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.     

3H-uridine incorporation by small lymphocytes of tolerant rats: relationship to T and B lymphocytes.

Tissue tolerance was induced in neonatal rats by the intravenous injection of bone marrow cells from adult allogeneic rat donors. After 6 to 8 weeks, lymphoid cells from rats in which tolerance had been induced were tested for mixed lymphocyte reactivity (MLR), 3H-uridine uptake, and the relationship of uridine incorporation to B and T lymphocytes. Lymph node (LN) and spleen (SPL) cells from the adult inoculated rats showed no reactivity in the MLR or normal lymphocyte transfer reaction (NLTRx), indicating that the animals were tolerant. After in vitro exposure to 3H-uridine, an abundance of small lymphocytes (SL) from these same tolerant rats were heavily labeled, in contrast to nontolerant controls, where relatively few SL were heavily labeled. In order to determine whether the heavily uridine-labeled cells were T cells or B cells, lymphoid cells from the LN and SPL of tolerant animals were exposed to either rabbit anti-AKR brain serum or rabbit anti-rat Ig conjugated with ferritin. The results showed that the heavily uridine-labeled SL of the tolerant rats were mainly Ig-positive cells.     

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.     

Antigenic relationship between bone marrow lymphocytes cortical thymocytes and a subpopulation of peripheral T cells in the rat: description of a bone marrow lymphocyte antigen.

Populations of rat bone marrow lymphocytes (BML) consisting of approximately 90 percent, “tnull” cells were prepared by density gradient centrifugation, passage through a column of fine glass beads, and treatment with anti-T cell and anti-B cell serum plus complement. Antisera to these bone marrow lymphocytes were raised in rabbits. After absorption with RBC and peritoneal exudate cells, the anti-BML sera were found by immunofluorescence to react selectively with “null” cells in bone marrow, with cortical thymocytes, and with a cortisone-sensitive subset of T cells in blood and in spleen, possibly in red pulp. The antigen that is common to these cell types is designated the rat bone marrow lymphocyte antigen (RBMLA). Lymphocytes that are positive fur KBMLA are negative for another lymphocyte-specific heteroantigen, rat musked thymocyte antigen (RMTA). As shown previously, RMTA is present on medullary thymocytes and ou cortisone-resistant T cells in white pulp of spleen, paracortex of lymph node and thoracic duct lymph. It is postulated that two developmentally and functionally distinct lines of T cells exist in peripheral lymphoid tissues of the rat, one derived from cortical thymocytes and one derived from medullary thymocytes. It is further postulated that the “null” population of bone marrow lymphocytes contains the lymphopoietic stem cells from which these two lines of T cells originate.     

Vitamin B6 deficiency and the lymphoid system: I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B₆-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate ³H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in ³H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for ³H-uridine incorporation.     

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.     

Circulating T and B lymphocytes of the mouse. II. Lifespan

The average lifespan of circulating lymphocytes was investigated by determining the percentage of labeling of thoracic duct lymphocytes (TDL⁵) from mice injected with tritiated thymidine (³HT) for various periods. Percentage of labeling of TDL from normal CBA mice, which consist of approximately 85% T cells and 15% B cells, was found to be directly proportional to the time of ³HT administration. This technique thus failed to demonstrate the presence of more than one population of lymphocytes. Less than 50% of TDL were labeled after ³HT injection for 8 weeks.Percentage of labeling of TDL from nude mice (which consist solely of B cells) was likewise found to be directly proportional to the duration of ³HT injection but occurred at a rate three to four times faster than in non-T cell-depleted CBA mice. Further experiments, in which a marker for B cells was used, allowed the rate of ³HT labeling of B cells to be studied in normal CBA mice. These data corroborated the findings in nude mice and indicated that, with regard to lifespan, thoracic duct B cells consisted of a single population with an average lifespan of 5–7 weeks. Similarly it was calculated that the average lifespan of thoracic duct T cells was in the order of 4–6 months.Studies on the rate of formation of TDL during prolonged thoracic duct drainage of normal CBA mice indicated that the percentage of newly formed cells increased rapidly after 24-hr drainage. The total numbers of newly formed cells, however, were found to remain relatively constant throughout the period of drainage investigated (up to 9 days) except for a transient increase during the second and third day. Newly formed small lymphocytes were found to consist of approximately equal proportions of T cells, B cells, and other “mononuclear” cells which lacked surface markers for either T or B cells. The great majority of large lymphocytes, in contrast, were found to be neither T cells nor B cells and probably belonged to the plasma cell line. In nude mice, production of newly formed lymphocytes during prolonged thoracic duct drainage was found to be very low in comparison with normal CBA mice.     

Mitogenic factor from inbred guinea pigs. II. Properties of the factor

Thymectomized adult rats which have been heavily irradiated and reconstituted with syngeneic bone marrow cells rapidly regain the ability to defend themselves against a primary infection with the intracellular bacterial parasite, Listeria monocytogenes. They do so by a cell-mediated immunological mechanism as evidenced by the protective immunity transferred adoptively by thoracic duct lymphocytes or peritoneal exudate cells from donors infected with this organism. But peritoneal exudate cells from thymus-derived donors convey only a fraction of the immunity transmitted by exudate cells from similarly infected intact rats. Since thymectomized irradiated animals can mobilize their cellular defenses more effectively when they are injected with a modest number of thoracic duct lymphocytes, an effect that cannot be duplicated with a massive infusion of bone marrow, it is argued that thymusdependent lymphocytes or T cells have an influential role in the development of cellular resistance to infection.     

