Br. J. Cancer (1981) 44, 68

BINDING OF PEANUT LECTIN TO GERMINAL-CENTRE CELLS:A MARKER FOR B-CELL SUBSETS OF FOLLICULAR LYMPHOMA?

M. L. ROSE*, J. A. HABESHAWt, R. KENNEDYt, J. SLOANE§, E. WILTSHAWIAND A. J. S. DAVIES*

From the *Chester Beatty Research Institute, Institute of Cancer Research, Fulham Road, London;tICRF Medical Oncology Group and Department of Pathology, St Bartholomew's Hospital,London; IDepartment of Medicine, Royal Marsden Hospital, Fulham Road, London; and

§Department of Pathology, Royal Marsden Hospital, Sutton, Surrey.

Received 5 January 1981 Accepted 17 AMarchi 1981

Summary.-The binding of horseradish-peroxidase-labelled peanut lectin (HRP-PNL) to cryostat sections of tonsil, lymphoma lymph nodes, reactive lymph nodes andmiscellaneous tumours demonstrated that PNL binds selectively to lymphocytes ingerminal centres. Lymph nodes from 21 patients with non-Hodgkin's lymphomaswere phenotyped as cell suspensions for PNL binding, and the following surfacemarkers: E rosetting, C3d, SIg, OK markers of T-cell subsets, Ig heavy-chain andlight-chain classes. There was a positive correlation between PNL binding and cellswith SIg and C3d receptors. 4/5 cases of centroblastic/centrocytic follicular lymphomahad a PNL+ SIg+ C3d+ phenotype. Both cases of centroblastic/centrocytic diffuselymphoma were PNL-. There was no correlation between PNL binding and heavy-or light-chain Ig class. PNL binding and presence of C3d receptors were not alwayspositively correlated, indicating that follicular cells may be either PNL+ SIg+ C3d+or PNL+ SIg+ C3d-. The binding pattern of PNL to 1 case of thymic hyperplasia and2 cases of malignant lymphoma lymphoblastic T type suggested that some but notall cortical thymocytes bind PNL.

THE LECTIN derived from the seeds ofthe peanut plant Arachis hypogaea (peanutlectin, PNL) binds to single terminalgalactose residues, but more avidly to thedisaccharide D-galactose 3' 1-3 D-N,acetylgalactosamine (Pereira et al., 1976).This sugar is part of the oligo-saccharidearray on cell surfaces, but it is commonlycovered by sialic acid. Thus PNL will notbind to most lymphocytes unless they arepre-treated with neuraminidase. However,cortical thymocytes of mouse and man(Reisner et al., 1976; 1979), small numbersof peripheral T cells in the mouse (Londonet al., 1978; Roelants et al., 1979) andgerminal-centre lymphocytes of mouse(Rose et al., 1]980) bind PNL withoutneuraminidase treatment. Experimentsfrom this laboratory have also revealed

that horseradish peroxidase-labelled PNL(HRP-PNL) binds to germinal-centrelymphocytes in human tonsil (Rose &Malchiodi, 1981). The selective binding ofPNL by human cortical thymocytes andfollicular lymphocytes prompted a searchfor the PNL-binding characteristics oflymphoblastic lymphomas of cortical T-cell type, and of follicular lymphoma. Inthis paper we describe the binding ofHRP-PNL to frozen sections of tonsil,lymphoma lymph nodes, reactive lymphnodes and some other non-lymphoidtumours, and the binding of fluorescein-isothiocyanate-labelled PNL (FITC-PNL)to cell suspensions from various tissuesand non-Hodgkin's lymphomas. Theresults confirm the restricted binding ofpeanut lectin to subsets of B and T

Address for reprints: Dr M. L. Rose, Chester Beatty Research Institute, Ftlliham Road, London SWV3 6JB.

IPNL BINDING TO GERlIINAL-CENTRE CELLS

lymphocytes, anid illustrate its potentialuse as a marker of follicular lymphoma, insection or in cell suspensions.

MATERIALS AND METHODS

Surgical biopsy specimens of the lymnphnodes studied ere ol)tained from patientsadmitted to the Royal Marsden Hospital,Fulham Road, London and Sutton, or theMedical Oncology Unit, St Bartholomew'sHospital, London. Conventional histologicaldiagnoses on paraffin-embedded sectionswere performied by either Dr John Sloane(Sutton) or by Dr A. E. Stansfeld (Bart'sHospital).Binding of HRP-PINTL to cr yostat sections.

