FC-GAMMA-RII/III AND CD2 EXPRESSION MARK DISTINCT SUBPOPULATIONS OF IMMATURE CD4-CD8-MURINE THYMOCYTES - INVIVO DEVELOPMENTAL KINETICS AND T-CELL RECEPTOR BETA-CHAIN REARRANGEMENT STATUS

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fcgamma receptors (FcgammaRII/III) and contain precursors for Tialpha/beta lineage T cells. Here we show that FcgammaRII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that FcgammaRII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN FcgammaRII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of FcgammaRII/III vs. CD2 (FcgammaRII/III+CD2-, FcgammaRII/III+CD2+, FcgammaRII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57Bl/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (FcgammaRII/III+CD2- --> FcgammaRII/III+CD2+ --> FcgammaRII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCR(low/-) stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for >48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, FcgammaRII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while FcgammaRII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. FCgammaRII/III-CD2+ are also more developmentally advanced than FcgammaRII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the FcgammaRII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, >90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing FcgammaRII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% FcgammaRII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (<3% FcgammaRII/III+). Thus, FcgammaRII/III expression defines an early DN stage preceding V(beta)(D(beta))J(beta) rearrangement, which in turn is followed by surface expression of CD2. Loss of FcgammaRII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.