Mitogen- and alloantigen-stimulated blastogenic responses of dinitrophenyl-modified human lymphocytes

The present study was designed to investigate alterations in the blastogenic response of dinitrophenyl (DNP)-modified normal human lymphocytes, Cells were isolated from heparinized blood and treated with dinitrofluorobenzene (DNFB) at ratios ranging from 10⁴ to 10¹¹ molecules per cell. Unmodified and DNP-modified lymphocytes were cultured in the presence of Concanavalin A, pokeweed mitogen or phytohaemagglutinin (PHA). Mixed lymphocyte cultures (MLC) were set up using unmodified and DNP-modified lymphocytes as both stimulators and responders. DNP-modified cells had normal blastogenic responses to all three lectins when treated with amounts of DNFB <10⁹ molecules/cell, but were totally unresponsive when treated with >3 x 10⁹ molecules. Cells treated with 10⁸-4 x 10⁹ molecules of DNFB had no reduction in viability as determined by trypan blue exclusion, but at ratios > 10⁹ molecules, incorporation of ³H-labelled amino acids, ³H-uridine and ³H-thymidine was decreased. MLC reactivity showed similar impairment with stimulator activity dropping off following treatment with 10⁹ and responder activity with 10¹⁰ molecules/cell. ¹²⁵I-labelled antibody directed against DNP was bound to modified cells suggesting that DNP groups may have been expressed on the cell surface. Concomitantly, there were changes in the expression of HLA antigens on DNP-modified cells, as shown by the detection of new antigens or an increase in reactivity of cells expressing previously identified antigens. Other studies, carried out in parallel with the present one, suggest that these alterations may be due to configurational and/or conformational changes in membrane proteins that are essential for mitogen responsiveness and MLC activity and also may define HLA specificities. The major point that emerges from the present study is that DNP, and inferentially TNP derivitization of lymphocytes, may not only bring about the expression of new antigenic determinants, but also may affect the intrinsic ability of lymphocytes to respond to an antigenic or a mitogenic stimulus.