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.     

Fate of H2-activated T lymphocytes in syngeneic hosts: II. Residence in recirculating lymphocyte pool and capacity to migrate to allografts

T.TDL—a purified population of lymph-borne H2-activated T lymphocytes—were transferred to syngeneic mice to examine their capacity to remain in the recirculating lymphocyte pool (RLP). Experiments with cells labelled with ³H-thymidine (³HTdR) or ⁵¹Chromium showed that although a considerable proportion of T.TDL joined the RLP (i.e., were mobilizable through a thoracic duct fistula) for several days after transfer, most of the cells soon left the pool. This applied whether the cells were transferred to normal mice or to “B” mice. Normal thoracic duct lymphocytes, by contrast, joined the RLP for long periods post-transfer.Studies with ³HTdR-labelled T.TDL showed that a small number of heavily labelled cells remained in the RLP for at least 3 months. Experiments with the θ-antigen as a cell marker suggested that a further small proportion of cells underwent division at some stage after transfer and then rejoined the RLP in expanded numbers.T.TDL showed a tendency to home to specific allografts of either skin, tumor cells or lymphoid cells. Although homing was specific it was very limited in extent.     

Nitric oxide production from a macrophage cell line: Interaction with autologous and allogeneic lymphocytes

The indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774. J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration ( < 10⁴ cells in 100 μl) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co-culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti-CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H-2^d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H-2^b B10BR; H-2^k) nonadherent lymphocytes, which were stimulated with conA or anti-CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co-cultured with SEA-reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti-CD3, proliferated well by the addition of L-arginine homologue, N^G-monomethyl-L-arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti-CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of N^G-monomethyl-L-arginine.     

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.     

Antigenic relationship between medullary thymocytes and a subpopulation of peripheral T cells in the rat: description of a masked antigen.

Using differentially absorbed rabbit antisera to rat thoracic duct cells, an antigen is described which normally is expressed on the surface of T cells in thoracic duct lymph and lymph node, but which exists in a masked form on medullary thymocytes and apparently not at all on cortical thymocytes. This antigen is termed the rat masked thymocyte antigen (RMTA). RMTA on medullary thymocytes can be unmasked mechanically by sectioning in a cryostat or enzymatically by treating with neuraminidase. Trypsin destroys or removes RMTA. Nearly all the T cells in thoracic duct lymph and lymph node are RMTA⁺, whereas only 58–66% of T cells in spleen are RMTA⁺. RMTA⁺ T cells, which are cortisone resistant, reside in the paracortex and periarteriolar sheath regions of lymph node and spleen. RMTA^? T cells, which are cortisone sensitive, appear to reside in the red pulp of spleen. The results suggest that (i) two antigenically distinct populations of T cells exist in the rat, RMTA⁺ and RMTA^? T cells, (ii) medullary thymocytes are the immediate precursors of RMTA⁺ T cells, and (iii) cortical thymocytes may be the immediate precursors of RMTA^? cells.     

Restoration of allograft responsiveness in B rats: IV. The divergent migratory behavior of lymphocyte populations mediating cardiac allograft rejection

The T lymphocyte-deprived (B) rat, produced by X-radiation and bone marrow reconstitution of adolescent thymectomized animals, exhibits a true immunological deficit and are unable to reject histoincompatible heterotopic cardiac allografts. A comprehensive survey of lymphocyte traffic in B recipients was performed to correlate the differential potency of specifically sensitized lymphocyte populations mediating re-establishment of immune responsiveness toward the graft, with their migratory and recirculatory behavior. ¹¹¹In-oxine-labeled thoracic duct lymphocytes (TDL) were retained in the peripheral blood and migrated from nonlymphoid organs to lymph nodes of B recipients in higher proportion than any other lymphoid population, particularly splenic lymphocytes (SL). Although all cell groups but TDL were sequestered in the spleen in equal and relatively large numbers, no differences were found between the lymphocyte populations tested in their capacity to accumulate in the grafts. In contrast, an increased avidity in the allograft of ¹²⁵IUdR-labeled TDL and lymph node (LNL) lymphoblasts, as compared to ¹²⁵IUdR-labeled SL, resembles closely the results of functional studies of the differential potency of adoptively transferred cells. We assume that specific cellular interactions induced by the accumulated ¹²⁵IUdR-labeled cells invoke nonspecific mechanisms for the recruitment of other uncommitted ¹¹¹Inlabeled lymphocytes which recirculate between blood and lymph and localize indiscriminately in the allograft amplifying its rejection. The latter lymphocytes can be “armed” by adherent cells residing in the lymphoid organs of graft recipients, particularly spleen, and subsequently increase the penetration of the foreign tissue. When radiolabeled lymphocytes were traced in B recipients experiencing rejection of their allografts following transfer of sensitized cells plus lymphokine, their migration patterns as well as blastogenic response in B hosts were similar to those observed during acute rejection of cardiac allografts in unmodified hosts. Thus the similarities between the rejection network brought by alloimmune cells into otherwise unresponsive animals and immunocompetent animals able to reject their grafts are stressed.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Nonrecirculating memory T lymphocytes in cellular resistance to infection

