Effect of triple costimulation blockade on islet allograft survival in sensitized mice.

BACKGROUND: Islet allograft rejection in sensitized recipients is difficult to control by costimulation blockade using anti-CD154 and cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig). Because leukocyte function antigen (LFA) 1 is highly expressed on memory T cells, adding an LFA-1 blockade may inhibit memory T-cell activities. We examined the effects on islet allograft survival of triple costimulation blockade in presensitized recipient mice. METHODS: C57BL/6 mice were sensitized by transplantation under the kidney capsule or intraperitoneal injection of Balb/c islets. Four weeks after transplantation, sensitization was confirmed by flow-cytometric detection of alloreactive antibodies. Diabetes was induced by a single intravenous injection of streptozotocin. Recipients were transplanted with 200 Balb/c islets under the right kidney capsule. Graft function was assessed by daily blood glucose and body weight records. Transplanted animals were divided into 3 treatment groups: group 1, control antibody; group 2, anti-CD154 and CTLA-4 Ig double therapy; group 3, anti-CD154, CTLA4Ig, and anti-LFA-1 triple therapy. Injections were administered every second day from day -2 to day 8. RESULTS: Naïve mice rejected islet allografts between days 7 and 29 (mean 16 +/- 6 d; n = 5), sensitized mice in group 1 between days 0 and 14 (mean 7 +/- 5 d; n = 8), in group 2 between days 4 and 16 (mean 8 +/- 4 d; n = 7), and in group 3 between days 4 and 26 (mean 11 +/- 7 d; n = 10). CONCLUSION: Triple costimulation blockade with anti-CD154, CTLA4Ig, and anti-LFA-1 was not sufficient to improve islet allograft survival in sensitized recipients.

The ADP/ATP ratio: A novel predictive assay for quality assessment of isolated pancreatic islets.

The current standard assays for islet product release criteria are unable to predict the outcome after clinical islet transplantation. Therefore, establishment of reliable and rapid assays enabling pre-transplantation prediction of islet potency is warranted. In the present study, we have evaluated the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) test, the glucose-stimulated insulin release, the loss of islets during the first 24 h in culture, and the insulin/deoxyribonucleic acid as predictive assays for the ability of isolated porcine islets to cure athymic mice with streptozotocin-induced diabetes. From the results presented, it is concluded that the measurement of the ADP/ATP ratio was the only test that correlated with transplantation outcome. In summary, we propose that the ADP/ATP assay is worthwhile as applied to human islet transplantation and seek to validate it as a rapid and potent predictor of transplant outcome.

Exendin-4 treatment improves metabolic control after rat islet transplantation to athymic mice with streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: Early islet graft survival is crucial in determining the outcome after clinical islet transplantation. Exendin-4 has anti-apoptotic and beta cell proliferative properties, which could improve islet graft survival and function. The aim of these studies was to evaluate the effect of exendin-4 on graft function after islet transplantation. MATERIALS AND METHODS: Rat islets were transplanted under the kidney capsule of diabetic athymic mice. First, we performed a dose-finding study and found that 30 islets just failed to cure diabetic mice. In the following two studies, we transplanted 30 islets and treated the mice that had received these islets with exendin-4 i.p. (100 ng/mouse) once daily for 1 week. Blood glucose levels and body weights were used as evaluation criteria. In the short-term study evaluation was done at day 8. This study was followed by a long-term study that was evaluated at 4 weeks. In this study, islets were precultured with exendin-4 (0.1 nmol/l) in addition to the treatment given to mouse-recipients of transplanted islets. The cured mice underwent an intraperitoneal glucose tolerance test (IPGTT). RESULTS: In the short-term study, 63% of exendin-4-treated mice achieved graft function compared with 21% of untreated mice (p = 0.033). In the long-term study, 88% of treated mice had functioning grafts compared with 22% of controls (p = 0.015). Cured mice showed a normal response in the IPGTT, comparable to that of healthy mice. Exendin-4-treated mice gained significantly more weight than their untreated counterparts. CONCLUSIONS/INTERPRETATION: Islet preculture and a short course of therapy with exendin-4 improves metabolic control after rat islet transplantation in athymic mice. The beneficial effect lasts beyond the treatment period.

Continued production of xenoimmune antibodies 6-8 years after clinical transplantation of fetal pig islet-like cell-clusters.

