Differential Expression of CD24-Related Epitopes in Mycosis FungoideslSCzary Syndrome: A Potential Marker for Circulating SCzary Cells

By Samuel J. Pirruccello and Molly S. Lang

In the hematopoietic system, the B-cell associated antigen CD24 is expressed at high density on B cells, B-cell precursors, and B-cell malignancies as well as at low density on peripheral blood polymorphonuclear leukocytes. The 42-Kd sialoglycoprotein has not been previously dem- onstrated to be expressed on T cells, thymocytes, or T-cell malignancies. We identified three patients with mycosis fungoides/Sbzary syndrome that showed low density ex- pression of the CD24-related epitope recognized by anti- body BA-1 on circulating T cells. All three patients had Sbzary cells by morphologic assessment and clonal T-cell populations in the peripheral blood by gene rearrangement

HE B-CELL associated antigen CD24 is a 42-Kd T sialoglycoprotein recognized by the prototypic anti- body BA- 1 The molecule has been successfully radiola- beled only on carbohydrate residues either by surface reduc- tion with tritiated sodium borohydride or by metabolic incorporation of tritium-labeled gluco~amine.~*~ CD24 has a broad banding profile on sodium dodecyl sulfate polyacryl- amide gel electrophoresis (SDS-PAGE), suggesting heavy glycosylation. The molecule also shows partial comigration with immunoglobulin G (IgG) heavy chains on SDS-PAGE, giving the impression of a multi-chain protein comple~ .~ .~

The function of CD24 is currently unknown. Previous studies reported an inhibition of Ig synthesis by pokeweed mitogen-stimulated B cells and by the CESS cell line after cross-linking CD24 with antibody:.’ However, Rabinovitch et a1 could show no change in B-cell free CaZ+ by cross- linking of CD24 alone or with costimulation of surface Ig. We found that CD24 antigen density increases as pre-B acute lymphoblastic leukemia (ALL) cells enter the cell cycle, but the molecule does not appear to deliver a prolifera- tion-related signal when cross-linked by antibody.’

In the hematopoietic system, the CD24 antigen is ex- pressed at high density on B cells and B-cell precursors as well as at low density on polymorphonuclear leukocytes.’ The antigen has proven to be a useful B-lineage marker in ALL and is a favorable prognostic indicator in pre-B ALL in children.’ The CD24 epitope recognized by monoclonal antibody (MoAb) BA-1 has also been demonstrated on renal tubular epithelium: on some nonhematopoietic tumors,” and on some normal epithelium.’’ In these tissues outside the hematopoietic system, it is currently unclear whether BA- 1 positivity represents epitope mimicry or true CD24 antigen expression. We report here the finding of limited CD24- related epitope expression on peripheral blood T lymphocytes from three patients with mycosis fungoides/Skzary syn- drome.

MATERIALS AND METHODS

Antibodies. Antibodies used for these studies included T6 (anti- CDl), T11 (anti-CDS), T3 (anti-CD3), T4 (anti-CD4), T1 (anti- CDS), T8 and T8-phycoerythrin (PE) (anti-CD8). I2 (anti-HLA- DR), J5 (anti-CDlO), B4 (anti-CD19), and B1 (anti-CD20) (Coulter Immunology, Hialeah, FL). Fluorescein isothiocyanate (F1TC)- conjugated goat antimouse, anti-r, and antid were obtained from

studies. In two of these patients, indirect immunofluores- cence with a panel of six anti-CD24 monoclonal antibodies demonstrated reactivity for two of six antibodies in one case and only one of six antibodies in the other. The biologic significance of CD24-related epitope expression on circulating T cells in mycosis fungoiddsbzary syndrome is unclear. However, these findings suggest that differen- tial, low density expression of CD24related epitopes (BA-1’. OKB2-) may be a useful phenotypic marker for identifying circulating Skary cells. 0 1990 by The American Society of Hematology.

Tag0 (Burlingame, CA). The hybridoma secreting MoAb BA-1 was the kind gift of Dr Tucker LeBien (University of Minnesota, Minneapolis), The anti-CD24 MoAbs LC66, ML5, and VIB-E3 were obtained from the Fourth International Workshop on Leuko- cyte Differentiation Antigens. MoAb OKB2 was obtained from Ortho Diagnostics (Raritan: NJ). Antibody 32D12 was the kind gift of Dr Steiner Funderud (Norwegian Radium Hospital, Oslo). Negative controls included a polyclonal mouse IgG, and IgG1-PE (Coulter). The IgM myeloma MOPC 104E was obtained from the American Type Culture Collection (ATCC; Rockville, MD) tissue bank.

