Lymphopoiesis in the rat. I. The effect of pool size on lymphocyte production

A method is described for measuring lymphopoiesis that enables the production of recirculating and non-recirculating small lymphocytes to be estimated simultaneously. Using this technique, experiments were undertaken to determine whether the production of recirculating cells is influenced by the number present in the recirculating lymphocyte pool. The results suggest that neither a massive lymphocyte transfusion nor depletion of the pool by whole body irradiation or chronic lymph drainage affect the rate at which recirculating small lymphocytes are generated.     

Evidence of extensive lymphocyte death in sheep Peyer's patches. II. The number and fate of newly-formed lymphocytes that emigrate from Peyer's patches

The emigration of newly produced lymphocytes from Peyer's patches (PP) of lambs was studied. Mesenteric lymph nodes (MLN) were excised from most animals a few weeks after birth, and then at 8 to 10 wk of age, the dividing cells in 3 to 4 m of the small intestine were labeled in situ with [3H]thymidine. An extracorporeal perfusion system was used to restrict the 15-min period of labeling to the perfused lengths of intestine, which included either the large continuous ileal PP or a number of smaller jejunal PP. One or 3 days later, the number of labeled cells in the perfused tissue and in other lymphoid organs was studied by autoradiography. In the perfused tissues, labeled lymphocytes accounted for 63.7% of ileal PP cells by 1 day and for 86.7% by 3 days compared with only 9.6% of lymphocytes in the perfused MLN. Labeled lymphoid cells in the perfused PP were nearly all in the follicles. Labeled lymphocytes that must have been produced in the segments of ileum or jejunum at the time of the perfusion, subsequently emigrated via the lymphatics, and were identified in the spleen, MLN, other lymph nodes, blood, jejunal PP, and at a lower frequency in the thymus, nonperfused ileal PP, and bone marrow. In lymph nodes, spleen, and nonperfused PP, more than 80% of the immigrant newly formed PP-derived cells were small- and medium-sized lymphocytes, and about 15% were large lymphocytes. The nature of the labeled cells in the lamina propria of the nonperfused small intestine was quite different in that approximately 50% were plasma cells as early as 24 hr after the cells were born in the perfused gut. It is proposed that terminal B cell differentiation was most likely initiated within the PP in response to the entry of antigen. It was estimated that at both 1 and 3 days after perfusion there were about 100 times more labeled cells in the perfused ileal PP than could be accounted for by emigration to other organs. It was concluded that these results provide additional support for the view that PP in lambs produce a tremendous number of lymphocytes, but relatively few leave their site of production; most apparently die in situ.     

The migration of lymphocytes across specialized vascular endothelium. IV. Prednisolone acts at several points on the recirculation pathways of lymphocytes

Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15–18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes. Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A second effect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

STUDIES ON LYMPHOCYTES

Continuous extracorporeal irradiation of the circulating blood (ECIB) of from 3 to 501/2 hr duration was used to study in the calf the differential depletion of lymphocytes from spleen, lymph nodes and thymus as compared to blood and thoracic duct lymph. The cell content of tissues was measured by planimetry and/or test point analysis. Lymphocyte depletion by ECIB from various lymphoreticular organs, and from different areas within a given organ, was less than in the circulating blood or the thoracic duct lymph and varied from one site of a lymphoreticular organ to another. The degree of depletion with time followed an exponential function with at least two components. The first component corresponded to a relatively rapid fall and the second to a very slow reduction in lymphocyte content. The former is related to the elimination of an easily mobilizable pool of lymphocytes while the latter corresponds to a more sessile mass of lymphocytes which exchange with blood lymphocytes very slowly. Elimination of the easily mobilizable pool of lymphocytes by ECIB from all tissues studied was observed within 10–15 hr, indicating that the rate of exchange with blood is similar for this group of cells in various lymphoreticular tissues. The size, however, of the easily mobilizable vs the more sessile pools of lymphocytes may vary considerably, the best estimates for the former being as follows (in per cent of total lymphocyte mass): lymph node medulla, less than 10%; lymph node cortex plus paracortical zone, 18% (depletion mainly paracortical); red pulp of the spleen, 37%; densely populated white pulp of the spleen, 55%; and loosely populated white pulp of the spleen, 60%. In comparison, the approximate fractions of lymphocytes originating fromthe easily mobilizable pools in various lymphoreticular tissues plus the cells already circulating a t the onset of EClB correspond to 64% for the thoracic duct lymph and 78% for the circulating blood respectively. These findings are discussed in relation to production, recirculation and life span of lymphocytes, and immune reactions.     

