Cellular Immunology 236 (2005) 167–170

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Immunologyellular

A comparison of Verotoxin B-subunit (Stx1B) and CD77 antibody to deWne germinal centre populations

S. Bailey a, C. Mardell a, L. Wheatland a, H. Zola b, P. J. Macardle a,¤

a Department of Immunology, Allergy and Arthritis, Flinders Medical Centre and Flinders University, Adelaide, SA, Australiab Child Health Research Institute, Women’s and Children’s Hospital, Adelaide, SA, Australia

Received 28 March 2005; accepted 23 May 2005Available online 12 September 2005

Abstract

We have directly compared the use of a CD77 antibody with the binding subunit of Shiga-like toxin 1, Verotoxin 1, and (Stx1B)for delineation on human tonsil cells. We determined that the Stx1B produced a greater intensity of staining than the CD77 anti-body, allowing three sub-populations of germinal centre cells to be seen. The populations express high, medium, and low levels ofglobotriaosylceramide as determined by the binding of the Stx1B reagent. The strong staining patterns of Stx1B suggest that it maybe useful in deWning germinal center B cell populations. 2005 Elsevier Inc. All rights reserved.

Keywords: CD77; Shiga-like toxin; Germinal centre; B-lymphocytes; Flow cytometry

1. Introduction

The subunit B of Verotoxin 1, a cytotoxin producedby Escherichia coli, binds speciWcally to globotriaosylcer-amide (Gb3). This glycolipid is also recognized as CD77,a B cell antigen normally restricted to germinal centrecells (GC) but also highly expressed on Burkitt’s lym-phoma cells and some follicular lymphomas (summa-rized in [1]). Expression of CD77 can be induced onnaïve tonsil B cells via threshold occupancy of CD40 [2],and appears to be expressed on GC B lymphocytes thatare programmed for apoptosis [3,4], suggesting thatCD77 has a critical functional role in the germinal centrecell reaction and aYnity maturation.

The expression of CD77 is useful in the diVerentialdiagnosis of Burkitt’s lymphoma, in staging of follicularlymphoma and in the delineation of centroblasts andcentrocytes. However, monoclonal antibodies to CD77

* Corresponding author. Fax: +618 82044158.E-mail address: Peter.Macardle@Xinders.edu.au (P.J. Macardle).

0008-8749/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.cellimm.2005.08.023

have not achieved popularity in these contexts, possiblybecause of issues with the intensity of staining.

The Shiga toxin family contains two types: Stx1,Verotoxin 1 (VT1), or Shiga-like toxin 1 (SLT1), andfollowing the same terminology: Stx2 (VT2, SLT2). AllStx members have a A–B structure in which the A-sub-unit has N-glycosidase activity and the B-subunit bindsto Gb3 (reviewed in [5]). Although binding of the wholetoxin molecule results in cell death [6] binding of theisolated B-subunit of Shiga-like toxin 1 does not.Hence, we examined the use of a recombinant constructof the B-subunit of Shiga-like toxin 1 (Stx1B) as a sub-stitute for CD77 monoclonal antibody. Recombinantbinding subunits of Verotoxin have been demonstratedto induce apoptosis in CD77 positive lymphoid cells[7], demonstrating a critical role for CD77 in germinalcentre development. However, as the intensity of stain-ing with CD77 monoclonal antibodies can be weak weexplored means to enhance the staining. Here, we pres-ent data of a direct comparison of Stx1B and the proto-typic CD77 monoclonal antibody 38.13 [8] on cells intonsil tissue. We show that the discrimination of

168 S. Bailey et al. / Cellular Immunology 236 (2005) 167–170

staining by the Stx1B reagent was greater than thatwith the monoclonal antibody and allowed clear recog-nition of populations of germinal centre cells. Addi-tionally, the use of a bacterial toxin, which is notrecognized by secondary anti-mouse reagents, allowedfor an extra Xuorescence reagent to be used without theneed for cross-blocking of murine epitopes.

2. Materials and methods

The Verotoxin construct Stx1B was was a generousgift from Dr. D. W. Acheson of Northern Medical Cen-tre, Boston, USA, and produced as described [10]. StxB1was biotinylated or coupled to Xuorescein isothiocya-nate according to standard methods.

2.1. Monoclonal antibodies

The following mAbs were used in this study: IgD-Biotin, CD38-FITC (Becton Dickinson, San Jose, CA),CD19-, CD10-, and CD23-FITC (Dako, Carpinteria,CA). The CD77 clone 38.13 was from ImmunoTech(Beckman Coulter, Melbourne, Australia). Isotype spe-ciWc negative control mAbs were selected from a panelagainst Salmonella and Clavibacter sps.

2.2. Human tonsillar cell preparations

All human material was obtained with the approvalfrom the Research Ethics Committee of the Women’sand Children’s Hospital, Adelaide, and from the Com-mittee on Clinical Investigation, Flinders Medical Cen-tre, Adelaide (82/2000). Tonsils were obtained followingroutine tonsillectomy, processed into single cell suspen-sions and used either fresh or following storage in liquidnitrogen.

Fig. 1. Comparison of Stx1B and CD77 antibody staining on tonsil Bcells. Tonsil cells were stained with CD19-FITC and biotin labelledStx1B or CD77 antibody, a minimum of 10,000 events were collectedon a FACScan Xow cytometer (BD, San Jose, CA) and analyzed withCellQuest software. The marker was placed on positive populations asdetermined by a negative control antibody (broken line). The negativecontrol antibody X63, an IgG1 isotype non-speciWc murine monoclo-nal antibody, was biotin conjugated in the case of the Stx1B histo-gram, and detected with SA–PE and with anti-mouse PE in the case ofCD77.