Immunofluorescence. Immunofluorescence assays were per- formed as described previ~usly.~ Briefly, peripheral blood mononu- clear cells were isolated from heparinized peripheral blood by Ficoll-Hypaque gradient centrifugation and washed twice with phosphate-buffered saline (PBS). The cells were washed with cold PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN,, and aliquoted at 2 x lo5 cells per tube. The cells were incubated with primary antibody for one-half hour at 4°C in 100 pL PBS/BSA/ NaN,. Cells were washed twice and incubated with FITC-goat antimouse (Fab,) for one-half hour at 4OC. For two-color immuno- fluorescence, cells were first incubated with IgM or BA-1 followed by goat antimouse FITC and then IgG,-PE or CD8-PE, respectively. After washing, the cells were analyzed on an EPICS C Flow Cytometer at 488 nm with a 5-W argon ion laser. Fluorescence was measured either on linear or log scale and percent positive cells were calculated after background subtraction using Epics software.

T-cell receptor gene rearrangement by South- ern blotting” was performed on Ficoll-Hypaque isolated mononu- clear cells or buffy coat preparations by standard techniques. Briefly, genomic DNA was isolated from the white blood cell preparations by

Southern blotting.

From the Department of Pathology and Microbiology, University

Submitted October 12,1989; accepted August 6, 1990. Supported by Grant LB.506, No. 89-44, Nebraska Department of

Health. Address reprint requests to Samuel J. Pirruccello, MD, Assistant

Professor, Department of Pathology and Microbiology, University of Nebraska Medical Center. 42nd St and Dewey Ave. Omaha, NE

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

of Nebraska Medical Center, Omaha.

68105-1065.

6 1990 by The American Society of Hematology. 0006-4971/90/761 I -O008$3.00/0

Blood, Vol76, No 1 1 (December 11, 1990: pp 2343-2347 2343

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2344

the method of Rakhshi et al." Aliquots of DNA were cut separately with EcnRI. Rumbil. and Hindlll and electrophoresed on 0.8% agarose gels at 40 V overnight. After denaturation in 0.2 N NaOH. 0.6 mol/l. NaCI. and neutraliration in 0.5 mol/!, Tris. pH 7.5. 1.5 mol/l. NaCI. the DNA was transferred to nylon membrana (Schleicher & Schuell. Keene. NH) by blotting in 20X SSC (3 mol/L SaCI. 0.3 mol/L sodium citrate. pH 7). The membrana were washed. baked. and then prehybridized with SX SSC. I X Denhardts solution. 0.02 mol/l. sodium sulfate. and 50% formamide overnight a t 42OC. The filters were then hybridized with a 770-bp TH constant region probe (Dr h e a l Masih. L'niversity of Nebraska Medical Center. Omaha) labeled by random primer extension (Dupont/NEN. Roston. MA)." After washing. the filters were sealed wet into plastic bags and allowed to e x p t e x-ray film from 4 to 40 hours at -7WC. f:or delination of germline patterns, placental DNA was used a s negative control.

Patient I was a 56-year-old woman who pmented with several nonhealing skin lesions in August 1986. Immunohistol- ogy of skin biopsy material yielded a working classification of peripheral immunoblastic lymphoma of T-cell type. The infiltrating lymphocyta had a cytotoxic suppresor T-cell phenotype being positive for CD2 (TII). CDR (TX. Leu-2) CD3 (Leu-4). CD7 (Leu-9). HLA-DR. and CD71 (OKT9). The tumor cells were negative for CD4 (Leu-3, OKT4). CDI (Leu-6). CD19 (I.eu-l2), and CD22 (Leu-14). The patient responded p o r l y to therapy and was later reclassified a s mycosis fungoida on October 16, 1987.

Patient 2 was a 58-year-old man who presented in July 19x8 with multiple skin lesions. Clinical workup yielded a working diagnosis of mycosis fungoida. Immunohistology of skin biopsy demonstrated a cytotoxic suppressor T-cell phenotype with expression of CD2 (Leu-5). CDI; (Leu-I). CD7 (1x11-9). CD8 (Leu-?). 1iI.A-DR. and CD4S (UCI!L-l). The infiltrating tumor cells were negative for

I4) .andCDl5 (Leu-MI). Patient 3 was a 51-year-old woman who presented with multiple

skin lesions in January 1989. Clinical workup yielded a working diagnosis of mycosis fungoides. Skin biopsy demonstrated infil- trating T cells with a helper inducer T-cell phenotype showing positivity for CD5 (Leu-I). CD3 (Leu-4). CD4 (Leu-3). Leu-X, HLA-DR. and CD71 (OKT9). The tumor cells were negative for

(Leu-14). and CDI 5 (Leu-M I ) .

