In Vivo Selection Unmasks a Dormant Pool of Repopulating Hematopoietic Clones

Long-term clonal tracking studies utilizing hematopoietic stem and progenitor cells (HSPCs) in nonhuman primates receiving myeloablative transplantation demonstrate a successive pattern of repopulation: short-term repopulating cells are succeeded by long-term clones. However, the duration of short-term repopulation and the numbers of clones contributing to either short or long-term repopulation are unclear. Here, we tracked >11,000 unique clones in 8 pigtail macaques for up to 9 years following myeloablative transplantation with autologous, lentivirus gene-modified CD34⁺ HSPCs. Seven of these animals received cells expressing the P140K mutant methylguanine methyltransferase transgene, which is resistant to the combination of O⁶-benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy, thus conferring a selective advantage to gene-modified cells in vivo. After transplantation and before in vivo selection with O6BG/BCNU, we observed a successive pattern of hematopoietic reconstitution, with short-term clones declining within 100 days after transplantation. Within the first year after transplant, the percent of persistent clones varied from animal-to-animal, ranging from 8% to 54% of clones detected at a >1% frequency, and remained stable in the absence of selective pressure. Importantly, when animals engrafted with P140K-expressing cells were administered O6BG/BCNU we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving ≤12x10⁶ CD34⁺ cells/kg at the time of transplant (n = 4), chemotherapy also induced a re-emergence of previously declined short-term repopulating clones or a stabilization (i.e. decreased fluctuation) of repopulating clones identified between 100 days and 1 year after transplant. However, in animals receiving robust cell doses, ≥35x10⁶ CD34⁺ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. In one animal receiving 22x10⁶ CD34⁺ cells/kg at transplant, chemotherapy administration beginning <1 year (253 days) after transplant induced clonal stability, which was maintained through two additional chemotherapy treatments. These data suggest that some short-term repopulating clones may have long-term repopulation ability, but revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess repopulating cells results in early dormancy of a large proportion of repopulating clones. Together, these findings suggest that previous estimates of HSPC frequency based on clone tracking are an underestimate of true graft repopulation potential.