Lymphocytes express specific antigen-independent contact interaction sites upon activation

Lymphocytes express specific antigen-independent contact interaction sites upon activation

CELLULAR IMMUNOLOGY 86, 14-32 (1984) Lymphocytes Express Specific Antigen-Independent Contact Interaction Sites upon Activation ALF HAMANN, DOROT...

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CELLULAR

IMMUNOLOGY

86,

14-32 (1984)

Lymphocytes Express Specific Antigen-Independent Contact Interaction Sites upon Activation ALF HAMANN,

DOROTHEE JABLONSKI-WESTRICH, AND HEINZ-G~NTER THIELE

ANDREAS RAEDLER,

Abteilung f: Immunologic. I. Medizinische IUinik d. Universitiit Hamburg, 2000 Hamburg 20, West Germany Received June 28, 1983; accepted January 10, 1984 Cell contact between lymphocytes can be observed in the form of clustering in autologous cultures of rat or mouse lymph node cells. Mutual binding takes place in the absence of adherent cells and is displayed by B cells as well as by T cells, with the exception of immature (Lyt 1,2+) T cells. Contact formation is related to activation of the lymphocytes since thymidine-incorpor#ing cells as well as plaque-forming cells are concentrated in the cluster cell fraction and the formation of clusters is greatly increased by periodate stimulation. The interaction is selective with respect to cell type (cells of other tissue origin are not bound) and differentiation (only activated lymphocytes and some of several lymphoid cell lines are able to interact). The reaction is not genetically restricted, but takes place even between different (but related) species. Neither antigen nor MHC structures are involved in contact formation. Protease treatment abolishes the ability to form clusters, but one part of the interacting receptor/acceptor structures is apparently trypsin resistant. The interaction is dependent on the presence of magnesium, whereas calcium ions have no supporting effect, Involvement of the cytoskeleton is shown by a partial inhibition of the cluster formation by cytochalasin B and azide. No indication for a lectin nature of the binding structures could be found by carbohydrate inhibition studies. The relation of this interaction mechanism to other models of physical interaction in the immune system as well as its possible function for signal exchange and local recruitment of activated cells is discussed.

INTRODUCTION The complexity of the regulating network in the immune system, involving several cooperating subpopulations of cells, raises the question of how the rare, corresponding partners specific for a given antigen, could find each other among thousands or millions of inappropriate cells. The transitory passage through lymphoid organs during continuous recirculation of lymphocytes represents one step in which distinct populations are sorted out from the circulation and brought together by specific homing and migration processes ( 1-3). That further cell recognition mechanisms may help to focus relevant cell populations locally is demonstrated by the phenomenon of local recruitment of antigen-specific lymphocytes in draining lymph nodes (4, 5). But in contrast to the process of recirculation of lymphocytes via high endothelial venules, for which reasonable data on functional and biochemical characteristics are available (6, 7), little is known about the mechanisms, by which cells engaged in an active immune response are selected 14 0008-8749/84 $3.00 Copyri&t Q 1984 by Academic Press, Inc. All rights of reproduction in any fom reserved.

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and concentrated locally. In this respect in vitro models of contact interaction could contribute to a better understanding of these phenomena. In the immune system contact interaction (adhesion, physical interaction) can be observed in different models: binding of target cells by cytolytic T cells, for which distinct non-antigen-specific cell interaction structures were described (8- 10); interaction between lymphocytes and accessory cells, such as dendritic cells (11) and macrophages (12, 13); or interaction between lymphocytes in the form of mutual binding and cluster formation (12, 14-16). The formation of lymphocyte clusters or contact between accessory cells and lymphocytes was demonstrated to be essential for the generation of an immune response (13, 14, 17, 18). It is presently not clear how far these contact mechanisms are related and in what way specificity for subpopulations or certain functional states of the lymphocytes may enable selection and focusing of relevant cell populations. We have analyzed in more detail the physical interaction between lymphocytes in cultures leading to clustering. We found that cluster formation represents the ability of lymphocytes to form specific cell contacts and that this property is acquired upon activation and transformation. Experiments were undertaken to characterize the functional and biochemical properties of the recognition mechanism and to determine its relationship to known structures involved in cell interaction. MATERIALS

AND

METHODS

Animals and cell lines. DA/Han rats, 8-12 weeks old, and Balb/c or CBA/Han mice, 6-10 weeks old, were used. The human leukemic cell lines, Reh (non-B/nonT), Molt 4 (T), Daudi (B), and U266 (myeloma), and the mouse lines, EL-4 (T), L1210 (T), BCLl (B), and P3x63-Ag 8.653 (myeloma), were cultured in RPM1 1640 containing lo-20% fetal calf serum. ’ EAC Karzel (mouse Ehrlich ascites) and the rat hepatoma Yoshida ALT 7974 were grown in ascites form. Antisera and chemicals. FITC-, TRITC-, or peroxidase-conjugated anti-Ig sera and biotin-avidin reagents were from Medac (Hamburg, GFR) or Nordic Immunology (Tilburg, The Netherlands). The following monoclonal antibodies were used: antimouse macrophage, clone M l/70 HL, anti-rat non-helper T lymphocytes OX 8HLK, anti-rat helper T lymphocytes W3/25 HL, anti-rat T-cell W3/ 13 HLK, all from Seralab (Camon, Wiesbaden, GFR); anti-Thy 1.2 was a gift from Dr. Opitz (WuppertalElberfeld, GFR); anti-Lyt 1.1 and Lyt 2.1 were from Cedarlane (Camon, Wiesbaden, GFR). Anti-Ia sera OX 6-HL (anti-rat Ia, cross-reacting with mouse strains I-AklS), H 116-32.R5 (I-Ak, Balb/c anti-CBA), 13/4-R5 (I-Ek,d, A.TH anti-A.TL), and antiH-2 D,K sera H 142-23, H lOO-27/55, H lOO-30/23 (H-2k,b,q,s,r, H-2k,‘, and H-2k,S, respectively, all Balb/c anti-CBA) were used. Anti-Ia and anti-H-2 sera were from Camon. The source of the monoclonal antibodies (except M l/70 and Lyt 1.1 and

