Immunologic risk stratification of pediatric heart transplant patients by combining HLA-EMMA and PIRCHE-II.

Human leukocyte antigen (HLA) molecular mismatch is a powerful biomarker of rejection. Few studies have explored its use in assessing rejection risk in heart transplant recipients. We tested the hypothesis that a combination of HLA Epitope Mismatch Algorithm (HLA-EMMA) and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II) algorithms can improve risk stratification of pediatric heart transplant recipients. Class I and II HLA genotyping were performed by next-generation sequencing on 274 recipient/donor pairs enrolled in the Clinical Trials in Organ Transplantation in Children (CTOTC). Using high-resolution genotypes, we performed HLA molecular mismatch analysis with HLA-EMMA and PIRCHE-II, and correlated these findings with clinical outcomes. Patients without pre-formed donor specific antibody (DSA) (n=100) were used for correlations with post-transplant DSA and antibody mediated rejection (ABMR). Risk cut-offs were determined for DSA and ABMR using both algorithms. HLA-EMMA cut-offs alone predict the risk of DSA and ABMR; however, if used in combination with PIRCHE-II, the population could be further stratified into low-, intermediate-, and high-risk groups. The combination of HLA-EMMA and PIRCHE-II enables more granular immunological risk stratification. Intermediate-risk cases, like low-risk cases, are at a lower risk of DSA and ABMR. This new way of risk evaluation may facilitate individualized immunosuppression and surveillance.

Incidence, characterization, and impact of newly detected donor-specific anti-HLA antibody in the first year after pediatric heart transplantation: A report from the CTOTC-04 study.

Data on the clinical importance of newly detected donor-specific anti-HLA antibodies (ndDSAs) after pediatric heart transplantation are lacking despite mounting evidence of the detrimental effect of de novo DSAs in solid organ transplantation. We prospectively tested 237 pediatric heart transplant recipients for ndDSAs in the first year posttransplantation to determine their incidence, pattern, and clinical impact. One-third of patients developed ndDSAs; when present, these were mostly detected within the first 6 weeks after transplantation, suggesting that memory responses may predominate over true de novo DSA production in this population. In the absence of preexisting DSAs, patients with ndDSAs had significantly more acute cellular rejection but not antibody-mediated rejection, and there was no impact on graft and patient survival in the first year posttransplantation. Risk factors for ndDSAs included common sensitizing events. Given the early detection of the antibody response, memory responses may be more important in the first year after pediatric heart transplantation and patients with a history of a sensitizing event may be at risk even with a negative pretransplantation antibody screen. The impact on late graft and patient outcomes of first-year ndDSAs is being assessed in an extended cohort of patients.

Re-examination of the lymphocytotoxic crossmatch in liver transplantation: can C4d stains help in monitoring?

C4d-assisted recognition of antibody-mediated rejection (AMR) in formalin-fixed paraffin-embedded tissues (FFPE) from donor-specific antibody-positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO- incompatibility status, histopathology and clinical-serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO-incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection-related causes of allograft dysfunction in DSA- and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA- recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

Persistent lack of human herpesvirus-6 specific T-helper cell response in liver transplant recipients.

BACKGROUND: Specific immunologic defects predisposing to human herpesvirus-6 (HHV-6), e.g. the role of HHV-6 specific T-helper cell memory response in liver transplant recipients, have not been assessed. METHODS: T-helper function (mitogen ConA response) as a marker of overall immunocompetence and T-helper response (memory response) specific to HHV-6 and cytomegalovirus (CMV) were assessed in 15 liver transplant recipients and compared with 25 healthy subjects. Samples were tested pretransplant, at 2 weeks, 1 month, 2-3 months, and 1 year posttransplantation. Stimulation index (SI) >3 was considered a positive response. RESULTS: Seven percent (1/15) of the transplant recipients at any time posttransplantation, as compared to 64% (16/25) of the healthy subjects, had a positive HHV-6 memory response (P = 0.00065). HHV-6-specific memory response in transplant recipients at 2 weeks (SI 1.43), 1 month (SI 1.1), and 2-3 months (SI 1.3) was significantly more suppressed than in healthy subjects (SI 17.5, P = 0.0001). Although transplant recipients as compared to healthy subjects also had a lower CMV-specific memory response posttransplant (P = 0.0439), CMV-specific memory response recovered significantly at 1 month (P = 0.03) and at 2-3 months (P = 0.027) as compared to that at 2 weeks. However, HHV-6 memory response was persistently absent up to 2-3 months with partial recovery at 1 year; 7% of the patients at 2-3 months, but 25% at 1 year had a positive HHV-6 specific memory response. Forty percent (6/15) of the patients developed HHV-6 viremia a mean of 4 weeks posttransplant. Patients with HHV-6 viremia had greater suppression of HHV-6 memory response at 1 month than those without viremia (mean SI, 0.96 vs. 1.3, P = 0.08). All but one of the patients had a positive ConA response. CONCLUSION: Prolonged suppression of HHV-6 memory response, but not overall T-helper cell function was documented and may play a role in the pathogenesis of HHV-6 infection in liver transplant recipients. Memory response to CMV after liver transplantation was significantly more robust than to HHV-6.

