1, resulted in indefinite rat islet xenograft survival in mice. Tolerance was species but not strain specific as long-term surviving recipients rejected third party BALB/c islet allografts but accepted a second rat islet xenograft from the same or different donor strain. Tolerance induction was associated with a large leukocyte infiltrate that did not exhibit features of immune deviation as intragraft T cell-specific cytokine gene expression was globally reduced. In particular, interleukin-4 gene expression was markedly suppressed. There was a complete inhibition of anti-donor IgG, IgG1, and IgM antibody in the serum of CTLA4-Fc/MR1- treated animals. Tolerance induction was associated with increased CD4+ T cell apoptosis as there was an increased proportion of annexin-V staining and Fas expressing CD4+ T cells and a decrease in CD4+ T cell Bcl-2 expression in the grafts and draining lymph nodes of CTLA4-Fc/MR1-treated recipients. CONCLUSION Combined costimulatory blockade was capable of producing tolerance to pancreatic islet xenografts. The induction of this tolerant state was associated with increased T cell apoptosis, whereas the maintenance phase of tolerance was associated with the accumulation of a large number of inactive lymphocytes within the graft.

Adoptive Transfer With In Vitro Expanded Human Regulatory T Cells Protects Against Porcine Islet Xenograft Rejection via Interleukin-10 in Humanized Mice

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ−/− mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

CD4+ T cells initiate pancreatic islet xenograft rejection via an interferon-gamma-dependent recruitment of macrophages and natural killer cells.

BACKGROUND In this study, the mechanisms by which CD4+ T cells interact with the innate immune system in xenograft rejection were investigated. METHODS Fetal pig pancreas (FPP) grafts were transplanted into female SCID mice. The FPP recipient SCID mice were reconstituted with exogenous leukocytes obtained from male BALB/c mice. RESULTS Although nonreconstituted SCID recipients or recipients reconstituted with CD4+ T cell-depleted leukocytes showed indefinite FPP graft survival with very few macrophages infiltrating their grafts, reconstitution of SCID recipients with as few as 2x10(5) CD4+ T cells was sufficient to induce rapid xenograft rejection. CD4+ T cells secreted interferon-gamma but not interleukin-4 and initiated the activation and accumulation of macrophages and natural killer cells, that were responsible for the rapid graft destruction. Suppression of interferon-gamma prolonged graft survival and suppressed the macrophages and natural killer cell accumulation and activation. CONCLUSIONS These results demonstrate that CD4+ T cell-dependent cellular xenograft rejection was a result of macrophage and natural killer cell accumulation and activation, but was not mediated by eosinophils. Consistent with this was the finding that interferon-gamma but not interleukin-4 was in part responsible for mediating this effect.

CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway.

BACKGROUND In this study, the role of cell-mediated cytotoxicity by human leukocytes against pig endothelial cells was examined in vitro. The aim was to determine which cell subsets were responsible for this phenomenon and which pathways were involved in cell lysis. METHODS Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in which cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding. RESULTS Human PBMC demonstrated specific lysis of porcine EC that was proportional to the effector: target ratio. CD4+ T cells accounted for >60% of this lysis, whereas CD8+ T cells accounted for <20%. CD4+ T cell-mediated lysis depended on direct recognition of porcine major histocompatibility complex class II molecules as inhibition of swine leukocyte antigen class II on porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated through the Fas/FasL pathway as addition of anti-Fas and/or anti-FasL antibody profoundly inhibited antiporcine lysis. In addition, FasL gene expression was detected in primed PBMC and CD4+ T cells by RT-PCR, whereas granzyme B gene expression was not. Primed CD4+ T cells demonstrated high level FasL protein by Western blotting and two-color FACS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human FasL induced apoptosis in Fas expressing porcine EC cells, demonstrating that human FasL interacted with and activated Fas on porcine EC cells. CONCLUSIONS In conclusion, human to pig cell-mediated cytotoxicity was mediated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptosis. These results suggest that direct cytotoxicity by xenoreactive CD4+ T cells may be one of several effector mechanisms involved in cellular xenograft rejection.

In vitro expanded human CD4+CD25+ regulatory T cells are potent suppressors of T-cell-mediated xenogeneic responses.

Background. Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Little is known of the effectiveness of CD4+CD25+ T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro. Methods. Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4+CD25− T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4+CD25− T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively. Results. Human Treg were expanded up to 3500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more potent at suppressing CD4+CD25− T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts. Conclusion. This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.

Elevated peripheral frequencies of Th22 cells: a novel potent participant in obesity and type 2 diabetes.

Objective Chronic low-grade inflammation has long been recognized as the central link between obesity and type 2 diabetes (T2D). The novel subset of T helper (Th) cells, Th22, plays an emerging role in chronic inflammation. We investigated the potential association between Th22 and the pathogenesis of obesity and T2D. Methods Ninety T2D inpatients (T2D group), 30 healthy participants with BMI ranged from 19 to 23.9 kg/m2 (CTL group) and 30 metabolically healthy obese controls with BMI ≥ 30 kg/m2 (MHO group) were employed in our study. Peripheral frequencies of Th22 and Th1 and Th17 cells were determined by flow cytometry based on their specific cytokine patterns. Cytokine levels in fresh plasma were quantified by ELISA. Results Compared to that in CTL group (1.18±0.06%, n = 28), peripheral frequency of Th22 cells was significantly increased in MHO group (1.88±0.10%, n = 30) and in T2D group (2.247±0.10%, n = 89). There was a consistent notable increase in plasma interleukin (IL)-22 of T2D patients [47.56 (30.55–76.89) pg/mL] as compared with that of MHO group [36.65 (29.52–55.70) pg/ml; *P<0.0001] and CTLs [36.33 (31.93–40.62) pg/mL; *P<0.0001]. Furthermore, other than Th1/Th17, previously frequently described participants in obesity and T2D, there was a strong correlation between Th22 frequency and the homeostasis model of assessment for insulin resistance index (r = 0.6771, *P<0.0001) and HOMA for β-cell function (r = −0.7264, *P<0.0001). Conclusions There were increased Th22 frequencies and IL-22 levels in obesity and T2D. Elevated Th22 and IL-22 also aided in the differentiation of MHO from T2D patients. The notable correlation implied that Th22 might play a more determinant role in both insulin resistance and β-cell impairment.

