Immunology Letters, 11 (1985) 101-105 Elsevier

lmlet 663

R E C O M B I N A N T I N T E R L E U K I N 2 I N D U C E S I M M U N O G L O B U L I N S E C R E T I O N

I N S T A P H Y L O C O C C U S A U R E U S C O W A N S T R A I N I A C T I V A T E D

H U M A N B - C E L L S

Rene DEVOS 2, Bettadapura JAYARAM 1, Peter VANDENABEELE 1 and Walter FIERS ~ ~Laboratory of Molecular Biology, State University of Ghent, Ledeganckstraat 35, and 2Biogent, Plateaustraat 22, B-9000 Ghent,

Belgium

(Received 12 July 1985) (Accepted 15 July 1985)

I. Summary

Human B-cells, exhaustively depleted for T- cells, were activated with Staphylococcus aureus Cowan strain I (SAC) and responded to recom- binant human interleukin 2 (rlL2) by secretion of immunoglobulin (Ig), as measured by a pro- tein A hemolytic plaque assay. The rlL2, howev- er, had to be present early, since addition later than 24 h after SAC-activation of the B-cells reduced the response to background levels. No clear dose response was observed and Ig- secreting cells (ISC) could be induced even with rlL2 at 0.5 U/ml. The monoclonal antibody anti- TAC prevented the rlL2-promoted induction of ISC. Ig production could be induced in SAC- activated cultures with supernatants of Xenopus laevis oocytes injected with sucrose-gradient- fractionated poly(A ÷) RNA derived from a stimulated human spleen cell culture. This activi- ty coincided with the IL2 mRNA activity and was well separated from the interferon-7 mRNA activity. Our results suggest that IL2 is not only a B-cell growth factor but also promotes the differentiation of activated human B-cells to- wards Ig secretion.

2. Introduction

T-cells are required for Ig secretion by human

Key words: recombinant interleukin 2 - Staphylococcus aureus

B-cells activated with Staphylococcus aureus par- ticles [1]. However, the concomitant activation of the T-cells with pokeweed mitogen (PWM) pro- vides a much stronger differentiation signal need- ed by the S. aureus Cowan strain I (SAC)- stimulated B-cells, indicating that T-cell-derived lymphokines play an important role [1, 2]. In- deed, it has been reported that T-cell-derived fac- tors different from human interleukin 2 (IL2) are involved in this differentiation [3-6] . However, a difficult question to resolve is whether IL2 is part of the differentiation signal by directly act- ing on the SAC-activated B-cells, or by acting on T-cells contaminating the B-cell preparations. Since IL2-receptors are expressed on SAC- activated human B-cells [7-11], we have inves- tigated the possible effect of purified Escherichia coil-derived rlL2 [12] on the differentiation of these cells. We report that recombinant IL2 (rlL2) is active at very low concentrations and has to be present from the start of the cultures to be most effective in promoting Ig secretion.

3. Materials and Methods

3.1. Isolation of human B-lymphocytes Human peripheral blood lymphocytes (PBLs)

were isolated from buffy coats (Red Cross Blood Centre, Ghent) by centrifugation over kymphoprep (Nyegaard, Oslo), and plastic-adherent cells were removed by incubation at 5 × 106 cells/ml for 2 h at 37 °C in RPMI 1640 medium supplemented

0165-2478 / 85 / $ 3.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division) 101

with 10070 fetal calf serum (FCS) in plastic flasks. Depletion of T-cells was accomplished by twice rosetting the cells with AET-treated sheep erythrocytes [13], followed by cytotoxic treatment with a panel of monoclonal antibodies, anti- Leu 1, anti-Leu 2a and anti-Leu 3a (Becton Dick- inson) and rabbit complement (Lowtox H rabbit complement, Cedarlane) [I]. Cells obtained in this manner contain less than 1°70 Leu 1 +-cells and do not respond to phytohemagglutinin (PHA).

3.2. B-cell cultures and enumeration of ISC 1 × 10 5 cells were cultured in f lat-bottom

microtitre trays in 0.2 ml RPMI 1640 medium with 10°70 FCS in the presence of 0.001°70 SAC (Calbiochem, Pansorbin) and rIL2 [12] or super- natant of interest. On day 5 or 6, the cells were washed with Balanced Salt solution and ISC- enumerated using a protein A hemolytic plaque assay as described by Tauris [14]. Plaques were developed in agarose, using goat anti-human Ig (M + G + A) (USB, Cleveland, OH) and guinea pig serum (pre-adsorbed with protein A- coupled sheep red blood cells - SRBC) as a source of complement. As a control for the PFC assay, 1 x l0 s B-cells were reconstituted with 1 x 104 T-cells, obtained after NH4CI lysis of the SRBC rosettes, and stimulated with 0.001% SAC + PWM (Gibco) at a final dilution of 1: 500.

