binding ofpeanut lectin to thymic cortex and germinal centres of

Immunology, 1981 42 583

Binding of peanut lectin to thymic cortex and germinal centres of lymphoid tissue

MARLENE L. ROSE & FIORELLA MALCHIODI* Chester Beatty Research Institute, Institute ofCancer Research, Royal Cancer Hospital, London

Acceptedfor publication 21 October 1980

Summary. The binding pattern of horseradish peroxi-dase conjugated peanut lectin (HRP-PNL) on frozensections of lymphoid tissue from man, mouse, rat,hamster, guinea-pig, rabbit, sheep and chicken hasbeen investigated. Binding of PNL was found to behighly species dependent; man, mouse and sheepshowed strong binding to lymphocytes in thymic cor-tex and germinal centres; lymphoid tissue from hams-ter, guinea-pig and rabbit did not stain withHRP-PNL and rat showed only lightly positive cells inthymic cortex and germinal centres; all lymphoidtissue from chicken, except the bursal cortex, boundPNL. Neuraminidase treatment of tissues which didnot bind PNL resulted in strongly PNL-positive cells.Double binding studies on murine Peyer's patcheswith fluorescein isothiocyanate conjugated PNL(FITC-PNL) and tetramethylrhodamine isothio-cyanate (TRITC)-anti-Thy- 1 2 or anti-immunoglobu-lin reagents revealed 3%-10% of PNL positive cells tobe Thy-1i2 positive and 70%-80% to bear surfaceimmunoglobulin.

INTRODUCTION

The binding ofpeanut lectin (PNL) to lymphoid tissue* Present address; Instituto di Anatomia ed Istologia Pato-

logica II, Policlinico Umberto I, Viale Regina Elena, Roma,Italy.

Correspondence; Dr M. L. Rose, Chester Beatty ResearchInstitute, Institute of Cancer Research, Royal Cancer Hospi-tal, Fulham Road, London SW3 6JB.00 1 9-2805/81/0400-0583$02.00t91981 Blackwell Scientific Publications

is currently a matter of interest to those concernedwith lymphocyte differentiation. Using fluoresceinisothiocyanate conjugated PNL (FITC-PNL) on cellsuspensions from murine tissues, it has been foundthat PNL binds to 65%-785% thymocytes and to smallnumbers (<20%) of cells in bone marrow, spleen,peripheral blood and lymph nodes (London, Berrih &Bach, 1978; Roelants, London, Mayor-Withey & Ser-ano, 1979). PNL-positive cells outside the thymushave been reported to be Thy- 1 positive or null cells(London et al., 1978; Roelants et al., 1979). I.t has beenshown that PNL-binding cells from mouse bone mar-row are enriched in spleen-colony-forming units(Reisner, Itzinovitch, Meshorer & Sharon, 1978). Thisobservation together with the finding that the PNL-binding thymocyte population is cortisone sensitive(London et al., 1978) and does not respond to PHA(Reisener, Linker-Israeli & Sharon, 1976) has led tothe suggestion that PNL binding is a marker for imma-turity among cells of the T-cell and haemopoietic-stem-cell series (London et al., 1978; Reisner et al.,1978). Studies on cell suspensions suffer the disadvan-tage that spatial relationships between cells are lost,thus valuable additional information may be gainedby using frozen tissue sections. The binding pattern ofhorseradish peroxidase conjugated PNL (HRP-PNL)on cryostat sections ofmouse lymphoid tissue revealedthat lymphocytes in germinal centres bind PNL (Rose,Birbeck, Wallis, Forrester & Davies, 1980). This find-ing in conjunction with the recent finding that somecells containing cytoplasmic immunoglobulin bindPNL suggests that some B cells bind PNL and raisesthe question whether these cells are immature.

583

Marlene L. Rose & Fiorella Malchiodi

Here the binding of HRP-PNL to cryostat sectionsof lymphoid tissue from man, mouse, rat, hamster,guinea-pig, rabbit, sheep and chicken have been inves-tigated. The results demonstrate that binding ofPNLis highly species dependent and places PNL binding insome biological perspective. Fortuitously mouse andman appear to show a similar pattern of binding,namely to the thymic cortex and to germinal centres.The phenotype of PNL-binding cells in murine

Peyer's patches has been investigated using doublelabelling with FITC-PNL and tetramethylrhodamineisothiocyanate (TRITC) labelled anti-Thy-[-2 or anti-immunoglobulin reagents. The results show that only3%-10% of PNL-positive cells are Thy-1i2 positive,and that 70%-80% of PNL-positive cells bear surfaceimmunoglobulin.

