BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

An extremes of outcome strategy provides evidence that multiple sclerosis severity is determined by alleles at the HLA-DRB1 locus.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system unsurpassed for variability in disease outcome. A cohort of sporadic MS cases (n = 163), taken from opposite extremes of the distribution of long-term outcome, was used to determine the role of the HLA-DRB1 locus on MS disease severity. Genotyping sets of benign and malignant MS patients showed that HLA-DRB1*01 was significantly underrepresented in malignant compared with benign cases. This allele appears to attenuate the progressive disability that characterizes MS in the long term. The observation was doubly replicated in (i) Sardinian benign and malignant patients and (ii) a cohort of affected sibling pairs discordant for HLA-DRB1*01. Among the latter, mean disability progression indices were significantly lower in those carrying the HLA-DRB1*01 allele compared with their disease-concordant siblings who did not. The findings were additionally supported by similar transmission distortion of HLA-DRB1*04 subtypes closely related to HLA-DRB1*01. The protective effect of HLA-DRB1*01 in sibling pairs may result from a specific epistatic interaction with the susceptibility allele HLA-DRB1*1501. A high-density (>700) SNP examination of the MHC region in the benign and malignant patients could not identify variants differing significantly between the two groups, suggesting that HLA-DRB1 may itself be the disease-modifying locus. We conclude that HLA-DRB1*01, previously implicated in disease resistance, acts as an independent modifier of disease progression. These results closely link susceptibility to long-term outcome in MS, suggesting that shared quantitative MHC-based mechanisms are common to both, emphasizing the central role of this region in pathogenesis.

Favourable outcome for patients with myeloid disorders treated with fludarabine-melphalan reduced-intensity conditioning and allogeneic bone marrow stem cell transplantation without the use of T-lymphocyte-depleting antibodies.

We report the use of reduced-intensity conditioning (RIC)-matched sibling allogeneic bone marrow stem cell transplantation as a method of establishing a graft-vs.-leukaemia (GvL) effect against myeloid disorders using a fludarabine-melphalan protocol without the use of T-lymphocyte-depleting antibodies. The 16 patients in this group had predominantly poor-risk acute myeloid leukaemia (AML) (n=10), AML/myelodysplasia (MDS) (n=2) and MDS (n=4). All but one patient achieved full haematopoietic engraftment. Thirteen of 16 patients are alive and in continued complete remission on completion of this study with a median follow-up of 426 d (range 83-1524). The actuarial 4 yr disease-free and overall survival is 79% for both. Only one patient relapsed following transplant, giving a relapse rate of 6% during the study period. The treatment-related mortality was 13% (n= 2). Overall, acute graft-vs.-host disease (GvHD) occurred in 53% (8/15), with acute GvHD grade II or above occurring in 47% (7/15). In the 13 evaluable patients, chronic GvHD occurred in 46% (6/13), with this being extensive in three patients. These results suggest that a GvL effect can be delivered against poor-risk myeloid disorders with a low non-relapse mortality using this fludarabine-melphalan RIC protocol.

Mother-to-child transmission of HIV infection and CTL escape through HLA-A2-SLYNTVATL epitope sequence variation.

Cytotoxic T lymphocytes (CTL) play a central role in containment of HIV infection. Evasion of the immune response by CTL escape is associated with progression to disease. It is therefore hypothesised that transmitted viruses encode escape mutations within epitopes that are required for successful control of viraemia. In order to test this hypothesis, escape through the dominant HLA-A2-restricted CTL epitope SLYNTVATL (p17 Gag residues 77-85 SL9) in the setting of mother-to-child-transmission (MTCT) was investigated. Initial data from two families in which the HIV-infected mother expressed HLA-A*0201 and had transmitted the virus to other family members were consistent with this hypothesis. In addition, analysis of the gag sequence phylogeny in one family demonstrated that CTL escape variants can be successfully transmitted both horizontally and vertically. To test the hypothesis further, a larger cohort of transmitting mothers (n=8) and non-transmitters (n=14) were studied. Variation within the SL9 epitope was associated with expression of HLA-A2 (P=0.04) but overall no clear link between variation from the SL9 consensus sequence and MTCT was established. However, the high level of background diversity within p17 Gag served to obscure any possible association between escape and MTCT. In conclusion, these studies highlighted the obstacles to demonstrating CTL escape arising at this particular epitope. Alternative strategies likely to be more definitive are discussed.

