Search

Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts.

We have analyzed the impact of anti-HLA antibodies present in the patients' circulation prior and/or following heart transplantation in a population of 108 pediatric recipients. Anti-HLA class I and class II antibodies were monitored by traditional CDC using donor and panel T and B lymphocytes and by SPA for detection of DSA. There was a highly significant correlation between the development of AMR and presence of CDC- or SPA-detected DSA. However, the fraction of the transplant population which remained AMR-free was much higher among patients with SPA-detected compared to CDC-detected DSA. Furthermore, long-term graft survival was negatively affected only by cytotoxic, complement-fixing anti-HLA class I antibodies developing following transplantation. Anti-HLA class I or class II antibodies detected by SPA had no effect on long-term survival rates.

The involvement of humoral response in allograft rejection has been suggested by both immunologic and histochemistry studies. In the present study, we explored the role of alloantibodies in a large cohort of heart allograft recipients followed for 15 years. Sequential samples of sera were obtained from 950 recipients of heart allografts before and after transplantation at the time when protocol endomyocardial biopsies were performed. The presence of anti–human leukocyte antigen (HLA) antibodies was investigated using complement mediated cytotoxicity and solid phase assay (SPA). Our data reveal an inverse correlation between the development of alloantibodies after transplantation and heart allograft survival. The 15-year graft survival was highest in patients who never developed alloantibodies (70%) or who displayed them only before transplantation (71%); graft survival in recipients who showed antibodies both before and after transplantation (56%), or only after transplantation (47%), was lower. The deleterious effect of antibodies on graft survival started 8 years after transplantation, suggesting that the production of de novo antibodies may have been triggered by some later event. We found that patients with de novo antibodies appearing more than 1 year after transplantation had the poorest survival. Furthermore, the development of de novo antibodies was preceded in 76% of these patients by cellular rejection grade 3 or higher, according to the International Society for Heart Transplantation (ISHT) grading criteria. Development of antibody-mediated rejection (AMR) had a significant negative impact on graft survival (16% in AMR + vs 63% in AMR – patients, p = 0.0008). Of the 23 patients with AMR, 21 displayed cytotoxic donor-specific antibodies (DSA) at the time of diagnosis, and in 18 of these cases SPA showed that they were directed against the donors' HLA. The data demonstrate that the detection of alloantibodies permits a better definition of AMR in heart allograft recipients. Identification of patients at risk for developing AMR is of great importance for early treatment of rejection episodes.

Ig-like transcript 3 (ILT3) is an inhibitory receptor expressed by tolerogenic dendritic cells. When human CD8+ T cells are allostimulated in the presence of recombinant ILT3-Fc protein, they differentiate into antigenic specific T suppressor (Ts) cells that inhibit CD4 and CD8 T cell effector function both in vitro and in vivo. ILT3-Fc–induced CD8+ Ts cells express high amounts of BCL6 that are crucial to their function. Knockdown of BCL6 from unprimed human T cells prevents their differentiation into Ts cells, whereas ex vivo overexpression of BCL6 converts CD8+ T cells into Ts cells. NOD/SCID mice transplanted with human pancreatic islets and humanized by injection of human PBMCs tolerate the graft and develop BCL6high CD8+ Ts cells when treated with ILT3-Fc before or after the onset of rejection. This indicates that ILT3-Fc acts through BCL6 and is a potent immunosuppressive agent for reversing the onset of allo- or possibly autoimmune attacks against pancreatic islets.

OBJECTIVE—The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action.RESEARCH DESIGN AND METHODS—NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L.RESULTS—Although human T-cell engraftment was similar in all groups, ILT3-Fc–treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks (P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8+ T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc–treated mice and control recipients rejecting the grafts.CONCLUSIONS—The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.

The interleukin-2 receptor alpha chain (IL-2Ra, CD25) plays a major part in shaping the dynamics of T cell populations following immune activation, due to its role in T cell proliferation and survival. Strategies to blunt the effector responses in transplantation have been developed by devising pharmaceutical agents to block the IL-2 pathways. However, such strategies could adversely affect the CD25(+)FOXP3(+)T regulatory (T reg) populations which also rely on intereukin-2 signaling for survival. The present study shows that a cohort of heart allograft recipients treated with Daclizumab (a humanized anti-CD25 antibody) display FOXP3 expression patterns consistent with functional T regulatory cell populations. High levels of FOXP3 were observed to correlate with lower incidence of and recovery from acute rejection, as well as lower levels of anti-donor HLA antibody production. Therefore, T reg populations appear fully functional in patients treated with Daclizumab, even when 5 doses were administered. By comparison, patients treated with fewer doses or no Daclizumab had a higher incidence of acute rejection, antibody production and graft failure. Therefore, our data indicates that Daclizumab treatment does not interfere with the generation of regulatory T cells and has a beneficial effect on heart allograft survival.