Lymphopoiesis in the rat. II. The effect of thymectomy

Lymphopoiesis with respect to recirculating and non-recirculating small lymphocytes was measured simultaneously in rats thymectomized as adults. Removal of the thymus at four to five weeks of age had a profound inhibitory effect upon the production of recirculating cells, whereas the formation of non-recirculating lymphocytes was only slightly depressed. Thymectomy had approximately the same impact of lymphopoiesis as thymectomy and exposure of the animal to a large dose of whole body X- and γ-irradiation. The latter finding, and the failure of a thoracic duct cell transfusion to augment lymphocyte production, accord with the view that the thymus is the principle intermediate source of recirculating small lymphocytes in the normal, unstimulated animal.     

B lymphocyte colony-forming cells in the SJL/J mouse.

Mercatoethanol-induced B lymphocyte cloning in semi-solid agar has been used to study lymphocyte colony formation by cells from the SJL/J mouse thymus. From the 3rd month of life, the SJL/J mouse thymus. From the 3rd month of life, the SJL thymus develops an increasing frequency of cells forming B lymphocyte colonies in agar. The peak frequency in 6- to 12-month-old mice was one colony per 1000 to 2000 cultured thymus cells. In contrast, 10 to 100 times lower frequencies were found in the thymus of five other inbred mouse strains. The rise in B lymphocyte colony-forming cells correlated well with the age-related rise in Ig-positive cells and approximately 50% of the colony cells reacted with anti-micron-serum indicating the B lymphocyte nature of the colony cells. Colony-forming cells from the thymus showed higher sensitivity than colony-forming spleen cells to cortisol and irradiation. Cell transfer experiments and thymus grafting suggested that the increased frequency of colony-forming cells in the thymus is caused by development of special thymus-seeking B lymphocytes in ageing SJL/J mice. Finally, B lymphocyte colony-forming cells were found to be more frequent in the thymus, spleen, and lymph nodes from healthy aged mice than in lymphoid organs from mice with spontaneous reticulum cell tumors.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Role of lymphocyte surface determinants in lymph node homing

Thoracic duct lymphocytes briefly incubated in vitro with trypsin and then infused into syngeneic rats are unable to migrate into lymph nodes. Trypsin-treated lymphocytes incubated at 37 °C in the absence of enzyme for 12 hr recovered their lymph node homing properties. In vitro recovery did not occur if the cells were cultured at 17 °C. Evidence was obtained that trypsin cleaved sialyglycoproteins from the surface of lymphocytes and that these determinants reappeared after the cells were maintained at 37 °C for 24 hr.Puromycin added to cultures of normal lymphocytes for 3 hr before infusion markedly reduced the recovery of donor cells in lymph nodes. The results suggest that surface determinants of recirculating lymphocytes essential for homing into lymph nodes may be rapidly turned over.     

