Interaction of MHC class II molecules with the invariant chain: role of the invariant chain (81-90) region.

Association of the invariant chain (Ii) with MHC class II alpha and beta chains is central for their functionality and involves the Ii CLIP(81-104) region. Ii mutants with an antigenic peptide sequence in place of the CLIP region are shown to form alphabetaIi complexes resistant to dissociation by SDS at 25 degrees C. This reflects class II peptide binding site occupancy, since substitution of the major anchor residue within the antigenic peptide sequence of one of these Ii mutants abolishes its capacity to form SDS-stable heterotrimers. Therefore, CLIP location within Ii is compatible with CLIP access to the class II binding groove. However, in wild-type Ii this access does not lead to a tight association, which seems to be affected by the Ii 81-90 region. This region, together with a region C-terminal of CLIP, is shown to contribute to Ii association with HLA-DR1 molecules. Thus, Ii mutants with non-HLA-DR1 binding sequences in place of the CLIP(87-102) region can still associate with HLA-DR1 molecules and inhibit peptide binding.     

The invariant chain is a phosphorylated subunit of class II molecules

The phosphorylation of the MHC, class II-associated invariant chain (gamma) is demonstrated in human B-lymphoblastoid, melanoma, and histiocytic lymphoma cell lines. Two-dimensional nonequilibrium gel electrophoresis of invariant chain and class II Ag immunoprecipitates isolated from [32P]orthophosphate-labeled cells demonstrates labeling of both free and class II-associated gamma, gamma s, and p41 forms of the invariant chain. The gamma 2/gamma 3 form of the invariant chain is not phosphorylated. Phosphoamino amino acid analysis of isolated invariant chain shows phosphorylation of serine residues. The isolation of invariant chain from 32P-labeled microsome preparations digested with proteinase K demonstrates that the phosphorylation occurs in the cytoplasmic tail. Limited proteolysis of [32P]orthophosphate-, [35S]cysteine-, and [35S]methionine-labeled invariant chain also indicates that the 32P-label is incorporated into the cytoplasmic domain. These results pinpoint serine residues at positions 9, 26, and 29 in the N-terminal cytoplasmic tail as potential sites for the phosphorylation of the invariant chain. Phosphorylation may be another mechanism by which the functions of invariant chain in class II-dependent immune responses are regulated.     

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss)

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.     

Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain

The intracellular transport and location of major histocompatibility complex (MHC) class II molecules and associated invariant chain (Ii) were investigated in a human melanoma cell line. In contrast to the class II molecules, which remain stable for greater than 4 h after synthesis, the associated Ii is proteolytically processed within 2 h. During or shortly after synthesis the NH2-terminal cytoplasmic and membrane-spanning segment is in some of the Ii molecules cleaved off; during intracellular transport, class II associated and membrane integrated Ii is processed from its COOH terminus in distinct steps in endocytic compartments. Immunocytochemical studies at the light and electron microscopic level revealed the presence of class II molecules, but not of Ii on the cell surface. Intracellularly both Ii and class II molecules were localized in three morphologically and kinetically distinct compartments, early endosomes, multivesicular bodies, and prelysosomes. This localization in several distinct endosomal compartments contrasts with the localization of class II molecules in mainly one endocytic compartment in B lymphoblastoid cell lines. As in these lymphoblastoid cell lines Ii is known to be rapidly degraded it is conceivable that the rate of proteolysis of the class II associated Ii and its dissociation from class II molecules modulates the retention of the oligomeric complex in endocytic compartments, and as a consequence the steady-state distribution of these molecules within the endosomal system.     

Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.

Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER.     

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.     

Isoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments

Newly synthesized class II molecules of the major histocompatibility complex must be transported to endosomal compartments where antigens are processed for presentation to class II-restricted T cells. The invariant chain (Ii), which assembles with newly synthesized class II alpha- and beta-chains in the endoplasmic reticulum, carries one or more targeting signals for transport to endosomal compartments where Ii dissociates from alpha beta Ii complexes. Here we show that the transport route of alpha beta Ii complexes is regulated selectively by two forms of Ii (p33 and p35) that are generated by the use of alternative translation initiation sites. Using a novel quantitative surface arrival assay based on labeling with [6-3H]-D-galactose combined with biochemical modification at the cell surface with neuraminidase, we demonstrate that newly synthesized alpha beta Ii molecules containing the Ii-p33 isoform can be detected on the cell surface shortly after passage through the Golgi apparatus/trans-Golgi network. A substantial amount of these alpha beta Ii complexes are targeted to early endosomes either directly from the trans-Golgi network or after internalization from the cell surface before their delivery to antigen processing compartments. The fraction of alpha beta Ii complexes containing the p35 isoform of Ii with a longer cytosolic domain was not detected at the cell surface as determined by iodination of intact cells and the lack of susceptibility to neuraminidase trimming on ice. However, treatment with neuraminidase at 37 degrees C did reveal that some of the alpha beta Ii-p35 complexes traversed early endosomes. These results demonstrate that a fraction of newly synthesized class II molecules arrive at the cell surface as alpha beta Ii complexes before delivery to antigen processing compartments and that class II alpha beta Ii complexes associated with the two isoforms of Ii are sorted to these compartments by different transport routes.     

Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus.

During the initiation of an immune response, antigen-presenting cells employ MHC class II antigens as key molecules to present small peptides to CD4-positive lymphocytes. The invariant chain (Ii; CD74) plays a critical role in this process by influencing the expression and peptide loading of the MHC class II molecules. Therefore, coordinate expression of these molecules is believed to play an important role in antigen presentation. This study explores the expression of these molecules in fetal tissues. Formalin-fixed, paraffin-embedded multi-organ tissue blocks from aborted fetuses (age range 7-22 weeks) were immunostained for Ii/CD74 and MHC class II antigens using commercially available monoclonal antibodies for Ii/CD74 (LN2) and MHC class II antigens (LN3), respectively. Coordinate staining for Ii/CD74 and MHC class II antigens was seen in the skin, proximal renal tubules, tips of small intestinal mucosa, and cells of the reticuloendothelial system, including the spleen and thymus. Expression of Ii/CD74, but not of MHC class II antigens, was seen in pulmonary alveolar epithelium in all cases and in testicular Leydig cells (11 of 11 testes examined). The distribution and intensity of staining did not change significantly with age. In conclusion, this study describes distribution of Ii/CD74 and MHC class II antigens in human fetal tissues. Coordinate expression of Ii/CD74 and MHC class II antigens was identified in most fetal tissues, but there were also notable exceptions. In all cases this took the form of expression of Ii/CD74 in the absence of MHC class II expression. Discordance was particularly striking in pulmonary alveolar epithelium and testicular Leydig cells. This suggests that the Ii/CD74 molecule has functional roles in addition to its role in antigen presentation.     

Transcriptional regulation of HLA class II and invariant chain genes

Class II (Ia) antigens are coded for by a family of genes located in the human MHC (HLA). These genes are regulated in a complex manner, being constitutively expressed, inducibly expressed, or not expressed, depending on the cell type examined. 6.1.6 is a variant of a normal B lymphoblastoid line that has lost expression of all class II molecules and has previously been shown to have a defect in the regulation of class II genes. In this report, we have examined those genes by Southern and Northern blotting and have found that 6.1.6 is severely deficient in mRNA for all class II genes examined, although the genes are structurally intact. P30, a partial revertant of 6.1.6, re-expresses mRNA for a subset of class II genes. mRNA for the class II-associated invariant chain is substantially reduced but not absent in 6.1.6.     

Invariable chain (Ii-chain) as a highly important molecular chaperone of histocompatibility class II molecules

The publications on the structural and functional features of the main molecular shaperon of li-chain were updated. In the 1990s the determination of molecular shaperon as a group of mutually unrelated protein molecules taking part in the assembly of other polypeptides was worked out. In humans the main isoform of li-chain is the protein with a mol. wt. of 31-33 kD. A great variety of functions of this shaperon is linked with definite amino acid sequences. In particular, the molecular shaperon of the molecules of the main histocompatibility complex-II has functions connected with the presentation of antigen and the differentiation of B lymphocytes.     

A human Ia cytoplasmic determinant located on multiple forms of invariant chain (gamma, gamma 2, gamma 3)

An antigenic determinant present in the cytoplasm, but not on the surface membrane of human Ia+ cells, is defined by a monoclonal antibody (VIC-Y1) and is shown by immunoprecipitation and by NEPHGE to be expressed by Ia oligomers. Immunoprecipitations of cellfree translates and of purified Ia subunits indicate that the VIC-Y1 determinant is located on the Ia gamma-(invariant) chain, as well as on two other related molecules, provisionally termed gamma 2 and gamma 3. Within our experimental conditions, the three forms of gamma-chains co-precipitate exclusively with Ia oligomers. As detected by VIC-Y1 and in the limits of our assays, gamma-chains could not be found at the cell surface; their tissue distribution, determined by cytoplasmic indirect immunofluorescence with VIC-Y1, closely resembles that of Ia antigens, with the possible exception of acute lymphatic leukemia cells (Ia+, gamma-chain-).     

