The Bw4/Bw6 difference between HLA-B*0802 and HLA-B*0801 changes the peptides endogenously bound and the stimulation of alloreactive T cells.

HLA-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 substitutions 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 endogenously 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.

Episodic evolution and turnover of HLA-B in the indigenous human populations of the Americas.

Nucleotide sequences were determined for the HLA-A, B and C alleles of three populations of Amerindians: the Havasupai tribe from North America, and the Guarani and Kaingang tribes from South America. All 15 Havasupai alleles are found in Eastern Hemisphere populations, whereas the Guarani and Kaingang each have six alleles that appear to be present only in the Western Hemisphere. Nine of the "new" alleles come from HLA-B, one comes from HLA-A and one from HLA-C: ten appear to be the result of recombination and one the result of point substitution. Of the 14 Guarani alleles and 16 Kaingang alleles, only four are held in common. Despite their differences, the three tribes possess comparable numbers of HLA class I alleles, revealing a trend for "allele turnover", in which new alleles tends to supplant older alleles rather than supplement them. Although many new HLA-B alleles have been produced in Latin America, their net effect has been to differentiate populations, not to increase allele diversity within a population. From sequence comparisons, the Amerindian subset of HLA class I allotypes appears to cover the overall ranges of peptide binding specificity, natural killer-cell interactions, and CD8 interactions, that are found in all HLA class I. The recombinations that produced the new alleles of the Kaingang and Guarani class I are predicted to have modulated these functional properties rather than radically change them. Exchange of Bw4 and Bw6 motifs by recombination are noticeably absent in the events forming new alleles in America, whereas they have been the most common of recombinations elsewhere.

A new HLA-B44 variant (B44BO [B*4408]) identified by serology.

Using HLA serology, we detected a new variant of HLA-B44- B44BO- in two families. This antigen reacts with B44 antisera and is negative with over one-third of B12 (B44, B45) sera but reacts with 50% antisera with a B62 component, especially if they contain anti-B57. The variant, B*4408, differs from the common B*4402 by 4 nucleotide substitutions in exon 2: 193, 206 and 209, which produce changes in the the alpha 1 domain at positions 41, 45 and 46 (TKE in B*4402 and AMA in B44BO); and nucleotide 213, a silent substitution. At each of these positions, B*4408 is identical to B*46 B*57 and may B*15 alleles. As anticipated from its predicted iso-electric point (5.71), one-dimensional isoelectric focusing studies showed that B44BO focuses at the same position as B*4402. The sequence and serological reactivity of this rare antigen allowed the identification of two likely epitopes shared by two different groups of HLA-B antigens.

Polymorphism in the alpha 1 helix of the HLA-B heavy chain can have an overriding influence on peptide-binding specificity.

Previously, we reported overlap in the repertoires of peptides endogenously bound by a group of HLA-B allotypes related to HLA-B7. Extending such analysis to four members of the B17 family and seven members of the B15 family shows that allotypes that share sequence identity in the alpha 1 helix of the class I heavy chain possess markedly similar peptide-binding specificities. Members of the B17 family share a preference for peptides with serine, threonine, or alanine at position 2 and aromatic residues at the carboxyl terminus. Strikingly, the presence of a segment of the B17 alpha 1 helix in B*1516 and B*1517 confers the B17-like peptide-binding motif. The strong influence of natural variation in the alpha 1 helix is exemplified by the differences in peptide-binding specificity of B15 allotypes related by conversion events that replaced segments of the alpha 1 helix. In contrast, evolutionary changes that are confined to the alpha 2 domain confer less dramatic change. They do not perturb the primary anchors of the peptide-binding motif but can modulate the specificity through development and diversification of secondary anchors. Our results, in combination with those obtained previously for other HLA-B allotypes, suggest a general trend whereby polymorphism in the alpha 1 helix is the overriding influence on peptide-binding specificity of HLA-B allotypes, while amino acid substitutions in the alpha 2 domain play a more modulatory role.

Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors.

The expression of KIR and CD94:NKG2 receptors was determined for more than 100 natural killer (NK) cell clones obtained from two blood donors who differ in their HLA class I and KIR genes. More than 98% of the clones were inhibited by individual autologous class I allotypes, and every clone was inhibited by the combination of autologous allotypes. The patterns of inhibition correlate with expression of inhibitory receptors of defined specificity. One donor possesses three class I ligands for KIR, and a majority of NK cells use KIR as their inhibitory receptor; the second donor possesses only a single ligand for KIR, and a majority of NK cells use the more broadly reactive CD94:NKG2a as their inhibitory receptor. Because of these differences, the first donor has subpopulations of NK cells that kill cells of the second donor, whereas the NK cells of the second donor are universally tolerant of cells from the first donor.

Expression of an unusual Bw4 epitope by a subtype of HLA-B8 [B*0802].

The primary structure of a variant HLA-B8 antigen has been determined by cDNA cloning and sequencing. The variant, B*0802 differs, from the common B*0801 subtype at positions 77-83 of the alpha 1 helix that determine the Bw4 and Bw6 public epitopes. Whereas B*0801 has the common Bw6 motif, B*0802 has the Bw4 motif found in B*13 and B*44 allotypes. Serological analysis of B cell lines expressing B*0802 and of a B*0802 transfectant made with the HLA-A,B negative cell line 721.221 shows that B*0802 reacts with Bw4-specific antibodies, but at a level much lower than expected for Bw4 positive HLA-B allotypes.

