Eur. J. Immunol. 1987.17: 1535-1538 B lymphocyte restimulations by factors and antibodies 1535

Short paper

John GordonA, Michelle J. MillsumA, Graeme R. Guy' and Jeffrey A. Ledbetter'

Department of ImmunologyA and Biochemistry', University of Birmingham, Birmingham and Oncogen Corporation', Seattle

1 Introduction

Synergistic interaction between interleukin 4 and anti-Bp50 (CDw40) revealed in a novel B cell restimulation assay*

Highly purified resting B lymphocytes stimulated for 3 days to high-rate DNA synthe- sis by a synergistic combination of phorbol dibutyrate and ionomycin soon returned to quiescence once those signals had been removed. The maintenance of DNA synthesis in such cultures was found to provide a sensitive assay for revealing the factors that interact with cycling B cells. Whereas several activities - namely, interleukin 4, anti- Bp50 and a low molecular weight B cell growth factor - were, by themselves, capable of prolonging DNA synthesis over a further day or so, no single factor was capable of sustaining the replication cycle out to day 6 of culture. By contrast, certain combina- tions of activities displayed significant synergy in the restimulation assay. The most striking observed was that between interleukin 4 and anti-Bp50 where, by day 6, their combined effect on maintaining DNA synthesis in 3-day stimulated cells was the same as having kept phorbol dibutyrate and ionomycin in the culture system. The implica- tions of these findings are discussed.

It is becoming clear that control of B lymphocyte growth is directed by a detailed interplay of several factors impinging on a number of receptors at appropriate times. Several activities have been described which exert their effects on already cy- cling B cells. These include interleukin 2 (IL2) and a high molecular weight B cell growth factor (hmw BCGF) for human B cells [ l , 21, while for murine B cells, IL5 and the C3d component of complement have been implicated [3, 41. At earlier stages in the human B cell growth programme, IL4 and a distinct low molecular weight BCGF (lmw BCGF) have been claimed to provide activation and progression signals, respectively [5, 61. Furthermore, by exploiting monoclonal antibodies as probes for potential receptor function, 3 distinct molecules have now been identified on the human B cell sur- face which, when ligated, amplify primary signals such as those delivered through surface immunoglobulin, CD20 or phorbol esters [7-91. Of these 3 molecules, the most potent in co- stimulation assays appears to be the 50-kDa surface protein (Bp50) described by the CDw40 panel of antibodies [9]. Although abundantly expressed on resting B cells and increas- ing on activation, there is, as yet, no information as to the natural ligand for this surface molecule. In this report we describe a novel and sensitive restimulation assay for human B cells which has allowed us to identify both Bp50 and IL4 as important molecules, not only in initiating B cell growth but also in sustaining it, particularly when their actions are paired together.

[I 62931

* This work was supported with grants from the Medical Research Council (U.K.) and the Leukemia Research Fund.

Correspondence: John Gordon, Department of Immunology, Univer- sity of Birmingham Medical School, Birmingham B15 2TJ, GB

Abbreviations: BCGF: B cell growth factor IL: Interleukin dThd: Thymidine PDB: Phorbol dibutyrate PMA: Phorbol 12-myristate 13-acetate

2 Materials and methods

2.1 Reagents

Phorbol 12-myristate 13-acetate (PMA) and phorbol dibuty- rate (PDB) were both purchased from Sigma (Poole, Dorset, GB). Ionomycin was obtained from Calbiochem (La Jolla, CA). Recombinant IL4 was a generous gift from S. Gillis and C. Henney (Immunex Corp., Seattle, WA). Recombinant IL2 was purchased from Genzyme (Norwalk, CT) and lmw BCGF was obtained as a partially purified preparation from Cellular Products (Buffalo, NY). The anti-Bp50 was a purified IgG, preparation from the G28-5 hybridoma used throughout at a concentration of 1 pg/ml which was previously found to be optimal in a PMA costimulation assay [lo].

