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Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro
CELLULAR
IMMUNOLOGY
32, 263-273
(1977)
Macrophage-Lymphocyte Clusters in the Immune Response to Soluble Protein Antigen in Vitro IV. Synergy of Lymphocytes OTTO BRXNDSTRUP The University
University
in the Formation
of Clusters
AND OLE WERDELIN
Institute of Pathological Anatomy, School of Medicine, of Copenhagen, Copenhagen, Denmark 2100 fl Received
November
4,1976
T-lymphocyte-enriched
lymph node lymphocytes from guinea pigs immunized with .produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD) -pulsed (peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on ‘the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes ~physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes. Mycobacterium
tuberculosis
INTRODUCTION Macrophages are of major importance for antigen-induced proliferation of lymphocytes (l-6). In the case of soluble protein antigens it is commonly assumed that the antigen after being taken up by a macrophage is processed and/or presented by that cell to reactive T lymphocyte+ which then initiate synthesis of DNA. Whether this requires direct cellular interaction is not settled, but it has been clearly demonstrated that lymphocytes interact physically with macrophages during the in vitro response to these antigens (1, 4, 5, 7-9). Recently, we reported observations of antigen-specific interaction between T-lymphocyte-enriched lymph node lymphocytes from immunized guinea pigs and antigen-pulsed peritoneal macrophages (10). The interaction took place in clusters (Fig. 3) consisting of an adherent macrophage, to 1 Abbreviations used: FCS, fetal calf serum; LNC, lymph node lymphocytes; LNL, LNC which have been passed through an adherence column; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; PEM, peritoneal exudate macrophages; PPD, purified protein derivative of tuberculin; RPMI-FCS, completely supplemented tissue culture medium 1640 containing 15% FCS ; T lymphocytes, thymus-dependent lymphocytes ; [1H] TdR, tritium-labeled thymidine. 263 Copyright 0 1977 by Academic Press, Inc. AlI rights of reproduction in any form reserved.
ISSN 0008-8749
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which one central lymphocyte adhered through a broad base while several other lymphocytes, the peripheral, adhered to the central one by uropods ( 11) . Autoradiographic studies demonstrated that the central lymphocyte was frequently undergoing DNA synthesis ( 12). We suggested that the cluster is initiated when a lymphocyte committed to the antigen makes contact with an antigen-pulsed macrophage (12). The interaction between lymphocyte and macrophage may induce attachment of other lymphocytes to the committed one, which then becomes the central cluster lymphocyte, and it may generate the signals that makes the central lymphocyte initiate synthesis of DNA. This scheme suggests several experiments. First, if lymphocytes capable of initiating clusters bind onto antigen-pulsed macrophages, it should be possible to separate them from other lymphocytes by incubating the whole population on antigen-pulsed macrophages and then removing the nonadherent cells. Second, if committed lymphocytes initiate clusters and are the only cells to start synthesis of DNA early during culture, it should be possibIe to confirm by autoradiography that all of the DNAsynthesizing lymphocytes attached to macrophages are located in the clusters. We report that populations of immune lymphocytes can be enriched in or deprived of cluster-initiating cells by mechanical manipulation of cultures containing antigenpulsed macrophages and that the majority of macrophage-attached DNA-synthesizing lymphocytes are located in the clusters in cultures with high numbers of lymphocytes. MATERIALS
AND
METHODS
Animds. Inbred and random-bred male and female guinea pigs of strain Ssc : AL and Heston, weighing about 400 g, were used. Imlzvnunization. Guinea pigs were immunized by intracutaneous injection of 0.1 ml of Mycobacterium tuberculosis (40 pg/O.l ml) in Marco1 paraffin oil in four sites on the abdomen. These animals were used in experiments 3 to 12 weeks later. Assay for clusters. The procedures for establishing cluster-producing cultures have been described in detail (10). Lymph nodes and peritoneal exudate cells (PEC) were obtained from immunized or nonimmunized guinea pigs, which 3 days previously had received an intraperitoneal injection of 10 ml of sterile paraffin oil. Inguinal and axillary lymph nodes were squeezed through a No. 100 stainless-steel mesh in completely supplemented RPM1 1640 tissue culture medium with 15% heat-inactivated fetal calf serum (RPMI-FCS), p roviding a suspension of lymph node cells (LNC) . After one wash, the cells were suspended in 5 ml of pure FCS and passed through a glass-bead adherence column and then through a nylon-wool column (13)) which retains dead cells and most cells that are not T lymphocytes. Column-passed LNC are referred to as LNL (lymph node lymphocytes). PEC were harvested by lavaging the peritoneal cavity with 20 ml of RPMI-FCS. After one wash, monolayers of PEC were produced in Lab-Tek tissue culture chambers (LabTek Products, Division of Miles Laboratories, Inc., Naperville, Illinois), type 4808, by adding 1.6 X lo6 PEC in 0.4 ml into each of the eight compartments of the Lab-Tek chamber. After incubation for 90 min in 5% COz in air at 37°C the monolayers were flushed four times with 37°C RPMI-FCS. These monolayers of peritoneal exudate macrophages (PEM) were then incubated with 0.4 ml of RPMI-
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FCS containing either 20 pg of PPD/ml or the equivalent volume of phosphatebuffered saline (PBS). After 60 min of incubation in 5% COz in air at 37°C the PEM were flushed four times with RPMI-FCS to remove soluble antigen. To each compartment was added 0.4 ml of RPMI-FCS containing 4 x lo5 syngeneic immune LNL for the standard assay. In some experiments, mixtures of syngeneic immune and nonimmune LNL were used in the numbers indicated under Results. In cultures used for autoradiography the PEC were exposed to 2500 rad before monolayer production for reasons discussed earlier (12). Irradiation does not influence the ability of PEM to support cluster formation (12). After cultivation for 20 hr in 5% COs in air at 37°C the cultures were terminated by adding 0.1 ml of 7.5% glutaraldehyde into each compartment ( IO). The cultures were stained with Giemsa stain and were prepared for histological examination, and the number of clusters was counted as described earlier (10). Assays for clusters were performed in quadruplicate. Manipulation of nzacrophage nzonolayers. Cultures were set up in Lab-Tek chambers as in the standard cluster assay with PPD- or PBS-pulsed macrophages and immune LNL. After 4 hr of cultivation in 5% COe in air at 37°C the cultures were flushed three times with 37°C warm RPMI-FCS to remove non-adherent cells. To these manipulated monolayers were added 4 x lo5 syngeneic immune LNL obtained from petri dishes (see below), 4 x lo5 syngeneic nonimmune LNL, or pure RPMI-FCS as indicated under Results. After 20 hr of cultivation, the cultures were terminated, and the clusters were counted. Manipulation of LNL. Macrophage monolayers were produced in S-cm plastic petri dishes by cultivation of 1.6 X lo5 PEC/cmZ on the bottom of the dishes. After 90 min of cultivation in 5% COn in air at 37”C, the dishes were flushed free of nonadherent cells with 37°C warm RPMI-FCS. The monolayers were then exposed to RPMI-FCS containing 20 tcg of PPD/ml or the equivalent volume of PBS. After 60 min of cultivation in 5% COs in air at 37°C the dishes were flushed free of soluble antigen with 37°C warm RPMI-FCS. Immune LNL, 1 X 106/ml of RPMI-FCS, were added in a number of 4 X 105/cm2 of the monolayer. After 4 hr of cultivation in 5% COZ in air at 37”C, the dishes were gently agitated for 30 set to bring the nonadherent cells into suspension. The medium was then aspirated, and the obtained cells were adjusted to 1 X lo6 LNL/ml by one wash and used in the assay for cluster formation with standard monolayers and with manipulated monolayers (see above). The concentrations of PEC and LNL in cultures in petri dishes were the same per square centimeter of cultivation area as used in the standard cluster assay in Lab-Tek tissue culture chambers. [3H] Thy&dine labeling of DNA-synthesizing cells in cultures and preparation of autoradiographs. The procedure has been described in detail (12). The cultures were labeled during the last hour of cultivation before fixation by adding 1 &i of [3H]TdR ( [methyZ-SH] thymidine, sp act 6.7 Ci/mmol, New England Nuclear Corp., Dreieichenhain, West Germany) per milliliter of medium. The microscopic slides with fixed adherent cells were washed in PBS and dipped in Ilford K-2 emulsion diluted with 2 vol of distilled water. The slides and smears were exposed at 4°C for 3 to 21 days, developed with Amidol, fixed with sodium thiosulfate, and stained with Giemsa stain. Counts of labeled cells in autoradiographs. The counts were performed in triplicate on coded culture slides. All clusters and all DNA-synthesizing lymphocytes
