Cytospin centrifuge, including holders, filter paper (Shandon)
Reagents for appropriate staining of micro-organism
J Prepare slides of macrophages infected in suspension culture, by making cytospin sample (600 r.p.m. for 10 min).
2 Stain slides with an appropriate method to visualize the organisms in question.
3 For adherent monolayers, prepare and stain replicate wells as will be used to evaluate T cell responses.
4 Determine both the percentage of infected macrophages and the number of organisms per 100 macrophages by microscopic examination. If the organisms are over-dispersed (as is common at low MOI) then a distribution should be established by scoring, for example, macrophages with zero, 1-3,4-5, 7-10, > 10 organisms.
In the case of pathogens which have been ttansfected to express reporter antigens it may be possible to develop direct methods for establishing antigen dosage in infected macrophages. For example, for our studies with OVA-transfected Leishmarcia, a capture ELISA was used to determine the amounts of OVA incorporated into macrophages following infection versus pulsing with soluble ovalbumin (17). By also determining the quantity of OVA per parasite and the numbers of parasites per macrophage, it is also possible to indirectly estimate the total level of antigen uptake.
An issue which continues to preoccupy and frustrate researchers is whether the presentation of antigens can occur from the various vacuolar compartments inhabited by different pathogens. Viable pathogens are known to make various modifications to the host vacuolar compartment, e.g. mycobacteria may exclude the proton ATPase, and hencc modify vacuolar pH, and ¡.eishmania may sequester MHC class II antigens (10). Often, these modifications occur only in the vacuoles surrounding live organisms, not those in which either dead organisms have been engulfed, or in which the organism has subsequently died. The issue then arises as to whether presentation observed from an infected macrophage population truly represents antigens derived from living, as opposed to dead organisms. A compelling discussion on this issue can be found in Wolfram el al (4). In their studies, optimal presentation of an abundant non-secreted leishmanial protease was achieved by killing the parasite within the vacuolar compartment using exposure to a leishmanicidal drug. These data, as with others previously published (17-19), suggested that to obtain access to antigen sequestered inside parasites, macrophages had to first destroy the parasite's membrane integrity. However, significant presentation was observed even with untreated macrophages infected with 'live' organisms. The leishmanial protease in question is abundant, and Wolfram et a!. calculated that death of a single intracellular organism would produce an intravacuolar antigen concentration of approx. 20 ixg/ml!
A major consideration with intracellular pathogens is the issue of antigen regurgitation, from either living or dead pathogens, and the possibility of representation by other, possibly non-infected cells in the culture. Two approaches are available to assess this functionally. First, supernatants from infected macrophages can be transferred to other fresh populations, i.e. used as 'soluble' antigen to pulse these secondary cultures (as described in Protocol 3). Secondly, the principles of MHC restriction can be utilized, whereby infected macrophages mismatched at the MHC are added to cultures containing uninfected macrophages syngeneic to the responder T cells. This latter approach has the advantage that because intimate contact is made between macrophages in the co-c:ulture, transfer of small quantities of antigen may be detectable. In addition, it has recently been appreciated that one potent means of antigen uptake is by the phagocytosis of apoptotic macrophages and acquisition of any antigens that they may contain. At least in the case of dendritic cells, this would appear an efficient way for cross-priming T cell responses (20). Protocol 9 describes one means of evaluating the level of antigen transfer between APC populations.
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