3.3. IL2 and interferon "I' (IFN-7) assay Human IL2 was assayed using the murine

CTLL2 line [15] and human IFN- 7 was assayed on human FS4-cells as described [16]. Stimula- tion of human spleen cultures with P H A / T P A , isolation and sucrose-gradient fractionation of poly(A +) RNA and injection of Xenopus laevis oocytes was as described previously [12, 16].

4. Results and Discussion

As reported by several laboratories [1, 2], a T- cell mitogen, such as PWM, and a B-cell mito- gen, such as SAC, act synergistically to activate human PBLs towards IgM and IgG production. B-lymphocytes stimulated by SAC alone do not

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secrete Ig in the absence of a T-cell signal, but the latter can be replaced by a lymphokine- termed B-cell differentiation factor [5] (BCDF) or B-cell-inducing factor (BIF) [3]. We have in- vestigated whether purified E. coil-derived hu- man rlL2 [12] could also substitute for T-cells in this process.

Fresh human PBLs depleted of adherent cells and exhaustively depleted of T-cells by a double rosetting with AET-treated SRBC and incubation with monoclonal antibodies (anti-Leu 1, anti- Leu 2a, anti-Leu 3a) and complement, were cul- tured in the presence of 0.001°70 SAC together with 2.5 U/ml rIL2 added on either day 0, day 1, day 2, or day 3. After 6 days of incubation, ISC were enumerated by the protein A-reverse hemo- lytic plaque-forming cell (PFC) assay. As con- trols, unfractionated PBLs (adherent cells re- moved), B-cells, and B-cells reconstituted with autologous T-cells were cultured alone or in the presence of PWM + SAC. Fig. la and Fig. 2a show that the response obtained for the B-cells in the presence of PWM + SAC - the most sensitive functional test for the presence of T- cells - was less than 20°70 of the response ob- tained with the reconstituted B + T-cell cultures. Addition of rlL2 on day 0 to the SAC-activated B-cells led towards a dramatic increase in ISC number (80-90°70 of B + T-cells in the presence of PWM + SAC) (Fig. lb). On the other hand, this number decreased when rlL2 was only add- ed on day 1, and was almost background (B-cells in the presence of SAC) after addition of rlL2 on day 2 or day 3. This result demonstrates that the early presence of rlL2 is necessary in this system to lead towards Ig-secreting B-cells. As shown in Fig. 2b, a final concentration of rlL2 up to 100 U/ml, when added on day 2, did not lead to ISC in SAC-activated B-cell cultures.

A low concentration of rlL2 (0.5 U/ml) was still sufficient to induce the differentiation of SAC-activated human B-cells (Figs. lc and 2b). Moreover, rlL2 at a final concentration of 0 . 5 -25 U/ml did not show a dose response in the PFC assay. The monoclonal antibody anti- Tac (a generous gift of Dr. L. Moretta), which recognizes the human IL2-receptor [17], was able to prevent the rlL2-induced Ig-secretion by SAC-

I

¢ J

x

L"-

x

a

6

5 -

4

3

2

1

U B

A B C A B C

B+T i

A B C

2.5 u r lL2/ml

0 2 3 -

day

day 0

i

- - I

I I

~'~. ~'~. c~. c~. "~. c~. c~. I

u rlL2/ml

Fig. 1. (a) Controls: induction of PFC with SAC and SAC + PWM in human unfractionated (U)-, T-cell-depleted (B)-, and recon- stituted (autologous T-cells added) (B + T) B-cell cultures. 1 x 105 unfractionated PBL or T-cell-depleted B-cells or T-cell-depleted B-cells plus I x 10 a autologous T-cells were cultured for 5 days at 37 °C without any addition (A), in the presence of 0.00107o SAC (B), or in the presence of 0.001% SAC plus PWM (1 : 500) (C). (b) Effect of time of addition of rlL2 to SAC-activated B-cells on the PFC response. 1 x 105 T-cell-depleted B-cells were cultured for 5 days at 37 °C in the presence of 0.00107o SAC (added on day 0) and 2.5 U/ml rlL2 added on day 0, 1, 2, 3 or not added ( - ) . (c) Effect of different concentrations of rlL2 added on day 0 to SAC-activated B-cells in the absence of anti-Tac antibody. 1 × 105 T-cell-depleted B-cells were cultured for 5 days at 37 °C in the presence of 0.001070 SAC and different amounts of rlL2 added on day 0, in the absence (open boxes) or presence (hatched boxes) of anti-Tac ascites fluid (1 : 1000). PFC were determined on day 5 using a reverse hemolytic plaque assay.

