Early outcomes for low-risk pediatric heart transplant recipients and steroid avoidance: A multicenter cohort study (Clinical Trials in Organ Transplantation in Children - CTOTC-04).

BACKGROUND: Immunosuppression strategies have changed over time in pediatric heart transplantation. Thus, comorbidity profiles may have evolved. Clinical Trials in Organ Transplantation in Children-04 is a multicenter, prospective, cohort study assessing the impact of pre-transplant sensitization on outcomes after pediatric heart transplantation. This sub-study reports 1-year outcomes among recipients without pre-transplant donor-specific antibodies (DSAs). METHODS: We recruited consecutive candidates (<21 years) at 8 centers. Sensitization status was determined by a core laboratory. Immunosuppression was standardized as follows: Thymoglobulin induction with tacrolimus and/or mycophenolate mofetil maintenance. Steroids were not used beyond 1 week. Rejection surveillance was by serial biopsy. RESULTS: There were 240 transplants. Subjects for this sub-study (n = 186) were non-sensitized (n = 108) or had no DSAs (n = 78). Median age was 6 years, 48.4% were male, and 38.2% had congenital heart disease. Patient survival was 94.5% (95% confidence interval, 90.1-97.0%). Freedom from any type of rejection was 67.5%. Risk factors for rejection were older age at transplant and presence of non-DSAs pre-transplant. Freedom from infection requiring hospitalization/intravenous anti-microbials was 75.4%. Freedom from rehospitalization was 40.3%. New-onset diabetes mellitus and post-transplant lymphoproliferative disorder (PTLD) occurred in 1.6% and 1.1% of subjects, respectively. There was no decline in renal function over the first year. Corticosteroids were used in 14.5% at 1 year. CONCLUSIONS: Pediatric heart transplantation recipients without DSAs at transplant and managed with a steroid avoidance regimen have excellent short-term survival and a low risk of first-year diabetes mellitus and PTLD. Rehospitalization remains common. These contemporary observations allow for improved caregiver and/or patient counseling and provide the necessary outcomes data to help design future randomized controlled trials.

Allospecific CD154+ B cells associate with intestine allograft rejection in children.

BACKGROUND: As a significant determinant of T- and B-cell cooperation, CD154 has been used to identify allospecific T-cytotoxic memory cells (TcM) for rejection risk assessment with high sensitivity or specificity but not for alloreactive B-cells, especially among recipients predisposed to acute cellular and humoral rejection, that is, children with intestinal transplantation (ITx). METHODS: Single blood samples from 32 pediatric ITx after lymphocyte depleting induction therapy were obtained within 30 days of protocol biopsies. Samples were assayed for allospecific CD154CD19 B cells and allospecific CD154 TcM in 16-hr live-cell mixed leukocyte reaction using multiparametric flow cytometry. Results were expressed as the immunoreactivity index (IR) or the ratio of donor- to third-party-induced CD154 B cells or TcM. The rejection threshold IR of B cells was determined by logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses. RESULTS: Biopsy-proven acute cellular rejection was present in 15 subjects (rejectors) and absent in 17 (nonrejectors). In archived serum samples from 16 of 32 subjects donor-specific anti-HLA antibodies (DSA) were assayed by Luminex bead array. DSA were absent in all 7 nonrejectors but present in 7 of 9 rejectors. The IR of allospecific CD154CD19 B cells more than or equal to 1.351 was associated with rejector status and was present in 13 of 15 rejectors (sensitivity 87%) and absent in 15 of 17 nonrejectors (specificity 88%). Excellent correlations were seen between CD154CD19 B cells and CD154 TcM (Spearman ρ=0.647, P=0.0001) but could not be tested independently for DSA, which was highly correlated with rejector status and with CD154 TcM. CONCLUSIONS: Allospecific CD154CD19 B cells identify rejection-prone children with ITx and can likely substitute for T-cell alloreactivity in estimating rejection risk in this rare subject population.

The impact of EBV load on T-cell immunity in pediatric thoracic transplant recipients.

