Long-lived Lymphocytes Research Articles

Studies of Non-Migrating, Long-Lived Lymphocytes in Rats

Abstract Rat lymph node lymphocytes were labeled by hind footpad injections of tritiated thymidine in the absence of an immune stimulus or after priming with a polymerized Salmonella flagellin. Fifty or more days later the rats were killed and the lymph node cells were fractionated on continuous albumin density gradients. The proportions of lymphocytes of lighter density (< 1.07 g/cm3) were greater in primed than in unprimed nodes. The density distributions of the “long-lived” cells, i.e., those with persistent label, were identical to those of the total lymph node cell population. After challenge with either antigenically identical or different flagellins, the density distribution of the persistently labeled cells demonstrated relative loss of cells in the density range from 1.068 to 1.072 g/cm3 in comparison to the bulk of lymph node cells, but not in patterns distinct for the specificity of the antigens used for challenge. Comparison of the density distributions of long-lived lymphocytes with flagellinbinding cells and with cells transferring adoptive immune reactions revealed partial overlap with these cells from primed but not from unprimed lymph nodes.

The immunologically specific retention of recirculating long-lived lymphocytes in lymph nodes stimulated by xenogeneic erythrocytes.

The lymph nodes of mice actively or adoptively immunized to sheep RBC and/or chicken RBC selectively retain long-lived lymphocytes after challenge with the appropriate antigen. This retention is demonstrable within 8 hr of the time of stimulation, though it probably begins even before this, and it is essentially complete within the first 24 hr. A similar selective retention is seen in nodes regional to the injection of some nonimmunogenic substances such as turpentine, but not others such as colloidal carbon or syngeneic RBC. In animals adoptively immunized to sheep and chicken RBC simultaneously, there is a preferential accumulation of the labeled long-lived lymphocytes of donors immunized to sheep RBC in lymph nodes challenged with sheep RBC, and a preferential accumulation of lymphocytes (labeled with a different radioisotope) from donors immunized to chicken RBC in lymph nodes challenged with this antigen. This immunologically specific component is demonstrable whether the antigen is given before or after adoptive immunization, suggesting that the only labeled cells capable of specific localization in this system are those cells that normally remain in the recirculating pool. In the present experiments, 31 out of 31 sets of antigenically stimulated lymph nodes have shown radiochemical evidence of immunological specificity in the distribution of donor lymphocytes between them, while corresponding sets of nonstimulated lymph nodes have shown only small random variations in the distribution of donor cells. Two different mechanisms are postulated whereby antigenic stimulation can alter the traffic of recirculating long-lived lymphocytes through stimulated lymph nodes. One affects recirculating cells of a particular immunological specificity, while the other affects recirculating cells without regard to their immunological specificity.

Thoracic duct lymph and lymphocyte studies in man using a thoracic duct "side-fistula".

A technique for cannulation of the cervical thoracic duct in man is described. It leaves the thoracic duct circulation unimpaired and allows repetitive lymph sampling without loss. This technique can be used to study the re-circulating pool of long-lived lymphocytes and thereby help to evaluate better the immunologic status of cancer patients. The technique of side-cannulation has been used in one patient. Thoracic duct lymphocytes labelled with 51Cr were reinfused IV and were recovered in the thoracic duct in a stable number over a 5-day study, thereby demonstrating a recirculating lymphocyte pool in man and the applicability of the side-cannulation to its study.

Studies of nonmigrating, long-lived lymphocytes in rats: I. Reactions after antigenic challenges in situ

