Cellular requirements for antigen processing by antigen-presenting cells: evidence for different pathways in forming the same antigenic determinants

In this report we examined the antigen-presenting cell (APC) requirements for activation of T-cell hybridomas specific for the protein antigen PPD (purified protein derivative of tuberculin). During the course of these studies we observed that glutaraldehyde fixation of Ia-positive A20.2JAD (A20) and P388D1 stimulator cells had different effects on T-cell activation. A20 cells fixed with glutaraldehyde stimulated the T cells in the presence of PPD as efficiently as nonfixed A20 cells. By contrast, glutaraldehyde treatment of Ia-positive P388D1 cells dramatically inhibited their ability to process and/or present PPD to T cells. This was not due to nonspecific effects on the P388D1 cells since cells prepulsed with PPD prior to glutaraldehyde treatment stimulated T cells as efficiently as non-glutaraldehyde-treated P388D1 cells. In addition, there was no apparent difference in "fixing" of the two cell types as determined by the uptake of radiolabeled thymidine. These observations suggested that P388D1, but not A20, cells required PPD internalization to form the relevant antigenic determinants. This was substantiated by showing that treatment of P388D1 cells with chloroquine prior to PPD pulsing eliminated their stimulatory capacity, but had no effect on P388D1 cells previously pulsed with PPD. Chloroquine treatment had no effect on stimulation by A20 cells. Since PPD internalization appeared not to be required for presentation by A20 cells, we next determined if isolated A20 plasma membranes would substitute for the intact cell. We observed that the isolated plasma membranes from PPD-pulsed A20 cells stimulated the T hybridoma cells, and that this stimulation was antigen-specific and was inhibited by anti-Ia monoclonal antibodies. Taken together, the results presented here suggest that for the PPD-specific T-cell responses examined here, different APC utilize distinct pathways to present the same antigenic determinant for T-cell recognition.