Originally researchers carrying out these experimentswould have to use particular antigenic peptides or wholemolecules presented by APCs of the appropriate type.Now it is possible to use synthetic MHCpeptide com-binations (Method Box 7.1).

Antigens are partially degraded into peptidesbefore binding to MHC moleculesThe processing of antigens to generate peptides that canbind to MHC class II molecules occurs in intracellularorganelles (Fig. 7.6). Phagosomes containing endocytosedproteins fuse with lysosomes where a number of proteasesare involved in breaking down the proteins to smallerfragments. The proteases include: cathepsins B and D; an acidic thiol reductase, -interferon-inducible

lysosomal thiol reductase (GILT), which acts ondisulfide-bonded proteins.

Alkaline agents such as chloroquine or ammoniumchloride diminish the activity of proteases in thephagolysosomes and therefore interfere with antigenprocessing.

In laboratory studies, the requirement for internaldegradation of antigen by APCs can be circumvented bythe use of synthetic peptides. This ability to use synthe-sized peptides of known sequences has enabled researchersto readily identify epitopes recognized by T cells withdifferent specificities.

The relative importance of different amino acids withina defined epitope can also be investigated by amino acidreplacements at different sites. Comparison of the effectsof amino acid substitution on MHC molecule binding andT cell reactivity has enabled conclusions to be drawn as to: which amino acid residues contact the MHC molecule;

and which amino acid residues contact the TCR

MHC CLASS I MOLECULES ASSOCIATEWITH ENDOGENOUS PEPTIDESMHC class I-restricted T cells (CTLs) recognize endoge-nous antigens synthesized within the target cell, whereas

MHC CLASS I MOLECULES ASSOCIATE WITH ENDOGENOUS PEPTIDES

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METHOD BOX 7.1Tetramers

Tetrameric complexes of MHC molecules may be used toidentify clones of T cells specific to presentation of a particularpeptide in a particular MHC molecule. These are usuallytetramers of MHC class I molecules, though MHC class IItetramers are being developed.

The tetramer assay allows the detection of antigen-specificcytotoxic T lymphocytes (CTLs) in ex-vivo cell preparationswithout the need for in-vitro expansion.

The basis of this technique is to directly stain antigen-specific T cells with the MHC moleculepeptide complex thatthey recognize via their T cell receptors (TCRs).

Because the affinity of single TCR molecules for the MHCmoleculepeptide is low, direct staining is not possible becauseof rapid dissociation. The formation of an MHC tetramer withfour copies of the MHC moleculepeptide complex results in avery enhanced avidity for the TCR.

MHC molecules of the appropriate allotype are fused to apeptide (bsp), which acts as a substrate for biotinylation. MHC-bsp and 2-microglobulin are expressed in Escherichia coli mixedwith the specific CTL epitopic peptide under folding conditions,and the MHC moleculepeptide complexes thus formed arepurified. The complexes are biotinylated and mixed at a ratio of4:1 with fluorescence-labeled streptavidin to produce thetetramers (Fig. 1). The binding of these labeled tetramers byantigen-specific CTLs can then be analyzed by the fluorescence-activated cell sorter (FACS, see Method Box 2.1, p. 32).

A class I MHC tetramer

Fig. 1 A tetrameric MHC molecule is produced bybiotinylating the MHC molecules via a linking peptide (bsp)and allowing them to bind to the tetravalent moleculeavidin a natural receptor for biotin.

biotin

antigenicpeptide MHC class I

bsp peptide

avidin

Antigen processing

Fig. 7.6 Exogenous antigens are internalized by APCs and arethen degraded by proteolytic enzymes in specializedintracellular compartments. Antigenic peptides associate withMHC class II molecules in vesicles that intersect the endocyticpathway on their way to the cell surface.

APC

antigen

internalizationpartial degradationin phagolysosome

MHC molecule

re-expressionof antigen atthe cell surface