STUDIES ON LYMPHOCYTES

Tritiated thymidine was administered to calves continually for 2 to 8 days via the thymic artery in an attempt to label intensively thymic lymphocytes. Heavily labeled cells which had migrated from the thymus were observed in the spleen, lymph nodes and Peyer's patches. Cell maps were made for the various lymphoid tissues and in all cases the majority of labeled thymic cells were found in the ‘thymus dependent areas’of the spleen and lymph nodes. The number of labeled thymic cells per thousand lymphocytes was highest in the ‘thymus dependent areas’. A few labeled thymic cells were seen in or near the post capillary venules. The labeling pattern in the Peyer's patches was different from that in the spleen and lymph nodes. Labeled thymic cells were not observed in the bone marrow. Heavily labeled cells were not detected in any of the lymphoid tissues of those calves which received continuous intravenous infusion of comparable amounts of tritiated thymidine.     

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm ³H/mg tissue or as the percentage change in [³H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena.All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.     

Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

The circulating life span, immunocompetence and 3H-uridine uptake of small lymphocytes from thymus-grafted, neonatally thymectomized rats.

By 7 weeks post-grafting, the number of small lymphocytes in the thoracic duct lymph (TDL) and blood of the thymus-grafted neonatally thymectomized adult rats had increased to 60% of the number of cells in sham controls, or 2-1/2 times thymectomized control values. This increasing consisted almost exclusively of long-lived, recirculating small lymphocytes and corresponded to a 60% recovery of cellular immunocompetence as measured by the mixed lymphocyte reaction (MLR). Associated with the return of cellular immunocompetence was an increased incorporation of 3H-uridine by the small lymphocytes. Cells from thymectomized animals grafted with lymph node fragments demonstrated no significant increase in lymphocyte numbers nor was there a return of immunocompetence as compared to thymectomized controls.     

3H-deoxythymidine incorporation in graft-versus-host disease in the Norway rat. II. Autoradiographic studies.

A sequential analysis was made of various areas within the lymph nodes and spleen of newborn Brown Norway (BN) rats suffering from graft-versus-host disease (GVHD) subsequent to an allogeneic injection of adult Lewis (L) lymph node cells (experimental). One micron thick autoradiographs were compared between such experimental and control littermates having received the same number of syngeneic adult BN cells. Both experimental and control animals received tritiated deoxythymidine (3HdT) one hour before killing. The autoradiographs revealed a 2.25 and 2.50 times higher thymidine labeling index of lymphocytes in the deep cortex of mesenteric lymph nodes and white pulp of the spleen, respectively, for experimental animals. The experimental effect occurred within one day. The majority of the labeled cells in experimental animals were large lymphoblasts with prominent nucleoli. The labeling index within these areas remained significantly higher than control values until day 8 in the spleen and through day 14 within the lymph nodes. However, differences in labeled cells present in high powered microscopic fields reached a peak on day 3 within compartments in experimental animals but fell significantly below control values by day 9 owing to a pronounced disappearance of both small and large lymphocytes from these areas, and a decreased intensity of individual cell labeling as the reaction progressed. In contradistinction the concentration of labeled cells present in high powered microscopic fields of lymph nodes' medulla became 3.13 times controls by day 4. Most of these labeled cells contained a more basophilic cytoplasm than those found in the deep cortex and some were distinctly plasma cell precursors. In contrast to the deep cortex their concentration remained approximately three times control values until death. The data indicates that the major proliferative events within the spleen and lymph nodes in neonatal rat GVHD are initially restricted to donor cell localization areas of these tissue compartments. Subsequently the GVHD-related events may be attributed to other areas and possibly cell types. Thus any proliferation contributing to splenomegaly in the latter stages of GVHD appears to occur in the red pulp and that contributing to lymph node enlargement a medullary response.     

Reaction of human lymphocytes with aggregated IgG columns. Effect on antibody-dependent cytotoxicity and EAC rosette formation.

The origin and life span of long-lived small lymphocytes in the bone marrow of mice have been evaluated by the use of radioautography, scintillation counting, and anti-theta serum. Thymus-deprived BALB/C mice and nude mice had a smaller percentage of long-lived lymphocytes in bone marrow and in thoracic duct lymph than sham-operated or normal littermates. Furthermore, the long-lived lymphocytes in the marrow of nudes have more varied—but generally shorter—life spans than long-lived lymphocytes from mice having a thymus. In thoracic duct lymph of nude mice a more homogeneous long-lived population—according to life span—was found.It was concluded that both long-lived T cells and long-lived B cells are normal residents in the bone marrow. Furthermore, it was concluded that cells of variable life spans comprise the B lymphocyte population: short-lived cells with life spans of 3–5 days and long-lived lymphocytes with life spans of weeks to months.     

