Equipment and reagents
• Flat-bottom 96-well tissue culture plates (Nunc)
• Recombinant murine IFN-y (Genzyme)
• Phosphate-buffered saline (PBS), comprising 10 mM sodium phosphate pH 7.5. 0.9% (w/v) NaCl
• Complete tissue culmre media: Dutch modified RPMI (Gibco) or DMEM, 4.5 mg/litre glucose (Gibco). Either medium should be supplemented with 1 mM L-glutamine, 2 mM sodium pyruvate, antibiotics, and 10% HI-FCS.
• Specific antigens for stimulation (e.g. soluble protein antigens such as ovalbumin, KLH), to be used over a 1-100 (ig/ml final concentration range
• 1% paraformaldehyde: make a 3% (w/v) stock of paraformaldehyde (Kodak) in PBS, as described in Chapter 2, and store at -20°C; prepare 1% stock in PBS using 3% stock as base
• A source of antigen-specific polyclonal T cells or a T cell hybridoma
1 Harvest IFN7 activated BMM0 (see Protocols 1 and 2) and wash once prior to assay.
2 Resuspend the cells at 106/ml in complete tissue culture medium for the final T cell assay.4
3 Make half-log10 dilutions (of the cell suspension), to 104/ml, using tissue culture medium.
4 Plate 100 jjlI of each dilution into flat-bottom 96-well tissue culture plates (Nunc, Falcon). Plate sufficient cells at each concentration to allow for assay in triplicate, and with valying antigen concentrations (see step 6).
5 Incubate plates at 37°C, 5% C03 for 2 h to allow macrophages to adhere.
6 Add 100 jxl of the antigen, of choice, serially diluted in complete tissue culture medium,
7 Incubate for 2-4 h with antigen at 37°C, 5% CO,.
8 Wash wells gently three times by removal/replacement of 200 warm complete medium. A multichannel pipette is most convenient for this procedure.
9 Wash once with serum-free tissue culture medium.
10 Add 50 p.1 of 1% (w/v) paraformaldehyde in PBS and incubate for 15 min at RT to fix the cells.
11 Remove paraformaldehyde by aspiration.
12 Add 50 fil of 0.1 M L-lysine in PBS to quench the fixation reaction (15 min at RT),
Protocol 3 continued
13 Wash fixed, antigen pulsed macrophages three times with complete tissue culture medium.
14 Add 100 n-1 of this medium after washing is complete.
15 Add 2-4 x 105 polyclonal antigen-specific T cells or 10s antigen-specific T cell hybridoma cells in 100 jj.1 complete medium.b
16 Incubate at 37°C, 5% C02 for 24 h (T cell hybridomas) or 90 h (polyclonal T cells).
17 Assess T celt responses/
1 We routinely prefer Dutch modified RPMI for use with primary T cell cultures, and DMEM for use with T cell hybridomas.
b It is important to also include cultures containing only T cells and antigen, at all doses used. For primary T cells, this control functionally assesses the degree of T cell purity, whereas for all types of T cells it excludes mitogenic activity in the antigen preparation. cTo assess T cell responses, either harvest culture supernatant fluid for IL-2 determination (T cell hybridomas) or pulse cells with 0.5 fj.Ci |3HjTdR (5 Ci/mol of specific activity, Amersham) for 6 h followed by scintillation counting (for polyclonal T cells).
Confirmation that observed T cell activity is due to class II-restricted presentation should be made. In the case of cloned antigen-specific T cells/hybridomas, MHC class II mismatched BMM0 can be used. More commonly, mAbs (--■ 20 p.g/ ml) specific for relevant class 11 gene products can be added to the cultures, 15 minutes prior to the addition of T cells (Protocol 3, step 15}, and left in for the duration of the assay.
3.1.4 Biochemical/cell biological aspects of class II processing
Having established the optimal cell and antigen concentrations, it is customary to determine the nature of the class II processing requirements for the antigen in question. This has historically involved the use of specific inhibitors of various lysosomal hydrolases, and modulators of intracellular pH. Common examples include chloroquine, pepstatin, and leupeptin (2). Novel inhibitors for various aspects of this pathway arc continually being developed, but if history does indeed repeat itself, it is worth bearing in mind that the specific inhibitor of today may well have other important effects tomorrow!
Protocol 4 outlines the basic scheme for assessment of the characteristics of antigen processing for MHC class II-restricted antigens.
Gene targeted mice are likely to have an increasing role in the study of macrophage APC function. Gene knockouts for various cathepsins already exist, and macrophages from such mice can be readily compared to wild-type control cells. The use of bone marrow-derived macrophages may be an important consideration when poor survival rates or neonatal mortality occur. Preliminary studies on cathepsin L knockout mice have again highlighted the differential processing which can occur in APCs from different tissue sites (13).
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