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Background: Testing for alloantibodies against human platelet antigens (HPAs) is essential for the clinical diagnosis of fetomaternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura, and platelet (PLT) refractoriness. Most of the methods currently used for HPA alloantibody detection rely on the availability of panels of HPA-typed PLTs and some rely on validated monoclonal antibodies (MoAbs) against the PLT glycoproteins. Recombinant β3 integrins displaying the HPA-1a (rHPA-1a) or HPA-1b (rHPA-1b) epitopes have been produced as an alternative source of antigen. The suitability of these integrin fragments was evaluated for the development of an HPA-1a alloantibody screening assay, using Luminex xMAP technology.Study Design and Methods: A 3-plex bead assay was developed by coupling biotinylated rHPA-1a, rHPA-1b, and recombinant glycoprotein VI to LumAvidin microspheres. Forty patient samples referred for FMAIT diagnostic testing, which were previously screened by the MoAb-specific immobilization of PLT antigens (MAIPA) assay, were used to assess the assay.Results: The rHPA-1a- and rHPA-1b-coupled beads were able to detect HPA-1a and HPA-1b alloantibodies in all patient samples tested that were previously confirmed to contain HPA-1-specific antibodies. Furthermore, HLA Class I antibodies did not cross-react with the coupled beads.Conclusion: The 3-plex bead assay can be used to detect HPA-1a antibodies with sufficient specificity and sensitivity for use in the clinical setting of FMAIT. The development of other recombinant integrin fragments with the use of Luminex xMAP technology may assist in providing more rapid HPA antibody detection, enabling prompt diagnosis of alloimmune PLT disorders. [ABSTRACT FROM AUTHOR]

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., (© 2021 by The American Society of Hematology.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Aims/objectives: To explore the impact of Human Leucocyte Antigen (HLA)-A and B epitope-matched platelets on the outcome of platelet transfusions in alloimmunised patients with aplastic anaemia (AA). The relevance of HLA-C epitope mismatches was also investigated., Background: Patients who become immunologically refractory (IR) to random platelet transfusions can experience an adequate rise in platelet count through the provision of HLA-compatible platelets using an antigen-matching algorithm. This approach has been shown to be effective in patients with a low calculated reaction frequency, but it is not always successful in highly sensitised patients. The use of HLA epitopes-selected platelets has been suggested as an alternative to the antigen matching approach., Methods: The effect of HLA epitope matching (both Eplets and Triplets) on the outcome of platelet transfusion was analysed in 37 highly immunised AA patients previously transfused with HLA-A and B antigen-matched platelets. Epitope matching was determined using the HLAMatchmaker programme. The outcome of the transfusions was assessed by the platelet count increments (PCIs) obtained 1 and 24 hours post-transfusions., Results: HLA-A and B epitope matching was equivalent to HLA antigen matching in raising platelet counts. There was no significant difference in PCI when HLA-C epitope mismatches were considered. In addition, transfusions with fewer than two antigen mismatches resulted in significantly higher PCIs compared to transfusions with more than two antigen mismatches., Conclusions: HLA epitope-matched platelet provision may represent a clinically effective transfusion strategy for patients IR to random platelet transfusions. Further prospective studies are required., (© 2019 British Blood Transfusion Society.)

The development of techniques to define the human leucocyte antigen (HLA) region has proven to be challenging due to its high level of polymorphism. Within a clinical laboratory, a technique for high-resolution HLA typing, which is rapid and cost effective is essential. NGS has provided a rapid, high-resolution HLA typing solution, which has reduced the number of HLA ambiguities seen with other typing methods. In this study, the One Lambda NXType NGS kit was tested on the Ion Torrent PGM platform. A total of 362 registry donors from four ethnic populations (Europeans, South Asians, Africans and Chinese) were NGS HLA typed across 9-loci (HLA-A, -B, -C, -DRB1,-DRB345 -DQB1 and -DPB1). Concordance rates of 91%-98% were obtained (for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) when compared to historical PCR-SSO HLA types, and the identification of uncommon alleles such as A*24:07:01 and C*04:82 were observed. A turnaround time of four days was achieved for typing 44 samples. However, some limitations were observed; primer locations did not allow all ambiguities to be resolved for HLA Class II where Exon I and IV amplification are needed (HLA-DRB1*04:07:01/04:92, HLA-DRB1*09:01:02/*09:21 and HLA-DRB1*12:01:01/*12:10). This study has demonstrated high-resolution typing by NGS can be achieved in an acceptable turnaround time for a clinical laboratory; however, the Ion Torrent workflow has some technical limitations that should be addressed., (© 2018 John Wiley & Sons Ltd.)