Purirnts.

CD4 (Leu-3). CDI (Leu-6). Leu-X. CD19 (Leu-12). CDZ2 (Leu-

CDX (Leu-2). CDI (Leu-6). CD7 (Leu-9). CD19 (!XU-12). CD22

RESULTS

A t t h e t ime of phenotypic analysis. a l l th ree pat ients had a high percentage of c i rculat ing lymphocytes with morphology consistent with Si.7ary cells on peripheral blood s m e a r (not shown). All three pat ients had monoclonal rearrangements of t h e p gene of t h e T-cell receptor in DNA isolated from peripheral blood white cells isolated by Ficoll gradients or from b u r y coat preparations. Representat ive blots a r c shown for pat icnts 2 a n d 3 in Fig I . These gene rearrangement s tudies confirmed t h e morphologic imprcssion of a circulat- ing population of mal ignant T cells.

T h e original phenotyping d a t a for t h e pat ients are pre- sented in T a b l e I . Pat ients I a n d 2 showed a predominance o f cytotoxic suppressor T cells. a n d patient 3 showed a predom- inance of helper inducer T cells consistent with t h e T-cell tumor phenotypes demonstrated by immunohistology o n skin biopsy. However. only patient 2 showed a n increase in peripheral blood lymphocytes expressing HLA-DR. which was present on all three pat ients t u m o r cell populations present in t h e skin lesions. A clear discordance was evident in

PIRRUCCELLO AND LANG

Flg 1. Southern blot arutyah of Fkdt-Hyp.qw iwlatul mono- nuclear colls from patient 2: Ian. B and a buffy coat preparation from patient 3, lane D demonstrating monoclorul rearrangement of the B gene of the T-cell receptor. lanes A and C consist of genomic DNA isolated from placenta for comparison of patient specimens with the germline Tb' pattorn. In lanes A and B DNA samples were cut with EcoRI. and in lanes C and D DNA samples ware cut with 88mHl before electrophoresis. After transfer samples wore probed with a 770-bp TB constant region probe.

a l l th ree cases for t h e percent RA-I (CD24) positive cells in comparison with t h e o ther R-cell markers CD19 a n d CD2O. a s well as t h e percentage of T cells present. O n a linear fluorescence scale. t h e mean channel fluorescence for RA-I was noted t o be considerably lower t h a n t h a t of t h e o ther T- a n d R-cell markers (not shown). Log scale fluorescence

Tabk 1. Cell Surf- Markrs of Porlphoral Blood Lymph0qt.r

A0 (Ab1 Plllmll 1 P n m i 2 PMml3

CD1 cT6) 1.2. 2.3 4.6 CD2 (T11) 85.0 99.0 93.1 CD3 (T3) 75.0 97.9 89.8 CD4 (T4) 28.4 6.2 79.1 CD5 (T1) 72.0 98.8 86.1 CD8 IT81 42.3 92.6 7.1 HLA-DR (121 9.9 33.5 3.3 CDlO (J5) 0 0.8 0.7 CD19 (84) 5.8 0.7 2.3 CD20 (B 1) 5.6 0.4 1.3 CD24 (BA-1) 27.5 37.0 48.3 I 4.6 0.6 1.7 A 2.6 0.7 1.4

* P w m t positive d s mensued on linear fluaescence scale.

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CD24 EXPRESSION ON SEZARY CELLS 2345

histograms are shown for patients 2 and 3 in Fig 2 and illustrate the pattern of BA-1 immunofluorescence. The mean fluorescence intensities for CD2 (not shown), CD4, and CD8 were 1.5 to 2 logs brighter than that obtained with BA- 1. For comparison with the low density expression of the BA-1 epitope, the lymphocytes from patients 2 and 3 were also examined for reactivity with the anti-CD24 MoAb OKB2. In contrast to the positive reactivity noted with BA-1, neither patient’s lymphocytes showed reactivity with anti- body OKB2 (Fig 2).

