Eur. J. Immunol. 1986.16: 1627-1630 CD23, a candidate B cell growth factor receptor 1627

Short paper

John Gordon', Adrian J. Webbm, Leonie Walker', Graeme R. Guy' and Martin RoweA

Departments of Immunology', Biochemistry' and Cancer StudiesA, University of Birmingham, Birmingham

Evidence for an association between CD23 and the receptor for a low molecular weight B cell growth factor*

Low molecular weight B cell growth factor (BCGF) and a monoclonal antibody (MHM6) to the 45-kDa, B lineage-restricted, CD23 activation antigen (BLAST-2; EBVCS) were found to be indistinguishable in their biological effects. Individually, both augmented DNA synthesis in activated, but not resting, B lymphocytes while no additional enhancement resulted from using the two agonists in combination. Fur- thermore, by increasing the expression of Tac, both MHM6 and BCGF promoted activated B cells to respond more vigorously to the late addition of recombinant interleukin 2. The presence of BCGF during B cell activations was found to down- regulate the expression of the CD23 antigen while the coating of activated cells with MHM6 antibody diminished their capacity to absorb BCGF activity. The findings demonstrate that CD23 and a low molecular weight BCGF deliver a comparable growth-promoting signal to activated B cells. A possible relationship between CD23 and the receptor for the low molecular weight BCGF is discussed.

1 Introduction

B lymphocytes require co-operation with other cell lineages in order to initiate, augment or maintain their activation to clonal expansion and expression of their effector function. Comple- ment components, interleukin 1 (IL 1) and T cell-derived fac- tors represent soluble mediators which contribute to these pro- cesses [ l , 21. For both mouse and man, a cloned T cell product has been described which exhibits pleiotropic effects on B cells at different stages of activation (3, 41. Termed B cell stimula- tory factor-1 (BSF-l), this molecule also acts to promote the growth of mast cells and some T lymphocytes [5]. There is some evidence in man for the existence of a second low molecular weight B cell growth factor (BCGF) which shares with BSF-1 the ability to synergize with minimal signals in activating resting B cells to DNA synthesis but also acts to maintain the long-term growth of BCGF-dependent B cell lines [6].

Clearly, for all such factors, the expression of their receptors will be pivotal in regulating the biology of their action. Fur- thermore, where the factor is ill-defined, identification of its receptor and the corresponding signal conveyed should prove valuable to further characterization. We have recently reported that a monoclonal antibody (MHM6) to the B cell- restricted, 45-kDa CD23 activation antigen [7-101 triggers DNA synthesis in activated B lymphocytes [l l] . Here, we

[I 57961

* This work was supported by the Medical Research Council (U.K.). Permanent address: Bristol Polytechnic, Department of Science, Bristol, BS16 lQY, GB.

Correspondence: John Gordon, Department of Immunology, The Medical School, Vincent Drive, Birmingham, B15 2TJ, GB

Abbreviations: BCGF: B cell growth factor 1L: Interleukin rIL2: Recombinant IL2 SAC: Staphylococcus aureus Cowan strain I dThd Thymidine EBV: Epstein-Ban virus TPA 12-O-Tetra- decanoyl-phorbol 13-acetate FITC: Fluorescein isothiocyanate

demonstrate that the actions of anti-CD23 mimic, both qual- itatively and quantitatively, that of a low molecular weight BCGF. Evidence is presented to suggest an association between CD23 and the receptor for this factor.

2 Materials and methods

2.1 Reagents

MHM6 recognizes the CD23, p45 antigen also referred to as BLAST-2; EBVCS [7-lo]. An IgG fraction from the ascites fluid was prepared by protein A affinity chromatography as previously described [l l] . BCGF was purchased from Cellular Products Inc. (Buffalo, NY) and represented the 12-kDa prod- uct of peripheral blood T lymphocytes stimulated with phy- tohemagglutinin (PHA) [6]. This preparation was deemed free of interleukin 1 (IL l ) , IL2 and interferon activities. Affinity- purified, monocyte-derived IL 1 and recombinant IL2 (rIL2) were purchased from Genzyme (Boston, MA) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) from Sigma (Poole, Dorset, GB). Fluorescein isothiocyanate (F1TC)-conjugated antibody to the IL 2 receptor (Tac antigen) was purchased from Becton Dickinson (Mountain View, CA). StuphyZococcus uureus Cowan strain I (SAC) was from Cal- biochem-Behring (Cambridge, GB).

