Technical Notes

Purification of Human Tonsil Plasma Cells:Pre-Enrichment Step by Immunomagnetic Selection of

CD311 CellsFrancisco Medina, Carmen Segundo, and Jose A. Brieva*

Servicio de Inmunologıa, Hospital Universitario Puerta del Mar, Cadiz, Spain

Received 28 June 1999; Revision Received 12 November 1999; Accepted 18 November 1999

Background: The advancement of knowledge about thebiology of human normal plasma cells (PC) is hamperedby their low frequency and difficult isolation. The aim ofthis study is to design a way of purifying these cells.Methods: To this end, advantage was taken of the factthat human tonsil PC expressed surface CD31 at higherlevels than the rest of tonsil B cells.Results: The immunomagnetic selection of CD311 cellsfrom tonsil B cells increased by a factor of 12 the propor-tion of PC, determined as CD38high cells. This methodrecovered half of the initial number of PC, and did notalter the PC functions, because IgG secretion was similar

in control B cell cultures as well as in cultures of B cellsobtained at successive steps of the selection procedure. Inaddition, CD38high cells pre-enriched by this techniquewere readily isolated by FACS sorting and clearly identifiedas PC.Conclusions: Therefore, immunomagnetic pre-enrichmentof CD311 cells is an efficient method that allows the com-plete purification of human functional PC. Cytometry 39:231–234, 2000. © 2000 Wiley-Liss, Inc.

Key terms: plasma cells; CD31; immunomagnetic selec-tion; human; tonsil

The terminal differentiation of B lymphocytes gives riseto very specialized cells that are capable of secretingspecific antibodies at very high rate. It is widely acceptedthat this functional B cell stage corresponds to the mor-phological entity known as the plasma cell (PC). A char-acteristic of human PC is their high expression of CD38molecules on the surface (CD38high) (1–3). In fact, ex vivoisolated B cells capable of spontaneous and high-rate Igsecretion in vitro, have been identified as CD38high cells(4–6). There are many open questions about the biologyof PC that require in vitro assays with highly purified cells.Nevertheless, obtaining such purity is a difficult task, be-cause of the low frequency at which PC are present in thedifferent lymphoid tissues (1–3,6), and the absence of anycharacteristic that allows a one-step purification protocol.

Tonsil is a lymphoid organ that provides large quantitiesof lymphoid cells, half of which are B lymphocytes.CD38high cells represent about 0.5–2% of the tonsil B cellfraction, and they have been identified as PC (1,6).

CD31 (PECAM 1) is a 130-KD glycoprotein belonging tothe Ig superfamily of surface molecules (7), that is ex-pressed on a variety of cells including endothelial cells,platelets, neutrophils, monocytes, NK cells, and certainlymphocytes (8). CD31 is an adhesion molecule that par-

ticipates in homo- as well as heterotypic cell interactions(9,10). Histochemical studies have recently demonstratedthat normal PC express CD31 on their membrane (11).The present study explores the possibility that this reac-tivity may be used to purify tonsil PC. The results showthat immunomagnetic selection of CD311 cells from hu-man tonsil B cell fractions increases by a mean factor of 12the proportion of PC. The method does not seem to alterPC Ig-secreting capacity and it provides a reasonable start-ing-point for complete purification of PC by sorting.

MATERIALS AND METHODSMaterials

Cycloheximide and collagenase-V were purchased fromSigma (St. Louis, MO). Fluorescein isotiocyanate (FITC)-labeled mAb against CD20, phycoerythrin (PE)-labeledmAb against CD31 and against CD38, and the correspond-

Grant sponsor: Fondo de Investigaciones Sanitarias of Spain; Grantnumbers: 96/2116 and 98/1182.

*Correspondence to: Jose A Brieva, Servicio de Inmunologıa, HospitalUniverdsitario Puerta del Mar, Avenida Ana de Viya 21, 11009 Cadiz,Spain.

E-mail: jabrieva@hpm.sas.cica.es

© 2000 Wiley-Liss, Inc. Cytometry 39:231–234 (2000)

ing isotypic negative controls were provided by BectonDickinson (San Jose, CA). Cy-chrome (CyC)-labeled mAbagainst CD38 and the corresponding negative controlwere from Caltag (San Francisco, CA). EDTA was pur-chased from Pharmacia Biotech (Uppsala, Sweden). Puri-fied mAb against CD31 was provided by Coulter (Miami,FL). Goat anti-mouse IgG magnetic microbeads, selectioncolumns of MS1 type and miniMACS magnet were ob-tained from Miltenyi Biotec (Auburn, CA). FITC-conju-gated rabbit-anti-human Igs was from Dako (Glostrup,Denmark). 96-well flat-bottom culture plates and U96Maxisorp plates used for ELISA were provided by Nunc(Roskilde, Demmark). Unconjugated and peroxidase-con-jugated goat F(ab)92 anti-human IgG, used in ELISA, werepurchased from Biosource (Camarillo, CA).