Blocks from unfixed surgical biopsy materialswere snap-frozen in liquid N2 and stored inliquid N2 before use. Embedded (Ames TCcompound) frozen blocks w,ere cryostat-sectioned at 4 ,um, and air-dried. HRP-PNL(prepared as previously described by Roseet at., 1980) was reacted with sections in a

concentration of 20 jug/ml in phosphate-buffered saline (PBS, pH 7.2). The reactionwas allow-ed to proceed for 30 min at 20°C,and w,as terminiated by wxashing sections x 3in a large excess of PBS. Sites of fixation ofHRP-PNL ere identified by developmentwith 3,4,3',4' tetra-aminobiphenyl hydro-chloride (B.D.H.).

Binldiny ofFITC-PNL to cell suspentsionis.FITC-PNL wras prepared as previously(lescribed (Rose et al.. 1980). Cell suspensionsfromn fresh surgical biopsy samples of lymphnodes wAere prepared in RPMI 1640, washedx 2. and cell concentration adjusted to107/ml. 100 ,ul of cell suspension was mixedwith 10() ,ul of FITC-PNL in PBS at a con-centr ation of 20 jug/ml. The resulting mixturewsras incubated at 4°C for 30 min and ashedx 3 in a large excess of PBS.Phenotypic analysis of cell suspensions.

Cell suspensions were analysed for content ofT and B cells by rosetting, and immuno-fluorescence techniques using standardizedparticle preparations and antisera as follo-ws:T cells ws ere quantitated using E rosettes(4 C). anti-HTLA (anti-human lymphocyteantigen serum raised against monkey thymusin rabbits, kindly provided by Drs M.Greaves, M. Roberts and G. Janossy) and insome instances monoclonal mouse antiseraagainst human T-cell subsets (OKT series

antisera Ortho diagnostics). B cells werequantitated using surface immunoglobulin(SIg) expression, with heteroantisera specificfor total immunoglobulin (Ig) (, y, a, 8heavy chains), and class-specific anti-Ig serafor [u, y, a, 8 heavy chains and K and A lightchains (Habeshaw et al., 1979). The expres-sion of C3d receptors by lymphocytes wasassessed concurrently, as previously de-scribed (Habeshaw et al., 1979). HLA and Taantigen expression was monitored with themonoclonal antisera W6/32 (anti-HLA-ABC)and Da2 (anti-HLA-D, Ia-like antigen).The phenotyping results, histological diag-

nosis, and HRP-PNL binding on frozensections were all assessed independently, andcombined after completion of the series.

RESULTS

PNL binding to tonsil and reactive lymphoidtissueHRP-PNL was found to bind to the

membrane of lymphocytes present ingerminal centres, but not to the coronalsmall lymphocytes of all the 10 tonsilsexamined (Fig. 1). Whole-cell suspensionsfrom tonsil indicated 10-20% of FITC-PNL-binding cells, whereas 25-40% of thesame cell population was SIg+ complement-receptor-positive. The PNL-binding cellpopulations found in reactive lymph nodesare shown in Table I. Where follicularhyperplasia was present, SIg+ C3d+ andPNL-binding cells appeared together, sug-gesting that follicular cells are SIg+C3d+ PNL+. The SIg positivity indicatesthat the cells concerned are B lympho-cytes.

PNL binding to lynmphomtia lymiph-nodecells, reactive lymtph nodes andmiscellaneous tumours

Preliminary experiments examined thebinding of HRP-PNL to frozen sectionsof a variety of malignant and non-malignant tissue (Table II). PNL wasfound to bind largely to lymphocytes offollicular lymphoma and reactive lymphnodes, and rarely to lymphocytes of otherlymphomas. The binding of HRP-PNLto oat-cell sarcoma and rhabdomyosar-

.9

0M. L. ROSE ET AL.

FIG. 1. Binding ofHRP-PNL to cryostat section of tonsil. GC = germinal centre; CO = corona. x 180.

TABLE I. PINL+ cells in reactive (non-neoplastic) lymph nodes% of total viable cells

Patient Age HistologyJW 27 Follicular hyperplasiaCY 25 Follicular hyperplasiaBK 21 Follicular reactionDHD 58 Sinus histiocytesEJ 47 Reaction to mammary

dysplasiaCP 16 Non-specific reactive

follicular hyperplasiaGA 65 Follicular reaction

E C3d SIg+ PNL30 37 45 20 FITC-PNL

18 40 40 17

33 24 42 +58 11 o30 +

51 14 40 + HRP-PNL*

ND ND ND +57 21 30 + J

* HRP-PNL oni cryostat sections. + is PNL+ lymphocytes; ± is lymphocytes PNL- but connectivetissue PNL+.