We have monitored the humoral immune responses of 10 type I diabetic patients, xenotransplanted with fetal porcine islet-like cell clusters for up to 8 years after xenotransplantation. We investigated the immunoglobulin subclass distribution as well as specificity differences of xenoreactive antibodies. Hemagglutintion tests, using pig erythrocytes, showed that some patients maintained higher titers of xenoreactive IgM antibodies during the entire follow up period, compared with pretransplant levels. In microcytotoxicity tests all but one patient tested showed higher than pretransplant levels of cytotoxic antibodies against pig peripheral blood mononuclear cells (PBMC) 6-8 years after transplantation. Levels of Gal alpha 1,3Gal specific antibodies, were also high. Antibody dependent cellular cytotoxicity (ADCC) activity against a Gal alpha 1,3Gal expressing human B cell line was detected in four patients while ADCC reactivity against adult pig islet cells was detected in only two patients, 6-8 years after transplantation. Immune sera collected 30 days and 1 year after transplantation showed positive staining of adult pig islet cells in fluoromicroscopy whereas sera from later time points did not. Western blot experiments showed that some patients had IgG1 antibodies reactive against epitopes on pig cells other than Gal alpha 1,3Gal, while xenoreactive IgM and IgG2 antibodies mainly reacted with Gal alpha 1,3Gal-containing epitopes as shown by absorption experiments. These results show that patients continue to produce higher than pretransplant levels of IgM and IgG2 xenospecific antibodies against Gal alpha 1,3Gal for extended time periods following xenotransplantation. Some patients also produce xenoreactive IgG1 antibodies directed against non-Gal alpha 1,3Gal epitopes.

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells.

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.

T cell receptor usage by HLA-DR3-specific T cell clones isolated from a renal allograft.

In order to evaluate the T cell receptor (TCR) usage by clones of human allograft infiltrating lymphocytes, this study utilized polymerase chain reaction (PCR) amplification of TCR transcripts from five clones which were previously shown to react with a human leucocyte antigen (HLA)-DR3 mismatch between a living related kidney donor and recipient. The five CD4+ (CD8-) clones, which were selected for TCR analysis, proliferated in response to HLA-DR3 and three of the clones were also cytotoxic against the same target cells. After identification of the TCRAV and TCRBV usage of the clones, the sequence of the TCR alpha and beta were determined by direct sequencing of the PCR product. The results indicate that several different TCRAV and TCRBV gene segments are used among the different clones, but the two clones that were both cytotoxic and proliferative in response to HLA-DR3 shared identical TCRAV27-J42-C and TCRBV13-D1-J1S2-C1 transcripts. The additional three clones showed various TCRAV and TCRBV transcripts, but evaluation of the CDR3 region of the TCR beta chain, corresponding to the peptide antigen binding sites, demonstrated shared amino acid motifs which resulted both from germline sequences and combinations of n-region and germline-derived codons. These results suggest that the repertoire for anti-HLA-DR3-reactive clones can include a diverse expression of TCR, but there may be selection for some clones, as well as conserved motifs in the CDR3 region of anti-DR3 specific clones.

15-Deoxyspergualin inhibits interleukin 6 production in in vitro stimulated human lymphocytes.

Experimental data show that relatively low concentrations of 15-deoxyspergualin (DSG) inhibit the induction of cytotoxic T lymphocytes (CTL) and the generation of antibody-producing cells. Considerably higher concentrations of DSG are required to inhibit proliferative responses. In this in vitro study, the effects of DSG on CTL induction, on proliferative responses induced by different stimuli, and on the production of interleukins IL-1, IL-2 and IL-6 and IFN-gamma (gamma-interferon) were assessed and compared with the effects of CsA (cyclosporine A) and/or FK506. We confirmed the suppressive action of DSG on the generation of CTL. Quite unexpectedly, however, we found that, although DSG did not affect the proliferative response to allogeneic lymphocytes or a superantigen, it did inhibit proliferation of peripheral blood leucocytes (PBL) stimulated with Staphyloccus aureus. DSG was active even when added on day 2 of in vitro culture, suggesting that DSG does not inhibit early events. The fraction of CD3+ lymphoblasts and the CD4/CD8 ratio was lower in cells stimulated by S. aureus in the presence of DSG, showing a selective effect on CD3+CD4+ responder T lymphocytes. The proportion of IL-2 receptor (CD25) positive cells was also reduced by DSG treatment. Moreover, we found that DSG inhibited the proliferation induced by PHA (phytohaemagglutinin) but not by Con A (concanavalin A). This effect of DSG was time-dependent, since PHA induced proliferation was not affected until day 4 after stimulation, and indicated that DSG may inhibit proliferation induced via a CD2- but not via a CD3-mediated pathway. DSG did not influence the production of IL-2 or IFN-gamma or the lipopolysaccharide induced production of IL-2 or IL-6. In contrast, the production of IL-6 was inhibited when cells were stimulated by allogeneic lymphocytes, S. aureus, PHA or Con A. This suggested to us that the DSG-suppressed IL-6 production could be the basis for the other observed effects. We tried to mimic the DSG effects with antibodies and indeed found that the IL-6 specific antibodies had similar effects. Furthermore, recombinant IL-6 completely overcame the suppressive effects of DSG on S. aureus and PHA induced proliferation, whereas addition of IL-6 to DSG treated PBL only partly restored the cytotoxic activity of lymphoblasts induced by allogeneic cells. Thus, the inhibitory effect of DSG on de novo synthesis of IL-6 could explain some of its immunosuppressive effects, but additional DSG-sensitive steps are obviously involved in CTL induction and differentiation.