Tonsil B Cell Isolation

Tonsils were obtained from subjects undergoing tonsil-lectomy for chronic tonsillitis. The tonsilar tissue waschopped to small pieces, which were washed severaltimes in HBSS and treated with collagenase-V (1 mg/ml inRPMI-1640) for 15 min, at 37°C in a shaking bath. Theresulting cell suspension was washed in culture medium,and separated into T and non-T cell populations by apreviously reported rosette technique (12). The non-T cellpopulation consisted mostly of B cells (.97% CD191cells), and was termed B cell fraction.

Isolation of CD311 Cells

CD311 cells were purified from the B cell fractions byan immunomagnetic technique. Briefly, B cells (up to100 3 106 cell/ml) were incubated with anti-CD31 mAb(10 mg/ml) in 2 mM EDTA 0.5% BSA-PBS, for 10 min in thedark at 4°C. After two washes in the same buffer, cells (upto 200 3 106 cell/ml) were incubated with goat anti-mouse magnetic microbeads, according to the manufac-turer’s instructions. After two washes, cells were resus-pended (up to 200 3 106 cell/ml) in the buffer. Aseparation column was placed in the magnet and 500 ml ofbuffer was applied at the top of the column and allowedto run through, the effluent being discarded. The cellsuspension was pipetted onto the column and allowed torun through. The effluent was collected as negative frac-tion. The column was washed three times with 1 ml ofbuffer and the effluents included in the negative fraction.At this point, the column was removed from the magnet,2 ml of buffer was applied at its top and the cells werefirmly flushed out, using the supplied plunger. The efflu-ent was collected as the positive cell fraction (CD311

cells).

Cell Culture and IgG ELISA

B cells were adjusted at 106 cells/ml, in a culture me-dium consisting of RPMI 1640 supplemented with 10%FCS, L-glutamine (10 mM) and gentamycine (0.05 mg/ml),and were cultured in 96-well plates, in a final volume of250 ml/well, at 37°C with 5% of CO2. In some experi-ments, cells were collected at different steps of the immu-nomagnetic isolation procedure (after either anti-CD31

mAb treatment or magnetic microbeads incubation), andwere also cultured in the same conditions. After 4 days,cell-free supernatants (SN) were collected, and IgG secre-tion was tested by ELISA in microtiter plates, as previouslyreported (13).

Cell Staining and Flow Cytometry Analysisand Cell-Sorting

For two- and three-color labeling experiments, 200 ml ofcells (at 0.5–5 3 106 cell/ml) were incubated with optimalconcentrations of mAb for 20 min in the dark at 4°C. Aftertwo washes, FACS analysis was performed on a FACSCali-bur cytometer (Becton Dickinson) equipped with an air-cooled argon ion laser emitting 15 mW at 488 nm. Theinstrument was equipped with three fluorescence detec-tor photomultiplier tubes, with green fluorescence (FITC)being collected through a 530/30 nm bandpass, red/or-ange (PE) through a 585/42 nm bandpass, and red (CyC)through a 650 nm longpass filter. Cell analysis was per-formed with CELLQUEST software (Becton Dickinson).Light scatter signals were recorded in linear mode andfluorescence signals in logarithmic mode. Data from 5000to 10,000 events/sample were collected.

Sorting of CD38high cells from BCD311 cell fraction, wasperformed on a FACStar-Plus cytometer (Becton Dickinson)equipped with an air-cooled argon ion laser emitting 100mW at 488 nm. Fluorescence detector photomultiplier tubesand filters were similar to those of FACSCalibur. Cells werepreviously labeled for CD20 (FITC) and CD38 (PE), as de-scribed, and CD38high cells were gated with LYSYS-II soft-ware (Becton Dickinson), and sorted in C-normal mode.

Plasma Cell Identification

Sorted CD38high cells (0.1 3 106 cells in 100 ml PBS)were cyto-centrifuged on slides and stained by the Giemsatechnique. Percentage of cells with PC morphology wasdetermined by optical microscopy. Cells containing intra-cytoplasmic Igs were detected on cytospin cell prepara-tions by a direct immunofluorescence technique. Positivecells were identified by fluorescence microscopy.

RESULTS AND DISCUSSIONTonsil B cells were triple-stained for CD20 (FITC), CD38

(CyC), and CD31 (PE) or the corresponding negative con-trols. As can be seen in Figure 1 (dot plot), three differentpopulations were distinguished by their pattern of CD20/CD38 expression, as has been previously reported (6,14):PC (CD38high), germinal center (GC) B cells (CD381

CD2011) and mantle zone (MZ) B cells (CD382 CD201).The heterogeneous expression of CD38 on tonsil B cellsmakes disadvantageous its use in an initial step for PCpurification. In an attempt to find an alternative strategy,the three B cell subsets were gated and their expression ofCD31 was analyzed. Whereas GC B cells were mostlynegative for CD31, and only a part of MZ cells showed lowquantities of surface CD31, PC were positive and most ofthem exhibited intense CD31 expression (Fig. 1, histo-grams and bars).