’ Abbreviations used: MHC, major histocompatibility complex; LNC, lymph node lymphocytes; CC, clustered cells; SC, single cells; PBL, peripheral blood lymphocytes; SRBC, sheep red blood cells; PBS, phosphate-buffered saline; FCS, fetal calf serum; DNP, dinitrophenyl; sIg, surface immunoglobulin; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; TCA, trichloroacetic acid; EDTA, ethylenediaminetetraacetic acid, EGTA, ethylene glycol bis@aminoethyl ether)-N,N’-tetraacetic acid.

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2.1) was ascites fluid. FCS was obtained from Medac; DNase I (EC 3.1.4.5.) was from Boehringer; sea-plaque agarose was from MCI (Rockland, Mass.); prostaglandin E2 and cytochalasin B were from Sigma (Munich, GFR); proteases were from Serva (Heidelberg, GFR); Percoll was from Pharmacia (Upsala, Sweden); r3H]thymidine (2 Ci/mmol) was from Amersham (Braunschweig, GFR); mannan (yeast) and thyroglobulin (porcine) were from Sigma; ovalbumin was from Serva. Asialo-fetuin was prepared from fetuin (10 mg/ml; kindly supplied by Professor R. Dernik, Hamburg) by treatment with 0.1 U/ml vibrio cholera neuraminidase (Behring, Mat-burg, GFR) for 24 hr at 37°C and inactivation of the enzyme for 3 hr at 70°C. Isolation of cluster-forming cells. Lymph node cells were prepared from peripheral and mesenteric lymph nodes by gentle teasing. Dead cells were removed by agglutination in high concentration phosphate buffer ( 19) and adherent cells by incubation for 1 hr 37°C in a glass-bead column or 2 hr in plastic tissue culture flasks. More than 98% of the cells were viable as determined by trypan blue exclusion. Cells were cultured for 3-5 days at a concentration of 2 X 1O6cells/ml in RPM1 1640 containing 20% FCS, 5 X 10e5 A4 2-mercaptoethanol, and 10 pg DNase on a slowly moving (8 mov./min) rocking platform (Bellco). Cell clusters were separated from single cells by lg sedimentation: 20 ml cell suspensions were carefully loaded on 5 ml 30% FCS and allowed to sediment for 30 min, after which time all but very small aggregates were found in the pellet. Rat cells ordinarily survived better and gave more clusters than mouse cells. For the latter, removal of dead cells before culturing turned out to be especially important. Electron microscopy. For electron microscopy clustered cells were isolated and fixed in 2.2% glutardialdehyde (Merck, Darmstadt, FRG) for 30 min at 4°C. They were then taken up in 2% agar (Behring-Werke, Marburg, FRG) and prepared for electron microscopy by postfixation in 1% 0~0~ for 2 hr at 4°C and dehydration in alcohols of increasing concentrations (35-100%). The material was embedded in Epon 8 12 and cut on a Reichert OMU 3 ultramicrotome. The ultrathin sections were contrasted with lead citrate/many1 acetate (20). The samples were viewed and photographed with a Zeiss electron microscope EM 9. IdentiJication of cell type. For immunofluorescence, cells were directly labeled by conventional antibodies (B cells) or indirectly labeled with monoclonal antibodies (1:50-1:500) as first and conventional second antibodies (mostly affinity purified, 1:5-l :20), in the presence of azide and 20% FCS. In order to exclude cross-reactivity of the second antibody, normal rat or mouse serum was added in some instances. For labeling of mouse T cells the monoclonal anti-Thy- 1.2 antibody was biotinylated (21) and after binding visualized by FITC-avidin. The cytotoxicity assay with Lyt antisera was carried out by sequential incubations with the antibody ( 1:10-l: 100) and rabbit complement, previously absorbed with thymocytes and spleen cells. Phagocytosis was tested by incubation for 1 hr at 37”C, with Indian ink dialyzed against PBS and diluted 1:5000, or with 1% sheep erythrocytes. Thymidine incorporation. DNA synthesis was measured by addition of 2.5-5 &i [3H]thymidine to 4 X lo6 lymph node lymphocytes in 2 ml after culturing for the indicated time. Five hours later clustered cells and single cells were separated by sedimentation. Cells were washed thoroughly with PBS and the number of viable cells was determined in each fraction. After precipitation and washing with trichloroacetic acid the pellets were solubilized with 1 M NaOH and radioactivity was counted in a scintillation counter.

