T lymphocytes with alpha beta receptors recognize antigen in association with the polymorphic products of the class I and class II loci of the major histocompatibility complex (MHC). This presentation of antigen results from the intracellular generation of protein fragments, and the binding and transport to the cell surface of these peptides in stable association with the MHC class I and class II molecules. Each class of MHC molecule appears specialized for capture of peptides present in a particular intracellular compartment. We describe here the structural basis of peptide-MHC molecule interaction, the differences in biochemical behavior that focus the two classes of MHC molecules on peptides of distinct size and location, and the cell biology of MHC molecule transport, peptide generation, and intracellular movement. The importance of conformational changes accompanying peptide binding that affect subunit stability of MHC molecules, and the relationship between these changes and the handling of proteins by intracellular chaperones, are emphasized as key features in the operation of the class I and class II presentation pathways.

The biochemistry and cell biology of antigen processing and presentation

Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases.

MHC class II molecules assemble in the endoplasmic reticulum in a chaperone-mediated fashion to form a nine-chain structure consisting of three alpha beta dimers associated with an invariant chain trimer. This complex is transported through the Golgi apparatus and into the endosomal system. The signal for endosomal targeting resides in the cytoplasmic tail of the invariant chain. Current evidence argues that the segregation of the class II-invariant chain complex from the constitutive pathway of membrane protein transport occurs in the trans-Golgi network. However, class II-invariant chain complexes that reach the cell surface are also rapidly internalized into endosomes. Within the endosomal system, probably in a late endosome/pre-lysosome, the invariant chain is degraded, releasing alpha beta dimers that bind peptides predominantly derived from endocytosed proteins. Evidence suggests that many of these peptides are actually generated in lysosomes. The precise mechanisms involved in forming class II-pept...

Assembly, Transport, and Function of MHC Class II Molecules

Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment. It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface. But certain mutations in the MHC can prevent presentation of antigens with class I molecules. In addition, mutations possibly in the MHC can affect presentation by class II molecules. Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters. This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.

Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters

HLA-DM induces clip dissociation from MHC class II αβ dimers and facilitates peptide loading

Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth-association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

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Simultaneous flow cytometric analysis of human t cell activation antigen expression and dna content

PRESENTATION of an exogenous protein antigen to helper (CD4+) T-lymphocytes by antigen presenting cells (APC) generally requires that the APCs degrade the native protein antigen into an immunogenic peptide, a process termed 'antigen processing', and that this peptide bind to a major histocompatibility complex (MHC) class II molecule1–3. The complex of peptide and MHC molecule on the APC surface provides the stimulatory ligand for the αβJ T cell receptor4. The intracellular pathways and molecular mechanisms involved in the generation of the peptide-MHC complex are not well understood. Here, we describe several mutant APCs which are altered in their ability to present native exogenous protein antigens but effectively present immunogenic peptides derived from these proteins. The lesions in these mutants are not in the class II structural genes, but they affect the conformation of mature class II dimers.

Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes.

Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium.

Point mutations that affect HLA-DR structure or expression have not previously been described. In the present study, we isolated such mutants by immunoselection of an ethyl methanesulfonate-mutagenized HLA-DR3 cell line with an anti-HLA-DR3 monoclonal antibody, 16.23. To facilitate analysis, we used a parent cell line with a preexisting deletion of one haplotype encompassing DR and DQ alpha and beta. The selection yielded two sets of mutants, one with defects in DR3 structure, the other with defects in different steps leading to DR expression. Of the expression-defective mutants, one had undergone a second deletion removing the remaining DR alpha gene but no other class II genes. It had a normal abundance of DR beta mRNA but had lost binding of DR monomorphic antibodies, indicating that DR beta chains do not form noncognate dimers. A second mutant had an abnormally large DR alpha mRNA, probably resulting from a splice site mutation. Several mutants had marked reductions in DR beta mRNA levels; in two of these, the lesion appeared to be transcriptional because the reduction in DR beta mRNA was paralleled by an altered methylation pattern of one of the DR beta genes. Other expression-defective mutants had different posttranscriptional defects. Some of the mutations were similar to those that have been found in mouse strains defective in I-E expression, whereas others have no known natural counterpart. The matrix of reactivities of anti-HLA class II monomorphic antibodies with these and similar mutants allowed us to define the gene products recognized by these antibodies. A set of seven mutants were "epitope defective," that is, they expressed normal or near normal levels of HLA-DR3 but no longer bound 16.23. Unexpectedly, each of the epitope mutants had decreased DR dimer stability. These mutants should be useful in localizing the DR3 alloepitope and in elucidating its contribution as a restriction element in the presentation of soluble antigen to immune T cells.

https://rupress.org/jem/article-pdf/162/4/1193/1095640/1193.pdf

HLA CLASS II REGULATION AND STRUCTURE Analysis with HLA-DR3 and HLA-DP Point Mutants

This study provides direct correlation via dual parameter flow cytometry (simultaneous assessment of immunofluorescence and DNA content) between mixed lymphocyte reaction (MLR) responder cell entry into the S/G2/M phases of the cell cycle with the kinetics of expression of two activation-associated cell surface proteins, Tac (IL 2 receptor) and 4F2 (unknown metabolic function). A small population of activated cells was identifiable by expression of both Tac and 4F2 antigens before peak DNA synthesis in the MLR. This population of activation antigen-positive cells expanded linearly in size from days 3 to 7 of culture. Treatment of immature MLR cultures with anti-4F2 Mab and complement (C) before DNA synthesis (treatment on day 3, peak DNA synthesis on days 5 to 6) resulted in blunted proliferation and activation antigen expression when the same culture was analyzed after maturation on day 6, indicating that the activated population had been previously detected and removed by anti-4F2 Mab + C. The 4F2 antigen was expressed on a greater percentage of cells in the MLR at all times (days 3 to 9) than was Tac, was present on virtually all S/G2/M phase responder cells, and a large fraction of cells remained intensely 4F2+ subsequent to peak DNA synthesis. In contrast, after initially preceding responder cell entry into the S phase of the cell cycle, the kinetics of Tac antigen expression closely paralleled the kinetics of responder cell proliferation. A subpopulation of cycling responder cells was noted in all MLR cultures studied that expressed Tac antigen weakly or not at all. Cells within both T4 and T8 cell subsets proliferate with similar kinetics in response to alloantigen. The possibility that activation antigens can be utilized to study effector cell generation in the MLR and that this flow cytometric technique may be utilized to analyze the response to various alloantigens is discussed.

Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction.

Tom Cotner

Papers

Simultaneous flow cytometric analysis of human t cell activation antigen expression and dna content

Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes.

Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium.

HLA CLASS II REGULATION AND STRUCTURE Analysis with HLA-DR3 and HLA-DP Point Mutants

Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction.