Consideration of noninherited maternal Ags as permissible HLA mismatches in cord blood donor selection.

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.

Clinical relevance of the HLA system in blood transfusion.

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.

Characterization of mesenchymal stromal cells derived from full-term umbilical cord blood.

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.

Primary alloproliferative TH1 response induced by immature plasmacytoid dendritic cells in collaboration with myeloid DCs.

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.

HLA-A*24020102L in the UK blood donor population.

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.

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood.

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.

Allele frequencies for an interferon-gamma microsatellite in a population of Brazilian leprosy patients.

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.

Allele frequencies for an interferon-y microsatellite in a population of Brazilian leprosy patients

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.

Major histocompatibility complex susceptibility genes and immune thrombocytopenic purpura in Caucasian adults.

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.

Identification of both myeloid CD11c+ and lymphoid CD11c- dendritic cell subsets in cord blood.

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.

A novel HLA allele, B*4104, identified in a cord blood donor.

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 HLA system in blood transfusion.

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.

Allele frequencies of polymorphisms of the tumour necrosis factor-alpha, interleukin-10, interferon-gamma and interleukin-2 genes in a North European Caucasoid group from the UK.

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.

Sustained expression of CD154 (CD40L) and proinflammatory cytokine production by alloantigen-stimulated umbilical cord blood T cells.

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.

HLA-A, -B and -DR antigen frequencies of the London Cord Blood Bank units differ from those found in established bone marrow donor registries.

Patients requiring allogeneic stem cell transplantation who do not have an HLA-matched related donor can sometimes obtain an unrelated donor by searching volunteer registries. The majority of donors in the registries are Caucasoid, which results in a lower probability of a non-Caucasoid patient finding a suitable donor. Cord blood is increasingly used as a source of haematopoietic stem cells for allogeneic bone marrow reconstitution and so far the London Cord Blood Bank has banked almost 3000 cord blood units. An analysis of the first 1500 units banked showed that more than 30% of the London Cord Blood Bank units are derived from UK ethnic minorities compared with only 2% of individuals recruited locally for the British Bone Marrow Registry (BBMR). The HLA types found in these cord blood units reflect their ethnic diversity and include: HLA-A34, A36, A80, B75, B61, B53, B78, B81 and B82. The units stored by the London Cord Blood Bank show an HLA profile which differs considerably from that of locally typed adult volunteers for the BBMR panel and this should help to increase the chances of obtaining acceptably HLA-matched donors for patients from ethnic minorities. Bone Marrow Transplantation (2000) 25, 475-481.

Parasitic infection with Trichuris trichiura influences plasma levels of soluble HLA class I.

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-A typing by reference strand-mediated conformation analysis (RSCA) using a capillary-based semi-automated genetic analyser.

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.