Cellular Recognition in Vitro by Mouse Lymphocytes

Abstract

The thymus is influential in establishing and maintaining elements of immune function throughout life (1, 2) and appears to be required for elicitation of delayed hypersensitivity reactions, for effective rejection of allografts (3, 4), for resistance to tumor induction (5, 6), and for response to certain classes of antigens (7,8). The objective of the studies to be reported here was to examine further the role of the thymus in cellular recognition. The susceptibility of whole and albumin gradient-separated subpopulations of cells taken from mice thymectomized at birth to stimulation in vitro by alloantigens and P H A was assayed. The capacity of thymus grafts of differing origins to restore cellular reactivity was also explored. These studies confirm that the thymus is required for normal levels of responsiveness of lymphoid cells to primary stimulation in vitro by alloantigens and PHA. Materials and MethodsExperimental Animals.--Mice of the inbred strains, C57BL/6J, A/J, BALB/c, DBA/2, and CBA/J, and the F~ hybrids of these strains, were derived from inbred stocks maintained in this laboratory or purchased directly from the Jackson Laboratories, Bar Harbor, Maine.Neonatal Thymectomy and Thymus Grafting Proceclure.--Thymectomy was performed within 24 hr of birth by the method described by Sjodin et al. (9). Macroscopic examination when the animals were sacrificed for an experiment verified the success of thymectomy.The thymus graft procedure was a modification of that of Metcalf et al. (10) in which a polyethylene trocar containing one lobe of newborn thymus tissue was inserted under the left kidney capsule under direct vision. The trocar was approximately the size of an 18 gauge needle and was mounted on a syringe containing 0.5 ml of air.

Potentiation of the T-Lymphocyte Response to Mitogens

“…These cells, together with those in band C, were responsive to PHA, as reported in earlier work (35,36). However, thev were much more stimulated by SUP and still more by SUP -k PHA.…”

“…Cell Separation.--Discontinuous bovine serum albumin (BSA) density gradients (34) were used to separate thymocytes into four bands, designated A, B, C, and D (from least to most dense) plus a pellet, P. As in earlier work (35,36), larger cells were found almost exclusively in A, B, and C, and macrophages in A and B. For removal of adherent cells (2, 6), 1-ml all quots of cell suspension, containing l0 T cells in MEM with 10% normal human serum or fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.), were incubated in Petri dishes (No.…”

Density Gradient Electrophoresis of Mouse Spleen Lymphocytes

“…These find ings are in agreement with the literature, where T lymphocytes separated by free-flow electrophoresis [13][14][15] or by other meth ods, are reported to be the responding cells [16][17][18][19]. Stimulating cells in our experiment were found in the low and intermediate mo bility fractions, where the majority of the cells were B lymphocytes and macrophages.…”

Immunologic Properties of Mouse Thymus Cells

“…The data in Tables II and III also show that the degree of PHA responsiveness in the B layer, although exceeding that of the other fractions and of whole thymus, is less than that found here and previously (15,16) cells were treated with anti-TL, -0, or -H-2 u in various experiments. The functional capacity of varying numbers of the resultant cell populations was then evaluated in terms of the ability to evoke GVH reactions in (CBA X C57BL/6)F1 hybrids, to recognize mitomycin-C-treated CBA alloantigens in vitro, or to be stimulated by PHA.…”