After demonstration of reactivity of BA-1 with lympho- cytes isolated from patients 2 and 3, we performed a number of control experiments to rule out the possibility that aggluti- nated antibody was causing a false positive immunofluores- cence signal. We performed indirect immunofluorescence of patients’ lymphocytes after centrifugation of BA- 1 ascites to remove potential aggregates, as well as assaying with sepa- rate aliquots of BA-1 ascites prepared both at different times and in different laboratories. All BA-1 antibody preparations showed similar fluorescence profiles on these two patients’ lymphocytes. We also performed two-color immunofluores- cence with BA-1-FITC and CDI-PE on the lymphocytes from patient 2 and could demonstrate co-expression of these two antigens (see Fig 3).

To further assess the selective CD24-related epitope expres- sion on these malignant T cells, we performed immunofluo- rescence studies with six of the seven antibodies comprising the anti-CD24 panel of the Fourth International Workshop on Leukocyte Differentiation Antigens. As seen in Table 2, of the six anti-CD24 monoclonals, lymphocytes from patient 2 reacted only with antibody BA-1. In contrast, the lympho-

F I

LOG GREEN FLUORESCENCE

Fig 2. Log scale fluorescence histograms of isolated peripheral blood lymphocytes from patient 2: A through D and patient 3: E through H illustrating low density expression of the BA-1 epitope. Histograms A end E represent mouse polyclonal IgG (negative con- trol). histogram B. CDB (T8). histogram F, CD4 (T4). histograms C and G, CD24 (BA-1) and histograms D and H, CD24 (OKB2).

IgM -FITC

l n 2

C D24 - F I TC Fig 3. Two-color fluorescence histograms demonstrating co-

expression of CD8 and the CD24 epitope recognized by antibody BA-1 on lymphocytes isolated from patient 2. Histogram A repre- sents the negative control with 100% of cells in quadrant 3 (double negatives). Histogram B represents the positive test with: 56% of cells in quadrant 1 (CD8+. BA-1-1. 39% of cells in quadrant 2 (CD8’. BA-l’),6% of cells in quadrant 3 (CDS-, BA-1 -1, and 0% of cells in quadrant 4 (CDS-, BA-1’).

cytes from patient 3 showed reactivity with MoAb BA-1 as well as with antibody VIB-E3.

DISCUSSION

The findings reported here demonstrate an unusual selec- tive expression of CD24-related epitopes on the surface of circulating T cells in mycosis fungoides/Sbzary syndrome. Stockinger et a1” reported expression of CD24 or the CD24-related epitopes recognized by VIB-E3 and HB8 on the surface of SCzary cells. In that study, the anti-CD24 monoclonals ALB9, LC66, CLB gran Blyl, VTB-CS, and ALla were all negative on the SCzary cells used for analysis. As with this previous report, it is unclear whether reactivity with antibody BA-1 represents expression of an altered CD24 molecule on these T cells or expression of CD24

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2346 PIRRUCCELLO AND LANG

Table 2. CD24 Epitope Expression of Peripheral Blood Lymphocytes

Ab Patient 2. Patient 3 t

BA- 1 37.0 66.3 LC66 0 1.3 32D 12 0 .1 2.9 ML5 0.5 2.5 VIBE3 5.2 59.4 OKB2 0 0

'Percent positive cells measured on a linear scale. Cells assayed for

tData represent assay of a separate specimen from that shown in patient 2 represent the same specimen reported in Table 1.

Table 1.

epitopes on other cell surface glycoproteins or glycolipids. The fact that VIB-E3 reacted with one of these patients' lymphocytes but not the other demonstrates heterogeneity of CD24 epitope expression even among this small sample of cases. We attempted radioimmunoprecipitation of the anti- gen recognized by BA- 1 from tritiated sodium borohydride- labeled cell lysates from patient 3 but were unable to demonstrate a protein or glycoprotein antigen. We are not sure whether this was due to the very low density expression of the BA-1 epitope or whether the epitope was dependent on tertiary or quaternary structure a t the membrane surface and was destroyed on cell lysis. These possibilities seem most likely since very few counts were precipitated with BA-1 in comparison with a radiolabeled pre-B ALL cell line lysate used as a positive control.

We have previously shown that all six antibodies used for these fluorescence studies radioimmunoprecipitate a glycopro- tein consistent with CD24.4 The positive immunofluores- cence with VIB-E3 on lymphocytes from patient 3 is consis- tent with the earlier report by Stockinger et all5 and the CD24 epitope expression detected with antibody BA-1. We have assayed peripheral blood lymphocytes obtained from patient 3 on four separate occasions. The patient has main- tained 75% or more CD4+ T cells, a consistent pattern of low density expression of the BA-1 epitope, and absence of the epitope recognized by antibody OKB2.

