J. Alejandro Madrigal

Molecular definition of an elusive third HLA-A9 molecule: HLA-A9.3

The HLA-A9 family has been characterized as possessing two well defined specificities; HLA-A23 and A24. Serological studies have suggested the presence of a third member of this family HLA-A9.3, however there is doubt surrounding the existence of this specificity. HLA-A23, A24, and the putative A9.3 proteins were analyzed biochemically by immunoprecipitation and isoelectric focusing. Both HLA-A24 and A9.3 have identical isoelectric points whereas A23 is different. We have sequenced cDNA encoding HLA-A23, A24, and A9.3. From the observed protein sequences, we found A9.3 to differ from A24 by two amino acid substitutions located in the α2 helix of the class I molecule. These substitutions are expected to significantly change the shape of the peptide binding cleft.

New HLA-A2 variants defined by monoclonal antibodies and cytotoxic T lymphocytes

Three HLA-A2 variants, A2-DW, A2-KC, and A2-Lee, were identified in three Chinese donors using a panel of monoclonal antibodies. A2-DW was negative with two of the ten HLA-A2 monoclonal antibodies tested, whereas A2-KC was negative with five of the ten and A-2 Lee was negative with one.Epstein-Barr virus-specific cytotoxic T cells generated from the A2-DW donor recognized and killed target cells prepared from the A2-KC donor, but did not recognize target cells from HLA-A2.1, −A2.2, or −A2.4 donors. In isoelectric focusing studies, A2-DW and A2-KC focus in identical positions more acidic than the other HLA-A2 antigens tested.

Polymorphic specificity of Q1/28, a monoclonal antibody that preferentially reacts with free class I heavy chains

The Q1/28 monoclonal antibody (mAb) reacts with class I HLA heavy chains, either when free or bound to /32-microglobulin (/32-m). Subsequent to the initial description of Q 1/28 by Quaranta and co-workers (1981), the target epitope for the antibody was mapped to the o~ 3 domain of the class I heavy chain (Engelhard et al. 1985) and found to be present on HLA-B7 but not HLA-A2 heavy chains (Parham et al. 1987). To further investigate the polymorphic specificity of Q 1/28 we measured its binding to a panel of CIR cells which had been transfected with different class I HLA-A and B genes (Storkus et al. 1987). CIR is a mutant human B cell line that expresses HLA-Cw4 and low levels of HLA-B35 as the only endogenous class I HLA products (Zemmour et al. 1992). Flow cytometric analysis (Table 1) showed Q1/28 bound to HLA-B7 and B27, but not to HLA-A2, A68, or A69 molecules. Low but significant binding of Q1/28 to untransfected CIR cells was also observed, indicating the endogenous Cw4 and/or B35 molecules were also reactive. Isoelectric focusing (IEF) of immunoprecipitates from biosynthetically radioloabeled B-cell lines confirmed that Q1/28 reacted with B27 but not A2 and also revealed positive reactions with A3, A11, and B35, and a negative reaction with A31 (Fig. 1). Transfectants of the LCL721 cell line, which expresses no endogenous class I HLA molecules, with HLA-E,G and with a hybrid of the A2 and HLA-F genes containing exon 3 of HLA-F were also examined for binding to Q1/28. Reactivity was observed with HLA-E and the A2/F construct but not with HLA-G. As HLA-A2 does not bind Q1/28, the positive reactivity with the A2/F hybrid indicates the ~3 domain of the HLA-F heavy chain is positive for the epitope. Q1/28 reactivity correlated with amino acid substitutions at positions 193,207, and 253 in the e~ 3 domain of

Serological Definition of HLA-A2 Variants

Variants of HLA-A2 have previously been defined by 1) using the highly specific ability of cytotoxic T lymphocytes to recognize HLA class I antigens on virus infected target cells (1), 2) differences in isoelectric focusing (IEF) points (2), 3) peptide mapping (3), and more recently, 4) different patterns of reactivity with monoclonal antibodies (MAb) (4).

A New DR Antigen, Associated with DQw7, Defined Serologically and by Restriction Fragment Length Polymorphism

The inheritance of a new DR antigen, COR, was studied in a two-generation family. The COR antigen has been inherited by two of four children clearly segregating with Al Cw7 B7 and DQw7. This family has now been typed in the last three International Histocompatibility Workshops, and only in the recent Tenth Workshop have any sera or monoclonal antibodies with COR reactivity been identified. These include 10W1008 (DR1++), 10W3010 (DR1+DR7+DR9+), and 10W3011 (DR1,2,7,9,w10, +), which all also react with DR“BON” (A. Cambon-Thomsen, personal communication).

Variants of HLA-Aw68 Recognized by Isoelectric Focusing

The family of related molecules called HLA-A2/28 includes all the subtypes of A2 and the two splits of A28, namely Aw68 and Aw69 (1). Moreover, Aw68 has been split serologically and by DNA sequencing into two further groups (2,3).

Biochemical Analysis of HLA Class II Antigens Using Transfected Cells

The class II products are expressed from a minimum of three sets of loci: HLA-DR, HLA-DQ, and HLA-DP. However, these loci are complex; for example, each haploid genome may express: one α and two or more β genes in the HLA-DR subregion, at least one α and one β gene in the HLA-DQ subregion and another α and β pair in the HLA-DP region (1,2).