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were counted. The DNA-synthesizing cells were classified as belonging to clusters or to the macrophage-attached non-cluster-involved compartment. RESULTS Cluster Production Procedures
by Lymphocytes
and Macrophages
Manipulated
by Various
Monolayers of macrophages from a nonimmune guinea pig were established in Lab-Tek chambers and the macrophages were pulsed with PPD. LNL, 4 X lo”, were added to the monolayers, the cultures were incubated for 20 hr as usual, and the clusters were counted. The lymphocytes derived from nonimmune or PPDimmune syngeneic guinea pigs and had been manipulated as indicated in Table 1. Immune LNL produced 407 clusters and nonimmune LNL produced 34 clusters. Immune LNL which had been preincubated on non-antigen-pulsed macrophages in a petri dish for 4 hr and then aspirated produced 382 clusters, but immune LNL which had been preincubated on PPD-pulsed macrophages for 4 hr and then aspirated produced only 80 clusters. One interpretation of this result is that some cells among the immune LNL which are necessary for the formation of clusters had been bound by the antigen-pulsed macrophages in the petri dish and were therefore removed from among the aspirated cells of the culture. To study this possibility, PPDpulsed macrophage monolayers in Lab-Tek chambers were manipulated by preincubation of 4 x lo5 immune LNL for 4 hr on the monolayer followed by removal of nonadherent cells by flushing with medium. Manipulated monolayers were then used for assay for clusters w.ith lymphocytes from nonimmune or immune guinea pigs (Table 1, lower part). While manipulated monolayers produced no clusters TABLE Effect on Cluster
Assay monolayer “manipulated””
No
Production Macrophages
Lymphocytes
No No No
Nonimmune Immune Immune, aspirated Immune, aspirated
Yes Yes Yes
None Immune, aspirated Nonimmune
1
of Manipulation Used in Cluster
of Lymphocytes Assay
added to assay monolayer
from PPD-pulsed from PBS-pulsed
macrophagesb macrophages”
from PPD-pulsed
macrophages
and
Number of clusters per culture (mean f SEM) 34f 407 f 80f 382 f
9 33 11 16
2* 420 f 395 f
1 22 26
a Assay monolayers were established in Lab-Tek chambers. The macrophages were pulsed with PPD. The monolayers that were not manipulated were used without further manipulation. The “manipulated” monolayers were preincubated for 4 hr with the standard number of immune LNL and were flushed free of nonadherent cells before use in the assay for clusters. b Immune LNL were added to petri dishes containing monolayers of PPD-pulsed macrophages and were incubated for 4 hr. The nonadherent cells were then aspirated from the dish, washed once, and used in standard numbers in the assay for clusters. c Immune LNL were added to petri dishes containing monolayers of non-antigen-pulsed macrophages and were incubated for 4 hr. The nonadherent cells were then aspirated from the dish, washed once, and used in standard numbers in the assay for clusters.
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FORMATION
themselves, both immune LNL aspirated from PPD-pulsed macrophage monolayers and nonimmune LNL produced about 400 clusters on the manipulated monolayer, even though these cells were unable to produce clusters on macrophage monolayers that were not manipulated (Table 1, upper part). One straightforward interpretation of these data is that cluster-initiating lymphocytes had been left bound to the macrophages of the manipulated monolayer during the preincubation period and that these lymphocytes were capable of producing clusters in synergy with the lymphocytes subsequently added to the culture. Number of Clusters Formed with Increasing Numbers of LNL
per Culture
If the clusters are produced by synergy between two populations of lymphocytes one would expect that an increase in the concentration of lymphocytes on the macrophage monolayer would result in an exponential increase in the number of clusters. To see if this were the case experiments were performed in which the number of lymphocytes varied. Immune LNL were used in numbers from 2 x lo5 to 1.6 x lo6 per culture. The clusters in each culture were counted and the number of clusters produced per 4 X lo5 was calculated (Fig. 1A). Increasing numbers of clusters per 4 X lo5 LNL were formed with increasing numbers of immune cells per culture, until a cell concentration of about 1.2 x lo6 per culture. The number of clusters per 4 x lo5 LNL did not increase further when even more immune cells were added. Experiments were also performed in which the number of immune LNL was chosen to be the standard number used in earlier experiments (4 X lo5 per culture). To these LNL were added from 2 x lo5 to 1.2 X lo6 syngeneic nonimmune LNL. Increasing numbers of clusters were produced with increasing numbers of added nonimmune cells until the total number per culture was about 1.2 X lo6 (Fig. 1B). The number of clusters produced per culture did not increase with cell concentrations above this number. The results of these two experiments demonstrate that the number of clusters produced by a certain number of lymphocytes, at cell concentrations lower than approximately 1.2 X lo6 per culture, depends not only upon the absolute number of immune lymphocytes, as shown previously (10, 12), but also on the cell concentration. Autoradiographic [3H] TdR
Studies of Cultures with Varying Numbers of LNL
Labeled with
In a preceding study (12) we made the observation that some of the DNA-synthesizing macrophage-attached lymphocytes in cluster-producing cultures were involved in clusters as the central lymphocyte even though the majority were attached to macrophages without being part of a cluster. In view of the above finding that the number of clusters produced in a culture depends upon the concentration of lymphocytes, it was of interest to study if a higher proportion of the cultures’ DNAsynthesizing lymphocytes would become involved in the clusters at high concentrations of lymphocytes. To that end we examined cultures with different numbers of immune LNL, from 2 X lo5 to 1.2 X 106, to see how many of the [3H]TdR-incorporating LNL of the cultures were located in clusters and how many were attached to macrophages without being involved in a cluster at each concentration of lymphocytes. This was done by counting all labeled lymphocytes in the cluster compartment and in the macrophage-attached compartment in autoradiographs of 20-hr
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Number lymphocytes per culture!xlW)
Number IK)n-immune LN4 added (x10? FIG. 1. A, The number of clusters developed in cultures with varying numbers of immune LNL. Cluster-forming cultures were established with 2 X lo6 to 1.6 X 10’ immune LNL- and PPD-pulsed macrophage monolayers. After 20 hr of cultivation the cultures were terminated, and the number of clusters was counted. Abscissa, number of immune LNL per culture; ordinate, number of clusters produced per 4 X 10’ LNL. B, the number of clusters formed by 4 X 10’ immune LNL to which were added varying numbers of syngeneic nonimmune LNL. Clusterforming cultures were established with 4 X 10” immune LNL supplemented with from 0 to 1.2 X 10’ syngeneic nonimmune LNL. After 20 hr of cultivation the number of clusters was enumerated. Abscissa, number of nonimmune LNL per culture; ordinate, number of clusters per culture.
