FcγRII/III and CD2 expression mark distinct subpopulations of immature CD4- CD8-murine thymocytes: In vivo developmental kinetics and T cell receptor β 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 Feγ receptors (FcγRII/III) and contain precursors for Tia/β lineage T cells. Here we show that FcγRII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that FcγRII/III expression is downregulated within the DN subpopulation and before the CD3⁻CD8^low SP stage in T cell receptor (TCR)-α/β lineage-committed thymocytes. DN FcγRII/III⁺ thymocytes also contain a small fraction of TCR-γ/δ lineage cells in addition to TCR-α/β progenitors. Fetal day 15.5 DN TCR-α/β lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of FcγRII/III vs. CD2 (FcγRII/III⁺ CD2 ⁻, FcγRII/ III⁺ CD2⁺, FcγRII/III⁻CD2⁺). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (FcγRII/III⁺ CD2⁻ → FcγRII/III⁺ CD2⁺ → FcγRII/III⁻ CD2⁺) that precedes the entry of DN thymocytes into the CD4⁺CD8⁺ double-positive (DP) TCRlow/⁻ 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, FcγRII/ III⁺ CD2⁻ DN thymocytes follow this same developmental pathway but are delayed by ~24 h before entering the DP compartment, while FcγRII/III⁻CD2⁺ display accelerated development by ∼24 h compared with total day 15.5 thymocytes. FcγRII/III⁻CD2⁺ are also more developmentally advanced than FcγRII/III⁺CD2⁻ fetal thymocytes with respect to their TCR β chain V(D)J rearrangement. At day 15.5 in gestation, β chain V(D)J rearrangement is mostly, if not entirely, restricted to the FcγRII/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 FcγRII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2⁻7⁻ mice (15% FcγRII/III⁺) versus rearrangement-competent heterozygous RAG-2⁺ /⁻ mice (< 3% FcγRII/III⁺). Thus, FcγRII/III expression defines an early DN stage preceding Vβ(D_β)J_β rearrangement, which in turn is followed by surface expression of CD2. Loss of FcγRII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.