Immunology 1996 89 120-125

Identification of a novel dendritic cell surface antigen defined by carbohydratespecific CD24 antibody cross-reactivity

L. A. WILLIAMS, A. D. MCLELLAN, K. L. SUMMERS, R. V. SORG, D. B. FEARNLEY& D. N. J. HART. Haematology/Immunology Research Group, Christchurch School of Medicine and Christchurch Hospital,

Christchurch, New Zealand

SUMMARY

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers andstimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen(HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus,skin and spleen. The human HSA homologue, CD24, is predominantly expressed by Blymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes hasnot yet been described. We have examined the expression of CD24 on human peripheral blood DCpopulations isolated directly or following in vitro culture. The CD24 antigen was absent fromblood DC; however, cross-reactive, sialylated carbohydrate epitopes were detected on DC withsome CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen,which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation/differentiation in vitro.

INTRODUCTION

Dendritic cells (DC) are potent stimulators of T-lymphocyteactivation but the properties or cell surface molecules specific toDC which contribute to this activity are ill defined. The mouseheat stable antigen (HSA) acts as a co-stimulatory molecule'and is expressed by DC obtained from blood,2 skin,3 liver4 andspleen.5 The human HSA homologue was identified as theCD24 antigen, a predominantly B-lymphocyte associatedsurface antigen also expressed by granulocytes.6 The CD24antigen has extensive carbohydrate structures attached to asmall, glycosyl-phosphatidylinositol (GPI)-anchored proteincore. Numerous CD24 monoclonal antibodies (mAb) havebeen described which identify several different protein core orcarbohydrate structure epitopes present on the molecule.79Selective expression of specific CD24 epitopes on differenthaemopoietic cells including plasma cells,'0 Sezary cells'" andactivated T lymphocytes12 suggested potential tissue specific

Received 5 March 1996; revised 21 May 1996; accepted 23 May1996.

Abbreviations: DC, dendritic cell; Lin, lineage marker; mAb,monoclonal antibody; GPI, glycosyl-phosphatidylinositol; PI-PLC,phosphatidylinositol-specific phospholipase C; HSA, heat stableantigen; ER, sheep erythrocyte; FITC, fluorescein isothiocyanate; PE,phycoerythrin; MFI, mean fluorescence intensity; MLR, mixedleucocyte reaction.

Correspondence: Professor D.N.J. Hart, Clinical Haematology,Christchurch Hospital, PO Box 151, Christchurch, New Zealand.

variations in CD24 glycosylation. The expression and functionof the CD24 antigen on human DC has received little attentionto date. Although the CD24 mAb, BA-I did not react withtonsil DC,13 binding of themAb VIB-E3 was reported oncultured DC isolated from human skin or differentiated fromCD34+ progenitors.'415 Both of these mAb have demonstratedspecificity for sialylated carbohydrate structures8'9 as part oflarger mucin-like structures bound to the protein core, whichreacts with the protein core specific CD24mAb SWA-l 1 andALB-9.7 We have identified cross-reactivity of the carbohy-drate epitope-specific CD24mAb VIB-E3, BA-i and HB-9with a cell surface molecule(s) termed CD24cre, expressed by Tlymphocytes and some human cell lines (Williams et al.,in press). These failed to stain with CD24 protein core-specificmAb SWA-11 and ALB-9 and revealed no CD24mRNA. The cross-reactive antigen(s) CD24cre was, like theCD24 antigen, sensitive to phosphatidylinositol-specific phos-pholipase C (PI-PLC) or non-specific protease, and mAbbinding was inhibited by bovine submaxillary mucin, indicatinga carbohydrate epitope expressed on a GPI-anchored protein.The expression of CD24 antigen on human peripheral bloodDC populations was therefore analysed using protein core andcarbohydrate epitope-specific CD24 mAb. Human blood DCexpressed no CD24 surface protein but CD24 carbohydrateepitope specificmAb reacted with a subpopulation of directlyisolated blood DC. This reactivity may identify a moleculedistinct from CD24cre reported on T lymphocytes and thisantigen has been provisionally designated DC-24.