MATERIALS AND METHODS

AnimalsThe following animals were used: 8-10-week-old malemice of the inbred strain CBA/Ca (bred in the ChesterBeatty Research Institute, London), 6-8-week-oldmale rats Wistar/P.Cbi (bred in the Chester BeattyResearch Institute, London), 10-week-old male NewZealand white rabbits (from Buxted Rabbits Ltd,Great Totease Farm, Uckfield, Sussex), 10-week-oldmale Durkin-Hartley guinea-pigs (from Porcelus,Heathfield, Sussex), 8-week-old male hamsters (CBRICream Syrian, bred in the Chester Beatty ResearchInstitute, London) and 6-week-old chickens (fromOrchards Farm, Kingshill, Great Missenden, Bucks).Lymphoid tissue from sheep was kindly provided byDr J. G. Hall of this Institute.

Human materialTonsils were from St Bartholomew's Hospital, Lon-don by arrangement with Dr J. Habeshaw.

LectinsPeanut (Arachis hypogaea) lectin (PNL) was preparedaccording to the method of Lotan, Skutelsky, Danon& Sharon (1975) as previously described (Rose et al.,1980). Ricinus lectin (RCA120, from Ricinus communis)a kind gift from Dr. J. A. Forrester ofthis Institute wasprepared essentially by the method of Nicolson &Blaustein (1972). The lectins were conjugated to hor-seradish peroxidase or to fluorescein isothiocyanate aspreviously described (Rose et al., 1980).

Binding ofHRP-PNL tofrozen sectionsLymphoid tissue was frozen in liquid nitrogen and 4pm thick sections were cut. The sections were air driedfor 10 min and a few drops ofHRP-PNL (final concen-tration 20 pg/ml in PBS) was added to each section andincubated at room temperature for 30 min. The sec-tions were washed three times in PBS and developed in3, 4, 3', 4-tetraaminobiphenyl hydrochloride (BDH).After washing again in PBS the sections were dehyd-rated and mounted.

Cell suspensionsCell suspensions from spleen or mesenteric lymphnode were prepared by gently squeezing the tissuethrough stainless steel sieves into cold medium 199.Clumps were allowed to settle and cells in the superna-tant were washed three times. Viability was assessed byexclusion of 0-2% trypan blue. Peyer's patches werecarefully excised from the small intestine and werewashed intact by vigorous shaking in a 10-ml syringewith 8 ml ofmedium 199. This procedure was repeateduntil the patches appeared free of adhering gut con-tents. They were then gently squeezed through stain-less steel sieves into medium 199 containing 10% FCSand clumps were allowed to settle for 10 min at roomtemperature. The cells in the supernatant wereremoved and washed in medium 199 containing 10%FCS.

Neuraminidase treatmentSections or cells were treated with neuraminidasebefore incubating with HRP-PNL or FITC-PNL.Neuraminidase (from Vibrio-cholerae, BehringwerkeA.G., Marburg/Lahn, W. Germany, 1 u./ml) at 0-01,0 005 or 0-001 u./ml in medium 199 were added tofrozen sections for 30 min at room temperature. Thesections were washed in PBS and stained withHRP-PNL. Lymphocytes (5x 107/ml) were treatedwith neuraminidase at 0 05, 0-01, 0-005 or 0-001 u./mlin medium 199 for 15 min at 37°. The cells were washedthree times in cold PBS before staining withFITC-PNL.

Staining of cells with FITC-PNLErythrocytes were prepared from heparinised bloodand washed three times in PBS before use. Onehundred microlitres of lymphocytes or erythrocytes at107/ml in PBS containing 0-01% sodium axide weremixed with l00pI of FITC-PNL in PBS/axide (50 pgPNL/ml). After 30 min incubation at 370 the cells werewashed three times and resuspended in 100 pl of

584

Peanut lectin binding to lymphoid tissue

PBS/azide. Cells were examined under fluorescenceoptics using a Zeiss epifluorescence condenser IV/F onstandard 18 microscope. Only viable cells with strongmembrane fluorescence were scored as positive. Twohundred cells per sample were examined.

