Eur. J. Immunol. 1990. 20: 1161-1165 A novel B cell activation pathway 1161

Messandro PoggiO, EMco Maggin, Roberta BiagiottP, Maria Grazia GiudiziO, Fabio AImerigognao, Nicoletta PeUao, Federico Caligaris-Cappio+ Sergio RomagnaniO and Lorenzo MorettaO.

Istituto Nazionale per la Ricerca sul Cancroo, Istituto di Oncologia Clinics e Sperimentale., University of Genoa and Centro Interuniversitario per la Ricerca sul Cancro (C.I.R.C.), Genoa, Laboratory of Clinical Immunology, Policlinico di Careggin, University of Florence, Florence and Istituto di CIinica Medica+, University of 'hrin, Torino

A novel pathway of human B cell activation initiated by CK226 surface antigen*

In this study we analyzed the effect of CK226 monoclonal antibody (mAb) on human B cell activation and proliferation. This mAb was shown to recognize a 75-kDa surface molecule expressed on bothTand B lymphocytes and to mediate T lymphocyte activation and proliferation. Flow cytometry analysis of B cell populations isolated from peripheral blood, tonsil and spleen showed that CK226 surface antigen is highly expressed on 40-80% of surface Ig+ cells. When purified B cells were cultured in the presence of CK226 mAb, up-regulation of major histocompatibility complex class 11 and CD23 surface structures and the de novo expression of CD25 antigen could be detected within 48 h. In addition, B cells underwent proliferation ([3H] thymidine uptake) in the absence of either Tcells or exogenous lymphokines. Proliferation was potentiated by the addition of suboptimal concentrations (0.5 ng/ml) of phorbol 12-myristate 13-acetate (PMA). Cells recovered at day 5 were surface Ig+ and no CD3+ cells could be detected. CK226-induced proliferation (either in the presence or in the absence of PMA) was not inhibited by anti-CD25 mAb. Addition of exogenous interleukin 2 to CK226-stimulated B cells resulted in further increase of B cell proliferation. On the other hand, CK226 mAb did not display a co-stimulatory effect with submitogenic concentrations of either anti-Ig antibody or Staphylo- coccus aureus Cowan strain I bacteria. In addition proliferation induced by mitogenic concentrations of the above stimuli was inhibited in a dose-dependent fashion by CK226 mAb.

1 Introduction

The mechanisms responsible for activation of resting B cells and their progression through the different steps of B cell maturation have been only partially defined [ 1-31. While mechanisms leading to B cell activation involve essentially signaling via cell membrane receptors (e.g. sIg) prolifera- tion and differentiation are mostly consequent to the action of a number of lymphokines (e.g. IL2, IL4) [l-41. B cell activation is physiologically triggered by specific soluble antigens; however, similar effect can be achieved with antibodies directed to sIg which thus mimic the antigedsIg interaction. Recently, it has been shown that antibodies against certain B cell surface molecules including CD20 and CD40 mediate B cell activation and proliferation in the presence of exogeneous lymphokines such as IL2, IL4 or E N - y [4-161. By the use of the CK226 mAb, we recently defined a 75-kDa surface molecule that is expressed on bothTand B lymphocytes. More importantly, upon binding with this mAb, CK226 molecule was found to mediate both T cell activation and proliferation in a manner similar to other triggering molecules expressed on the T cell surface [17]. In the present report, we show that CK226 mAb induce a similar effect on resting B cells leading to (a) an up-regulation of MHC class 11 and CD23 surface antigen expression and the novel expression of CD25 (IL2R); (b)

TI 82731

* This work was supported in part by grants awarded by the Consiglio Nazionale della Ricerca (CNR) and Associazione Italiana per la Ricerca sul Cancro (AIRC).

Correspondence: Alessandro Poggi, Laboratorio di Immunopato- logia, Istituto Nazionale per la Ricerca sul Cancro,Viale Benedetto XV, n. 10, Genova, Italy

Abbreviation: SAC: Staphylococcus aureus Cowan strain I

an autonomous proliferation occurring in the absence of exogenous lymphokines or T cells; (c) a synergistic effect with exogenous IL2; and (d) an inhibitory effect on the B cell proliferation induced by anti-IgM antibody or Staphy- lococcus aureus Cowan strain I bacteria.