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CD24 mAb cross-reactivity on dendritic cells

MATERIALS AND METHODS

Monoclonal antibodiesThemAb CMRF-31 (CD 14, immunoglobulin G2a (IgG2a)) andCMRF-42 (immunoglobulin M, IgM) were produced andcharacterized in this laboratory. The FMC-63 hybridoma(CD19, IgG2a) and the isotype controlmAb X63 (IgGl), Sal4(IgG2b) and Sal5 (IgG2a) were a gift from Dr H. Zola (FlindersMedical Centre, Adelaide, Australia) and HuNK-2 (CD16,IgG2a) and HuLym3 (CD48, IgG2a) from Prof I.F.C. McKen-zie (Austin Research Institute, Melbourne, Australia). TheOKT3 (CD3, IgG2a), L243 anti-(HLA-DR, IgG2a) and OKM 1(CD 1 Ib, IgG2b) hybridomas were obtained from the AmericanType Culture Collection (ATCC, Rockville, MD). TheCD24mAb SWA- 11 (IgG2a) was a gift from Dr E. Weber(University of Zurich, Zurich, Switzerland), VIB-E3 (IgM)from Dr W. Knapp (University of Vienna, Vienna, Austria)and HB-9 (IgM) from Dr T. Tedder (Duke University,Durham, NC). Phycoerythrin (PE)-conjugated anti-HLA-DRand isotype control mAb were purchased from Becton Dickin-son (Mountain View, CA).

Cell preparationPeripheral blood mononuclear cells (PBMC) were obtainedfrom buffy coats of whole blood by Ficoll/Hypaque (Pharmacia,Uppsala, Sweden) gradient separation. PBMC were depleted ofT lymphocytes by rosetting with neuraminidase-treated sheeperythrocytes (ER). DC were isolated from this ER- PBMCpopulation directly by depletion of lineage marker (CD3,CD Ilb, CD14, CD16, CD19) positive cells (lin+) using theMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany)or by sorting on a fluorescence activated cell sorter (FACS)Vantage (Becton Dickinson) (16,17). Alternatively, ER- PBMCwere subjected to 16 hr in vitro culture (2 x 107 cells/mL),followed by enrichment of low density cells via Nycodenz(d = 1-068 g/cm3; Nycomed Pharma, Oslo, Norway) gradientcentrifugation, prior to depletion of lin+ cells as described above,to obtain a more mature or activated DC population.17"18

In some experiments, directly isolated DC were cultured inmedium (RPMI-1640 medium supplemented with 10% fetalcalf serum, 2mM L-glutamine, 100 pg/mL streptomycin and60 U/mL penicillin (Life Technologies, Auckland, NZ), or withthe addition of interferon-7y (IFN-y; 500 U/mL, Boehringer-Ingelheim, Ingelheim, Germany) or granulocyte-macrophagecolony-stimulating factor (GM-CSF; 500 U/mL, Sandoz Pharma,Basel, Switzerland). These populations were examined bytrypan blue exclusion to establish viability following the cultureperiod.

ImmunostainingCells (5 x 104/test in 0 5% bovine serum albumin/phosphate-buffered saline (BSA/PBS) were incubated with primarymAbfollowed by fluorescein isothiocyanate-conjugated sheep anti-mouse-immunoglobulin (FITC-SAM-Ig; Silenus Labora-tories, Hawthorn, Australia) prior to analysis on a FACSVantage. Dual labelling utilized primary incubations with mAband FITC-SAM-Ig, followed by incubation (O min) on ice in10% mouse serum prior to addition of PE-conjugated mAb.Results are presented as four-log cycles of arbitrary fluores-cence units versus cell number or four-log scale dot plots ofFITC versus PE fluorescence. In some cases, histograms of

fluorescence measurements within gated HLA-DR+ cells areshown.

