In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C-mediated lysis and panning. While the majority (79.0 ± 12.0%) of DN cells were TCRγδ+ as shown by staining with mAb TCRδ-1, a minor fraction (6.7 ± 4.7%) expressed TCR αβ as revealed by staining with mAb BMA031. Within the γδ+ DN fraction, most cells reacted with mAb TiγA which delineates a Vγ9JpCγ1 epitope, whereas a minor fraction stained with mAb δTCS-1 which identifies a Vδ1Jδ1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIl-2 and/or rIl-4) are provided. © 1990.
