Specific areas of lymphocyte depletion, termed thymus-dependent areas, have been delineated in neonatally thymectomized C3H/Bi and F1 (C57BL x C3H/Bi) mice. They occur within the lymphoid follicles of the spleen immediately surrounding the central arterioles, and constitute the mid and deep cortical zones of the lymph nodes. These depleted areas appear in healthy thymectomized mice as early as 3 wk after operation but, in mice which survive for more than 6 to 7 wk, the thymus-dependent areas are repopulated by rapidly dividing pyroninophilic cells, the majority of which are immature plasma cells. Syngeneic thymus cells, labeled in vitro with tritiated adenosine localize preferentially in the thymus-dependent areas after intravenous injection. Similarly labeled spleen cells also accumulate in these areas but, in addition, are distributed at the periphery of splenic follicles and in the outer cortical zone of the lymph nodes. Many more spleen than thymus cells enter the lymphoid tissues and the spleen appears to be the primary target. The apparent paradox that syngeneic thymus cells are less efficient than spleen cells in restoring neonatally thymectomized mice to normality is discussed in the light of these results and possible routes by which the migrating cells could enter the lymphoid tissues are considered. The origin of the plasma cells which repopulate the lymphocyte depleted areas is also discussed. It is concluded that the normal thymus produces cells which contribute directly to the migratory or circulatory lymphocyte population but that there also exists another source of supply for the plasma cell series. These two systems may function synergistically so that the thymus may control, directly or indirectly, the balance of cell populations within the body.

Normal offspring were obtained from mice with orthotopic ovarian grafts of tissue that had been frozen and stored at -79°C . Tissue so treated showed a remarkable capacity for reorganization and function, but the number of oocytes surviving was small and the reproductive life of the females bearing the grafts was curtailed in each of the four strains of mice used.The most successful method of preservation involved soaking the tissue for 30 to 40 min in a medium consisting of 12 % glycerol in horse serum before slow cooling to -79°C . Oocyte destruction was increased when the concentration of glycerol was reduced to 8 %, when the tissue was soaked for 1 to 2 hr in 15% glycerol in horse serum, and when the tissue was stored at -79e C for longer than 24 hr. Soaking in glycerol solutions at room temperature for 1 \ hr without subsequent freezing also eliminated many oocytes. No viable grafts were obtained after 'twostage' rapid cooling.Preservation of the fertility of mice with grafts of ovarian tissue has proved to be more difficult than maintenance of cyclic cornification of the vagina. The problems involved are discussed.

SUMMARY Existing methods for the production of lymphocytes from the small intestine have proved unsatisfactory when applied to the mouse. We report here a new method for the production of highly pure suspensions of lymphoid cells from the epithelial layer and lamina propria of mouse small intestine. The production and purification methods are described in detail. At least ten million lymphocytes are obtainable from each small intestine from either the epithelium or lamina propria and the cell suspensions are shown to be little contaminated by non-lymphoid cells. Preliminary analysis of the two cell types indicates that they belong either to two separate populations or to one population in very different stages of differentiation. The use of purified lymphoid cells from the epithelium and lamina propria of the small intestine may enable examination of the generation of cytotoxicity towards gut epithelial cells; this may be important in the development of inflammatory bowel diseases.With increasing interest being shown in the immunology of the gut, it has become necessary to produce pure populations of lymphoid cells from the intestine to examine their immune effector function. Potentially the small intestine can yield lymphocytes both from the epithelial layer, the so-called intraepithelial lymphocytes, and from the lamina propria. So far, the majority of studies have been carried out on human small or large bowel tissue where two methods of extraction have been tried: mechanicall-3 and enzymic.3-5 Mechanical and enzymic extraction techniques have also been tried with varying success on experimental animal intestinal tissue.A-8 In the vast majority of these studies, 'mucosal' lymphocytes have been extracted with little regard to their origin from the epithelial layer or lamina propria. In view of the observation8s that cytotoxic T cells in the epithelial layer appear to have a different cytotoxic potential from those in the lamina propria, it is necessary to examine separately lymphoid cells from the two layers when assessing effector function.We report here an efficient, economical, and reproducible technique for producing lymphoid

Lymphocytes separated from the epithelial layer of mouse small intestine, IEL, were tested for their NK cytotoxicity against Yac-1 targets. There was little NK activity in a 4 hour assay, but high activity in an 18 hour assay, and the NK activity of IEL did not parallel that in the spleen in any of the mouse strains tested. Furthermore, IEL exerted a suppressor activity on mouse spleen NK activity. Specific T-cell cytotoxicity appeared in IEL in mice immunized with an intraperitoneal injection of P-815 tumor cells. By contrast with IEL, LPL had little NK or NK suppressor activity, but higher levels of specific T-cell cytotoxicity in tumor-immunized mice than intraepithelial lymphocytes. A high proportion of IEL had granules that stained with Giemsa and Astra blue. Furthermore many IEL carried Lyt-2+ phenotype and no other T-cell surface antigen. Intraepithelial lymphocytes appeared, therefore, to have staining and phenotype characteristics of both granular NK cells and suppressor cells. It was clear that the intestinal mucosa contained populations of immune effector cells that were heterogeneous in nature and function.