These findings provide some insight into the epitope specificity of recognized anti-CD24 MoAbs. Stockinger et all5 organized a subclustering scheme of anti-CD24 antibod- ies based on differences in epitope expression on various cell lines and by cross-blocking studies. Engel et all6 reported a clustering scheme of CD24 epitopes by measuring time- dependent epitope loss from B cells following activation. We previously reported that anti-CD24 MoAb 32D12 recognizes a distinct epitope from the other anti-CD24 MoAbs VIB-C5, VIB-E3, LC66, BA-1, ML5, and OKB2 based on immuno- fluorescence studies with the Nalm 6 cell line.4 We demon- strate here that antibodies BA-1 and VIB-E3 also recognize distinct epitopes based on the BA-l+, VIB-E3-and BA-l+, VIB-E3+ phenotypes in patients 2 and 3, respectively. Radioimmunoprecipitation studies suggest that CD24 repre- sents a heavily glycosylated protein*.' and raises the possibil- ity that differences in epitope expression may be the result of selective expression of specific glycosyl transferases. More

rigorous studies of CD24 protein and carbohydrate structure will be required to address this issue.

These findings raise a more clinically relevant possibility related to immunophenotyping and monitoring of peripheral blood in mycosis fungoides. All three patients reported here demonstrated low density expression of the antLCD24 epitope recognized by antibody BA-I . More detailed immunofluores- cence studies on the lymphocytes isolated from patient 2 as well as repetitive studies on patient 3 showed a consistent BA-1 positive OKB2 negative phenotype. The malignant T cells demonstrated on skin biopsy in patients 1 and 2 were derived from a cytotoxic suppressor T-cell lineage, while those of patient 3 represented a helper inducer T-cell lineage. The malignant T-cell phenotypes were, except for HLA-DR expression in patients 1 and 3, concordant with the predomi- nant T-cell subset present in the circulation. Further, we demonstrated coexpression of BA-1 on the CD8 positive peripheral blood T cells from patients 2. We have subse- quently seen a fourth patient with a working diagnosis of mycosis fungoides/Stzary syndrome. In this patient we could also demonstrate a circulating helper inducer T-cell population that was BA-1 + and OKB2- (not shown). How- ever, gene rearrangement studies have not been performed to confirm the presence of a circulating clonal T-cell population in this fourth patient. These findings suggest that regardless of the functional T-cell phenotype, differential expression of the CD24 epitopes recognized by antibody BA-1 and OKB2 may be a useful marker for circulating SCzary cells.

These chronic T-cell malignancies frequently express nor- mal mature T-cell phenotype^.'^ Confirmation of the pres- ence of circulating malignant SCzary cells therefore requires the demonstration of a monoclonal population by T-cell receptor gene rearrangement." Involvement of the periph- eral blood in mycosis fungoides has been shown to predict a poorer survival and a higher frequency of lymphomatous and visceral tumor inv01vement.l~~~~ A phenotypic marker for circulating Stzary cells would, therefore, be a useful diagnos- tic adjunct in the management of these patients.

We attempted to demonstrate the clonal nature of the CD24 positive T cells using cells stored on patient 3 by BA-1-immunomagnetic bead selection and Southern blot- ting. Unfortunately, the few cells surviving the freeze thaw in 10% dimethyl sulfoxide (DMSO) were greater than 80% CD24 positive and we could not obtain enough CD24 negative cells after separation for DNA extraction. The only other DNA source available on this patient for comparison was from a buffy coat preparation. Due to neutrophil contamination this DNA preparation was not adequate for demonstrating clonal enrichment after BA- 1 selection. There- fore, the ultimate usefulness of this phenotypic trait will require confirmation of these findings in a larger series of mycosis fungoides patients by correlation with gene rearrange- ment studies and demonstration of true monoclonality in the CD24+ T-cell population.

ACKNOWLEDGMENT

The authors thank Martin Bast and the Lymphoma Study Group for help with immunohistologic patient data, and Karen Spiegel for preparation of the manuscript.

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CD24 EXPRESSION ON SEZARY CELLS

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SJ Pirruccello and MS Lang cellsfungoides/Sezary syndrome: a potential marker for circulating Sezary Differential expression of CD24-related epitopes in mycosis 

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