Detection of donor-specific-antibodies by solid phase assay and its relevance to complement-dependent-lymphocytotoxicity cross-matching in kidney transplantation.

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.

HLA antibodies in pediatric heart transplantation.

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.

New-onset graft dysfunction after heart transplantation--incidence and mechanism-related outcomes.

BACKGROUND: Graft dysfunction (GD) after heart transplantation (HTx) is a major cause of morbidity and mortality. The impact of different pathophysiologic mechanisms on outcome is unknown. In this large, single-center study we aimed to assess the incidence of GD and compare the outcomes with different histopathologic mechanisms of rejection. METHODS: We analyzed a data set of 1,099 consecutive patients after their HTx at Columbia University Medical Center between January 1994 and March 2008, and identified all patients hospitalized with new-onset GD. Based on the histopathologic data, patients were divided into GD-unexplained (Group-GD-U), GD-antibody-mediated rejection (Group-GD-AMR), GD-cardiac allograft vasculopathy (Group-GD-CAV) and GD-acute cellular rejection (Group-GD-ACR) groups. We compared the in-hospital and 3-, 6- and 12-month mortality across these groups using the chi-square test. We also compared the 3-, 6- and 12-month survival curves across groups using the log-rank test. RESULTS: Of 126 patients (12%) identified with GD, complete histology data were available for 100 patients. There were 21, 20, 27 and 32 patients identified in Group-GD-U, Group-GD-AMR, Group-GD-CAV and Group-GD-ACR, respectively. The in-hospital mortality rates were 52%, 20%, 15% and 6%, respectively. The in-hospital mortality rate was significantly higher in Group-GD-U compared with all other groups (p = 0.0006). The 3-, 6- and 12-month survival rate was also significantly lower in Group-GD-U compared with all other groups. CONCLUSION: A significant proportion of patients presenting with new-onset GD have unexplained histopathology. Unexplained GD is associated with a significantly higher mortality rate. New diagnostic tools are necessary to better understand and detect/predict this malignant phenotype.

Pre- and posttransplantation allosensitization in heart allograft recipients: major impact of de novo alloantibody production on allograft survival.

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.

BCL6 is required for differentiation of Ig-like transcript 3-Fc-induced CD8+ T suppressor cells.

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 BCL6(high) 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.

Alloantibodies in heart transplantation.

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.

Relevance of different antibody detection methods for the prediction of antibody-mediated rejection and deceased-donor kidney allograft survival.

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.

Polymorphism and linkage disequilibrium of immunoglobulin-like transcript 3 gene.

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.

Immunoglobulin-like transcript 3-Fc suppresses T-cell responses to allogeneic human islet transplants in hu-NOD/SCID mice.

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.

Soluble Ig-like transcript 3 inhibits tumor allograft rejection in humanized SCID mice and T cell responses in cancer patients.

Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

Alloantibodies and the outcome of cadaver kidney allografts.

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.

The relationship of nodular endocardial infiltrates (Quilty lesions) to survival, patient age, anti-HLA antibodies, and coronary artery disease following heart transplantation.

BACKGROUND: Quilty lesions are mononuclear cell infiltrates identified in human heart transplant biopsies. The biologic significance of Quilty lesions remains undetermined. METHODS: We monitored acute rejection by biopsy and lymphocyte growth assay (LGA) as well as transplant-related coronary artery disease (TRCAD) by yearly angiogram in 285 recipients of primary heart allografts. Patients showing Quilty lesions on biopsies during the first year posttransplant were compared with patients without such lesions. Recipients' sera were obtained at the time of biopsy and tested for anti-HLA Class I and II antibodies. RESULTS: The actuarial survival of patients who developed Quilty lesions was significantly better than those who did not (P=.0074). Patients with Quilty lesions were younger and more likely to have a biopsy diagnosis of acute rejection (P=.002) and positive LGA (P<.0001) during the first posttransplant year. Among patients who do not form anti-HLA Class II antibodies, those with Quilty lesions were more likely than patients without Quilty lesions to develop TRCAD 5 years posttransplantation (P=.04). There was no correlation of Quilty status with the number of HLA donor-recipient mismatches or posttransplant development of anti-HLA antibodies. CONCLUSIONS: Quilty formers showed improved survival and are more likely to be diagnosed with acute rejection on biopsy and have positive LGAs. Allograft recipients who do not form anti-HLA Class II antibodies but do form Quilty lesions are more likely to develop TRCAD by 5 years posttransplantation than those who do not form Quilty lesions.

Flow cytometric analysis of normal and reactive spleen.

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.

Anti-HLA antibodies in heart transplantation.

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.

Logarithmic correction to the probability of capture for dissipatively perturbed Hamiltonian systems.

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.