In Colchester, Britain's oldest recorded town, during the Roman period there were areas which were clearly used solely as cemeteries. One of the most significant is at Butt Road, which includes a late Roman probable Christian cemetery with an associated building, apparently a church, that overlies and developed from a pagan inhumation cemetery. DNA was extracted from the long bones (femurs) of 29 individuals, mostly from a large complex of burials centered on two timber vaults. These were thought to comprise a number of family groupings, deduced from osteological analysis, stratigraphical and other considerations. The use of a modified version of the silica-based purification method recovered nanogram quantities of DNA/gram of bone. Two-stage amplification, incorporating primer-extension preamplification-polymerase chain reaction, permitted simultaneous amplification of both mitochondrial and nuclear DNA. Sequence-specific oligonucleotide probes yielded human leukocyte antigen (HLA)-DR typing of seven samples, with four revealing the infrequent HLA-DR10 genotype. Examination of the control region of mitochondrial DNA (mtDNA) by direct sequencing revealed polymorphisms yet to be reported in the modern population. HLA-DRB typing and mtDNA analysis affirmatively supported kinship among some, if not all, individuals in the "vault complex" and demonstrate a continental European origin of the individuals investigated.

In cord blood (CB) transplantation, virtual 6/6 HLA matches, whereby the donor-recipient mismatch is identical to the CB noninherited maternal Ag (NIMA), have similar outcomes to inherited 6/6 matches. In the UK-British Bone Marrow Registry (BBMR), 4707 of the total 21 020 CB donors have the NIMA defined. Retrospective searches of these donors, for 1-3 NIMA matches, identified a virtual 6/6 match for 31.4% of 274 European Caucasoid (EC) and 25.4% of 67 other ethnicity (OE) patients. Patients weighing ⩽50 kg were also evaluated for a single graft with adequate cell dose. In 125 EC patients, 6/6 HLA matches were identified for 24.0% and virtual 6/6 matches were identified for a further 21.6%. The remaining EC patients had a 5/6 (30.4%) or a 4/6 (22.4%) match. In OE patients, 6/6 HLA matches were identified for 9.3% and virtual 6/6 matches were identified for a further 18.7%. The remaining OE patients had a 5/6 (30.2%) or a 4/6 (37.2%) match. Searches were also performed using the 26 735 Bone Marrow Donors Worldwide CB with defined NIMA and yielded comparable increases. Considering NIMA as permissible mismatches in donor selection therefore increased the availability of a 6/6 match in this cohort.

A series of analogues of the (S)-2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) receptor agonist BnTetAMPA (5b) were synthesized and characterized pharmacologically in radioligand binding assays at native and cloned AMPA receptors and functionally by two-electrode voltage clamp electrophysiology at the four homomeric AMPA receptors expressed in Xenopus laevis oocytes. The analogues 6 and 7 exhibit very different pharmacological profiles with binding affinity preference for the subtypes GluA1 and GluA3, respectively. X-ray crystal structures of three ligands (6, 7, and 8) in complex with the agonist binding domain (ABD) of GluA2 show that they induce full domain closure despite their low agonist efficacies. Trp767 in GluA2 ABD could be an important determinant for partial agonism of this compound series at AMPA receptors, since agonist efficacy also correlated with the location of the Trp767 side chain.

Background: Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant β3 integrins, displaying the biallelic HPA-1 epitopes (rHPA-1), have been shown to detect HPA-1a alloantibodies implicated in FMAIT. This report describes a multicenter validation of the beads using the results of well-characterized samples to define the optimum parameters for analysis of a large cohort of 498 clinical samples., Study Design and Methods: Fifty-one blinded quality assurance (QA) samples were tested by six laboratories to standardize the rHPA-1 bead assay and to develop an algorithm for sample classification. Five laboratories retrieved samples from 498 independent FMAIT cases, previously tested by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) assay, from their local archives for testing with the rHPA-1 beads. The results were evaluated using a mathematical algorithm developed to classify the samples., Results: The QA samples gave a mean concordance of 94% between the bead and MAIPA assays, while 97% concordance was observed with the FMAIT samples. Of the 15 discrepant samples, seven were positive by the beads but negative by MAIPA, while the contrary was observed for eight samples. Overall, the bead assay achieved 98% sensitivity for HPA-1a antibody detection in FMAIT and 98.7% specificity compared to the local MAIPA., Conclusion: The rHPA-1 bead assay is a rapid 3-hour assay for the sensitive detection of HPA-1 antibodies. Its ease of use would enable prompt detection of maternal HPA-1a antibodies in suspected FMAIT cases, which is important supportive evidence for treatment by transfusion with HPA-1b1b PLTs., (© 2015 AABB.)