cultures to which had been added [3H]TdR 1 hr prior to their termination. The result of one experiment is shown in Fig. 2A. With increasing numbers of immune LNL per culture, increasing proportions of the cultures’ labeled lymphocytes were located in the cluster compartment and decreasing proportions were located in the macrophage-attached compartment. The percentage of clusters containing one or more labeled central lymphocytes was approximately constant in cultures with different numbers of immune LNL. The absolute number of DNA-synthesizing
SYNERGY
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P 5
ioo @ I %I .P 8 6 f p 50 z 5 m h E 8 2
02
OA
Number of Lymphocytes
per culkdx
10-d
Ncimhcr of Lymphocytes
per culture
(x10-S)
I12
FIG. 2. A, Distribution of labeled macrophage-attached lymphocytes involved in clusters and not involved in clusters in cultures with varying numbers of cells. Cluster-forming cultures were established with 2 X 10” to 1.2 X 10’ immune lymphocytes per culture. After 19 hr of cultivation the cultures were labeled with 1 &i/ml of r3H]TdR. The cultures were terminated after 20 hr of culture and processed for autoradiography. Labeled macrophage-attached lymphocytes were scored as involved or not involved in clusters. Open bars, labeled macrophage-attached lymphocytes involved in clusters; closed bars, labeled macrophage-attached lymphocytes not involved in clusters. B, Distribution of labeled macrophage-attached lymphocytes involved in clusters and involved in clusters in ‘cultures containing either 4 X lo” immune lymphocytes or 4 X 10” immune lymphocytes plus 1 X lo6 syngeneic nonimmune lymphocytes. After 19 hr of cultivation, the cukures were labeled with 1 &i/ml of [‘H]TdR. The cultures were terminated after 20 hr of culture and processed for autoradiography. Labeled macraphage-attached lymphocytes were scored as involved or not involved in clusters. Open bars, labeled macrophage-attached lymphocytes involved in clusters; closed bars, labeled macrophage-attached lymphocytes not involved in clusters.
lymphocytes was directly proportional to the number of immune cells in these cultures. Autoradiographic studies were also performed using cultures with 4 x 10” immune LNL supplemented or not supplemented with 1.0 x loo syngeneic nonimmune LNL and labeled with [ 3H] TdR during the last hour of culture (Fig. ZB). In the
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FIG. 3. Compartments of lymphocytes in cluster-producing cultures. The antigenpulsed macrophages (Mac) adhere to the bottom of the culture vessel. To the left is a cluster. The enlarged central lymphocyte (CL) is attached to the macrophage through a broad area on the cells’ surface membranes. The peripheral lymphocytes (PL) are attached through uropods to the central lymphocyte without contaot with the macrophage. To the right is a macrophage with two lymphocytes (ML) attached to it. Above are ‘(free” lymphocytes (FL) not attached to macrophages. Some Iymphocytes are drawn larger to denote blast forms.
cultures supplemented with nonimmune LNL a considerably higher proportion of the cultures’ labeled lymphocytes were located in the cluster compartment and a corresponding lower proportion were located in the macrophage-attached compartment (Fig. 2B). DISCUSSION T-lymphocyte-enriched lymph node lymphocytes from PPD-immune guinea pigs produce clusters of a unique structure (Fig. 3) when incubated on monolayers of PPD-pulsed syngeneic macrophages (10, 11). In the standard assay, 4 X l@ lymphocytes from a PPD-immune guinea pig produce from 150 to 1500 clusters on a monolayer of PPD-pulsed macrophages, usually about 500, and each cluster contains a mean of 10-11 lymphocytes (10). Thus, only a few percent of the cultures’ lymphocytes are involved in the clusters. Most of the remaining lymphocytes of the culture are “free cells” not adherent to macrophages or other lymphocytes, and the remaining are adherent to macrophages but not involved in clusters (12). By mechanical manipulations, the lymphocytes of these cultures could be separated into two populations, each containing members with apparently different roles in the production of the clusters. The separation was carried out by aspirating the cells that were not adherent from cultures of PPD-immune lymphocytes which had been incubated for 4 hr on monolayers of PPD-pulsed macrophages. At this time clusters had not yet developed (Werdelin, Braendstrup, and Pedersen, to be published). The aspirated immune cells were, like nonimmune lymphocytes, incapable of producing significant numbers of clusters in the standard assay. The aspiration procedure left the macrophages attached to the culture vessel together with some lymphocytes which adhered to the macrophages. When such manipulated monolayers were incubated for an additional 20 hr they did not produce clusters. However, when 4 x lo5 of the aspirated immune lymphocytes were added to the mampulated monolayer, or when 4 X lo5 nonimmune lymphocytes were added, clusters were produced in the course of 20 hr of incubation in numbers equal to the numbers produced in the standard assay of the immune lymphocytes used in the experiment (Table 1). We interpret this result to mean that two populations of lymphocytes produce the clusters in synergy. Members of the one population make contact with the antigen-pulsed macrophages early during culture and become relatively firmly bound to them. This macrophage-bound lymphocyte initiates the cluster
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by somehow attaching other lymphocytes to itself and becomes the central cluster lymphocyte. In the late phase of the culture period, increasing proportions of these cells engage in synthesis of DNA (12). These lymphocytes are present only in immune animals. The members of the other population attach themselves to the central lymphocyte as peripheral lymphocytes during 20 hr of culture. These cells are present in both immune and nonimmune animals. Since the formation of clusters is antigen-specific (10) the question arises : Which of the cluster lymphocytes are committed to the antigen ? The evidence points to the central lymphocyte. First, this cell is the only one of the cluster lymphocytes which is in contact with the antigen on the macrophage, and, second, the central lymphocyte is the only one regularly stimulated to DNA synthesis. Thus, in our view, the central lymphocyte initiates the cluster by specific interaction with the antigen-pulsed macrophage surface and by unknown mechanisms attaches and retains noncommitted bystander lymphocytes to its surface for interaction, For convenience, the term recruitment is used below for this. Earlier observations (12). on autoradiographs of [3H] TdR-labeled cultures have shown that the cluster-producing standard culture contains three compartments of DNA-synthesizing lymphocytes (Fig. 3). One contained the lymphocytes attached to macrophages and involved in the clusters as central lymphocytes, one contained those attached to macrophages but not ‘involved in the clusters, and one compartment contained “free” nonadherent cells. It was suggested that the compartment of free DNA-synthesizing lymphocytes was derived from lymphocytes stimulated to DNA synthesis during interaction with macrophages either inside or outside clusters. The existence of two compartments of DNA-synthesizing macrophage-attached lymphocytes, one recruiting and one not recruiting other lymphocytes as peripheral lymphocytes, may reflect the activation of two different populations of lymphocytes committed to the same antigen. Alternatively, it may reflect the