THE KINETICS OF LYMPHOCYTE RECIRCULATION WITHIN THE RAT SPLEEN

The migration of lymphocytes from the blood into the splenic pulp and the release of lymphocytes from the spleen into the blood was studied by isolating the rat spleen and perfusing it with 15 ml of recirculating, oxygenated blood. When thoracic duct lymphocytes labelled with tritiated uridine were added to the initial perfusate the concentration of these cells fell exponentially for 2–3 hr and then rose to a flat secondary peak. From this pattern it was inferred that small lymphocytes entered the spleen at a rate proportional to their instantaneous concentration in the perfusate, traversed the splenic pulp and re-entered the perfusate with a minimum transit time of 2–3 hr. The rate of release of small lymphocytes from the spleen was not influenced by the prevailing concentration of small lymphocytes in the perfusate but probably reflected the rate of migration into the spleen over a period earlier than 2 hr before. The rate of exchange of small lymphocytes between the blood and the intact spleen in vivo was estimated to be about 84 × 10⁶ cells/hr. The size of the intrasplenic pool of recirculating small lymphocytes was probably 400–500 × 10⁶ cells. The rate of migration of small lymphocytes into the spleen was not affected by prior irradiation of the spleen donor. When either of two antigenic materials were added to the perfusate no inhibition of lymphocyte migration into the spleen was noted although the release of lymphocytes from the spleen was diminished by the addition of a large dose of sheep erythrocytes.     

A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat

Cell surface molecules involved in lymphocyte adhesion to high endothelial cell venules (HEV) of Peyer's patches (PP) have been studied in the rat by using a mouse monoclonal anti-HEBFPP (1B.2) antibody. We previously showed that rat thoracic duct lymph contains a high endothelial cell binding factor termed HEBFPP, which in vitro blocks lymphocyte binding sites of HEVPP but not HEVLN. Monoclonal 1B.2 antibody was produced by fusing P3U1 myeloma cells with spleen cells of a mouse immunized with this material. Immunoprecipitation studies with 125I surface-labeled rat thoracic duct lymphocytes (TDL) showed that the antibody recognized an 80-kilodalton protein. This antigen was present in the majority of TDL, spleen, LN, and PP cells but was found on few (5 to 10%) thymus and bone marrow cells (indirect immunofluorescence). Treatment of TDL with 1B.2 antibody blocked their ability to bind in vitro to HEVPP; antibody treatment did not interfere with TDL adhesion to HEVLN. Analysis of 1B.2 antigen isolated from lymph and detergent lysates of TDL by antibody-affinity chromatography showed that this material had the capacity to block lymphocyte binding sites of HEVPP but not HEVLN. In contrast, material with such blocking activity was not isolated from detergent lysates of thymocyte, a population deficient in HEV-binding cells. The results indicate that the 1B.2 antigen is a component of the lymphocyte surface recognition structure mediating adhesion to HEVPP and provide further evidence that distinct adhesion molecules of rat TDL mediate interaction with high endothelium of LN and PP.     

Lymphocyte adhesion to cultured Peyer's patch high endothelial venule cells is mediated by organ-specific homing receptors and can be regulated by cytokines

Adhesion of lymphocytes to high endothelial venule (HEV) cells is the first step in the migration of these cells from blood into lymph nodes and Peyer's patches (PP). In the present study, we isolated and cultured HEV cells from PP of the rat and assessed their capacity to interact with lymphocytes. Flow cytometric analysis with a rat HEV-specific mAb KJ-4 revealed that greater than 90% of the cultured cells were stained by the antibody. Furthermore, confluent monolayers of PP HEV cells retained the capacity to support the adhesion of lymphocytes from spleen, thoracic duct, and lymph nodes but not binding of immature cells from thymus and bone marrow, which are deficient in cells capable of binding to HEV in vivo. In addition, intraepithelial lymphocytes that preferentially migrated into mucosal lymphoid tissues were also enriched in cells that adhered to the endothelial monolayers. The binding process required energy, was calcium-dependent, and could be inhibited by cytochalasin D, trypsin, and mixed glycosidase. Interestingly, pretreatment of PP HEV cells with rTNF, IFN-gamma, or granulocyte-macrophage CSF significantly increased the endothelial adhesiveness for thoracic duct lymphocytes in a time- and dose-dependent manner. In contrast, stimulation of lymphocytes with phorbol ester or TNF resulted in the rapid modulation of the surface expression of the PP homing receptor and decrease in lymphocyte binding to normal or TNF-stimulated HEV cells. The adhesion of lymphocytes to normal or cytokine-stimulated HEV cells can be blocked by pretreatment of lymphocytes, but not HEV cells, with the PP homing receptor-specific 1B.2.6 antibody. Taken together, these experiments provide strong evidence that the interaction between lymphocytes and cultured HEV cells are mediated by adhesive mechanisms that regulate lymphocyte entry into PP in vivo and that cytokines can promote HEV adhesiveness for lymphocytes through increased expression of organ-specific ligands on HEV cells.     

