Formation of donor-specific antibodies depends on the epitope load of mismatched HLAs in lung transplant recipients: A retrospective single-center study.

The development of donor-specific antibodies (DSA) has a significant impact on graft outcome in solid organ transplantation. Mismatched HLAs are recognized directly and indirectly by the recipient immune system. Both pathways occur in parallel and result in the generation of plasma cells, DSA, cytotoxic and T helper lymphocytes. Here, we present the results of an analysis of the epitope load of mismatched HLAs in a cohort of 220 lung transplant recipients using two in silico algorithms, HLAMatchmaker and PIRCHE-II (Predicted Indirectly ReCognizable HLA Epitopes). De novo DSA (dnDSA) were detected by single antigen bead assays. The percentage of recipients who developed dnDSA was significantly higher in the group of patients who received lung transplants with a mismatching score above the detected threshold than in the group of patients who received lung transplants with a mismatching score below the threshold. In a multivariate Cox proportional hazard analysis, the PIRCHE-II score appeared to be a superior predictor of dnDSA formation. In addition, PIRCHE-II technology was shown to be useful in predicting separate dnDSA1 and dnDSA2 formation. We conclude that both algorithms can be used for the evaluation of the epitope load of mismatched HLAs and the prediction of DSA development in lung transplant recipients.

Pretransplant HLA typing revealed loss of heterozygosity in the major histocompatibility complex in a patient with acute myeloid leukemia.

INTRODUCTION: Chromosomal abnormalities are frequent events in hematological malignancies. The degree of HLA compatibility between donor and recipient in hematopoietic stem cell transplantation is critical. PURPOSE OF THE STUDY: In this report, we describe an acute myeloid leukemia case with loss of heterozygosity (LOH) encompassing the entire HLA. MATERIALS AND METHODS: HLA molecular typing was performed on peripheral blood (PB) and buccal swabs (BS). Chromosomal microarray analysis (CMA) was performed using a whole genome platform. RESULTS: Typing results on PB sample collected during blast crisis demonstrated homozygosity at the -A, -B, -C, -DR, and -DQ loci. A BS sample demonstrated heterozygosity at all loci. A subsequent PB sample drawn after count recovery confirmed heterozygosity. The CMA performed on PB samples collected during and after blast crisis revealed a large terminal region of copy-neutral LOH involving chromosome region 6p25.3p21.31, spanning approximately 35.9 Mb. The results of the CMA assay on sample collected after count recovery did not demonstrate LOH. CONCLUSIONS: LOH at the HLA gene locus may significantly influence the donor search resulting in mistakenly choosing homozygous donors. We recommend confirming the HLA typing of recipients with hematological malignancies when homozygosity is detected at any locus by using BS samples, or alternatively from PB when remission is achieved.

Class II Human Leukocyte Antigen Epitope Mismatch Predicts De Novo Donor-Specific Antibody Formation After Liver Transplantation.

Formation of de novo donor-specific antibodies (dn-DSAs) has been associated with longterm immunologic complications after liver transplantation (LT). We hypothesized that human leukocyte antigen (HLA) epitope/eplet mismatch (MM) is a marker of immunogenicity and a risk factor for dn-DSA formation. Sera from 80 LT recipients were prospectively screened for dn-DSA by a Luminex single-antigen test (One Lambda, Inc., Canoga Park, CA) at 1, 2, 3, 6, 12, 18, 24, and 36 months after LT. HLA typing of the recipients and donors was performed using polymerase chain reaction (PCR)-SSP and PCR-SSOP Luminex low-resolution methods (One Lambda, Inc.). The HLAMatchmaker computer algorithm was used for identification of MM eplets at HLA-DRB1 and -DQA1/B1 loci. Luminex single-antigen bead solid phase assay was used for antibody analysis. Standard immunosuppression included thymoglobulin-rituximab induction and tacrolimus maintenance. There were 27 (34%) patients who developed dn-DSA. There were no episodes of antibody-mediated rejection, and 9 (11%) developed acute cellular rejection (ACR). A positive crossmatch status and a higher number of HLA-A, -B, -DR, and -ABDR MMs were not associated with dn-DSA formation. Patients developing dn-DSA had a significantly higher number of total (38 ± 2.7 versus 28 ± 2.3; P = 0.01) and antibody-verified (AbVer; 14 ± 1.1 versus 10 ± 1; P = 0.015) class II MM eplets. By a multivariate regression analysis, the number of class II MM eplets was strongly associated with risk of class II dn-DSA formation (odds ratio [OR], 1.2; P < 0.01). Patients with ACR had a significantly higher number of total (20.2 ± 1.3 versus 13.9 ± 0.9; P < 0.01) as well as AbVer (10.7 ± 1.1 versus 7.5 ± 0.6; P = 0.03) class I MM eplets. In conclusion, donor-recipient HLA epitope MM is associated with a risk of dn-DSA formation and rejection after LT. However, further studies are required to evaluate the clinical utility of epitope matching in LT.