activation of only one population of lymphocytes committed to the antigen of which some members are incapable of recruiting other lymphocytes due to suboptimal culture conditions, with respect to the availability of recruitable peripheral lymphocytes. This last possibility was examined in the present study. We found that the use of increasing numbers of immune lymphocytes per culture until about 1.2 x lo6 cells per culture yielded increasing numbers of clusters per 4 x 10” lymphocytes and that the use of 4 X lo5 immune lymphocytes supplemented with increasing numbers of syngeneic nonimmune lymphocytes until about 1.0 x lo6 cells per culture yielded increasing numbers of clusters per culture (Fig. 19, This indicates that only with cell concentrations of at least 1.2 x 106/cmZ (the bottom area in Lab-Tek tissue culture chambers) are sufficient numbers of recruitable lymphocytes present to be recruited to all prospective macrophage-attached central lymphocytes as peripheral lymphocytes to form recognizable clusters. The optimal cell concentration for cluster production may be determined not only by the proximity of the members of the synergizing lymphocyte populations, but also by the amount of nutritive factors in the small volume of culture medium used in the cluster assay. Apart from the concentration of lymphocytes per unit area of the culture chamber, the number of clusters that are produced depends on the density of the macrophage monolayer. When this falls below a certain density the number of clusters is reduced (10). A similar principle may govern the antigen-induced lymphocyte transformation in the response to soluble protein antigens, as studied by others. The amount
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of radiolabeled thymid,ine incorporated by peripheral blood leukocytes is dependent upon the cell concentration in the culture (5, 14-16), with high cell concentration resulting in larger thymidine incorporation per million cells. It has been suggested that th.is is so because interaction with macrophages is needed for all antigen-reactive lymphocytes to be activated by antigen (1, 5, 15, 17). It has been consistently demonstrated that the shape of the culture vessel, at a constant cell concentration, influences the amount of thymidine incorporation. Thus, the use of round-bottom tubes, which tends to minimize the distance between the lymphocytes and macrophages of the culture, resulted in significantly more thymidine incorporation than did the use of flat-bottom tubes (14, 15). It remained to be examined whether the steep increase in numbers of clusters which resulted from the use of increasing numbers of lymphocytes in the clusterproducing cultures was caused by more intense recruitment of lymphocytes to activated macrophage-attached lymphocytes. This was examined in autoradiographs of cultures with increasing concentrations of immune lymphocytes to which [3H]TdR had been added 1 hr before the culture was terminated. Increasing proportions of labeled macrophage-attached lymphocytes were found to recruit other lymphocytes to produce clusters, whereas decreasing proportions of labeled macrophage-attached lymphocytes were found attached singly to macrophages (Figs. 2A and B). The proportion of labeled clusters, defined as clusters with one or more labeled centrally placed lymphocytes (12)) was approximately the same using different concentrations of lymphocytes. Similar results were obtained from counts on autoradiographs prepared from cultures of immune lymphocytes supplemented with syngeneic nonimmune lymphocytes. Thus, it appears that an inverse relationship, dependent upon the ceil concentration, exists between recruiting and nonrecruiting activated macrophage-attached lymphocytes. We suggest from these results that the majority of T lymphocytes which are triggered to DNA synthesis during interaction with antigen-pulsed macrophages pass through a stage where they recruit other lymphocytes if these are available. The mechanism of this recruitment is unknown. Probably membrane changes induced in the central lymphocyte in clusters during interaction with the macrophage are responsible, as discussed in the accompanying paper (18). Attachment of uropods of lymphocytes to lymphoblasts may be a general phenomenon, since this has also been observed in mixed leukocyte cultures and PHAstimulated cultures (19,20). The present data indicate that the percentage of cluster-initiating lymphocytes in a population of lymph node lymphocytes@enriched in T lymphocytes is two to three times higher than we concluded from our earlier studies. Thus, PPD-immune cluster-initiating lymphocytes comprise, in guinea pigs immunized with M. tuberculosis from 3 to 12 weeks earlier, about 0.3 to 1.070 of the column-passed lymph node cells. A possible application of the cluster phenomenon is for quantitation of antigen-binding T lymphocytes. As shown previously (lo), the number of clusters in a series of cultures containing the same numbers of lymphocytes is directly proportional to the number of immune lymphocytes in the cultures. A count of the number of macrophage-lymphocyte clusters produced by a known number of lymphocytes on a macrophage monolayer pulsed with a given antigen might therefore be used to calculate the percentage of specific antigen-binding cells or a subpopulation of these. A second possible application is the use of monolayers of antigenpulsed macrophages as specific immunoabsorbents. As shown, a popuIation of im-
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mune lymphocytes can be largely deprived of antigen-binding cells, and a population enriched in members capable of binding antigen can be recovered together with macrophages. Studies are in progress to further characterize cell populations fractionated in this way. ACKNOWLEDGMENT This investigation was supported by Statens Laegevidenskabelige Forskningsr%d, Grants 5124170 and 512-6502.
REFERENCES 1. Cline, M. J., and Swett, V. S., J. Exp. Med. 128, 1309, 1968. 2. Oppenheim, J. J., Leventhal, B. G., and Hersh, E. M., J. Imrnunol. 101, 262, 1968. 3. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44, 1970. 4. Hanifin, J. M., and Cline, M. J., J. Cell Biol. 46, 97, 1970. 5. Hersh, E. M., and Harris, J. E., J. Immunol. 100, 1184, 1968. 6. Rosenstreich, D. L., and Rosenthal, A. S., J. Immulzol. 110, 934, 1974. 7. Sulitzeanu, D., Kleinman, R., Benezra, D., and Gery, I., Nature New Biol. 229, 254, 1971. 8. Lipsky, P., and Rosenthal, A. S., J. Exp. Med. 142, 138, 1975. 9. Salvin, S. B., Sell, S., and Nishio, J., J. Zmmunol. 107, 655, 1971. 10. Werdelin, O., Brzndstrup, O., and Pedersen, E., J. Exj. Med. 140, 1245, 1974. 11. Nielsen, M., Jensen, H., Braendstrup, O., and Werdelin, O., J. Exp. Med. 140, 1260, 1974. 12. Brzndstrup, O., Andersen, V., and Werdelin, O., Cell. Immunol. 25,207, 1976. 13. Rosenstreich, D. L., and Wilton, M., In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 113. Academic Press, New York, 1975. 14. Moorhead, J. F., Connolly, J. J., and McFarland, W., I. Immunol. 99, 413, 1967. 15. Leventhal, B. G., and Oppenheim, J. J., In “Proceedings of the Third Annual Leukocyte Culture Conference (W. 0. Rieke, Ed.), p. 13. Meredith, Des Moines, 1969. 16. Valentine, F. T., and Lawrence, H. S., Fed. Proc. 27, 265, 1968. 17. Waldron, J. A., Horn, R. G., and Rosenthal, A. S., I. Zmmunol. 111, 58, 1973. 18. Brandstrup, O., Andersson, P., Jensen, H., and Werdelin, O., Cell. Immzmol. 32, 274, 1977. 19. McFarland, W., and Heilman, D. H., Nature (London) 205, 887, 1965. 20. McFarland, W., Heilman, D. H., and Moorhead, J. F., J. Exp. Med. 124, 851, 1966.
