Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex A alpha A beta dimers from the endoplasmic reticulum/cis-Golgi compartment.

Invariant chain (Ii) is a nonpolymorphic, non-major histocompatibility complex (MHC)-encoded glycoprotein that rapidly associates with newly synthesized class II MHC alpha and beta chains in the rough endoplasmic reticulum. This oligomerization of Ii, alpha, and beta and their cotransport within the cell led to speculation that Ii was an essential alpha beta transport protein. However, direct tests failed to show an absolute requirement for Ii in class II MHC molecule transport. More recently, it has become clear that different class II alpha beta chain combinations vary greatly in their efficiency of cell-surface expression, based largely on the allelic origin of the alpha and beta amino-terminal regions. Because the previous tests of Ii for a role in class II molecule expression utilized efficiently expressed alpha beta combinations, we have reexamined this question with several haplotype-mismatched murine A alpha and A beta chain combinations of various potentials for cell-surface expression. Using a transient expression assay in Ii-negative COS cells, we find that many inefficiently expressed alpha beta combinations show marked augmentation of surface expression upon cosynthesis of Ii. This effect is absent or minimal with evolutionarily coselected, haplotype-matched chains that give efficient expression alone. Biochemical studies show that at least one component of the Ii effect is an increased egress of already formed alpha beta dimers from the rough endoplasmic reticulum/cis-Golgi. We suggest that these results reflect the interaction of Ii with the peptide-binding domain of the poorly expressed class II molecules, either aiding in maintenance of a transportable conformation or competing with endoplasmic reticulum retention proteins, and thus enhancing movement to the cell surface. These results suggest a complex and variable role for trans-associated alpha and beta chains in the immune responses of MHC heterozygotes and provide a method for examining Ii interaction with class II MHC molecules independent of measurement of peptide presentation to T cells.

Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.

HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.

Creation of an HLA-A2/HLA-Aw69 alloantigenic determinant on an HLA-A3 molecule by site-directed mutagenesis.

The HLA-A2 and HLA-Aw69 molecules share an antigenic determinant not expressed by HLA-Aw68 and HLA-A3. Comparison of the amino acid (aa) sequences of these molecules and previous studies of the antigenic determinant expressed by different HLA-A2 X HLA-A3 hybrid molecules had established that three aa at positions 95, 97, and 107 were possibly involved in the formation of this determinant. The HLA-A3 gene was therefore mutagenized to replace successively at these positions the HLA-A3-specific aa by the HLA-A2 residues. A single substitution at position 107 of a glycine by a tryptophan residue is sufficient for full expression by HLA-A3 molecules of the HLA-A2/Aw69 shared antigenic determinant without modification of the other serological reactivities characteristic of the HLA-A3 molecules. Previous studies of ethyl methanesulfonate mutants having shown the involvement of aa 161 in this determinant, we assume that the two aa residues 107 and 161 are close to each other.

Serological and structural analysis of HLA class I molecules: beta 2-microglobulin interacts with the two external domains of the HLA class I heavy chain.

The serological reactivities of HLA class I molecules were studied in relation to structural modifications of these molecules, including shuffling of external exons and exchange of human beta 2-microglobulin for beta 2-microglobulin from different species. Two major clusters (I and II) of monomorphic and polymorphic antigenic determinants could be delineated. beta 2-Microglobulin participates in the formation of the two clusters, indicating that the light chain interacts tightly with the two external domains of the HLA class I heavy chain. However, external molecules can modify these interactions and alter the antigenic structure of the overall molecule. Thus, fixation on HLA class I molecules of the Fab fragment of a monoclonal antibody directed at antigenic determinants associated with cluster II resulted in enhanced fixation of a monoclonal antibody (B10.6) related to cluster I. The structural and functional implications of these results are discussed.

Transfection of murine LMTK- cells with purified HLA class I genes. VII. Association of allele- and locus-specific serological reactivities with respectively the first and second domains of the HLA-B7 molecule.

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2Kd, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the B7 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.

The association between murine beta 2-microglobulin and HLA class I heavy chains results in serologically detectable conformational changes of both chains.

HLA-A3-, HLA-B7-, and HLA-CW3-transfected L cells, maintained in medium supplemented with murine serum so as to ensure that the human heavy chains were associated with murine beta 2-microglobulin, were subjected to a systematic serologic analysis for an evaluation of the structural consequences of such an heterologous association. The hybrid molecules exhibited alterations of their serologic reactivities that suggest the occurrence of structural modifications of both light and heavy chains. Thus, reactivity of HLA-A3-, HLA-B7-, and HLA-Cw3-transfected L cells with a monoclonal antibody (B1.1G6) directed at a human beta 2-microglobulin specific antigenic determinant was observed; this implies structural modifications of murine beta 2-microglobulin after its association with HLA class I heavy chains. Conversely, a profound reduction of the reactivity of the same transfectants with a monoclonal antibody (W6/32) directed at a monomorphic heavy chain related epitope was observed. The W6/32 reactivity was restored after replacement of the murine by the human light chain, indicating that the conformation adopted by the HLA class I heavy chain depends on the origin of the beta 2-microglobulin associated. Therefore it appears that the complex interactions that develop between the extracellular domains (including the one formed by the light chain) markedly influence the overall structure and the antigenic properties of HLA class I molecules.