232 MEDINA ET AL.

The differential high expression of CD31 on CD38high cellswas used as a pre-enrichment step for PC isolation. Accord-ingly, positive immunomagnetic selection of CD311 cellswas performed in tonsil B cell fractions. The populationobtained by this method was highly purified in CD311 cells(.98%), as expected. The analysis of CD20 and CD38 ex-pression in this cell population revealed that the proportionof CD38high cells was markedly enriched by this method (Fig.2). In addition to PC, Figure 2 shows that a small subset ofCD311 T cells (CD202) was selected as well, and that someMZ B cells (CD201 CD382) remained in the CD31 selectedpopulation. CD38high cell figures were increased from 1.6%6 0.2 in the initial B cell fraction to 18.9% 6 4.6 in thepurified CD311 cells (mean 6 SEM; n 5 8), representing anincrease by a mean factor of 12.2 (6 2.8) times. In compar-ison, the purification of CD38high cells by using CD38 as theinitial selection marker enriched this population only threetimes (from 2.1% 6 0.05 to 6.41% 6 0.4; n 5 3). In conse-quence, although the high expression of CD38 is the mostspecific marker for human PC (1–3), its use at the initialpurification step of these cells is hampered by the highnumber of CD381 cells (GC B cells) present in the B cellpreparation. Therefore, the use of the CD31 selectionmethod provides a pre-enrichment step for human tonsil PCpurification better than those previously reported (6). Fur-thermore, 45.7% 6 8.9 (mean 6 SEM; n 5 8) of the CD38high

cells present in the initial B cell fraction were recovered afterthe CD31 cell-selection step. Thus, this method is reasonablyefficient for the enrichment of tonsil CD38high cells.

Although PC pre-enrichment was clearly achieved bythe described technique, the method might have an ad-verse effect on PC functioning. Accordingly, experimentswere conducted to assess whether CD31 cell-selectionaltered Ig secretion by these cells. To this end, tonsil B

cells either nonmanipulated or recovered after the treat-ment with anti-CD31 mAb alone or followed by the treat-ment with the magnetic microbeads, were cultured and

FIG. 2. Comparison of CD20 CD38 expression in human tonsil B cells(upper dot plot) and after immunomagnetic selection of CD311 cells(lower dot plot).

FIG. 1. B cells from tonsil were triple stained with anti-CD20-FITC, anti-CD38-CyC, and anti-CD31-PE mAbs (or the corresponding negative controls) andthey were analyzed by flow cytometry. Left panel: Dot plot represents a typical pattern of CD20/CD38 expression in human tonsil B cells. Middle panel:Different B cell subsets (PC, GC, and MZ) were gated and the CD31 expression by at least 5000 cells of each gate was recorded. Histograms show arepresentative example of CD31 expression (solid line). Negative controls are represented as dotted line. Right panel: Bar diagrams represent the resultsof CD31 expression by GC and MZ B cells and PC, expressed as percentage of CD311 cells (upper) and the mean fluorescence intensity (MFI, lower) insix experiments (mean 6 SEM).

233CD31 PRE-SELECTION OF HUMAN PLASMA CELLS

the IgG secreted into the supernatants was monitored. AsFigure 3 shows, these treatments did not significantlymodify in vitro IgG production by these cell fractions. Theaddition of cycloheximide (10 mg/ml) to these culturesinhibited more than 95% of the IgG secretion, indicatingthat these cells were capable of active production. There-fore, the CD31 selection method provided perfectly func-tional PC.

Finally, to confirm that CD38high cells pre-enriched bythe described technique actually were PC, CD31-selected,CD38high cells were purified by FAC sorting. Figure 4 (dotplot) shows that sorted populations were highly enrichedin CD38high cells (. 97%, n 5 5). Stained cytospin prep-arations of these cells showed their morphological fea-tures (Fig. 4, upper photograph), and cytoplasmic Ig stain-

ing was strongly positive (Fig. 4, lower photograph),demonstrating that the purified cells were typical PC.

In conclusion, CD31 pre-enrichment is an efficient andreproducible method that provides a reasonable starting-point for the isolation of functional PC from human tonsil.Complete purification of PC can be achieved by the com-bination of this method with the subsequent selection ofCD381 cells, either by FACS-sorting or immunomagnetictechnique.

ACKNOWLEDGMENTSWe thank Paco Tamayo for photographical technical

assistance.

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FIG. 3. B cells were collected at different steps of the immunomagneticisolation procedure (after either anti-CD31 mAb treatment or magneticmicrobeads incubation). These fractions and control B cells were culturedfor 4 days. Cell-free supernatants were collected, and IgG was quantifiedby ELISA. Values were expressed as the percentage of control IgG pro-duction. The results represent the mean (6 SEM) of three experiments.IgG production of control B cells in these experiments was 3118 6 1322ng/ml.

FIG. 4. Left panel: Dot plot representing CD38/CD20 expression byCD38high cells sorted from CD311 cell fractions. Right panel: Giemsastaining (upper photograph) and intracytoplasmic Ig immunofluorescentstaining (lower photograph) of cytocentrifugal preparations of sortedCD38high cells.

234 MEDINA ET AL.