Localization of an antibody to CD74 (MHC class II invariant chain) to human B cell lymphoma xenografts in nude mice

The tumor-specific localization of an anti-CD74 Ab, LL1, was demonstrated in nude mice bearing xenografts of human B-cell lymphoma. This Ab, conjugated to radionuclides emitting Auger electrons, including ¹²⁵I and ¹¹¹In, was previously reported to kill tumor cells in vitro effectively and specifically. The cytotoxic potency of this Ab is due to its uptake and catabolism at a very high level, which also affected the Ab biodistribution experiments. Thus, Ab localization to the tumor was only detected if a “residualizing” radiolabel was used, meaning a label that is trapped within cells, usually within lysosomes, after catabolism of the Ab to which it was conjugated. Similar results were obtained with three different residualizing labels: ¹¹¹In conjugated via the chelators benzyl diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), or ¹³¹I-dilactitol-tyramine, a residualizing form of iodine. The Ab protein dose could be high, 0.5 mg/mouse, without causing a decrease in specific tumor uptake, probably reflecting the high capacity for uptake. Moreover, tumors of moderate size were found to cause rapid, specific removal of the Ab from the blood, also a result of catabolic processes. This induced blood clearance naturally affected the Ab localization experiments, but this factor could be circumvented by increasing the Ab protein dose. Using a different Ab, anti-(mature MHC class II), the ability of Ab to penetrate relatively large solid tumors was investigated. Complete saturation of antigenic sites was observed in tumors up to 0.3 g in size, but quite high Ab protein doses were required, 5.0 mg/mouse. These results provide a rationale for attempting therapy with radiolabeled LL1. Received: 4 November 1999 / Accepted: 19 January 2000     

Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II- associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild- type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.     

Biosynthetic relationships of the chondroitin sulfate proteoglycan with Ia and invariant chain glycoproteins

As a first step to elucidating the role of the Ia-associated chondroitin sulfate proteoglycan (CSPG) in the biology of the Ia antigens, we have studied several aspects of the interactions of the CSPG with the Ia/invariant chain glycoproteins. These studies revealed that at any time point, only 2 to 5% of Ia molecules were associated with the CSPG, and that this fraction included Ia molecules that were expressed at the cell surface. Pulse-chase studies indicated that the association of Ia molecules and the CSPG was rapid and short lived. Newly synthesized [35S]sulfate-labeled CSPG molecules were detected in association with Ia molecules immediately after a 15-min pulse, but were barely detectable after a 30-min chase, and were completely undetectable after a 60-min chase. Similarly, newly synthesized [3H]leucine-labeled Ia molecules associated with the CSPG were detectable immediately after a 20-min pulse, and after a 75-min chase, but could not be detected in association with the CSPG after a 300-min chase. Virtually no CSPG that was similar in size to that associated with Ia molecules was found free in the cells or was secreted into the media. The results in this report are compatible with the hypothesis that some or all of the Ia molecules associate transiently with the CSPG, or that a small fraction of Ia molecules associate permanently with the CSPG in a short-lived complex. These studies tend to favor a role for the CSPG in the biosynthesis of Ia rather than in intracellular trafficking or in intercellular communication.     

Murine thyroid follicular epithelial cells can be induced to express class II (Ia) gene products but fail to present antigen in vitro

To determine whether thyroid follicular epithelial cells (TFEC) might be involved in the induction of autoimmune thyroiditis, they were tested for their potential to express Ia antigens, and for their ability to present antigen in vitro. Results showed that Ia antigens, absent on normal TFEC, could be readily induced with interferon gamma, as detected by immunofluorescence. Maximal expression of Ia antigens in over 50% of TFEC was observed after 4 days of culture in the presence of IFN-gamma, and was quantitatively comparable to spleen cells by cytofluorometric analysis. Moreover, primary TFEC in culture secreted thyroglobulin (tg) and interleukin 1. However, TFEC consistently failed to stimulate various populations of T cells. These included lymph node cells sensitized to tg, a T-cell clone specific for azo-benzene-arsonate tyrosine (ABA), and a hybridoma specific for beef insulin. Likewise, Ia-positive TFEC did not stimulate T-cell hybridomas restricted to the class II alloantigen I-Ab, while stimulating a hybridoma specific for the class I alloantigen Kb. T-cell unresponsiveness could not be explained by inhibitory activity of TFEC, released either into the culture supernatant or exerted by cell contact. The data indicate that Ia-positive TFEC failed to serve as class II-restricted antigen-presenting cells (APC) in vitro and thus argue against a primary role for these cells in the inductive phase of thyroiditis.     

Recognition of class II molecules by human T cells

The HLA-D region of individuals with the DRw11, w52, DQw3 haplotype encodes multiple molecular products of three distinct subregions, DR, DP, and DQ. Since each molecule can carry multiple stimulatory epitopes, the repertoire of allogeneic T-cell responses to determinants of this haplotype can be quite large. In the present experiments, alloreactive cloned T-cell lines recognized six distinct epitopes associated with DRw11, DRw52, DQw3 haplotypes. Panel studies established that three epitopes were DRwll-like and three were DRw52-like. Blocking with monoclonal antibodies showed that two DRw11-like epitopes were carried by DR-subregion products and one DRwll-like epitope was carried by DQ-subregion molecules. DRw52-like epitopes were detected on separate DR subregion-encoded molecules. One of them carried both DRwl1-and DRw52-like epitopes, the other carried two of the DRw52-like epitopes. These epitopes, which represent functional units that trigger T-cell responses, can be detected at the present time only with the methods used in this report. Conventional allogeneic T-cell responses represent the summation of responses to multiple epitopes encoded by different D-subregion genes.