Definition of a new HLA-B7 subtype (B*0704) by isoelectric focusing, family studies and DNA sequence analysis.

During screening of potential bone marrow donors, a previously undescribed banding position for the serologically defined HLA-B7 antigen was identified in three unrelated families using one dimensional isoelectric focusing and class I specific Western blot analysis. The isoelectric point of the new variant is more acidic than the two HLA-B7 variants that had been defined before. In each family the new B7 variant was found linked to HLA-A2 and -Cw7. Cloning and sequencing of full-length clones of complementary DNA showed that the new allele (B*0704) differs from B*0702, the common allele encoding HLA-B7, by three nucleotide substitutions within the codon for residue 156 of the mature heavy chain. As a result of these differences amino acid 156 is changed from arginine to aspartic acid, a difference consistent with the isoelectric points. The group of three nucleotide substitutions that distinguish B*0704 from B*0702 is present in other HLA-B alleles.

The HLA-A,B,C genotype of the class I negative cell line Daudi reveals novel HLA-A and -B alleles.

Daudi, a lymphoblastoid B cell line derived from an African Burkitt lymphoma does not express HLA-A,B,C antigens at the cell surface. Although HLA-A,B,C heavy chains are made normally they do not assemble into functional molecules because beta 2-microglobulin is absent. Previous serological analysis of somatic cell hybrids indicated that the HLA haplotypes of Daudi encoded HLA-A1, A10(A26), B17, and B16(38) antigens. Here we describe the application of molecular methods: ARMS-PCR, cDNA cloning and sequencing, immunoprecipitation and gel electrophoresis, to define the class I genotype of the Daudi cell line which is HLA-A*0102, A*6601, B*5801, B*5802, Cw*0302 and Cw*0602. With the exception of the B38 antigen, which is not a product of the alleles defined, the genotype is consistent with the serological description. Two previously undiscovered alleles emerged from this analysis: A*0102 and B*5802. The A*0102 allele differs from A*0101 by 5 nucleotide substitutions within exon 2 where it has a motif shared with A*30 alleles; the B*5802 allele differs from B*5801 by 3 substitutions in exon 3 where it has a motif shared with B*14 alleles. Subtyping HLA-A1 alleles showed A*0102 was well represented amongst individuals typed serologically as A1 in an African population but was absent from caucasoids. B*5802 has been found in a second individual. Thus the novel A and B alleles are not specific to the Daudi tumor. Overall, this analysis of a single East African cell illustrates the power of molecular methods to define new class I HLA alleles in non-caucasoid populations.

HLA-B16 antigens: sequence of the ST-16 antigen, further definition of two B38 subtypes and evidence for convergent evolution of B*3902.

The ST-16 antigenic specificity of the HLA-B locus is defined as a B39 variant of Mexican-Americans. Nucleotide sequencing of cDNA shows the ST-16 allele (B*3905) differs from B*39011 by a single substitution that substitutes tyrosine for aspartic acid at position 74 of the mature class I heavy chain. The complete coding region sequence for the common caucasoid allele encoding the B38 antigen has been determined. This B*3801 allele differs from B*3802 at two nucleotide substitutions within the Bw4 sequence motif. B*3801 and B*3802 may have been derived independently from B*39011 by conversion events with B alleles donating distinctive Bw4 motifs. A novel allele B*39022 derived from a Colombian Indian differs from the B*39021 allele of Japanese origin at two widely separated silent substitutions. Comparison of sequences for the known B16 alleles suggest that B*39021 and B*39022 were independently derived by recombination from B*39013 and B*39011 respectively.

A small test of a sequence-based typing method: definition of the B*1520 allele.

Santamaria et al. (Human Immunology 1993 37: 39-50) describe a method of sequence-based typing (SBT) for HLA-A, B and C alleles said to give "unambiguous typing of any sample, heterozygous or homozygous, without requiring additional typing information". From SBT analysis, which involves determination of partial sequences of mixed alleles, these investigators reported that cell lines KT17 (HLA-B35,62) and OLGA (HLA-B62) from the reference panel of the 10th International Histocompatibility Workshop express novel variants of HLA-B15 (B1501-MN6) and HLA-B35 (B3501-MN7) respectively. To study further the novel alleles, we cloned and sequenced full-length HLA-B cDNA clones isolated from the KT17 and OLGA cell lines. We find that KT17 expresses B*3501, as assigned by SBT, and B*1501, the common allele encoding the B62 antigen. We were unable to confirm that KT17 expresses the novel B1501-MN6 variant identified by SBT. For OLGA our analysis confirms the partial sequences obtained by SBT. Thus OLGA expresses B*1501 and a novel HLA-B allele. The complete sequence of the latter shows it is a hybrid having exons 1 and 2 in common with B*1501 and other B15 subtypes and exons 3-7 in common with B*3501 and related molecules including B*5301 and B*5801. The novel allele has been designated B*1520 because of its sequence similarity with the B15 group; furthermore, serological analysis shows that the B*1520 product does not express epitopes in common with either B35, B53 or B58. The B*1520 heavy chain has a similar isoelectric point to A*3101; B*1520 was undetected by previous applications of isoelectric focusing because B*1520 and A31 are both expressed by OLGA. In conclusion, HLA-B typing of two cell lines by cDNA cloning and sequencing gives concordant results with SBT for three of the four alleles. The cause of the discrepancy for the fourth allele is unknown, however, this finding indicates that the novel HLA-A, B and C sequences emerging from SBT studies need independent verification.