Immunology 1978 35 643

Activation of human B lymphocytes induced by Robinia pseudoacacia lectin in the presence of T cells A.

SHARIF*, J. LETHIBICHTHUYt, Y. BROCHIERt, GOUSSAULT* & R. BOURRILLON* *Laboratoire de Biochimie, Centre de Recherches sur les Proteines, Faculte de Medicine Lariboisie're-Saint-Louis, 45 rue des Saints-Peres, 75006 Paris and tLaboratoire d'Immunologie, Inserm U80 (Prof. J. Traeger) Hdpital Edouard Herriot, 69374 Lyon Cedex 2, France

Received 5 January 1978; acceptedfor publication 6 March 1978

Summary. Robinia pseudoacacia seed lectin is a potent human lymphocyte activator which is capable of activating pure T cells but not pure B lymphocytes. However, when B and T cells were cultured together, the thymidine incorporation was found to be higher than that expected from B- or T-cell cultures alone. Killing of T cells by anti-human-Tlymphocyte antigen (HTLA) serum and complement at the time of thymidine incorporation was found to be unable to suppress completely the thymidine uptake whereas treatment by anti-human-B-lymphocyte and monocyte antigen (HBLMA) serum reduced the response to some extent. Moreover, stimulated lymphoblasts were shown to bear B-cell markers (surface Ig and complement receptors) in

about the same proportion as B lymphocytes present in the cultures. These results show that B cells proliferate in the presence of T cells and Robinia lectin. Finally, activation of B cells by Robinia lectin in the presence of T cells led to their maturation to plasma cells in the same way as PWM.

INTRODUCTION

Among the different mitogens which activate the in vitro proliferation of lymphocytes, some are regarded as specific for T or B cells and others as mixed activators (Greaves & Janossy, 1972). Human B lymphocytes behave somewhat differently from other B cells in that they do not respond to mitogens which are able to trigger murine spleen B lymphocytes, such as lipopolysaccharide (LPS),

ABBREVIATIONS USED IN THE TEXT T-lymphocytes. Thymus-derived lymphocytes; B-lymphocytes, Bursa equivalent-derived lymphocytes: PHA, Phytohaemagglutinin phaseolus vulgaris; Robinia lectin, Robinia pseudoacacia lectin; PWM, Pokeweed mitogen; HTLA, Human T-lymphocyte antigen; HBLMA, Human B-lymphocyte and monocyte antigen; NHS, Normal human serum; ALG, Anti-lymphocyte globulins; EAC, Erythrocyte-antibody complement; RCF, Rosette forming cells; FCS, Foetal calf serum. Correspondence: Professor R. Bourrillon, Laboratoire de Biochimie, Centre de Recherches sur les Proteines, Faculty de Medecine Lariboisiere-Saint-Louis, 45 rue des SaintsPeres, 75006 Paris, France. 0019-2805/78/1000-0643$02.00 A) 1978 Blackwell Scientific Publications

dextran sulphate or tuberculin (Gronowicz & Coutinho, 1975). There are convincing results which show that they are activated neither by phytohaemagglutinin (PHA), Concanavalin A nor even Pokeweed mitogen (PWM) in the absence of T cells (Geha, Rosen & Merler, 1974; Clot, Massip & Mathieu, 1975; Brochier, Samarut, Gueho & Revillard, 1976c; Keightley, Cooper & Lawton, 1976; Weksler & Kuntz, 1976; Gmelig-Meyling, Uytdehaag & Ballieux, 1977; Insel & Merler, 1977; Romagnani, Maggi, Amadori, Giudiz & Ricci, 1977). 643

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644

We reported previously that a lectin extracted from Robinia pseudoacacia seeds was a mitogen for human lymphocytes as potent as PHA (Sharif & Bourrillon, 1975) and, more recently, that this lectin was able to stimulate pure T cells but not pure B lymphocytes separated from blood or tonsils (Sharif, Brochier & Bourrillon, 1977) although it bound equally well to both types of cells. In the present work, we investigated whether or not the addition of T cells to B lymphocytes could allow them to respond to Robinia lectin. We found that a consistent number of blasts induced by Robinia possess B-cell markers and that part of the thymidine incorporation should be attributed to B lymphocytes; moreover, this B-cell activation led to the maturation of some B-cells to plasma cells.

MATERIALS AND METHODS

Lectins Robinia lectin was isolated by the method previously described (Bourrillon & Font, 1968). Briefly, a saline extract from Robinia pseudoacacia seeds was fractionated with ammonium sulphate and then on DEAE-cellulose. The homogeneous lectin had a molecular weight of 75,000 and contained 10-7 % sugars. PHA was obtained commercially from Difco and ALG from Institut Merieux, Lyon, France.

Lymphocyte suspensions Blood taken from normal healthy volunteers was defibrinated and lymphocytes were separated by centrifugation (20 min, 400 g, 40) on Ficoll-Isopaque (6.3-9.9 % final concentration; density 1 -080). Lymphocytes recovered at the interface were diluted in Hank's saline, spun down (12 min, 400 g), and then washed twice in Hank's saline (10 min, 250 g). Fresh tonsils used directly after the operation were teased with forceps into Hank's saline. After two washes, tissue debris was eliminated by filtration on a nylon column (Leukopak FT 42, Fenwall) which also removed most of the adherent cells. Tonsil cells were then centrifuged on Ficoll-Isopaque and washed three times. Fractionation after rosette formation Equal volumes of lymphocytes (12 x 106/ml) and sheep erythrocyte (E; 2-4 %) suspensions in RPMI

1640 (Eurobio) containing 15% foetal calf serum were mixed, incubated for 15 min at 370, spun down for 5 min at 200 g, and incubated at 4°. Since it had previously been shown that 1 h incubation was necessary for reaching the maximum number of rosettes, cells were either incubated for 1 h and used immediately or incubated overnight. In this case, control cells were also kept overnight at 40 and no alteration was ever seen in their response to mitogens. After another centrifugation on FicollIsopaque, the rosette-forming lymphocytes (T) were recovered at the bottom and washed three times after red cell lysis for 10 min in 0-87 % NH4C1 at room temperature. Lymphocytes recovered in the upper phase (B) were washed three times and used as a source of B-enriched lymphocytes either immediately, if they were pure enough, or after another cycle of rosette formation with fresh erythrocytes.

Cell markers Antisera specific for human T-lymphocyte antigen (HTLA) or human B-lymphocyte and monocyte antigen (HBLMA) were prepared according to a previously described technique (Brochier, AbouHamed, Gueho & Revillard, 1976a). Briefly, antiHTLA serum was prepared from horse antilymphocyte globulins (ALG). ALG were thoroughly absorbed on human plasma, AB red cells, polymerized placenta, a mixture of lymphoblastoid cell lines 4098 and 1788, and finally non-T tonsil cells. Anti-HBLMA serum was prepared in the rabbit by immunization with cell line 4098; the serum was absorbed sequentially with polymerized placenta. AB red cells, polymerized Ig, liver, and finally thymocytes. The criteria of specificity of these sera have been described in an earlier publication (Brochier et al., 1976a). Both antisera were specific for the corresponding lymphocytes in the cytotoxic test performed in the presence of complement according to the two-step technique: 10 ,ul of antiserum (or dilution in PBS or Hank's saline) were mixed with 5 ,ul of lymphocyte suspension (5 x 106/ml in Hank's saline+ 15 % AB serum) in plastic microplates (Linbro IS-MRC-96) for 20 min at room temperature; 10 ,ul of fresh rabbit serum used as a source of complement were added for 30 min at 37°. The percentage of dead cells was counted after the addition of 5 [1 of 0-5 % eosin Y (Gurr) in distilled water; no less than 200 cells were counted. The cytotoxic index was calculated according to the following formula:

Activation of human B lymphocytes by Robinia lectin

Cytotoxic index= % dead cells with antiserum % dead cells in control 100- % dead cells in control. An aliquot of anti-HTLA globulins was labelled with fluorescein isothiocyanate according to the method described by Demon, Holmes & Deinhardt (1965). Surface immunoglobulins (SIg) were detected using an anti-light chain serum obtained by immunizing rabbits with light chains purified from pooled human sera. This serum was labelled with fluorescein isothiocyanate as above (Demon et al., 1965). For fluorescein staining, 8 x 105 lymphocytes were incubated in 20 jl of Hank's saline containing 4% bovine serum albumin (BSA) 0,1 % sodium azide and fluoresceinated antiserum at the dilution 1/16 for 30 min at 00. After three washes in Hank's saline, BSA wet mounts were prepared and examined under an orthoplan Leitz microscope equipped with Ploem epiluminescence. Lymphoblasts were identified under phase contrast. Cells with receptors for complement were identified by the rosette technique described by Bianco, Patrick & Nussenzweig (1970). 2 x 105 lymphocytes were incubated with 107 sheep erythrocytes coated with rabbit anti-sheep erythrocyte 19S antibodies and mouse complement in 200 til of Hank's saline + 15 % FCS for 45 min at 37°. Cells were smeared and stained with May-Grunwald Giemsa. Cells binding three or more erythrocytes were counted as rosette forming cells (RFC). Plasma cells were identified by the presence of cytoplasmic immunoglobulins (CIg) on cytocentrifuged smears fixed 4 min in ethanol. The smears were washed for 15 min in PBS, incubated with a fluoresceinated polyvalent anti-human Ig serum (Institut Pasteur, Paris), previously absorbed on human tonsil cells, for 30 min at room temperature in a humidified atmosphere, washed 45 min in PBS and mounted in glycerol-PBS (9/1 in volume). -

Lymphocyte cultures Lymphocyte cultures were grown in microplates (Linbro IS-FB-96-TC) according to methods already published (Brochier, 1971). Cells recovered as above were resuspended in RPMI 1640 medium supplemented with 15% human AB serum or foetal calf serum (FCS) and antibiotics (vacomycin, 20 jig/ml; gentamycin, 20 jig/ml; amphotericin B, 2 jig/ml). To each well was added 0-2 ml of a 106/ml

645

lymphocyte suspension. PHA (50 t±g/ml), Robinia lectin (5 ng/ml) and ALG (160 iug/ml) were added in a volume of 20 [l. 3H-thymidine (CEA Saclay, specific activity 1 Ci/mmol, 2 IiCi/well) was added at day 2 and cells were harvested at day 3 with a semiautomatic harvesting machine onto glass-fibre filter discs. Discs were allowed to dry, put into glass counting vials, and solubilized with 0-2 ml of soluene 350 (Packard). Three millilitres of scintillator (0-6% PPO and 0-01 % POPOP in toluene) were added to each vial and the radioactivity was counted in a liquid scintillation spectrometer. Corrections for quenching were done by automatic external standard and results were expressed as disintegrations per minute (d.p.m.) ±standard error (SE). Cultures were carried out at least in triplicate. When necessary, cells were blocked for division by X irradiation for 90 s (4500 rad).

RESULTS Reconstitution of the B-cell response In a first series of experiments we confirmed our previous results (Sharif et al., 1977) that purified B lymphocytes did not respond to Robinia lectin (Table 1; Fig. 1). In the four cases studied, addition of T cells to the unresponsive B-cell suspensions caused a thymidine incorporation higher than that of T cells alone or that observed in T cells after addition of an equivalent number of irradiated B cells. In only one experiment (Exp. No. 3 Table 1) was the addition of irradiated B cells found to promote thymidine incorporation in the T cells. Another two similar experiments were performed in serum-free medium; in both cases the thymidine incorporation in the mixture of T and B cells was found to be higher than expected. In one of these experiments, we harvested the supernatant of T-cell cultures which were either stimulated by Robinia lectin or not and used this to culture unresponsive B cells; no stimulation occurred. In the experiment summarized in Fig. 1, different quantities of purified T cells were added to unresponsive B cells; a small increase of thymidine incorporation was seen with as few as 5 % of T lymphocytes and the maximum obtained with 25 or 50 %. Nevertheless, no increase of thymidine incorporation was seen when the mixture of T and B or T and X-irradiated B cells were cultured in the presence of ALG which had already been shown to be T cell-specific (Brochier et al., 1976c).

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Table 1. Reconstitution of the proliferation response of tonsil B lymphocytes to Robinia lectin in the presence of T lymphocytes Cultured cells Mitogen

Exp. no.