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.     

Immune mechanism of rats on Nippostrongylus brasiliensis in vitro. II. The influence of lymphocytes and peritoneal cells

Nippostrongylus were collected from the intestines of rats 6 days p.i. and kept under sterile conditions in cultures. Serum, lymphocytes and peritoneal cells of immune or non-infected animals were added in various combinations to the culture media. The culture media were changed 2-3 times in an experimental period of 10 days, resp. serum and cells were added. The lymphocytes were isolated from the peripheral blood or from the mesenterial lymph nodes whereas the mononuclear cells were obtained from the peritoneal cavity. Serum and lymphocytes from the peripheral blood both from immune and non-infected rats, had no increased lethal effect on Nippostrongylus. The highest lethality rate of adults (65-68%) was achieved in cultures with peritoneal cells and lymphocytes from the lymph nodes of sensitized rats. Serum of infected or non-infected animals had no influence on adult Nippostrongylus in cultures with these cell combinations. In the controls without any cell-supplements the survival rate of the parasites was up to 88%.     

The bronchoalveolar lymphocyte: Studies on the life history and lymphocyte traffic from blood to the lung

Although recent work has shed some light on the identity and function of lymphocytes that reside in the bronchoalveolar air space (lung lymphocytes), little is known about the origin and life history of these cells. To determine the proportion of recently divided lung lymphocytes, DA-strain rats were labeled in vivo for 3 days with tritiated thymidine ([³H]dTR). Autoradiographs of lavaged lung and peritoneal cells indicated that a large fraction (44–77%) of lung lymphocytes was labeled and that these values were comparable to the proportion of labeled lymphocytes in peritoneal exudates (61–74%). To determine if some newly divided lung lymphocytes might come via the blood, additional experiments were performed in which rats were labeled ([³H]dTR) in vivo for 7 days. Lymphocytes were then obtained in labeled rats by thoracic duct drainage and were adoptively transferred (by vein) into syngeneic recipients. The percentage recovery of labeled lymphocytes in lung aspirates of recipient rats was determined from cell counts and autoradiographs. These results demonstrate that blood may be a source of recently divided lymphocytes but they do not indicate the relative contribution blood makes toward these cells in the lung.     

Some endocrine aspects of the thymus gland.

In studies of the mouse thymus, lymphocyte mitoses are seen to be most frequent in the thymus cortex. There is evidence from thymic grafts that a hypothetical factor, thymopoietin, may stimulate mitosis of thymic lymphocytes. It is a factor which is postulated to act in conjunction with the PAS-positive mesenchymal reticular cells and epithelial reticular cells of the cortex. The thymus medulla is necessary for the integrity of thymic grafts, and may also elaborate a secretion for maintaining the cellular functions of the gland. Thymectomy has been used as a gauge for judging normal thymic function and results, in the mouse, in lymphopenia, degeneration of spleen and lymph nodes, delayed rejection of skin allografts, reduced ability of spleen cells to mount the graft versus host reaction, and reduced primary immune response to certain antigens. Correction of these deficiencies offers a means of evaluating various thymic extracts and grafts. Lymphocytosis-stimulating hormone (LSH) is known to maintain the peripheral lymphoid organs and cause lymphocytosis in the thymectomized animal. Diffusion chamber studies of thymic grafts also show restored lymphoid tissue by a cell-free factor (CIF). These two factors may be the same and probably represent the basis of the highly purified lymphocyte-stimulating proteins, LSHr and LSHh, which restore the L/P ratio in thymectomized animals and may stimulate lymphopoiesis in spleen and lymph nodes. LSHr, unlike LSHh, increases the total lymphocyte count. LSHr has been found to increase the humoral antibody response in neonatal mice both by the PFC technique and by direct hemolysis of sheep erythrocytes. Homeostatic thymic hormone (HTH) is a thymic extract of small molecular weight and contains nucleic acid. In the thymectomized guinea pig it has been found to maintain normal levels of lymphocytes in the blood, spleen and lymph nodes, to restore antibody titers to typhoid H antigen and to restore the toxic allergic reaction. Thymic humoral factor (THF) is of smaller molecular weight (less than 1,000) and probably is not a protein. It also enhances lymphoid proliferation in neonatally thymectomized mice. There is evidence that THF participates in humoral antibody formation because it stimulates PFC formation from neonatally thymectomized mice after inoculation with sheep erythrocytes. Its effects on cell-mediated immunity are seen from findings that injection of THF restores the ability of thymectomized mice to reject skin allografts. THF enables spleen cells from thymectomized or neonatal animals to mount the graft versus host reaction, and causes maturation of bone marrow cells and spleen or lymph node cells so that they can participate in the graft versus host reaction. It has been reported to stimulate lymphocytes to kill isogeneic tumor cells in vitro. Thymosin is protein extracted from the thymus. It has been found to alleviate leukopenia slightly and provide some improvement in lymphoid histology in thymectomized mice...     

