CD45 isoform transitions on multinegative human thymocytes differentiating in vitro mimic patterns predicted for selective events in vivo

Abstract

Experiments were designed to test the idea that the patterns of CD45 isoform expressed by differentiating human multinegative (MN) thymocytes (CD3^- 4^- 8^- 19^- )are regulated by the type of growth or activation stimuli delivered. We have proposed that within the thymus, CD45RA expression is fundamental to maintenance of the thymic generative lineage while a transition to CD45RO indicates entry into the path of intrathymic death. It seems likely that the signal transduction pathways leading to positive selection are different from those leading to negative selection. Upon culture, MN thymocytes proliferate in response to interleukin-2 (IL-2) or anti-CD2/28 and differentiate as defined by acquisition of CD3 as well as CD4 and/or CD8. Even at day 9 of culture, 30–40% of cells remain multinegative. DNA analysis indicates that both CD3^- and CD3⁺ cells are actively cycling. Although the CD3^- set includes a substantial number of cycling cells, it continues to express almost exclusively the CD45RA isoform. Among CD3⁺ progeny, 53–80% have acquired CD45RO while maintaining high expression of CD45RA, although 10–47% are able to maintain exclusive expression of CD45PA despite their more differentiated state. In contrast, stimulation with PHA/PMA, which gives a vigorous proliferative response, appears to inhibit acquisition of CD3 and thus differentiation, while forcing a premature transition from CD45RA to CD45RO. These in vitro systems appear to permit generative thymic development while maintaining a cycling multinegative subset, thus mimicking thymic development in vivo, permitting an exploration of the events in positive and negative selection of human thymocytes. The maintenance of CD45RA expression with co-expression of CD45RO by CD3⁺ progeny in cultures supplemented with IL-2 or anti-CD2/28, in contrast to the loss of CD45RA after PHA stimulation, shows that the regulation of CD45 isoform expression is closely linked to the nature of the developmental signals received by a thymocyte.