2 Materials and methods

2.1 mAb

The CK226 mAb (IgM) and the CK248 (IgM, anti-CD28) were obtained as previously described [17]. Anti-CD25 (anti-IL2R, IgGI), anti-CD19 (Leu-12, IgGI), anti-CD20 (Leu-16, IgG1) , anti-CD2 (Leu-5b, IgGI), anti-CD7 (Leu- 9, IgG2&, anti-CD3 (Leu-4, IgGI) and goat anti-human sIg (anti-sIg) were purchased from Becton Dickinson (Moun- tain View, CA); anti-HLA DR mAb (Dl-12, IgG2a) was a generous gift of Dr. R. Accolla (University of Verona, Italy); anti-CD23 mAb (anti-B6) was from Coulter Immu- nology (Hialeah, FL); FITC-conjugated goat anti-mouse IgG was purchased from Cappel (Cochranville, PA) and PE-conjugated goat anti-mouse IgM was from Southern Biotechnology (Birmingham, AL).

2.2 Cells

B cells were isolated from tonsils (spleen or peripheral blood) obtained at routine tonsillectomy (from Surgical Staff Ospedale Giannina Gaslini, Genova, Italy) by nega- tive selection of E-rosetting positive cells [18].The purity of B cell preparation (99%) was assessed by direct immuno- fluorescence using either specific anti-B or anti-T lympho- cytes mAb (anti-CD19, anti-CD20, anti-sIg, anti-CD2, anti-CD7 and anti-(333). Resting B cells of high buoyant density were obtained through Percoll (Pharmacia, Upp-

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 0014-2980/90/0505-1161$02.50/0

1162 A. Poggi, E. Maggi, R. Biagiotti et al. Eur. J. Immunol. 1990. 20: 1161-1165

sala, Sweden) density gradients of 60%. As detailed elsewhere [18], such populations were 99% pure with regard to B cells and cells residing in the GO phase of cycle. All cultures were performed at 37 “C in a humidified COz rich atmosphere in 0.2 ml of RPMI 1640 medium (Fakola, Freiburg, FRG) supplemented with 10% FCS, L-glutamine and penicillin-streptomycin (Gibco Laboratories, Grand Island, NY) in 96 U-bottom microplates (Greiner, Nurtin- gen, FRG) at 106/ml.

2.3 Indirect immunofluorescence and FCM analysis

The techniques used have been described in detail else- where [19]. Briefly, aliquots of lo6 cells were stained with specific mAb followed by FITC-goat anti-mouse TgG or IgM. Control aliquots were stained with the fluorescent reagent alone. The samples were analyzed on a FACStar flow cytometer (Becton Dickinson) gated to exclude nonviable cells. Analysis of either PBMC or tonsil and spleen cell suspensions, for the co-expression of CK226 antigen and CD2 or CD3 or CD7 or sIg was performed applying two-color cytofluorometric technique as described [19]. The simultaneous analysis of green and red immuno- fluorescence was obtained from a single laser exciting both FITC and PE at 488 nm.

2.4 Proliferation assay

Highly purified resting B cells of high buoyant density were cultured in triplicate wells as described above either alone or with 0.5ng/ml of PMA (Sigma, St. Louis, MO) and another stimulus, for varying culture intervals. CK226 mAb was used at various concentrations ranging from 5 up to 500 ng/ml; SAC bacteria was obtained from Calbiochem- Behring (Cambridge, GB) and used at 1 : 20 to 1 : 12500 diluted; anti-human Ig anti-IgM was an IgG F(ab’)z preparation from goat antiserum to human p chains (Cappel) and used at 3 to 100 pg/ml. DNA synthesis was measured at times indicated by pulsing cultures with 1 pCi = 37 kBq [3H]dThd (Amersham Int., Amersham, GB) 18 h before harvesting. Each value represents the mean of triplicate cultures. SD were 2%. In co-stimulatory experiments lymphokines such as IL2 (Cetus Corporation, Emeryville, CA), IL4 (Immunex Corp., Seattle, WA) or IFN-y (Biogen, Geneva, Switzerland) were added to the cultures after 24 h of stimulation with CK226 mAb, and cell proliferation was evaluated at different times as indi- cated.

