Hirohito Ichii

Islet Transplantation in Type 1 Diabetes Mellitus Using Cultured Islets and Steroid-Free Immunosuppression: Miami Experience

Following the success obtained with transplantation of fresh human islets under steroid‐free immunosuppression, this trial evaluated the transplantation of islets that had undergone a period of in vitro culture and the potential of tumor necrosis factor (TNF‐α) blockade to improve islet engraftment. Subjects included 16 patients with type 1 diabetes mellitus (T1DM); half were randomly assigned to receive Infliximab immediately preceding initial infusion. Immunosuppression consisted of daclizumab induction and sirolimus/tacrolimus maintenance. Out of 16 subjects 14 achieved insulin independence with one or two islet infusions; adverse events precluded completion in two. Without supplemental infusions, 11/14 (79%) subjects were insulin independent at 1 year, 6/14 (43%) at 18 months; these same subjects remain insulin independent at 33 ± 6 months. While on immunosuppression, all patients maintained graft function. Out of 14 patients, 8 suffered chronic partial graft loss, likely immunological in nature, 5 of these received supplemental infusions. Currently, 11 subjects remain on immunosuppression, 8 (73%) are insulin independent, two with supplemental infusions. Insulin independent subjects demonstrated normalization of HbA1c, fructosamine and Mean Amplitude of Glycemic Excursions (MAGE) values. No clinical benefit of infliximab was identified. These results demonstrate that transplantation of cultured human islet allografts results in reproducible insulin independence in all subjects under this immunosuppressive regimen, comparable to that of freshly transplanted islets (Edmonton protocol).

Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells

Naïve T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8+ T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8+ T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA‐binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA‐binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining β‐cell viability.

Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells.

After immunization with T cell-dependent antigens, the high-affinity B cells selected in germinal centers differentiate into memory B cells or long-lived antibody-forming cells. However, a role for germinal centers in development of these B lineage cells is still controversial. We show here that Bcl6-deficient B cells, which cannot develop germinal centers, differentiated into IgM and IgG1 memory B cells in the spleen but barely differentiated into long-lived IgG1 antibody-forming cells in the bone marrow. Mutation in the V-heavy gene was null in these memory B cells. Therefore, Bcl6 and germinal center formation are essential for somatic hypermutation, and generation of memory B cells can occur independently of germinal center formation, somatic hypermutation, and Ig class switching.

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells

OBJECTIVE To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8+ T cells

Central memory CD8+ T cells (TCM) are considered to be more efficient than effector ones (TEM) for mediating protective immunity. The molecular mechanism involved in the generation of these cells remains elusive. Because Bcl6 plays a role in the generation and maintenance of memory CD8+ T cells, we further examined this role in the process in relation to TCM and TEM subsets. In this study, we show that TCM and TEM were functionally identified in CD62L+ and CD62L− memory (CD44+Ly6C+) CD8+ T cell subsets, respectively. Although TCM produced similar amounts of IFN-γ and IL-2 to TEM after anti-CD3 stimulation, the cell proliferation capacity after stimulation and tissue distribution profiles of TCM differed from those of TEM. Numbers of TCM were greatly reduced and elevated in spleens of Bcl6-deficient and lck-Bcl6 transgenic mice, respectively, and those of TEM were constant in nonlymphoid organs of these same mice. The majority of Ag-specific memory CD8+ T cells in spleens of these mice 10 wk after immunization were TCM, and the number correlated with Bcl6 expression in T cells. The proliferation of Ag-specific memory CD8+ T cells upon secondary stimulation was dramatically up-regulated in lck-Bcl6 transgenic mice, and the adoptive transfer experiments with Ag-specific naive CD8+ T cells demonstrated that some of the up-regulation was due to the intrinsic effect of Bcl6 in the T cells. Thus, Bcl6 is apparently a crucial factor for the generation and secondary expansion of TCM.

Rescue purification maximizes the use of human islet preparations for transplantation

The relative inefficiency of the islet purification process may hamper obtaining enough islets for transplantation even with adequate pre‐purification counts. In this study, we determined the effect of an additional purification step on total islet yields and pancreas utilization at our center.

Shipment of Human Islets for Transplantation

The use of regional human islet cell processing centers (ICPC) supporting distant clinical islet transplantation programs (CITP) has proven successful in recent clinical trials. Standardization of islet shipping protocols is needed to preserve cell product identity, quantity, quality and sterility, and to meet criteria for transplantation. We evaluated the use of gas‐permeable bags for human islet preparation shipment from a single ICPC to two remote CITPs. Product release tests (counts, purity, viability, sterility and potency) were performed at both centers using identical protocols to determine adequacy for transplantation. Thirty‐five islet preparations were shipped either immediately after isolation (n = 20) or following culture (n = 15). Islet recovery rate after shipment was higher in cultured preparations, when compared to those not cultured (91.2 ± 4.9% vs. 72.9 ± 4.7%, respectively; p < 0.05), though the overall recovery rate based on isolation and pre‐transplant counts was comparable (72.9 ± 4.7% vs. 70.4 ± 3.5%, respectively; p = N.S.). All preparations met product release criteria for transplantation. Additional experiments showed that gas‐permeable bags led to improved recovery and potency, when compared to 50‐mL conical tubes or to non‐gas‐permeable bags for shipment. Collectively, our data demonstrate that the use of gas‐permeable bags is efficient for clinical‐grade and should be preferred also for the shipment of research‐grade islet preparations.

Rapamycin impairs in vivo proliferation of islet beta-cells

Background. Progressive graft dysfunction is commonly observed in recipients of islet allografts treated with high doses of rapamycin. This study aimed at evaluating the effect of rapamycin on pancreatic islet cell proliferation in vivo. Methods. The murine pregnancy model was utilized, since a high rate of &bgr;-cell proliferation occurs in a well-defined time frame. Rapamycin (0.2 mg/kg/day) was given to C57BL/6 mice for 5–7 days starting on day 7.5 of pregnancy. Cell proliferation was evaluated by detection of bromodeoxyuridine incorporation by immunohistochemistry. Results. Pregnancy led to increased &bgr;-cell proliferation and islet yield with skewing in islet size distribution as well as higher pancreatic insulin content, when compared to that of nonpregnant females. These effects of pregnancy on &bgr;-cell proliferation and mass were significantly blunted by rapamycin treatment. Minimal effect of rapamycin was observed on islet function both in vivo and in vitro. Rapamycin treatment of islets in vitro resulted in reduced p70s6k phosphorylation, which was paralleled by increased ERK1/2 phosphorylation. Conclusions. Rapamycin treatment reduces the rate of &bgr;-cell proliferation in vivo. This phenomenon may contribute to impair &bgr;-cell renewal in transplanted patients and to the progressive dysfunction observed in islet graft recipients.

Current status of islet cell transplantation

Despite substantial advances in islet isolation methods and immunosuppressive protocol, pancreatic islet cell transplantation remains an experimental procedure currently limited to the most severe cases of type 1 diabetes mellitus. The objectives of this treatment are to prevent severe hypoglycemic episodes in patients with hypoglycemia unawareness and to achieve a more physiological metabolic control. Insulin independence and long term-graft function with improvement of quality of life have been obtained in several international islet transplant centers. However, experimental trials of islet transplantation clearly highlighted several obstacles that remain to be overcome before the procedure could be proposed to a much larger patient population. This review provides a brief historical perspective of islet transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, and outlines current challenges and future directions in clinical islet transplantation.