The role of humoral immunity in causing antibody-mediated rejection (AMR) of organ allografts has been extensively documented. For this reason, negative complement-dependent cytotoxicity (CDC) cross-matches between recipient sera and donor T and B lymphocytes have become a mandatory requirement for cadaveric kidney transplantation. However, the significance of donor-specific antibodies (DSAs) detectable only by flow cytometry (FC) or solid phase assays (SPA) but not CDC is still controversial. We have performed a retrospective analysis of FC cross-matching results in 80 consecutive cadaver kidney allograft recipients. Antibodies against HLA class I and class II antigens were measured by CDC and SPA in sequential samples of sera obtained prior to transplantation. The preoperative cross-match was performed by CDC using magnetically sorted T and B cells from donor spleen. Sera obtained from each patient before and at the time of transplantation were included in the final cross-match. The sample of serum obtained at the time of transplantation was cross-matched retrospectively by FC and analyzed for anti-HLA antibody specificity on high resolution SPA. The actuarial kidney allograft survival at one year was 98%. Two of these eighty patients lost the graft, one due to AMR, the other for reasons unrelated to DSAs. Donor-specific antibodies were detected by FC in 17 of 80 patients, yet only 6 of 17 had an early episode of AMR. This episode was successfully reversed by desensitization therapy using intravenous immunoglobin (IVIG) and plasmapheresis. Flow cytomery cross-matching showed 95% specificity but only 35% sensitivity for prediction of AMR (p = 0.002). There was a significant correlation between high panel reactive antibodies (PRA) and positive FC cross-matching (p = 0 .0001), as well as high PRA and AMR (p = 0.0004 by CDC and 0.0011 by Luminex). Reversible AMR occurred 12-30 days post-transplantation in 8 patients. Of these 8 patients, 3 had no detectable DSAs in spite of C4d positivity, 4 had C4d deposition in conjunction with anti-HLA antibodies, and 1 patient had DSAs (anti-MICA) yet no C4d deposition. We conclude that early initiation of desensitization protocols can prevent transplant failure and that retrospective FC cross-matches may facilitate the diagnosis of AMR. Extensive analysis of patients' sera using a comprehensive set of tests may contribute to early treatment and better understanding of the mechanism underlying humoral rejection.

Although transplantation tolerance cannot be yet reliably achieved in humans, there is evidence that active immunosuppression contributes to the maintenance of quiescence. However, the mechanism that underlies quiescence and the precise identity of regulatory cells are not completely understood. We have demonstrated that allograft recipients who remain rejection-free display allospecific T-suppressor cells (Ts). Ts express the CD8 + CD28 – phenotype, recognize major histocompatibility complex (MHC) class I antigens, and suppress the up-regulation of costimulatory molecules induced by CD40 ligation of donor antigen presenting cells. The presence of Ts is inversely correlated with T cell alloreactivity to donor MHC peptides, alloantibody production, and rejection. Monitoring of Ts has been successfully used in our studies for tailoring immunosuppression in kidney and liver allograft recipients.

Dendritic cells are crucial to the activation as well as suppression of the immune response. Previous reports have illustrated that APC interacting with antigen-specific T suppressor cells become tolerogenic, inducing T helper anergy. To characterize the molecular changes occurring in tolerogenic APC, the mRNA profile of KG-1 dendritic cells exposed to allospecific T helper and T suppressor cells were analyzed. This study now provides evidence that immature dendritic cells stimulated by T suppressor cells differentiate into mature dendritic cells with a distinct phenotype. The identification of Ts induced pathways of dendritic cell differentiation is critical to the development of new therapeutic strategies.