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Periodate stimulation. Suspensions of 5 X 10’ cells/ml were prepared from lymph nodes, washed in K+-free PBS, and incubated for 15 min at 4°C with 0.5 mM sodium periodate if not stated otherwise. After washing, dead cells and adherent cells were removed as described above. The same procedure was applied for human peripheral blood lymphocytes, except that adherent cells were removed only after overnight incubation. Borohydride reduction of periodate-stimulated cells was carried out by incubation with 10 mA4 borohydride in PBS at 0°C (22) after 2 days of culture. Purification of stimulated T cells. In order to obtain purified stimulated T cells, periodate-treated cells were incubated overnight in plastic tissue culture flasks after removal of dead cells. B cells were then removed from the nonadherent cell fraction by panning (23) on petri dishes coated with rabbit anti-mouse Ig (affinity-purified) antibody. Less than 2% B cells or cells with surface-bound immunoglobulin were present in this preparation. Cluster formation was determined after a further 2 days of culture. Plaque-forming cells. Antibody-secreting cells were enumerated by a hemolytic plaque assay (24). Lymphocytes (0.5-2 X 106) were plated together with 1% ovalbuminor DNP-coated sheep red blood cells and 3.3% guinea pig serum in 0.45% agarose (final concentrations). For indirect (IgG) plaques, a rabbit anti-rat-IgG serum, which inhibits IgM plaques completely, was added. Ovalbumin was coupled onto red blood cells by the chromium chloride method (25), using a concentration of 2 mg ovalbumin/ml. DNP was coupled to SRBC via DNP-modified rabbit anti-SRBC F(abh (4 DNP groups/F(ab)Z). DNP modification was carried out according to Good et al. (26). Influence offree hapten on clusterformation. Spleen lymphocytes of rats immunized several times with ovalbumin or DNP-keyhole limpet hemocyanin were prepared as described above and cultured for 2 days. The cells were dissociated and allowed to reaggregate in the presence of various concentrations of DNP-alanine. After 3 hr, clustered cells were separated from single cells. Both fractions were thoroughly washed and cultivated for another day, to permit recovery of the cells from side effects of the hapten, before assaying for plaque-forming cells in each fraction. DNP-alanine (Serva) was dissolved in complete medium and sterilized by filtration. At the highest concentration used, 5 mM, viability of treated cells was slightly decreased. The efficiency of the hapten concentrations used in inhibiting antigen binding by the anti-DNPIgG produced by the rat cells was tested by adding it in a further plaque assay. DNPspecific plaques were inhibited by 100% at 0.05 mM DNP-alanine and by 90% at 0.005 mM DNP-alanine, whereas ovalbumin-specific plaques remained unaffected. Reaggregation test. The ability of clustering cells to reaggregate in the presence of antisera or other potentially inhibiting agents was tested as follows: Cluster-forming cells were isolated as described above, suspended in medium +20% FCS + 10 pg DNase I, and dissociated by vigorous vortexing or by forcing them through a pipet. Cells (5 X 105) were mixed with the agent to be tested which was dissolved in complete medium. Stock solutions of sugars were made 300 rnM in water to maintain isotonicity. The cells were allowed to reaggregate for 3 hr on a rocking platform in horizontally mounted small tubes. Separation of reaggregated and single cells was performed by sedimentation for 30 min after underlaying with 0.5-l ml of lo-20% Percoll. Coaggregation. The ability of different cell types to form either mixed aggregates with clustering lymphocytes or to show sorting out was tested using FITC-labeled clustering cells (27) and unlabeled cell line cells in two different approaches: (1) cells,

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which behave or grow as single cells, were added in a small number (2 X lo5 cells) to dissociated cluster cells (8 X 1O5cells). After reaggregation and separation of clusters it was determined whether the added cells were included in the clusters or whether they were left behind in the single-cell fraction. (2) Cells forming clusters in culture or growing as aggregates were thoroughly dissociated, mixed in equal amounts with dissociated cluster lymphocytes, and allowed to aggregate. Thirty to sixty small clusters (6-20 cells per cluster) were microscopically examined and categorized with respect to the ratio of the two cell types. Ten different categories were established. If the cells form mixed aggregates, the frequency of clusters within the ten categories follows a Gaussian distribution. If sorting out takes place, the aggregates are composed primarily of one cell type and a bimodal distribution is found. Protease treatment. Isolated clustering cells were treated for 30 min at 37°C with the proteases dissolved in PBS and after washing and dissociation they were allowed to reaggregate in complete medium for 1 hr. RESULTS Characterization

