An epitope-based approach of HLA-matched platelets for transfusion: a noninferiority crossover randomized trial.

Platelet transfusion refractoriness results in adverse outcomes and increased health care costs. Managing refractoriness resulting from HLA alloimmunization necessitates the use of HLA antigen-matched platelets but requires a large platelet donor pool and does not guarantee full matching. We report the first randomized, double-blind, noninferiority, crossover trial comparing HLA epitope-matched (HEM) platelets with HLA standard antigen-matched (HSM) platelet transfusions. Alloimmunized, platelet-refractory, thrombocytopenic patients with aplastic anemia, myelodysplastic syndrome, or acute myeloid leukemia were eligible. HEM platelets were selected using HLAMatchMaker epitope (specifically eplet) matching. Patients received up to 8 prophylactic HEM and HSM transfusions provided in random order. The primary outcome was 1-hour posttransfusion platelet count increment (PCI). Forty-nine patients were randomized at 14 UK hospitals. For intention to treat, numbers of evaluable transfusions were 107 and 112 for HEM and HSM methods, respectively. Unadjusted mean PCIs for HEM and HSM methods were 23.9 (standard deviation [SD], 15) and 23.5 (SD, 14.1), respectively (adjusted mean difference, -0.1; 95% confidence interval [CI], -2.9 to 2.8). Because the lower limit of the 95% CI was not greater than the predefined noninferiority limit, the HEM approach was declared noninferior to the HSM approach. There were no differences in secondary outcomes of platelet counts, transfusion requirements, and bleeding events. Adequate 1-hour PCI was more frequently observed, with a mean number of 3.2 epitope mismatches, compared with 5.5 epitope mismatches for inadequate 1-hour increments. For every additional epitope mismatch, the likelihood of an adequate PCI decreased by 15%. Epitope-matched platelets should be considered to support HLA alloimmunized patients. This trial was registered at www.isrctn.com as #ISRCTN23996532.

Next generation sequencing of 11 HLA loci characterises a diverse UK cord blood bank.

The introduction of next generation sequencing (NGS) for stem cell donor registry typing has contributed to faster identification of compatible stem cell donors. However, the successful search for a matched unrelated donor for some patient groups is still affected by their ethnicity. In this study, DNA samples from 714 National Health Service (NHS) Cord Blood Bank donors were typed for HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 by NGS. Analysis of the ethnic diversity showed a high level of diversity, with the cohort comprising of 62.3% European and 37.7% of either multi-ethnic or non-European donors, of which 12.3% were multi-ethnic. The HLA diversity was further confirmed using PyPop analysis, 405 distinct alleles were observed in the overall NHS-CBB cohort, of which 37 alleles are non-CWD, including A*31:14N, B*35:68:02, C*14:23 and DQA1*05:10. Furthermore, HLA-DQA1 and HLA-DPA1 analysis showed 12% and 10%, respectively, of the alleles currently submitted to IMGT, confirming further diversity of the NHS-CBB cohort. The application of 11 HLA loci resolution by NGS revealed a high level of diversity in the NHS-CBB cohort. The incorporation of this data coupled with ethnicity data could lead to improved donor selection, contributing to better clinical outcomes for patients.

Immune reconstitution following umbilical cord blood transplantation: IRES, a study of UK paediatric patients.

To obtain a qualitative as well as quantitative view immune reconstitution following umbilical cord blood (UCB) transplantation of paediatric patients, we utilised a broad panel of flow cytometry markers to monitor the phenotypes of lymphoid and myeloid cells at 1-12 months post-transplant. Samples were received from 46 patients with a median age of 3.3 years and survival was 76% at 1 year. Monocytes were at similar or higher median levels than in adult controls at all times tested, with a high CD16+ proportion in the first 3 months. NK cells were also within adult ranges, with a CD56++ high proportion in the first 6 months. B cell recovery was seen from 2 months in most patients and T cells from 3 months, both were delayed with anti-thymocyte globulin (ATG) treatment. CD4:CD8 ratios were high in the first 6 months, and the proportion of T cells with recent thymic emigrant and naïve phenotypes rose from 3 months. NK and plasmacytoid dendritic cell numbers remained at reduced levels in patients not surviving to 1 year. Our results can serve as a useful reference for detailed monitoring of immune reconstitution in paediatric recipients of UCB.

Enhancement of the immunoregulatory potency of mesenchymal stromal cells by treatment with immunosuppressive drugs.

BACKGROUND AIMS: Multipotent mesenchymal stromal cells (MSCs) are distinguished by their ability to differentiate into a number of stromal derivatives of interest for regenerative medicine, but they also have immunoregulatory properties that are being tested in a number of clinical settings. METHODS: We show that brief incubations with rapamycin, everolimus, FK506 or cyclosporine A increase the immunosuppressive potency of MSCs and other cell types. RESULTS: The treated MSCs are up to 5-fold more potent at inhibiting the induced proliferation of T lymphocytes in vitro. We show that this effect probably is due to adsorption of the drug by the MSCs during pre-treatment, with subsequent diffusion into co-cultures at concentrations sufficient to inhibit T-cell proliferation. MSCs contain measurable amounts of rapamycin after a 15-min exposure, and the potentiating effect is blocked by a neutralizing antibody to the drug. With the use of a pre-clinical model of acute graft-versus-host disease, we demonstrate that a low dose of rapamycin-treated but not untreated umbilical cord-derived MSCs significantly inhibit the onset of disease. CONCLUSIONS: The use of treated MSCs may achieve clinical end points not reached with untreated MSCs and allow for infusion of fewer cells to reduce costs and minimize potential side effects.

Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation.

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues, with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However, the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper, we report that suppression of mitogen-induced T cell proliferation by human UC-, bone marrow-, and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation, indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays, an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore, we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation.

Efficient expansion of mesenchymal stromal cells from umbilical cord under low serum conditions.

BACKGROUND: Mesenchymal stromal cells (MSC) are of clinical interest for their potential use in regenerative medicine and immunotherapy. Originally derived from bone marrow (BM), MSC have now been isolated from most tissues, including umbilical cord (UC) and UC blood (UCB). If MSC from UC are biologically equivalent to those from BM, they would be attractive as a readily available and non-invasive source for cellular therapies. METHODS: Sections of UC were separated into vascular and Wharton's jelly (WJ) fractions, which were then digested individually to release MSC that were isolated by plastic adherence in a 10% fetal calf serum (FCS) medium, or a low serum medium designed for multipotent adult progenitor cells (MAPC). The resulting perivascular (PV) and WJ MSC lines were assayed for expression of characteristic markers and differentiation and immunosuppressive properties. RESULTS: MSC lines were readily derived from most UC tested. Cells grown in MAPC medium (MM) tended to be smaller and more elongated and expressed more nestin, but did not differ substantially in their growth rate, expression of other markers and differentiation capacity. All UC lines tested were adipogenic but poorly osteogenic, and were equivalent in their ability to suppress T-cell proliferation induced by phytohemagglutinin (PHA), activation beads and allostimulation. CONCLUSIONS: UC is a convenient, efficient source of MSC that can be expanded under low serum conditions for application on future studies of tissue regeneration and immunosuppression.

Analysis of CD4+ T-Cell responses to a novel alpha-fetoprotein-derived epitope in hepatocellular carcinoma patients.

PURPOSE: Alpha-fetoprotein (AFP) is a tumor-associated antigen in hepatocellular carcinoma and is a target for the development of cancer vaccine. Four immunodominant AFP-derived HLA-A*0201-restricted peptides have been identified and the administration of these peptides with an adjuvant has stimulated AFP-specific CTL responses in hepatocellular carcinoma patients. However, no AFP-derived CD4 T-cell epitope has yet been reported and the status of AFP-specific CD4(+) T-cell responses in hepatocellular carcinoma patients is not fully understood. The aim of this study was to analyze naturally occurring CD4(+) T-cell responses to AFP. EXPERIMENTAL DESIGN: We analyzed the ability of CD4(+) T cells to recognize an HLA-DR-restricted AFP-derived epitope in 41 hepatocellular carcinoma patients and 24 non-hepatocellular carcinoma control patients using intracellular cytokine assays for IFN-gamma. RESULTS: Here, for the first time, we report the identification of an AFP-derived CD4(+) T-cell epitope that is recognized by circulating lymphocytes from hepatocellular carcinoma patients in association with HLA-DR. The absence of detectable responses in healthy donors and patients with chronic liver disease suggests that AFP-specific CD4(+) T cells in the responder patients had been previously expanded in vivo in response to the tumor. The anti-AFP CD4(+) T-cell response was only detected in hepatocellular carcinoma patients with normal or mildly elevated serum AFP levels who were in the early stage of disease. CONCLUSION: Our data will be instrumental in the development of cancer vaccine using AFP-derived immunogens.

The London Cord Blood Bank: analysis of banking and transplantation outcome.

Cord blood units (n = 5500) stored at the London Cord Blood Bank, including 59 units transplanted into a high risk and heterogeneous group of patients, were analysed. Transplant outcome data was available for 44 patients with a median clinical follow-up of 14 months (range 3-44 months). Over 40% of the collected units were of ethnic minority origin with a median volume of 79 ml (range 40-240 ml) and a median total nucleated cell (TNC) count of 11.9 x 10(9)/l (range 10.0-24.8 x 10(9)/l). The average patient's weight was 28 kg (range 5-80 kg) and the median age was 8 years (range 0.7-40 years). The median number of nucleated cells infused was 4 x 10(7)/kg (range 1.10-16 x 10(7)/kg). Neutrophil engraftment of 0.5 x 10(9)/l was observed in 33 (74+/-%) patients with an average time of 28 days (range 11-60). The Kaplan-Meier estimate of acute graft-versus-host disease (grade II >) at day 100 was 37 +/- 7% and in 27 (62%) patients, it was grade I or absent. The overall survival and disease-free survival at 2 years was 49 +/- 8% and 41 +/- 8%, respectively. Two years after transplantation the survival rate was 69% and 54% for patients receiving a 6/6 or 5/6 HLA matched units, respectively. Infection was the main cause of transplanted related mortality in these patients.

Cord blood dendritic cells: subsets, functional characteristics and in vitro generation.

Dendritic cells (DCs) form a heterogeneous population of cells capable of stimulating naive T cells and initiating primary immune responses. This well-known function of DCs has offered the possibility of developing clinical protocols for their use in immunotherapy to tumours. DCs may also play a critical role in the induction of peripheral immunological tolerance, which could have important implications in the treatment of autoimmunity or in the outcome of clinical transplantation. Recent reports have indicated that cord blood transplantation is associated with a reduced incidence of graft versus host disease. Thus studies on the identification and characterisation of DCs present in, or derived from cord blood will help to understand their role, not only in neonatal immunity, but also in the outcome of cord blood transplantation.