Laser capture microdissection as a new tool to assess graft-infiltrating lymphocytes gene profile in islet transplantation.

Innovative tolerogenic protocols in transplantation would take advantage of the development of new tools capable of evaluating the impact of these treatments on the immune system. These assays have potential for clinical application. Currently, many of these studies are based on the analysis of peripheral lymph nodes and blood-derived cells, where the percentage of alloantigen-specific cells can be low or even unpredictable. We combined a laser capture microdissection (LCM) technique with real-time PCR (RT-PCR) to evaluate gene profile of islet-infiltrating lymphocytes. Donor Lewis rats islets were transplanted under the kidney capsule in diabetic Brown Norway rats. Administration of anti-LFA1 mAb or anti-CD28 F(Ab)' was able to prolong islet survival, while the combined treatment resulted in indefinite survival. The analysis of gene expression profile for IL-2, IFN-gamma, and IL-10 production of graft-infiltrating cells revealed high IL-2, IFN-gamma, and IL-10 in untreated rats; on the contrary, the combined treatment selectively abrogated IL-2- and IFN-gamma-producing cells infiltrate. The comparison between cytokine profile in periphery (even during an allogenic extra stimulus) and in the graft revealed the dichotomy between graft and peripheral cytokine assessment. We thus propose that direct analysis of graft-infiltrating cells should be used whenever possible to evaluate the effects of a new immunomodulatory protocol.

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.

AIMS/HYPOTHESIS: Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. METHODS: Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. RESULTS: By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). CONCLUSIONS/INTERPRETATION: These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

Islet allotransplantation in type 1 diabetic patients.

Islet transplantation was proposed more than 10 years ago as treatment for normalising glucose homeostasis in type 1 diabetic patients. Since the beginning it has aroused great interest among diabetic patients being an easy procedure, burdened by minor complications: islet transplantation in fact consists on a transhepatic percutaneous injection under local anaesthesia. The initial clinical outcomes not came up to expectations, being low the insulin independence rate and the long term graft function in recipients. Recently, thanks to the introduction of new immunosuppression strategies, clinical data greatly improved: insulin independence was reached in all recipients and maintained in more than 70% of them 2 years from the transplant. The need of an immunosuppression therapy limits the indication of islet transplantation to diabetic patients already immunosuppressed for a previous organ transplant or to patients with brittle diabetes, that is not controlled also with the new strategies of insulin treatment, with a poor quality of life and an increased rate of diabetic complications. Other problems are represented by the progressive decrease of graft function during long term follow up, and by the low number of organ donors that limits the number of transplantation feasible per year.

CD28 and LFA-1 contribute to cyclosporin A-resistant T cell growth by stabilizing the IL-2 mRNA through distinct signaling pathways.

In clinical transplantation, the occurrence of cyclosporin A (CsA)-resistant production of IL-2 in vitro correlates with graft rejection in vivo. In this study we investigated the role of the costimulatory molecules CD28 and LFA-1 in this process in the setting of TCR-induced proliferation of primary T lymphocytes in vitro. Co-stimulation with ICAM-1 and B7.2 led to strong and CsA-resistant proliferation, which was found to be largely IL-2 dependent. All of the known calcineurin-dependent events, such as induction of NF-AT and NF-kappaB or stress-activated protein kinase activation, were markedly modulated by CsA independently of costimulation. In contrast, both ICAM-1 and B7.2 enhanced the half-life of the inducible IL-2 transcript in a CsA-resistant manner. LFA-1- but not CD28-induced IL-2 mRNA stabilization required the integrity of the actin-based cytoskeleton, suggesting that the two costimulatory molecules impact on qualitatively different signaling pathways. This is further suggested by the demonstration that LFA-1 and CD28 acted synergistically to confer CsA resistance in a model of co-stimulation using superantigen-pulsed dendritic cells. We propose that IL-2 transcript accumulation and subsequent T cell proliferation at the low transcriptional rate imposed by CsA are the result of co-stimulation-dependent stabilization of IL-2 mRNA.