Cell-mediated immune response to lymphocytic choriomeningitis and vaccinia virus in rats.

The parameters of cell-mediated immune responses of rats to infection with lymphocytic choriomeningitis virus or vaccinia virus were assessed by measuring primary footpad swelling, increased weights of the local lymph nodes, increased numbers of lymphocytes per lymph node, and the course of virus-specific cytolytic activity by these lymphocytes. Except for lack of a defined swelling caused by vaccinia virus injected into the hind footpads of rats, the kinetics of all these responses correlated and were in accord with the usual time course of cellular immune responses. Starting 3 days after infection, peaking at 5 to 7 days, and disappearing after 10 to 12 days, the responses by rats to both viruses were comparable to those found in mice. The phagocytes of these infected rats inhibited the growth of Listeria monocytogenes in vivo, indicating activation of the macrophages by virus-specific cellular immunity. The rat cytotoxic lymphocytes were thymus derived as judged by various criteria: inactivation by an absorbed rabbit anti-rat brain antiserum plus C, susceptibility to anti Thy 1.1 plus C, restriction of the lytic activity within inbred strains and probably by the Ag-B locus, and the kinetics of the response. The cytotoxic T lymphocytes were virus specific since they killed only target cells infected with the same virus but not uninfected cells, or targets that were infected with an unrelated virus.     

WOUND HEALING AND COLLAGEN FORMATION : VI. The Origin of the Wound Fibroblast Studied in Parabiosis

Healing skin wounds were studied in a series of parabiotic rats. The femurs of one parabiont of each pair were shielded whilst both animals were given 800 r from a Co⁶⁰ source. The animals were wounded 3 days after irradiation. Each animal with partially shielded marrow was then given tritiated thymidine intraperitoneally daily while the cross-circulation was arrested by clamping. After the thymidine-³H had cleared the blood, the clamp was released. Animals were sacrificed, and wounds were prepared for radioautography 1, 2, and 6 days after wounding. In the wounds of the shielded animals thymidine-³H was observed in epidermis, endothelium, leukocytes, fibroblasts, and mast cells. Only neutrophilic leukocytes, monocytes, and lymphocytes were labeled, as determined by light and electron microscope radioautography, in the wounds of each nonshielded parabiont. None of the many fibroblasts present were found to contain label in the wounds of the nonshielded parabionts through the 6 day period. These observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells.     

Migration of recently divided B and T lymphocytes to peritoneum and lung

It is recognized that a population of newly divided (or young) cells migrate preferentially to inflamed foci. It has been shown that a large proportion of lymphocytes residing in the bronchoalveolar airspaces of rat are recently divided cells and that blood may be an important source of these cells. To further delineate how blood may contribute to lymphocyte subpopulations in inflamed peritoneum and lung, a comparison of the capacity of recently divided T and B cells to migrate from blood to inflamed peritoneum and lung was made. To label young lymphocytes, DA strain donor rats were given Initiated thymidine by vein in vivo for 7 days. After thoracic duct drainage, the following labeled cell populations were adoptively transferred by vein into syngeneic recipients: (i) unseparated thoracic duct lymphocytes (TDL), (ii) enriched T cells (>90%) or B cells (>80%) recovered after passage of TDL through nylon columns, and (iii) thoracic duct lymphocytes (> 99% B cells) obtained from “B rats” that were prepared by X irradiation, thymectomy, and bone marrow reconstitution. T and B cells were identified by specific heterologous antisera. The percentage recovery of labeled lymphocytes in the recipients with inflamed peritoneum or lung aspirates was determined from cell counts and autoradiographs. The studies indicated that (a) both labeled T and B cells migrated to inflamed peritoneum and lung; (b) labeled B cells migrated to peritoneum and lung better than did labeled TDL or T cells; and (c) labeled lymphocytes did not migrate to unstimulated peritoneum. The enhanced migration of newly divided B lymphocytes to inflamed peritoneum and normal lung (a site that is likely under chronic antigenic stimulation) was unexpected, but may provide additional information on the relative contribution of these subpopulations in the immune inflammatory response.     

