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Thymosin fraction 5 and a more purified acidic fraction of thymosin at a 100-fold lower concentration elevated cGMP levels, but not cAMP levels, in murine thymocytes. When both thymus and spleen lymphocytes were fractionated via a BSA gradient procedure, both cAMP and cGMP basal values varied depending on the density of the subpopulations. Thymosin Fr5 did not elevate cAMP in any thymus subpopulation of lymphocytes obtained from the BSA density gradients. The cGMP elevation due to thymosin Fr5 in thymocytes was maximal in the most buoyant thymocytes, and no elevation of cGMP was detected in nude mouse spleen lymphocytes. These results suggest that the cGMP elevation may be an early event in the thymosin-mediated differentiation of a more mature subpopulation of thymocytes. They also suggest that utilization of subpopulations may be necessary for the complete determination of the effects of agents on cyclic nucleotide values of lymphocytes.

The enzyme 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) was studied in bone marrow cells from various strains. The bone marrow cells of congenitally athymic (nude) and normal BALB/C mice consisted of 2 subpopulations of nucleated cells, larger cells of 10-14 microns and smaller cells of 7 microns in size. About 75% of the 20 alpha SDH activity was located in the larger cells. Bone marrow cells from young neonatally thymectomized or congenitally athymic mice had an increase portion of big cells and contained twice as much 20 alpha SDH activity as control mice. Liver cells from 1-day-old mice and spleen cells from mice lethally irradiated and bone marrow reconstituted, both of which contained primarily stem cells, erythroid, myeloid and pre B lymphocytes, had essentially no 20 alpha SDH. Following stimulation with PHA marrow cells from athymic nude mice responded to the mitogen in terms of [3H]thymidine incorporation and stimulation of 20 alpha SDH activity. Bone marrow cells after stimulation with PHA were labelled with fluorescein conjugated anti-theta antibodies. With the aid of fluorescence activated cell sorter (FACS II) it was found that the fluorescent cells had 2.5-5 fold more 20 alpha SDH activity than the unlabelled cells. The results support the suggestion that the 20 alpha SDH activity is located in the marrow pre T lymphocytes.