Background Allospecific CD154+T-cytotoxic memory cells (CD154+TcM) predict acute cellular rejection (ACR) after liver or intestine transplantation (LTx, ITx) in small cohorts of children and can enhance immunosuppression management, but await validation and clinical implementation. Methods To establish safety and probable benefit, CD154+TcM were measured in cryopreserved samples from 214 children <21 years (NCT#1163578). Training set samples, n=158, were tested with research-grade reagents and 122 independent validation set samples were tested with cGMP-manufactured reagents after assay standardization and reproducibility testing. Recipient CD154+TcM induced by stimulation with donor cells were expressed as a fraction of those induced by HLA-non-identical cells in parallel cultures. The resulting immunoreactivity index (IR) if > 1 implies increased rejection-risk. Results Training and validation set subjects were demographically similar. Mean coefficient of test variation was <10% under several conditions. Logistic regression incorporating several confounding variables identified separate pre-transplant and post-transplant IR thresholds for prediction of rejection in respective training set samples. An IR ≥ 1.1 in post-transplant training samples, and IR ≥1.23 in pre-transplant training samples predicted LTx or ITx rejection in corresponding validation set samples in the 60-day post-sampling period with sensitivity, specificity, positive and negative predictive values of 84%, 80%, 64%, and 92%, respectively (AUC 0.792), and 57%, 89%, 78%, and 74%, respectively (AUC 0.848). No adverse events were encountered due to phlebotomy. Conclusions Allospecific CD154+T-cytotoxic memory cells predict acute cellular rejection after liver or intestine transplantation in children. Adjunctive use can enhance clinical outcomes.
Cell‐mediated immunity to CMV, if known, could improve antiviral drug therapy in at‐risk children and young adults with LT and IT. Host immunity has been measured with CMV‐specific T cells, which express IFNγ, but not those which express CD154, a possible substitute for IFNγ. CMV‐specific CD154+ T cells and their subsets were measured with flow cytometry after stimulating PBL from recipient blood samples with an overlapping peptide mix of CMV‐pp65 antigen for up to 6 hours. CMV‐specific CD154+ T cells co‐expressed IFNγ in PBL from three healthy adults and averaged 3.8% (95% CI 3.2%‐4.4%) in 40 healthy adults. CMV‐specific T cells were significantly lower in 19 CMV DNAemic LT or IT recipients, compared with 126 non‐DNAemic recipients, 1.3% (95% CI 0.8‐1.7) vs 4.1 (95% CI 3.6‐4.6, P < .001). All T‐cell subsets demonstrated similar between‐group differences. In logistic regression analysis of 46 training set samples, 12 with DNAemia, all obtained between days 0 and 60 from transplant, CMV‐specific T‐cell frequencies ≥1.7% predicted freedom from DNAemia with NPV of 93%. Sensitivity, specificity, and PPV were 83%, 74%, and 53%, respectively. Test performance was replicated in 99 validation samples. In 32 of 46 training set samples, all from seronegative recipients, one of 19 recipients with CMV‐specific T‐cell frequencies ≥1.7% experienced DNAemia, compared with 8 of 13 recipients with frequencies <1.7% (P = .001). CMV‐specific CD154+ T cells are associated with freedom from DNAemia after LT and IT. Among seronegative recipients, CMV‐specific T cells may protect against the development of CMV DNAemia.
