Although islet transplantation is an effective treatment for Type 1 diabetes, primary engraftment failure contributes to suboptimal outcomes. We tested the hypothesis that islet isolation and transplantation activate innate immunity through TLR expressed on islets. Murine islets constitutively express TLR2 and TLR4, and TLR activation with peptidoglycan or LPS upregulates islet production of cytokines and chemokines. Following transplantation into streptozotocin-induced diabetic, syngeneic mice, islets exposed to LPS or peptidoglycan had primary graft failure with intra-and peri-islet mononuclear cell inflammation. The use of knockout mice showed that recipient CD8 1 T cells caused engraftment failure and did so in the absence of islet-derived DC. To mimic physiological islet injury, islets were transplanted with exocrine debris. Transplantation of TLR2/4 À/À islets reduced proinflammatory cytokine production and improved islet survival. Stressed islets released the alarmin high-mobility group box protein 1 (HMGB1) and recombinant HMGB1 (rHMGB1) induced NFkB activation. NFkB activation was prevented in the absence of both TLR2 and TLR4. rHMGB1 pretreatment also prevented primary engraftment through a TLR2/4-dependent pathway. Our results show that islet graft failure can be initiated by TLR2 and TLR4 signaling and suggest that HMGB1 is one likely early mediator. Subsequent downstream signaling results in intra-islet inflammation followed by T-cellmediated graft destruction.Key words: Chemokines . Diabetes . Innate immunity . Islet cell transplantation IntroductionIslet transplantation holds promise as a definitive therapy for Type 1 diabetes, but the long-term results have been disappointing, as progressive loss of graft function is observed in the majority of patients [1]. The islet mass is already marginal shortly after transplantation and thus susceptible to become insufficient when subsequently exposed to negative local influences. Recent estimates indicate that less than 30% of islets stably engraft, a result that explains the requirement for infusing large numbers of islets and for repeat islet infusions to maintain insulin-free euglycemia [2]. Mechanisms underlying early islet loss following à These authors have contributed equally to this work. 2914transplantation remain poorly defined but apoptotic cell islet cell death associated with peri-and intra-islet graft inflammation have been described previously [3,4].TLR are a family of pattern recognition receptors that bind to PAMP or to endogenous ligands released by damaged cells (damage-associated molecular patterns, DAMP). Among the latter group are HSPs, high-mobility group box protein 1 (HMGB1), heparan sulfate, hyaluronan fragments, and fibronectin [5]. Regardless of the source of the specific ligand, TLRtransmitted signals activate innate immunity by inducing chemokine and cytokine release and through upregulating costimulatory molecule expression, among a multitude of other effects [6]. Recent studies revealed the importance of islet-expressed TLR...

Abstract. Chemokines and their receptors play a pivotal role in the initiation and amplification of the immune response. Investigated was their differential expression after syngeneic and allogeneic islet transplantation. During the 7 d after transplantation, the chemokines MCP-1, MCP-2, RANTES, MIG, IP-10, I-TAC, and two CC chemokine receptors CCR2 and CCR5 were highly expressed in allografts when compared with isografts. Disrupting the CCR2 and CCR5 pathways individually resulted in prolongation of the survival time 16.1 Ϯ 0.4 and 15.8 Ϯ 0.9 d, respectively, of fully major histocompatibility complex-mismatched islet grafts compared with wildtype controls (11.2 Ϯ 1.0 d). Blockade of both receptors had no synergistic effect. Rapamycin-treated wild-type recipients rejected their grafts at 17.4 Ϯ 2.2 d, in contrast to rapamycintreated CCR2Ϫ/Ϫ recipients at 38 Ϯ 8.6 d (P ϭ 0.025). The disruption of the CCR2 and CCR5 signaling, alone or in combination, moderately prolong islet allograft survival. However, the combination of low-dose immunosuppression and targeting of CCR2 greatly augmented islet graft survival.Leukocyte infiltration of pancreatic islets is the characteristic feature in acute rejection after islet transplantation. In addition to adhesion molecules, such as selectins and integrins, the complex process of extravasation and influx of leukocytes subsets into the site of tissue injury is mediated to a significant extent by the expression of specific chemokines and chemokine receptors. The chemokine superfamily is separated into four general branches (CL, CCL, CXCL, and CX3CL chemokines) on the basis of cysteine residues within their primary amino acid sequence. The biologic actions of chemokines are mediated through their interaction with a large family of G protein-coupled receptors (i.e., C, CC, CXC, and CX3C receptors) (1). During the development of allograft rejection of vascularized organs, a variety of genes of the chemokine system are prominently expressed (2,3). Targeting chemokine receptors, including CCR1, CCR5, CXCR3, and CX3CR1, with either knockout animals or monoclonal antibodies has resulted in delayed rejection of vascularized heterotopic cardiac transplants (3,4). Recently, the role of CCR2, CCR5, and CXCR3 has also been demonstrated in an islet transplantation model, with conflicting results (5,19,20). To date, little is known of the effect of CCR2 blockade in allograft rejection in combination with low-dose immunosuppression and the simultaneous blockage of CCR2 and CCR5. We investigated the differential chemokine and chemokine receptor expression in syngeneic and allogeneic islet grafts. On the basis of the initial screening, we investigated the importance of the CCR2 and CCR5 pathways in mediating islet allograft rejection. Materials and Methods Mice and the Diabetic ModelAnimals were treated in strict compliance with regulations established by the Institutional Animal Care and Use Committee. Mice were born and housed under specific-pathogen-free conditions. The recipients were render...