Rapid and strong de novo donor-specific antibody development in a lung transplant recipient: Short communication/case report.

A 66-yo female patient (typed B*39:01, 44:02) underwent first left single lung transplant (typed B*81:01, 15:17) on 02/07/2016 with negative for DSA in current and historical samples. On 02/17/2016 strong de novo DSA (MFI=15,200, C1q+) to B81 were detected. The recipient has two children typed B*07:02, 44:02 B*27:03, 39:01, and had received multiple vaccinations. Twinrix, Zostavax and MMR vaccines contain viruses grown on live human lung fibroblasts (MRC-5, typed B*07:02, 44:02, and WI-38, typed B*08:01, 58:01). Each dose of vaccine used for injection is known to contain protein components of fibroblasts including HLA. Most likely rapid de novo DSA development is due to booster effect produced by five exposures to mismatched B locus alleles which share the following epitopes: 70IAQ, 65QIA, 65QIA+76esn, 69aa+80n, and 163ew+73te. The later three consist of paired non-self and self eplets. Although likelihood of bystander effect produced by multiple vaccinations is low its impact cannot be ruled out.

Prospective Monitoring of Donor-specific Anti-HLA Antibodies After Intestine/Multivisceral Transplantation: Significance of De Novo Antibodies.

BACKGROUND: Presence of circulating donor-specific antibodies (DSA) may be associated with worse clinical outcomes after intestine/multivisceral transplantation. METHODS: In 79 intestine/multivisceral recipients, sera were prospectively screened for DSA by Luminex Single antigen test at 1, 3, 6, 9, 12, 18, 24, and 36 months after transplantation. Standard immunosuppression included thymoglobulin-rituximab induction and tacrolimus-prednisone maintenance. C4d staining was performed retrospectively on biopsies in patients that developed acute rejection (AR). RESULTS: Twenty-two (28%) patients developed de novo DSA at a median posttransplant period of 3 (1-36) months. De novo DSA were observed in 10 of 40 liver-including and 12 of 39 liver-excluding transplants (P = 0.57). Occurrence of AR was slightly higher in patients with de novo DSA (45% vs 33%, respectively; P = 0.41). Similarly, chronic rejection (14% vs 5%; P = 0.21) and graft loss due to AR (18% vs 7%; P = 0.14) were numerically higher in patients with de novo DSA. Only 35% patients experiencing AR had circulating de novo DSA at the time of AR. Antibody-mediated rejection was diagnosed in 6 patients based on C4d staining, of these 2 patients had circulating de novo DSA at the time of biopsy. CONCLUSIONS: De novo DSA formation, particularly early in the posttransplant course may be associated with trends toward worse outcomes. However, its significance in the pathophysiology of AR remains uncertain. Studies focusing mechanisms of DSA-related graft injury and intragraft DSA detection might provide further insight into this issue.

Meningeal relationships to the spinal nerves and rootlets: a gross, histological, and radiological study with application to intradural extramedullary spinal tumors.