The influence of strong transplantation antigens (Ag-B) on lymphocyte migration in vivo.

The tissue localization of syngeneic thoracic duct lymphocytes was compared to that of allogeneic cells in four rat strain combinations differing at the Ag-B locus (HO → DA, DA → HO, AO → HO, HO → AO). Dual isotope labeling with [³H]uridine and [¹⁴C]uridine was applied in order so that the distribution of allogeneic and syngeneic cells could be followed in one recipient. During the first couple of hours after iv injection, allogeneic lymphocytes usually migrated as easily into the various tissues as did syngeneic cells. However, after 24 and 48 hr, a reduced amount of label associated with allogeneic cells was often measured in the tissues. This reduction differed in magnitude in the different strain combinations and was most pronounced in the lymph nodes. A reduced number of allogeneic cells also appeared in the thoracic duct. By contrast, no reduced localization of allogeneic lymphocytes was measured in the draining popliteal lymph nodes late after sc injection. In preimmunized animals allogeneic cells were rapidly removed from the blood and therefore failed to localize in the lymphoid tissues. Furthermore, the lymph node localization of allogeneic cells was more like that of syngeneic cells in splenectomized rats, as well as in irradiated recipients (when the irradiation was given shortly before cell transfer). It is concluded that transplantation antigens play no essential role in the interaction between recirculating lymphocytes and the venous endothelium at the sites where the large-scale physiological emigration of the cells takes place (the HEVS of the lymph nodes and the marginal zone vessels of the spleen). The elimination of allogeneic cells is found later; it probably takes place in the lymph nodes and spleen. Possible mechanisms responsible for this rapid removal of allogeneic lymphocytes in nonimmunized recipients are discussed.     

Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte.

The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately 10(4) fold by the addition of 2 X 10(6) normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG8 myeloma tumor cells secreting IgG1. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth-supporting factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cell abolishes their growth-enhancing properties. Thymus cells can be syngeneic and allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-8 week old C3H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of XB3-AG8 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shown that these B cells divide every 18 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immonoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.     

ESTIMATION OF THE MIGRATION OF THORACIC DUCT LYMPHOCYTES TO NON-LYMPHOID TISSUES

The distribution of radioisotopes in tissues was measured following i.v. injection of labelled thoracic duct lymphocytes into syngeneic rats. The rate of elution of an isotope from the labelled cells and the subsequent fate of the eluted isotope were shown to be the most important factors limiting the usefulness of such isotopes for measuring cell localization particularly in non-lymphoid tissues. Comparison of labelling procedures using [³H] and [¹⁴C]uridine, [³H] and [¹⁴C]leucine, [⁷⁵Se]-L-selenomethionine, [^99mTc]sodium pertechnetate and [⁵¹Cr]sodium chromate in vitro and [³H]thymidine in vivo showed that ⁵¹Cr had the fewest disadvantages in the present context. Using ^5ICr-labelled cells, the radioactivity was measured in a wide range of non-lymphoid tissues, and estimates of cell traffic were obtained. In skin, for example, the results indicate a cell flux in the range of 10⁴-10⁵ lymphocytes/gm/hr. Evidence is presented which suggests that the early substantial localization of labelled cells in the lung is not an artefact due to sequestration or embolization of traumatized cells but probably reflects a slow intravascular transit time through this capillary bed. The primary lymphoid organs, thymus and bone marrow were shown to include a subpopulation of lymphocytes which belong to the recirculating pool. The thymus always contained a greater concentration of radioactivity at 24 hr than all non-lymphoid tissues except liver and kidney (approx. 0-1% of the recirculating lymphocyte pool) and the bone marrow was capable of temporarily accepting a substantial proportion (approx. 25%) of the injected cells.     

