Expression of β₂m‐Free HLA Class I Heavy Chains in Neuroblastoma Cell Lines

Abstract

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with beta 2-microglobulin (beta 2m) by neuroblastoma (NB) cell lines IMR-32 and LA-N-1. The cells, which express barely detectable amounts of beta 2m-free (L31-positive molecules) and beta 2m-complexed HLA class I antigens (W6.32- and BBM.1-reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon-gamma (rIFN-gamma) treatment led IMR-32 and LA-N-1 cells to almost exclusively express beta 2m-complexes HLA class I heavy chains. Surface beta 2m-free MHC class I molecules displayed a molecular mass of approximately 45 kDa and did not bind exogenously added beta 2m. No changes in the synthesis of either HLA class I and beta 2m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post-translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of beta 2m-free class I heavy chains is a feature of other cell types, such as NB cells.

Multiple defects of the antigen-processing machinery components in human neuroblastoma: immunotherapeutic implications

“…Furthermore, rIFN-g treatment induced surface b 2 m upregulation in all cell lines (Marozzi et al, 1993) and surface b 2 m-free HC upregulation in two of them (Pickl et al, 1993). A possible explanation for these findings is that b 2 m and HC dissociate from some HLA-A, B and C complexes that do not contain peptides and are therefore unstable.…”

Expression of distinct conformations of free HLA-Cw4 heavy chains in transfected neuroblastoma cells

“…In IMR-32 cells, INF-y is capable of trans-activating [32m and HLA class I mRNAs, consequently inducing the surface expression of complexed W6.32-positive (but not of ~32m-free) HLA class I molecules (Marozzi et al_ 1993). Figure 2A shows the northern blot analysis of total RNA from three of the transfectants cultured for one day with IFN-y, using HLA-Cw4 (panel 1) and [~2m (panel 2) cDNAs as hybridization probes.…”

“…Filters were then incubated with 125I-L31 mAb, and the binding revealed by autoradiography (Marozzi et al_ 1993). Total RNA, extracted from approximately 107 IMR-32 cell clones as described (Chomczynski and Sacchi 1987), was fiactionated by formaldehyde agarose gel electrophoresis and blotted onto nitrocellulose filters (Amersham, Buckinghamshire, UK).…”

“…Total RNA, extracted from approximately 107 IMR-32 cell clones as described (Chomczynski and Sacchi 1987), was fiactionated by formaldehyde agarose gel electrophoresis and blotted onto nitrocellulose filters (Amersham, Buckinghamshire, UK). The hybridization was carried out with 3zP-labelled probes as previously described (Marozzi et al 1993).…”

“…Resting T lymphocytes express negligible levels of free chains, but cell activation induces their expression in human (Schnabl et al 1990;Grassi et al 1991), and mouse lymphocytes of HLA-Cw4 transgenic mice (Aiuti et al 1995). HLA class I free chains are also expressed to different extents on some B, T and myeloid cell lines, on cell line-derived clones representing distinct stages of differentiation (Madrigal et al 1991;Pickl et al 1993), and on neuroblastoma cell lines upon in vitro differentiation (Marozzi et al 1993).…”