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Preincubation of lymphocytes with des-Tyr1-gamma-endorphin (DT gamma E) inhibits the reaction between some HLA alloantisera and their corresponding antigens. One HLA-A2-specific antiserum was found which could detect a subtype of the HLA-A2 antigen on DT gamma E-treated lymphocytes from some donors. Comparison with the HLA-A2 subtypes as defined by a combination of cytotoxic T lymphocyte typing and biochemistry showed a complete correlation with the previously described HLA-A2.3 subtype.

Human alloimmune cytotoxic T cells, sensitized selectively against the HLA-A2 antigen, were tested on a panel of selected target cells. Five HLA-A2 positive outlier cells could be identified. These outlier cells were only weakly lysed by HLA-A2 specific CTLs, although they were serologically indistinguishable from the other HLA-A2 positive, strongly lysed target cells. Furthermore, it was found that the outlier cells were poor cold target inhibitors in contrast to the other HLA-A2 positive target cells, which showed adequate inhibition of specific lysis of HLA-A2 positive target cells. Population studies indicate that the frequency of such HLA-A2 outlier cells may be approximately 10%.

The population of HLA-A2-positive individuals, currently considered serologically homogeneous, can be divided into three subtypes on the basis of antigen recognition by various HLA-A2-specific cytotoxic T lymphocytes (CTLs). When these three types of HLA-A2 antigens were analyzed biochemically, they were found to be distinct. Isoelectric focusing (IEF) of HLA antigens digested with neuraminidase (NANAse) suggested that the difference(s) reside in the polypeptide backbone of the HLA-A2 heavy chain. Biochemical analysis distinguishes three distinct categories of HLA-A2 antigens: (1) a major subtype, designated HLA-A2.I, (2) a minor subtype, designated HLA-A2.II, possessing a more basic isoelectric point (IEP) and (3) a minor HLA-A2 subtype more acidic in its IEP than HLA-A2.I, designated HLA-A2.III. A fourth HLA-A2 subtype could be defined by discordance between cell-mediated lympholysis (CML) typing and biochemical analysis. The latter HLA-A2 antigen was defined as a variant by CTL, but was biochemically indistinguishable from the major subtype HLA-A2.I.

A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule.

Alloimmune CTLs specifically recognizing the HLA-A2.3 subtype could be made besides the previously described HLA-A2.1 and A2.2 subtype-specific CTLs. Examination of the fine specificity of 15 different CTLs directed against distinct HLA-A2 subtypes demonstrated further complexity of antigenic epitopes present on the A2 molecule. First, epitopes could be defined which are unique for the HLA-A2.1, A2.2, A2.3, and A2.4 subtypes. Second, epitopes could be defined which are shared between the HLA-A2.1, A2.2 and A2.4 subtypes, but which are not shared by the A2.3 subtype. Analysis of the reactivity patterns of CTLs directed against the HLA-A2.2 and A2.4 subtypes indicated that the observed cytotoxic response was dependent on the HLA type of the responder cell. Biochemical analysis demonstrated the existence of isoelectric point variation in A2 heavy chains which deviated from the expected pIs for the A2 subtypes as described previously. Individuals were identified who possessed A2 heavy chains typical for the A2.3 subtype antigen although the CTL analysis demonstrated the presence of an A2.1 subtype antigen.

The clinical response of schizophrenic patients to treatment with gamma-type endorphins was found to be associated with certain HLA class I antigens (Bw22, B15, B13). Moreover, pretreatment of lymphocytes from healthy donors with des-Tyr1-gamma-endorphin (DT gamma E) inhibits the complement-dependent cytotoxicity between alloantisera and those HLA antigens, of which the frequency was increased among schizophrenic patients, who respond well to the gamma-endorphin therapy. Also for the opiate antagonist, naloxon interactions with HLA class I antigens could be demonstrated. Using the inhibition assay with DT gamma E it was possible to detect a subtype of HLA-A2, which, until now, was only defined by cytotoxic T lymphocytes and biochemistry. These data suggest an interaction between the HLA class I antigens and the receptor(s) for DT gamma E and naloxon, which may support the hypothesis that HLA class I antigens play a role in many recognition processes. Their role in immunological recognition would then be only a specialized form of a more general function.

A comprehensive analysis of human alloimmune cytotoxic T lymphocytes (CTLs) specific for the HLA-A2 antigen identified 11% of HLA-A2 positive cells as outliers. In total, 11 unrelated serologically indistinguishable, but distinguishable by cell-mediated lympholysis (CML) HLA-A2 positive outlier cells were identified. The outlier cells could be subdivided in two subgroups according to reactivity patterns obtained with CTLs directed against the HLA-A2 antigen of outlier cells and their inhibitory capacity in specific competitive inhibition experiments. Thus, the serologically defined HLA-A2 specificity can be divided into at least three subtypes using CTLs specific for the HLA-A2 antigen. Moreover, CTLs specific for an HLA-A2 subtype could be induced when responder cells expressed a different HLA-A2 subtype antigen. On the basis of several family studies, we conclude that the subtype HLA-A2 antigens are inherited in a codominant way.