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., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

HLA alloimmunization induced by pregnancy, multiple transfusions or transplantation is responsible for some of the serious complications seen in patients receiving blood and blood products. These complications are primarily the result of antibody and antigen triggering an acute immunological reaction, which in some cases can be fatal e.g. TRALI. Some adverse reactions are triggered by HLA antibodies present in the patient whereas others are initiated by antibodies or HLA reactive cells present in the transfused product. The introduction of universal leucodepletion for the prevention of vCJD transmission has resulted in a significant reduction in these reactions by eliminating the main source of alloimmunization, but residual cellular components or platelets are still able to activate the immune system and induce the development of HLA reactive antibodies or T cells. However, the use of more sensitive and specific techniques to detect HLA antibodies and antigens has not only improved the investigation of transfusion reactions and their subsequent diagnosis, but it has also facilitated the implementation of a number of measures such as the use of HLA antibody negative products to further reduce their development., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

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.

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.

Background: The outcome of clinical transplantation and a number of disease susceptibilities show very strong associations with genetic variants within the major histocompatibility complex, particularly in the human leukocyte antigen (HLA) genes. A problem with many association studies is the lack of sufficient DNA to perform multiple genetic analyses, particularly with transplantation outcomes where donor and recipient DNA are often in short supply. This study assesses whether a multiple-strand displacement whole genome amplification (WGA) method could generate sufficient template of high quality to perform unbiased amplification for analysis of the HLA-A, -B, -C, -DRB1, and -DQB1 genes., Study Design and Methods: A panel of DNA samples from various biological sources was subjected to WGA reaction using Phi29 DNA polymerase. The HLA genotypes were subsequently determined using standard polymerase chain reaction (PCR)-based methods including sequence-specific oligonucleotide probes (PCR-SSOP, Luminex, Luminex Corp.) and sequence-based typing (PCR-SBT). WGA products and original DNA samples were used to determine the sensitivity of the Luminex assay; in addition, reamplified WGA products were also genotyped., Results: The WGA templates, as well as serially amplified DNA for two successive rounds, yielded HLA genotypes fully concordant with those determined for the original DNA samples. WGA products and original DNA gave reproducible HLA-DQB1 genotypes with 100 to 10 ng of template. Purification of the WGA products was required for successful PCR-SBT, but not for the PCR-SSOP method., Conclusion: Our study suggests that WGA can be a reliable method for generating unlimited DNA for medium- or high-resolution HLA typing using the techniques described above.

Background: Multipotent mesenchymal stromal cells (MSC) are of interest for their potential to repair bone and cartilage, and also their immunosuppressive properties. Umbilical cord blood (UCB) is reported to contain MSC, and therefore may be a useful source of these cells for clinical applications., Methods: We evaluated protocols for isolating MSC from UCB and characterized the surface phenotype, differentiation potential and immunoregulatory properties of the cells obtained., Results: Ten of 25 UCB units processed yielded MSC-like colonies, with depletion of lineage+ cells providing a higher efficiency. Only two of the cultures could be expanded satisfactorily; the remainder failed to proliferate. One culture generated transformed lines that were grossly aneuploid, had up-regulated TERT transcripts and had lost CD90 expression and the capacity to differentiate. The two propagated UCB-MSC lines were similar to those from bone marrow but were not identical to each other, with differences seen in expression of surface markers and cytoskeletal proteins. Both underwent osteogenesis, but at different rates and to different degrees, while neither generated adipocytes. When added as a third party to a mixed lymphocyte culture, both suppressed proliferation., Discussion: MSC-like cells can be isolated from UCB, but at low efficiencies, and they exhibit a variety of morphologies, growth rates and differentiation potentials and can transform in culture.

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.