The role of the indirect allorecognition pathway in acute allograft rejection has been documented both in organ recipients and in experimental models. However, it is unknown whether self-restricted recognition of donor alloantigens also contributes to chronic allograft rejection. The aim of this study was to determine the relationship between allopeptide reactivity, epitope spreading, and chronic rejection. Using synthetic peptides corresponding to the hypervariable region of 32 HLA-DR alleles, we have followed the specificity of self-restricted T cell alloresponses to the donor in a population of 34 heart allograft recipients. T cells from sequential samples of blood collected from the patients up to 36 mo after transplantation were studied in limiting dilution analysis for allopeptide reactivity. The incidence of coronary artery vasculopathy (CAV) was significantly higher in patients who displayed persistent alloreactivity late after transplantation than in patients who showed no alloreactivity after the first 6 mo after transplantation. Both intra- and intermolecular spreading of epitopes was observed with an increased frequency in patients developing CAV in less than 2 yr, compared with patients without CAV; this suggests that diversification of the immune response against the graft contributes to chronic rejection. These data provide a strategy for identifying patients at risk of developing CAV and a rationale for therapeutic intervention aimed to prevent the progression of the rejection process.

Transplant rejection is mediated by the direct and indirect pathways. To explore the role of the indirect recognition pathway in the rejection of liver allografts, T cells obtained from peripheral blood were expanded in medium containing IL-2 and tested in LDA for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial investigations of 17 recipients showed that T-cell reactivity to donor HLA-DR peptides was strongly associated with acute rejection episodes. In recipients carrying a graft that was mismatched by two HLA-DR alleles, a single donor antigen was targeted during primary rejection, although allopeptide reactivity against the second HLA-DR antigen was observed during subsequent episodes of acute rejection. The finding that allopeptide reactivity occurs early following transplantation and is predictive of rejection is consistent with the notion that processing of donor alloantigens by recipient APCs activates the indirect T-cell recognition pathway that plays a major role in initiating and amplifying allograft rejection.

Autonomous Hamiltonian systems with a homoclinic orbit connecting a saddle point to itself are analyzed under small symmetric perturbations, which correspond to dissipative perturbations for conservative systems. Since the differential equations are autonomous, phase plane variables are used. The usual asymptotic expansion of a nearly homoclinic orbit is shown to fail as the surrounding saddle regions are approached. A non-linear analysis of the surrounding saddle regions is derived. By matching (to sufficiently high order) the asymptotic expansion of the nearly homoclinic orbit to the solution in the surrounding saddle regions, we obtain the change in the Hamiltonian from one saddle approach to the next. In this way, we derive a new higher-order logarithmic correction to the well-known Melnikov integral for the leading order change of the Hamiltonian.

To identify patients with increased risk of chronic lung allograft rejection, we assessed the utility of an in vitro biopsy-derived lymphocyte growth assay and serum anti-HLA antibody screening as a complement to currently available methods of monitoring lung allograft recipients. Lymphocyte growth assay was performed on bronchoscopic fragments of tissue cultured in medium with rIL-2. Seventy-nine biopsies from 31 lung transplant recipients were tested by lymphocyte growth assay, and results were correlated with histopathology findings. Positive lymphocyte growth was found in 12/26 (46%) episodes of acute rejection, 5/44 biopsies without rejection (11%), and 0/9 episodes of bronchitis. Positive lymphocyte growth was seen in 7/16 (44%) grade A1 rejections and in 5/10 (50%) grade A2 rejections, as opposed to only 5/44 (11%) grade A0 (no rejection) biopsies (P < 0.01 for both A1 and A2 with respect to A0). Actuarial probability of remaining free from obliterative bronchiolitis (OB)* tended to be higher in patients who did not exhibit lymphocyte growth in biopsies. Sequential samples of sera obtained at the time of the biopsy were screened for lymphocytotoxic anti-HLA antibodies. Twenty-two of 44 recipients (50%) developed anti-HLA antibodies during the first postoperative year, exhibiting greater than 10% reactivity to an HLA reference panel of lymphocytes in four or more consecutive serum samples. Actuarial survival of lung allograft recipients with anti-HLA antibodies (n = 22) was lower than in those without anti-HLA antibodies (n = 22; P = 0.03). Of the 22 antibody producers, 7/12 died as a consequence of OB. Of the 22 non-antibody-producers, 1/2 deaths occurred as a consequence of OB. Anti-HLA antibodies were present in 9/11 instances of OB (82% sensitivity) and in 13/33 patients without OB (61% specificity; P = 0.03). These data indicate that lung transplant recipients with positive lymphocyte growth and anti-HLA antibodies are at an increased risk of chronic allograft rejection.