Detection of HLA-specific IGG antibodies using single recombinant HLA alleles: the MonoLISA assay.

BACKGROUND: Because of the presence of confounding antigens, the assignment of HLA antibody specificity is difficult in highly sensitized patients, and the definition of an acceptable HLA mismatch requires a significant workload per patient. We describe a new ELISA method, monoLISA, for detection of immunoglobulin (Ig)G HLA antibody using single recombinant HLA class I monomers bound to microtiter plates. METHODS: HLA-A2 and -B8 monomers were synthesized and used as screening targets for 85 sera from renal patients. The sera contained various IgG and IgM HLA-specific antibodies, including anti-A2 and anti-B8,defined in a conventional complement-dependent cytotoxicity test (CDC). Investigations were performed to determine possible effects on antibody binding of differential monomer peptide presentation as well as lack of glycosylation. RESULTS: A good correlation was found between CDC-defined specificities and the reactivity observed with HLA monomers. MonoLISA attained means of 100% sensitivity and 92.5% specificity compared with CDC. Neither the presence of different peptides, nor the absence of glycosylation of the monomer affected the ability of monoLISA to detect antibody. CONCLUSION: This study demonstrates that the mono-LISA method for HLA antibody detection is valid. Because this has the potential to reduce the work involved in screening sensitized patients awaiting transplantation for HLA antibodies, resources aimed at increasing the number of constructed monomers would be well targeted.

Six unrelated HLA-DR-matched adults recognize identical CD4+ T cell epitopes from influenza A haemagglutinin that are not simply peptides with high HLA-DR binding affinities.

Influenza A/Beijing/32/92 (H3N2) haemagglutinin (HA)-specific short-term CD4+ T cell lines were generated from six unrelated HLA-DR0701, 1501 positive adults (aged 27-60 years) 3 months following administration of an influenza subunit vaccine containing HA A/Beijing/32/92. Epitope recognition was examined using 118 HA A/Beijing/32/92-specific 16mer peptides which overlapped by 11 residues and which spanned the entire molecule. Following influenza vaccination the donors recognized identical HA peptides. The selected peptides represented HA regions which have been free from extensive drift mutation since the emergence of human H3N2 influenza A strains. Using DAP DR7.0701 cells (a murine cell line expressing HLA-DR0701) as antigen-presenting cells the majority of CD4+ T cell responses were shown to be HLA-DR0701 restricted. The relationship between HA peptide recognition and relative strength of HA peptide-HLA-DR0701 binding was then explored in a competition assay with biotinylated CLIP peptide. Although peptides representing dominant HA epitopes bound to DR0701, the relationship between relative strength of binding and immunodominance was complex, and many strongly binding peptides, particularly those with glycosylation sites and showing inter-strain variation, were not recognized. These results illustrate the control HLA class II exerts over CD4+ T cell HA epitope selection in unrelated adult humans. Immunodominance appears not to be directly related to the relative strength of HA peptide-HLA class II binding, and thus reflects complex interactions between antigen processing, intracellular competition for HLA binding, TCR repertoires and repeated exposure to different strains of influenza A viruses.

A correlation between HLA-C matching and donor antirecipient CTL precursor frequency in bone marrow transplantation.

A newly developed, reliable, DNA-based method for typing for alleles of the HLA-C locus has been applied in the context of unrelated, volunteer donors for bone marrow transplantation. Some donors matched for HLA-A, -B, -DR, and -DQ have been found to generate in vitro high frequencies of CTL reactive with the recipient's cells. Here we demonstrate that there is a highly significant correlation of the frequencies of CTL precursors and incompatibility at the HLA-C locus. These data indicate that HLA-C locus incompatibility should be avoided in unrelated donor bone marrow transplantation.