DIFFERENCES IN THE REQUIREMENT OF ANTIGEN-PRESENTING CELLS FOR LONG-TERM GROWTH AMONG CYTOMEGALOVIRUS-SPECIFIC TH CLONOTYPES

Functional and molecular studies of in vivo-activated T lymphocytes in volved in normal and abnormal immune responses, i.e., cells infiltrati ng tissues affected by autoimmune processes, require their previous in vitro expansion. Problems such as unavailability of specific antigen( s) (Ag) and/or the requirement of large amounts of autologous peripher al blood mononuclear cells (PBMC) as feeders have prompted the develop ment of alternative expansion methods that circumvent the use of antig en-presenting cells (APC) and/or Ag. We have previously shown that cyt omegalovirus (CMV)-specific T cell lines and clones can be efficiently propagated in long-term cultures by stimulation with agonistic monocl onal antibodies (mAb) coated onto polystyrene particles in the absence of APC. However, this and other stimulation protocols may skew the re pertoire of clonotypes that proliferate in response to nominal Ag and APC. Here we show that polyclonal populations of CMV-primed and interl eukin-2 (IL-2)-stimulated PBMC undergo the same clonotypic selection w hen induced to grow both by continuous stimulation with CMV and an ant i-CD3 mAb presented by APC. This selection, reproduced in three indepe ndent expansion experiments, involved the dominant growth of two CMV-s pecific, IL-2-secreting CD4(+) clonotypes sharing J alpha, J beta, V a lpha and V beta genes and highly homologous T cell receptor (TcR) junc tional sequences. The dominant growth of these 2 clonotypes required a direct T cell/APC interaction since when coated onto polystyrene part icles the same mAb induced the selective expansion of another IL-2-sec reting CD4(+) CMV-specific clonotype expressing a different, yet homol ogous, TcR heterodimer (used same V alpha and V beta genes), which was underrepresented before expansion (5 vs. 58%). T cell clones belongin g to the subdominant clonotype proliferated significantly faster in re sponse to stimulation with anti-CD3 mAb coated onto beads than in resp onse to stimulation with CMV or anti-CD3 mAb presented by APC, possibl y due to long-term expansion without APC or antigen. In contrast, neit her the dominant clonotypes nor unprimed T cells were able to undergo CD3-mediated expansion in the absence of APC. We conclude that quantit ative differences in growth competency among nominal Ag-activated T-he lper (Th) clonotypes in response to antigenic stimulation can be repro duced by stimulation through the TcR in the absence of TcR occupancy b ut only in the presence of APC and that certain clonotypes do not requ ire APC for long-term growth in vitro. These data have practical impli cations for the isolation and repertoire characterization of in vivo-a ctivated T cells from tissues affected by inflammatory, i.e. autoimmun e, phenomena.