ORIGINAL PAPER Kelly L. Arnett ? Wen Huang ? Nicholas M. Valiante Linda D. Barber ? Peter Parham The Bw4/Bw6 difference between HLA-B*0802 and HLA-B*0801 changes the peptides endogenously bound and the stimulation of alloreactive T cells Received: 29 October 1997 AbstractmHLA-B*0801 is unique among HLA-B allotypes in having dominant amino acid anchors at positions 3 and 5 of the peptide-binding motif. HLA-B*0802 is a variant of HLA-B*0801 in which the Bw6 sequence motif is replaced by a Bw4 sequence motif. This change, involving substitu- tions at positions 77, 80, 81, 82, and 83 of the B*08 heavy chain, is probably the result of a single evolutionary event of interallelic conversion. Moreover, the difference between B*0802 and B*0801 is sufficient to stimulate a cytotoxic T-cell response. To assess further the functional impact of the Bw4 motif on a B8 background, we compared the peptide-binding specificity of the B*0801 and B*0802 allotypes by sequencing the mixture of peptides endogen- ously bound to B*0802 and 12 individual peptides purified from that mixture. The HLA-B*0802 allotype, while able to bind some peptides bound by B*0801, has a broader repertoire of endogenously bound peptides than B*0801: the peptides bound by B*0802 are more variable in length and exhibit greater diversity in the carboxyl-terminal amino acid which interacts with the F pocket. Key wordsmHLA-B8 subtypes ? Peptide-binding specificity ? Endogenous peptide ? Alloreactivity Introduction Of the human HLA class I loci, HLA-B is the most polymorphic, with nearly 200 HLA-B alleles defined to date (Bodmer et al. 1997). HLA-B polymorphism is con- centrated at sites of functional importance within the a1 and a2 domains of the protein, including positions that determine peptide-binding specificity. New HLA-B alleles evolve from existing alleles, primarily by events of inter- allelic gene conversion in which a short segment of one allele is replaced by a homologous region of another. The conversion events which have generated new alleles tend to occur in five regions of the a1 and a2 domains (Parham et al. 1997). Of these, the region encompassing the Bw6 and Bw4 epitopes, amino acids 77±83, appears to be most frequently involved. There are minimally 14 pairs of HLA-B allotypes in which the two partners are identical at all positions except those forming the Bw4 and Bw6 epitopes. Among HLA-B allotypes there are four defined variants of the Bw4 epitope (Table 1); a fifth variant is found only in HLA-A allotypes. The most common Bw4 variant is that present in B*5301 and B*2702, with more than half of the Bw4-positive HLA-B alleles having this motif. The second most common variant is that present in B*2701, B*13, and B*0802. The Bw4 epitope is a ligand for one of the inhibitory class I receptors of natural killer (NK) cells (Cella et al. 1994; Gumperz et al. 1995). Furthermore, the presence of the Bw4 epitope in B*5301 provides a resis- tance to severe malaria not afforded by its Bw6-containing partner, B*3501 (Hill et al. 1992). Thus the conversions which replace the Bw6 epitope with Bw4 can have impor- tant effects upon the immunological functions of HLA-B molecules. A previous study of HLA-B*0801 peptide-binding spe- cificities revealed that this allotype is unique among HLA-B molecules in that it has dominant anchors at positions 3 and 5 of the peptide-binding motif (DiBrino et al. 1994; Malcherek et al. 1993; Sutton et al. 1993). All other HLA-B allotypes analyzed have anchors at position 2 and at the carboxyl-terminus. HLA-B8, a common human class I antigen, is usually associated with the Bw6 public epitope (Imanishi et al. 1992). Recent studies have identi- fied two Bw4-positive B8 variants of the common B*0801: HLA-B*0802 (Arnett et al. 1995), and HLA-B*0803 (E. Petersdorf, personal communication). To access the func- tional effects of the Bw4 epitope on the unusual peptide- binding background of HLA-B8, we compared the peptide binding-specificity of B*0801 and B*0802 and examined the capacity of the difference between B*0801 and B*0802 to stimulate alloreactive T cells. K.L. Arnett ? W. Huang ? N.M. Valiante ? L.D. Barber ? P. Parham ( ) Departments of Structural Biology and Microbiology & Immunology, Stanford University, Stanford, CA 94305, USA Immunogenetics (1998) 48: 56 ± 61 Ó Springer-Verlag 1998