Modulation of gene expression during Vα14i NKT-cell development. (A) C57BL/6 thymocytes were stained for CD1d tetramer, TcRβ, NK1.1, and CD44. CD1d tetramer⁺ TcRβ⁺ cells were subdivided into 3 populations based on CD44 and NK1.1 expression and sorted. CD44^low NK1.1^– cells were defined as stage 1, CD44^high NK1.1^– as stage 2, and CD44^high NK1.1⁺ as stage 3. (B) Gene tree clustering using similarity measure standard correlation. Genes were selected for analysis if their average expression level deviated from that of Stage 1 by at least a factor of 1.8 in 2 out of 2 experiments (597 genes met this criteria). Each column represents data from the indicated developmental stage. Each row represents a gene significantly induced (red) or repressed (green) during the development of Vα14i NKT cells. A color bar shows the magnitude of gene expression changes as a ratio of expression in Stage 1 versus Stage 2 and Stage 3. Genes names, probe ID, and normalized values for each gene can be found in Table S1. (C) Total RNA was prepared from Vα14i NKT cells at each of the 3 defined stages of development and quantitative real-time PCR was performed using primers and probes specific for granzyme B (GzmB), Fas ligand (CD95L), CCR5, IFNγ, CxCR3, CD122, CD38, T-bet, RANTES, and perforin. The amount of each transcript in the 3 developmental stages was determined by quantitative PCR with normalization to the amount of HPRT mRNA in each sample. Results are shown as mean ± standard deviation. Data are representative of at least 3 independent experiments.