Clin. exp. Immunol. (1990) 80, 420-425

Jacalin:

a

lectin mitogenic for human CD4 T lymphoctyes

N. PINEAU, P. AUCOUTURIER, J. C. BRUGIER & J. L. PREUD'HOMME Laboratory of Immunology and Immunopathology (CNRS URA 1172), Poitiers University Hospital, Poitiers, France (Acceptedfor publication 4 December 1989)

SUMMARY The major protein component of seeds from jackfruit is the lectin jacalin. Jackfruit crude extracts are known to stimulate human lymphocytes, but the mitogenic properties of purified jacalin have not been studied in detail so far. Study of the proliferative response of cell populations from normal human peripheral blood to purified jacalin showed it to be mitogenic through an interaction with lymphocytes by its lectin-binding site, as shown by inhibition by IgA. Jacalin failed to stimulate B cells to proliferate and to undergo plasma cell maturation. It induced a proliferation of CD4 (and not CD8) lymphocytes, as shown by phenotypic analysis ofcells recovered after culture and by studies of the response of isolated T cell subpopulations. The proliferative response to jacalin was autologous monocyte-dependent. The kinetics of jacalin-induced DNA synthesis, expression of CD25 and interleukin-2 secretion was shifted by comparison with that induced by phytohaemagglutinin. The reason for the restricted responsiveness of CD4 T cells is presently unclear; jacalin bound to all blood cells and did not significantly co-cap with CDl, CD2, CD3, CD4, CD8 and CD38, and jacalin response was neither enhanced nor inhibited by antibodies to these surface antigens. Keywords human CD4 lymphocytes proliferative

INTRODUCTION The major protein component of seeds from jackfruit (Artocarpus heterophyllus), is a Galfl-1-+3GalNAC specific lectin, jacalin, whose reactivity with human immunoglobulins, especially IgA I and IgD, is well documented (reviewed by Aucouturier et al., 1989). The mitogenic properties of this lectin were less studied. Jackfruit seed crude extract (JCE) was first reported to induce both human lymphocyte (mostly T cell) proliferation and B cell terminal differentiation (Bunn-Moreno & CamposNeto, 1981). More recently, JCE was found to stimulate only T cells and to inhibit immunoglobulin production, possibly by activating suppressor T lymphocytes (Saxon, Tsui & MartinezMaza, 1987; Gattass, Ghobrial & Bunn-Moreno, 1988). JCE also enhances the production of interferon-gamma (IFN-y) by human T cells (Crane et al., 1984). JCE contains jacalin and a variety of other molecules (Aucouturier et al., 1987, 1989). To our knowledge, there has been no study of the mitogenic properties of purified jacalin, a molecule of about 50 kD, of known amino acid and carbohydrate content, made up of two types of non-covalently linked subunits, an unglycosylated 12 kD and a glycosylated 15 kD subunit (Aucouturier et al., 1987). JCE can be fractionated by diethylaminoethyl (DEAE) chromatography (Moreira & Ainouz, 1981) into fractions containing variable proportions of jacalin with different ratios

response

lectin jacalin

of the glycosylated and unglycosylated subunits, JCE from different geographic origins being possibly non-identical (Aucouturier et al., 1987, 1988). We therefore studied the mitogenic properties of purified jacalin. MATERIALS AND METHODS

Purification ofjacalin Jacalin was purified from jackfruit seeds collected in the Indian Ocean island La Reunion, by DEAE-Trisacryl (IBF, Villeneuve La Garenne, France) chromatography as previously described (Aucouturier et al., 1987, 1988, 1989). Purity was controlled by SDS-PAGE followed by Coomassie brilliant blue and silver (Bio-Rad, Richmond, CA) staining and by Western blot analysis using peroxidase-coupled F(ab')2 fragments of purified anti-jacalin rabbit IgG antibodies. Isolation of mononuclear cell populations Peripheral blood mononuclear cells (PBMC) from healthy adults were isolated by Ficoll (Lymphoprep; Nycomed, Oslo, Norway) centrifugation. B cells were enriched by two T cell depletion steps using the 2-aminoethylisothiouronium hydrobromide (Sigma, St Louis, MO) activated sheep red blood cell rosette method (Saxon et al., 1976). The percentage of residual CD3-positive cells was always below 1 %. T enriched cells were obtained after one cycle of E rosetting. They contained virtually no surface immunoglobulin (SIg) bearing B cells and 2-5% monocytes. Blood lymphocytes from four patients affected with

Correspondence: J. L. Preud'homme, CNRS URA 172, CHUR La Miletrie, BP 577, F 86021 Poitiers Cedex, France.