Double staining ofcells with PNL and anti-immunoglo-bulin or anti-thy-1-2 reagentsOne hundred microlitres of lymphocytes from Peyer'spatches (107/ml) were mixed with 50 p1 of FITC-PNLin PBS (100 ,ug/ml) and either 50 pl of rabbit anti-mouse immunoglobulin (diluted 1/8, polyvalent anti-serum from Miles Laboratories) or 50 p1 of PBScontaining 2 pg of biotin-conjugated anti-thy- 1-2(Becton-Dickinson, Calif.) and left on ice for 30 min.The cells were washed twice and resuspended in 100 p1of PBS. They were further mixed with 50 pl ofFITC-PNL(50 pg/ml in PBS) and either TRITC-labelled goat anti-rabbit immunoglobulin (diluted 1/8,Nordic Laboratories) or 50 p1 of PBS containing 2 jugof TRITC-labelled avidin (Bectin-Dickinson, Calif.)and left on ice for 30 min. They were washed threetimes in PBS and resuspended in 100 pl of PBS beforeexamination under fluorescent optics.

Control experiments using unlabelled PNL revealedthat PNL does not interfer with the binding of anti-immunoglobulin or anti-thy- I 2 to the cell surface.

RESULTS

Binding ofPNL to frozen sections of lymphoid tissue ofdifferent speciesThe binding ofHRP-PNL to the thymus-, spleen- andgut-associated lymphoid tissue of seven differentspecies is shown in Table 1.The pattern ofPNL binding is clearly species depen-

dent. Man, mouse, and sheep appear to be similar;lymphocytes in the thymic cortex are positive (Fig. 1)and so are lymphocytes in the germinal centres oflymphoid tissue in human tonsil (Fig. 2), murinePeyer's patches (Fig. 3) and sheep mesenteric lymphnode (Fig. 4). So far we have no evidence that germinalcentres in different organs bind PNL to differentdegrees. The corona or mantle of small lymphocytessurrounding the germinal centre is clearly not bindingPNL (Figs 2-4). However some interfollicular den-dritic cells are positive (Figs 2 and 3) as are Hassall'scorpuscles in human and murine thymus, and there arestrongly-positive eosinophils or macrophages in sheep(Fig.4). A second group of animals (hamster, guinea-

pig and rabbit) failed to show binding of PNL tolymphocytes in the lymphoid organs studied, althoughsome dendritic cells, epithelial cells and smooth musclewere slightly stained. In contast, all the lymphocytes ofthe chicken thymus, spleen and caecal tonsil appearedto bind PNL. In the bursa of Fabricius, the medullabut not the cortex was positive (Fig.5). It is interestingthat the basement membrane associated epithelium atthe cortico-medullary border is strongly positive as isthe basement membrane of the plical epithelium. Thebinding of HRP-PNL to rat thymic cortex and ger-minal centres was very weak compared with mouse,man and sheep. The ability of erythrocytes from thevarious species to bind FITC-PNL was also examined(Table 1). Only chicken and rabbit proved PNL posi-tive. Thus, there was no correlation between PNLbinding of lymphocytes and erythrocytes.

Specificity of lectin binding to murine germinal centres

PNL binds to a terminal non-reducing galactose resi-due. However, the sugar inhibition studies of Pereira,Kabat, Lotan & Sharon (1976) show that certain dis-accharides are bound with much greater affinity by thelectin than is galactose alone. In particular the disac-charide D-galactose #1 -+3 DN-acetylgalactosamine isover 50 times more effective (on a molar basis) thanD-galactose as an inhibitor of glycoprotein precipi-tation by PNL. We have previously reported that PNLbinding to germinal centres is abrogated in the pre-sence of 0 IM galactose but not 0 IM glucose (Rose etal., 1980). Ricinus lectin (RCA120, from R. communis)also binds to galactose but it was found that HRP-ricinus lectin bound to all lymphocytes in Peyer'spatches and it did not distinguish the germinal centresas does PNL. Binding of HRP-ricinus was abrogatedby 0 IM galactose but not 0- IM glucose.Thus the interaction of PNL with galactose alone

appears not sufficiently strong to give a staining reac-tion under the conditions here described. The require-ments for staining are probably that terminal galac-tose residues are in a 1-.3# linkage with N-acetylD-galactosamine in an oligosaccharide array.

