Equipment and reagents9
• 4 ml polypropylene culture tubes (Falcon 2063)
• '6 + 2' bone marrow-derived macrophage (BMM0, see Protocol 1)
* Complete tissue culture medium: supplemented DMEM (see Protocol i) with 10% FCS
• Recombinant murine IFN-y (Genzyme)
1 Adjust BMM0 to 106/ml in complete tissue culture medium.
2 Aliquot 3 x 106 per tube to the required number of polypropylene culture tubes.
3 Add recombinant murine IFN-y to a final concentration of 100 U/mLb
5 Resuspend BMM0 by gently flicking tube.
6 Remove an aliquot of cells and determine levels of class II expression by flow cytometry (using methods described in Chapter 3).
7 Wash remaining BMM0 twice in complete tissue culture medium, prior to use in functional assays.
a All possible caution should be taken to avoid LPS contamination in culture medium, reagents, glassware, etc.
b If recombinant IFN7 is not available, 10% (v/v) supernatant from ConA-stimulated splenocytes (5 M-g/ml ConA for 72 h. with a cell concentration of 5 x 106/ml will usually suffice. However, other cytokines present may have adverse effects on macrophage function.
The time at which class II should be induced may vary depending on the experimental design. For example, in a direct assay of processing function, optimal levels of class II are usually desired prior to antigen uptake, such that class II
transcription and subcellular redistribution are not limiting factors. In contrast, in studies aimed at addressing more specific questions, class II induction may be delayed until after antigen uptake. Such was the method adopted to determine whether newly synthesized class II molecules were able to be transported to the parasitophorous vacuole surrounding Leishmania parasites (10).
LPS contamination should be avoided in all work. High concentrations of LPS may inhibit class II expression to a degree, and also regulate co-stimulatoiy ligand expression (3). LPS will also, even in nanogram levels, synergize with IFN7 to activate microbicidal activity and the production of NO. NO may be an inhibitor of T cell proliferation (11).
3.1.3 Pulsing macrophages with protein antigens to measure class ll-restricted processing
Performing checkerboard titrations is the best initial approach to establishing the optimal conditions for later, more refined experimental designs. Checkerboard titrations may also prove valuable for evaluating the homogeneity of an APC population. A sigmoid titration curve, with a linear relationship between macrophage number versus T cell activity (e.g. proliferation, cytokine production) extending for 1-2 logs in macrophage number, is generally indicative of the population displaying uniform function. In contrast, a rapid loss of functional activity within a small titration range may indicate that APC function actually resides in a minor subset of cells which is rapidly titrated out. Antigen dose responses are also informative, and will generally produce sigmoid or bell-shaped curves. Protocol 3 describes a method for simultaneously analysing antigen dose and optimal macrophage number. Subsequent experiments are best performed with both parameters at cell/antigen concentrations giving responses just below maximum. In contrast, when macrophages are to be modified, e.g. by infection, numbers should be ideally used that give 50% maximal T cell activity (allowing both enhancement and depression of function to be observed).
The above protocol allows simultaneous determination of the optimal macrophage number and antigen dose required for T cell recognition. The fixation steps (steps 10-13) are not an absolute requirement and unfixed antigen pulsed macrophages can be taken directly from step 8 to step 14. However, fixation provides the most reliable way of terminating processing, and hence for the analysis of processing kinetics (by varying the duration of incubation prior to fixation; step 7 above). Nevertheless, fixation may have some deleterious effects on APC efficiency, probably due to the fixation sensitivity of certain co-stimulatoiy molecules. It should also be noted that extremely low levels of aldehyde fixation may enhance APC-T cell interactions by the formation of Schiff bases (12). Fixation also obviates any interference in T cell response by secreted products of macrophages, e.g. NO or PGE2. If fixation is not used, inhibitors of these metabolites (5 |xg/ml indomethacin and 0.4 mM NMMA, respectively) should be added to media from step 7.
If the efficiency of macrophages compared to other APC is to be compared then this protocol will only be suitable if all populations are adherent (e.g. a comparison with trans fee ted fibroblasts). If this criterion is not satisfied, other antigen pulsing procedures will be required (e.g. see Protocol 7).
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