Acquired resistance of rats to intracellular infection with Listeria monocytogenes rests on the cooperation between sensitized mediator lymphocytes and effector macrophages. Large numbers of specific T lymphoblasts, capable of transferring resistance to recipients, appear in central lymph shortly (3–6 days) after subcutaneous infection of rats. In contrast, late-phase immunity is poorly transferrable with thoracic duct lymphocytes (TDL) despite high levels of specific resistance observed in spleen, liver, and testes of actively immunized animals. That late-phase immunity is mediated partly by resident, nonrecirculating T cells is attested to by the ineffective transfer of resistance from preinfected to normal partners of parabiotic rats. Transfer studies with thoracic duct cells and peritoneal cells from the stimulated and unstimulated peritoneal cavity seem to suggest that resident T cells mediating late-phase resistance are the progeny of lymphoblasts that extravasated during early phase. Assays measuring the proliferative response upon antigen stimulation in vitro support the concept of a gradual redistribution within the animal of memory T cells.     

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.     

Effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat

The first step in the migration of lymphocytes out of the blood is adherence of lymphocytes to endothelial cells (EC) in the postcapillary venule. It is thought that in inflammatory reactions cytokines activate the endothelium to promote lymphocyte adherence and migration into the inflammatory site. Injection of IFN-gamma, IFN-alpha/beta, and TNF-alpha into the skin of rats stimulated the migration of small peritoneal exudate lymphocytes (sPEL) into the injection site, and these cytokines mediated lymphocyte recruitment to delayed-type hypersensitivity, sites of virus injection, and in part to LPS. The effect of cytokines on lymphocyte adherence to rat microvascular EC was examined. IFN-gamma, IFN-alpha/beta, IL-1, TNF-alpha, and TNF-beta increased the binding of small peritoneal exudate lymphocyte (sPEL) to EC. IFN-gamma was more effective and stimulated adherence at much lower concentrations than the other cytokines. IL-2 did not increase lymphocyte adherence. LPS strongly stimulated lymphocyte binding. Treatment of EC, but not sPEL, enhanced adhesion, and 24 h of treatment with IFN-gamma and IL-1 induced near maximal adhesion. Lymph node lymphocytes, which migrate poorly to inflammatory sites, adhered poorly to unstimulated and stimulated EC, whereas sPEL demonstrated significant spontaneous adhesion which was markedly increased by IFN-gamma, IL-1, and LPS. Spleen lymphocytes showed an intermediate pattern of adherence. Combinations of IFN-gamma and TNF-alpha were additive in stimulating sPEL-EC adhesion. Depletion of sPEL and spleen T cells by adherence to IFN-gamma stimulated EC decreased the in vivo migration of the lymphocytes to skin sites injected with IFN-gamma, IFN-alpha/beta, TNF-alpha, poly I:C, LPS, and to delayed-type hypersensitivity reactions by 50%, and significantly increased the migration of these cells to normal lymph nodes, as compared to unfractionated lymphocytes. Thus the cytokines and lymphocytes involved in migration to cutaneous inflammation in the rat stimulate lymphocyte adhesion to rat EC in vitro, and IFN-gamma stimulated EC appear to promote the selective adhesion of inflammatory site-seeking lymphocytes.     

The mediator of cellular immunity: XI. Origin and development of MIF producing lymphocytes

Thoracic duct lymphocytes obtained from rats infected with Listeria monocytogenes were characterized with respect to size, turnover and their capacity to release macrophage migration inhibitory factor (MIF). Cells responsive to Listerial antigens (LMA) in the MIF assay were identified in lymph during the first week of an immunizing infection. These were immunoblasts or large lymphocytes, as evidenced by their sedimentation with S phase lymphocytes at unit gravity. When labeled cells from the lymph of Listeria-infected donors were infused into similarly infected recipients, donor S phase lymphocytes localized rapidly, and in substantial numbers, in peritoneal exudates induced by the unrelated organism, F. tularensis. Within this immigrant population were cells which conferred immunity against L. monocytogenes and released MIF in cultures containing LMA. Exudates harvested 36 hr or 61 hr after stimulation contained labeled lymphocytes that were smaller than the S phase cells recovered during the early post-induction period. The observed shift of radioactivity from large to smaller lymphocytes was parallelled by a shift MIF production to exudate fractions containing smaller cells. The MIF producing cells in exudates of advancing age also exhibited increasing resistance to inhibition by vinblastine. These findings suggest that MIF is released by a family of lymphocytes—large, medium and small. LMA-responsive lymphocytes are delivered to the thoracic duct soon after their formation, at a stage in development when they can be stimulated to release only low levels of MIF. These mediator producing cells circulate briefly in the blood and differentiate fully only after they extravasate into inflammatory foci.     

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.