420

Human CD4 cell stimulation by jacalin chronic lymphocytic leukaemia (CLL) and high lymphocytes counts were also studied. These cells were monoclonal SIgbearing B cells and the percentage of CD3-positive lymphocytes in the cell suspensions obtained from these patients was below 1%. Monocytes were eliminated from PBMC by three steps (Wernet & Kunkel, 1973; Mossalayi et al., 1985): PBMC were applied to a Sephadex GlO (Pharmacia, Uppsala, Sweden) column and incubated at 370C for 30 min. The cells recovered by washing with warm medium were incubated for 40 min at 250C with 5 mM L-leucine methyl ester (Sigma) in medium RPMI 1640 and washed. Finally, the cell suspension was incubated at 370C for 1 h in Petri dishes coated with fetal calf serum (FCS). Nonadherent cells were recovered. They contained less than 1 % monocytes. Monocyte-enriched suspensions were obtained by adherence on FCS-coated Petri dishes (1 h at 37 C) and resuspension by flushing with a Pasteur pipette. CD4 and CD8 enriched cell populations were isolated by incubation with anti-CD4 or anti-CD8 antibody (OKT4, OKT8; Ortho, Raritan, NJ) and panning on Petri dishes coated with rabbit anti-mouse IgG antibodies adsorbed on human IgG, according to Wysocki & Sato (1978). In addition, T cells depleted in CD4 lymphocytes by panning were studied. Cell cultures Cells were cultured for 1 to 7 days in medium RMPI 1640 supplemented with 10% FCS, 25 mm HEPES, 2 mM Lglutamine, 100 U/ml penicillin and 100 jg/ml streptomycin (GIBCO, Grand Island, NY), in 96-well microtitre plates in a 5% CO2 incubator. Proliferative responses induced by jacalin (2 to 2000 Mg/ml), pokeweed mitogen (PWM, 10 Mg/ml) and phytohaemagglutinin (PHA, 10 pg/ml) (GIBCO) were measured by 3Hthymidine incorporation. Preliminary experiments showed the optimal jacalin concentration to be 200 Mg/ml and the time of culture that yielded the most reproducible results to be 4 days. Hence, experiments described below were performed under these conditions, except where specifically stated. All samples were tested in triplicate. Inhibition of jacalin-induced proliferation by antibodies to surface antigens was searched for by pre-incubating PBMC in microplates (2 x 105 cells/well) either for 30 min at room temperature or for 1 h at 37°C with monoclonal antibodies (MoAbs) (Ortho) extensively dialysed against RMPI medium and adjusted at 5 Mg/ml. The following MoAbs were used: antiCD1 (OKT6), CD2 (OKT 11), CD 3 (OKT3), CD4, CD8 and CD38 (OKT1O). Then, the cells were cultured for 4 days in the presence of jacalin as above. To study the relationship between the lectin-binding site and lymphocyte activation, PBMC were pre-incubated for 30 min at room temperature with increasing concentrations of two different purified monoclonal IgA1 and cultured for 4 days with jacalin. IL-2 production was measured with PBMC cultured in medium RPMI 1640 containing 2% FCS in the presence of jacalin or PHA. After 1 to 7 days, supernatants were collected and tested for IL-2 activity by the CTLL2 cell assay, according to Gillis et al: (1978), using recombinant IL-2 as a standard. Lymphocyte markers The number of cytoplasmic immunoglobulin (cIg) containing cells was determined in 7 day jacalin and PWM-stimulated cultures of unseparated PBMC by immunofluorescence with