IMMUNOLOGY
32, 263-273
(1977)
Macrophage-Lymphocyte Clusters in the Immune Response to Soluble Protein Antigen in Vitro IV. Synergy of Lymphocytes OTTO BRXNDSTRUP The University
University
in the Formation
of Clusters
AND OLE WERDELIN
Institute of Pathological Anatomy, School of Medicine, of Copenhagen, Copenhagen, Denmark 2100 fl Received
November
4,1976
T-lymphocyte-enriched
lymph node lymphocytes from guinea pigs immunized with .produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD) -pulsed (peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on ‘the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes ~physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes. Mycobacterium
tuberculosis
INTRODUCTION Macrophages are of major importance for antigen-induced proliferation of lymphocytes (l-6). In the case of soluble protein antigens it is commonly assumed that the antigen after being taken up by a macrophage is processed and/or presented by that cell to reactive T lymphocyte+ which then initiate synthesis of DNA. Whether this requires direct cellular interaction is not settled, but it has been clearly demonstrated that lymphocytes interact physically with macrophages during the in vitro response to these antigens (1, 4, 5, 7-9). Recently, we reported observations of antigen-specific interaction between T-lymphocyte-enriched lymph node lymphocytes from immunized guinea pigs and antigen-pulsed peritoneal macrophages (10). The interaction took place in clusters (Fig. 3) consisting of an adherent macrophage, to 1 Abbreviations used: FCS, fetal calf serum; LNC, lymph node lymphocytes; LNL, LNC which have been passed through an adherence column; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; PEM, peritoneal exudate macrophages; PPD, purified protein derivative of tuberculin; RPMI-FCS, completely supplemented tissue culture medium 1640 containing 15% FCS ; T lymphocytes, thymus-dependent lymphocytes ; [1H] TdR, tritium-labeled thymidine. 263 Copyright 0 1977 by Academic Press, Inc. AlI rights of reproduction in any form reserved.
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which one central lymphocyte adhered through a broad base while several other lymphocytes, the peripheral, adhered to the central one by uropods ( 11) . Autoradiographic studies demonstrated that the central lymphocyte was frequently undergoing DNA synthesis ( 12). We suggested that the cluster is initiated when a lymphocyte committed to the antigen makes contact with an antigen-pulsed macrophage (12). The interaction between lymphocyte and macrophage may induce attachment of other lymphocytes to the committed one, which then becomes the central cluster lymphocyte, and it may generate the signals that makes the central lymphocyte initiate synthesis of DNA. This scheme suggests several experiments. First, if lymphocytes capable of initiating clusters bind onto antigen-pulsed macrophages, it should be possible to separate them from other lymphocytes by incubating the whole population on antigen-pulsed macrophages and then removing the nonadherent cells. Second, if committed lymphocytes initiate clusters and are the only cells to start synthesis of DNA early during culture, it should be possibIe to confirm by autoradiography that all of the DNAsynthesizing lymphocytes attached to macrophages are located in the clusters. We report that populations of immune lymphocytes can be enriched in or deprived of cluster-initiating cells by mechanical manipulation of cultures containing antigenpulsed macrophages and that the majority of macrophage-attached DNA-synthesizing lymphocytes are located in the clusters in cultures with high numbers of lymphocytes. MATERIALS
AND
METHODS
Animds. Inbred and random-bred male and female guinea pigs of strain Ssc : AL and Heston, weighing about 400 g, were used. Imlzvnunization. Guinea pigs were immunized by intracutaneous injection of 0.1 ml of Mycobacterium tuberculosis (40 pg/O.l ml) in Marco1 paraffin oil in four sites on the abdomen. These animals were used in experiments 3 to 12 weeks later. Assay for clusters. The procedures for establishing cluster-producing cultures have been described in detail (10). Lymph nodes and peritoneal exudate cells (PEC) were obtained from immunized or nonimmunized guinea pigs, which 3 days previously had received an intraperitoneal injection of 10 ml of sterile paraffin oil. Inguinal and axillary lymph nodes were squeezed through a No. 100 stainless-steel mesh in completely supplemented RPM1 1640 tissue culture medium with 15% heat-inactivated fetal calf serum (RPMI-FCS), p roviding a suspension of lymph node cells (LNC) . After one wash, the cells were suspended in 5 ml of pure FCS and passed through a glass-bead adherence column and then through a nylon-wool column (13)) which retains dead cells and most cells that are not T lymphocytes. Column-passed LNC are referred to as LNL (lymph node lymphocytes). PEC were harvested by lavaging the peritoneal cavity with 20 ml of RPMI-FCS. After one wash, monolayers of PEC were produced in Lab-Tek tissue culture chambers (LabTek Products, Division of Miles Laboratories, Inc., Naperville, Illinois), type 4808, by adding 1.6 X lo6 PEC in 0.4 ml into each of the eight compartments of the Lab-Tek chamber. After incubation for 90 min in 5% COz in air at 37°C the monolayers were flushed four times with 37°C RPMI-FCS. These monolayers of peritoneal exudate macrophages (PEM) were then incubated with 0.4 ml of RPMI-
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FCS containing either 20 pg of PPD/ml or the equivalent volume of phosphatebuffered saline (PBS). After 60 min of incubation in 5% COz in air at 37°C the PEM were flushed four times with RPMI-FCS to remove soluble antigen. To each compartment was added 0.4 ml of RPMI-FCS containing 4 x lo5 syngeneic immune LNL for the standard assay. In some experiments, mixtures of syngeneic immune and nonimmune LNL were used in the numbers indicated under Results. In cultures used for autoradiography the PEC were exposed to 2500 rad before monolayer production for reasons discussed earlier (12). Irradiation does not influence the ability of PEM to support cluster formation (12). After cultivation for 20 hr in 5% COs in air at 37°C the cultures were terminated by adding 0.1 ml of 7.5% glutaraldehyde into each compartment ( IO). The cultures were stained with Giemsa stain and were prepared for histological examination, and the number of clusters was counted as described earlier (10). Assays for clusters were performed in quadruplicate. Manipulation of nzacrophage nzonolayers. Cultures were set up in Lab-Tek chambers as in the standard cluster assay with PPD- or PBS-pulsed macrophages and immune LNL. After 4 hr of cultivation in 5% COe in air at 37°C the cultures were flushed three times with 37°C warm RPMI-FCS to remove non-adherent cells. To these manipulated monolayers were added 4 x lo5 syngeneic immune LNL obtained from petri dishes (see below), 4 x lo5 syngeneic nonimmune LNL, or pure RPMI-FCS as indicated under Results. After 20 hr of cultivation, the cultures were terminated, and the clusters were counted. Manipulation of LNL. Macrophage monolayers were produced in S-cm plastic petri dishes by cultivation of 1.6 X lo5 PEC/cmZ on the bottom of the dishes. After 90 min of cultivation in 5% COn in air at 37”C, the dishes were flushed free of nonadherent cells with 37°C warm RPMI-FCS. The monolayers were then exposed to RPMI-FCS containing 20 tcg of PPD/ml or the equivalent volume of PBS. After 60 min of cultivation in 5% COs in air at 37°C the dishes were flushed free of soluble antigen with 37°C warm RPMI-FCS. Immune LNL, 1 X 106/ml of RPMI-FCS, were added in a number of 4 X 105/cm2 of the monolayer. After 4 hr of cultivation in 5% COZ in air at 37”C, the dishes were gently agitated for 30 set to bring the nonadherent cells into suspension. The medium was then aspirated, and the obtained cells were adjusted to 1 X lo6 LNL/ml by one wash and used in the assay for cluster formation with standard monolayers and with manipulated monolayers (see above). The concentrations of PEC and LNL in cultures in petri dishes were the same per square centimeter of cultivation area as used in the standard cluster assay in Lab-Tek tissue culture chambers. [3H] Thy&dine labeling of DNA-synthesizing cells in cultures and preparation of autoradiographs. The procedure has been described in detail (12). The cultures were labeled during the last hour of cultivation before fixation by adding 1 &i of [3H]TdR ( [methyZ-SH] thymidine, sp act 6.7 Ci/mmol, New England Nuclear Corp., Dreieichenhain, West Germany) per milliliter of medium. The microscopic slides with fixed adherent cells were washed in PBS and dipped in Ilford K-2 emulsion diluted with 2 vol of distilled water. The slides and smears were exposed at 4°C for 3 to 21 days, developed with Amidol, fixed with sodium thiosulfate, and stained with Giemsa stain. Counts of labeled cells in autoradiographs. The counts were performed in triplicate on coded culture slides. All clusters and all DNA-synthesizing lymphocytes