3 Results

3.1 Expression of CK226 antigen on B cell populations from different sources

The expression of CK226 antigen was studied in different lymphoid populations isolated from peripheral blood, tonsil and spleen. Double-fluorescence analysis revealed that CK226+ cells represented 40%-80% of sIg+ cells. In addition, the expression of CK226 antigen appears to be higher on B than on T cell populations. Thus, double- fluorescence analysis showed that cells displaying the highest reactivity with CK226 mAb were sIg+ (CD3+ cells

were characterized by mediudow reactivity with CK226 mAb, Fig. 1).

3.2 CK226 mAb induces “de novo” expression of CD25 antigen and up-regulates surface MHC class II and CD23 antigens on normal B cells

Normal B cells express constitutively MHC class I1 and CD23 surface antigens while antigens such as CD25 are expressed only following B cell activation [20-231. We investigated whether CK226 mAb was able to modulate the expression of these surface molecules. To this end, highly purified B cells were cultured for 48 h with CK226 mAb, and the expression of CD25, MHC class 11 and CD23 antigens was analyzed by FCM using FACS. As shown in Fig. 2, CK226 mAb induced both up-regulation of MHC class11 and CD23 surface antigens and the de novo expression of CD25 antigen. Thus, CK226 mAb appears to regulate the expression of surface structures relevant to B cell activation and proliferation.

3.3 CK226 mAb induces T cell-independent B cell proliferation

It is well known that some degree of B cell proliferation in the absence of either Tcells or exogenous lymphokines can be induced by either anti-IgM antibody or SAC bacteria [l-161. In order to assess whether CK226 mAb could promote B cell growth, we first studied its ability to induce DNA synthesis in human B cells. B cells were pulsed with the CK226 mAb, and [3H]dThd uptake analyzed in the presence or absence of suboptimal concentrations of PMA (0.5 ng/ml) [24]. As shown in Fig. 3A, a noticeable pro- liferation was achieved with the antibody alone; however,

anti - sIg anti -CD3

Figure 1. -0-color FACS analysis of PBMC using CK226 mAb in conjunction with anti-sIg or anti-CD3 reagents. Cells were stained with CK226 mAb followed by goat anti-mouse PE-IgM and then with FITC-anti-human sIg (A) or FITC-anti-CD3 mAb (B). Samples were analyzed on a FACStar gated to exclude dead cells. Based on control samples, the contour plot was divided into quadrants representing unstained cells (lower left), cells with only red fluorescence (upper left), cells with only green fluorescence (lower right), and cells with both green and red fluorescence (upper right). About 10% of CK226+ cells were sIg+ (A) whereas > 80% were CD3+ (B). It is noteworthy that CK226+sIg+ cells expressed CK226 antigen with a higher fluorescence intensity that CK226+CD3+ cells [compare the upper right quadrants of (A) and (B). Results are expressed on the contour plot as log green fluorescence (FITC) intensity vs. log red fluorescence (PE) intensity.

Eur. J. Immunol. 1990.20: 1161-1165 A novel B cell activation pathway 1163

this effect was potentiated by the addition of PMA (Fig. 3C). I t should be noted that the amount of antibody necessary to induce B cell proliferation (5 ng/ml) was 100-1000 times lower than doses required to induce T cell proliferation (0.5-5 pg/ml, not shown). Maximal B cell proliferation was detectable at day 3-4 and was strongly reduced by day 5-6. That proliferating cells were indeed represented by B cells was indicated by surface marker analysis. Cells recovered at day 5 and 7 were homogeneous- ly composed of sIg+ lymphocytes, while CD3+ cells were undetectable (Fig. 4). Since CK226 mAb induced de nova

expression of CD25 antigen it was possible that the observed proliferation could be, at least in part, consequent to production of IL2 (although no CD3+ cells could be detected). In order to assess this point, we investigated whether anti-CD25 mAb could inhibit the CK226-induced B cell proliferation. As shown in Fig. 3B and D, anti-CD25 mAb had no inhibitory effect on the CK226-induced cell proliferation (either in the presence or absence of PMA). On the other hand, in control cultures the anti-CD25 mAb strongly inhibited T cell proliferation induced by CK226 mAb (not shown).