Effect of neuraminidase on tissue sections from hamster,guinea-pig and rabbitThe lack of binding of PNL to frozen sections oflymphoid tissue from hamster, guinea pig or rabbitdenotes either that the PNL-binding sugars arecovered, or that they are not present on the cell sur-

585


Table 1. Summary of binding pattern ofPNL on lymphoid tissue and erythrocytes of different species

Lymphoid areas which bind HRP-PNL FITC-PNL

Group Species Thymus Gut-associated lymphoid tissue Peripheral lymphoid tissue Erythrocytes

I Man Cortex Tonsil-GC* Axillary lymph node-GC NegativeMouse Cortex Peyer's patch-GC Spleen-GC Negative

Lymph nodes-GCSheep Cortex Not done Mesenteric lymph node-GC Negative

II Hamster All negative Peyer's patch negative Spleen, mesenteric lymph Negativenode negative

Guinea-pig All negative Peyer's patch negative Spleen, lymph node Negativenegative

Rabbit All negative Peyer's patch negative, Spleen, lymph node Positiveappendix negative negative

III Chicken Cortex and medulla Bursal medulla, caecal tonsil Spleen PositiveIV Rat Cortex slightly positive Peyer's patch, GC slightly Mesenteric lymph node, Negative

positive GC, slightly positive

*GC = germinal centre.The binding of HRP-PNL to cryostat sections is as described in Materials and Methods. In all instances except man and

sheep at least three animals ofeach species were examined. Six tonsils and one human thymus, and lymphoid tissue from onesheep was investigated. In each experiment a positive control for this technique (mouse thymus) was included.

_u i ._

FIgure 1. Binding ofHRP-PNL to mouse thymus, x 288. C, cortex; M, medulla.

586


~: itV: ., , 1... a,I .,X - # I -.s

ie r~, .. >

A~~~~~~~

COFigure 2: Binding of HRP-PNL to human tonsil, x 198. GC, germinal centre; CO, corona; arrow points to interfolliculardendritic cells.

face. The disaccharide sequence D-galactose ,B1 -p3 DN-acetylgalactosamine is often masked by sialic acid.To distinguish between these possibilities frozen sec-tions were treated with neuraminidase for 30 min atroom temperature before PNL staining. The effect of

neuraminidase was concentration dependent, above0 005 u./ml all lymphocytes, indeed all cells becamestrongly positive. Thus it appears in those specieswhich are apparently PNL negative the galactose resi-dues to which PNL binds are covered by sialic acid.

Figur3.BndinofHPPNLtomusePyerpatc ,x205.O,germinacentre;O,corona

F'iglue 3. Binding of HRP-PNL to mouse Peyer's patch, x 205. GC, gerTninal centre; CO, corona.

587


,. _ .

.W T...-401

t.

al, .l a

Figure 4 Binding of HRP-PNL to sheep mesenteric lymph node, x 304. GC, germinal centre.

Presence of immunoglobulin and Thy-1-2 on the surfaceof PNL-positive cells

Surface Ig. Cell suspensions ofmouse Peyer's patchlymphocytes were doubly labelled with FITC-PNLand rabbit anti-mouse Ig (polyspecific) followed by

TRITC-anti-rabbit. The number ofPNL-binding cellsand the number of cells bearing surface Ig was esti-mated (Table 2). The cells staining brightly withFITC-PNL were then examined for TRITC staining.It was found that about 20% of FITC-PNL bindingcells bore no TRITC (that is surface Ig) label and the

Figure 5. Binding of HRP-PNL to bursa of Fabricius, x 250. C, cortex; M, medulla; arrow points tojunction; arrow points to PE, plical epithelium.

,corticomedullary

588


Table 2. Binding of PNL, anti-Ig and anti-Thy-1-2 to Peyer'spatch lymphocytes and the binding of anti-Ig and anti-Thy-I-2to PNL + cells

Positive binding to Peyer's Positive binding toReagents patch lymphocytes (%) PNL+ cells (%)

PNL 24-4, 29-0, 35-0Anti-Ig 60 0, 47 0, 55-0 78.0, 80-0, 70 0Anti-Thy-1-2 24-0, 14-0 3 4, 10-0

Values are individual results from experiments done on dif-ferent days.