The Importance of Tissue Factor Expression by Porcine NICC in Triggering IBMIR in the Xenograft Setting

Background. In islet transplantation, tissue factor (TF) has been reported to be involved in triggering the instant blood-mediated inflammatory reaction (IBMIR), which causes early massive loss of islets transplanted intraportally. TF is synthesized and secreted by several cell sources including islets and inflammatory cells such as neutrophils, monocytes, and platelets. In this study, we investigated whether xenografts-mediated IBMIR could be inhibited by selectively inhibiting TF production by islets using small interfering RNA (siRNA)-mediated TF gene knockdown. Methods. Porcine neonatal islet cell clusters (NICC) were transfected with siRNA specific for TF or a nonspecific siRNA. TF gene and protein expression were analyzed by real-time polymerase chain reaction and fluorescence-activated cell sorting, respectively. The effect of TF knockdown on IBMIR was evaluated using an in vitro tubing loop model of human blood-NICC interactions. Results. TF siRNA transfection of NICC resulted in reduced TF gene and protein expression. TF siRNA transfected NICC showed a significant reduction in the formation of blood clots, platelet activation, thrombin generation, and complement activation after exposure to human ABO compatible blood in vitro. In addition, there was reduced neutrophil infiltration within blood clots containing TF siRNA transfected NICC. Conclusions. TF expression on porcine NICC is an important initiator of IBMIR in islet xenotransplantation. This study identifies porcine TF as a potential target for inhibiting this response.

IL-10 is required for human CD4 + CD25 + regulatory T cell-mediated suppression of xenogeneic proliferation

Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Human CD4+CD25+ regulatory T cells (Tregs) are capable of suppressing the CD4+ T cell‐mediated xenogeneic response in vitro. However, the precise mechanisms that are involved remain to be identified. In this study, we analyzed whether interleukin‐10 (IL‐10) is required for Tregs to suppress xenogeneic responses in vitro by small interfering RNA (siRNA)‐mediated IL‐10 knockdown. After siRNA transfection, Tregs were analyzed for IL‐10 gene and protein expression and their phenotype. Mixed lymphocyte reactions (MLRs) were performed by stimulating human CD4+CD25− T cells with allogeneic or pig peripheral blood mononuclear cells (PBMCs) in the presence or absence of Tregs in a coculture or transwell system. The production of effector cytokines by xeno‐ or alloreactive CD4+CD25− T cells, or suppressive cytokines by Tregs, was examined using enzyme‐linked immunosorbant assay (ELISA). We showed that IL‐10 knockdown resulted in a substantially reduced IL‐10 production by Tregs, leading to impaired Treg‐mediated suppression of xeno‐ but not alloreactive CD4+ CD25− T‐cell proliferation. However, IL‐10 knockdown had no effect on Treg phenotype, their suppression of effector cytokine production by xeno‐ or alloreactive T cells and the production of the Treg‐suppressive cytokine, transforming growth factor‐β (TGF‐β). This study shows that IL‐10 is required for human Tregs to suppress xenogeneic but not allogeneic proliferation in vitro.

CD8+ T cells are capable of rejecting pancreatic islet xenografts

BACKGROUND In this study, the capacity of CD8+ T cells to act as a potential effector mechanism in pancreatic xenograft rejection was examined. METHODS The fate of pancreatic islet xenografts was studied in mice deficient in MHC class II molecules and CD4+ T cells. Fetal pig pancreas (FPP) or Wistar rat islets (RI) were transplanted into nondiabetic or streptozotocin-induced diabetic I-A knock-out (CII K/O) mice. RESULTS CII K/O mice were capable of rejecting both RI and FPP grafts. RI graft survival was not prolonged compared with wild type C57BL/6 controls. However, FPP grafts did survive longer in CII K/O recipients than in C57BL/J6 mice. Both RI and FPP graft rejection were CD8+ T-cell phenomena in CII K/O mice, as anti-CD8 monoclonal antibody prolonged graft survival, there were increased CD8+ T cells in the grafts and spleens of CII K/O recipients, and cell-mediated cytotoxicity was a CD8+ T-cell phenomenon associated with activation of the perforin/granzyme B system. By contrast, RI and FPP graft rejection was a CD4+ T cell-dependent phenomenon in wild type C57BL/6 mice with graft survival prolonged by anti-CD4 monoclonal antibody. There were increased numbers of CD4+ T cells, and cell-mediated cytotoxicity was a CD4+ T-cell phenomenon associated with activation of the Fas/FasL lytic pathway. CONCLUSIONS The results demonstrate that, in the absence of CD4+ T cells, CD8+ T cells were capable of rejecting both rat and pig pancreatic islet xenografts.