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 low-expression allele HLA-A*24020102L was identified on a likely haplotype bearing B*55, Cw*01 during the investigation of nine cases (four families and five unrelated subjects) of HLA-A*24 lacking the A24 specificity. A search was made of 70,007 HLA-A, HLA-B, and HLA-C DNA-based typed largely northwestern European Caucasoid blood donors on the British Bone Marrow Registry and Welsh Bone Marrow Donor Registry panels for phenotypes possessing A*24, B*55, Cw*01. Fifty three were found, and 12 of these were subsequently shown to possess A*24020102L by sequence-based typing or polymerase chain reaction with sequence-specific primers. This indicated that the likely A*24, B*55, Cw*01 haplotype has a frequency of 0.000378 in the UK and that approximately 22.6% of these will possess A*24020102L. Consequently, HLA-A*24020102L, B*55, Cw*01 is a principal A*24020102L-bearing haplotype, and the minimum carriage and gene frequencies of A*24020102L are 0.017% and 0.000086, respectively, in UK blood donors., (Copyright 2004 Blackwell Munksgaard)

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.

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.

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.

A group of Brazilian leprosy patients and controls were genotyped for a CA-repeat microsatellite polymorphism within the interferon (IFN)-gamma gene. A significantly higher frequency of alleles 5-7 was observed in this patient population, indicating that IFN-gamma gene polymorphism may contribute to the course of leprosy post-infection.

Immune thrombocytopenic purpura (ITP) is a heterogeneous disorder with wide variability in response rates to treatments including corticosteroids, splenectomy and intravenous immune globulins. The nature of the underlying predisposing causes for this autoimmune disorder are not known. We have HLA typed 71 adult Caucasian patients with chronic primary ITP, and compared the data with 750 control samples. In this association study, we were not able to identify a significant immunogenetic susceptibility factor for ITP with HLA class I and class II alleles. However, it appeared that there might be an association between HLA-A2 and ITP, particularly in female patients, who are the predominantly affected group; and HLA-A2 was also present at increased frequency in patients with chronic ITP progressing to splenectomy. These findings are reviewed in the context of other similar reported HLA studies in ITP. Further studies based on larger groups of patients will be necessary to identify genetic susceptibility factors for this disease.

Dendritic cells (DCs) are the most potent antigen-presenting cells described to date. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have recently been described as being exclusively of the immature CD11c- lymphoid DC subset. Using an alternative method of enrichment, based on a negative selection system, both lymphoid (HLA-DR+ CD123+++ CD11c- CD33-) and myeloid (HLA-DR++ CD123+ CD11c+ CD33+) DCs were identified in CB. Although the majority of CB DCs showed a lymphoid phenotype, a significant number of CD11c+ myeloid DCs (25.6% +/- 14.5%, n = 13) were also present. Other markers, such as CD80 and CD83, were negative in both subsets. Analyses of the allostimulatory capacity of both subsets showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the inverted CD11c+/CD11c- ratio observed in CB DCs (1:3) with respect to adult blood DCs (3:1) remains to be explained.

Results from a number of HLA typing methods prompted the DNA sequencing (SBT) of a cord blood sample at the HLA-B locus. A novel HLA-B allele, B*4104, was identified. This was confirmed by sequencing the mother of the cord blood unit for HLA-B.

The human leukocyte antigen (HLA) system, originally discovered as the result of a transfusion reaction, is now known to play a crucial role in many areas of clinical medicine. The main function of the HLA molecules is to present antigenic peptides to the immune system and in this way regulate the induction of immune responses. This is a highly regulated process which requires a close interaction between the HLA molecules, the antigenic peptide and the T cell receptor.HLA molecules are also known to be associated with a variety of autoimmune, non-autoimmune and infectious diseases and to restrict the antibody response to certain antigens and to vaccines. It is likely that the mechanism responsible for this restriction is the preferential presentation of antigen-derived peptides to T cells. Furthermore, HLA antigens, in contrast to most polymorphic molecules, have the ability to activate the immune system using two different pathways of T cell activation, the direct and indirect pathways. As a result of these features, HLA antigens and antibodies are responsible for some of the serious clinical complications of blood transfusion, and have an important influence on the outcome of solid organ and haemopoietic stem cell transplantation., (Copyright 2000 Harcourt Publishers Ltd.)