Enzyme digestion and inhibition studiesSamples of 2-5 x 105 directly isolated DC were incubated in1 mL PBS with either 500 jyg/mL pronase (Calbiochem, SanDiego, CA) or 1 U/mL phosphatidylinositol-specific phos-pholipase C (PI-PLC, Boehringer-Mannheim, Mannheim,Germany) at 370 for 60min. Following enzyme treatment,cells were washed twice in PBS and stained as described above.Results are expressed as percentage decrease of mean fluores-cence intensity (MFI) or percentage of cells positive afterenzyme digestion. Inhibition studies were carried out byimmunofluorescent labelling (as described above) in thepresence or absence of l0-Ogg or 100 jg bovine submaxillarymucin9 (Sigma, St Louis, MO). Results are also expressed aspercentage decrease in MFI or positive cells.

Mixed leucocyte reaction (MLR)Directly isolated lin- DC were labelled with CD24mAb HB-9,followed by incubation with FITC-SAM-Ig as described above.Positive and negative cells were separated by FACS and sorteddirectly into round bottom, 96-well plates (Falcon, FranklinLakes, NJ) in graded doses (1 x 103_- x 104 cells/well). DCwere co-cultured with 1 x 105 allogeneic ER' T lymphocytes ina final volume of 200 jpL for 5 days. Cultures were pulsed with0-5,Ci [3H] thymidine (Amersham International, ArlingtonHeights, IL) per well for the final 16 hr, before harvesting andliquid scintillation counting. All assays were performed intriplicate.

RESULTS

Expression of CD24 determinants on human peripheral blood DC

Directly isolated peripheral blood DC, prepared by depletionof lin+ cells, did not express the CD24 core proteindeterminants detected by the mAb SWA- (n =6, Fig. a).Similarly, lin- cultured low density DC did not stain withSWA- 11 (n = 4, Fig. 1 b). Both types of DC preparationslikewise failed to react with the CD24 core protein reactivemAb, ALB-9 (data not shown).The carbohydrate epitope-specific CD24mAb HB-9 (Fig. 1),

BA-I and VIB-E3 (not shown) labelled a subpopulation ofdirectly isolated, lin-, HLA-DR+ cells in all six preparationstested (Fig. la). The proportion of cells reacting withthesemAb varied between individual preparations and com-

prised 20-90% of the HLA-DR+ population. The labelling wasreduced or absent on lin- cultured low density DC (n =4,Fig. Ib). The reactivity of these IgM CD24mAb was clearlydistinguished from non-specific or Fc receptor-mediatedbinding by several IgM mAb controls, which failed to stainDC (data not shown). Furthermore, the staining of CD24carbohydrate specific mAb, but not control CD48 mAbHuLym3, was inhibited (>90%) in the presence of 10upgbovine submaxillary mucin (Table 1) as a source of sialylatedcarbohydrate structures confirming specificmAb reactivity.9

These results demonstrate that directly isolated DC show no

expression of CD24 surface antigen, and yet they express

sialylated carbohydrate structures which cross react withcertain CD24 mAb.

CC 1996 Blackwell Science Ltd. Immunology, 89, 120-125

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Figure 1. Expression of CD24 determinants on human blood dendritic cells. Directly isolated DC (a) or cultured low density DC (b)were dual labelled with CD24 carbohydrate (HB-9) or protein core (SWA-l 1) specific mAb and anti-HLA-DR-PE or isotype matchedcontrols. Results shown are 4-log scale dot plots of FITC versus PE fluorescence with percentage positive cells indicated. Data are

representative of at least four experiments.

CD24 cross-reactive determinants on blood DC are

down-regulated following in vitro culture

The CD24 mAb, VIB-E3 and HB-9 bound to directlyisolated HLA-DR+ DC but showed no or only limited stainingof cultured low density DC (Fig. 1). To investigate thisdifference further, directly isolated DC were cultured inmedium, with or without the addition of IFN-y (500 U/mL)or GM-CSF (500 U/mL) for 48 hr and then analysed. Asubstantial reduction in VIB-E3 or HB-9 binding to HLA-DR+, lin- DC was observed following 48 h culture (n = 4,Fig. 2). The culture-induced down-regulation of the VIB-E3 or

HB-9 cross-reactive antigens on the HLA-DR+ population was

unaffected or only minimally affected by the presence of eitherGM-CSF or IFN-y (Fig. 2). Cell viability was not affected byeither the culture period or the presence of cytokines.