of Clustering

Cells

In syngeneic cultures of rat or mouse lymph node lymphocytes slowly shaken on rocking platforms, soon after initiation of the culture a small percentage of the cells is found to form clusters. The clusters are initially small and labile but grow with time and at 3 to 6 days of culture they may comprise hundreds to thousands of cells (Fig. 1). At this time 5-20% of the viable cells are included in the clusters. These can be dissociated mechanically by vortexing or repeated forcing through a pipet. The dissociated cells reaggregate readily during rocking for l-3 hr. Cluster formation, or reformation after dissociation, at least for the time the cells remain viable, proceeds equally well in medium without fetal calf serum or in autologous serum. Clustered cells, isolated by sedimentation over a 30% FCS cushion and dissociated, reassociate with high efficiency (usually 50-80% are found reaggregated). Interestingly the cells in the single-cell fraction form new aggregates after cultivation for several hours. The clustering phenomenon can hardly be ascribed to adhesion of lymphocytes to adherent cells, since these have been efficiently removed in all experiments. Thus, cells phagocytosing carbon particles (rat LNC) or labeled by the monoclonal macrophage-specific antibody Ml/70 (mouse LNC) were found to represent less than 1% of such cultures. Morphologically the cells found in early clusters strongly resemble small lymphocytes which are only loosely aggregated and maintain contact by relatively small surface areas (Fig. 2a). The clustering cells of 3-day cultures (Fig. 2b) represent mostly large, blastlike lymphocytes with numerous protrusions. A portion of the cells is in intimate mutual contact whereas others seem to be only loosely connected by interdigitating processes. Occasionally plasma cells are found within the clusters. Immunofluorescence labeling of Thy 1.2 and surface Ig in Balb/c mouse lymphocyte clusters indicates the simultaneous presence of T and B cells in the aggregates (not shown). Estimations of the percentage of cells of the T- or B-cell lineage were carried out by immunofluorescence analysis with anti-Ig antibodies, biotinylated monoclonal anti-Thy 1.2, and the rat W3/25 and OX8 monoclonal antibodies, as well as by cytotoxicity with monoclonal anti-Lyt 1.1 and 2.1 antibodies. As shown in Table 1,

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FIG. 1. Small clusters of rat lymph node lymphocytes in a 3-day culture. Bright field illumination, bar = 50 pm.

the ratio of T to B cells is roughly the same as that found in unfractionated lymph node cell cultures or fresh lymph node cells. With regard to the subpopulations of T cells, it was found that, compared with fresh lymphocytes, in cultures the Lyt If

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FIG. 2. Electron microscopic picture of clustered rat lymph node cells. (a) One day of culture, small lymphocytes with limited contact zones. (b) Three days of culture, mainly large lymphoblastoid cells, showing intimate contact with one another. Bar = 5 pm.

cell compartment was increased and Lyt 2+ cells were decreased. This obviously reflects a differential proliferation of these cells in the culture system and does not relate to the clustering ability of these cells, since their proportion in the single and the clustered cell fractions is roughly the same. Interestingly, this is not the case for Lyt 1,2+ cells. This subpopulation is the only one which is not included appreciably in the clusters. The simultaneous presence of both B and T cells is apparently not a prerequisite for cluster formation. Thus, clusters are formed in cultures of lymph node cells from nude mice which do not possess mature T cells (not shown). Furthermore, clusters

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2-Continued.

are found in purified, activated T lymphocytes obtained by periodate stimulation (2 1% clustering cells) as well as in an unseparated mixture of activated B and T cells (15% clustering cells).

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TABLE I Percentage of Lymphocyte Subpopulations in the Cluster Cell Fraction Clustered cells

Single cells

Fresh LNC

Lyt+ (V” Sk+ (B)

56 40

73 36

57 40

Lyt 1+ Lyt 2+ Lyt 1,2+

86 10 4

70 3 27

58 18 24

W3/25+ (helper) OX 8+ (non-helper) Sk+ (B)

40 23 33

Subpopulation Mouse % of total cells 96 of T cells

Rat % total cells

29 19 51

Note. Rat and mouse lymph node lymphocytes were separated into clustered and single cells after 3 days of culture and the proportions of subpopulations determined by cytotoxic assays(Lyt 1,2) or immunofluorescence (sIg, W3/25, and OX 8). For comparison, the values obtained for fresh, unseparated lymph node lymphocytes (LNC) are also shown. Given is the mean of triplicate determinations. a Numbers of T ceils as determined by immunofluorescence with biotinylated anti-Thy 1.2 and FITCavidin were essentially the same.

In conclusion, physical interaction seems not to be the property of a special subpopulation of lymphocytes but rather seems to be expressed by mature cells in a particular functional state.

day 3

day 6

FIG. 3. Thymidine incorporation of clustered cells. Three- or six-day cultures of rat lymph node cells were incubated for 5 hr with [)H]thymidine. After separation and enumeration of clustered and single cells, the TCA-insoluble radioactivity per viable cell was determined in each fraction. The ratio of incorporated thymidine in clustered cells versus incorporated thymidine in single cells is shown. n , 0, 0, A, different experiments; CC, clustered cells; SC, single cells.