Introduction Acute rejection currently accounts for 11% of the deaths of allograft recipients within the first six months of transplantation. Though endomyocardial biopsy is the most common method of detecting rejection, the procedure is highly invasive and expensive. MicroRNAs are RNA sequences that regulate gene expression and immune function and can be detected through tests of sera in blood. Previous studies have identified a correlation between microRNAs and acute rejection. This study hopes to address the usage of certain microRNAs as possible non-invasive biomarkers of acute cardiac cellular rejection. Methods We measured the expression of six different microRNAs (miR-146a, miR-155, miR-21, miR-30b, miR-30c, and miR-9) in 4 patients with rejection and in 16 healthy individuals. Using a Student’s t -test, miR-21 demonstrated a statistically significant difference in expression and was selected to test for expression in 12 heart allograft patients who did and did not experience acute cellular rejection, as defined by an ISHLT endomyocardial biopsy grade of 2R/3A on the day of serum collection. Chi-square analysis was used to compare expression levels between the two groups of patients and determine the accuracy of miR-21 levels in distinguishing between rejection and quiescence. Results Analysis of microRNA-21 levels showed significant up-regulation in patients with acute cellular rejection compared to patients without rejection. Patients with a minimum of 40% up-regulation were considered to be predictive for rejection. Rejection was predicted with 80% sensitivity, 100% specificity, a positive predictive value of 100% and negative predictive value of 98%, demonstrating high accuracy in determining rejection (P = 0.0001). Conclusions The study has shown that differential expression of microRNA-21 occurs in heart allograft patients experiencing rejection. Although further research will be done to determine the exact mechanism through which miR-21 affects rejection, it shows great promise as a non-invasive biomarker of cardiac transplant rejection.

Hamiltonian systems are analyzed with a double homoclinic orbit connecting a saddle to itself. Competing centers exist. A small dissipative perturbation causes the stable and unstable manifolds of the saddle point to break apart. The stable manifolds of the saddle point are the boundaries of the basin of attraction for the competing attractors. With small dissipation, the boundaries of the basins of attraction are known to be tightly wound and spiral-like. Small changes in the initial condition can alter the equilibrium to which the solution is attracted. Near the unperturbed homoclinic orbit, the boundary of the basin of attraction consists of a large sequence of nearly homoclinic orbits surrounded by close approaches to the saddle point. The slow passage through an unperturbed homoclinic orbit (separatrix) is determined by the change in the value of the Hamiltonian from one saddle approach to the next. The probability of capture can be asymptotically approximated using this change in the Hamiltonian. The well-known leading-order change of the Hamiltonian from one saddle approach to the next is due to the effect of the perturbation on the homoclinic orbit. A logarithmic correction to this change of the Hamiltonian is shown to be due to the effect of the perturbation on the saddle point itself. It is shown that the probability of capture can be significantly altered from the well-known leading-order probability for Hamiltonian systems with double homoclinic orbits of the twisted type, an example of which is the Hamiltonian system corresponding to primary resonance. Numerical integration of the perturbed Hamiltonian system is used to verify the accuracy of the analytic formulas for the change in the Hamiltonian from one saddle approach to the next. (c) 1995 American Institute of Physics.

Conservative dynamical systems with a homoclinic orbit are analyzed under a small dissipative perturbation. The asymptotic expansions of a nearly ho- moclinic orbit are shown to fail as the surrounding saddle regions are approached. A weakly nonlinear analysis of the surrounding saddle regions is derived assuming the dissipation can be approximated by linear damping in the neighborhood of the saddle point. By matching (to sufficiently high order) the asymptotic expansion of the nearly homoclinic orbit to the solutions in the surrounding saddle regions, the change in the Hamiltonian (the energy dissipation) over a complete nearly homoclinic orbit is obtained. We show there is a new higher-order logarithmic correction to the well-known Melnikov integral for the change of the Hamiltonian from one saddle approach to the next. Numerical computations of this change in the Hamiltonian for a double-well potential are shown to compare favorably with the asymptotic result.

We have analyzed the impact of anti-HLA antibodies present in the patients' circulation prior and/or following heart transplantation in a population of 108 pediatric recipients. Anti-HLA class I and class II antibodies were monitored by traditional CDC using donor and panel T and B lymphocytes and by SPA for detection of DSA. There was a highly significant correlation between the development of AMR and presence of CDC- or SPA-detected DSA. However, the fraction of the transplant population which remained AMR-free was much higher among patients with SPA-detected compared to CDC-detected DSA. Furthermore, long-term graft survival was negatively affected only by cytotoxic, complement-fixing anti-HLA class I antibodies developing following transplantation. Anti-HLA class I or class II antibodies detected by SPA had no effect on long-term survival rates.