Table 3. Effect of neuraminidase on the percentage of FITC-PNL binding lympho-cytes

Concentration of neuraminidase u./ml

Mouse Organ 0 0 001 0 005 0-01 0 05

CBA Peyer's patch 28-0 40-0 95-0 ND * NDSpleen 4 0 34-0 ND > 95-0 > 95-0Mesenteric LN 7 0 42-0 99.0 > 95 0 99 0

BALB/c Peyer's patch 20-0 ND > 95-0 > 95 0 > 95-0Spleen 4 0 32-0 > 95 0 > 95-0 ND

ND = not done.Values are the mean from duplicate samples.

remaining FITC-PNL positive cells although positivefor TRITC were only lightly labelled. When cells stain-ing strongly with TRITC (that is, cells strongly Igpositive) were examined for FITC (PNL) binding,only 2% bore PNL.

It appears therefore that the large majority ofPNL-binding cells in Peyer's patches are Ig positive but thestaining with polyspecific anti-Ig reagent is patchy andweak compared with the bright ring fluorescence ofthePNL-negative cells.

Thy-1-2. Cell suspensions of mouse Peyer's patcheswere doubly labelled with FITC-PNL and biotin-con-jugated anti-Thy-1 -2 followed by TRITC-avidin. Thenumber of PNL-binding cells and the number of cellsbearing Thy-1-2 was estimated (Table 2). The cellsstaining brightly with FITC-PNL were then examinedfor TRITC staining and 3%-10% were positive. Itappears therefore that a minority ofPNL-positive cellsin Peyer's patches bear Thy-i -2, and that at least 90%of Thy- 1-2 positive cells do not bind PNL.

Effect of neuraminidase on B cells

The above results show that cells staining strongly forsurface Ig are almost entirely PNL negative. Neura-minidase treatment before incubation with FITC-PNL was found to increase the number ofcells bindingFITC-PNL in a concentration dependent manner(Table 3). Thus at low concentrations of neuramini-dase (0-001 u./ml) 40% and 34% of Peyer's patch andspleen lymphocytes were found to bind FITC-PNL.However, at and above 0 005 u./ml, neuraminidasetreatment resulted in >95% very bright and aggre-gated PNL-binding cells. Similar results were obtainedusing lymphoid tissue from BALB/c mice. It appearstherefore that if high enough concentrations of neura-minidase are used all lymphocytes bind FITC-PNL.

DISCUSSION

The biological significance of PNL binding is un-known. Clearly, PNL is useful as a probe to detect the

589


sequence of sugars (galactose,I 3 D N-acetylgalac-tosamine) which bind PNL. Whether the presence ofsuch sugars on the cell surface denotes functionalimmaturity however is a question which requires criti-cal investigation. Here we have sought to view PNLbinding in biological perspective by studying the dis-tribution of HRP-PNL on cryostat sections of lym-phoid tissue from animals of different species.

Seven different mammals and one bird were studied.Two interesting points emerge from this particularstudy. One is that PNL binding is highly species depen-dent. The second is that the mammals which bind PNLstrongly (man, mouse and sheep) show the same pat-tern of binding, namely PNL binds predominantly tothymic cortex and germinal centres. That it is corticaland not medullary thymocytes which bind PNLaccords with previous reports that the PNL-bindingpopulation of murine and human thymis is cortisonesensitive and unresponsive to PHA (Reisner et al.,1976; London et al., 1978; Reisner, Biniaminov,Rosenthal, Sharon and Ramot, 1979). We have pre-viously reported that PNL binds to lymphocytes inmurine germinal centres (Rose et al., 1980). The pre-sent report that it binds similarly to human germinalcentres suggests that the 10%-15% of large PNL-bind-ing cells recovered in cell suspension from humantonsil by Ballet, Fellous, Sharon, Reisner & Agrapart(1980) are germinal centre cells. The observation thatlymphoid tissue from Wistar rats binds PNL onlyweakly and that the hamsters, guinea-pigs and rabbitsused in this study do not bind PNL may reflect a strainrestriction rather than a species restriction. However,there is as yet no published evidence of strain differ-ences in murine lymphoid tissue with regard to PNLbinding. It is curious that the binding pattern ofPNLin the chicken is a mirror image of that found inmouse, man and sheep namely all lymphocytes exceptthe bursal cortex bind PNL. Lymphocytes in bursalcortex are said to be less mature than those in themedulla (Grossi, Lydyard & Cooper, 1977). Thechicken thus demonstrates that PNL binding is not amarker of functional immaturity per se. These obser-vations do not exclude the possibility that PNL bind-ing may be a marker for immaturity in the mammalswhose tissues bind PNL.The fact that PNL binds strongly to lymphocytes in