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Clustering and Lymphocyte Activation Since the cluster-forming cells ultrastructurally have the features of blast cells, it was investigated whether mutual adhesivity is correlated with the state of activation of the cells. In a first set of experiments the incorporation of thymidine was studied. If the clustered cells were separated by sedimentation after 3 to 6 days of culture and the single and clustered cell fractions were allowed to incorporate [3H]thymidine for 5 hr, the radioactivity incorporated per viable cell was much higher in the cluster cell fraction (Fig. 3). This effect became more pronounced with time of culture. The correlation between activation and clustering was further analyzed by means of mitogen stimulation. Since lectins are not suited for this purpose because of their agglutinating properties, periodate was used for stimulation. Figure 4 shows the effect of different concentrations of periodate on the percentage of cells found in clusters after 2 days of culture. The optimal stimulating dose was 0.5 mM, whereas at 1 rnM the percentage of clustered cells decreases again, thus excluding a purely chemical effect of periodate on the cell surface’s adhesivity. Furthermore, reduction of remaining reactive aldehyde groups by borohydride just before reaggregation did not affect the clustering ( 17% reaggregation in borohydridetreated cluster cells versus 18% in untreated cells). The kinetics of cluster formation and DNA synthesis are shown in Fig. 5. DNA synthesis is still low at 20 hr after stimulation, displaying a sharp peak then at Day 2, whereas cluster formation is induced earlier, at 20 hr approaching two-thirds of the maximal value of 75% of cells found in the clusters. At Day 5 a second increase in the percentage of clustering cells (as well as in the thymidine incorporation on a per cell basis) can be observed, probably representing cells of a late proliferating cell subset. Further evidence for a correlation between functional activity and mutual recognition resulting in clusters, came from the distribution of plaque-forming cells in the clustering and the single-cell fractions. The lymph node lymphocytes of rats immunized several times in vivo with ovalbumin or DNP-hemocyanin were cultured and after 3 days separated in a clustered and a single cell fraction. In a direct or indirect plaqueforming assay against ovalbumin or DNP-coated SRBC, a pronounced enrichment

0

0.25 0.5 1 mM Periodate FIG. 4. Influence of periodate stimulation on the degree of cluster formation. Unseparated rat lymph node cells were treated with the concentrations of periodate indicated and the adherent cells removed. After 2 days of culture clustered and single cells were separated and the percentage of cells found in the cluster fraction was determined.

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ET AL.

3

4

5 chys

FIG. 5. Kinetics of cluster formation and thymidine incorporation after periodate stimulation. Rat lymph node cells were stimulated by 0.5 mM periodate and aliquots cultured in tubes. After various times the percentage of cluster-forming cells (CC) (A) and the radioactivity incorporated per total culture (0) were determined.

of specific antibody-producing cells was found in the clustered cell fraction. The same is true for DNP-specific cells (Table 2).

Specljkity of the Cell-Cell Contact From the above described experiments it cannot be concluded whether clustering of the cells is a specific process, mediated by selective receptor/acceptor structures, or whether it merely reflects some state of unspecific stickiness of the cells. To answer this question, the ability of activated lymphocytes to bind cells of different origins was examined by coaggregation experiments. Clustered cells of 3- to 6&y cultures of rat or mouse lymph node lymphocytes were isolated and labeled with FITC. After addition of a small amount of the cells to be tested (Fig. 6) the dissociated cluster cells were allowed to reaggregate and the number of unlabeled cells bound by the newly formed clusters was determined. For cells growing as aggregates or forming clusters in culture by themselves, the test for sorting out or coaggregation with clustering lymphocytes was slightly modified: Equal numbers of dissociated cells of each cell type (one of them labeled with FITC) were mixed and allowed to reaggregate. The percentage of labeled cells was determined for a number of individual, small clusters and the frequency of clusters of a given composition plotted (Fig. 7). With combinations of cells having functional cooperating receptor/acceptor sites, coaggregation takes place and the frequency of each cell type TABLE 2 Relation of Plaque-Forming Cells in Clustered Cells to That in Single Cells

Expt

Ratio (PFC per lo6 clustering cells/ PFC per IO6 single cells)

Target antigen

A=Y

1 2 3 4

2.7 3.6 3.4 8.5

Ovalbumin Ovalbumin Ovalbumin DNP

Direct Direct Indirect Indirect

Note. Spleen lymphocytes from in viva immunized rats were analyzed for IgM (direct) or IgG (indirect) plaque-forming cells (PFC) after 3 (Experiment 1) or 2 days (Experiments 2-4) in culture.

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Hep

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EL-4

Reh

U266

25

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EL-4

EAC

FIG. 6. Inclusion of different cell types in reaggregates of clustering lymphocytes. Clustered rat or mouse lymph node cell cultures were isolated, labeled with FITC, and after dissociation reaggregate in the presence of a small amount (20%) of different types of unlabeled single percentage of unlabeled cells in the reformed clusters was determined and compared to that mixture before aggregation.

cells from allowed to cells. The of the cell

in the aggregates follows a Gaussian distribution. With inappropriate cell pairs, sorting out takes place and a bimodal distribution or even pure aggregates of each cell type are found. The following results were obtained in these experiments: Mutual adhesivity of the activated lymphocytes is not due to an unspecific stickiness, since several other cell types are excluded completely from the clusters or form only separate clusters. This n 1’0

E!&ClCBA

0

so

100% CBA

LN-CC

P Rat I Mm

10

rLN -Molt

4

mLN -LIZ10

10

rLN - L1210

mLN-BCLI

0 mtLN-CC

50

ioox mouse LN-CC

0

100 %

%I rut LN-CC

FIG. 7. Coaggregation of different cell types with clustering lymphocytes. Clustered cells from rat or mouse lymph node cultures (rLN, mLN) or from stimulated human PBL were isolated, labeled with FITC, and allowed to reaggregate in combination with dissociated, unlabeled cells of various cell lines or clustering cells of other origin. The percentage of labeled cells in each of 30-60 small clusters was determined and the frequency distribution of clusters falling in the different categories was plotted. Ordinate: Number of clusters found in the respective range of cellular composition. Abscissa: Categories of cellular composition, expressed as percentage of the cell type stated on the right.