Chronic rejection is the major threat to both heart and renal allograft survival. We have explored the possibility that some patients with anti-donor HLA antibodies (Ab1) develop specific anti-idiotypic antibodies (Ab2) which suppress the production of Ab1, and subsequently, the progression of chronic rejection. Analysis of Ab2 in sera obtained from Ab1 producers showed that 22% of heart and 18% of kidney recipients produced Ab2. The 4- and 5-year actuarial graft survivals in Ab2 producers were 100% and 83%, respectively, compared to 57% in patients who formed Ab1 but not Ab2 (p < 0.004). Patients carrying the DR2 alleles, DRB1*1501, *1502 or *1601 were at a lower risk of producing anti-donor HLA antibodies.

We have investigated the HLA-class I and class II polymorphism in a population of 83 Romanians using conventional serology together with PCR amplification and oligonucleotide typing of HLA-class II genes. Romanians show a higher frequency of HLA-A11, B13, B18, B37, B39, B51 and DR2 than other European populations. HLA-DRB1*1501 and 1601 account for the high frequency of the serologic specificity DR2. In Romanians, HLA-DR2 is in linkage disequilibrium with HLA-B18 and HLA-Bw52 rather than with HLA-B7 as is the case in other Europeans. Unexpected HLA-DR2 haplotypes include HLA-DRB1*1502, DQA1*0102, DQB1*0601; HLA-DRB1*1602, DQA1*0102, DQB1*0502. Other unusual haplotypes include HLA-DRB1*0405, DQA1*03, DQB1*0302; HLA-DRB1*1305, DQA1*0103, DQB1*0603; and HLA-DRB1*1405, DQA1*0101, DQB1*05032. Analysis of the genetic distance between Romanians and other Europeans who have been studied serologically are consistent with the hypothesis that Romanians descend from Roman ancestors who colonized Dacia between the 1st century B.C. and 1st century A.D.

The presence of complement fixing anti-human leukocyte antigen (HLA) antibodies in the circulation of organ transplant recipients may result in heart allograft rejection. Here, we assessed the clinical impact of pre- and post-transplantation allosensitization on long-term survival of heart allografts. Sequential samples of sera from heart allograft recipients were screened pretransplantation for panel reactive antibodies using the complement-dependent cytotoxicity test. Patients were monitored post-transplantation for donor specific anti-HLA class I and class II antibodies. Kaplan-Meier graft survival plots were generated to analyze the effect of anti-HLA antibodies on transplantation outcomes. Statistical analysis showed that the post-transplantation development of alloantibodies was a significant risk factor that was associated with low long-term survival rates; in contrast, recipients' gender, age, previous transplantations, and degree of HLA matching with the donor had no effect on long-term survival. The presence in pretransplantation sera of antibodies against more than 10% of the HLA reference panel (PRA >10%) was associated with AMR and with a relatively lower rate of graft survival after 1 year but did not affect 10-year survival. The present data underline the importance of monitoring the development of anti-HLA antibodies as a tool for early diagnosis and treatment of AMR.

Presensitizing alloantibodies may represent a grave danger in organ transplantation, increasing the risk of antibody-mediated rejection (AMR) and graft loss. However, not all antibodies are harmful to the graft. In our study of a cohort of 325 deceased-donor renal allograft recipients, the patients were determined eligible to receive an allograft based on a negative complement-dependent cytotoxicity (CDC) crossmatch (XM). Yet at the time of transplantation, many candidates displayed donor-specific antibodies (DSA) by more sensitive methods, such as solid-phase assays (SPA, Luminex) or flow cytometry crossmatch (FCXM). The majority of the patients who were DSA positive by either SPA (67%) or FCXM (66%) presented an AMR-free clinical course posttransplantation. Among the patients who developed AMR (N = 29), 76% proved clinically manageable and did not lose the graft. Analysis of the DSA mean fluorescence intensities (MFI) of Luminex indicated no statistically significant difference between patients who experienced AMR episodes and those who did not. Importantly, many of the patients with AMR did not test positive for DSA by SPA (20/29) or FCXM (14/29). Despite false-positive and false-negative results, the detection of DSA by SPA or FCXM was positively associated with AMR, but not with actuarial graft survival. The field of organ transplantation has always struggled to reconcile two opposing goals: improving transplantation outcome while increasing access to transplantation. SPA and FCXM appear to be oversensitive and defining patients as "sensitized" according to these methods would block access to transplantation for many candidates who would otherwise benefit greatly from receiving the allograft. Nevertheless, SPA and FCXM are invaluable tools, assisting clinicians in gauging AMR risk and tailoring immunosuppression of the posttransplantation immunological monitoring accordingly.