2.2 B cell activations

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 [12]. Low buoyant density cells were collected above 55% Percoll gradients. All cultures were performed in flat-bottom microwells (growth area = 0.32 cm') laced at 37°C in a humidified C02-rich atmosphere with 10 cells per 200 pl of growth medium comprising 10% fetal calf serum, 1 unit/ml IL 1 , 2 mM L-glutamhe, 5 X lo-' M 2-mercaptoethanol and antibiotics in RPMI 1640. TPA or SAC was included as

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0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1986 0014-2980/86/1212-1627$02.50/0

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indicated in the text. DNA synthesis was determined over times indicated by pulsing wells with 50 p1 of [3H]thymidine ([3H]dThd) at 10 pCi/ml= 370 kBq/ml and determining the amount of radioactivity incorporated. Expression of surface antigens was determined by taking cells from cultures, wash- ing, staining either directly or indirectly with FITC-conjugated antibodies and analyzing on a fluorescence-activated cell sor- ter (FACS IV, Becton Dickinson). In indirect tests, a FITC- conjugated sheep anti-mouse immunoglobulin was used as the second layer.

J. Gordon, A. J. Webb, L. Walker et al. Eur. J. Immunol. 1986.16: 1627-1630

3 Results

3.1 Monoclonal antibody to CD23 mimics low molecular weight BCGF

The results depicted in Fig. 1 illustrate that, with respect to its ability to promote DNA synthesis in B cells activated with TPA, the MHM6 antibody was indistinguishable from low molecular weight BCGF. Moreover, no additional stimulation followed as a result of the two agents being present in combi- nation. In contrast, the augmentation of DNA synthesis result- ing from the inclusion of rIL2 at initiation of culture was strictly additive with both the BCGF- and the MHM6-pro- moted effects. These findings demonstrate that MHM6 and BCGF are delivering similar growth-promoting information to the same population of cells whereas rIL2 is transmitting a quite distinct signal, possibly to a separate subset.

3.2 MHM6 and BCGF promote a late response to IL 2 by increasing Tac expression

We investigated the consequence of delaying for 48 h the addi- tion of rIL2 into TPA-stimulated cultures which had been driven with either MHM6 or BCGF. The results illustrated in Fig. 2 show that in the absence of rIL2 both the MHM6 and BCGF effects began to wane after 74 h, returning to back-

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01 I 1 I 1 :

250 5 0 10 2 0.4 008 0 20 10 5 2.5 13 0 6 0

m MHM6 LgG(w/m l ) BCGF (%)

Figure I. Augmentation of B cell stimulation by MHM6 and BCGF. Purified resting B cells were cultured at lo5 per 200 p1 of medium in the presence of TPA (2 ng/ml), MHM6 and/or BCGF as indicated. [3H]dThd incorporation was assessed between 48 and 64 h with results given as means of triplicate determinations which never varied by more than 10% of each other. (a) (B) MHM6 alone; (0) MHM6 + BCGF (20%); (0) MHM6 + rIL2 (100 unitdml). (b) (0) BCGF alone; (B) BCGF + MHM6 (250 pg/ml); (0) BCGF + rIL2 (100 unitdml). In the absence of TPA, the response of resting B cells to any one or combination of agents never exceeded 2000 cpm.

30 48 66 74 90 116 144 30 48 66 74 90 116 144

lma hours post-activation

Figure 2 . MHM6 and BCGF priming of B cells for an IL2 response. Cultures were initiated with TPA (2 ng/ml) as for Fig. 1 in the absence (0, 0) or presence of (a) MHM6 IgG (50 pghl). (0, B) or (b) BCGF (10%) (0, B). rIL2 (100 unitdml) was either added (arrow) at 48 h (B, 0) or omitted from culture (0, 0). The hourly rate of DNA synthesis over the times indicated was assessed by determining the incorporation of [3H]dThd during these intervals.

linear fluor. intensity UXm

Figure3. Induction of IL2 receptors by MHM6 and BCGF. Cells were cultured as for Fig. 1 and stained for IL2 receptors (Tac) after 64 h: (---) control cells; (-) TPA-activated cells; (m) TPA- activated cells + MHM6 IgG (50 pg/ml); (-) TPA-activated cells+BCGF (10%). Data generated from 5 X lo4 cells on a FACS IV.