Monitoring the patient off immunosuppression. Conceptual framework for a proposed tolerance assay study in liver transplant recipients.

The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.

Clinical relevance of in vitro propagation of activated lymphocytes from endomyocardial biopsy samples of pediatric heart transplant recipients.

In vivo activated T-lymphocytes can be cultured from endomyocardial biopsy samples of human cardiac allografts, sometimes even in the absence of histological rejection. We investigated the clinical relevance of this "lymphocyte growth assay" in pediatric heart transplant recipients. Specifically, we wished to determine if: (i) positive lymphocyte growth from EMB samples in the absence of significant rejection identifies a patient as being at increased risk for the development of acute rejection; (ii) withdrawal or major dose reduction of corticosteroids in the presence of lymphocyte growth results in high risk of rebound rejection; and (iii) presence of lymphocyte growth during acute rejection helps predict the response to treatment. Cultures were performed on 789 consecutive EMB samples from 65 pediatric heart transplant recipients in media containing 30 U/ml of recombinant IL-2. T-lymphocytes were cultured from 16% of EMB samples with low grade rejection (grade 0-1b) and from 34% of EMB samples with grade 2-4 rejection. EMB samples obtained early post-transplant (<180 days) were significantly more likely to yield positive lymphocyte growth compared to biopsies obtained late for any given rejection grade. Lymphocyte growth was comparable between patients managed with cyclosporine or tacrolimus based immunosuppression. For 227 EMB samples without rejection, a subsequent EMB sample was obtained within 12 weeks. Lymphocyte cultures were positive in 47 of these 227 EMB samples (21%), and in 19 out of 47 (40%) cases acute rejection (grade 2-4) was present on the follow-up EMB sample. By contrast, of 180 biopsies without growth, only 29 (16%) showed rejection at the next EMB (p<0.0001). When a follow-up biopsy was performed within 12 weeks of corticosteroid withdrawal, "rebound rejection" was observed in 3 out of 10 (30%) cases where the previous EMB sample yielded positive lymphocyte growth and in 4 out of 38 (11%) cases when it did not (p=0.29). The presence of lymphocyte growth in association with rejection was also predictive of whether rejection would resolve following high dose intravenous corticosteroid therapy (persistent rejection in 33 out of 50 (66%) cases with positive growth, versus 25 out of 80 (31%) cases without growth (p<0.0001)). Thus, positive lymphocyte growth is strongly associated with higher grade of rejection and earlier time from transplantation. Lymphocyte growth in the absence of rejection indicates high risk for rejection within the next 12 weeks. Growth in association with acute rejection indicates high probability of persistence of rejection following treatment with high dose corticosteroids.

Heat shock protein-induced T-lymphocyte propagation from endomyocardial biopsies in heart transplantation.

BACKGROUND: Recent studies have shown that heat shock proteins can be recognized by T cells during various immunologically mediated inflammatory processes. Injurious stimuli to cells induce an increased production of heat shock proteins which could lead to their cell surface expression and subsequent recognition by the immune system. We have postulated that allograft infiltrating cells may recognize heat shock proteins, especially during rejection. METHODS: This hypothesis was tested by incubating heart transplant biopsy specimens from 89 heart transplant recipients with soluble Mycobacterium tuberculosis extracts, a source of heat shock proteins or recombinant mycobacterial heat shock protein 65. T cell phenotypes were determined by flow cytometry. RESULTS: Mycobacterium tuberculosis extract can induce lymphocyte propagation from heart transplant biopsy specimens especially during rejection. A highly significant correlation was seen between Mycobacterium tuberculosis extract and interleukin-2-induced lymphocyte growth and an accelerated growth was seen for cultures incubated with Mycobacterium tuberculosis extract + interleukin-2. A second series of experiments has also shown the propagation of lymphocytes induced by recombinant mycobacterial heat shock protein-65. T-cell phenotype analysis of biopsy propagated lymphocytes has shown higher frequencies of CD8 cells in Mycobacterium tuberculosis extract and heat shock protein-65 propagated lymphocytes from early posttransplantation biopsy specimens, whereas, later on, most cultures showed a predominance of CD4 cells. T-cell receptor gamma delta cells were more frequently found in biopsy-derived lymphocyte cultures from long-term survivors, especially after propagation with Mycobacterium tuberculosis extract and heat shock protein-65. These gamma delta cells expressed primarily the delta 1 rather than the gamma 9 phenotype. CONCLUSIONS: These observations provide first evidence for the presence of heat shock protein-reactive lymphocytes in cellular infiltrates of transplants undergoing rejection.