B* +B(RX)t 0 Robinia 0 Robinia 0 Robinia

1 2 3

1018$ 938 1018 938 1820 1194

(433)§ (103) (433) (103) (610) (191)

T +T(RX) 878 68801 470 3074 2459 5055

(46) (14336) (78) (686) (236) (781)

T +B(RX) 917 68539 492 2258 1232 16338

(296) (11589) (89) (491) (108) (2604)

T +B 784 99559 526 6438 1041 18230

(192) (8445) (160) (491) (356) (4420)

T(RX) +B(RX) 606 906 400 428 736 1452

(206) (200) (90) (64) (160) (526)

* 105 of each type of cell was added in each well. t R(X) =X-irradiated cells (10o cells/well). t 3H T-incorporation expressed in d.p.m. § S.E. Purity of the cell suspension: No. I (0% HTLA and 97% HBLMA positive cells), No. 2 (2% HTLA and 98% HBLMA), No. 3 (0% HTLA and 95% HBLMA).

150[-

E

100oo

c

0 0 0

50 I

0

5 % T Lymphocytes in B lymphocytes

Figure 1. Effect of the addition of T cells to B lymphocytes cultured in the presence of ALG or Robinia lectin. (0), T Lymphocytes +B-lymphocytes, unstimulated; (O), T lymphocytes +X-irradiated B lymphocytes, unstimulated; (O), T lymphocytes +B lymphocytes, ALG; (U), T lymphocytes +X-irradiated B lymphocytes, ALG; (A), T lymphocytes + B lymphocytes, Robinia lectin; (A), T lymphocytes +X-irradiated B lymphocytes, Robinia lectin. Varying amounts of T cells w-ere added to B or X irradiated B (4 500 rad) cell suspensions in such a way that the total number of cells was always 2 x 106 cells per well. The cells were then cultured in the presence of absence of Robinia lectin or ALG.

647A

Activation of human B lymphocytes by Robinia kectin Table 2. Culture of B and T peripheral blood lymphocytes in serum-free medium Cultured cells Ex. no.

Mitogen T +Bt B

T*

expectedl 1 2

0 Robinia 0 Robinia

426§ 8366 135 3029

(28)1¶

704 830 170 201

(1773) (39) (268)

(84) (23) (67) (56)

observed 328 (41) 8231 (328) 155 (50) 2097 (13)

565 4598 180 1615

* 1 x 106 cells/well. t 5 x 105 T cells +5 x 105 B cells/well.

EH Expected value =2

thymidine in T +'H thyminide in B 2

§ d.p.m. 1¶ S.E.

Evidence for the contribution of B cells to the cell proliferation response In order to obtain direct confirmation that B cells were contributing to the thymidine incorporation, unfractionated lymphocytes of blood or tonsil were cultured. At the time of addition of tritiated thymidine, T or B cells were killed by anti-HTLA or anti-HBLMA sera respectively in the presence of complement (Table 3). Most of the response was suppressed by anti-HTLA serum but a consistent thymidine incorporation persisted. Anti-HBLMA serum inhibited the thymidine incorporation in about the same proportion as that remaining after anti-HTLA treatment. Similar results were obtained

with cultures stimulated by PHA which had already been shown to activate B cells in the presence of T lymphocytes (Brochier et al., 1976c). Surface markers on lymphobhsts In two experiments we investigated the presence of

SIg and complement receptors in lymphoblasts generated after 3 days of culture in the presence of Robinia lectin (Table 4). In both cases a significant number of blasts carrying these B cell markers was found, which was correlated to the number of lymphocytes bearing these markers in unstimulated cultures. In accordance with results of Table 3 the majority of lymphoblasts bore the HTLA.

Table 3. Killing of T or B cells by anti-HTLA or anti-HBLMA serum and complement at day 2 of the culture Control Lymphocyte

origin

Blood Tonsils

Robinia lectin

PHA

Treatment

Living cells (%) NHS +C anti-HTLA +C anti-HBLMA +C NHS +C anti-HTLA +C anti-HBLMA +C

100 8 48 100 51 54

d.p.m. (SE) 560 1988 441 2749 1751 924

(67) (28) (85) (74) (107) (145)

Living cells (%) 100 28 85 100 53 51

d.p.m. (SE) 226,146 27924 163,837 186,488 25111 109,390

(19664) (3180) (1850) (5198) (774) (2672)

Living cells (%) 100 19 68 100 54 61

d.p.m. (SE) 168,384 55698 86886 110,834 38967 79612

(6844) (3651) (3618) (4640) (1628) (3178)

Human peripheral or tonsil lymphocytes were cultured in the presence or absence of Robinia lectin or PHA. After 48 h the cells were treated with normal horse serum (NS), anti-HTLA or anti-HBLMA sera and complement (C) according to the cytotoxicity technique described in materials and methods and washed three times. The cell viability was tested and the cells were incubated at 370 in a new medium containing 3H-thymidine for a further 24 h.