ground levels by 116 h. Where cultures had been sup- plemented with rIL 2, however, both sets of stimulations were extended considerably. Furthermore, the late response of acti- vated cells to rIL2 was dependent on the earlier presence of MHM6 or BCGF. We asked whether these effects were being controlled at the level of receptor expression. As seen from Fig. 3, the expression of Tac-antigen was significantly enhanced when either MHM6 or BCGF had been included in the TPA stimulations. Not only was the mean intensity of expression higher but the number of cells positive for Tac also increased from 17% to 38% and 36% in the presence of BCGF and MHM6, respectively. These findings are compatible with the notion that BCGF and MHM6 recruit more cells to become responsive to IL2 by increasing the level of functional IL2 receptors on these cells.

3.3 BCGF down-regulates CD23 expression

Next, we explored the influence of BCGF on the expression of the CD23 antigen. Simple blocking of MHM6 binding to a CD23' cell line by cold competition with the low molecular weight BCGF was not seen (Fig. 4). Other sources of BCGF,

Eur. J. Immunol. 1986.16: 1627-1630 CD23, a candidate B cell growth factor receptor 1629

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801

'19(*1 linear fluor. intensity

Figure 4 . Lack of blocking of MHM6 by BCGF. Cells from a Burkitt lymphoma line carrying the EBV (a kind gift from A. Rickinson, Birmingham) were incubated with 1 ml of 40% BCGF per 5 X lo6 cells at 0°C for 4 h. The cells were washed thoroughly in ice-cold medium and stained with MHM6 ascites (1 : 250) in an indirect technique using FITC-labeled sheep antibody to mouse immunoglobulin as the second layer: (Go(>) BCGF-included, MHM6 + FITC antibody; (-) BCGF omitted, MHM6 + FITC antibody; (-) FITC antibody only.

Linear f luor intensity

Figure 5. Down-regulation of CD23 by BCGF. Cells were cultured as for Fig. 1 and stained for CD23 after 48 h using MHM6 in an indirect technique: (---) control cells; (-) TPA-activated cells; (0-0-0) TPA-activated cells + BCGF (10%). Nonspecific control staining is indicated (-).

including supernatants from the MP6 T cell hybridoma (a gift from A. Rosen) and from Namalwa cells stimulated with PHA (a source of high molecular weight BCGF), similarly failed to interfere with MHM6 binding to its target antigen (data not shown). However, it is clear from Fig. 5 that the presence of low molecular weight BCGF during stimulations of cells at 37°C resulted in a substantial down-regulation of CD23 expression so that by 48 h > 75% of the MHMG-binding sites were no longer available to cells which had seen BCGF in comparison to cultures where the factor had been omitted.

3.4 MHMC inhibits BCGF absorption

Fig. 6a reveals that low buoyant density tonsillar B cells were highly efficient at absorbing out BCGF activity as was a Bur- kitt lymphoma line carrying the Epstein-Barr virus (EBV) genome. Both cell populations were strongly positive for CD23 expression (Fig. 4 and not shown). By contrast, high density B cells exhibited very poor absorbing capacity and were, likewise, low expressors of CD23 (see Fig. 5). The results illustrated in Fig. 6b reveal that the prior coating of low

0 1 , I I 1 I I

20 10 5 2.5 20 10 5 2.5 1.25

mm BCGF Figure 6. Inhibition of BCGF absorption by MHM6. BCGF activity was assessed in this experiment by its ability to augment the stimula- tion of resting B cells cultured as for Fig. 1 except that fixed SAC (1 : lo6) was used in place of TPA. To absorb BCGF activity, cells were cultured at 2 X 107/ml of 20% BCGF for 2 h at 0°C with constant shaking. The cells were then spun down, the supernatant removed and passed through a 0.22-pm filter before testing. For antibody coating prior to absorption, cells were suspended in MHM6 ascites (1 : 250) for 2 h, washed extensively in ice-cold medium and resuspended in fresh medium. (a) (A) Unabsorbed BCGF; (0) absorbed with high-density (>62.5% Percoll) B cells; (A) absorbed with low-density (<55% Percoll) B cells; (M) absorbed with Burkitt lymphoma cells (see Fig. 4). (b) (A) Unabsorbed BCGF; (A) absorbed with low-density B cells; (0) absorbed with low-density B cells coated with MHM6 at 0°C; (0) absorbed with low-density B cells coated with MHM6 at 37°C.

buoyant density B cells with MHM6 significantly reduced their capacity to absorb the BCGF activity. The outcome was the same whether the incubations with MHM6 had been per- formed at 0°C or at 37°C. We have subsequently shown that MHM6 alone is unable to modulate its antigen at 37°C over short-time intervals (unpublished observations). The inhibi- tion was specific in as much as BK 19.9, which is also an IgGl antibody and binds to a similar number of determinants as MHM6 on activated B cells [13], failed to have any influence on the cells' absorbing capacity for BCGF. Furthermore, cells coated with MHM6 antibody absorbed as much IL2 activity as uncoated cells (data not shown).