421

tetramethylrhodamine (TRITC) conjugated rabbit F(ab')2 fragments polyvalent for human immunoglobulin (prepared in our laboratory). SIg-bearing cells were counted using the same antiimmunoglobulin conjugate, as previously described (Preud'homme & Labaume, 1976). The cells were studied for T cell antigens using the MoAbs listed above and an anti-CD25 MoAb (anti-Tac, a kind gift of Professor T. A. Waldmann) by indirect immunofluorescence with TRITC-conjugated F(ab')2 fragments of rabbit IgG anti-mouse IgG (extensively adsorbed on human immunoglobulin,. prepared in our laboratory). Differential redistribution experiments were performed as previously described (Preud'homme et al., 1972): PBMC were stained at 0°C by FITC-conjugated jacalin, then incubated at 37°C for 1 h, and restained at 0°C with anti-CDl, CD2, CD3, CD4, CD8 and CD38 MoAbs followed by the TRITCconjugated anti-mouse IgG F(ab')2 fragments. In the reciprocal experiments, the cells were first stained in the cold for these various T cell antigens, incubated for 1 h at 37°C, and restained at 0°C by FITC-conjugated jacalin. RESULTS The mitogenic response of normal unseparated PBMC to jacalin was 33 7+15 x 103 ct/min (ct/min in unstimulated control cultures subtracted, means and s.d. from 52 experiments). This response was related to the IgA-binding property of jacalin since it was inhibited by IgA1 in a dose-dependent manner. B-T cell specificity ofjacalin stimulation T cell enriched cells (which contained a small percentage of monocytes) displayed a proliferation similar to that of whole PBMC (not shown). T cell depleted cells failed to proliferate. Since T cell depleted cells are enriched in monocytes which could have been in excess and play an inhibitory role, they were further depleted of monocytes and the response of B cells supplemented with known percentages (1%, 5%, 10%, 20% and 50%) of monocytes was assayed. No proliferative response was observed at any monocyte concentration. CLL B lymphocytes did not respond to jacalin either. The number of cIg-containing cells was determined after culture of PBMC in the presence of variable amounts ofjacalin. No significant stimulation of B cell terminal maturation was observed, in contrast to control cultures in the presence of PWM (Table 1).

Stimulation of T cell subpopulations and monocyte requirement Most cells recovered from 7-day cultures of unseparated PBMC with jacalin were CD4-positive T cells (Table 2). In 4-day cultures, results were similar and CD4-positive cells were large cells, whereas CD8-positive cells were small lymphocytes. In contrast, PBMC stimulated by PHA and, to a lesser extent, those cultured without mitogen were enriched in CD8 lymphocytes. Monocyte depletion of whole PBMC resulted in an almost complete abrogation of the response to jacalin. T cell subpopulations were therefore examined with and without monocytes. As shown in Table 3, CD4 and CD8 lymphocytes obtained by panning with the relevant monoclonal antibodies and containing 97 6+1 5 CD4 cells and 2-7+1 4% CD8 cells for CD4enriched cells, and 92+1% CD8 and 106 + 4.7 CD4 cells for CD8-enriched cells, respectively, failed to respond to jacalin in the absence of monocytes. When increasing percentages of

N. Pineau et al.

422

Table 1. Percentages of clg-containing cells (mean + s.d.) in jacalin stimulated 7-day cultures of unfractionated PBMC

Mitogen concentration

Jacalin

(pg/ml)

None

2

10

20

50

100

200

PWM 10

No.ofexperiments cIg

9

3

9

3

11

12

11

7

1-47+1 19

1-71+203

078+03

205+ 17

1 55+143

190+209

102+1-6

10-8+5-53

clg, cytoplasmic immunoglobulins. Table 2. Surface phenotype ofjacalin-stimulated PBMC (7-day cultures, percentages, mean + s.d.)

Fresh cells

Jacalin

PHA

Medium alone

92-5+ 1-9

98 2+3 5

98 7+0 9

96-5+2 6

(4)

(4)

(4)

(4)

59-4+7-9

92-0+4 2

40 0+9-8

57 0+8 2

(7)

(7)

(7)

(4)

31-1+88

10-6+56

60-8+11 1

465+107

(7)

(7)

(7)

(4)

Effect ofjacalin on IL-2 secretion and CD25 expression Jacalin induced both the secretion of IL-2 and the expression of CD25. Comparison of the kinetics of the responses to jacalin and PHA showed a one day shift for DNA synthesis and IL-2 secretion, which peaked on day 2 and day 3, respectively, in PHA-stimulated cultures and on day 3 and day 4 after stimulation by jacalin (Fig. 1). Similarly, the percentage of cells expressing CD25 plateaued from day 3 to day 6 in the PHA controls whereas it kept slowly increasing from day 4 to day 6 with jacalin.

