Cancer Immunol. Immunother. 8, 225-230 (1980) ancer mmunolggyand mmunotherapy

© Springer-Verlag 1980

Original Articles

A Unique Antigen (Sigma) on Sezary Cells

A m a n u l l a h K h a n , Sandra L. F. Burt , N o r w o o d O. Hill, and J. M. Hill

Department of Immunotherapy, Wadley Institutes of Molecular Medicine, 9000 Harry Hines Blvd., Dallas, Texas 75235, USA

Summary. L ymphapheresis was performed on a patient with Sbzary syndrome. The Sbzary cells were purified by removing E-rosette-forming and Fc receptor-bearing cells. Antiserum against these purified Sbzary cells was raised in rabbits. This antiserum had cytotoxicity against Sbzary cells as well as against normal peripheral blood lymphocytes. Absorption was carried out with chronic lymphocytic leukemia (CLL) and normal lymphocytes. The absorbed antiserum maintained cytotoxicity against Sbzary cells but lost cytotoxicity against CLL and normal peripheral blood lymphocytes.

Indirect immunofluorescence assay showed that the antiserum reacted against purified Sbzary cells and a high percentage (66%) of peripheral blood mononuclear cells from five patients with Sbzary syndrome. It also reacted against 5.7% of normal lymphocytes, 8% of CLL cells, 5% of the lymphocytes from a patient who had undergone splenectomy, 2% oflymphocytes from apatient with multiple myeloma, 5% of lymphocytes from a hairy cell leukemia patient, and 1% of acute lymphocytic leu- kemia cells (T cell). The antiserum did not react against thymocytes but reacted against 34.6% of the bone marrow lymphocytes. This unique marker was designated as sig- ma (~r) antigen. It was suggested that Sbzary syndrome may represent proliferation or malignant transformation of normally present ~ antigen-positive lymphocytes.

Introduction

I t has been suggested that S6zary cells are a subset of

T cells [ 2 - 4 , 6, 8, 15, 23]. The present work was un- der taken to see whether these cells possess a un ique anti- gen or marke r on their surface. Pat ients with S~zary synd rome can be m a n a g e d by lymphapheres is . This pro- cedure relieves symptoms and lowers the peripheral b lood coun t [5, 17]. Lymphoey tes ob ta ined f rom a pat ient with S+zary syndrome, who was undergo ing lymphapheres is

for therapeut ic reasons, were f ract ionated to ob ta in pure S~zary cells. A n t i s e r u m against these cells was raised in

rabbi ts and a un ique marke r (antigen) was demons t ra t ed on the surface of S~zary cells. This marke r was designated as the s igma (or) antigen.

Materials and Methods

Lymphapheresis. The diagnosis of S6zary syndrome was based on a typical clinical picture and the demonstration of S6zary cells by light microscopy and electron microscopy [7, 14, 16, 18, 21]. Lympha- pheresis was performed with the aid of a continuous-flow centrifuge [10] (Celltrifuge-Aminco). Each procedure lasted 3}'2-4 h and yielded white blood cells that were rich in lymphocytes. These cells were subjected to further fractionation.

Isolation of Sbzary Cells. Phagocytic cells were removed with a mag- net after being allowed to ingest iron filings at 37 ° C for 30 min [12]. The remaining cells were resuspended in Hank's balanced salt solution (HBSS) and further purified by density gradient according to the Ficoll-Hypaque method [ 1]. Mononuclear cells staying at the interface were retained.

Eighty-nine percent of the peripheral blood lymphocytes of this patient reacted with an anti-T cell serum [20]. The antiserum was kindly supplied by Dr. H. Toben, Wayne State University, Detroit, Mi- chigan. However, only 14% of his lymphocytes formed E-rosettes. The inability of S~zary cells to form E-rosettes was exploited in the separa- tion procedure. The E-rosette-forming cells were removed by immu- noabsorbent erythrocyte (E) monolayers. This method removed 85%-90% non-Fc receptor-bearing cells. These monolayers were prepared in 100 × 15 mm polystyrene petri plates (Falcon) that had previously been treated with poly L-lysine [9]. Twenty milliliters of lymphocyte suspension (I × 107 cells/ml) was applied per plate. The plates were incubated at 37 ° C for 30 rain, with agitation at 10-min in- tervals, followed by a 30-min incubation at the same temperature without agitation. The supernatant, consisting of nonadherent S6zary cells and B lymphocytes, was collected.