4 Discussion

The findings above reveal that the growth-regulating signal transmitted to activated B cells through CD23 is indistinguish- able from that delivered by a low molecular weight BCGF. Furthermore, this same BCGF was found to down-regulate the expression of CD23 while coating of the antigen with MHM6 antibody interfered with the ability of cells to absorb the BCGF activity. While the data fall short of establishing CD23 as the receptor for this BCGF they do indicate that possibility. However, the observation that BCGF failed to block MHM6 binding while MHM6 only partially blocked BCGF absorption demonstrates that different epitopes at least may be recognized by the two agents. An alternative interpre- tation would be that, while not actually serving as the recep-

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tor, CD23 and the action of BCGF are functionally linked. For example, CD23 and the BCGF receptor may represent alter- native surface structures for delivering the same type of growth signal. Another possibility is that ligation of CD23 might elicit the production of autocrine BCGF [12] which would have the appearance of mimicking the BCGF added.

The relationship between the low molecular weight BCGF used in this study and BSF-1 has yet to be determined. There is some evidence that the receptor for BSF-1 might be associated with the lymphocyte function antigen, LFA-1 [14]. Interest- ingly, however, the 45-kDa Lyb-2 antigen has also been impli- cated as its ligation delivers to murine B cells a growth-pro- moting signal which is augmented by IL2 but not by a low molecular weight BCGF [15, 161. It is therefore tempting to suggest that Lyb-2 in mouse and CD23 in man represent com- parable structures. As CD23 is not significantly expressed on resting cells [13] and the major targets for murine BSF-1 are small B lymphocytes [17] there may exist a low molecular weight activity distinct from BSF-1 serving as a B cell pro- gression factor and acting through a 45-kDa receptor in both species. This notion is strengthened by our observation that dense B lymphocytes failed to absorb the BCGF used in this study.

We have recently shown that the MHM6 monoclonal antibody exerts its influence on B lymphocytes in the early GI phase of the cycle and that IL 1 then assists these cells through S phase where they become arrested in G2 + M [ll] . The present study has demonstrated that rIL2 is capable of prolonging the cycle of cells which have been triggered through CD23 so that, even after several days, cells are still entering S phase. The phy- siological basis for this late response to rIL2 appeared to relate to the ability of both BCGF and MHM6 to increase the number of (presumably functional) IL 2 receptors on activated B cells. Taken together with our previous observation, these findings indicate that IL2 may control a restriction point in the G2 phase of the human B cell cycle, a notion compatible with the studies of Melchers and Lernhardt on cycling murine B lymphocytes [2], We are currently investigating the precise interrelationship of IL1, IL2 and BCGF, together with their receptors, in sustaining the growth of human B cells over sev- eral replication cycles. Furthermore, the mechanism through which CD23 conveys its progression signal to activated B cells is also being explored.

In conclusion, we have shown that CD23, one of the first lineage-restricted antigens to appear on the activation of human B cells, conveys growth information which is identical to that elicited by a T cell derived, low molecular weight BCGF. The major sequel to receptor ligation is the pro- gression of activated cells through the cycle resulting in an enhanced expression of IL 2 receptors via which further

J. Gordon, A. J. Webb, L. Walker et al. Eur. J. Immunol. 1986.16: 1627-1630

growth signals can be deIivered. Whatever its precise function, CD23 seems pivotal in controlling B cell replication. It follows that the deregulation of CD23 expression may be one means by which B lymphocytes gain oncogenic potential. It is prob- ably significant that CD23 was first identified through its high constitutive expression on cells transformed by the EBV [9, lo]. Given that of B cells infected by EBV only those expressing CD23 proceed to transform [18], it will be of con- siderable interest to determine whether there is a relationship between the autocrine factors released by immortalized B lym- phocytes and the CD23 antigen [12, 191.

Received September 18, 1986.

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