INTRODUCTION: Juxtapositional tumors of the spinal nerve roots have been noted to not only interact with the roots at various vertebral levels, but also differ among patients. Therefore, the aim of the current study was to elucidate the potential for variation among the relationships of the meningeal layers at different nerve levels. METHODS: In 20 unembalmed adult cadavers and five fetal specimens, the spinal nerve roots from the cervical, thoracic, and lumbar regions were harvested with their associated meningeal layers and subjected to microdissection, histological analysis, or radiological imaging using 9.4-T MRI. RESULTS: As the nerve rootlets passed from the cord, they received their root sheath covering from the pia. After crossing the subarachnoid space to reach the apertures in the dura, they received two additional looser sheaths, an outer from the dura and an inner from the arachnoid. The pia mater always ended proximal to the arachnoid, and the pia and arachnoid layers extended more distally along the roots with caudal descent. Although the dorsal and ventral roots generally exited through separate dural openings, a single dural opening was also observed, often in the lower spinal regions. Thin intradural septations almost always separated the dorsal and ventral rootlets. The left and right sides frequently differed within individuals. CONCLUSIONS: On the basis of our study, variations of the meninges surrounding the spinal nerve roots are common, but themes do exist. Such data support surgical observations of the different interactions between tumors in these regions with surrounding neural tissues.

Methodological aspects of anti-human leukocyte antigen antibody analysis in solid organ transplantation.

Donor human leukocyte antigen (HLA)-specific antibodies (DSA) play an important role in solid organ transplantation. Preexisting IgG isotype DSA are considered a risk factor for antibody mediated rejection, graft failure or graft loss. The post-transplant development of DSA depends on multiple factors including immunogenicity of mismatched antigens, HLA class II typing of the recipient, cytokine gene polymorphisms, and cellular immunoregulatory mechanisms. De novo developed antibodies require special attention because not all DSA have equal clinical significance. Therefore, it is important for transplant clinicians and transplant immunologists to accurately characterize DSA. In this review, the contemporary immunological techniques for detection and characterization of anti-HLA antibodies and their pitfalls are described.

Crossmatch-positive liver transplantation in patients receiving thymoglobulin-rituximab induction.

BACKGROUND: Positive crossmatch (CM) in liver transplantation (LT) is associated with worse outcomes. Role of induction immunosuppression in this setting remains to be studied. METHODS: One thousand consecutive LT patients receiving rabbit antithymocyte globulin±rituximab induction were studied. Pretransplantation sera of 55 CM-positive (CM) patients were tested for C1q-fixing donor-specific antibodies (DSA). Diagnosis of antibody-mediated rejection required presence of diffuse vascular C4d expression on liver biopsies. RESULTS: CM was positive in 112 (11%) recipients. Antibody-mediated rejection was observed in 3 (0.03%) patients, whereas acute cellular rejection (ACR) occurred in 31 (3%) patients. CM status was associated with a higher incidence of ACR (9% in CM vs. 2% in CM-negative [CM]; P<0.01) and chronic rejection (4% in CM vs. 1% in CM; P<0.01). Graft survival was slightly lower in CM patients (at 1 year; 85% in CM vs. 89% in CM; P=0.26). Patients with autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis as a group had a tendency toward CM status as well as developing ACR. Upon multivariate analysis, CM status was the strongest predictor of ACR (B=1.14; P=0.02). Only half of CM patients harbored C1q-fixing DSA. Presence of C1q-fixing DSA was not associated with increased incidence of ACR. CONCLUSIONS: In LT, CM status is associated with an increased incidence of acute rejection, chronic rejection, and slightly worse graft survival. With the use of rabbit antithymocyte globulin±rituximab induction, overall low rejection rates can be achieved in CM LT.

The effectiveness of the combination of rituximab and high-dose immunoglobulin in the immunomodulation of sensitized kidney transplant candidates.

Kidney transplantation faces many challenges not the least of which is the presence of pre-formed HLA antibodies. At our institution, we have used a combination of methods to immunomodulate sensitized patients. Most recently, this has been attempted with a combination of immunoglobulin (IVIG) and rituximab (Rituxan; Genetech, CA, USA). A total of 31 patients were followed for up to one yr following treatment with IVIG (2 gm/kg on day 1 and day 30) and rituximab (1 g - day 15). Antibody levels were followed serially at designated time points via solid-phase single-antigen beads (SAB) method (One Lambda, Inc., Canoga Park, CA, USA). Concentration of antibodies was based on median fluorescence intensity (MFI). The majority of patients had both class I and class II antibodies (79%). Our results showed that this protocol appeared to be patient and antibody specific. The most pronounced MFI reduction in antibodies occurred within the 30- to 100-d period post-treatment. Calculated panel-reactive antibodies decreased but rebound tended to occur by 104 d after antibody MFI nadir. Because of this rebound, it can be inferred that the patients did not show a durable increase in their potential for transplantation. The search for a more effective method to immunomodulate patients continues.

Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab.

BACKGROUND: Preexisting donor-specific antibodies against human leukocyte antigens are major risk factors for acute antibody-mediated and chronic rejection of kidney transplant grafts. Immunomodulation (desensitization) protocols may reduce antibody concentration and improve the success of transplant. We investigated the effect of desensitization with intravenous immunoglobulin and rituximab on the antibody profile in highly sensitized kidney transplant candidates. METHODS: In 31 transplant candidates (calculated panel-reactive antibody [cPRA], 34%-99%), desensitization included intravenous immunoglobulin on days 0 and 30 and a single dose of rituximab on day 15. Anti-human leukocyte antigen antibodies were analyzed before and after desensitization. RESULTS: Reduction of cPRA from 25% to 50% was noted for anti-class I (5 patients, within 20-60 days) and anti-class II (3 patients, within 10-20 days) antibodies. After initial reduction of cPRA, the cPRA increased within 120 days. In 24 patients, decrease in mean fluorescence intensity of antibodies by more than 50% was noted at follow-up, but there was no reduction of cPRA. Rebound occurred in 65% patients for anti-class I antibodies at 350 days and anti-class II antibodies at 101 to 200 days. Probability of rebound effect was higher in patients with mean fluorescence intensity of more than 10,700 before desensitization, anti-class II antibodies, and history of previous transplant. CONCLUSIONS: The desensitization protocol had limited efficacy in highly sensitized kidney transplant candidate because of the short period with antibody reduction and high frequency of rebound effect.

Impact of positive flow cytometry crossmatch on outcomes of intestinal/multivisceral transplantation: role anti-IL-2 receptor antibody.

BACKGROUND: Positive crossmatch may be associated with an increased risk of acute rejection (AR) and worse overall outcomes after intestinal/multivisceral (MV) transplantation. However, the evidence from published studies in this setting is sparse and contradictory. This study reports the impact of positive flow cytometry crossmatch on clinical outcomes after intestinal/MV transplantation and the use of anti-interleukin (IL)-2 receptor antibody as a maintenance immunosuppressant. METHODS: Records of all intestinal/MV transplants from 2003 to 2010 were reviewed. Flow cytometry was used to evaluate T- and B-cell crossmatch status. Standard immunosuppression included rabbit anti-thymocyte globulin-rituximab induction with tacrolimus and steroid maintenance. From 2008 onwards (second era), monthly anti-IL-2 receptor antibody was added to the maintenance immunosuppression in patients receiving liver-excluding transplants. RESULTS: Of 131 intestinal/MV transplants, 27 (21%) had a positive crossmatch. Positive crossmatch was not associated with an increased incidence of AR and graft loss (30% and 37% vs. 29% and 47%; P=0.94 and 0.35, respectively). This effect was maintained in liver-excluding transplants. Overall rate of AR decreased from 39% to 22% in the second era. In liver-excluding transplants, there was a significant decrease in AR from 75% to 44% with the use of anti-IL-2 receptor antibody therapy. CONCLUSIONS: With rabbit anti-thymocyte globulin-rituximab induction, positive crossmatch status is not associated with worse outcomes after intestinal/MV transplantation. Use of anti-IL-2 receptor antibody as a part of maintenance immunosuppression may be beneficial in liver-excluding transplants.

Early findings of prospective anti-HLA donor specific antibodies monitoring study in pancreas transplantation: Indiana University Health Experience.