The major threat to long-term survival of heart allograft recipients is the development of graft atherosclerosis, which seems to be a manifestation of chronic rejection. To assess the role of anti-HLA antibodies in heart allograft rejection we studied 107 patients and compared the survival of recipients who formed anti-HLA antibodies with the survival of recipients who developed no antibodies. At 4 years the actuarial survival was 90% in the nonproducer group and 38% in antibody-producers (P = 0.038). We further explored the possibility that HLA antigens from the injured graft are released into the circulation and can be found in the serum either free or complexed with anti-HLA antibodies. This hypothesis was confirmed by the finding that the frequency of sera containing soluble HLA antigens from the graft or immune complexes of HLA alloantigens with anti-HLA antibodies was significantly higher in patients who rejected compared with patients with successful heart allografts (P less than 0.05). Following depletion of soluble HLA antigens, anti-HLA antibodies became detectable in 53% and 74% sera obtained during the first and second year posttransplantation, respectively, from patients undergoing chronic rejection. Long-term survivors showed a significantly lower (P less than 0.001) frequency of anti-HLA antibodies in sera depleted of HLA antigens. Lastly, studies of anti-anti-HLA-A2 and A3 antibodies in recipient sera suggest that quiescence is maintained by antiidiotypic antibodies.

To assess the significance of antibodies detected by complement-dependent cytotoxicity (CDC), solid phase (SPA) and flow cytometry (FC) assays we compared their predictive value in 354 consecutive cases of deceased-donor kidney transplantation. Pre-transplantation screening of anti-HLA class I and class II antibodies was performed by CDC and SPA. The direct crossmatch between recipients' sera and donors' T and B cells was performed by CDC followed by FC and SPA ("virtual cross-match"). The past history of antibodies displayed by the recipient was not considered a contraindication for transplantation even when it showed DSA. A side-by-side comparison of the correlation between graft loss, history of DSA and cross-match results indicated that sensitivity was 5%, 16% and 17% while specificity was 99%, 93% and 86% in CDC, SPA, FC crossmatches respectively. There was no significant difference between the 3 year survival of primary and secondary kidney allografts. We conclude that screening and cross-matching the sera by CDC provides reliable results and optimizes the patient's chances to receive a transplant. SPA and FC, however, are of great importance for identifying patients which require close monitoring by biopsy and serology for early diagnosis and treatment of acute antibody mediated rejection (AAMR).

Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor molecule expressed by dendritic cells, monocytes, and endothelial cells. Upon upregulation of ILT3 expression, antigen presenting cells (APCs) become tolerogenic, triggering the differentiation of antigen-specific CD8(+) and CD4(+) regulatory T cells. To analyze the polymorphism of ILT3, we screened DNA from a panel of 150 healthy subjects for single nucleotide polymorphisms (SNPs) within genomic region encoding the extracellular domain (exons 1-8). Here we report the identification of 15 SNPs, including nine nonsynonymous, three synonymous base-pair substitutions, and three intronic, including one deletion polymorphism within 3.6 kb of the ILT3 genomic region. Analysis of three physically linked SNP in healthy individuals indicates that c.356-41-46del, a 6-base-pair (bp) deletion located in intron 3/4, is predominantly associated with c.678A allele, a nonsynonymous SNP located in exon 5. Linkage studies in five nuclear families showed that these two minor alleles co-segregate. Our results demonstrate that ILT3 is highly polymorphic and may be associated with susceptibility to immune disorders.