thymic cortex and germinal centres perhaps suggestsbiological affinity. Indeed these two structures shareseveral biological features; their cells are mitoticallyactive, cell death is common and the cells are outsidethe pool of recirculting lymphocytes. All these possibi-

lities should be considered as reasons for PNL-positi-vity before the idea is accepted that it reflects commonmembrane perturbations concerned with maturation.PNL-positivity cannot however be an indispensableconsequence ofcell division because bone marrow andfoetal liver, both active sites of cell division, are < 5%and 16% respectively PNL positive (London et al.,1978; Rose et al., 1980). Also stimulation of spleencells in vitro with mitogen did not result in an obviousincrease in PNL positivity (Rose et al., 1980) but itmay be that PNL-positivity is transiently expressedduring a small and specific part of the cell cycle.The binding of PNL to lymphocytes within ger-

minal centres provides an opportunity to study thesurface properties of the lymphocytes in cell suspen-sion using a double marker system. Such studies onPeyer's patches reveal 70%-80% to be surface Ig posi-tive and 3%-10% are thy-1 2 positive. It is interestingthat the PNL-binding cells which bind anti-immuno-globulin antiserum show only patchy weak staining.This is unexpected in view of the fact that, like Weiss-man (1975), we find strong binding of anti-immuno-globulin antiserum to germinal centres of frozen sec-tions of Peyer's patches. This apparent discrepancysuggests the binding on tissue sections may be due toantigen-antibody complexes on the surface of dendri-tic reticulum cells. The weak Ig positivity of germinalcentre lymphocytes may reflect either the fact they area rapidly dividing population or that they are in adifferent state of maturation from lymphocytes out-side the germinal centre. The present results confirmprevious reports (London et al., 1978; Roelants et al.,1979; Newman & Boss, 1980) that strongly Ig-positivecells are PNL negative. The failure of Ig-positive lym-phocytes to bind PNL can either be due to masking ofthe PNL-binding sugars by sialic acid or by absence ofthe relevant sugars. Unlike Newman & Boss (1980) wefind that treatment of lymphocytes (either as a cellsuspension or in frozen sections) with neuraminidasemakes all lymphocytes bind PNL strongly, but theeffect is concentration dependent. Thus at low concen-trations (0-001 u./ml) only a proportion of lympho-cytes become PNL positive. This effect may representdifferential resistance or accessibility of sialic acid onthe surface of T and B cells to neuraminidase. Weconclude that all lymphocytes bear the appropriatesugars to bind PNL but that in the majority of lym-phocytes it is not available because of further substitu-tion by sialic acid.The suggestion that PNL binding is a marker for

immature T cells and haemopoietic stem cells (London

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Peanut lectin binding to lymphoid tissue 591

et al., 1978; Reisner et al., 1979) clearly needs to bemodified in view of the results reported here and thefinding that cells containing cytoplasmic immunoglo-bulin but not surface immunoglobulins, presumptivepre-B cells, bind PNL (Newman & Boss, 1980). It mustbe remembered that many non-lymphoid elementsshow strong membrane binding to PNL and it is diffi-cult to describe them as immature. For example thepresent study has revealed PNL binding to some inter-follicular dendritic cells, epithelial cells in the thymus,macrophages, some endothelial cells and smooth mus-cle. It is likely that a number of structurally differentmacromolecules can bind PNL (Alexander, Living-stone, Yates & Sage, 1978) and the fact that diverse celltypes bind PNL does not necessarily denote biologicalaffinity. It is necessary to establish whether they sharethe same PNL-binding macromolecule. There aremany anti-galactosyl lectins (Uhlenbruck, Vaith, Kar-duck, Haupt & Muller, 1979) yet we have shown thatonly PNL and not ricin distinguishes germinal centresfrom the surrounding lymphocytes. This resultdemonstrates that the interaction of PNL with galac-tose alone is not sufficiently strong to allow discri-mination of germinal centres and that interation withthe disaccharide is probably necessary. In particular itis of interest to those concerned with B-cell differentia-tion to discover whether the PNL-binding macro-molecule on pre-B cells is the same as that found onlymphocytes in the germinal centre.