BACKGROUND: Immunologic monitoring of pediatric transplant (Tx) recipients, who are at increased risk of Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disease, is an important goal in clinical transplantation. Here, we investigated the impact of EBV load on T-cell immunity from pediatric Tx recipients, using clinically applicable tests for improved assessment of T-cell immune competence. METHODS: Thirty-five asymptomatic pediatric thoracic Tx patients were categorized into three groups according to their EBV load levels as follows: undetectable viral load (UVL), chronic low viral load (LVL) and chronic high viral load (HVL). Global and EBV-specific T-cell immunity were assessed by ATP release using Cylex Immuknow and T Cell Memory assays. RESULTS: UVL patients exhibited normal ATP release to Concanavalin A (ConA) and phytohemagglutinin (PHA; 190+/-86 ng/mL, 328+/-163 ng/mL) and detectable EBV-specific (37+/-34 ng/mL) ATP responses. LVL patients displayed significantly stronger responses to ConA (373+/-174 ng/mL), PHA (498+/-196 ng/mL) and EBV (152+/-179 ng/mL), when compared with UVL or to HVL patients (ConA 185+/-114 ng/mL, PHA 318+/-173 ng/mL, and EBV 33+/-42 ng/mL). Moreover, HVL patients displayed significant inverse correlation between CD4+ T-cell ATP levels and EBV loads. CONCLUSIONS: Evaluation of global and EBV-specific T-cell immunity provides a rapid assessment of patients' immune competence. It is still unclear whether selective oversuppressed ATP release by CD4+ T cells reflects HVL patients at risk of posttransplant lymphoproliferative disease. Further longitudinal studies will determine the importance of Immuknow test in identifying asymptomatic HVL patients vulnerable to EBV complications.

Immunogenicity and immunomodulatory effects of amnion-derived multipotent progenitor cells.

This is the first study on the immunologic properties of a clinically relevant population of cells derived from the amnion of human placenta. Unlike other cells from the amnion, these amnion-derived multipotent progenitor cells (AMP cells), from human amnion, grow in serum-free conditions and have never been cultured in the presence of medium containing animal-derived components. This study reports the immunologic characteristics of AMP cells and their roles as immunomodulators. Characterization of AMP cells revealed the presence of major histocompatibility complex (MHC) class I but the lack of class II antigens and absence of co-stimulatory molecules B7-1 and B7-2. The nonclassical human leukocyte antigen (HLA)-G was expressed at low levels on cultured AMP cells. Expression was significantly increased after interferon-gamma (IFN-gamma) treatment. Cultured peripheral blood mononuclear cells did not respond to irradiated AMP cells, indicated by lack of proliferation as measured by standard mixed lymphocyte reaction. Culturing AMP cells with IFN-gamma did not reverse this result and did not upregulate class II expression. The AMP cells were shown to have immunomodulatory capabilities by inhibiting peripheral blood mononuclear cell proliferative responses to mitogen, alloantigen, and recall antigen, but the AMP cells were unable to inhibit preactivated T-cell blast response to growth factor media. This immunomodulatory effect of AMP cells was found to be dependent on cell-to-cell contact.

Pancreas transplantation under alemtuzumab (Campath-1H) and tacrolimus: Correlation between low T-cell responses and infection.