Toll-like receptors (TLRs) provide an important link between innate and adaptive immune system. We hypothesized that the recognition of endogenous TLR4 ligands is occurring at the time of transplantation, and these innate signals drive the inflammation and affect alloimmune responses. We confirmed that early after transplantation of allogenic islets, transcripts for TLR4 as well as potential ligands were released or upregulated. In an allogenic islet transplantation model, genetic disruption of TLR4 on donor islets had no effect on allograft survival, whereas TLR4 deficiency in recipients lead to prolonged graft survival. Low dose rapamycin-treatment of TLR4 −/− recipients induced permanent engraftment of 45% islet graft (p=0.005) compared to WT recipients. This prolonged graft survival was dependent on the presence of CD4 + CD25 + Foxp3 + Treg. Naïve CD4 + CD25 − T cells cultured with the TLR4 ligand lipopolysaccharide showed enhanced IL-4, IL-6, IL-17, IFNγ secretion and inhibited TGFβ induced Foxp3 + Treg generation. Thus, inhibition of recipient TLR4 activation at the time of transplantation decreases proinflammatory signals and allows Treg generation.

Previously we have shown that adenovirus-mediated gene transfer and expression of vIL-10 are able to prolong cardiac allograft survival, through the inhibition of the immune response to both alloantigen and adenoviral antigens. In the current study, we have defined further mechanisms of Ad.vIL-10-mediated prolongation of cardiac allograft survival. E1-and E3-deleted adenoviral vectors encoding b-galactosidase or vIL-10 were transferred into grafts at the time of transplantation, chemokine and chemokine receptor expression were evaluated by a pathway-specific cDNA array, and the results were confirmed with real time RT-PCR on selected genes. Ischemic injury, alloantigen and adenovirus vector induced the expression of multiple pro-inflammatory chemokines in the grafts, which likely amplify allograft rejection. Most of these Th1-related chemokine genes were inhibited or down-regulated by Ad.vIL-10 administration, which may help to decrease leukocytic infiltration and improve graft survival. Among the potent Th1 type chemokines inhibited were the CXCR3 ligands CXCL9 and CXCL10, which could directly inhibit vector-mediated gene expression in myoblasts, although targeting CXCR3 or its ligands did not prolong allograft survival with vIL-10 gene transfer. Ad.vIL-10 administration also induced the expression of the Th2-associated chemokines eotaxin-2 and MIP-1 c , suggesting Th1 to Th2 immune deviation. These results demonstrated that the vIL-10 gene transfer inhibits chemokine expression, preventing stimulation of innate and adaptive immunity.

T he interaction of the MHC molecules and their associated antigenic peptides with the T cell receptor is the pivotal step in the cognate response to foreign antigen (1). Synthetic peptides that correspond to polymorphic and nonpolymorphic regions of the MHC molecule have been demonstrated to be effective inhibitors of the allergic, autoimmune, and alloimmune responses in vitro and in vivo (2,3). Immunomodulatory polymorphic peptides that bind within the groove of the MHC molecule may influence the subsequent response of the T cell by altering the recognition of the MHC-peptide complex. Modification of antigenic peptide sequences by a single amino acid has been shown to change the T cell response qualitatively resulting in partial T cell activation and the production of T cell anergy (4,5). The use of these altered peptide ligands has proved to be an effective means of ameliorating autoimmune diseases in small animal models (6,7). In addition, polymorphic MHC classes I and II peptides, which are known to elicit an antigenic response, inhibit the alloimmune response in vivo after administration via oral, intrathymic, and intranasal routes (8 -10). Manipulation of the immune response by modification of known antigenic peptides is limited by the need to identify the specific autoantigens in the case of autoimmune diseases and by an exhaustive number of potential allogeneic peptides in the case of transplantation.Several peptides derived from nonpolymorphic regions of both MHC classes I and II have been shown to inhibit T cell responses in vitro (11). It is interesting that the binding sites and mechanisms of action of these peptides are remarkably different from each other and from that of polymorphic MHC peptides. The inhibitory effect of the nonpolymorphic MHC class I peptides (ALLOTRAP) and their rationally designed analogues correlated with the induction of heme oxygenase-1 and inhibition of TNF-␣ production in vivo (12). These peptides have been shown to prolong survival in several different small animal transplant models (13-15) and modify disease in a murine model of Crohn's disease (16). Boytim et al. (17) demonstrated that a synthetic MHC class II peptide that corresponds to the ␣1 helix of DQA03011 (DQ 65 to 79) inhibits T cell proliferation in an allele-independent manner in vitro through binding to proliferating cell nuclear antigen and competitive inhibition of binding of p21 to proliferating cell nuclear antigen (18). We have shown previously that a peptide, HLA-DQA1, derived from a conserved region of the HLA-DQ ␣ chain (62 to 77), is an efficient inhibitor of the human, rat, and mouse MLR (19). HLA-DQA1 requires the interaction of T cells and antigenpresenting cells (APC) for its inhibitory action on T cells. HLA-DQA1 peptide induced apoptosis in B cells, macrophages (20), and dendritic cells (our unpublished observations) via a nonclassical, caspase-independent mechanism. In addition to inducing apoptosis in APC, HLA-DQA1 renders T cells unresponsive to further stimulation. We have shown that a lo...