_~I0

~ J

e s x

a U B B+T b

day 0 day 1 day 2 day 3

i

A B A B A B ~ o~ ~ooo~ -oo o~ "-OOO

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Fig. 2 (a) Controls: as described in the legend to Fig. 1 (a), - no additions (A) or 0.00107o SAC + PWM (1 : 500) added (B). (b) Effect of rlL2 added at different times on the PFC response observed in SAC-activated B-cell cultures. 1 x 105 T-cell-depleted B-cells were cultured for 5 days at 37 °C in the presence of 0.00107o SAC and 1, 10 or 100 U/ml rlL2 added on day 0, 1, 2, or 3. PFC were determined on day 5.

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activated B-cells (Fig. lc). We believe this inhibi- t ion is due to the b ind ing of the anti-Tac molecules to the IL2-receptor present on the ac- tivated B-cells (and not by neutra l iza t ion of the protein A on the SAC particles) since irrelevant mouse ascites fluid used as a control did not in- hibit the r lL2- induced response.

Incuba t ion of unf rac t iona ted PBLs or B-cells with r lL2 in the absence of SAC did not lead to- wards ISC (not shown). Neither did the addi t ion of T-cells to the B-cell preparat ions in the pres- ence of SAC lead to a dramat ic increase of PFC compared to the response obta ined in the pres- ence of P W M + SAC (Table 1) [1]. The effect of r lL2 on induc t ion of ISC is therefore more likely to be directly on the SAC-activated B-cells rather than by acting on con tamina t ing T-ceils by in- ducing the p roduc t ion of other factors. It has been shown [7-11] that the IL2-receptor is ex- pressed on SAC-activated h u m a n B-cells and that these cells proliferate in the presence of r lL2 [15]. Our data show that r iL2 at a very low con- cent ra t ion can also promote the different iat ion of these SAC-activated h u m a n B-cells when add- ed at the start of the culture. Maraguchi et al. [7] recently showed that r lL2 induced the product ion of IgG and IgM in h u m a n tonsi l lar B-cells preactivated with SAC for 2 days. Differ- ent culture condi t ions and a different cell source might be responsible for this late-acting activity of rlL2. We also compared the abili ty of r lL2 to promote Ig-synthesis in SAC-activated B-cells

75

"~ 5[ ×

E 2s

3[- I-1 18s I e2F IL A ) A26°

5 10 15 20 fraction number

t, o 3_g 2 1

Fig. 3. Induction of PFC in SAC-activated human B-cell cul- tures by supernatants of iV. laevis oocytes injected with sucrose-gradient-fractionated poly(A*) RNA derived from a stimulated human spleen cell culture. Poly(A') RNA was iso- lated from human splenocytes stimulated for 48 h with PHA/TPA; after sucrose-gradient centrifugation, appropriate fractions were injected into X. laevis oocytes [12, 16]. After 72 h incubation at 23" °C, the bathing medium was removed and assayed for IL2 and IFN-'r activity. Induction of ISC in- sert was assayed using 20/~1 oocyte bathing medium added on day 0 to 1 × 105 human T-cell-depleted B-cells in the pres- ence of 0.001°70 SAC. PFC were determined on day 5.

with IL2 derived from st imulated h u m a n spleen superna tan t and purif ied according to Welte et al. [18]. Such preparat ions were found to be very active in our PFC assay (data not shown). Con- sidering the low amoun t s of IL2 sufficient to promote different iat ion of SAC-activated B-cells, the PFC- induc ing activity present in BIF prepa- rations purif ied according to Ralph et al. [3],

Table 1 The effect of T-cells on the PFC number in SAC and PWM + SAC-activated human B-cell cultures

B-cells B- + T-cells

SAC SAC + PWM (PFC/2 x 10 4 cultured B-cells)

SAC SAC + P W M

Exp. 1 45 15 6 309 30 15 9 585

Exp. 2 37 109 118 712 39 121 81 720

1 × 10 ~ purified human B-cells either alone or in the presence of 1 x 104 autologous T-cells from 2"donors (exp. 1 and exp. 2) were cultured for 5 days in the presence of SAC or PWM + SAC. PFC numbers were measured twice per one fifth of the culture.