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is most clearly seen in combinations with cells of another tissue origin, e.g., hepatoma and EAC cells (Fig. 6) or epithelial cells (HeLa, Fig. 7), but also with combinations of lymphoid cell lines of the same or different species (mouse CC/P3x63-Ag8; rat CC/EL-4, CC/Reh, CC/U266, CC/Molt 4). Some cell lines show an intermediate reactivity and only one, BCLl, behaves as if the receptor/acceptor structures are functionally identical with those of activated mouse lymph node lymphocytes. Furthermore it can be seen that only a minor percentage of freshly prepared lymph node lymphocytes or thymocytes is included in the clusters. In Fig. 7 the results of allogeneic or xenogeneic combinations of activated lymph node lymphocytes are also depicted. It can be seen, that cells from different strains of mice as well as from mouse and rat completely coaggregate with each other, whereas between rat and human clustering lymphocytes, a partial sorting out takes place. Thus, the interacting structures seem to be functionally identical in different strains of mice and have only slowly changed during evolution.

Clues to the Interaction Structures Further experiments were undertaken to characterize the receptor/acceptor structures in more detail and to elucidate their relationship to known cell surface structures. First, the sensitivity of the interacting structures towards proteases was tested. As shown in Table 3, reaggregation of clustering lymphocytes is prevented by previous protease treatment. The ability to interact is reexpressed after 24 hr of cultivation. Interestingly, one part of the corresponding receptor and acceptor structures is trypsin resistant, since trypsinized cells can be passively incorporated into clusters of untreated cells. Material cleaved off by the proteases used proved not to be inhibitory in a reaggregation test (not shown). Divalent cations are known to be essential for a variety of cell contact phenomena. As shown in Table 4, this is also the case for the interaction of activated lymphocytes, since EDTA and EGTA inhibit their reaggregation and dissociate existing clusters. The inhibition is fully reversible by addition of magnesium, but not of calcium. Agents, which act on cytokinetic elements either directly (cytochalasin B) or by cutting their energy supply (sodium azide), were found to reduce the clustering and the stability of aggregates markedly (Table 4). TABLE 3 Influence of Protease Treatment on Cluster Formation Treatment

Reaggregation (% of controls)

Papain Chymotrypsin Trypsin Trypsin, assay after 24 hr recovery at 37°C Trypsin-treated cells mixed 1:1 with untreated cells”

14 19 9 90 94

Note. Clustered cells of 3day cultures were separated, washed in PBS, and incubated for 30 min at 37°C with 1 mg/ml protease. After washing, the cells were allowed to reaggregate in complete medium for 1 hr and the number of cells found in clusters was determined. 0 Untreated cells were labeled with FITC and were not counted.

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TABLE 4 Influence of Divalent Cations and Cytoskeleton-Directed Drugs on the Reaggregation of Clustering Cells Treatment EDTA EGTA EGTA EGTA

(2 mA4) (0.5 mM) + 1.5 mM Ca*+ + 1.5 mM Mg*+

Cytochalasin B (5 &ml) Azide (10 mM)

Reaggregation (W of controls) 11 18” 22” 100” 68 52

Note. Isolated clusters from 3-day cultures were dissociated and allowed to reaggregate in the presence of the different agents noted. Reformed clusters were separated and the number of cells contained therein was determined. Essentially the same values were observed, if the dissociation of intact clusters by the agents EDTA, NaNs, and cytochalasin B was measured. ’ Reaggregation of washed cells was carried out in Hanks’ salt solution without Ca*’ and Mg*+ containing 1% bovine serum albumin.

Recently, two drugs were found to influence the migration of lymphocytes, prostaglandin E2 (28), and prednisolone (29). It was of interest therefore, to see whether these agents would interfere directly with the cell surface receptor/acceptor structures relevant for the contact between activated lymphocytes. However, in concentrations of l-20 ng or 0.5-50 pg respectively no inhibition was found. Cell surfaces are armed with a variety of carbohydrate structures, some of them being specific for certain cell populations and differentiation stages. A series of inhibition studies with carbohydrates was undertaken to learn whether sugar residues would act as ligands for lectinlike binding structures in our system. The following carbohydrates and glycoconjugates were added to dissociated cluster cells and the degree of reaggregation was compared with that of the control: glucose, galactose, mannose, fucose, cu-methylmannoside, N-acetylglucosamine, N-acetylgalactosamine, lactose, melibiose, raffinose and stachyose, in concentrations of 150, 50, and 15 rnA4 each; mannan (yeast), porcine thyroglobulin, asialo-fetuin, and ovalbumin at 10, 1, and 0.1 mg/ml each. No inhibition was found with the high-molecularweight glycoconjugates nor were significant influences of sugars measurable (8-l 5 determinations), though the variability in the latter experiments was higher than usual. Products of the main histocompatibility complex are cell surface structures playing a central role in several systems of functional interaction between cells of the immune system. Therefore it was important to see whether physical interaction between activated lymphocytes as represented by the clustering phenomenon can be inhibited by antibodies directed against histocompatibility antigens. Six monoclonal, nonagglutinating IgG antibodies against class I and class II antigens (see Materials and Methods) were tested alone or in mixtures for inhibition of the reaggregation of clustering cells. No significant differences could be found in the reaggregation of cells of the appropriate strain (H-zk) versus cells of the inappropriate strain (H-zd) in antiserum dilutions of 1: 10 to 1: 1000. Though the periodate stimulation and the coaggregation experiments yielded strong arguments against a role of antigens as bridging elements in the clustering of lym-