LO^. green rluorescence intensity i a . u . i

a U

7

1

0 0- I

I 4 8 I

Figure 2. Expression of HLA class 11, CD23 and CD25 surface antigens fol- lowing B cell triggering with CK226 mAb. Highly purified resting B cells of high buoyant density isolated from ton- sils were cultured either in medium alone (A, B, C) or in the presence CK226mAb (10 ng/ml; D, E , F) for48 h and analyzed by FACS for the expres- sion of HLA cclass I1 (A, D), CD23 (B, E) and CD25 (C, F) surface anti- gens. CK226 mAb-induced activation leads to the up-regulation of HLA class I1 and CD23 antigens [compare (A) with (D) and (B) with (E)] and the de novo expression of CD25 antigen [compare (C) with (F)]. Results are representative of four different experi- ments.

a U

DAV DAV

a U

DAY DAV

Figure 3. CK226 mAb induces B cell proliferation both in the absence (A, B) and in the presence (C, D) of PMA. Tonsil B cells were cultured (105 cells/well) for different intervals with 10 ng/105 cells of CK226 mAb either in the presence (*) or in the absence of (+) saturating amounts of anti-CD25 mAb. (A) and (C) show B cell proliferation in the absence of exogenous IL2 whereas in (B) and (D) 50 U/ml of IL2 was added after 24 h of culture. In control samples (0) cells were cultured in the presence of 0.5 ng/W cells CK248 mAb (anti-CD28) [19]. Cell proliferation was evaluated by [3H]dThd uptake after an 18-h pulse (cpm x

1164 A. Poggi, E. Maggi, R. Biagiotti et al. Eur. J. Immunol. 1990.20: 1161-1165

Log. green fluorescence (a.u.)

Figure 4. Phenotypic analysis of CK226-stimulated B cell popula- tions.%nsil B cells stimulated with CK226 mAb were analyzed by direct immunofluorescence after 5 days (A, B) or 7 days of culture (C, D) for the expression of CD3 antigen (A, C) or sIg (B, D). No CD3+ cells could be recovered after CK226-induced B cell proliferation (similar results were obtained using anti-CD7 or anti-CD2 mAb, not shown) whereas virtually all cells were sIg+ although with a wide range of fluorescence intensity.

tory effect of anti-CD25 mAb on the B cell proliferation induced by CK226 mAb and IL2 (Fig. 3B and D).

3.5 CK226 mAb regulates B cell responses to anti-IgM antibody or SAC bacteria

Since human B cell activation and proliferation can be induced by either anti-IgM antibody or SAC bacteria [ 10-121, we investigated possible relationships existing between these pathways of activation and that initiated by CK226 mAb. To this end, the effect of CK226 mAb was analyzed in a co-stimulation assay using submitogenic concentrations of either anti-IgM antibody or SAC bacte- ria. In these experiments CK226 mAb did not co-stimulate either in anti-IgM or SAC assays (not shown). In addition, as shown in Fig. 5, CK226 mAb inhibited in a dose- dependent fashion B cell proliferation induced by mitogen- ic concentrations of either anti-IgM antibody or SAC bacteria.