Cells bearing Fc receptors were removed by antibody-coated sheep erythrocyte (EA) monolayers [9]. These monolayers were pre- pared in poly L-lysine-treated polystyrene petri dishes. The cell sus- pension of S6zary cells and B lymphocytes obtained from the E mono- layers was then applied to the EA monolayers. The incubation proce- dure was the same as for the E monolayers. This procedure removed

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90%--98% of the Fc-bearing cells. The nonadherent cell population consisted predominantly of S~zary cells. The yield of S~zary cells ranged from 12% to 40% of the S6zary cells present in the initial sample.

Preparation and Testing of Antiserum. The anti-S~zary cell serum was prepared in New Zealand white rabbits by IV injection of 5 × 108 purified S~zary cells suspended in 1-3 ml of normai saline on days 1, 14, and 48. Fourteen days after the last injection the animals were bled to collect the serum. The serum was inactivated at 56 ° C for 30 min. It was absorbed three times with normal peripheral blood lymphocytes and five times with CLL cells at concentrations of 5 x 108 cells/ml anti- serum during each absorption. The antiserum was tested for cyto- toxicity by a modified Terasaki method [ 19], before, during, and after absorption, against normal lymphocytes, CLL cells, and purified Stza- ry ceils. Normal rabbit serum was also absorbed in a similar manner and was used as a control in the cytotoxicity and immunofluorescent studies.

Indirect Immunofluorescence. The absorbed antiserum was tested by indirect immunofluorescence against lymphocytes obtained from dif- ferent sources. Briefly, the lymphocytes were washed once in phos- phate-buffered saline (PBS) then resuspended (3 × 106 cells/ml) in HBSS.

Aliquots (1 ml) were transferred to microtest tubes (Falcon). Cells were then washed once with HBSS supplemented with 10% fetal calf serum (FCS) and 2% sodium azide. Twenty microliters of antiserum was added to each tube containing cells. Absorbed normal rabbit serum (20 ~1) was substituted for the antiserum in the control tubes in every experiment. The cells were resuspended and incubated for 30 min at 4 ° C. Following incubation, the ceils were washed twice with HBSS containing FCS. Twenty microliters of fluorescein-conjugated goat anti-rabbit 7S globulins (Meloy) was added to the cells, which were incubated for 30 min at 4 ° C. One milliliter of HBSS with FCS was added to the cells. The cell suspension was layered over 2 ml FCS and spun at 40 g for 10 rain. The cells were washed twice in HBSS with FCS and slides were prepared. The fluorescence was detected under a Leitz Ortholux microscope with epifluorescence [20].

Double Labeling for Surface Ig and ~r Antigen. In the double labeling procedure, surface Igs were stained by a direct immunofluorescence method with anti-human (Igs) rhodamine-labeled IgG obtained from Cappel Lab, Inc., in addition to indirect immunofluorescence with anti-Stzary serum. The normal peripheral blood lymphocytes were prepared and treated in the same manner as for indirect immuno- fluorescence, except that 20 ~t rhodamine-conjugated IgG fraction of goat anti-human IgG, IgA, IgM was added to the cells. This antiserum was added along with the fluorescein-conjugated goat anti-rabbit 7S globulins. The rest of the procedure was the same as described for indirect immunofluorescence. Fluorescein and rhodamine fluores- cence were visualized simultaneously and could be easily distinguished from each other due to difference in color [t3].

Collection of Lymphoeytes for Testing. Human peripheral blood and bone marrow lymphoeytes were obtained by Ficoll-Hypaque differ- ential sedimentation of heparinized samples [1]. Thymic tissues re- moved during cardiac surgery in children were used to collect thy- mocytes. Pieces of tissue were cut with sterile scalpels and scissors then teased with needles. The large fragments of tissue were allowed to settle by gravity and the supernatant was aspirated through a 21-gauge needle into a syringe and then expelled into plastic test tubes. The cells were washed and resuspended in Hank's solution at a concentration of 5 x 106 cells/ml. These cells were then used for indirect immunofluo- rescence with the antiserum.

A. Khan et al.: A Unique Antigen (Sigma) on Stzary Cells

Results

Lymphapheresis. The patient had 70% S~zary cells in his peripheral blood. During a 3y2-h run of lymphapheresis, 4 × 10 a° lymphocytes were collected. Eighty-three percent of these were Sbzary cells. Following the purification procedure, 4 × 108 S+zary cells were obtained.

Cytotoxicity. The anti-S+zary serum was cytotoxic to normal peripheral blood lymphocytes, S~zary cells, and CLL cells (Table 1). During absorption, the antiserum gradually lost its cytotoxicity and was no longer toxic to normal peripheral blood lymphocytes or CLL cells at the end of the absorption process (Table 1). It was still cy- totoxic to Stzary cells. The normal rabbit serum was nontoxic to lymphocytes and Stzary cells.

