Translation in Xenopus laevis oocytes of hybrid selected LEW rat RT1.B alpha- and beta-chain transcripts results in serologically discrete class II polypeptide chain complexes.

Using the non-crossreactive mAb MRC-OX3 and MRC-OX6, two serologically distinct RT1.B-specific (I-A equivalent) alpha, beta heterodimers have previously been described by us as residing at the cell surface of LEW rat spleen cells. The two-chain elements were suggested to represent stable conformation isomers, diverged by dissociation of the mature gamma-chain from a mAb MRC-OX6 reactive biosynthetic intermediate, composed of terminally glycosylated alpha-, beta- and gamma-chains. In this study we addressed the question of whether or not the presence of terminally glycosylated invariant gamma-chain was obligatory for the formation of the two MRC-OX3 and MRC-OX6 reactive two-chain complexes. The synthesis of RT1.B-specific alpha, beta heterodimers was therefore initiated, in the absence of accompanying invariant gamma-chains, by microinjecting hybrid-selected RT1.B alpha- and beta-specific mRNA into oocytes of Xenopus laevis for translation. Class II molecules produced were analyzed by affinity chromatography of radioactive-labeled oocyte detergent lysates using the appropriate monoclonal immunoadsorbents for identification. Although rat gamma-chain mRNA was excluded in this assay system, distinct MRC-OX3 and MRC-OX6 reactive two-chain complexes were detected by two-dimensional gel electrophoresis. These findings clearly indicate that the formation of the two RT1.B-specific alpha, beta heterodimers is independent of the presence of the rat invariant gamma-chain.

Synthesis and expression of MHC class II molecules in the absence of attached invariant chains by recombinant-interferon-gamma-activated bone-marrow-derived macrophages.

Pure populations of in vitro propagated bone marrow-derived macrophages are constitutively Ia negative. Co-culturing of these cells with recombinant interferon-gamma (rIFN-gamma) resulted in the appearance of high amounts of Ia antigens at the cell surface of essentially all cells. The continuous presence of the stimulus was a prerequisite for sustained Ia expression because removal of the stimulus resulted in rapid decline of surface Ia. Two-dimensional (2D) gel analysis (1D isoelectric focusing, 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of class II molecules synthesized by rIFN-gamma-stimulated bone marrow macrophages (BMM phi) revealed that, in contrast to class II complexes hitherto described, BMM phi-derived I-A and I-E subregion-encoded subunits are synthesized without invariant chains. The invariant chain-deficient alpha,beta heterodimers are expressed at the cell surface in high proportions demonstrating that their correct assembly and transport to the cell surface is accomplished in the absence of invariant chains. The lack of invariant chains appears not to be due to a failure of rIFN-gamma to induce transcription of the gamma-chain gene because rIFN-gamma-induced, in contrast to uninduced, BMM phi accumulate high levels of invariant chain-specific transcripts as evidenced by Northern blot analysis. These findings suggest that translation of gamma-chain-specific mRNA is blocked in BMM phi for as yet unknown reasons. Alternatively, newly synthesized gamma chains might have escaped their regular intracellular maturation pathway as a result of unidentified modifications mediated by altered post-translational processing mechanisms.

Biochemical aspects of constitutive and facultative expression of MHC class II molecules at the pre- and posttranslational level.

Biosynthetic properties of Ia antigens derived from B cells, from T cell clone BK-BI-2.6.C6 and from rIFN-gamma induced bone marrow macrophages (BMMO) were evaluated before and after translation. Gross features of B and T cell Ia were found to be very similar. At the pretranslational level no obvious differences were noticed. Following translation in both cases alpha, beta heterodimers associate noncovalently with invariant proteins of the gamma-chain group. These proteins, in particular the principal gamma-chain (p32) become the target of extensive posttranslational modification by sialic acid. The effect is much more pronounced in T cells than in B cells. Unlike in B cells, at the cell surface the bulk of terminally glycosylated gamma-chain (p35) remains attached to the alpha, beta isotypic complexes. This finding renders such T cells very useful for studies on the possible role of the invariant chain during antigen presentation. In BMMO rIFN-gamma elicited high levels of transcripts of polymorphic alpha, beta and invariant gamma chains. However, translation of the polymorphic subunits and their expression at the cell surface occurred in substantial amounts in the total absence of invariant chains. This finding argues against the notion of gamma being required for assembly of alpha, beta isotypes and their intracellular transport to and expression at the plasma membrane.