Cytokine gene polymorphisms affecting cytokine production may influence rejection and graft-versus-host disease following solid organ and haemopoietic stem cell (HSC) transplantation, respectively. Polymorphisms in the regulatory regions of several cytokine genes have been described; for example, tumour necrosis factor-alpha (TNF-alpha) has a G/A substitution at position -308, interleukin-2 (IL-2) has a T/G substitution at position -330 and interleukin-10 (IL-10) has substitutions at positions -1082(G/A), -819(C/T) and -592(C/A). Microsatellites associated with cytokine production have been detected in the first intron of the IFN-gamma gene and flanking the TNF-alpha gene. In this study, we have genotyped a single panel of healthy Northern European Caucasoids living in the south-east of England for the above-mentioned polymorphisms and compared the results to those published for other populations. A PCR method using sequence-specific primers (SSP) was developed for genotyping the IL-2 polymorphism, and the ABI PRISMtrade mark 310 genetic analyser was used to detect the TNF-alpha and IFN-gamma microsatellites. The allele frequencies of all the studied polymorphisms were consistent with those reported for other UK Caucasoid populations, but differences were observed when compared to other Oriental, African and Caucasoid groups. If these cytokine polymorphisms prove to have functional consequences, then any differences across population groups may have significant clinical relevance in disease and in the outcome of solid organ and HSC transplantation.

Recent data suggests that graft-versus-host disease (GVHD) is initiated by host APCs. Blockade of CD40:CD154 interactions between APCs and T cells in vivo induces T cell tolerance to host alloantigen and dramatically reduces GVHD. Because allogeneic cord blood (CB) transplantation results in a lower incidence and severity of acute GVHD compared with bone marrow transplantation, we have investigated whether CB T cells can express CD154 in response to stimulation by allogeneic monocyte-derived dendritic cells (MDDC) and have used 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling in combination with intracellular cytokine analysis to assess the proliferation and cytokine profiles of alloantigen-responsive cells. CB T cells stimulated with allogeneic MDDC showed stronger proliferation than adult blood T cells. Surface CD154 expression was detected in the actively dividing CFSElow populations of both the CD4+ and CD4- subsets and was brightest in cells that had divided the most. Assessment of supernatants from MDDC-stimulated CB and adult blood T cells showed no significant difference in the levels of either IFN-gamma or TNF-alpha, but CB T cell supernatants did show a significant lack of detectable IL-2. Intracellular cytokine analysis revealed that dividing CB T cells had been primed to produce IFN-gamma, TNF-alpha, and IL-2 on restimulation. Further phenotype analysis showed that 75% of CB T cells producing IFN-gamma were CD8+. These data suggest that MDDC-stimulated CB T cells express functional CD154 and provide enough costimulation for dendritic cells to prime naive CD8+ CB T cells and induce type 1 cytokine production.

High levels of sHLA-I (soluble HLA--class I) have been correlated with rejection episodes in solid organ transplant recipients and with graft versus host disease in bone marrow recipients. Studies of human infection with parasitic worms of the gut have suggested that certain individuals may be genetically predisposed to intense infection. In this study, the influence of parasitic helminth infection on levels of sHLA-I in plasma was investigated in 155 HLA typed individuals from St. Lucia, exposed to the gut parasite Trichuris trichiura. The results confirmed previous findings showing increased levels of sHLA-I in HLA-A9, and in this case HLA-A23 positive individuals. However, HLA-A9 positive individuals with high worm burden had significantly lower levels of sHLA-I in their plasma compared with HLA-A9 positive subjects with low worm burden. These results suggest that the intensity of T. trichiura infection influences the ability of HLA-A9 positive subjects to maintain high levels of sHLA-I.

HLA typing of class I loci by reference strand-mediated conformation analysis (RSCA) using a slab gel genetic analyser has been described. This study adapted the method for use in the capillary based ABI PRISM 310. Control DNA samples were used to create a database of mobility values for 37 HLA-A alleles. The technique was validated by comparing RSCA and sequence-specific oligonucleotide probe (SSOP)/sequence-specific primer amplification (SSP) HLA-A locus typing results from 214 cord blood samples. Of the samples tested, 6.5% required confirmatory typing by SSP, compared with a repeat rate of 10-40% for SSOP. In 200 samples where no SSP was necessary, there was 100% concordance between RSCA and previous results. The ABI PRISM 310 RSCA method defines HLA-A types at medium resolution and is quick and easy to implement.