HLA class I genes: from structure to expression, serology and function.

HLA class I genes have been isolated from phage and cosmid libraries and assayed by transfection into murine L cells. The transfection step proved to be very important because of the large number of genes (and pseudogenes) in this family. All functional genes characterized so far in this way are "classical" class I genes, i.e. members of the HLA-A, -B or -C families. Three of these have been sequenced (HLA-A3, -Aw24; HLA-Cw3) in addition to the pHLA 12.4 pseudogene. Sequence comparisons indicate, in particular, extreme conservation of the 3' non-coding region between allelic HLA-A locus genes; the general organization of all these genes (8 exons) is very similar. Restriction mapping around the functional genes has been performed to investigate the degree of conservation (e.g. between HLA-A3 regions from 2 different individuals) and examine allelism at the DNA level (e.g. between HLA-A3 and HLA-Aw24 regions). Exon shuffling experiments followed by serological analysis of the expressed product indicate that, as expected, specificities are determined by the first two domains of the molecule. However, further constructs show that as soon as a single exon is exchanged most specific reactivities disappear. CTL analysis of murine cells expressing HLA molecules has run into many difficulties but still holds promise for the study of structure-function relationships in this system.

Expression of an HLA-Bw6-related specificity by the HLA-Cw3 molecule.

Radioimmunoassay of HLA-transformed mouse L cells expressing A3, A24, B7, or Cw3 HLA class I molecules with a set of monomorphic monoclonal antibodies distinguishes between A3-A24 and B7-Cw3 patterns of reactivity. Analyses with Bw6-specific monoclonal antibodies and a human alloantiserum demonstrate the expression by the HLA-Cw3 molecules of a Bw6 public specificity related to but not identical with that expressed by the HLA-B7 molecules. Exon-shuffling experiments and inhibition studies of monoclonal antibody cell-surface fixation indicate that similar parts of B7 and Cw3 molecules account for their serological cross-reactivity.

Transformation of LMTK- cells with purified HLA class I gene. VI. Serological characterization of HLA-B7 and AW24 molecules.

Serological characterization of HLA-B7 and HLA-AW24 class I molecules following transfection of murine LMTK- cells with purified HLA class I genes was performed using human alloantisera. Induction by murine alpha interferon of the expression of class I molecules was required to obtain unambiguous identification of these molecules which appear serologically identical to the HLA-B7 and HLA-AW24 molecules expressed at the surface of human peripheral blood lymphocytes of 20 unrelated individuals. Analysis of the transformed cells with 8 different anti-HLA class I monoclonal antibodies results in the definition of 3 separate clusters of antigenic determinants shared by all HLA class I molecules. These studies further suggest the existence of locus-specific serological reactivities associated either with the HLA-A or with the HLA-B and C gene products.

Transformation of LMTK- cells with purified class I genes. V. Antibody-induced structural modification of HLA class I molecules results in potentiation of the fixation of a second monoclonal antibody.

A potentiation phenomenon was observed with HLA-A3 and CW3 transformed murine L cells between anti-HLA class I B10.6 (potentiated) and B10.8 (potentiating) monoclonal antibodies (m.Ab.). Further studies of this phenomenon with these transformed L cells indicated that: 1) no significant specific binding of B10.6 m.Ab. to HLA-A3 and CW3 transformed L cells could be demonstrated by conventional radioimmunoassay or cytofluorometric study in the absence of B10.8 m.Ab.; 2) potentiation of the fixation of B10.6 m.Ab. was induced by other anti-HLA class I m.Ab., which all reacted with the same cluster of antigenic determinants; 3) potentiation reflects an increased specific fixation of B10.6 m.Ab. to HLA class I molecules implicating its combining site; 4) potentiation was mediated by B10.8 Fab fragments. These results indicate that potentiation of the fixation of B10.6 m.Ab. to the HLA-A3 and CW3 molecules expressed by the transformed L cells reflects conformational changes of these molecules after interaction with B10.8 m.Ab.

Absence of cell surface fixation of a monoclonal antibody detectable by conventional immunoassays does not exclude expression of and interaction with the corresponding antigenic determinant.

No specific binding of anti-HLA class I B.10.6 monoclonal antibody (mAb) could be demonstrated by cell surface radioimmunoassay and cytofluorographic studies at the surface of murine transformed L cells expressing HLA-A3 or Cw3 molecules. However, specific interaction of this antibody with these molecules at the surface of these transformed cells was indirectly established, since it inhibited specifically the binding to the same HLA class I molecules of other anti-HLA class I mAb. Therefore, the absence of detectable binding of mAb, in conventional immunoassays, does not exclude expression by these cells of the corresponding antigenic determinant.

Transformation of LMTK- cells with purified HLA class I genes. II. Serologic characterization of HLA-A3 and CW3 molecules.

The expression of two different HLA class I genes was observed after transformation of LMTK- cells. The corresponding class I molecules reacted differentially with monomorphic monoclonal antibodies (m.Ab). Absorption and elution studies of the human alloantibodies reacting with the transformed cells and cellular radioimmunoassay of these cells with polymorphic m.Ab resulted in the identification of HLA-A3 and CW3 molecules. These transformed cells were used to immunize C3H mice and induce the production of xenogeneic antisera, which, following absorption, showed polymorphic reactivity with human cells, suggesting that some of these sera could be used as typing reagents.