4 Discussion

In this study we describe a novel pathway of activation of human resting B cells initiated by the 75-kDa CK226 surface molecule. As previously described, CK226 antigen is not restricted to B cells but is also expressed on a subset of T lymphocytes and promotes Tcell activation and prolifer- ation [17] .The activity of CK226 mAb on resting B cells and its ability to induce the de novo expression of activation antigens such as CD25 is reminiscent of the B cell activation induced by anti-IgM antibody or SAC bacteria [21-231. The

ao - 26 -

..... physiological role of CK226 molecule and the relationship existing between this molecule and the antigen-dependent (or other alternative) pathway of B cell activation is unclear

.............. '"(... ............. ....... ............ .......... I. ..... ..... .-.. I.. ..... - at present. Antibody-mediated capping of sIg did not affect

the surface expression of CK226 antigen. This experiment provided evidence for an independent mobility for CK226

0 10 ao 10 40 SO i o antigen and sIg, and suggested that the two structures are not physically linked. On the other hand, co-stimulation exDeriments using CK226 mAb and anti-sIg antibody or

't-----------._____________

CKPP6 mAb concmtrrtion <n*g/ml>

Figure5 CK226 mAb regulates B cell responses to anti-IgM antibody or SAC bacteria. Highly purified B cells were cultured with various concentrations of anti-CK226 mAb (0) (2,4,8,10 and 50 ng/ml) in the presence of optimal concentrations of anti-IgM antibody (*; 100 pglml) or SAC bacteria (+; 1 : 500 final dilution). CK226 mAb inhibited in a dose-dependent fashion the B cell proliferation induced by mitogenic concentrations of either anti- IgM antibody or SAC bacteria.

3.4 CK226 mAb is co-stimulatory with IL2

Given the de novo expression of IL2R following cell triggering with CK226 mAb, it was important to determine whether cells could efficiently proliferate in response to exogenous IL2 [23]. To this end, purified B cells were cultured in the presence of CK226 mAb, IL2 was added after 24 h of culture and cell proliferation was evaluated at different time intervals. As shown in Fig. 3B, IL2 strongly enhanced the CK226-induced proliferation. A modest potentiating effect could also be observed with IL4 or IFN-y (not shown). These data suggest that CK226 mAb induces functional receptors for IL2 (and other lympho- kines). This concept was further reinforced by the inhibi-

SAC bacteria shiwed that a functional relaGonship eksts between the pathways of B cell activation initiated by these stimuli. Thus, addition of CK226 mAb strongly inhibited B cell proliferation induced by anti-IgM mAb or SAC bacte- ria. Similarly to these agents, CK226 induced B cell proliferation in the absence of detectable Tcells or exoge- nous interleukins. On the other hand, B cell signaling via CK226 is distinguishable from that mediated by lympho- kineheceptor interaction. Indeed, lymphokines promoting B cell proliferation only acted on B cells that had been activated. Although CK226 and lymphokines could act synergistically in inducing B cell proliferation, this syner- gistic effect only occurred when CK226 mAb was added prior to or simultaneously with lymphokines. Indeed, CK226 mAb induced the expression of functional surface receptors which allowed B cell response to exogenous IL2. These data indicate that CK226-activated B cells are susceptible to amplification of B cell proliferation mediated by Tcell-derived lymphokines. It should be noted, however, that B cell proliferation induced by CK226 mAb alone is clearly independent from T cells or their products. This is indicated by the fact that (a) no Tcells could be detected during and at the termination of culture of CK226-

Eur. J. Immunol. 1990. 20: 1161-1165 A novel B cell activation pathway 1165

stimulated B cells; (b) neither IL2 nor IL4 could be detected in culture SN (not shown); (c) anti-CD2S mAbdid not inhibit the CK226 mAb-induced proliferation (Fig. 3A and C), and (d) the amounts of CK226 mAb needed to induce T cell responses were approximately 100-1000-fold higher than those required for B cell proliferation. Taken together these data further support the concept that CK226 mAb induces an autonomous B cell proliferation.

In conclusion, our data demonstrate the existence of a new pathway through which B cells can be activated.The natural ligand of CK226 molecule remains to be identified. What- ever the natural ligand may be, it is interesting that in contrast to other “triggering” surface molecules, CK226 mediates cell activation and cell cycle progression in bothT and B lymphocytes.

We thank Ms. C. Miriello for preparation of the manuscript.

Received January 25, 1989.

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