coma was seen as strong cytoplasmic stain-ing, and distinct from the membranestaining of PNL to lymphocytes. Cryostatsections from PNL+ follicular lymphomashow large areas of PNL-binding lympho-cytes (Fig. 2). PNL was also found to bindto follicular dendritic cells, connectivetissue and macrophages to varying degreesin different lymphomas, but it is notpossible to say whether it was membraneor cytoplasmic binding. Electron-micro-

scope studies have revealed HRP-PNLbinding to the membrane of lymphoblastsfrom human tonsil (Lydyard & Birbeck,personal communication) and the fact thatlarge numbers of FITC-PNL-binding cellscan be recovered in cell suspensions fromlymph nodes from patients with follicularlymphoma demonstrates that the bindingof PNL to follicular lymphocytes is mem-brane binding. Where possible, retrospec-tive surface marking was established for

70

PNL BINDING TO GERMINAL-CENTRE CELLS

TABLE II.-Binding offrozen sections of lympAcells, reactive lymph nolaneous tumours

TumourHodgkin's lymplhomaT-cell lymphomaReactive hyperplasiaNodular follicularlymphoma

Diffuse lymphomaOther lymphomas an(ltumourst

Wilms' tumour

Nexan

HRP-PNL to the lymphoma patients of this serieswoma lymphnode (Table III).odes and miscel- These results clearly j ustified a more

detailed study of the surface phenotypeNo. associated with large numbers of PNL+

containing lymphocytes. FITC-PNL binding was

;0ne(l PNLo therefore undertaken on cell suspensionsnine(l cytes from lymphoid organs of a number of8 1* other patients in conjunction with descrip-37 tion by other markers (Table IV). The

results establish some interesting corre-6 5 lates of PNL binding with histology and

0 o with phenotype.141 CPtC'T only + PIVL- lymphom,a8

* Small discrete germinal cenitre at edge of node.t Oat-cell carcinoma an(l rhabdomyosarcoma.I Carcinoma of the larynx, leg sarcoma, angio-

blastoma, breast carcinoma, "secondary" carcin-oma, non-endemic Burkitt's lymphoma, immuno-blastic sarcoma, undifferentiated sarcoma, meta-static carcinoma, high-grade malignant lymphoma,malignant lymphoma lymphocytic, reactive sinushistiocytosis, oat-cell carcinoma, rhabdlomyo-sarcoma.CT = connective tissue.

None of the cells from the nodes orblood of lymphocytic lymphoma patientsbound PNL. The single case of T CLL(Patient EB) showed slight (8%) PNLbinding of lymph-node cells. Patient AB,with MLL, who showed a phenotypicprofile of SIgA-positive B cells expressingC3d receptors, did not bind PNL, evidence

a~~~q~ .. . ...a emm.

FIG. - g ofHf l n fo a

FIG. 2.-Binding of HRP-PNL to cryostat section of lymph node from a patient with CB/CC/F. x 140.

71

72 M1. L. ROSE ET AL.

TABLE III.-HRP-PNL positivity in lymphoma: retrospective correlation with surfacemarking

HistologyCB/CC/FCB/CC/D)MLLMLLMLLPlasmacytomaMLHgU

E3412

2854

64

C3d13626

03

1512

SIg58719070882220

SIg class HR1'-PNL*y / K +8 K -

/I K _

/I K _

8C K -

(X K + tpC _

* Binlding to lymphocytes unless otherwise statedl.t Connective tissue was especially positive.

MLIB = malignant lymphoma immunoblastic; MLHgU = maligniant lymplioma lighi-gra(le unclaisified;MLCC = malignant lymphoma centrocytic; MLL = malignant lymphoma lympliocytic; ML/LB :B = malignantlymphoma lymphoblastic B type; ML/LB :T = malignant lymphoma lymphoblastic T type; CB/CC/F =centroblastic and centrocytic follicular; CB/CC/D = centroblastic and centrocytic diffuse; HD = Hodgkin's(lisease; LP = lymphocyte predominant; ML/CC/SC = malignant lymphoma centrocytic small-cell;TdT = terminal (leoxynucleotidyl transferase; pc= polyclonal.