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phocytes, it was nevertheless desirable to test this possibility directly. For this we monitored the inclusion of plaque-forming cells specific for DNP in the clusters and the influence of soluble hapten during reaggregation. Table 5 shows that the inclusion of the plaque-forming cells in clusters is not inhibited by free hapten in the culture fluid, as would be expected in the case of antigen bridging. DISCUSSION Cell-cell recognition is a constitutive step of a variety of cellular interactions in the immune system. In phenomena as diverse as selective homing, cooperation between lymphocytes and accessory cells, and interactions of cytolytic T cells or natural killer cells with their targets, antigen-independent recognition processes linked to cell contact can be observed. Cell contacts between lymphocytes in culture were observed earlier in the form of clustering and were suggested to represent a correlate to functional interactions (1416). However, it was not clear which cell populations were involved, how specific the interaction is, and what kind of structures may be responsible for the interaction. We found, that the formation of aggregates by cultivated lymphocytes is independent of adherent cells and is not restricted to a particular subpopulation of lymphocytes but is shown by T cells as well as B cells. Only one type of T lymphocytes was found not to be included in clusters, the Lyt 1,2+ subpopulation. This fraction is thought to consist mainly of immature T lymphocytes, whereas functionally competent cells are predominantly found in the Lyt l+ (helper) or Lyt 2+ (suppressorjcytotoxic cells) fraction (30). The proportions of the subpopulations present in the 3-day old lymph node cultures are slightly changed compared with that of fresh lymph node cells. The increase in the Lyt I+ cell fraction and decrease in the Lyt 2+ cell fraction compare well with the reported findings for the autologous mixed-lymphocyte reaction in which nonT cells act as stimulators and predominantly Lyt If or OKT4+ cells were identified as responder cells (3 1, 32). Furthermore we also found the B cells of the clusters to be in a blast stage. This led us to conclude that all differentiated populations of mature lymphocytes can express the property of mutual binding. Maturity, however, is not the only preTABLE 5 Influence of Free Hapten on Clustering of Antigen-Specific Cells PFC per lo6 cells DNP-alanine (mkf) during reaggregation 0.05 0.5 5

Clustered cells 64 52 46 105

k + + f

22 22 19 52

Single cells 26+ 8 252 6 26k 7 60-t 19

Note. Spleen lymphocytes from in viva immunized (with DNP-hemocyanin) rats were cultured for 2 days, dissociated, and allowed to reaggregate in the presence of different concentrations of the hapten DNPalanine. The reformed clusters were separated by sedimentation, and the cells were washed and cultured for a further day before assaying for IgCi plaque-forming cells.

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requisite, since only a small percentage of freshly prepared lymph node lymphocytes forms aggregates. Electron microscopic evaluation reveals a predominantly blastlike appearance of the cells found in clusters of 3-day cultures, pointing to a correlation between activation and cluster formation. Indeed this hypothesis is supported by several functional criteria: (1) Thymidine incorporation is much higher in cells found in clusters than that in single cells. (2) Functionally active cells, i.e., antibody-synthesizing cells, are concentrated in the clusters. This indicates that not only initial stages of activation show an increased ability for mutual binding but at least in the B-cell line also the final ones. Indeed, plasma cells are occasionally detected electron microscopically in the clusters. (3) Stimulation by periodate leads to a drastic increase in the percentage of clusterforming cells. Periodate is thought to act by cross-linking cell surface molecules through formation of aldehyde groups on carbohydrate residues reacting with amino groups of other surface molecules (33). Since borohydride reduction of cells already stimulated was not found to affect reaggregation, it can be supposed that the aldehyde groups on the cell surface induced by periodate treatment are not by themselves responsible for agglutination of the cells. From these experiments it can be concluded that physical interaction, i.e., mutual binding, is among the functional capabilities newly acquired by lymphocytes upon activation. It has to be mentioned that cluster formation precedes DNA synthesis as it was also reported for rabbit leukocytes (15). On the other hand, the adhesive stage may be a transient event in the cells’ development. The quick reformation of new aggregates after removal of the clustered cells in contrast to the rather constant percentage of clustered cells in a given culture could indicate this. The selective enrichment of activated lymphocytes in the clusters and the exclusion of the immature Lyt 1,2+ population may give a first clue as to the specificity of the interaction mechanism. Coaggregation experiments between fresh lymphocytes and the cluster cell fraction further confirmed this specificity, in showing that only a small percentage of fresh lymphocytes will be included in clusters, the vast majority being sorted out. This means that quiescent lymphocytes obviously lack the respective receptor, as well as acceptor sites which could be bound by the receptors present on activated lymphocytes. The suggestion that the expression of such interacting structures is limited to certain differentiation stages is further strengthened by the finding that among a series of cell lines tested so far only one, BCL, , a B-lymphoblastoid cell line, was found to coaggregate perfectly with clustering cells, as would be expected in the case of functional receptor/ acceptor identity. Several other lymphoid cell lines show only an intermediate degree of coaggregation. The reason for the sorting out cannot be explained by a lack of adhesion mechanisms, since cells of most of the cell lines show self-clustering. Thus, the more or less complete sorting out in separate clusters points to the existence of additional, functionally different receptor/acceptor systems for physical interaction in some of the cell lines tested. It should be noted that cells of different tissue origin (epitheloid or liver cell derived) tested so far do not coaggregate at all. This points to a strict “tissue” specificity of this interaction, comparable with the adhesion mechanisms of embryonal cells in mammals (34).