Table 1. Inhibition of CD24mAb bindingsubmaxillary mucin

to DC with bovine

Mucin MFI mAb positive cellsmAb (pg) (% decrease) (% decrease)

VIB-E3 1-0 83-4 ± 4-8 72 ± 10-5(CD24) 10 0 99 2 ± 1 1 97-7 + 2 1HB-9 1.0 88-2 ± 0-8 90 6 ± 0-07(CD24) 100 98-5 ± 02 97-6 + 08HuLym3 10 0 ± 0 1 85 ± 2-6(CD48) 10 0 7-3 + 10-3 0 ± 0

Directly isolated DC were stained with CD24 (VIB-E3, HB-9)or CD48 (HuLym3) mAb in the presence or absence of bovine

submaxillary mucin (10 jpg, 10-0pg). Data are expressed as the

percentage i SEM in MFI and positive staining of HLA-DR positivecells in the presence of mucin, relative to control (PBS). Resultsrepresent staining above isotype controls and were obtained from two

experiments.

Sensitivity of blood DC CD24 determinants to enzyme digestion

To determine whether the CD24 cross-reactive determinant on

DC is anchored to the membrane via a GPI-linkage, as forCD246 and CD24cre (Williams et al., in press), DC were

isolated directly and treated with PI-PLC. Following PI-PLCdigestion, equal numbers of cells were dual labelled with theCD24mAb VIB-E3 and HB-9, together with anti-HLA-DR-PE. In two separate experiments, no decrease in HB-9 bindingand only a partial reduction in VIB-E3 staining was detected,despite a distinct loss of the binding of the CD48 positivecontrol mAb HuLym3 to its GPI-linked target antigen(Table 2).The relative lack of sensitivity of the CD24mAb cross-

reactive epitope(s) to PI-PLC cleavage raised the possibilitythat the CD24mAb were reacting with carbohydrate deter-minants attached to membrane glycolipids, rather thanglycoproteins. However, as shown in Table 2, treatment ofdirectly isolated blood DC with non-specific protease (pronase)completely abolished CD24 mAb binding. Thus, theCD24mAb HB-9 and VIB-E3 recognize a sialylated carbohy-drate structure(s), attached to a protein antigen on DC, whichdoes not appear to be (or is only partially) anchored to the cellsurface by a PI-PLC sensitive GPI-linkage. We have desig-nated this antigen DC-24.

Allostimulatory activity of DC-24+ and DC-24-, lin- directlyisolated blood DC subpopulations

The CD24mAb, VIB-E3 and HB-9 identify a subpopulation ofHLA-DR+ cells within lin-, directly isolated blood DCpreparations (Fig. 1). In order to determine the associationbetween expression of DC-24 and the allostimulatory activityof DC, the allogeneic T-lymphocyte proliferation in a MLRinduced by HB-9+ and HB-9- lin- cells was analysed.As shown in Fig. 3, both HB-9+ lin- and HB-9- lin- cells

had allostimulatory activity. Based on HB-9 reactivity alone,neither population had consistently greater allostimulatory

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HB-9 Table 2. Reduction in CD24 determinant expression on DC followingcleavage with PI-PLC or non-specific protease

MFI mAb positive cellsmAb Specificity (% decrease) (% decrease)

PIPLCVIB-E3 CD24 36-0 ± 13 7 33 0 ± 4 0HB-9 CD24 7 2 ± 0-6 13 5 ± 15 6HuLym3 CD48 61 3 ± 12 2 42-8 ± 0-4PronaseVIB-E3 CD24 917 ± 10 966 ± 085HB-9 CD24 96 5 ± 49 97 6 ± 0-78HuLym3 CD48 73 6 i 291 93-1 ± 1-3L243 HLA-DR 0 ± 0 ND

Directly isolated DC were labelled with CD24 or controlmAbfollowing incubation with or without PI-PLC or pronase. Data areexpressed as percentage decrease of MFT ( ± SEM) and ofmAb positivecells (± SEM) for electronically gated HLA-DR+ cells followingenzyme digestion. Isotype control values were subtracted from eachenzyme treatment or PBS control. Data were obtained from twoexperiments.