TABLE IV.-Percentage of FITC-PNL lymphocytes inwith phenotype

HistologyCB/CC/F*CB/CC/F +HDCB/CC/FCB/CC/FCB/CC/FCB/CC/DjCB/CC/DML/LB :BML/LB :BMLIBMLIBMLCCMLLMLLMLL"T" CLLIL/LB :T

31L/LBB:TPlasmacytomaLennert'slymphoma

ML/CC/SC

E24301

2026

112

26338

128144

5580

C3M SIg76

35 3741 7039 3012 667 98

26 7150 803 97

16 329 55

34 880 702 80

44 942 34 4

70t I 14 15 22

80 7 6

12 13 84

malignant lymphoma-correlation

0/

SIg class FITC-PNL Biopsyy only 3 NodePc 36 Nodex A 66 Node(x 8A20 Nodey,ut A 70 Nodey it 8K 4 Blood8 K 0 Node

i K 7 Node/I 8 K 97 NodetL y A 8 K 8 Nodey 8 K 0 Nodey a y 0 Node/ K 0 NodeayY K 1 Blood0X K I Nodepc 8 Node

I Pleuraleffusatel

0 Nodet8 KCya K 45 Stomach

Pc 0 Node0 Node

* Recurrent. t OKT6+, TdT+. I Leukaemic. Cy = Cytoplasmic.For key to abbreviations, see Table III.

that the PNL-binding site cannot be thesame as the SIgA or C3d binding sites.The 2 cases of T-cell lymphoblastic

lymphoma (KF, LT) (Table IV) did notshow PNL binding, though 80% of theirlymphocytes expressed thymic cortical-lymphocyte antigen (OKT6+) and were

TdT+. This suggests that not all corticalthymocytes bind PNL, a possibility whichis supported by the profile of a thymusgland (Table V) where there is a dis-

crepancy between the number of corticalthymocytes (84%) and the number of cellsbinding PNL (46%o).One case of immunoblastic lymphoma

(GC, Table IV) showed only small num-

bers (8%) of PNL-binding cells, thoughsome of the B cells in this patient expressedC3d receptors. Patient MC (Table III)with a similar profile to GC, also failed toshow PNL positivity of lymphocytes inlymph-node sections stained with HRP-

l:atientCCSDJBHBLODPMGMIC

Age1947256259482_3

l'atientJYJNLPGBHSSDSDCBLFGCJBLCHBJIABEBKF

LTPMGFS

FR

Age37663453694747921-

476478624472645

74851

61

P'NL I3LN.DING TO GERMINAL-CENTRE CELLS

0() Bill(illng

4696 (H1TLA 90)844312

+

2. Cryostat sect'i(HRP-P1NL (lefinite 'fOllieular" staining iII

eortex with groups otf l'NI,+ cells.

PNL. One lymnphoblastic lymphomia of B-cell type (CB, Table IV) had only 7 o

lymphocytes binding FITC-PNL, despitethe presence of complement receptors onmost B lymphocytes. In Patient JY(Table IV) with follicular lymphorna,PNL-binding cells were not detected.This biopsy sample was from a patienttreated for recurrent disease over 2 years.Her phenotype was atypical in 2 previousbiopsy samples, the tumour cells lackedC3d receptors and did not express light-chain Ig.

In the single case of CB/CC/diffuselymphoma (SD, Tables III & IV) little or

no binding of PNL could be shown, eitherby HRP-PNL in section, or by FITC-PNLin suspension. This patient was leukaemicin addition to showing nodal involvement,and blood lymphocytes on 9 repeat testsdid not bind PNL. One centrocytic lym-phoma (LC, Table IV) did not show

FITC-PNL positivity, though the SIg+C3d+ phenotype characteristic of theselesions (Habeshaw et al., 1979) was clearlyexpressed.

PNYL+ lynmphomias

Clear positive binding of PNL wasfound in 4/5 cases of follicular lymphoma(CB/CC/F), in plasmacytoma, and inlymphoblastic lymphoma of B-cell type. Ineach positive case studied in suspension,the positivity ranged from 20% to 97°, ofthe lymph-node cells. In tissue sections,positivity in follicular lymphoma (CB/CC/F) was restricted to the nodules, andwas not expressed on lymphocytes in

surrounding internodular tissue. All PNL+follicular lymphomas showed a B-cellprofile (SIg+ C3d+) characteristic of thislesion (Habeshaw et al., 1979). PNL posi-tivity was also present in B lymphoblasticlymphoma (LF, Table I V) in which com-plement receptors were not expressed. Noobvious correlation between SIg isotypeand PNL binding could be establishedfrom this series.

I)1SC USSION

iDiscussion of these results centresaround the PNL reactivity of follicularB-cell populations, and the usefulness ofFITC-PNL or HRP-PNL as markers inmalignant lymphoma.

