Wayne Shumway

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. III, Typing for 24 alleles of HLA-DP

The second exon of HLA-DPB includes five polymorphic segments with extensive sharing of sequences between alleles. In order to facilitate assignment of specificities in heterozygous individuals, we have used group-specific amplification of two nonoverlapping sets of DPB alleles (here called group A and group B) with especially designed primers. Group A and group B polymerase chain reaction products were hybridized with sequence-specific oligonucleotide probes generating easily recognizable patterns which defined 24 distinct HLA-DPB alleles. We also established a routine procedure for distinguishing HLA-DP homozygosity from failed amplification in one of the alleles. Our results showed that when only one allele was detected, failure of amplification had occurred in less than 4% of the cases. DNA typing with this method correlated well with primed-lymphocyte typing for HLA-DP in the Tenth Workshop, as determined by us in assays performed on the workshop B-cell lines. Two normal panels of unrelated subjects were tested to obtain population frequencies. We conclude that this method is simple, relatively quick, and accurate. It is the method of choice for studies to determine the role of HLA-DP alleles in T cell reactions, in various diseases, and in transplantation.

Donor-specific antibody against denatured HLA-A1: Clinically nonsignificant?

Pre-transplant screening of a woman with end-stage renal disease (ESRD) showed no anti-human leukocyte antigen (HLA) alloantibodies by anti-human globulin-complement-dependent cytotoxicity (AHG-CDC; class I) or enzyme-linked immunosorbent assay (class II). Following a negative AHG-CDC crossmatch, an HLA*01:01+ deceased donor (DD) kidney was transplanted in September 2005. Subsequent screening of pre-transplant serum by LABScreen Single Antigen (SA) array showed strong reactivity versus A*01:01. Despite that reactivity, at 5 years post-transplant, the patient has a serum creatinine of 1.6 mg/dl and has never experienced humoral or cellular rejection. Retrospective flow-cytometric crossmatch of pre- and post-transplant sera versus DD cells was negative. Rescreening of multiple pre- and post-transplant sera revealed anti-A1 reactivity persisting from the first through the last samples tested. The patients anti-A1 was almost two fold more reactive with denatured A*01:01 FlowPRA SA beads after denaturation with acid treatment (pH 2.7) than with untreated beads. Parallel results were observed with pH 2.7 treated versus untreated A1+ T cells in FXM. These data highlight the difficulty in interpreting screening results obtained using bead arrays, because of antibodies that appear to recognize denatured but not native class I HLA antigens. We suggest that such bead-positive, flow cytometric crossmatch negative antibodies are not associated with humoral rejection, may not necessarily be detrimental to a graft, and deserve further evaluation before becoming a barrier to transplantation.

New Alleles of the HLA-B15 Family

In a previous study of B locus alleles by sequence-specific oligonucleotide probe (SSOP) hybridization, we observed 18 novel patterns in a panel of 360 individuals. Four of these novel patterns were caused by alleles of the human leukocyte antigen (HLA)-B15 group, and three were available for this study. These alleles were found in Oriental, Latin American, African American, and Caucasian individuals. In addition, we analyzed a Caucasian subject who was found by serology to have an unusual B15 specificity. We sequenced these four samples by performing amplification from genomic DNA using polymerase chain reaction primers designed to obtain HLA class I products that included exon 2 and exon 3 as well as the intervening intron. The amplified segments were cloned and identified by colony hybridization with nonradioactive SSOP. Nucleotide sequences were obtained using an automated DNA sequencer. The allele B*1530 differs from B*1501 by a substitution of Asp for Asn in position 114 and Ser for Tyr in codon 116. The new allele B*1531 differs from B*1502 at amino acids 94, 95, and 152. The variant B*1524 was found to have N-77, I-80, A-81, L-82, R-83. A similar motif exists in B locus alleles that have the supertypic specificity Bw4 and in B*1513, B*1516, B*1517, and B*1523; it is likely to have been generated by gene conversion. Finally, the novel allele B*1527 is similar to B*1501 except for the presence of Phe instead of Tyr at position 99. Because this change exists also in B*1506, it is possible that B*1506 was derived from B*1501 through B*1527. It is of interest that a similar substitution (Cys for Tyr at position 99) distinguishes A*0201 from A*0207 and is known to determine an epitope recognized by T cells. Thus, B*1527 may also carry a change that is functionally relevant in cell-mediated immunity.