Requirements for the processing of antigens by antigen-presenting B cells. I. Functional comparison of B cell tumors and macrophages.

We have conducted functional studies to compare antigen-presenting B cell tumors to macrophages with regard to the requirements for some type of antigen “processing” events to take place before antigen presentation. The capacity of antigen-specific MHC-restricted T cell hybridomas to produce interleukin 2 (IL 2) when antigen was presented by an appropriate accessory cell was used as the assay system for these studies. After the binding of antigen by A20-1.11 B lymphoma cells or macrophages at 4°C, a lag period of 45 to 60 min at 37°C was required before antigen presentation could be detected. The compartmentalization of the antigen relevant for triggering T cells was studied by comparing pronase digestion of cell surface antigen with paraformaldehyde fixation. B cells and macrophages that were pronase treated immediately after antigen was allowed to bind at 4°C failed to present antigen whether or not they were subsequently fixed with paraformaldehyde. In contrast, cells that had been incubated for 2 hr at 37°C before pronase treatment presented antigen if they were not fixed after the pronase treatment, but they failed to present antigen if they were fixed immediately after the pronase treatment. These data suggest that immediately after binding at 4°C, all the relevant antigen is located on the cell surface and can be removed by pronase treatment. After the antigen-pulsed cells are incubated at 37°C for 2 hr, antigen is in two distinct compartments. There is a cell surface compartment that is still susceptible to removal by pronase, and if the cells are fixed after the pronase treatment, the antigen-presenting capacity of the cells is destroyed. However, if the cells are not fixed, the second compartment that is resistant to pronase digestion can be expressed on the cell surface after the pronase treatment and thereby restore the antigen-presenting capacity to the cells. As was the case with macrophages, B cells could present cross-reactive determinants present on native ovalbumin (N-OVA) and denatured ovalbumin (D-OVA) to a cloned OVA-specific T cell hybridoma, DO11; this suggested that, like macrophages, B cell antigen processing may involve partial antigen degradation. Further evidence for this came from the finding that chloroquine, an inhibitor of lysosomal proteolysis, if present during the antigen-processing phase, blocked the presentation of that antigen but not that of a second antigen whose processing was allowed to proceed before chloroquine treatment. In addition, monensin, which inhibited the intracellular recycling of membrane receptors, also inhibited antigen presentation. These data are interpreted as supporting the concept that there is some active antigen-processing events that must take place after antigen binding before accessory cells can present antigen. Furthermore, as measured by function, the same requirements for antigen processing that have been demonstrated for macrophages have also been shown for B cell tumors.