The significance of donor-specific antibodies (DSA) is not well known in the setting of pancreas transplantation. Since December 2009, we prospectively followed pancreas transplant patients with single-antigen-luminex-bead testing at one, two, three, six, and then every six months for the first two yr. Thirty-five of the 92 patients that underwent pancreas transplantation (13 pancreas-alone [PTA], 20 with a kidney [SPK], and two after a kidney [PAK]) agreed to participate in study. Median age at transplant was 45 yr and follow-up was 23 months. Majority were Caucasian (n = 33) and male (n = 18). Rabbit anti-thymocyte globulin induction was used. Median HLA-mismatch was 4.2 ± 1.1. Eight patients (7SPK, 1PAK) developed post-transplant DSA at median follow-up of 76 d (26-119), 1 SPK had pre-formed DSA. Seven patients had both class I and class II DSA, one with class I and one with class II only. Mean peak class I DSA-MFI was 3529 (±1456); class II DSA-MFI was 5734 (±3204) whereas cumulative DSA MFI (CI + CII) was 9264 (±4233). No difference was observed in the patient and donor demographics among patients with and without DSA. One patient in non-DSA group developed acute cellular rejection of pancreas. From our data it appears that post-transplant DSA in pancreas allograft recipients may not impact the early-pancreatic allograft outcomes. The utility of prospective DSA monitoring in pancreatic transplant patients needs further evaluation and long-term follow-up.

Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site.

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.

The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients.

The aim of this study was to evaluate the utility of donor-specific antibodies (DSA) and flow cytometry crossmatch (FCCM) as tools for predicting antibody-mediated rejection (AMR) in desensitized kidney recipients. Sera from 44 patients with DSA at the time of transplant were reviewed. Strength of DSA was determined by single antigen Luminex bead assay and expressed as mean fluorescence intensity (MFI). T- and B-cell FCCM results were expressed as mean channel shift (MCS). AMR was diagnosed by C4d deposition on biopsy. Incidence of early AMR was 31%. Significant differences in the number of DSAs (p = 0.0002), cumulative median MFI in DSA class I (p = 0.0004), and total (class I + class II) DSA (p < 0.0001) were found in patients with and without AMR. No significant difference was seen in MCS of T and B FCCM (p = 0.095 and p = 0.307, respectively). The three-yr graft survival in desensitized patients with DSA having total MFI < 9500 was 100% compared to 76% with those having total MFI > 9500 (p = 0.022). Desensitized kidney transplant recipients having higher levels of class I and total DSA MFI are at high risk for AMR and poor graft survival. Recipient DSA MFI appears to be a more reliable predictor of AMR than MCS of FCCM.

Early steroid withdrawal in repeat kidney transplantation.

BACKGROUND AND OBJECTIVES: Kidney re-transplantation (KRT) candidates are considered at high risk for graft failure. Most of these patients are kept on a chronic steroid maintenance (CSM) regimen. The safety of early steroid withdrawal (ESW) remains unanswered in KRT. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study was aimed at comparing the outcomes of ESW and CSM in KRT. Retrospective analysis of 113 KRT patients (ESW, n=59; CSM, n=54) was performed. All patients received rabbit anti-thymocyte globulin/steroid induction and were maintained on mycophenolate/tacrolimus (±steroids). RESULTS: One- and 5-year patient survival for the ESW and the CSM group were not significantly different (98 versus 96% and 91 versus 88%, respectively; P=0.991). No significant difference was seen in the graft survival for both groups at 1 and 5 years (98 versus 93% and 80 versus 74%, respectively; P=0.779). Mean 1- and 5-year estimated GFR was not statistically different between the groups (P=0.773 and 0.790, respectively). The incidence of acute rejection at 1 year was 17 and 22% in ESW and CSM patients, respectively (P=0.635). Compared with the ESW group, patients in the CSM group were more likely to be hyperlipidemic (P=0.044), osteoporotic (P=0.010), post-transplant diabetics (P=0.051) and required more medications to control BP (P=0.004). CONCLUSIONS: ESW seems to be a reasonable approach in KRT recipients because the short and intermediate patient survival, graft survival, and graft function is comparable to CSM immunosuppression.

Tolerance induced by anti-CD3 immunotoxin plus 15-deoxyspergualin associates with donor-specific indirect pathway unresponsiveness.

