Assessment of the biochemical characteristics of class IIrestricted antigen processing

Equipment and reagents

Method

1 Prepare BMM0 and put them into culture as described in Protocol 3, steps 1-5.

2 Add enzyme inhibitors or modifiers of cellular function diluted in complete tissue culture medium alone or in combination to the adherent monolayers."

3 Incubate the macrophage monolayers (5% C02 for 1 h at 37°C).

4 Add antigen diluted in complete tissue culture medium containing the inhibitor to treated cells (see Protocol 3).b

5 Assess efficiency of treatment by determining the influence on antigen-specific T cell stimulation (see Protocol 3).

1 Optimal concentration should be empirically determined by titration with appropriate diluent controls.

b It is important to ensure that all antigens and wash buffer contain the appropriate inhibitor until the fixation step (Protocol 3, step 10) is reached.

3.1.5 Processing of soluble antigens for class i-restricted presentation

Various methods have been devised for the introduction of soluble antigens into the cytosol and hence the conventional 'endogenous' class I processing pathway. Perhaps that most frequently used is osmotic lysis of pinosomes (14). Protocol 5 describes a method for the loading of soluble antigens into macrophages, using this technique.

Controversy still exists as to whether 'exogenous' protein antigens can be processed for class I-restrictcd presentation by BMM0, in the abscncc of obvious means for cytosolic entry. In most cases where this has been achieved directly, the very high concentrations of soluble antigen required may allow peptide contaminants to directly load class I molccules (15). Functional testing for peptide contaminants can be made by adding high concentrations of soluble antigen to fixed macrophages, and/or by dialysing protein antigens before use. Coupling of soluble antigens to latex beads, thereby introducing antigen by phagocytosis may improve processing efficiency, though whether this proceeds via conventional TAP-dependent pathways after 'bursting' of occasional phagosomes (15)

• C02 incubator

• Complete tissue culture medium (see

• Inhibitors of antigen processing, e.g. chloroquine, pepstatin, leupeptin (Sigma)

• Soluble antigen (e.g. ovalbumin) for which antigen-specific T cells are available

Protocol 2)

or via a novel route (16) requires confirmation by the use of inhibitors or gene targeted macrophages. In contrast to BMM0, DC appear efficient at processing exogenous antigen for class I-restricted recognition, possibly as a consequence of their ability to perform constitutive macropinocytosis. In our hands, class I processing of soluble antigens by macrophages is always inefficient, and DC are usually suspected when individual preparations of cells show otherwise unexplained high efficicncy of class I processing {Kaye, unpublished). The outcome of assays in which stimulation of class I-restricted responses arc measured generally is assessed by monitoring IL-2 production (using CD8" T cell hybridomas) (19) or CTL activity of primed T cells against appropriate target cells (14).

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