2.2 Restimulation assay

Highly enriched, resting B cells were isolated from tonsils by negative selections and buoyant density gradients of > 62.5% Percoll (Pharmacia, Uppsala, Sweden) as described in detail elsewhere [7]. B cells were cultured for 3 days at 37°C in a humidified C02-rich atmosphere with lo6 cells/ml of growth medium comprisin! 10% fetal calf serum (FCS), 1 mM L- glutamine, 5 X 10- M 2-mercaptoethanol and antibiotics in RPMI 1640 supplemented with PDB (1 ng/ml) and ionomycin (0.8 pg/ml). On day 3, cells were taken (= lo8) and washed 4 times in 50 ml vol. of RPMI 1640 containing 1% FCS. The inclusion of FCS in the washings was found to be essential as, without it, activated cells adhered to the walls of the washing vessels. Washed cells were then replated at 105/200 p1 of growth medium in flat-bottom microwells (growth area = 0.32 cm2) with the additions and for the times indicated in Sect. 3. DNA synthesis was determined by pulsing wells with 50 p1 of [3H]thymidine ([3H]dThd) at 10 pCil ml = 370 kBq/ml and determining the amount of radioactivity incorporated.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0014-2980/87/1010-1535$02.50/0

1536

3 Results

J. Gordon, M. J. Millsum, G. R. Guy and J. A. Ledbetter Eur. J. Immunol. 1987.17: 1535-1538

3.1 Restimulation assay

We have previously shown that PMA and calcium ionophores act synergistically to promote growth in resting B cells [ll]. Essentially identical kinetics were observed for initiating DNA synthesis in high-density tonsillar B cells when PDB was sub- stituted for PMA in a synergistic combination with ionomycin (Fig. 1). The initial peak of DNA synthesis was found to occur on day 3 which, as shown previously [12], corresponds to the first cohort of cells entering S-phase. The ability of PDB to replace PMA in this response provided the potential for a novel restimulation assay as the binding of the former, but not the latter, phorbol ester is highly reversible [13]. This potential was assessed by taking PDB plus ionomycin-treated cells at day 3 of culture, washing them extensively and replating them

l I 4

1

1 2 3 4 5

Day of culture

Figure 1. Kinetics of PDB plus ionomycin stimulations. High-density tonsillar B cells were plated at 105/200 p1 of culture medium for the times indicated and pulsed for the final 8 h with [3H]dThd. DNA synthesis in control cultures never exceeded 537 cpm at any time. Cultures were supplemented with ionomycin: 0.8 pg/ml (A); PDB: 1 ng/ml (A); PDB plus ionomycin (0); PMA (0.2 ng/ml) plus ionomy- cin (W).

Table 1. Kinetics of DNA synthesis in washed and replated cells

Additions ['HIdThd incorporation (cpm)") Exp. 1 Exp. 2 Exp. 3

Day4 Day 5 Day 6 Day6 Day6

CM 23842 5659 247 459 376 PDB 38486 8417 376 - - Ionomycin 27213 6364 156 - - PDB plus ionomycin 72 647 103 501 76 578 65 768 71 937

a) Cells cultured for 3 days with PDB (1 nglml) plus ionomycin (0.8 pg/ml) were washed and replated as described in Sect. 2.2 with either control medium (CM), PDB (1 ng/ml), ionomycin (0.8 pglml) or a combination of the two. DNA synthesis was as- sessed at days 4,5 and 6 of culture as indicated by an 8-h [3H]dThd pulse. Results given are means of triplicate determinations.

in either control medium, PDB or ionomycin alone or the two agents back together in synergistic combination. The outcome of this was that over the next three days, cells cultured without stimulants, or with either agent alone, soon returned to quies- cence whereas cells recultured with PDB and ionomycin together maintained a high level DNA synthesis over the whole period (Table 1). Day 6 results are given for three experiments to indicate the reproducibility of the assay.

3.2 Influence of growth-promoting activities

The ability of IL4, lmw BCGF and anti-Bp50 to modify the kinetics of DNA synthesis in 3-day stimulated cells was fol- lowed over a further 3 days of culture. As seen in Fig. 2, each alone at optimal concentration was capable of delaying the swift return to quiescence but could not sustain the stimulation beyond a total culture period of 5 days. When IL4 was titrated into the assay in combination with either of the other two activities, a different picture emerged so that by day 6, signifi- cant DNA synthesis was now occurring with combinations of IL4 plus lmw BCGF and IL4 plus anti-Bp50. Indeed, the latter combination yielded a level of DNA synthesis approach- ing that achieved under optimal conditions, i.e. when cells were recultured with PDB plus ionomycin (Fig. 3).