In a search for a noncirculating mechanism of immunologic memory, popliteal lymph node cells were labeled with tritiated thymidine after a primary immune stimulus with polymerized Salmonella flagellins and were challenged with a variety of antigens 45 days later. The appearance of increased numbers of labeled large lymphoid cells in situ, i.e. lymphoblasts and plasmablasts, 48 hr after an antigenic stimulus, and their ability to produce antibody were used as criteria for judging the response of noncirculating, long-lived small lymphocytes to the challenge. Little or no effect of specific or nonspecific antigens was seen on the numbers of persisting labeled small lymphocytes in situ. There were increases in the numbers of and in the mean grain counts of labeled large lymphoid cells after all challenging stimuli, including BSA and SRBC. A small but significantly greater increase in proportion of labeled large lymphoid cells appeared after immunologically specific challenges than after nonspecific challenges. Virtually none of those large cells derived from long-lived lymphocytes were capable of causing specific bacterial adherence. In contrast, cells labeled with 125I-UdR 2 hr before challenge could cause immunologically specific bacterial adherence. Most were found when a secondary challenge was given, but some cells were also found after a primary challenge. We conclude that this work makes unlikely those theories of immunologic memory involving sequential development of a single cell line. We reopen the question whether some of the precursors of functional plasma cells are a continuously dividing cell line in a secondary response, pointing out that this is quite consistent with the current information implicating a two-cell system in immunologic memory.

Radioautographic Study of Cellular Migration Using Parabiotic Rats

Abstract Leukocyte exchange between the hemopoietic tissues of parabiotic rats was studied subsequent to giving multiple injections of 3H-thymidine to one member of each pair while arresting the cross-circulation. Cell types that migrated from one parabiont to the other were segmented granulocytes, small, medium and large lymphocytes, immunoblasts, monocytoid cells, macrophages or their immediate precursors, and plasma cells. Evidence for the transformation of circulating cells to other cell types was rarely seen. The long-lived small lymphocytes were equilibrated between parabionts, suggesting that this is a single pool of cells with respect to kinetic behavior and recirculation. There was no evidence for a trephocytic function of lymphocytes. A small number of bone marrow lymphocytes coursed directly to lymph nodes and spleen. Evidence is given for a limited recirculation of short-lived lymphocytes of thoracic duct lymph (TDL), as well as for long-lived cells. Only a few immunoblasts of TDL recirculated. The majority of cells that entered the white pulp of the spleen were long-lived small lymphocytes, while the majority of immigrant cells to the red pulp were monocytoid cells and granulocytes. Many small lymphocytes originated in splenic red pulp and entered the blood. No immigrant cells to the thymic cortex were noted, although some small lymphocytes and monocytoid cells entered the medullary areas. Immigrant cells to the bone marrow (less than 2% of the cells in marrow) included monocytoid cells, small lymphocytes, and plasma cells. Evidence for the direct transformation of a circulating cell into a committed blast, based on reduction in grain count, was noted only in bone marrow.

Studies of non-migrating, long-lived lymphocytes in rats. 3. Decreased retention in lymph nodes of gnotobiotic rats.

Tritiated thymidine was injected into the hind foot pads of isolated, gnotobiotic rats and of conventional rats after a stimulus with <i>Salmonella flagellin </i>or after no specific antigenic stimulus. The popliteal nodes were examined 30, 37, and 45 days after labeling. The rate of disappearance of stably labeled, long-lived small lymphocytes was more rapid in nodes from isolated rats than in nodes from conventional rats. Prior immunization made no difference. Labeled plasma cells persisted in high proportion in isolated rats, but in greater numbers in conventional rats.

Studies of non-migrating, long-lived lymphocytes in rats and mice. II. Effects of anti-lymph node and anti-thymus antisera.

Lymphocytes in mouse and rat popliteal lymph nodes were labeled with tritiated thymidine after an immunologic stimulus. Thirty days later the animals were given one or a series of injections of heterologous anti-lymph node or anti-thymus serum. The proportion of persistently labeled lymphocytes in the popliteal nodes was determined one or two days later. The proportion of long-lived lymphocytes in the nodes did not change even when a significant lymphopenia was produced. Since the nodes were depleted of small lymphocytes, labeled cells must have emigrated. These data support the hypothesis that non-migrating lymphocytes are not qualitatively different from the circulating lymphocyte pool.