Aim Pre-sensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allograft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. In this study, our aim is to determine whether kidney deceased donor recipients with preformed DSA detectable in serum obtained prior to transplant are at an increased risk of graft failure. Methods We studied 640 patients who received a kidney transplant from a deceased donor between January 1, 2009 and December 31, 2014 at Columbia University Medical Center. Sera collected from the recipients prior to transplantation were tested for DSA by CDC and SPA with single antigen coated beads. All patients were transplanted with negative CDC crossmatch. Results We analyzed the actuarial graft survival in patients with a primary allograft ( n = 525) and in patients with a secondary allograft ( n = 115). Kidney allograft survival was significantly higher in primary allograft recipients (85%) than in secondary allograft recipients (71%) at 6 years. ( P = 0.02) Forty-six out of 525 (8.7%) primary kidney allograft recipients and sixty-two out of 115 (53.9%) secondary kidney allograft recipients had SPA-detectable DSA in sera collected prior to transplantation. Six years following transplantation of primary kidney allografts the actuarial graft survival was 83% and 85%, respectively, regardless of whether DSA were detectable in recipients’ pre-transplantation sera. ( P = 0.39) In recipients of secondary kidney allografts with and without SPA-detectable DSA, the actuarial graft survival was 67% and 77% at 6 years, respectively. ( P = 0.12) Conclusions The presence of SPA-detectable DSA in recipients of kidney allografts from deceased donors is associated with lower long term (6 year) actuarial graft survival although the difference does not reach statistical significance.

Spleen is surgically removed for both non-neoplastic and neoplastic pathologies. A significant proportion of splenectomy specimens require distinguishing between reactive and neoplastic conditions (eg lymphoma). To establish a 'normal' reference range for the spleen lymphocyte subsets, fresh samples of benign, reactive spleens obtained from adult patients (N=12) and samples of normal spleen obtained from cadaveric transplant donors (N=14) were analyzed using three- and four-color flow cytometry. Study of pan-B, -T, and -NK marker expression revealed that the frequency of T cells is higher and that of B cells is lower in reactive (non-neoplastic) compared to normal (cadaveric) spleen. Furthermore, our study established a frame of reference for cell markers commonly used for immunophenotyping of lymphoma, and identified discrete lymphocyte subsets, such as early plasma cells and T cells carrying the phenotype of the NK/T subset. These results will facilitate an accurate interpretation of the flow cytometric analysis of human spleen lymphocytes.

We have analyzed the relationship between the development of transplant-related coronary artery disease (TRCAD) and the following potential risk factors: (a). number of HLA mismatches between recipient and donor; (b). production of anti-HLA antibodies; (c). growth of lymphocytes infiltrating the graft; and (d). frequency of biopsy proven episodes of acute rejection. The study population consisted of 285 adult heart allograft recipients who were monitored over a period of two years or more. The results demonstrate a significant correlation between TRCAD, generation of anti-HLA class II antibodies and potential of lymphocytes infiltrating the graft to proliferate ex-vivo in medium containing IL-2. Humoral and cellular immune responses to HLA-DR antigens expressed by the graft seem to underlie the development of TRCAD.

Human regulatory CD8+CD28− T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific, and results in Th anergy. To study the population of CD8+CD28− T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients and 12 normal controls. A significant expansion of CD8+CD28− T cells was found in transplant recipients as compared with normal individuals ( p = 0.005). Expression of CD38, human leukocyte antigen-DR, and perforin positive cells within the CD8+CD28− subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28− T cells from patients without rejection as compared with patients in rejection ( p = 0.005), indicating that the memory-like CD8+CD28−CD27+ T-cell subset comprises regulatory cells, which play a protective role for the graft. CD8+CD28− T cells isolated from transplant patients did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes that occur in patients with heart transplants and opens new avenues for the induction of specific immunosuppression in transplantation.

Aim The aim of this study was to evaluate the prognostic value of the C1q-SPA assay in recipients of kidney from deceased donors. Methods The study population consisted of 123 recipients transplanted at Columbia University Medical Center between January 1 and December 31, 2012. None of the patients showed DSA that could be detected by complement dependent lymphocytotoxicity. Sera obtained from each patient prior to transplantation were evaluated for donor-specific anti-HLA IgG antibody (DSA) by Luminex single-antigen bead (SAB) testing (SAB-IgG). SAB-IgG positive sera were further tested by Luminex SAB-based assay detecting C1q-binding HLA antibodies (SAB-C1q). Results The one year kidney allograft survival was 96.6% in this patient population. Four recipients had an episode of AMR although 27 showed DSA detectable by SAB-IgG. C1q testing shows DSA in 7 sera among the SAB-IgG positive results (7/27, 26%), 3 of which were in patients with AMR and 4 in patients without AMR. Conclusions There was a positive correlation between the presence of C1q detectable DSA in pre-transplantation sera and development of AMR. However, the correlation between SAB-IgG, SAB-C1q and rejection was weaker, suggesting the need of further evaluation of methods that can predict the risk of AMR, without affecting the patients’ chances of receiving a transplant.