The results given in Table 2 reveal the effects seen with other combinations of activities in the restimulation assay. Apart from IL4 together with lmw BCGF or anti-Bp50, the only supra-additive combination observed was that between anti- Bp50 and lmw BCGF. Interestingly, IL2, which displayed a modest effect by itself, was, at best, additive with the other growth-promoting molecules (Fig. 3 and Table 2). All effects were deemed to be directly on B cells as, even by day 6 of culture, no CD3 or sheep erythrocyte rosette-positive cell among 500 scored was detected.

I14 X 10' I.

/ \

8 12

10

8 o U 3

6 % s L

O<\J

0

4 5 6 Day of culture

rn

Figure 2. Influence of individual factors in restimulation assay. Cells stimulated for 3 days with PDB plus ionomycin were washed and replated as in Fig. 1 and followed over the times indicated with a 16-h [3H]dThd pulse. Washed cells were supplemented with PDB plus ionomycin (0); IL4, 1000 unitdm1 (0); G28-5 (anti-BpSO), 1 pg/ml (A); Imw BCGF, 10% (v/v) (V); control medium (0).

Eur. J. Immunol. 1987.17: 1535-1538 B lymphocyte restimulations by factors and antibodies 1537

(murine) is based on neoplastic cells presumably altered in their growth requirements [ 141; lipopolysaccharide-based acti- vations (murine) automatically select those 30% of splenic B lymphocytes which respond [4]; assays based on measuring DNA synthesis in low-buoyant density tonsillar B cells, con- sidered “pre-activated” [2], are irreproducible as the constitu- tion of such populations is extremely varied (unpublished observations); assays based on Ig-mediated triggering contain the inherent limitations previously discussed. Although the PDB plus ionomycin regime might be considered “artificial”, our earlier observation that physiological signalling of B cells utilizes the second message system on which these two agonists impinge largely counteracts that particular argument [ 161.

Table 2. Effect of combinations on the restimulation assay

[’HIdThd incorporation (cpm)”’ Control IL4 IL2 BCGF Anti-BpSO

Control 417 6156 3717 4305 2937

- 7675 5998 - - 17951

IL 2 BCGF

IL 4 - 8657 23835 91 142 - - - - -

a) Cells were cultured for 3 days with PDB plus ionomycin, washed and reset in the activities shown for a further 3 days before receiv- ing a 16-h [3H]dThd pulse as described in Fig. 2. Results given as cpm and represent the means of triplicate determinations. IL4, 1000 uiml; IL2, 200 u/ml; BCGF 10% (v/v); anti-Bp50, (G28-5), 1 pg/ml.

7 x lo-‘

6

2

c 1 - c 1 - - O ~ c J , ~ 1

0 O-0 0-0-0- ’A‘A-A\A,Jl

A

4000 2000 1000 500 250 0

IL4; units/rnl 116295.31

Figure 3. As for Fig. 2 but washed cells cultured for 6 days in total with a [3H]dThd pulse for the final 16 h. Supplements on replating were: G28-5 (anti-BpSO), 1 pg/ml (0); Imw BCGF, 10% (v/v) (A); IL2, 200 unitshl (0); control medium (0).

4 Discussion

Restimulation assays have been designed to determine the constitution of what have generally been termed “BCGF 11”- activities [14]. For the human system, stimulation of resting B cells for 3 days with Staphylococcus aurew Cowan strain I (SAC) has commonly provided the targets for assessing “BCGFII” by a measure of DNA synthesis a further 3 days later [l]. We have found such SAC-based assays insensitive and irreproducible (unpublished observations), possibly reflecting the negative growth signalling which can be trans- mitted to cycling B cells via surface Ig [15] which could be further exacerbated by a difficulty in removing the tenacious SAC particles from the activated B cell surface. In this report we have exploited our original observation that PMA and cal- cium ionophore, in combination, provide the most potent mitogenic signal available for human B cells, driving virtually every resting tonsillar B cell into cycle [12], by substituting PDB for PMA and rendering the stimulations reversible. We believe that this approach fulfills many of the criteria desired for an assay based on pre-activated cells which have not been met in previous systems. For example, the BCLl assay

While we have seen that a wide spectrum of activities can contribute to the restimulation assay, no single factor has pro- vided any significant effect beyond a day or two. In addition to those factors described, negative single activities include the recombinant factors IL l a , IL lp , IL 5 and interferon-y (unpublished observations). In striking contrast, certain com- binations have sustained the cycle out to an additional 3 days of culture. While the synergy observed between IL 4 and Imw BCGF is of interest, particularly when given the effects claimed for these molecule on CD23 expression [5, 171, the outstanding combination of activities for maintaining DNA synthesis was that of IL4 and anti-Bp50. The latter observa- tion clearly raises many questions. In particular, (a) what is the effect of this combination on actual growth ? (b) where in the cell cycle do these agents act? and (c) what is the natural ligand for Bp.50 ? Hopefully, with the availability of a sensitive reliable assay, coupled with the use of recombinant factors and functional antibodies, it should not be long before these, and many other questions concerning B cell replication, are finally answered.