ACKNOWLEDGMENTS

We are very grateful to Professor A. J. S. Davies andDr J. A. Forrester for their helpful comments on thismanuscript. This work was supported by the MedicalResearch Council.

REFERENCES

ALEXANDER S.S., LIVINGSTONE L.R., YATES L.P. & SAGE H.J.(1978) The binding of lectins to components of plasmamembranes from porcine submaxillary lymph node lym-phocytes. Biochim. biophys.Acta, 512, 350.

BALLETT J.J., FELLOUS M., SHARON N., REISNER Y. & AGRA-PART M. (1980) Reactivity of human lymphoid and lym-phoblastoid cells with peanut agglutinin: detection of ablood cell subset which lacks detectable membrane HLA.Scand.J.Immunol. 11, 555.

GROSSI C.E., LYDYARD P.M. & COOPER M.D. (1977) Onto-geny of B cells in the chicken. II. Changing patterns ofcytoplasmic IgM expression and of modulation require-ments for surface IgM by anti-p antibodies. J.Im-munol,119, 749.

LONDON J., BERRIH S. & BACH J.F. (1978) Peanut agglutinin.I. A new tool for stuyding T lymphocyte populations.J.Immunol. 121, 438.

LOTAN R., SKUTELSKY E., DANON D. & SHARON N. (1975)The purification, composition and specificity of the anti-Tlectin from peanut (Arachis hypogaea). J.Biol.Chem. 250,8518.

NEWMAN R.A. & Boss M.A. (1980) Expression of bindingsites for peanut agglutin during murine B lymphocytedifferentiation. Immunology, 40, 193.

NICOLSON G.L. & BLAUSTEIN J. (1972) The interaction ofRicinus communis agglutinin with normal and tumour cellsurfaces. Biochim.biophys.Acta. 266, 543.

PEREIRA M.E.A., KABAT E.A., LOTAN R. & SHARON N. (1976)Immunochemical studies on the specificity of the peanut(Arachis hypogaea) agglutinin. Carb.Res. 51, 107.

REISNER Y., LINKER-ISRAELI M. & SHARON N. (1976) Sepa-ration of mouse thymocytes into two subpopulations bythe use of peanut agglutinin. Cell.Immunol. 25, 129.

REISNER Y., ITZINOVITCH L., MESHORER A. & SHARON N.(1978) Haemopoietic stem cell transplantation usingmouse bone marrow and spleen cells fractionated bylectins. Proc. natn. Acad. Sci. U.S.A. 75, 2933.

REISNER Y., BINIAMINOV M., ROSENTHAL E., SHARON N. &RAMOT B. (1979) Interaction of peanut agglutinin withnormal human lymphocytes and with leukemic cells.Proc. natn.Acad.Sci. U.S.A. 76, 447.

ROELANTS G.E., LONDON J., MAYOR-WITHEY K.S. & SERANOB. (1979) Peanut agglutinin. II. Characterisation of theThy. I Tla and Ig phenotype of peanut positive cells inadult, embryonic and nude mice using double fluores-cence. Europ.J.Immunol. 9, 139.

ROSE M.L., BIRBECK M.S.C., WALLIS V.J., FORRESTER J.A. &DAvIES A.J.S. (1980) Peanut lectin binding properties ofgerminal centres of mouse lymphoid tissue. Nature, 284,364.

UHLENBRUCK G., VAITH P., KARDUCK D., HAUPT H. &MULLER W.E.G. (1979) Anti-galactan lectins. In: Pro-tides of Biological Fluids, 27th Colloquium. (Ed. byH.Peeters), p.595. Pergamon Press, Oxford.

WEISSMAN I.L. (1975) Development and distribution of im-munoglobulin-bearing cells in mice. Transplantn.Rev. 24,159.

binding ofpeanut lectin to thymic cortex and germinal centres of

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