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were counted. The DNA-synthesizing cells were classified as belonging to clusters or to the macrophage-attached non-cluster-involved compartment. RESULTS Cluster Production Procedures
by Lymphocytes
and Macrophages
Manipulated
by Various
Monolayers of macrophages from a nonimmune guinea pig were established in Lab-Tek chambers and the macrophages were pulsed with PPD. LNL, 4 X lo”, were added to the monolayers, the cultures were incubated for 20 hr as usual, and the clusters were counted. The lymphocytes derived from nonimmune or PPDimmune syngeneic guinea pigs and had been manipulated as indicated in Table 1. Immune LNL produced 407 clusters and nonimmune LNL produced 34 clusters. Immune LNL which had been preincubated on non-antigen-pulsed macrophages in a petri dish for 4 hr and then aspirated produced 382 clusters, but immune LNL which had been preincubated on PPD-pulsed macrophages for 4 hr and then aspirated produced only 80 clusters. One interpretation of this result is that some cells among the immune LNL which are necessary for the formation of clusters had been bound by the antigen-pulsed macrophages in the petri dish and were therefore removed from among the aspirated cells of the culture. To study this possibility, PPDpulsed macrophage monolayers in Lab-Tek chambers were manipulated by preincubation of 4 x lo5 immune LNL for 4 hr on the monolayer followed by removal of nonadherent cells by flushing with medium. Manipulated monolayers were then used for assay for clusters w.ith lymphocytes from nonimmune or immune guinea pigs (Table 1, lower part). While manipulated monolayers produced no clusters TABLE Effect on Cluster
Assay monolayer “manipulated””
No
Production Macrophages
Lymphocytes
No No No
Nonimmune Immune Immune, aspirated Immune, aspirated
Yes Yes Yes
None Immune, aspirated Nonimmune
1
of Manipulation Used in Cluster
of Lymphocytes Assay
added to assay monolayer
from PPD-pulsed from PBS-pulsed
macrophagesb macrophages”
from PPD-pulsed
macrophages
and
Number of clusters per culture (mean f SEM) 34f 407 f 80f 382 f
9 33 11 16
2* 420 f 395 f
1 22 26
a Assay monolayers were established in Lab-Tek chambers. The macrophages were pulsed with PPD. The monolayers that were not manipulated were used without further manipulation. The “manipulated” monolayers were preincubated for 4 hr with the standard number of immune LNL and were flushed free of nonadherent cells before use in the assay for clusters. b Immune LNL were added to petri dishes containing monolayers of PPD-pulsed macrophages and were incubated for 4 hr. The nonadherent cells were then aspirated from the dish, washed once, and used in standard numbers in the assay for clusters. c Immune LNL were added to petri dishes containing monolayers of non-antigen-pulsed macrophages and were incubated for 4 hr. The nonadherent cells were then aspirated from the dish, washed once, and used in standard numbers in the assay for clusters.
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FORMATION
themselves, both immune LNL aspirated from PPD-pulsed macrophage monolayers and nonimmune LNL produced about 400 clusters on the manipulated monolayer, even though these cells were unable to produce clusters on macrophage monolayers that were not manipulated (Table 1, upper part). One straightforward interpretation of these data is that cluster-initiating lymphocytes had been left bound to the macrophages of the manipulated monolayer during the preincubation period and that these lymphocytes were capable of producing clusters in synergy with the lymphocytes subsequently added to the culture. Number of Clusters Formed with Increasing Numbers of LNL
per Culture
If the clusters are produced by synergy between two populations of lymphocytes one would expect that an increase in the concentration of lymphocytes on the macrophage monolayer would result in an exponential increase in the number of clusters. To see if this were the case experiments were performed in which the number of lymphocytes varied. Immune LNL were used in numbers from 2 x lo5 to 1.6 x lo6 per culture. The clusters in each culture were counted and the number of clusters produced per 4 X lo5 was calculated (Fig. 1A). Increasing numbers of clusters per 4 X lo5 LNL were formed with increasing numbers of immune cells per culture, until a cell concentration of about 1.2 x lo6 per culture. The number of clusters per 4 x lo5 LNL did not increase further when even more immune cells were added. Experiments were also performed in which the number of immune LNL was chosen to be the standard number used in earlier experiments (4 X lo5 per culture). To these LNL were added from 2 x lo5 to 1.2 X lo6 syngeneic nonimmune LNL. Increasing numbers of clusters were produced with increasing numbers of added nonimmune cells until the total number per culture was about 1.2 X lo6 (Fig. 1B). The number of clusters produced per culture did not increase with cell concentrations above this number. The results of these two experiments demonstrate that the number of clusters produced by a certain number of lymphocytes, at cell concentrations lower than approximately 1.2 X lo6 per culture, depends not only upon the absolute number of immune lymphocytes, as shown previously (10, 12), but also on the cell concentration. Autoradiographic [3H] TdR
Studies of Cultures with Varying Numbers of LNL
Labeled with
In a preceding study (12) we made the observation that some of the DNA-synthesizing macrophage-attached lymphocytes in cluster-producing cultures were involved in clusters as the central lymphocyte even though the majority were attached to macrophages without being part of a cluster. In view of the above finding that the number of clusters produced in a culture depends upon the concentration of lymphocytes, it was of interest to study if a higher proportion of the cultures’ DNAsynthesizing lymphocytes would become involved in the clusters at high concentrations of lymphocytes. To that end we examined cultures with different numbers of immune LNL, from 2 X lo5 to 1.2 X 106, to see how many of the [3H]TdR-incorporating LNL of the cultures were located in clusters and how many were attached to macrophages without being involved in a cluster at each concentration of lymphocytes. This was done by counting all labeled lymphocytes in the cluster compartment and in the macrophage-attached compartment in autoradiographs of 20-hr
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Number lymphocytes per culture!xlW)
Number IK)n-immune LN4 added (x10? FIG. 1. A, The number of clusters developed in cultures with varying numbers of immune LNL. Cluster-forming cultures were established with 2 X lo6 to 1.6 X 10’ immune LNL- and PPD-pulsed macrophage monolayers. After 20 hr of cultivation the cultures were terminated, and the number of clusters was counted. Abscissa, number of immune LNL per culture; ordinate, number of clusters produced per 4 X 10’ LNL. B, the number of clusters formed by 4 X 10’ immune LNL to which were added varying numbers of syngeneic nonimmune LNL. Clusterforming cultures were established with 4 X 10” immune LNL supplemented with from 0 to 1.2 X 10’ syngeneic nonimmune LNL. After 20 hr of cultivation the number of clusters was enumerated. Abscissa, number of nonimmune LNL per culture; ordinate, number of clusters per culture.