Radiation Recovery of Long-Lived Lymphocytes in the Rat

Rats were given 300 rads total body irradiation and the recovery time of the long-lived lymphocyte pool was determined. The mobilizable lymphocyte pool was measured by thoracic duct lymphocyte drainage. The cell content of the superficial cervical and mesenteric lymph nodes was also determined. Additionally, the transformation response of thoracic duct lymphocytes to pokeweed mitogen was tested to assess the recovery of immunologic competence of the irradiated animals. It was found that the lymphocyte pool was in process of recovery at one month and completely recovered by two and three months after 300 rads total body irradiation. The transformation response of the thoracic duct lymphocytes of animals was tested after irradiation and was equal to that of unirradiated control animals. The slow recovery of the lymphocyte pool, which is thought to involve a two-step bone marrow-thymus pathway of reconstituting cells, is consistent with the normal kinetics of the long-lived lymphocyte pool.

The Separation and 51Chromium Labeling of Human Lymphocytes With In Vivo Studies of Survival and Migration

Abstract This study looks at the application of 51Cr labeling of lymphocytes as a method of obtaining in vivo information about the lymphocyte in human beings. Lymphocytes were separated from whole blood by methods based on isopycnic and rate zonal centrifugation techniques and the conditions for 51Cr uptake by the separated lymphocytes standardized to enable a known amount of radioactivity to be injected into the subjects under study. The uptake of the label into various sites in the body was studied by the means of surface probes linked synchronously to a digital printout device and the survival in the circulation estimated by scintillation counting of blood samples taken at various times after injection of the label. The in vivo studies of survival and migration in 10 normal subjects show an initial rapid clearance of cells from the circulation associated with an uptake of cells into spleen and liver sites, and to a lesser extent, into sites over bone marrow and the abdomen. Survival of the circulating lymphocytes after this period appears to be relatively short, with a half-life of 1.7 days. As the available evidence suggests, this short life may be due to the differential trapping of short-lived lymphocytes in the circulation at the expense of the long-lived lymphocytes. Kinetic interpretations of the data indicate an inverse exponential uptake of cells into the sites studied, and the decline over the organs appears to follow the death rate of the cells in the body as a whole. Comparisons with studies in patients having chronic lymphatic leukemia show a relative inability of leukemic lymphocytes to leave the circulation and enter some sites in the body. These preliminary studies indicate the potential of 51Cr labeling as a useful clinical research tool in the study of lymphocytes in human beings.

Studies on the life history of lymphocytes. I. The life-span of cells responsive in the mixed lymphocyte interaction.

The life history, within the rat, of lymphocytes responsive to histocompatibility isoantigens in the mixed lymphocyte interaction was examined by the use of in vivo labeling with tritiated thymidine and radioautography. Lymphocytes in the peripheral blood and H-ARC (mitotic figures in the MLI) were compared with respect to the frequency of labeled cells and the median grain count. The following conclusions were drawn from this study: (a) Although some can be considered long-lived, the majority of H-ARC are the products of recent divisions in the body. (b) Adult thymectomy does not eliminate the production of long-lived lymphocytes, some of which are H-ARC. Hence, in addition to direct origin in the thymus, H-ARC, as well as other lymphocytes of the long-lived lymphocyte population, may derive from already existing thymus-derived cells in the circulation and thymus-dependent areas of the secondary lymphoid tissues. (c) Sublethal X-irradiation (600 R) in combination with adult thymectomy does not eliminate the capacity to produce some long-lived lymphocytes, however, few if any are H-ARC. (d) H-ARC and other long-lived lymphocytes appear to go through a series of rapid multiple divisions before they enter the circulation. Thereafter, long-lived lymphocytes appear to undergo intermittent single divisions which decrease both the frequency and median grain count of labeled cells gradually with time. On the other hand, labeled H-ARC maintain a more stable grain count despite a rapid decrease in frequency with time. This is taken to indicate that H-ARC are less likely to undergo occasional single divisions during their life-span, but may undergo periodic rapid sequential divisions. A speculative model is developed from these data on the life history of H-ARC which may be of predictive value in future studies and which can be tested against known facts.