Aim To determine whether the expansion of γδ T cells observed in pediatric heart allograft recipients associates with transplant outcomes. Methods We studied a population of 58 pediatric heart transplant recipients under the auspices of the Institutional Review Board. We performed Ab determinations by solid phase assays (SPA-IgG, “LSAB”). Flow cytometry staining was performed to determine the percentage of lymphocytic lineage markers: CD3, CD4, CD8, CD19, CD16& 56, CD45, TCRγδ, TCRαβ. Results Pediatric heart allograft recipients often require total or partial thymectomy at the time of their first heart operation, which leads to lifelong alterations in their T cell repertoire. While the prevailing population of circulating T cells in non-thymectomized children carries the TCRαβ TCR (96 ± 3%), we observed a significant expansion of TCRγδ T cells by Flow Cytometry in 37 of 58 patients studied. Within the population with high circulating fraction of gd T’s (8–20% of lymphocytes, n = 37), 23 patients (23/37, or 62%) developed anti-HLA antibodies detected by SPA-IgG (LSAB), while 14 did not. In patients with normal TCRγδ fractions ( Conclusion Thymectomy in pediatric heart recipients which results in the expansion of γδ T cells is associated with lesser risk of alloantibody development. The finding that patient with low γδ T cells are more likely to produce anti HLA Abs suggests that thymectomy results in a breakdown of immune regulation. Th2 impairment may result in insufficient cytokine production to fully support antibody production in these patients. Although mechanistic studies are needed to understand the causation relationships underpinning this phenomenon, the association alone suggests that monitoring of γδ T cell expansion may constitute a useful tool for Ab development prediction in pediatric heart transplant patients.

Specific immunosuppression of host’s immune response to donor HLA antigens has been a major goal to clinical transplantation. Recent evidence has been accumulating to show that a distinct population of T cells expressing the CD8 + CD28 − phenotype display suppressor function and inhibit Th activation and proliferation by modulating the APC function. To assess the presence of Ts in transplant recipient’s circulation, we have developed a flow cytometry method that measures the expression of costimulatory molecules on donor APC exposed to recipient Th and Ts. Our results demonstrate that quantitation of the capacity of CD8 + CD28 − T cells from patient circulation to suppress the activation of costimulatory molecules (CD80, CD86) on donor APC offers a reliable tool for monitoring specific immunosuppression against the graft in solid organ transplantation.

Aim The aim of this study was to establish whether the presence of DSA detected by Luminex based solid-phase assay (SPA) correlates with the cross-match results performed by complement-dependent cytotoxicity (CDC) in deceased donor kidney transplantation. Methods Between January 1, 2012 and December 31, 2012, lymphocytes from a total of 398 deceased donors were cross-matched by our laboratory. For each donor, sera from an average of 6.24 patients were selected for direct cross-matching by CDC, amounting to a total of 2,485 cross-matches. Pre-transplant sera were screened by CDC on HLA reference panel, and by LABScreen using HLA class I and class II antigen coated beads. Results Of the 2,485 patients cross-matched, 870 had DSA and should have been excluded as potential transplant recipients on this basis. However, 39.3% (342/870) of the DSA positive population showed no cytotoxic antibodies to the putative donor reflecting the non-Complement fixing nature of the antibodies detected by SPA and/or their low affinity for their target antigen. Since 60.7% (528/870) of patients with DSA showed positive CDC cross-matches, there was still a highly significant correlation (P Conclusions In this study, we have shown that 39.3% of patients with DSA would have been denied the transplant if it was solely based on the virtual cross-match result. Furthermore, there was no statistically significant difference between graft function at one year in patients with or without DSA.

Aim To establish the impact of anti-HLA antibodies formed prior and/or following heart transplantation in a cohort of 137 pediatric recipients, transplanted between January 2000 and December 2011. Methods Anti-HLA class I and class II antibodies were monitored by traditional CDC methods using donor and panel T and B lymphocytes and SPA for detection of DSA. Endomyocardial biopsies were performed by protocol and whenever rejection was suspected. Results The allograft survival was 89% at 10 years in patients who had no lymphocytotoxic antibodies either before or after transplantation, yet dropped to 55% in patients who developed anti-HLA class I antibodies (p Conclusions All AMR episodes occurring in patients from this study population have documented evidence of complement binding antibodies and C4d deposition in the graft, reinforcing the conclusion that an integrated study of pathologic, immunologic and functional changes is required for timely and effective treatment of AMR.