2.3. ImmunoXuorescence and Xow cytometry

Surface expression was studied as described previ-ously [11]. Cells were stained with pre-determined dilu-tions of antibody or an isotype-matched negative

Fig. 2. Tonsil B cells were puriWed by T-cell depletion using monoclo-nal antibodies to T-cell antigens and cells removed with magneticbeads (Dynal Biotech, Vic., Australia). Simply, total tonsil cells wereincubated with a cocktail of T-cell Mabs, washed and incubated withanti-mouse labeled Dynal beads (M-450) and labeled cells removedaccording to the manufacturers instructions. The B cells (>97% byimmunoXuorescence) were then stained with a combination of IgD,CD38, and either Stx1B or CD77 antibody. Regions were set on eachof the four populations as indicated and the staining given by Stx1B orCD77 antibody expressed as a histogram on these populations.

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S. Bailey et al. / Cellular Immunology 236 (2005) 167–170 169

control followed by dilutions of either biotinylated horseanti-mouse immunoglobulin (Caltag, San Francisco,CA) and phycoerythrin–streptavidin (SAPE, Sigma, St.Louis, MO) or anti-mouse immunoglobulin-FITC (Sile-nus, Australia) or anti-mouse immunoglobulin–PE(Southern Biotechnology, Birmingham, AL). The anti-mouse reagents were F(ab�)2 products. Two-color analy-sis was carried out with the addition of either FITC- orPE-directly conjugated antibodies and three color analy-sis was performed using FITC-conjugated and phycoer-ythrin-labeled antibody following indirect labeling withbiotinylated secondary antibody labeled with streptavi-din–Quantum Red (SAQR, Sigma, St. Louis, MO). Freebinding sites of the secondary antibodies were blockedusing normal mouse immunoglobulin (Sigma). Flowcytometry was performed on a FACScan with CellQuestsoftware (BD, San Jose, CA) and reanalyzed usingWinMDI Version 2.8 (J. Trotter, The Scripps ResearchInstitute).

3. Results and discussion

Tonsil cells were labeled with a range of monoclonalantibodies and the expression of CD77 and Stx1B com-pared. In preliminary experiments the Stx1B and CD77were carefully titrated to determine the concentrationthat gave the greatest diVerence between negative andpositive populations. Hence, each reagent was used at itsoptimal concentration. In all the experiments carried outthe staining given by the CD77 antibody was consider-ably weaker than that seen for Stx1B. In Fig. 1, the pop-ulations of CD19 positive tonsil B cells labeled witheither Stx1B or CD77 are shown. In the case of Stx1B,two distinct B cell populations were seen with about 16%of the total B cell population being dual positive forCD19 and Stx1B. In contrast, the staining seen with theCD77 antibody was considerably weaker making it diY-cult to delineate distinct populations. Hence, althoughthe proportions of Stx1B and CD77 positive cells weresimilar among tonsil B cells, (n D 10 tonsils), 16 § 7.5%

for Stx1B positive cells and 13.7 § 5.9% for the CD77antibody positive populations; the diVerence in the dis-tribution by Xuorescent intensity was considerable.

We then examined the distribution of Stx1B andCD77 antibody staining on puriWed tonsil B cell popula-tions based on the expression of IgD and CD38. In thecomposite Fig. 2, panel (A) shows the placement ofregions for IgD+CD38¡ve (A), IgD+CD38+ve (B),IgD¡CD38+ve (C), and IgD¡CD38¡ve (D). These popu-lations correspond to naïve, pre-germinal centre, germi-nal centre, and memory cells, respectively [9]. Thecolumns (Fig. 2B) show the expression of CD77 orStx1B in each of the regions. Of particular interest is thedistinct populations seen with Stx1B compared to thatseen with the CD77 antibody. In both panels (B) and (C)the overall staining with CD77 antibody or Stx1B washigher than that seen in negative populations (see A andD as examples). However, the level of staining with theCD77 antibody was comparatively low. Hence, the stain-ing pattern seen with Stx1B demonstrated at least threepopulations present in germinal centre cells, aStx1B weak/negative population (labeled 1) a Stx1Bmoderate population [2], and a Stx1B strong population[3]. Similarly, the percentage of Stx1B positive cells wasgreater than the CD77 positive cells in the pre-germinalcentre cells (B).

As expected Stx1B and CD77 antibody staining waslimited to germinal centre cells and was negative onCD23 positive tonsil B cells (Fig. 3). However, this againemphasized the contrast in intensity of staining forStx1B and CD77 antibody.

Hence, our data demonstrates that Stx1B may be auseful reagent to complement the phenotypic character-ization of germinal centre cells based on CD77 expres-sion. Additionally, as Stx1B is not detected byanti-mouse antibodies, used to detect primary monoclo-nal antibodies, it is a useful reagent in multicolor analy-sis. To this end, we have labeled Stx1B with Xuoresceinisothiocyanate or biotin succinimide ester, using stan-dard techniques, and used the reagent in multiparameterXow cytometry.

Fig. 3. PuriWed populations of tonsil B cells were stained with a directly conjugated CD23 antibody (Dako Cytomation) and with either Stx1B orCD77 antibody as in Fig. 2. Data are expressed in 5% probability plots.

17% 7%

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77

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170 S. Bailey et al. / Cellular Immunology 236 (2005) 167–170

Acknowledgment

We gratefully acknowledge Dr. D.W. Acheson,Northern Medical Centre, Boston, USA, for supplyingthe Stx1B used in this study.

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