ULTRASTRUCTURAL ANALYSIS OF MOUSE THYMOCYTE SUBPOPULATIONS

To understand the lineage relationship and to define morphological cha racteristics of each thymocyte subset, we have performed ultrastructur al analysis of highly purified thymocyte subpopulations. By flow cytom etry, five subpopulations were sorted based on the expression of CD4 a nd CD8 and on cell size (forward scatter): large and small CD4(+)8(+), CD4(-)8(-), CD4(+)8(-), and CD4(-)8(+) thymocytes. Small CD4(+)8(+) t hymocytes were the smallest among lymphoid cells, and had a round and smooth cell outline with condensed nuclei, the cytoplasm was scanty an d the cell organelles were not developed, suggesting the majority of t his subset might be inactive by morphological criteria. CD4(+)8(-) thy mocytes appeared to be similar to peripheral CD4(+) T cells. The CD4(- )8(-) thymocyte subset contained morphologically immature cells in ter ms of cell size, presence of cell surface villi, and euchromatic appea rance of the nucleus. CD4(-)8(+) thymocytes, heterogeneous in cell siz e, nuclear chromatin contents and amount of cytoplasm, could be divide d into two distinct types. Type 1 CD4(-)8(+) thymocytes were intermedi ate in size, and therefore similar to peripheral mature CD8(+) T cells . Type 2 CD4(-)8(+) thymocytes were large and irregular in shape (larg e CD4(-)8(+)) with irregular-shaped and euchromatic nuclei. Large CD4( -)8(+) cells were, thus, considered to be at the transitional stage fr om CD4(-)8(-) to CD4(+)8(+). At least two groups of large CD4(+)8(+) c ells were ultrastructurally classified by the nuclear chromatin conten t. Large CD4(+)8(+) cells with heterochromatic nuclei were round with a smooth cell membrane, whereas large CD4(+)8(+) cells with euchromati c nuclei were spherical with projections. Cytological features of hete rochromatic large CD4(+)8(+) cells are similar to those of small CD4()8(+) thymocytes except for cell size. Euchromatic large CD4(+)8(+) ce lls could be regarded as active blasts potentially leading to mature c ells. Taken together, this is the first report that describes the ultr astructural characteristics of each thymocyte subset highly purified b y flow cytometry.