Progression of clonality in splenocytes of infected IBMI-huNOG. (A) Occupancy of HTLV-1–infected clones in the spleen. Abundant integration sites of HTLV-1 provirus were amplified by IL-PCR and subcloned into plasmids. The number of integration sites in each splenic DNA sample was determined by quantitative PCR using the clone-specific nucleotide sequence for each integration site. Results from 8 individual HTLV-1–infected mice are shown as pie charts. Size of the slice is proportional to the relative abundance of T-cell clones successfully amplified by IL-PCR, while data of minor clones with less than 0.1% occupancy were omitted. Gray regions represent clones with undefined integration sites. n, number of integration sites determined by nucleotide sequence of cloned PCR fragments in each mouse. (B) PVLs of specifiedT-cell populations. Splenocytes from HTLV-1–infected mice (n = 5) were sorted into CD25⁻ or CD25⁺ CD4 T cells and CD8⁺ T cells. Genomic DNA isolated from each T-cell population was analyzed for PVL by real-time PCR using primers for the pX region of HTLV-1. (C) Comparative analysis of viral transcripts in CD25⁻ and CD25⁺ CD4 T-cell populations. Splenocytes from HTLV-1–infected mice (n = 5) are identical to those in mentioned above. The expression levels of tax (left) and HBZ (right) were analyzed by quantitative RT-PCR and were normalized to that of HPRT1. Results are presented as the fold change compared with the value in CD25⁻ CD4 T cells. (D) Detection of common T-cell clones in the CD25⁻ and CD25⁺ CD4 T-cell populations. Clonal occupancy in both CD25⁻ and CD25⁺ populations are presented as pie charts. Two abundant common clones were analyzed for occupancy. Identified integration sites are listed in supplemental Table 5. The purity of each sorted population was >95% (supplemental Figure 3).