cultures to which had been added [3H]TdR 1 hr prior to their termination. The result of one experiment is shown in Fig. 2A. With increasing numbers of immune LNL per culture, increasing proportions of the cultures’ labeled lymphocytes were located in the cluster compartment and decreasing proportions were located in the macrophage-attached compartment. The percentage of clusters containing one or more labeled central lymphocytes was approximately constant in cultures with different numbers of immune LNL. The absolute number of DNA-synthesizing
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P 5
ioo @ I %I .P 8 6 f p 50 z 5 m h E 8 2
02
OA
Number of Lymphocytes
per culkdx
10-d
Ncimhcr of Lymphocytes
per culture
(x10-S)
I12
FIG. 2. A, Distribution of labeled macrophage-attached lymphocytes involved in clusters and not involved in clusters in cultures with varying numbers of cells. Cluster-forming cultures were established with 2 X 10” to 1.2 X 10’ immune lymphocytes per culture. After 19 hr of cultivation the cultures were labeled with 1 &i/ml of r3H]TdR. The cultures were terminated after 20 hr of culture and processed for autoradiography. Labeled macrophage-attached lymphocytes were scored as involved or not involved in clusters. Open bars, labeled macrophage-attached lymphocytes involved in clusters; closed bars, labeled macrophage-attached lymphocytes not involved in clusters. B, Distribution of labeled macrophage-attached lymphocytes involved in clusters and involved in clusters in ‘cultures containing either 4 X lo” immune lymphocytes or 4 X 10” immune lymphocytes plus 1 X lo6 syngeneic nonimmune lymphocytes. After 19 hr of cultivation, the cukures were labeled with 1 &i/ml of [‘H]TdR. The cultures were terminated after 20 hr of culture and processed for autoradiography. Labeled macraphage-attached lymphocytes were scored as involved or not involved in clusters. Open bars, labeled macrophage-attached lymphocytes involved in clusters; closed bars, labeled macrophage-attached lymphocytes not involved in clusters.
lymphocytes was directly proportional to the number of immune cells in these cultures. Autoradiographic studies were also performed using cultures with 4 x 10” immune LNL supplemented or not supplemented with 1.0 x loo syngeneic nonimmune LNL and labeled with [ 3H] TdR during the last hour of culture (Fig. ZB). In the
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FIG. 3. Compartments of lymphocytes in cluster-producing cultures. The antigenpulsed macrophages (Mac) adhere to the bottom of the culture vessel. To the left is a cluster. The enlarged central lymphocyte (CL) is attached to the macrophage through a broad area on the cells’ surface membranes. The peripheral lymphocytes (PL) are attached through uropods to the central lymphocyte without contaot with the macrophage. To the right is a macrophage with two lymphocytes (ML) attached to it. Above are ‘(free” lymphocytes (FL) not attached to macrophages. Some Iymphocytes are drawn larger to denote blast forms.
cultures supplemented with nonimmune LNL a considerably higher proportion of the cultures’ labeled lymphocytes were located in the cluster compartment and a corresponding lower proportion were located in the macrophage-attached compartment (Fig. 2B). DISCUSSION T-lymphocyte-enriched lymph node lymphocytes from PPD-immune guinea pigs produce clusters of a unique structure (Fig. 3) when incubated on monolayers of PPD-pulsed syngeneic macrophages (10, 11). In the standard assay, 4 X l@ lymphocytes from a PPD-immune guinea pig produce from 150 to 1500 clusters on a monolayer of PPD-pulsed macrophages, usually about 500, and each cluster contains a mean of 10-11 lymphocytes (10). Thus, only a few percent of the cultures’ lymphocytes are involved in the clusters. Most of the remaining lymphocytes of the culture are “free cells” not adherent to macrophages or other lymphocytes, and the remaining are adherent to macrophages but not involved in clusters (12). By mechanical manipulations, the lymphocytes of these cultures could be separated into two populations, each containing members with apparently different roles in the production of the clusters. The separation was carried out by aspirating the cells that were not adherent from cultures of PPD-immune lymphocytes which had been incubated for 4 hr on monolayers of PPD-pulsed macrophages. At this time clusters had not yet developed (Werdelin, Braendstrup, and Pedersen, to be published). The aspirated immune cells were, like nonimmune lymphocytes, incapable of producing significant numbers of clusters in the standard assay. The aspiration procedure left the macrophages attached to the culture vessel together with some lymphocytes which adhered to the macrophages. When such manipulated monolayers were incubated for an additional 20 hr they did not produce clusters. However, when 4 x lo5 of the aspirated immune lymphocytes were added to the mampulated monolayer, or when 4 X lo5 nonimmune lymphocytes were added, clusters were produced in the course of 20 hr of incubation in numbers equal to the numbers produced in the standard assay of the immune lymphocytes used in the experiment (Table 1). We interpret this result to mean that two populations of lymphocytes produce the clusters in synergy. Members of the one population make contact with the antigen-pulsed macrophages early during culture and become relatively firmly bound to them. This macrophage-bound lymphocyte initiates the cluster
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by somehow attaching other lymphocytes to itself and becomes the central cluster lymphocyte. In the late phase of the culture period, increasing proportions of these cells engage in synthesis of DNA (12). These lymphocytes are present only in immune animals. The members of the other population attach themselves to the central lymphocyte as peripheral lymphocytes during 20 hr of culture. These cells are present in both immune and nonimmune animals. Since the formation of clusters is antigen-specific (10) the question arises : Which of the cluster lymphocytes are committed to the antigen ? The evidence points to the central lymphocyte. First, this cell is the only one of the cluster lymphocytes which is in contact with the antigen on the macrophage, and, second, the central lymphocyte is the only one regularly stimulated to DNA synthesis. Thus, in our view, the central lymphocyte initiates the cluster by specific interaction with the antigen-pulsed macrophage surface and by unknown mechanisms attaches and retains noncommitted bystander lymphocytes to its surface for interaction, For convenience, the term recruitment is used below for this. Earlier observations (12). on autoradiographs of [3H] TdR-labeled cultures have shown that the cluster-producing standard culture contains three compartments of DNA-synthesizing lymphocytes (Fig. 3). One contained the lymphocytes attached to macrophages and involved in the clusters as central lymphocytes, one contained those attached to macrophages but not ‘involved in the clusters, and one compartment contained “free” nonadherent cells. It was suggested that the compartment of free DNA-synthesizing lymphocytes was derived from lymphocytes stimulated to DNA synthesis during interaction with macrophages either inside or outside clusters. The existence of two compartments of DNA-synthesizing macrophage-attached lymphocytes, one recruiting and one not recruiting other lymphocytes as peripheral lymphocytes, may reflect the activation of two different populations of lymphocytes committed to the same antigen. Alternatively, it may reflect the