HLA‐C heavy chains free of ß₂‐microglobulin: distribution in normal tissues and neoplastic lesions of non‐lymphoid origin and interferon‐γ responsiveness

Abstract

Lacking monospecific antibodies to HLA-C, the expression and synthesis of these molecules have been difficult to evaluate. Using biochemical and flow cytometry approaches, the present report demonstrates that the reactivity of the murine monoclonal antibody L31 is restricted to naturally occurring HLA-C (HLA-Cw1 through -Cw8), HLA-B8 and HLA-B51 heavy chains not associated with beta2-microglobin (beta2m). This is due to two properties of HLA-C heavy chains: (a) they share the L31 epitope which distinguishes them from all the HLA-A and most HLA-B molecules; (b) they accumulate intracellularly, in a beta2m-free form, in much greater amounts than most L31-reacting HLA-B heavy chains. On the basis of this restricted reactivity, a representative panel of normal and neoplastic human tissues and cells derived from HLA-B8- B51- individuals was selected and employed to assess the tissue distribution, surface expression and IFN-gamma responsiveness of beta2m-free HLA-C heavy chains. At variance from antibody W6/32 to beta2m-associated heavy chains, L31 stains normal and neoplastic tissues with a ground-glass pattern and weakly binds to the surface of viable cells, even after treatment with interferon gamma (IFN-gamma). Thus, beta2m-free HLA-C heavy chains are, for the most part, located intracellularly. In spite of their distinct cellular localization, L31- and W6/32-reacting molecules have an overlapping tissue distribution, undergo concordant changes upon transformation and are upregulated in their synthesis by IFN-gamma to a similar extent. These observations demonstrate a coordinate regulation of HLA-C with HLA-A and -B molecules. In addition, they indicate that the assembly of HLA-C is impaired in most body districts and IFN-gamma is unable to completely reverse this impairment. The present results are consistent with a low surface expression of HLA-C and with a privileged role of these molecules in signaling class I loss to cytotoxic effectors in pathological conditions.

A Single Bottleneck in HLA-C Assembly

“…The HLA-A, -B, and -C transfectants in 221 were obtained through the courtesy of several investigators (see "Acknowledgments") and are referenced elsewhere (14). Epstein-Barr virus-transformed, HLA-typed, and homozygous B cells are also described elsewhere (12,14).…”

“…For immunoprecipitation, purified antibodies were covalently linked to Affi-Gel (Bio-Rad). The isoelectric focusing (IEF) and Western blot techniques (reducing conditions in all cases) used have been described (11,12,14). In vitro assembly experiments were performed exactly as described in the presence of the NCPERIITL Cw1 ligand (11).…”

“…Consistent with these biochemical features, ␤ 2 m-free HLA-C heavy chains associate primarily with calnexin and, in much smaller amounts, with members of the peptide-loading complex, including TAP and tapasin (11). Thus, it appears that ␤ 2 m-free HLA-C heavy chains accumulate not only at the calnexin/␤ 2 m checkpoint in ␤ 2 m-defective mutants (as would be expected if they just failed to bind ␤ 2 m), but also at subsequent steps, on several distinct chaperones acting in sequence, and even in the absence of obvious defects in the antigen-processing/peptide-loading machinery (11,12).…”

Biosynthesis of HLA-C heavy chains in melanoma cells with multiple defects in the expression of HLA-A, -B, -C molecules

“…Pulsechase studies have shown that the L31 epitope becomes 'hidden' upon assembly with β 2 m, this process being more efficient in HLA-B than HLA-C heavy chains. HLA-A heavy chains are essentially unreactive with L31 (Setini et al, 1996;Giacomini et al, 1997). HC-10 was raised against denatured HLA-B locus heavy chains and cross-reacts with certain HLA-C and -A products including B8 and A25 (Stam et al, 1986).…”

“…The remaining bands in the low (acidic) region of the gel were assigned to HLACw*0701. In contrast to HLA-A and -B alleles (Hajek-Rosenmayr et al, 1989;Setini et al, 1996), HLA-Cw7 displayed a multiplicity of irregularly spaced bands, five of which (marked as Cw7 in (Setini et al, 1996;Giacomini et al, 1997), it does not represent an artifact resulting from plasmid construction or transfection. The nature of this component was not further investigated.…”