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Figure 2. CD24 carbohydrate determinant expression by DC followingin vitro culture. Directly isolated DC were cultured for 48 hr in mediumalone and with GM-CSF (500 U/mL) or IFN- (500 U/mL) and thendual labelled with anti-HLA-DR-PE and CD24mAb (VIB-E3, HB-9)or isotype controls. Results are shown as immunofluorescence profilesof isotype control (dashed line) or CD24 mAb (solid line) staining ofelectronically gated HLA-DR+ cells. Mean fluorescence intensity(MFI) and percentage of positive cells are indicated. Data are

representative of four experiments.

activity. However, the lin- HB-9- population contained a

higher proportion of contaminating HLA-DR- cells than thelin- HB-9+ population in all three experiments (Fig. 3). Thus,the actual number of HLA-DR+ stimulator cells tested was

lower in the HB-9- population compared to the HB-9+population. When corrected for HLA-DR+ cell number, itbecomes clear that the HB-9- HLA-DR+ DC are the superior

stimulators. In one particular experiment (Fig. 3c) bothpopulations, HB-9+ and HB-9-, contained a similar propor-

tion of HLA-DR+ cells and the HB-9- DC were more potentstimulators than the HB-9+ DC population. The presence ofthe HB-9 mAb itself had no effect as inclusion of HB-9 in theMLR did not affect T-lymphocyte proliferation (data not

shown).

DISCUSSION

The specific immunophenotypic identification of DC in humanperipheral blood is problematical due to an absence of

DC-specific lineage markers and several studies have suggestedthe presence of a heterogeneous DC population in blood DCpreparations.'6'920 Directly isolated DC, prepared usingminimal manipulation, require activation or further differentia-tion induced by in i'tro culture to express the co-stimulatorymolecules CD80 and CD8617 as well as the DC-associatedsurface markers CMRF-44'7,2' and CD83. 1720,22,23 In contrast,cultured low density blood DC reveal a more activated or

differentiated phenotype and show expression of CD86,CMRF-44 and CD83.17 23. Thus, these two DC preparationsprobably represent successive stages in DC activation/differen-tiation, which also correlates with an increase in stimulatoryactivity for T lymphocytes.'9'24.

Both directly isolated and cultured low density blood DC,lacked surface expression of CD24 protein core epitopes.However, the CD24 mAb VIB-E3, HB-9 and BA- 1, which haveknown specificity for sialylated carbohydrate structures(89'Williams et al., in press), reacted with a subpopulationof directly isolated DC, whereas no or only limited staining ofcultured low density DC was observed. This suggests thepresence of a CD24 mAb cross-reactive carbohydrate-depen-dent epitope on a novel molecule(s) expressed at an earlier stageof DC differentiation. The cross-reactive epitope was carbohy-drate dependent as consistent inhibition of labelling in thepresence of competing sialylated carbohydrates, such as thosepresent in bovine submaxillary mucin,9 was observed. Theantigen, designated here DC-24, was sensitive to non-specificprotease and is, therefore, likely to be associated with a cellsurface protein. Previous studies identifying CD24 on humanDC' 145 may have detected a similar cross-reacting epitope as

that identified here as only the carbohydrate binding VIB-E3 mAb was used. Our experiments indicate that DC do not

express the CD24 antigen. No data exists to suggest humanCD24 has co-stimulatory activity as attributed to mouse HSA'but the absence of CD24 on human blood DC makes it unlikelythat it contributes to DC co-stimulation.

C 1996 Blackwell Science Ltd. Immunology, 89, 120-125

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Figure 3. Allostimulatory activity of blood DC populations identified by reactivity with CD24mAb HB-9. Directly isolated DC were

separated on the basis of HB-9 positivity by FACS sorting and co-cultured with 1 x 105 allogeneic T lymphocytes for 5 days. Graphsrepresent [3H] thymidine uptake (c.p.m. + SD) in response to graded doses ofHB-9', HB-9- or unseparated cell populations for threeindividual experiments. The dual labelling of the same preparations with HB-9 and anti-HLA-DR mAb is shown on the left as 4-logscale dot plots of FITC versus PE fluorescence and the percentage positive cells in each quadrant is indicated. The % HLA-DR+positive cells within the sorted cells used in each MLR as stimulation was (a) 81% of HB-9+, 7% of HB-9-, (b) 88% of HB-9+, 29% ofHB-9- and (c) 95% of HB-9', 80% of HB-9-.