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w h i c h c o n t a i n IL2 , can p r e s u m a b l y be a sc r ibed

to IL2 itself . Moreove r , the P F C - i n d u c i n g ac t iv i -

ty p resen t in s u p e r n a t a n t s o f Xenopus laevis oo- cytes in j ec ted wi th h u m a n s p l e e n - d e r i v e d

p o l y ( A +) R N A f r a c t i o n a t e d on sucrose g rad i en t s

c o i n c i d e d wi th the IL2 m R N A ac t iv i ty (Fig. 3) ( l l S ) a n d was well s e p a r a t e d f r o m IFN-3, m R N A

(15S). Never the less , we s h o u l d e m p h a s i z e tha t o u r resul ts do n o t exc lude the exis tence o f o t h e r

T-ce l l -der ived B C D F act ivi t ies . F u r t h e r m o r e , it is

poss ib le tha t B-cells ac t iva t ed by S A C do n o t

represen t a f u n c t i o n a l e q u i v a l e n t o f B-cel ls un-

d e r g o i n g an an t igen i c s t i m u l a t i o n a n d the re fo re

m a y n o t be a val id sys tem on wh ich to base an

assay for such factors .

Acknowledgements

We w o u l d like to t h a n k A n n e m i e Devreese a n d

W i l m a B u r m s for t e chn ica l ass is tance. We are

a l so g ra te fu l to C h r i s t i n e Vermei re a n d Bas van

O o s t e r h o u t for the i r ed i t o r i a l help. Pe ter Van-

d e n a b e e l e t h a n k s the I W O N L for a fe l lowship .

Th i s w o r k was s u p p o r t e d by Biogen , N.V.

References

[1] Falkoff, R. J. M., Zhu, L. P. and Fauci, A. S. (1982) J. Immunol. 129, 97-102.

[2] Saiki, O. and Ralph, P. (1982) J. Immunol. 127, 1044-1047.

[3] Ralph, P., Welte, K., Levi, E., Nakoinz, I., Litcofsky, P. B., Mertelsmann, R. H. and Moore, M. A. S. (1984) J. Immunol. 132, 1858-1862.

[4] Butler, J. L., Falkoff, R. J. M. and Fauci, A. S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2475-2478.

[5] Hirano, T., Teranishi, 1". and Onoue, K. (1984) J. lm- munol. 132, 229-234.

[6] Muraguchi, A., Kehrl, J. H., Butler, J. L. and Fauci, A. S. (1984) J. Clin. Immunol. 4, 337-347.

[7] Muraguchi, A., Kehrl, J. H., Longo, D. L., Volkman, D. J., Smith, K. A. and Fauci, A. S. 0985) J. Exp. Med. 161, 181-197.

[8] Tsudo, M., Uchiyama, T. and Uchino, H. (1985) J. Exp. Med. 160, 612-617.

[9] Jung, L. K. L., Hara, T. and Shu Man Fu (1984) J. Exp. Med. 160, 1597-1602.

[10] Waldmann, T. A., Goldman, C. K., Robb, R. J., Depper, J. M., Leonard, W. J., Sharrow, S. O., Bongiovanni, K. E, Korsmeyer, S. J. and Greene, W. C. (1984) J. Exp. Med. 160, 1450-1466.

[11] Mingari, M. C., Gerosa, E, Carra, G., Accolla, R. S., Moretta, A., Zubler, H. R., Waldmann, T. A. and Moretta, L. 0984) Nature (London) 312, 641-643.

[12] Devos, R., Plaetinck, G., Cheroutre, H., Simons, G., De- grave, W., Tavernier, J., Remaut, E. and Fiers, W. (1983) Nucleic Acids Res. II, 4307-4323.

[13] Madsen, M., Johnson, H. E., Hansen, P. W. and Chris- tiansen, S. E. (1980) J. Immunol. Methods 33, 323-336.

[14] Tauris, P. (1983) Scand. J. Immunol. 18, 241-248. [15] Mingari, M. C., Gerosa, E, Moretta, A., Zubler, R. H.

and Moretta, L. 0985) Eur. J. lmmunol. 15, 193-196. [16] Devos, R., Cheroutre, H., Taya, Y. and Fiers, W. (1982)

J. Interf. R. 2, 409-420. [17] Leonard, W. J., Depper, J. M., Uchiyama, T., Smith, K.

A., Waldmann, T. A. and Greene, W. C. (1982) Nature (London) 300, 267-269.

[18] Welte, K., Wang, C. Y., Mertelsmann, R., Venuta, S. and Moore, M. A. S. 0982) J. Exp. Med. 156, 454-464.

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