648

A. Sharif et al.

Table 4. Surface markers on lymphoblasts at day 3 of culture Origin of cells

Markers

Mitogen Blood

Tonsil

SKg

0 Robinia

235* 21.5t

385* 24 Ot

HTLA

0 Robinia

65.0* 66-5t

54-5* 64 Ot

EAC-RFC

0 Robinia

14 * 12 t

22 * 28 t

* Percentage of total cells. t Percentage of lymphoblasts. Tonsil and blood lymphocytes were cultured for three days in RPMI medium containing 15% of FCS. SIg and HTLA of both stimulated or unstimulated cells were detected by immunofluorescence according to the procedure described in Materials and Methods.

Generation of plasma cells In four cases, unfractionated cells were cultured for 7 days and stained for cytoplasmic immunoglobulins (Table 5). In all four cases a significant number of plasma cells was found in the cultures stimulated by Robinia lectin. In cultures of blood lymphocytes stimulated by ALG, no plasma cells were generated.

Table 5. Generation of cytoplasmic Ig-containing cells at day 7 of culture

Mitogen Cell origin 0

Robinia

ALG

Blood

2-0*

10 0

0.5

Tonsil 1

1-3

3-8

ndt

Tonsil 2

2-6

5-9

nd

Tonsil 3

00

9-2

nd

* Percentage of cells stained by fluoresceinated polyvalent anti-human Ig serum. t Not done because of a high level of agglutination. Cells were incubated for seven days in RPMI medium containing 15% FCS.

DISCUSSION Robinia pseudoacacia seed lectin is a mitogen for human lymphocytes as potent as PHA (Sharif et al., 1975). In the present study we have confirmed our previous finding that it activates pure T cells but not pure B cells (Sharif et al., 1977). However, when B and T cells were cultured together, the thymidine incorporation was found to be higher than that expected in cultures of B or T cells alone or than that of T cells cultured with Xirradiated B cells. Although in one experiment (Exp. No. 3, Table 1) the B cells were found to promote thymidine incorporation in the T cells, we assumed that B cells might be activated by Robinia lectin in the presence of T cells. Direct evidence that the B cells were contributing to the proliferation response was that the thymidine incorporation diminished after the B cells had been killed by anti-HBLMA serum and complement at day 2 of the culture. We found that, in accordance with this result, treatment by anti-HTLA did not completely suppress the thymidine incorporation. However, most (80 to 90%) of the proliferation response could be attributed to T lymphocytes. Further confirmation of B-lymphocyte activation lies in the presence of SIg or complement receptors on the surface of a number of lymphoblasts equivalent to the proportion of B cells present in the culture suspensions. Finally, B cell- activation by Robinia lectin in the presence of T cells led to maturation to cells containing intracytoplasmic 1g. The number of plasma cells was approximately the same as that obtained in cultures stimulated by PWM or Nocardia water soluble mitogen (MWSM) (Lethibichthuy, Ciorbaru & Brochier, 1978). These results show that B cells are definitely activated by Robinia lectin in the presence of T cells. Such T cell-dependent human B-cell activation has already been observed with other phytomitogens (Clot et al., 1975; Brochier et al., 1976 c; Weksler & Kuntz, 1976; Romagnani et al., 1977; Keightley, 1976; Gmelig-Meyling et al., 1977; Insel & Merler, 1977). The mechanism of B-cell recruitment remains to be elucidated. We can only say that such recruitment can occur in the absence of serum in the medium and that preliminary experiments were unable to show that any stimlatory T-cell factors were released in the supernatant medium. Nonetheless, Robinia lectin can be considered as a helpful tool, in addition to the other phytomitogens and to NWSM which is a T-independent human B-cell activator (Brochier,

Activation of human B lymphocytes by Robinia lectin Bona, Ciorbaru, Revillard & Chedid, 1976b), for studying the T-cell control of human B lymphocyte differentiation.

ACKNOWLEDGMENTS We would like to thank Miss Font for providing Robinia lectin, Mrs V. Buisson and Mrs M. Blanc for their technical assistance. This work was supported by INSERM grants (ATP 76-59 et 10-74-31) and by the 'Fondation pour la Recherche Medicale Fran9aise'.

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