T-cell differentiative capacity of haematopoietic stem cells immortalized in vitro with radiation leukemia virus

The differentiative capacity of a unique haematopoietic cell type has been investigated. These cells have been found to be a common target in vitro to infection and immortalization by radiation leukemia viruses (RadLVs). Many continuous lines of these cells have been generated. Since RadLV retroviruses are known to be strictly T-cell-tropic in vivo, we have questioned whether these cells are precursors of T cells. To this end, the RadLV-induced C1-V13D cell line has been inoculated into thymus of sublethally irradiated syngeneic CBA/H mice and tested for capacity to proliferate and differentiate, i.e. express T-cell markers. When inoculated in high number, C1-V13D cells can induce a thymic tumour within 14 to 21 days. Expression of T-cell markers on these cells was determined by fluorescence-activated cell sorting (FACS) analysis, after gating out C1-V13D cells on the basis of their high 90 degree scatter and their high forward scatter. They can also be distinguished by their unique expression of RadLVgp70 envelope (env) protein, B220, CD44, and aberrant expression of a class I epitope. Explanted primary thymomas from many animals showed no evidence of T-cell marker expression on C1-V13D cells upon reisolation. However, when C1-V13D was further passaged intrathymically, there was clear expression of Thy-1, CD4, CD8, and TCR-alpha beta on C1-V13D cells reisolated from these tumours. Two-colour FACS analysis and fluorescent antibody staining have confirmed the acquisition of T-cell surface markers by C1-V13D cells growing in this environment. Northern analysis confirmed endogenous expression of T-cell receptor beta chain genes in C1-V13D cells isolated after the third passage. All data indicate that RadLV preferentially infects a unique haematopoietic precursor cell in spleen which can differentiate along the T lineage once located within the thymic environment. The cell lines described here represent valuable models for studying T-cell differentiation from lymphoid stem cells, and for dissecting the early events in leukemogenesis.