Immunological identification of the protein-producing masculine differentiation of the Wolffian duct in the fetal mouse: western blot analysis and determination of the biological activity.

Recently, we identified a protein in the developing male reproductive tract of the fetal mouse which induced Wolffian duct differentiation in vitro in the absence of testis or testosterone. In this paper, we further evaluated the masculinizing role of this protein using an immunological approach. Thus, we purified a 72K protein from the 18-day-old male fetal reproductive tracts, prepared an antibody against the protein, and determined the role of this protein in producing masculine differentiation of the Wolffian duct using the polyclonal antibody against the protein. We demonstrate that the antibody reacted with the purified 72K protein, producing only one immunoreactive band around the 72K region in Western blot analysis. However, it reacted with 72K and 63K proteins present in the 18-day-old male reproductive tract. Both of these immunoreactive bands disappeared when the antibody was pretreated with the purified antigen. Nonimmune immunoglobulin G (IgG) produced no reactive bands with the extract of the male reproductive tracts. The IgG preparation of the immune serum specifically prevented Wolffian duct differentiation when tested in organ culture containing 13-day-old fetal male or female reproductive tracts in the presence of testis or testosterone (1 micrograms/ml) in a dose-dependent manner. No effect was found on the development of other organs, namely testis, Mullerian duct, and urogenital sinus, that were included in the organ culture. Nonimmune IgG, as expected, produced no effect on Wolffian duct differentiation. Western blot analysis of male and female reproductive tracts indicated a difference in their reactivities with this antibody. In females, the reactivity to the proteins around the 72K and 63K regions was very weak compared to that found with male proteins in those regions. Both of the above bands of the female reproductive tract disappeared when the antibody was pretreated with an excess of female reproductive tract extract, but in males, only the 63K band disappeared, whereas its 72K band remained. In conclusion, it appears that a 72K protein of the male reproductive tract plays a role in the Wolffian duct differentiation of the fetal mouse.

Role of prostaglandins in the testosterone-dependent wolffian duct differentiation of the fetal mouse.

Recent observations from this laboratory indicated a role of prostaglandin E2 (PGE2) in masculine differentiation of the external genitalia of the fetal mouse, induced by fetal testosterone. In this communication, we further investigated the role played by PGE2 in the testosterone-induced differentiation of the internal genital tract (Wolffian duct) of the fetal mouse. Using in vitro organ culture bioassay of Wolffian duct differentiation, we determined the effect of a PG-depleting agent, namely, anti-PGE2 antibody, and of inhibitors of PG synthesis for their ability to prevent Wolffian duct differentiation in the presence of testosterone. We demonstrated that anti-PGE2 antibody inhibited Wolffian duct differentiation in a dose-dependent manner in embryonic male explants containing fetal testes. At 1:10 dilution, the antibody inhibited the appearance of the entire Wolffian duct as well as growth of the specimen. At 1:100 dilution, however, only development of the Wolffian duct was prevented, as indicated by the absence of regions of the Wolffian duct or by the presence of epithelial disintegration throughout the ductal lumen. The antibody at 1:1000 dilution produced no significant effect on the appearance of the Wolffian duct. PGE2 (10 micrograms/ml) replacement in the medium prevented Wolffian duct disintegration induced by anti-PGE2. We next determined whether the testosterone-dependent Wolffian duct differentiation requires ongoing PG synthesis within the reproductive tract and analyzed the effects of the compounds inhibiting PG synthesis at the level of phospholipase A2 (PLA2) namely, cortisone and dexamethasone-and of those inhibiting at the level of cyclooxygenase-namely, aspirin and indomethacin. We have demonstrated that both PLA2 and cyclooxygenase inhibitors inhibited Wolffian duct differentiation in the male explant, i.e., in the presence of testis. These compounds also prevented the appearance of the Wolffian duct in female explants induced by exogenous testosterone. PGE2 added in the medium blocked the anti-masculinizing effects of PG synthesis inhibitors both in the male and female specimens. Thus, it appears that PG synthesis plays a role in the testosterone-induced masculine differentiation of the Wolffian duct.