activation of only one population of lymphocytes committed to the antigen of which some members are incapable of recruiting other lymphocytes due to suboptimal culture conditions, with respect to the availability of recruitable peripheral lymphocytes. This last possibility was examined in the present study. We found that the use of increasing numbers of immune lymphocytes per culture until about 1.2 x lo6 cells per culture yielded increasing numbers of clusters per 4 x 10” lymphocytes and that the use of 4 X lo5 immune lymphocytes supplemented with increasing numbers of syngeneic nonimmune lymphocytes until about 1.0 x lo6 cells per culture yielded increasing numbers of clusters per culture (Fig. 19, This indicates that only with cell concentrations of at least 1.2 x 106/cmZ (the bottom area in Lab-Tek tissue culture chambers) are sufficient numbers of recruitable lymphocytes present to be recruited to all prospective macrophage-attached central lymphocytes as peripheral lymphocytes to form recognizable clusters. The optimal cell concentration for cluster production may be determined not only by the proximity of the members of the synergizing lymphocyte populations, but also by the amount of nutritive factors in the small volume of culture medium used in the cluster assay. Apart from the concentration of lymphocytes per unit area of the culture chamber, the number of clusters that are produced depends on the density of the macrophage monolayer. When this falls below a certain density the number of clusters is reduced (10). A similar principle may govern the antigen-induced lymphocyte transformation in the response to soluble protein antigens, as studied by others. The amount
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of radiolabeled thymid,ine incorporated by peripheral blood leukocytes is dependent upon the cell concentration in the culture (5, 14-16), with high cell concentration resulting in larger thymidine incorporation per million cells. It has been suggested that th.is is so because interaction with macrophages is needed for all antigen-reactive lymphocytes to be activated by antigen (1, 5, 15, 17). It has been consistently demonstrated that the shape of the culture vessel, at a constant cell concentration, influences the amount of thymidine incorporation. Thus, the use of round-bottom tubes, which tends to minimize the distance between the lymphocytes and macrophages of the culture, resulted in significantly more thymidine incorporation than did the use of flat-bottom tubes (14, 15). It remained to be examined whether the steep increase in numbers of clusters which resulted from the use of increasing numbers of lymphocytes in the clusterproducing cultures was caused by more intense recruitment of lymphocytes to activated macrophage-attached lymphocytes. This was examined in autoradiographs of cultures with increasing concentrations of immune lymphocytes to which [3H]TdR had been added 1 hr before the culture was terminated. Increasing proportions of labeled macrophage-attached lymphocytes were found to recruit other lymphocytes to produce clusters, whereas decreasing proportions of labeled macrophage-attached lymphocytes were found attached singly to macrophages (Figs. 2A and B). The proportion of labeled clusters, defined as clusters with one or more labeled centrally placed lymphocytes (12)) was approximately the same using different concentrations of lymphocytes. Similar results were obtained from counts on autoradiographs prepared from cultures of immune lymphocytes supplemented with syngeneic nonimmune lymphocytes. Thus, it appears that an inverse relationship, dependent upon the ceil concentration, exists between recruiting and nonrecruiting activated macrophage-attached lymphocytes. We suggest from these results that the majority of T lymphocytes which are triggered to DNA synthesis during interaction with antigen-pulsed macrophages pass through a stage where they recruit other lymphocytes if these are available. The mechanism of this recruitment is unknown. Probably membrane changes induced in the central lymphocyte in clusters during interaction with the macrophage are responsible, as discussed in the accompanying paper (18). Attachment of uropods of lymphocytes to lymphoblasts may be a general phenomenon, since this has also been observed in mixed leukocyte cultures and PHAstimulated cultures (19,20). The present data indicate that the percentage of cluster-initiating lymphocytes in a population of lymph node lymphocytes@enriched in T lymphocytes is two to three times higher than we concluded from our earlier studies. Thus, PPD-immune cluster-initiating lymphocytes comprise, in guinea pigs immunized with M. tuberculosis from 3 to 12 weeks earlier, about 0.3 to 1.070 of the column-passed lymph node cells. A possible application of the cluster phenomenon is for quantitation of antigen-binding T lymphocytes. As shown previously (lo), the number of clusters in a series of cultures containing the same numbers of lymphocytes is directly proportional to the number of immune lymphocytes in the cultures. A count of the number of macrophage-lymphocyte clusters produced by a known number of lymphocytes on a macrophage monolayer pulsed with a given antigen might therefore be used to calculate the percentage of specific antigen-binding cells or a subpopulation of these. A second possible application is the use of monolayers of antigenpulsed macrophages as specific immunoabsorbents. As shown, a popuIation of im-
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mune lymphocytes can be largely deprived of antigen-binding cells, and a population enriched in members capable of binding antigen can be recovered together with macrophages. Studies are in progress to further characterize cell populations fractionated in this way. ACKNOWLEDGMENT This investigation was supported by Statens Laegevidenskabelige Forskningsr%d, Grants 5124170 and 512-6502.
REFERENCES 1. Cline, M. J., and Swett, V. S., J. Exp. Med. 128, 1309, 1968. 2. Oppenheim, J. J., Leventhal, B. G., and Hersh, E. M., J. Imrnunol. 101, 262, 1968. 3. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44, 1970. 4. Hanifin, J. M., and Cline, M. J., J. Cell Biol. 46, 97, 1970. 5. Hersh, E. M., and Harris, J. E., J. Immunol. 100, 1184, 1968. 6. Rosenstreich, D. L., and Rosenthal, A. S., J. Immulzol. 110, 934, 1974. 7. Sulitzeanu, D., Kleinman, R., Benezra, D., and Gery, I., Nature New Biol. 229, 254, 1971. 8. Lipsky, P., and Rosenthal, A. S., J. Exp. Med. 142, 138, 1975. 9. Salvin, S. B., Sell, S., and Nishio, J., J. Zmmunol. 107, 655, 1971. 10. Werdelin, O., Brzndstrup, O., and Pedersen, E., J. Exj. Med. 140, 1245, 1974. 11. Nielsen, M., Jensen, H., Braendstrup, O., and Werdelin, O., J. Exp. Med. 140, 1260, 1974. 12. Brzndstrup, O., Andersen, V., and Werdelin, O., Cell. Immunol. 25,207, 1976. 13. Rosenstreich, D. L., and Wilton, M., In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 113. Academic Press, New York, 1975. 14. Moorhead, J. F., Connolly, J. J., and McFarland, W., I. Immunol. 99, 413, 1967. 15. Leventhal, B. G., and Oppenheim, J. J., In “Proceedings of the Third Annual Leukocyte Culture Conference (W. 0. Rieke, Ed.), p. 13. Meredith, Des Moines, 1969. 16. Valentine, F. T., and Lawrence, H. S., Fed. Proc. 27, 265, 1968. 17. Waldron, J. A., Horn, R. G., and Rosenthal, A. S., I. Zmmunol. 111, 58, 1973. 18. Brandstrup, O., Andersson, P., Jensen, H., and Werdelin, O., Cell. Immzmol. 32, 274, 1977. 19. McFarland, W., and Heilman, D. H., Nature (London) 205, 887, 1965. 20. McFarland, W., Heilman, D. H., and Moorhead, J. F., J. Exp. Med. 124, 851, 1966.