Identity and Proliferation of Small Lymphocyte Precursors in Cultures of Lymphocyte-rich Fractions of Guinea Pig Bone Marrow

Radioautography with ³H-thymidine was used to examine the proliferative activity of bone marrow lymphoid cells and to identify the precursor cells of small lymphocytes in short-term cultures of lymphocyte-rich marrow fractions. High concentrations of small lymphocytes (nuclear diameters less than 8.0 µ in smears) together with large lymphoid ("transitional") cells (nuclear diameters greater than 8.0 µ) were separated from suspensions of guinea pig bone marrow by centrifugation in linear sucrose-serum density gradients. When such lymphocyte-rich marrow fractions were cultured in vitro the labeling and mitotic indices following either continuous or terminal exposure to ³H-thymidine indicated that the large lymphoid cells were confined mainly to the pre-DNA-synthetic (G₁) and early DNA-synthetic (S) phases at first, but proceeded subsequently through S phase and mitosis. From these data tentative values were derived for the in vitro duration of G₁ (12 hours) and S (13.7 hours). Further cultures were followed radioautographically after a 1-hour pulse of ³H-thymidine at 6-7 hours of culture. The absolute numbers of labeled large lymphoid cells declined during the subsequent 21 hours but, simultaneously, labeled small lymphocytes appeared and increased progressively in absolute numbers to 44.4 ± 8.1 per cent of the initial numbers of labeled large lymphoid cells. The mean grain count of labeled small lymphocytes was half that of the initially labeled large lymphoid cells. Very few labeled undifferentiated cells other than large lymphoid cells were observed. The results demonstrate that lymphocyte-rich marrow fractions are capable of sustaining the production of small lymphocytes in short-term cultures and that the immediate precursors of marrow small lymphocytes are contained within a population of large lymphoid cells.