Chemokines fail to up-regulate beta 1 integrin-dependent adhesion in human Th2 T lymphocytes.

Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of P- and E-selectin ligands and selected chemokine receptors. Here we investigated the expression and function of beta 1 integrins in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands laminin 1 and fibronectin in response to chemokines such as RANTES and stromal cell-derived factor-1, and this process is paralleled by the activation of the Rac1 GTPase and by a rapid burst of actin polymerization. Selective inhibitors of phosphoinositide-3 kinase prevent efficiently all of the above processes, whereas the protein kinase C inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting Rac1 activation and actin polymerization. Notably, chemokine-induced adhesion to beta 1 integrin ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving phosphoinositide-3 kinase, Rac1, and actin turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced beta 1 integrin-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.

Upregulation of integrin alpha6/beta1 and chemokine receptor CCR1 by interleukin-12 promotes the migration of human type 1 helper T cells.

CD4(+) T helper 1 (Th1) cells and Th2 cells are distinguished based on the pattern of cytokines they are able to produce. Selectin ligands and chemokine receptors are differentially expressed in Th1 and Th2 cells, providing a basis for tissue-specific recruitment of helper T-cell subsets. However, the modes and mechanisms regulating tissue-specific localization of Th1 and Th2 cells are still largely unknown. Here, we show the preferential expression on Th1 cells of the integrin alpha6/beta1, which is distinctly regulated by the Th1-inducing cytokines interleukin-12 (IL-12) and interferon-alfa (IFN-alpha). The pattern of integrin alpha6/beta1 regulation closely mirrors that of the chemokine receptor CCR1. Analysis of signal transducer and activator of transcription 4 (Stat4) activation by IL-12 and IFN-alpha shows distinct signaling kinetics by these cytokines, correlating with the pattern of CCR1 and integrin alpha6/beta1 expression. Unlike IFN-alpha, the ability of IL-12 to generate prolonged intracellular signals appears to be critical for inducing integrin alpha6/beta1 upregulation in Th1 cells. The expression and upregulation of CCR1 and alpha6/beta1 integrin promotes the migration of Th1 cells. These findings suggest that the exquisite regulation of integrin alpha6/beta1 and CCR1 may play an important role in tissue-specific localization of Th1 cells.

Human natural killer lymphocytes directly recognize evolutionarily conserved oligosaccharide ligands expressed by xenogeneic tissues.

BACKGROUND: In discordant xenogeneic species combinations, vascularized transplants are hyperacutely rejected, due to binding of xenoreactive natural antibodies (XNA) to selected tissues of the graft, followed by activation of the complement and coagulation cascades. A major epitope recognized by human XNA is the terminal disaccharide Gal alpha(1,3)Gal. Poorly defined, early cell-mediated events also contribute to recognition and rejection of discordant xenografts, and we have suggested a role of natural killer (NK) lymphocytes in this process. METHODS: Human NK cells were used as effectors in functional assays of adhesion to and lysis of xenogeneic discordant endothelial cells in vitro. Adhesion and lysis inhibition experiments were performed using a large panel of carbohydrates, as well as F(ab')2 fragments of human XNA. COS cells transduced with the porcine alpha-galactosyltransferase were also used as targets for NK cell adhesion. RESULTS: We demonstrate that XNA-reactive carbohydrate epitopes expressed by xenogeneic cells, including Gal alpha(1,3)Gal, are also directly recognized by human NK cells. First, selected carbohydrates in solution displace with comparable efficiency both XNA and NK cell binding to xenogeneic endothelium; second, XNA F(ab')2 fragments selectively inhibit human NK cell adhesion to porcine endothelium, but not to human endothelium; third, unstimulated NK lymphocytes adhere selectively to COS-7 cells expressing the porcine glycosyltransferase that encodes the Gal alpha(1,3)Gal epitope. CONCLUSIONS: Collectively, our findings suggest that humoral and cellular components of the natural immune response against heterologous species independently evolved recognition patterns directed against overlapping carbohydrate determinants.