BACKGROUND: Alemtuzumab induction and tacrolimus-based immunosuppression has been effective in pancreas transplantation. Despite the encouraging results of this minimalistic approach to immunosuppression, infection still remains a significant cause of morbidity. The Cylex ImmuKnow [corrected] assay was used in this study to compare pancreas recipient clinical states (stable, rejection, infection) with T cell responses. METHODS: Blood samples were taken from pancreas recipients pretransplant and at approximately three-month intervals posttransplant for analysis of T cell responses. When possible, T cell responses were also quantified during changes in clinical status (infection or rejection). RESULTS: A range between 100-300 ng/ml adenosine triphosphate (ATP) was found in stable patients (mean 194+/-123, n = 51) with good graft function and no infection or rejection. A low T cell response was highly correlated with infectious states. The fourteen patients with infections/posttransplant lymphoproliferative disease had a mean ATP of 48 ng/ml. Risk hazard analysis showed that patients with ATP levels <100 ng/ml were four to seven times more susceptible to infection compared to stable patients. Four patients with rejection showed a T cell response of 550 ng/ml ATP, which was statistically significant compared to stable patients, although the sampling numbers (9) were too small to be conclusive. CONCLUSION: The Cylex ImmuKnow [corrected] assay is a valuable tool to more precisely modulate immunosuppression in pancreas transplant patients. In particular, the assay is extremely useful in detecting overly immunosuppressed patients vulnerable to infections.

Monitoring immune function during tacrolimus tapering in small bowel transplant recipients.

Long term use of immunosuppressants impacts the cardiovascular system and increases the risk of infection and malignancy. To effectively reduce immunosuppression in a transplant recipient a tool is needed to directly monitor the level of immune function. The Cylex(R) Immune Cell Function Assay, approved by the FDA for the assessment of cell-mediated immunity, shows promise as an objective measure of a transplant recipient's immune function. In a blinded retrospective study, the immune function was compared to clinical courses and histological examinations of biopsies of 20 small bowel transplant recipients during periods of immunosuppressant tapering. Eight patients with no major adverse events or changes of immunosuppressive therapy had moderate to low immune function and were categorized as immunologically and clinically stable. Twelve patients displaying strong immune responses were immunologically and clinically volatile requiring addition of steroids and or OKT3. Results validate the clinical utility of the Cylex Immune Cell Function Assay as an objective tool for assessing immune function. By evaluating immune function, physicians now can identify those patients who are candidates for minimization of immunosuppressant therapy, manage the timing and rate of immunosuppressant weaning and be forewarned of increased patient risk.

Propagation of activated T lymphocytes from endomyocardial biopsy samples of cardiac allografts: influence of the addition of recombinant interleukin-4 to the culture environment.

In vivo, activated T cells can be propagated from endomyocardial biopsy (EMB) samples of cardiac allografts in cultures containing recombinant interleukin-2 (rIL-2). However, T cells are sometimes not propagated in such cultures, even when rejection is present, and at other times the yield of lymphocytes is too small to allow further studies of these graft-infiltrating cells. The current study investigated the effects of the addition of recombinant interleukin-4 (rIL-4) to the culture environment. Cultures were performed on 532 consecutive EMB samples from 120 adult and pediatric heart transplant recipients. Each sample was divided into multiple fragments. Half of the fragments were cultured in media containing 30 U/mL of rIL-2 and the remaining half were cultured under identical conditions but with the addition of 200 U/mL of rIL-4. After 14 days, cell counts were performed, the cell phenotypes were assessed by flow cytometry, and donor specificity and cytotoxicity were assessed using the primed lymphocyte test (PLT) and cell-mediated lympholysis (CML) assay, respectively. Lymphocyte growth occurred in 18% of grade 0-1a EMB in the presence of rIL-2 and in 29% of grade 0-1a EMB in the presence of rIL-2/rIL-4 (p = 0.02). For higher-grade EMB (equivalent to grade >or=1b), the proportion of positive cultures (approximately 39%) was similar in both conditions. For positive cultures, there was a 5-fold increase in the number of cells in the rIL-2/4 cultures compared to rIL-2 alone (1.6 x 10(6) versus 3.4 x 10(5)). Flow cytometry revealed an increase in the proportion of CD8+ cells in the rIL-2/4 cultures (42% versus 23%, p = 0.004). Proliferative responses to donor antigens (as assessed by using the PLT) were comparable between the two groups, but donor-specific cell-mediated cytotoxicity was enhanced on addition of rIL-4. Hence, addition of rIL-4 enhances the propagation of donor-specific T cells from heart biopsy samples, especially in the presence of minimal rejection. This will provide a greater quantity of material for further studies of graft-infiltrating cells.