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The aims of this study were (a) to find a regime of immunization with cholera toxoid in rats which would establish a high density of antitoxin containing cells (ACC) in the lamina propria of the intestine and (b) to determine the origin of the ACC. The best cellular response was achieved by a single i.p. dose of toxoid in FCA followed by an intraintestinal boost 2 wk later. ACC appeared in the thoracic duct lymph 2 days after boosting, reaching a peak of about 200,000 ACC/h at 3--4 days. This was followed by the appearance of large numbers of ACC in the intestine. The i.p. dose of toxoid by itself gave rise to very few ACC in the gut or thoracic duct lymph, but it had clearly primed the gut immune system for a secondary response. Priming was also achieved by the prolonged oral intake of toxoid. The importance of the intestinal route for boosting was shown by the failure of i.p. challenge to give an ACC response in the intestine after i.p. priming and the small response it provoked after oral priming. ACC among thoracic duct lymphocytes (TDL) and in the lamina propria contained predominantly IgA. Two observations indicated that the major source of the lamina propria ACC was from cells that emerged in the thoracic duct lymph after intraintestinal challenge. Firstly, the establishment of a thoracic duct fistula immediately before challenge prevented the appearance of ACC in the intestine. Secondly, many ACC appeared in the intestine of normal rats after the injection of TDL rich in ACC. Although homing of ACC precursors to the gut was not antigen-dependent, the distribution of ACC in the lamina propria was considerably influenced by the site of the intestinal challenge, the density of ACC being greatest at or distal to the site of injection of toxoid into the lumen of the gut.

The lamina propria of the intestine contains large numbers of IgA-producing plasma cells which arise from precursors in the gut-associated lymphoid tissue (GALT) 1 in response to antigens in the lumen of the gut (1). The precursors are large lymphocytes which travel from the GALT to the blood in the thoracic duet lymph and then migrate into the lamina propria where they secrete IgA antibody (2-4). The mechanism of this selective migration into the lamina propria is not known and, in particular, there is controversy about the role of antigen in determining the distribution of IgA-secreting cells along the intestine.It has been reported that radiolabeled large lymphocytes from adult syngeneic donors will migrate into the intestinal lamina propria of neonatal rats (5) and into fetal intestine grafted under the kidney capsule of adult mice (6). These experiments with "antigen-free" gut are held to show that factors other than antigen determined the gut-homing of large lymphocytes. On the other hand, Ogra and Karzon (7) showed that local immunization of a segment of human colon with polio vaccine was followed by the appearance of specific IgA antibody which was entirely confined to the immunized segment. Husband and Lascelles (8) reported similar findings in sheep with two Thiry intestinal loops. When different antigens were administered into each loop, IgA antibody against each of the antigens appeared only in the correspondingly immunized loop and never in both loops. In these studies antigen appeared to be decisive in determining the localization of the blood-borne IgA precursors. Similar contradictions were reported by Pierce and Gowans (4). In immunized rats, the density of cells in the lamina propria producing IgA antibody against cholera toxoid was always highest in that region of the intestine which had been challenged with antigen. On the other hand, the small intestine of nonimmunized rats rapidly accumulated anti-toxin-containing cells (ACC) after an intravenous injection of lymphocytes obtained from the thoracic duet of immunized donors.In an attempt to reconcile these contradictory reports the colonization of the lamina propria with specific antibody-producing cells has been studied under more defined conditions using Thiry-Vella intestinal loops in rats and cholera toxoid as antigen. The results indicate that the accumulation of ACC in the gut is probably the result of both antigen-dependent and independent processes.

The experiments presented in this paper support the idea that the output of small lymphocytes from the thoracic duct of the rat (about 10 9 /day) is normally maintained by a large-scale re-circulation of cells from the blood to the lymph. It has been shown that the main channel from blood to lymph lies with in the lymph nodes and that small lymphocytes enter the nodes by crossing the walls of a specialized set of blood vessels, the post-capillary venules. In order to trace the fate of small lymphocytes, cells from the thoracic duct of rats were incubated for 1 h in vitro with tritiated adenosine. This labelled the RNA of about 65% of the small lymphocytes and more than 95% of the large lymphocytes; it also labelled the DNA of a proportion of the large lymphocytes. The mixture of small and large labelled lymphocytes was transfused into the blood of two groups of rats which belonged to the same highly inbred strain as the cell donors. At various times after the transfusions the thoracic ducts in one group of rats were cannulated to determine the proportion of labelled cells which could be recovered in the lymph; at corresponding times, the rats in the other group were killed and autoradiographs prepared from their tissues to determine the location of the labelled cells. The radioactive label in the RNA of small lymphocytes was stable enough to ensure that the labelled small lymphocytes which were recovered in the lymph several days after a transfusion were those which had originally been transfused into the blood. When the thoracic duct was cannulated 20 to 27 h after a transfusion, about 70% of the labelled small lymphocytes which had been transfused into the blood could be recovered from the thoracic duct over a 5-day period of lymph collection. During the first 36 to 48 h after cannulation, while the total output of small lymphocytes was falling rapidly, the proportion of labelled cells in the lymph remained approximately constant. The pool of the animal’s own cells with which the labelled cells had mixed contained between 1·5 and 2 × 10 9 small lymphocytes; this was identified as the re-circulating pool. An autoradiographic study showed that after their transfusion into the blood the labelled small lymphocytes ‘homed’ rapidly and in large numbers into the lymph nodes, the white pulp of the spleen and the Peyer’s patches of the intestine. The concentration of labelled cells in other tissues was trivial in comparison. Labelled small lymphocytes were seen penetrating the endothelium of the post-capillary venules in the lymph nodes within 15 min of the start of a transfusion; they were traced into the cortex of the nodes and finally into the medullary lymph sinuses. Labelled small lymphocytes did not migrate into the adult thymus but a few entered the thymus of newborn rats. It was concluded that the re-circulating pool of small lymphocytes was located in the lymphoid tissue, the thymus excepted, and that the rapid ‘homing’ of cells into the lymph nodes had its basis in the special affinity of small lymphocytes for the endothelium of the post-capillary venules. The interpretation of these experiments was not complicated by the presence of large, as well as of small lymphocytes in the suspensions of labelled cells which were transfused. Other experiments, in which the large lymphocytes alone were labelled with tritiated thymidine, showed that most of them migrated from the blood into the wall of the gut where they assumed the appearance of primitive plasma cells; very few divided to form small lymphocytes.

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