Martin Tibudan

Improved coating of pancreatic islets with regulatory T cells to create local immunosuppression by using the biotin-polyethylene glycol-succinimidyl valeric acid ester molecule

BACKGROUND We showed that T regulatory (Treg) cells can be attached to the surface of pancreatic islets providing local immunoprotection. Further optimization of the method can improve coating efficiency, which may prolong graft survival. In this study, we compared the effectiveness of two different molecules used for binding of the Tregs to the surface of pancreatic islets. Our aim was to increase the number of Treg cells attached to islets without compromising islets viability and function. METHODS The cell surface of human Treg cells and pancreatic islets was modified using biotin-polyethylene glycol-N-hydroxylsuccinimide (biotin-PEG-NHS) or biotin-PEG-succinimidyl valeric acid ester (biotin-PEG-SVA). Then, islets were incubated with streptavidin as islet/Treg cells binding molecule. Treg cells were stained with CellTracker CM-DiL dye and visualized using a Laser Scanning Confocal Microscope. The number of Treg cells attached per islets surface area was analyzed by Imaris software. The effect of coating on islet functionality was determined using the glucose-stimulated insulin response (GSIR) assay. RESULTS The coating procedure with biotin-PEG-SVA allowed for attaching 40% more Treg cells per 1 μm(2) of islet surface. Although viability was comparable, function of the islets after coating using the biotin-PEG-SVA molecule was better preserved than with NHS molecule. GSIR was 62% higher for islets coated with biotin-PEG-SVA compared to biotin-PEG-NHS. CONCLUSION Coating of islets with Treg cells using biotin-PEG-SVA improves effectiveness with better preservation of the islet function. Improvement of the method of coating pancreatic islets with Treg cells could further facilitate the effectiveness of this novel immunoprotective approach and translation into clinical settings.

Application of Digital Image Analysis to Determine Pancreatic Islet Mass and Purity in Clinical Islet Isolation and Transplantation.

Pancreatic islet mass, represented by islet equivalent (IEQ), is the most important parameter in decision making for clinical islet transplantation. To obtain IEQ, the sample of islets is routinely counted under a microscope and discarded thereafter. Islet purity, another parameter in islet processing, is routinely assessed by estimation only. In this study, we validated our digital image analysis (DIA) system by using the software of Image Pro Plus and a custom-designed Excel template to assess islet mass and purity to better comply with current good manufacturing practice (cGMP) standards. Human islet samples (60 collected from a single isolation and 24 collected from 12 isolations) were captured as calibrated digital images for the permanent record. Seven trained technicians participated in determination of IEQ and purity by the manual counting method (manual image counting, Manual I) and DIA. IEQ count showed statistically significant correlations between the Manual I and DIA in all sample comparisons (r > 0.819 and p < 0.0001). A statistically significant difference in IEQ between Manual I and DIA was not found in all sample groups (p > 0.05). In terms of purity determination, statistically significant differences between assessment and DIA measurement were found in high-purity 100-μl samples (p < 0.005) and low-purity 100-μl samples (p < 0.001) of the single isolation. In addition, islet particle number (IPN) and the IEQ/IPN ratio did not differ statistically between Manual I and DIA. In conclusion, the DIA used in this study is a reliable technique to determine IEQ and purity. Islet sample preserved as a digital image and results produced by DIA can be permanently stored for verification, technical training, and information exchange among islet centers. Therefore, DIA complies better with cGMP requirements than the manual counting method. We propose DIA as a quality control tool to supplement the established standard manual method for islet counting and purity estimation.

Impact of culture medium on CD4+ CD25highCD127lo/neg Treg expansion for the purpose of clinical application

A recently discovered population of lymphocytes, called T regulatory cells (Tregs), is characterized by expression of transcription factor Forkhead box P3 (FoxP3). These cells have been successfully used as therapeutic treatments and prophylaxis for graft-versus-host disease (GVHD) and diabetes and might become an attractive alternative to traditional immunotherapy. Here we evaluated how the type of culture medium and the type of serum can influence yield and quality of Tregs after in vitro expansion. We compared Treg fold of expansion and their phenotypical characteristics including expression of FoxP3, CD25, CD127, CD62L and CD45RA in three commercially available culture media (RPMI 1640 (Cellgro; Manassas VA, USA), SCGM (Cellgenix; Freiburg, Germany), and X-VIVO 20 (Lonza; Walkersville, MD, USA)) with addition of human serum (HS, 10%) or fetal bovine serum (FBS, 10%). Among the tested media, X-VIVO 20 supplemented with HS produced the highest yield after 17days of in vitro expansion (a median of 86-fold expansion, range 30-1365) and highest level of FoxP3 expression (a median of 66.8% of positive cells, range 56-84.8%) in CD4(+) CD25(hi)CD127(lo/neg) FACS sorted polyclonal Tregs. There was no difference in Tregs yield whether HS or FBS serum was used. In conclusion, the yield of the ex vivo expanded Tregs is related to the type of media applied. Supplementation of the culture with FBS or human serum is equally beneficial.

Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders

Adoptive transfer of T regulatory cells (Tregs) is of great interest as a novel immunosuppressive therapy in autoimmune disorders and transplantation. Obtaining a sufficient number of stable and functional Tregs generated according to current Good Manufacturing Practice (cGMP) requirements has been a major challenge in introducing Tregs as a clinical therapy. Here, we present a protocol involving leukapheresis and CD4+ cell pre-enrichment prior to Treg sorting, which allows a sufficient number of Tregs for a clinical application to be obtained. With this method there is a decreased requirement for ex-vivo expansion. The protocol was validated in cGMP conditions. Our final Treg product passed all release criteria set for clinical applications. Moreover, during expansion Tregs presented their stable phenotype: percentage of CD4+CD25hiCD127− and CD4+FoxP3+ Tregs was > 95% and > 80%, respectively, and Tregs maintained proper immune suppressive function in vitro. Our results suggest that utilization of leukapheresis and CD4 positive selection during Treg isolation improves the likelihood of obtaining a sufficient number of high quality Treg cells during subsequent ex-vivo expansion and they can be applied clinically.

BETA-2 Score Allows for Convenient and Precise Monitoring of Islet Function and Early Detection of Islet Dysfunction

Introduction BETA-2 was developed to more conveniently and precisely assess islet graft function than commonly used beta-score, which requires a stimulation test. After validation of BETA-2 in our cohort of patients in relation to 90-min glucose in a mixed meal tolerance test and beta-score, we decided to assess the practical utility of BETA-2 in monitoring islet allograft function in individual patients. Methods We retrospectively analyzed BETA-2 calculations during clinical evaluations in islet allotransplantation (ITx) recipients with up to 3 islet infusions. We specifically looked in reference to previously established BETA-2 cut-offs for the detection of glucose intolerance <18 and insulin independence >13. Results We analyzed 298 BETA-2 scores calculated in 14 patients (7 women and 7 men) with an average age of 42.9 ± 9.5 years. In these patients, BETA-2 correlated well with islets function. Four patients, who experienced stable, long-term insulin independence after only one transplant, had a BETA-2 continuously over 18 (Fig 1). Figure. No caption available. Another 3 patients required a second transplant to reach the same outcome with the same BETA-2 characteristic (Fig 2). Figure. No caption available. In those patients, despite only discrete changes in fasting glucose level, the BETA-2 decreased to below 18 and continued gradually declining. This drop in BETA-2 always predicted islet dysfunction, the need for insulin support and subsequent transplant, which became clinically obvious as BETA-2 dropped below 13 (Fig 3 and 4). Figure. No caption available. Figure. No caption available. In the remaining patients, even when BETA-2 eventually reached peak above 18 after subsequent transplant, islet function gradually declined without obvious reason within a year or 2 with BETA-2 below 13 requiring re-introduction of insulin support. (Fig 4). One of these patients has not been able to stop insulin completely despite 3 subsequent transplants but BETA-2 has never increased above 18, which confirms utility of BETA-2. 12% (14/121) of BETA-2 measurements above 18 were made during the early period after iTx, when insulin was administered irrespectively of beta-cell function, in order to facilitate islet engraftment. In 80.2% of cases (142/177) where BETA-2 value was above the cut-off of 13, patients were off-insulin. The remaining 19.8% were on insulin due to failing islet function with dropping BETA-2 or supporting post infusion islet engraftment as described above. BETA-2 calculations below 13 were in 94.4% (113/121) in patients receiving insulin. Remaining 6.6% of BETA-2 calculations (8/121) were below the cut-off of 13, yet patients were still off insulin due to their non-compliance to the recommendation to resume insulin therapy based on clinically evident suboptimal glucose control. Conclusion BETA-2 score, based on only a single fasting blood sample is a very reliable tool for islet function assessment and allows early detection of islet graft decline before obvious clinical symptoms. This work was supported by the Illinois Department 370 of Public Health Grant “Pancreatic Islet Transplantation” US Public Health Service Grant DK-020595 to the University of Chicago Diabetes Research Training Center.