Received July 15, 1987.

5 References

1 Mingari, M. C., Gerosa, F. , Moretta, A, , Zubler, R. H. and Moretta, L., Eur. J . Immunol. 1985. 15: 193.

2 Ambrus, J. L., Jurgensen, C. H., Brown, E. J. andFauci, A. S . , J . Exp. Med. 1985. 162: 1319.

3 Kinashi, T., Harada, N., Severinson, E., Tanbe, T., Sideras, P., Konishi, M., Azuma, C., Tominaga, A., Bergstedt-Lindqvist, S., Takahashi, M., Matsuda, F., Jaoita, Y., Takatsu, K. and Honjo, T., Nature 1986. 324: 70.

4 Melchers, F. and Lernhardt, W., Proc. Natl. Acad. Sci. USA 1985. 82: 7281.

5 Defrance, T., Aubry, J. P., Rousset, F., Vanbervliet, B., Bon- nefoy, J. y . , Arai, N., Takebe, y . , Yokata, T., Lee, F., Arai, K., De Vries, J. and Banchereau, J. , J . Exp. Med. 1987. 165: 1459.

6 Gordon, J., Webb, A. J., Walker, L., Guy, G. R. and Rowe, M., Eur. J . lmmunol. 1986. 16: 1627.

7 Gordon, J., Rowe, M., Walker, L. and Guy, G. R., Eur. J . lmmu- nol. 1986. 16: 1075.

8 Pezzuto, A., Dorken, B., Moldenhauer, G. and Clarke, E. A. J . Immunol. 1987. 138: 98.

9 Clark, E. A. and Ledbetter, J. A., Proc. Natl. Acad. Sci. USA 1986. 83: 4494.

10 Gordon, J., Webb, A. J., Walker, L., Guy, G. R., Paulie, S., Ehlin-Henriksson, B. and Rosen, A., in McMichael, A. J., Bever- ley, P. C. L., Cobbold, S. , Crumpton, M. J., Gilks, W., Gotch, F. M., Hogg, N., Horton, M., Ling, N., MacLennan, I. C. M., Mason, D. Y., Milstein, C., Spiegelhalter, D. and Waldmann, H.

1538 J. Gordon, M. J. Millsum, G. R. Guy and J. A. Ledbetter

(Eds.), Leukocyte Typing Ill, White cell differentiation antigens, Oxford University Press, Oxford 1987, p. 426.

11 Guy, G. R., Bunce, C. M., Gordon, J., Michell, R. H. and Brown, G., Scand. J . Immunol. 1985.22: 591.

12 Walker, L., Guy, G. R., Brown, G., Rowe, M., Milner, A. E. and Gordon, J., Immunology 1986. 58: 583.

13 Driedger, P. E. and Blumberg, P., Proc. Natl. Acad. Sci. USA 1980. 77: 567.

Correction

Vol. 17, Number 7, July 1987 V. K. Milsauskas, S . G. Kaminsky and I. Nakamura

Page 1045, Table 1

Eur. J. Immunol. 1987.17: 1535-1538

14 Howard, M. and Paul, W. E., Annu. Rev. Immunol. 1983.1: 307. 15 Gordon, J., Melamed, M. D., Ley, S. C. and Hughes-Jones, N.

16 Guy, G. R., Gordon, J., Michell, R. H. and Brown, G., Biophys.

17 Guy, G. R. and Gordon, J., Proc. Natl. Acad. Sci. USA 1987, in

C., Immunology 1984. 52: 89.

Biochem. Res. Commun. 1985. 131: 484.

press.

Class I H-2Db determinants are not involved in hybrid resis- tance to parental H-2b/Hh-lb bone marrow allograft

Lines 3, 5 and 7 should read “in vivo” instead of “in vitro”. Please replace with the enclosed new Table 1.

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