We have also established that the carbohydrate-specificCD24mAb cross-react with a GPI-linked protein antigen,CD24cre, sensitive to cleavage with PI-PLC, which is found on

peripheral blood T lymphocytes (Williams et al., in press). Itis not yet possible to conclude for certain whether theCD24cre and DC-24 antigens are related, but the DC-24antigen on DC was not as sensitive to PI-PLC cleavage as isthe CD24cre antigen on T lymphocytes. No reduction in HB-9epitope expression, but at least some decrease in VIB-E3staining of DC, was observed following PI-PLC digestion(Table 2). The different sensitivity of HB-9 and VIB-E3binding to DC following PI-PLC treatment might imply thepresence of more than one CD24 cross-reactive antigen;however, the glycoprotein antigen detected by theseCD24mAb could well demonstrate heterogeneity with respectto GPI-linkage structures within the DC population. Indeed,the CD48 antigen on DC, recognized by themAb HuLym3,demonstrated some resistance to digestion with PI-PLC (50%reduction) compared to the CD48 antigen present on Tlymphocytes and granulocytes (90% reduction; Williams et al.,in press). Conflicting results in relation to PI-PLC sensitivitysuggest that this type of heterogeneity may also apply tomouse HSA.The reactivity of CD24mAb was diminished or absent in

cultured low density DC preparations and was lost following48 hr culture of directly isolated DC in medium, IFN-y, GM-CSF (Figure 2), tumour necrosis factor-a (TNF-a) or

interleukin-12 (IL-12) (not shown). The downregulation ofDC-24 expression and its absence or only limited expressionon cultured low density DC correlates with a process ofactivation or differentiation characterized by up-regulation ofCD28/CTLA-4 ligands17 and the antigens recognized byHB1 5a1718,20'22'23 and CMRF-44 mAb.17"18'22 The subpopu-lation of directly isolated HB-9+ blood DC, therefore, appears

to represent DC in a lesser state of activation or differentiation,whereas the HB-9- DC have a more activated or maturephenotype. When corrected for the number ofHLA-DR+ cellsit became obvious that the HB-9-cells were superior instimulating T-lymphocyte proliferation in an allogeneicMLR. Thus, DC-24- blood DC represent a more mature or

activated DC population than the DC-24+ population.A correlation of increased stimulatory activity with progres-

sive DC differentiation/activation has been reported previouslyfor cultured DC. 19'24 The kinetics of DC-24 expression would,then, argue against a role in T-lymphocyte co-stimulation.Although we have no information as to the function of the DC-24 molecule in lin- allostimulatory cell populations, similaractivation induced down-regulation of antigen expression was

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CD24 mAb cross-reactivity on dendritic cells 125

observed for receptors which mediate antigen uptake such asthe mannose receptor27 and Fc receptors.28 Interestingly, DCdifferentiation/maturation upon culture is characterized by adecrease in antigen uptake and processing capability and theacquisition of T-lymphocyte co-stimulatory potential.29 Furthercharacterization of the molecule expressing the sialylatedCD24-cross-reactive carbohydrate epitope(s) on DC by con-ventional biochemical and cloning techniques will provide thetools for more extensive functional analysis in DC populations.

ACKNOWLEDGMENTS

The authors would like to thank Dr E. Weber, Professor T. Tedder, DrT. Le Bien and Dr W. Knapp for generously supplying the CD24antibodies used in this report. We would also like to thank Mr A. Raefor FACS analysis, Dr B. D. Hock for helpful discussions and Ms S.Banks for assistance with this manuscript.

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NOTE ADDED IN PROOF

WILLIAMS L.A., HOCK B.D. & HART D.N.J. (1996) Human Tlymphocytes and haemopoietic cell lines express CD24 associatedcarbohydrate epitopes in the absence of CD24 mRNA or protein.Blood, in press.

:3 1996 Blackwell Science Ltd, Immunology, 89, 120-125