Immunologically Specific Retention of Long-Lived Lymphoid Cells in Antigenically Stimulated Lymph Nodes

Abstract The lymphoid cells selectively retained in lymph nodes that have been stimulated by xenogenic erythrocytes include cells of the long-lived subpopulation. This selective retention appears to be real and not an artifact resulting from the reutilization of radioactively labeled DNA or its split products. Lymph nodes draining skin allografts also show a selective retention of long-lived lymphoid cells, although this retention is of a lesser magnitude and occurs at a somewhat slower rate. While most of this selective retention is related to factors other than the immunologic specificity of the long-lived cells involved, we have demonstrated that a small portion of it reflects the accumulation of specific memory cells. This has been done by using an experimental design based on: a) the adoptive immunization of mice to two different antigens (BALB/c mice immunized to DBA/2 and C57BL/Ks histocompatibility antigens), b) the labeling of long-lived lymphocytes of the donor mice with either 3H-thymidine or 14C-thymidine following immunization, and c) the simultaneous counting of both radioisotopes in each set of stimulated lymph nodes.

The mediator of cellular immunity. I. The life-span and circulation dynamics of the immunologically committed lymphocyte.

Thoracic duct cells from rats which have survived an infection with Listeria monocytogenes can confer a high level of antimicrobial resistance upon normal recipients. The cells which confer protection appear in the thoracic duct during the 1st wk of the immunizing infection, at a time when newly formed lymphocytes are being added to the lymph in substantially increased numbers. The protective cells differ in at least two respects from the majority of small lymphocytes in central lymph: they have a rapid turnover rate and a short life-span in the circulation. Evidence was also obtained that lymphopoiesis affecting the long-lived small lymphocyte, which belongs to the recirculating pool, is not increased during an acute Listeria infection.

Subpopulations within “short-lived” and “long-lived/rd lymphocytes resolved by isopycnic sedimentation in Ficoll solutions coupled with a double radioisotopic labeling technique

The distribution of “short-” and “long-lived” cells among the subpopulations of the cells in lymphoid tissues of the rat has been examined by means of isopycnic density gradient centrifugation in solutions of Ficoll. Employing a double isotope counting technique, the “long-lived” cells were labeled by their ińcorporation of multiple doses of tritiated thymidine over a 14 day period while the “short-lived” cells in the same animals were similarly labeled by the incorporation of 14C-labeled thymidine over a 40 h period. The reported tissue distribution of short-lived and long-lived lymphocytes was confirmed. In addition, the existence of discrete subpopulations of cells having varying apparent half lives in some of the tissues was demonstrated.

A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization.

A population of lymphoid cells from several animal species, including man, was identified through a membrane receptor which binds sheep red blood cells treated with antibody and complement. When cells from different lymphoid organs were incubated with EAC at 37 degrees C, only part of the lymphocytes (named CRL) bound EAC and formed rosettes, and this interaction was shown to be C3-dependent. Mouse lymphoid cells could be specifically depleted of CRL by allowing them first to interact with EAC and then submitting the mixture to ultracentrifugation in a gradient of BSA. After ultracentrifugation, a population of cells containing 95% or more of non-CRL were recovered from the upper layers of the gradient. In addition to their different abilities to bind EAC, CRL and non-CRL from mouse lymphoid organs could be distinguished by the following properties: (a) CRL adhered preferentially to nylon wool at 37 degrees C in the presence of mouse serum. (b) After differential flotation in a gradient of BSA, a significantly higher proportion of CRL were recovered from the upper layers of the gradient. (c) The population of CRL contained most of the lymphocytes bearing immunoglobulin determinants on their membranes. (d) The distribution of CRL was quite different among lymphocytes obtained from various lymphoid organs, and they were never found in the thymus. (e) The membrane receptor for EAC was not detected in plaque-forming cells of mice which had been previously immunized with burro red cells. CRL and non-CRL could not be distinguished by their life span, as they were found in similar proportions among long-lived and short-lived lymphocytes from mouse peripheral lymph nodes. The function of this receptor on the membrane of certain lymphoid cells may be related to (a) the trapping and localization of antigen in lymphoid organs or (b) the localization of lymphoid cells in inflammatory sites.