Donor-induced and third-party-induced proliferation of T-helper and T-cytotoxic (Tc) cells and their naïve and memory subsets was evaluated simultaneously in single blood samples from 77 children who received steroid-free liver transplantation (LTx) after induction with rabbit anti-human thymocyte globulin. Proliferation was measured by dilution of the intravital dye carboxyfluorescein succinimidyl ester (CFSE) in a 3-to 4-day mixed lymphocyte response coculture. The ratio of donor/third-party-induced proliferated (CFSE low ) T-cells was reported as the immunoreactivity index (IR) for each subset. Rejectors were defined as those who experienced biopsy-proven acute cellular rejection within 60 days of the assay. IR Ͼ 1 signified increased risk of rejection, and IR Ͻ 1 implied decreased risk. Demographics for 32 rejectors and 45 nonrejectors were similar. Proliferated CFSE low T-cells and subsets were significantly higher among rejectors compared with nonrejectors. In 33 of 77 randomly selected children, logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses showed that the IR of Tc cells was best associated with biopsy-proven rejection (sensitivity Ͼ 75%, specificity Ͼ 88%). Sensitivity and specificity were replicated in the remaining 44 children who composed the validation cohort. IR of CFSE low Tc cells correlated significantly with IR of proinflammatory, allospecific CD154ϩ Tc cells (r ϭ 0.664, P ϭ 0.0005) and inversely with IR of allospecific, anti-inflammatory, cytotoxic T lymphocyte antigen 4 -positive Tc cells (r ϭ Ϫ0.630, P ϭ 0.007).In conclusion, proliferative alloresponses of Tc cells can identify rejection-prone children receiving LTx. Liver Transpl 15: 978-985, 2009.
Background Operational tolerance after retransplantation of the intestine has never been reported. Purpose To two recently described intestine transplant recipients with operational tolerance, we now add a third. Methods Review of case record and immunological testing to confirm donor‐specific hyporesponsiveness in multiple immune cell compartments. Results Re‐transplanted with a multivisceral liver‐ and kidney‐inclusive intestine allograft at age 12 years, this recipient self‐discontinued immunosuppression 14 years after the retransplant and has been rejection free for 2 years thereafter. As in the two previous reports, immunological testing demonstrated decreased donor‐specific inflammatory response of T‐cytotoxic memory cells and B‐cells, decreased presentation of donor antigen by B‐cells and monocytes, absence of donor‐specific anti‐HLA antibodies, circulating FOXP3 + T‐helper cells, and intact cellular and humoral immunity to cytomegalovirus and Epstein–Barr virus. Additionally, our recipient demonstrated enhanced donor‐activation‐induced apoptosis of alloreactive T‐cytotoxic memory cells. Conclusions Despite variable paths to tolerance which include graft versus host disease in two previous cases, and rejection‐related loss of the primary isolated intestinal allograft in our recipient, the three cases with operational tolerance are bound by common themes: a relatively large donor antigenic load transmitted during intestine transplantation, and donor‐specific hyporesponsiveness. Cell‐based assays suggest enhanced donor‐induced apoptosis of recipient T‐cells and circulating T‐regulatory cells as mechanistic links between antigenic load and donor‐specific hyporesponsiveness.
PurposeEnhanced B-cell presentation of donor alloantigen relative to presentation of HLA-mismatched reference alloantigen is associated with acute cellular rejection (ACR), when expressed as a ratio called the antigen presenting index (API) in an exploratory cohort of liver and intestine transplant (LT, IT) recipients.MethodsTo test clinical performance, we measured the API using the previously described 6-hour assay in 84 LT and 54 IT with median age 3.3 years (0.05-23.96). Recipients experiencing ACR within 60 days after testing were termed rejectors.ResultsWe first confirmed that B-cell uptake and presentation of alloantigen induced and thus reflected the alloresponse of T-helper cells, which were incubated without and with cytochalasin and primaquine to inhibit antigen uptake and presentation, respectively. Transplant recipients included 76 males and 62 females. Rejectors were tested at median 3.6 days before diagnosis. The API was higher among rejectors compared with non-rejectors (2.2 ± 0.2 vs 0.6 ± 0.04, p-value=1.7E-09). In logistic regression and ROC analysis, API ≥ 1.1 achieved sensitivity, specificity, positive and negative predictive values for predicting ACR in 99 training set samples. Corresponding metrics ranged from 80-88% in 32 independent post-transplant samples, and 73-100% in 20 independent pre-transplant samples. In time-to-event analysis, API ≥ 1,1 predicted higher incidence of late DSA after API measurements in LT (p=0.011) and graft loss in IT recipients (p=0.008), compared with recipients with API<1.1, respectively.ConclusionEnhanced donor antigen presentation by circulating B-cells predicts rejection after liver or intestine transplantation as well as higher incidence of DSA and graft loss late after transplantation
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