It is clear from previous studies ofmouse and man that PNL binding isexpressed by cells of both T and Blineage. In T cells, PNL binding isrestricted to cortical lymphocytes inmouse and man, and to small numbers ofperipheral T cells in the mouse (Londonet al., 1978). However, whereas PNL bindsto 80% of murine thymocytes, and histo-logically this appears to correlate withall cortical thymocytes (Rose & Malchiodi,1981), the results presented here (TableV) and other reports (Reisner et al., 1979)suggest that PNL does not bind to allcortical thymocytes in man.Lymphocytes (presumptive B cells) in

germinal centres but not in the coronabind PNL. This has been demonstrated inmtouse (Rose et al., 1980) and here forhuman tonsil and other lymph nodes.Moreover, in the present series substantialnumbers of PNL+ cells were found con-currently with similar numbers of Ig+cells (Table IV). It is known that theperipheral lymphoid tissue of unstimu-lated mice contains <500 PNL+ cells,and that Ig+ cells are PNL- (Newman &Boss, 1980; Rose & Malchiodi, 1981).However, in the mitotically active Peyer'spatches, which contain germinal centresand 300o PNL+ cells, double-labellingstudies have revealed that 80% of thePNL+ cells bear surface Ig (Rose &

TABLE V.-PNVL positivity in thymrus1. Cell stuspel.si(ii

MI1arkersFITC-INLE rosettesOKT6HLA (XV6/32)Ta (D)a2)'TITSlu

73

74 MT. L. ROSE ET AL.

Malchiodi, 1981). It may be assumed thatin the present series of non-T-cell lym-phomas the majority of PNL+ follicularcells are also Ig+, and the question ariseswhether such cells share the other folli-cular phenotype of C3d positivity. It hasbeen shown in this paper that C3d posi-tivity does not always correlate with PNLpositivity. It may be that PNL positivityidentifies only one of the follicular B-cellsubsets. This subset can present as aSIg+ C3d- cell (as in Patient LF, TableIV) or as a cell expressing C3d receptors(as in Patients LP and GB, Table IV).One unexpected feature was the un-

equivocal positivity of a case of plasma-cytoma, in which the plasma-cell compo-nent of the lesion was PNL+. Plasma cells,like cortical thymocytes and germinal-centre lymnphocytes, are not activelyrecirculating lymphocytes. They may beonly temporarily sessile while in a certainstate of differentiation. We would like tosuggest that PNL may be a marker ofsessile lymphoid populations of T or Bclass. This may have important implica-tions in the prognosis of lymphoma, asthe tendency of PNL+ tumours to becomeleukaemic should be less marked than forPNL- tumours of equivalent phenotype.

This work wvas supported by grants to thoInstitute of Cancer Research, Royal Cancer Hos-

pital, from the Medical Research Council and theCancer Research Campaign. We are grateful toMarjorie Butt for typing the manuscript.

REFERENCES

HABESHAW, J., CATLEY, P. F., STANFIELD, A. G. &BREARLEY, R. L. (1979) Surface phenotyping,histology and the nature of non-Hodgkin'slymphoma in 157 patients. Br. J. Cancer, 40, 11.

LONDON, J., BERRIN, S. & BACH, J. F. (1978)Peanut agglutinin. I. A new tool for studying Tlymphocyte populations. J. Immunol., 121, 438.

NEWMAN, R. A. & Boss, M. A. (1980) Expression ofbinding sites for peanut agglutinin during murineB lymphocyte differentiation. Immunology, 40,193.

PEREIRA, AI. E. A., KABAT, E. A., LOTAN, R. &SHARON, N. (1976) Immunochemical studies in thespecificity of the peanut (Arachis hypogaea)agglutinin. Carbohydr. Res., 51, 107.

REISNER, Y., BINIAMINOV, M., ROSENTHAL, E.,SHARON, N. & RAMOT, B. (1979) Interaction ofpeanut agglutinin with normal human lympho-cytes and with leukaemic cells. Proc. Natl Acad.Sci. U.S.A., 76, 447.

REISNER, Y., LINKER-ISRAELI, M. & SHARON, N.(1976) Separation of mouse thymocytes into twosubpopulations by the use of peanut agglutinin.Cell. Immunol., 25, 129.

ROELANTS, G. E., LONDON, J., MAYOR-WITHEY,K. S. & SERANO, B. (1979) Peanut agglutinin. II.Characterisation of the Thy 1, T]a and Ig pheno-type of peanut posive cells in adult, embryonic andnude mice using double immunofluorescence.Eur. J. Immuntol., 9, 139.

ROSE, M. L., BIRBECK, AM. S. C., WTALLIS, V. J.,FORRESTER, J. A. & DAVIES, A. J. S. (1980)Peanut lectin binding properties of germinalcentres ofmouse lymphoid tissue. NVature, 284, 364.

ROSE, M. L. & MALCHIODI, F. (1981) Binding ofpeanut lectin to thymic cortex and germinalcentres of lymphoid tissue. Immuniology, 42, 583.