6-8+2 8 (6)

1 0+0 (3)

0+0 (3)

0-7+0 6 (3)

DISCUSSION

Cells cultured with

CD2

CD4

CD8 SIg

surface antigens, capped and restained with jacalin yielded similar negative results.

Number of experiments is indicated in parentheses.

autologous, but not of allogeneic monocytes were added, the mitogenic response of CD4 lymphocytes was restored. The optimal concentration of monocytes was 10%. In contrast to CD4 cells, isolated CD8 lymphocytes did not respond to jacalin in the presence of monocytes, the marginal stimulation observed corresponding likely to contamination by residual CD4 lymphocytes. Control experiments with PHA showed a significant response of both isolated CD4 and CD8 lymphocytes. This mitogenic response was further increased by the addition of monocytes.

Effect of pre-incubation of PBMC with antibodies to surface antigens PBMC pre-incubated with antibodies to CD1, 2, 3, 4, 8 or 38 were cultured for 4 days with and without jacalin, without removal of the MoAb. As expected, anti-CD3 and anti-CD38 antibodies were stimulatory. In cultures containing jacalin, no significant additive effect could be detected nor any inhibition of the jacalin response. Pre-incubation with a mixture of anti-CD2 and anti-CD3 MoAb had no effect on the jacalin-induced response either, in contrast to PHA controls (Table 4). Diferential redistribution ofjacalin and T cell surface antigens When PBMC stained at 0°C with FITC-conjugatedjacalin were incubated at 37°C for 1 h, capping was observed on most cells. Restaining in the cold with antibodies to CDl, CD2, CD3, CD4, CD8 and CD38 and TRITC-tagged anti-murine IgG F(ab')2 fragments did not show any significant co-capping. The reciprocal experiments in which the cells were first stained for these

Only a few studies performed with unpurified extracts were devoted to jacalin mitogenic activity. We therefore undertook a study of the response of normal human PBMC to purified jacalin. It was found to be essentially a T lymphocyte activator and we observed no activation of B cells. This is at variance with the first report (Bunn-Moreno & Campos-Neto, 1981) but agrees with a more recent work showing an inhibition of B cell maturation into plasma cells (Saxon et al., 1987). There are several possible explanations for these discrepancies. It was suggested that differences in the human immunoglobulin binding specificity of JCE could be the consequence of geographic variability (Kobayashi et al., 1988) and the same might apply to mitogenic specificity. Botanical differences may also play a role. Among the species of Artocarpus cultivated for their edible fruits, the botanists clearly distinguish the jack (A. heterophyllus, most common synonyms A. integrifolia and A. integra) from the breadfruit (A. altilis) and the champedak (A. integer) (Quisumbing, 1951; Jarret, 1959; Purseglove, 1968; Barrau, 1976). However, identification of the jack is difficult and natural or artificial hybridization between the jack and the champedak is known to occur. It could result in seed extracts with immunological properties different from those of jackfruit seeds. It must also be pointed out that the present study was performed with purified jacalin and not with crude extracts. We showed (unpublished results) that JCE contains mitogenic molecules other than jacalin (unreactive with IgA and antijacalin antibodies and without the characteristic subunit composition of jacalin). Contrarily to jacalin, the response to these molecules was inhibited by anti-CD2 and anti-CD3 antibodies and their subpopulation specificity might be different. Variations in such non-jacalin molecules may explain differences between crude extracts.

Human CD4 cell stimulation by jacalin

423

Table 3. Proliferative response to jacalin (ct/min x 103, mean and s.d.) of CD4 and CD8 lymphocytes with and without monocytes

Mitogen T cells

CD4

Corresponding unseparated PBMC CD8

Corresponding unseparated PBMC

Monocytes (%)

experiments

None

Jacalin

PHA