The kinetics of antigen-reactive cells during lymphocyte recruitment

Lymphocyte recruitment, the increased traffic of lymphocytes from blood to lymph which occurs within antigenically stimulated lymph nodes, was monitored in the efferent lymph of single lymph nodes in sheep after immunization with allogeneic lymphocytes or purified protein derivative. Specific antigen-reactive cells were assayed by their ability to proliferate in vitro in the presence of the priming antigen. During lymphocyte recruitment such cells were no longer detected in the efferent lymph draining either the immunized node or a nonstimulated node remote from the region of antigen administration. These results probably reflect the selective removal of specific lymphocytes from the recirculating pool. Alternatively, the findings could involve a state of specific unresponsiveness of the cells.     

Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

Quantitative evaluation of the total number and distribution of lymphocytes in young pigs.

In young pigs, the spleen, thymus and all lymph nodes were dissected out and weighed. The relative content of lymphoid cells was determined from histological sections. The number of nucleated cells was evaluated by two different methods: firstly, by measuring the DNA content of samples of lymphoid tissue and dividing by the DNA content of a single nucleus; and, secondly, by counting all lymphoid cells in histological sections of defined volumes of these organs. The number of lymphoid cells in tonsils, gut, bone marrow and lung were determined using histological evaluations and the volumes or weights of these organs. The resulting average number of lymphocytes was 321 times 10 (9) for a pig of 26 kg body weight. The lymphocytes showed the following distribution in lymphoid and non-lymphoid organs: thymus 44%, spleen 9%, mesenteric lymph nodes 17%, cervical lymph nodes 9%, other peripheral lymph nodes 3%, gut-associated lymphocytes 5%, tonsils 2%, bone marrow 5%, blood 3%, lung 0.2% and an estimated figure of 3% for all other tissues.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Peyer's patches export lymphocytes throughout the lymphoid system in sheep

The lymphocyte output from small intestine containing either the long continuous ileal Peyer's patch (PP) or several smaller jejunal PP was examined in young lambs. Most studies were done in 2-mo-old lambs, 1 mo after removal of mesenteric lymph nodes (MLN). Extracorporeal perfusion of part of the intestine and addition of fluorescein isothiocyanate to the perfusate led to the labeling, in their normal microenvironment, of a regionally defined population of cells. One day later considerable numbers of emigrant lymphocytes were identified by fluorescence microscopy in the spleen, MLN and peripheral lymph nodes, jejunal PP, and bone marrow. In nonperfused ileal PP and thymus the labeling indexes were low. The highest labeling index was in the blood where 3.7% of the lymphocytes were labeled. A similar organ distribution of emigrant cells was found on day 3. When MLN were included in the perfused region more emigrants were identified. In some animals the intestinal lymphatic draining the perfused ileum was cannulated. Continual lymph drainage caused a dramatic decrease in the labeling indexes in other lymphoid organs. A substantial number of lymphocytes leave both ileal PP and jejunal PP via lymphatics and travel to all other lymphoid organs. However, the number of emigrant lymphocytes compared with the total number of labeled lymphocytes in the perfused tissue was about 10 times greater after perfusing gut with the jejunal PP than after ileal PP perfusion. We conclude that relatively more lymphocytes emigrate from the jejunal PP than from the ileal PP.