Kinetics of Kupffer cells as shown by parabiosis and combined autoradiographic/immunohistochemical analysis

Leucocytes from syngeneic rats were labeled with tritiated thymidine and donor and recipient rats were connected by a bilateral arteriovenous shunt. Based on the time-dependent label index and labeling intensity, it was concluded that Kupffer cells, the resident macrophages of the liver, have a half-life of 12.4 days and originate from monocytes undergoing one mitosis within 8.4 days after immigration into the liver. The labeled cells were easily identified as Kupffer cells by their selective immunoreactivity with the monoclonal antibody Ki-M2R which is specific for phagocytosing macrophages in the rat. The applicability of combined autoradiography/immunohistochemistry for the identification of other poorly defined macrophage subpopulations is shown.     

Circulating T and B lymphocytes of the mouse. I. Migratory properties

Studies on the identity of thoracic duct lymphocytes (TDL⁴) from normal and T cell-depleted mice indicated that as many circulating B lymphocytes were produced by healthy T cell-depleted mice as normal mice. Proportions of T and B cells from the thoracic duct of CBA mice changed markedly during the first 4 days of drainage from 82% T cells and 16% B cells at 12 hr to approximately equal proportions of both classes after 3 days. In absolute terms, T cells were mobilized rapidly by thoracic duct drainage and B cells very slowly. Histologically, this was reflected in a rapid depletion of the T cell-dependent areas of the lymphoid organs. The B cell-dependent areas, in contrast, became depleted of lymphocytes only after drainage for a week or more.The homing properties of circulating lymphocytes were investigated using TDL from normal and T cell-depleted mice as relatively pure sources of T and B cells, respectively. Four hours after injection of ⁵¹Cr-labeled T and B cells, a large proportion of both cell classes were found in the spleen. By 24 hr, many T cells had left the spleen and appeared in the lymph nodes. Such redistribution by B cells, however, was minimal.Intravenously injected T and B cells, labeled with tritiated uridine (³HU), localized specifically in the T and B cell-dependent areas, respectively, of the lymphoid tissues.³HU-labeled T cells were found to recirculate rapidly from blood to lymph. Labeled B cells, in contrast, recirculated only very slowly.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

Cellular events in tolerance. V. Detection, isolation and fate of lymphoid cells which bind fluoresccinated antigen in vivo.

The binding of tolerogen to specific receptors of lymphocytes and the subsequent fate of such cells was directly studied in Lewis rats injected with fluorescein-labeled sheep gamma globulin (F-SGG). This tolerogen produced unresponsiveness both in SGG-specific T cells (carrier tolerance) and F-specific antibody-forming cell precursors. The former (T-cell tolerance) was still significant more than 60 days after tolerogen whereas tolerance in the latter (B-cell tolerance) had waned by that time.Cells which have bound the tolerogen (antigen-binding cells, ABC) in vivo were detectable by direct immunofluorescence of washed spleen cell suspensions from rats injected with F-SGG up to 7 days previously. These cells were isolated using antifluorescein affinity columns, and shown to contain immunocompetent precursors for F- and SGG specific responses.The frequency of such ABC was between 30 and 80 per 10⁵ spleen, lymph node or bone marrow cells; no ABC were detected in the thymus. Both Ig positive and Ig negative cells were found to be ABC; Ig negative ABC usually showed a “capped” fluorescent pattern whereas Ig positive ABC generally were “spotted.”By 10 days after injection, ABC were not detectable in the spleen, lymph nodes, thymus or bone marrow of tolerant rats. Furthermore, reinjection of F-SGG after this time did not label any cells. This suggests that antigen-binding cells are not present at this time or that such cells, if available, lack receptors. In contrast, rats previously injected with a lower non-tolerogenic dose of F-SGG or an immunogenic form (F-SGG on bentonite) possessed cells at these later times which could be labeled with F-SGG. Thus, ABC remain detectable following immunogen or a subtolerogeic dose of F-SGG, but disappear in tolerant rats.By approximately 40 days after initial high dose tolerogen injection (when B cell tolerance has started to wane), cells capable of binding a second dose of F-SGG again became detectable. It is suggested that high doses of F-SGG are bound by specific lymphocytes (identifiable as ABC) and that these cells either fail to regenerate new receptors or die. As tolerance begins to wane, either new receptors or new cells are generated.     