The Response of Long- and Short-Lived Small Lymphocytes of the Rat to Pokeweed Mitogen

Summary Small lymphocytes of the thoracic duct lymph, mesenteric lymph node, spleen and thymus from Lewis and Wistar rats were exposed to PWM in vitro. The results of radioautographic studies reveal that both the long- and short-lived small lymphocytes transformed in the presence of PWM, but the short-lived populations of the thoracic duct lymph, mesenteric lymph node and spleen were more responsive. In contrast, the short-lived cells of the thymus transformed less than the long-lived small lymphocytes of the same organ. The responsiveness of short-lived small lymphocytes from a given tissue reflected the number of cells from this tissue required to initiate graft-versus-host reactions.

Repopulation of Irradiated Lymph Nodes by Recirculating Lymphocytes

The effects of local versus total-body irradiation of lymph nodes were compared in rats in which the long-lived lymphocytes had been labeled with tritiated thymidine (TTH) prior to irradiation. Thr...

Effect of Antilymphocytic Serum on Rat Lymphocytes

Summary The results of this radioautographic study demonstrated that ALS destroys large numbers of the long-lived small lymphocytes. Morphologic evidence of destruction was apparent in measenteric lymph nodes and was reflected by reduced numbers of long-lived cells in the thoracic duct lymph. In addition, this agent stimulated some long-lived small lymphocytes to transform into large blast cells. Little, if any, change was noted in the cell content of the thymus and bone marrow, tissues known to contain large numbers of short-lived small lymphocytes. A maximum reduction in the number of long-lived cells was seen by 24 hr after the first injection of ALS and subsequent daily injections for 7 days did not appear to enhance the effect. Although blood lymphocyte levels returned to normal within 6 weeks after cessation of ALS treatment, the number of small lymphocytes in TDL reached only 50% of the control levels. Throughout the experiments normal numbers of short-lived small lymphocytes continued to appear in the TDL of ALS-treated rats. The results are discussed with respect to the immunosuppressive action of ALS.

The Reactivity of Long-Lived Lymphocytes to Typhoid Vaccine in situ in Rat Popliteal Lymph Nodes

Summary The cells of the popliteal lymph nodes of rats were labeled with tritiated thymidine for 4 days after primary or secondary injections of OT or after a secondary injection of typhoid vaccine. Thiry-one days after the last dose of tymidine, rats of each group were challenged with typhoid vaccine. Increased numbers of heavily-labeled large lymphocytes and mitotic figures were found in the popliteal nodes for 2 days after challenge with typhoid vaccine regardless of the antigen given prior to labeling. This in situ reaction to typhoid vaccine was demonstrated as long as 7 months after labeling. Reasons indicating that these large lymphoid cells were derived from labeled, long-lived small lymphocytes are discussed. We believe these results confirm the validity of the method of following tritiated thymidine-labeled lymphocytes for long periods of time.

Blood: Studies of Leukocyte Kinetics in Chronic Lymphocytic Leukemia

Abstract Intravenously administered H3TdR was used to study peripheral blood leukocyte kinetics of ten patients with CLL in relapse and one in partial remission. Two distinct patterns were found. The first pattern indicates the presence of a small, proliferating population of lymphocytes which is present in the blood for a relatively brief time. This pattern was found in patients with no organomegaly and with relatively lower peripheral lymphocyte counts. In the second pattern almost all of the cells present were non-proliferating and long-lived. The patients with the latter pattern had organomegaly. These studies support the concept that there is a progressive widespread accumulation of long-lived lymphocytes in CLL often leading to splenic pooling and, in some cases, hypersplenism.

CHANGES IN THE LIFE-SPAN OF CIRCULATING SMALL LYMPHOCYTES IN MICE AFTER TREATMENT WITH ANTI-LYMPHOCYTE GLOBULIN

The blood and lymphoid tissues of mice which had been injected with anti-lymphocyte globulin (A.L.G.) proved to be depleted particularly of long-lived small lymphocytes. The blood later became repopulated predominantly by shortlived small lymphocytes. This change in the composition of the circulating small-lymphocyte population, which was observed in intact animals and in mice thymectomised as adults, was not reflected in the absolute blood lymphocytecount. These findings indicate one mechanism by which A.L.G. exerts its prolonged immunosuppressive effects.