Assessment of simple indices based on a single fasting blood sample as a tool to estimate beta cell function after total pancreatectomy with islet autotransplantation - a prospective study

We investigated six indices based on a single fasting blood sample for evaluation of the beta‐cell function after total pancreatectomy with islet autotransplantation (TP‐IAT). The Secretory Unit of Islet Transplant Objects (SUITO), transplant estimated function (TEF), homeostasis model assessment (HOMA‐2B%), C‐peptide/glucose ratio (CP/G), C‐peptide/glucose creatinine ratio (CP/GCr) and BETA‐2 score were compared against a 90‐min serum glucose level, weighted mean C‐peptide in mixed meal tolerance test (MMTT), beta score and the Igls score adjusted for islet function in the setting of IAT. We analyzed values from 32 MMTTs in 15 patients after TP‐IAT with a follow‐up of up to 3 years. Four (27%) individuals had discontinued insulin completely prior to day 75, while 6 out of 12 patients (50%) did not require insulin support at 1‐year follow‐up with HbA1c 6.0% (5.5–6.8). BETA‐2 was the most consistent among indices strongly correlating with all reference measures of beta‐cell function (r = 0.62–0.68). In addition, it identified insulin independence (cut‐off = 16.2) and optimal/good versus marginal islet function in the Igls score well, with AUROC of 0.85 and 0.96, respectively. Based on a single fasting blood sample, BETA‐2 score has the most reliable discriminant value for the assessment of graft function in patients undergoing TP‐IAT.

Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype

The first clinical trials with adoptive Treg therapy have shown safety and potential efficacy. Feasibility of such therapy could be improved if cells are cryopreserved and stored until optimal timing for infusion. Herein, we report the evaluation of two cell-banking strategies for Treg therapy: 1) cryopreservation of CD4+ cells for subsequent Treg isolation/expansion and 2) cryopreservation of ex-vivo expanded Tregs (CD4+CD25hiCD127lo/- cells). First, we checked how cryopreservation affects cell viability and Treg markers expression. Then, we performed Treg isolation/expansion with the final products release testing. We observed substantial decrease in cell number recovery after thawing and overnight culture. This observation might be explained by the high percentage of necrotic and apoptotic cells found just after thawing. Furthermore, we noticed fluctuations in percentage of CD4+CD25hiCD127- and CD4+FoxP3+ cells obtained from cryopreserved CD4+ as well as Treg cells. However, after re-stimulation Tregs expanded well, presented a stable phenotype and fulfilled the release criteria at the end of expansions. Cryopreservation of CD4+ cells for subsequent Treg isolation/expansion and cryopreservation of expanded Tregs with re-stimulation and expansion after thawing, are promising solutions to overcome detrimental effects of cryopreservation. Both of these cell-banking strategies for Treg therapy can be applied when designing new clinical trials.

Total Pancreatectomy with Islet Autotransplantation for the Ampullary Cancer. A Case Report

Total pancreatectomy (TP) leads to pancreatic exocrine deficiency and poorly controlled diabetes despite insulin treatment [1]. Simultaneous islet autotransplantation (IATx) can prevent diabetes in 30–40% of patients and improve glycemic control with insulin supplementation in an additional 30% of individuals leading to improvement in quality of life in the majority of patients [2]. Total pancreatectomy with islet transplantation (TPIAT) has been offered mostly as a last resort procedure to highly selected patients with chronic or recurrent acute pancreatitis and intractable pain despite medical, endoscopic, and other conventional surgical interventions. Although patients with advanced benign tumors who require TP also may receive IATx, application of TPIAT in the setting of pancreatic malignancy remains controversial due to the risk of contamination of the islet prep with tumor cells and possible dissemination of malignancy [1]. Nevertheless, encouraging results in single cases of IATx have been reported in those patients with pancreatic cancer, who required TP due to complications after initial Whipple procedure [3–7]. Here we present for the first time, a case report of a patient with a small, early-stage ampullary cancer without lymph node involvement, who developed disseminated neoplastic disease 6 months after TPIAT. Method