Origin and nature of the cells participating in the popliteal graft versus host reaction in mouse and rat.

The origin and nature of the cells accumulating in the popliteal lymph node graft versus host reaction (GVHR) of mice and rats were studied by karyotypic analysis, immunofluorescence, and radioautography after [³H]thymidine incorporation. At all times explored, the proliferating cells represented at most 10% of the cells, and among them the frequency of mitoses of donor origin was about 50, 15, and 4% on the third, seventh, and fifteenth days, respectively. Using alloantisera, no significant numbers of donor T cells could be detected among the resting lymphocytes. On the seventh day, the enlarged nodes contained at most 2% of donor cells and about two-thirds of T and one-third of B lymphocytes, i.e., a cell composition comparable to that of normal lymph nodes, except for an increased proportion of lymphoblasts and plasmablasts. Some plasmablasts secreted antibodies against sheep red blood cells, probably reflecting polyclonal activation of B cells. Popliteal GVHRs in T-depleted F₁ mice were of comparable intensity, with no increase in the proportion of donor cells, and the enlarged nodes contained a high proportion of B lymphocytes and plasmablasts of host origin.     

Comparative migration of B- and T-Lymphocytes in the rat spleen and lymph nodes.

B-lymphocytes were obtained either by thoracic duct cannulation of thymectomized, irradiated rats or by isolation of complement-receptor-bearing lymphocytes from normal rats. They were labeled in vitro with [³H]-leucine and injected iv into syngeneic recipients from which samples of spleen and lymph node were taken at intervals from 15 min to 48 hr after injection. The sites of initial localisation of B- and T-lymphocytes were identical suggesting that the cells migrated into both organs by a common entrance. The two cell types remained closely associated for several hours in the paracortex of lymph nodes and at the periphery of the periarteriolar lymphoid sheath of the spleen. After 1–6 hr, B-cells segregated from T-cells by moving on into the adjacent part of the lymphocyte corona in the follicular area. By 24 hr, B-cells were evenly distributed throughout the corona. A definite minority of B-cells but no T-cells were seen within the germinal centres. In the spleen, T-cells moved into the central area of the periarteriolar sheath before returning to the blood. The immunological significance of the routes of B- and T-cell migration is discussed.     

Natural killer cell activity in the rat. V. The circulation patterns and tissue localization of peripheral blood large granular lymphocytes (LGL)

Large granular lymphocytes (LGL) and T cells were separated from blood by centrifugation on discontinuous gradients of Percoll, were labeled with [3H]uridine or [111In]oxine, and were injected i.v. into syngeneic euthymic or athymic nude rats. The tissue distribution of these labeled cells was monitored for up to 24 hr after transfer by scintillation counting of tissue homogenates and autoradiography of tissue sections. In normal euthymic rats, the main sites of LGL localization were the alveolar walls of the lungs and spleen red pulp; however, they were not detectable in the major traffic areas of T lymphocyte recirculation, the spleen white pulp, and lymph nodes. Furthermore, the density of labeled LGL was very low in the small intestine, thymus, kidney, and liver, although on a per-organ basis, about 10% of the injected radioactivity was found in the liver by 24 hr post-injection. When 111In-labeled LGL were injected i.v. into rats with an indwelling thoracic duct cannula, they completely failed to enter the thoracic duct lymphocyte (TDL) population over an observation period of 6 days. This finding was markedly different from the results obtained with T cells and was consistent with the lack of natural killer and antibody-dependent cellular cytotoxicity activity observed among TDL, even in rats pretreated with the biological response modifier, poly I:C. LGL in athymic nude rats also failed to recirculate between blood and lymph. However, in contrast to normal euthymic animals, a significant increase in the localization of radiolabeled LGL to lymph nodes was observed in nude rats between 30 min and 24 hr. Taken as a whole, these findings define the areas within the lungs and spleen in which blood LGL normally localize, and clearly demonstrate that LGL do not normally recirculate between blood and lymph.