Peritransplant treatment with anti-CD3 immunotoxin plus deoxyspergualin induces tolerance to kidney allografts in most rhesus macaque recipients. Tolerant recipients maintain normal function for years without evidence of chronic rejection. Indirect alloantigen presentation is implicated in chronic rejection. Accordingly, we determined if anti-CD3 immunotoxin plus deoxyspergualin induced rejection-free tolerance associates with suppression of anti-donor indirect pathway responses. Tolerant recipients exhibited an early decrease in direct anti-donor responses with recovery to baseline levels by 3 years posttransplantation. In contrast, tolerant monkeys were unresponsive to donor antigens presented by the indirect pathway. Recipients that rejected their allografts retained vigorous direct and indirect anti-donor responses. Therefore, following temporary donor-specific hyporesponsiveness, direct responses recover in tolerant recipients >1.5 years after transplantation. However, tolerant recipients tested at 1.9-4 years posttransplant are specifically unresponsive to donor antigens presented by the indirect pathway. Thus, the rejection-free state of tolerant recipients may depend on mechanisms regulating indirect pathway responsiveness.

Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient management.

Each year, 55 000 organ transplants are performed worldwide. Cumulatively, the number of living organ recipients is now estimated to be over 300 000. Most of these transplant recipients will remain on immunosuppressive drugs for the remainder of their lives to prevent rejection episodes. Controlled doses of these drugs are required to prevent over-medication, which may leave the patient susceptible to opportunistic infection and drug toxicity effects, or under-dosing, which may lead to shortened graft survival because of rejection episodes. This paper describes the result of a multicenter study conducted at the Universities of Pittsburgh, Alabama and Maryland to evaluate an in vitro assay (CylexTM Immune Cell Function Assay) for the measurement of global immune response in transplant patients receiving immunosuppressive therapy. The assay uses a whole blood sample to maintain the presence of the drug during incubation. Following overnight incubation of blood with phytohemagglutinin (PHA), CD4 cells are selected using paramagnetic particles coated with a monoclonal antibody to the CD4 epitope. The CD4-positive cells are targeted as major immunosuppressive drugs are designed to specifically inhibit T-cell activation which has been implicated in rejection. The data generated at these three sites were submitted in support of an Food and Drug Association (FDA) application for the use of this assay in the detection of cell-mediated immunity in an immunosuppressed population. The assay was cleared by the FDA on April 2, 2002. This cross-sectional study was designed to establish ranges for reactivity of this bioassay in the assessment of functional immunity for an individual solid organ recipient at any point in time.

Stable alpha- and beta-islet cell function after tolerance induction to pancreatic islet allografts in diabetic primates.

Pancreatic islet transplantation (PIT) is an attractive alternative for type 1 diabetic patients. PIT is not yet an effective clinical reality due in part to early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. Precise and durable alpha- and beta-cell function is essential for the success of PIT. Therefore, it is important to establish whether PIT can produce adequate long-term metabolic control, especially in the absence of chronic immunosuppressive therapy (CIT). In the present study, the stability of functional alpha- and beta-cell mass and metabolic function was assessed in streptozotocin (STZ)-induced diabetic primates following PIT in the absence of CIT. Diabetes was induced in rhesus macaques with STZ, 140 mg/kg. Hyperglycemia was reversed rapidly by PIT coupled with a 14-day tolerance induction protocol based on F(Ab)2-IT and DSG (n = 7). Two diabetic animals received the tolerance induction protocol without PIT. Acute rejection was presented in three animals at 70, 353 and 353 days post transplant in the tolerance induction protocol, whereas the controls [F(Ab)2-IT or DSG alone] showed early 10-day function but all lost islet function by days 15-70. One recipient [F(Ab)2-IT or DSG] died euglycemic after a surgical procedure on day 187. At 2 years, three animals studied had a normal FIM evaluated by oral glucose tolerance test, mixed meal test, acute insulin response to glucose, glucose disposal rate, and hyperinsulinemic hypoglycemic clamp. PIT in STZ-induced diabetic primates resulted in restoration of normal alpha- and beta-cell function. Operational tolerance induction was achieved with only peritransplant administration of F(Ab)2-IT and DSG sparing the animals from chronic exposure of diabetogenic immunosuppressive drugs. These results offer an exciting new potential for treatment of type 1 diabetes mellitus.