30

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AL.

However, the interaction of the activated lymphocytes is not strictly strain or species specific. We found allogeneic mouse-mouse as well as mouse-rat combinations to coaggregate in a manner indistinguishable from a syngeneic combination, whereas between rat and human cells a partial sorting out takes place. A similar situation was reported for the interaction of lymphocytes with high endothelial cells (35). Several groups studied the contact interaction of lymphocytes with accessory cells. Lymphocytes were found to bind to macrophages and furthermore to activated lymphocytes already bound by the macrophages. These reactions were antigen-independent and genetically not restricted ( 12). An additional, antigen-dependent and genetically restricted mechanism could be demonstrated (12, 13, 39). Mutual binding was also observed between lymphocytes and dendritic cells (11, 36). Too little data about the characteristics of these interaction systems are presently available to ascertain their relationship to the model studied by us, but one is tempted to speculate that all these antigen-independent interaction phenomena may indeed be closely related. Their functional role can be thought to provide a supporting matrix (36) for the functional cooperation of accessory cells and lymphocytes, and between different subpopulations of lymphocytes, actively engaged in an immune response. Thus, the physical link would stabilize and intensify the process of signal exchange and regulation. Furthermore, we would like to hypothesize that the acquisition of “adhesive” properties of lymphocytes upon stimulation could be operative in the well-known phenomenon of trapping of antigen-specific lymphocytes in local lymph nodes. A binding step, which is activated by antigen recognition, but is antigen independent in its further course as also known from the interaction of cytolytic T cells with their targets (37, 9, lo), may help to focus appropriate populations of specific lymphocytes locally. Another set of experiments was performed in order to characterize the molecular structures involved in the contact interaction and to study their eventual relationship to other mechanisms of cell interaction. First, the protease digestion experiments revealed that at least a part of the interacting structures comprises a polypeptide and that the structures are resynthesized after 24 hr of further incubation. The finding that trypsinized cells can passively be incorporated into clusters indicates a different sensitivity towards proteases of the structures, functioning either as receptor or acceptor sites, one of them being resistant to trypsin. Thus, these sites seem at least to reside in different domains of the putative interaction molecule if they are not located in different molecules. In any case, both entities appear on the lymphocyte only after activation, since unstimulated lymphocytes neither aggregate by themselves nor are they passively included in clusters of activated cells to an appreciable extent. The dependency of the aggregation of activated lymphocytes on divalent cations is a feature characteristic for some, but not all, cell contact mechanisms of diverse cellular models (34, 38) and also for the antigen-independent binding step of cytolytic T lymphocytes to their target cells (37). Interestingly, the latter system shares, with the model studied here, the supporting effect of magnesium and the insufficiency of calcium ions. Antigen bridging was considered earlier by Mosier ( 14) as a possible mechanism for the clustering of lymphocytes. This, however, is excluded by the finding that periodate stimulation leads to strongly increased clustering. It can be assumed that

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31

for the vast majority of the mitogen-stimulated lymphocytes the nominal antigen is not present in the culture. Furthermore, antigen-mediated binding should be inhibited in the presence of free antigen. We found, however, that the addition of free hapten during the reaggregation period did not hinder the inclusion of hapten-specific plaqueforming cells into the clusters. That anti-idiotypic antibodies or complement bridges are not involved in the contact formation between activated lymphocytes can be concluded from the fact that pure, activated T cells as well as cells in serum free media are able to form clusters. For the antigen-dependent binding of lymphocytes to macrophages, class II histocompatibility antigens have been shown to be involved (12, 39). In the case of contact interaction of activated lymphocytes, however, we can rule out a participation of these structures since the interaction is not inhibited by antibodies against class I or II antigens and also takes place between lymphocytes of MHC-different mouse strains or lymphocytes of mouse and rat. In several systems of cell-cell interaction (40) as well as in the binding of macrophages and natural killer cells to appropriate targets (41, 42) carbohydrate structures were identified as acceptor structures. Furthermore, membrane lectins could be detected in mouse spleen and thymus lymphocytes and in human activated lymphocytes (43, 44). However, glycoconjugates and single sugars, including those which were inhibitory for these lymphocyte membrane lectins, did not hinder the clustering of activated lymphocytes. Thus, there is no indication so far, that lectinlike bonds are involved in this cell interaction system. In conclusion, the experiments presented here characterize the contact interaction of activated lymphocytes as a unique, highly specific mechanism which is not mediated by the main surface structures involved in functional interactions. Instead, it displays some features of cell-cell contact systems which are not related to immune recognition. Further experiments are required to elucidate the functional meaning of the antigenindependent interaction mechanism under study and to clarify the bearing on other, possibly related interaction systems. ACKNOWLEDGMENTS Cell lines were a kind gift of Dr. L. Gtirtler (Munich), Dr. W. L&old (Hannover), Dr. H. Lemke (Kiel), and Dr. K. Wielckens (Hamburg). We would like to thank Dr. R. Amdt for helpful discussions and A. Ladak for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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