Indirect Immunofluorescence Test. Table 2 gives the re- sults of indirect immunofluorescence with the antiserum after absorption. Purified Stzary cells (nonrosetting) from two patients showed 98% and 85% immunofluorescence. Fig. 1 demonstrates the immunofluorescence of Stzary cells. The absorbed normal rabbit serum used as control failed to give immunofluorescence. The antiserum was also tested against normal peripheral blood lymphocytes and lymphocytes obtained in various diseases (Table 2). Peripheral blood from 15 normal volunteers showed im- munofluorescence ranging from 1 %-1 t%. The mean percentage of cells showing immunofluorescence was 5.7 _+ 3.4 Fig. 2 shows a normal lymphocyte positive for immunofluorescence. Again, no immunofluorescence was detected with normal rabbit serum.

Mononuclear cells obtained from the peripheral blood of five patients with Stzary syndrome contained 48%-90% cells positive for immunofluorescence (mean 66%). Two patients with CLL were also tested. Both of them showed 8% immunofluorescence of cells. Other immunologic parameters of lymphocytes were also stud- ied in CLL patients. One patient had 17,858 absolute lymphocytes/mm 3 in his peripheral blood. Rosetting cells were as follows: EAC 10% and E-rosettes 8%. The other patient had 21,800 absolute lymphocytes/mm 3 with 7% EAC and 2% E-rosette-forming cells. One patient with id- iopathic thrombocytopenic purpura, who had undergone splenectomy, showed 5% cells with immunofluorescence. One patient with multiple myeloma and a normal per- ipheral blood count showed 2% immunofluorescence with antiserum. One patient with hairy cell leukemia gave 5% immunofluorescence. A patient with acute lymphocytic leukemia (T cell) had a peripheral WBC count of 598,000/mm 3. Eighty-seven percent of the peripheral blood white cells consisted of blasts and 88% formed E-rosettes. Only 1% of his white cells gave positive immu- nofluorescence with the antiserum. Each experiment in-

A. Khan et al.: A Unique Antigen (Sigma) on S~zary Ceils

Table 1. Cytotoxicity of anti-S6zary serum at various stages of absorption

Cells tested Volume and Cytotoxicity dilution of antiserum

Unab- Absorbed sorbed 3 x N a serum 3 x CLL

Absorbed 3 x N a 5 x CLL

Normal 9 ~xl 1 : 1 dilution 8 b 2 b 1 u Lymphocytes 6 ~1 1 : 1 dilution 8 1 1 (1.5 x 10 3 ) 3 ~1 1:1 dilution 8 1 1

1 rxl 1:1 dilution 8 1 1 1 ~1 1 : 2 dilution 8 1 1 1 rxl 1 :4 dilution 8 1 1

Stzary 9 ~1 1 : i dilution 8 8 8 Lymphocytes 6 ~1 1 : 1 dilution 8 8 8 (1.5 x 10 3 ) 3 93 1:1 dilution 8 8 8

1 p~l 1 :1 dilution 8 6 8 1 vl 1 : 2 dilution 8 8 8 1 ~1 1 :4 dilution 8 8 8

CLL 9 ~1 1:1 dilution 8 1 1 Lymphocytes 6 ~1 1 : 1 dilution 8 1 1 (1.5 x 103 ) 3 VI 1:1 dilution 8 2 1

1 p,1 1 : 1 dilution 8 2 1 1 ~1 1 : 2 dilution 8 1 1 1 ~xl 1 :4 dilution 8 8 1

a Serum was absorbed with normal and chronic lymphocytic leukemia cells (5 x 10Scells/ml serum) during each absorption u Code: 1, negative: same viability as negative control; 2, negative: 10%-19% dead lymphocytes; 4, weakly positive: 20%-39% dead lymphocytes; 6, positive: 40%-79% dead lymphocytes; 8, strongly positive: 80%-100% dead lymphocytes. Normal rabbit serum did not show cytotoxicity against normal lymphocytes, CLL cells, and S~zary cells

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Table 2. Percentage of peripheral blood lymphocytes reacting with the antiserum by indirect immunofluorescence

Peripheral blood No. Percent tested positive

Mean + SD b

Normal a 15 5.7 _+ 3.4 S~zary syndrome 5 66 Chronic lymphocytic leukemia 2 8 Idiopathic thrombocytopenic purpura 1 5 Multiple myeloma 1 2 Hairy cell leukemia 1 5 Acute lymphocytic leukemia (T cell) 1 1

a Controls substituting absorbed normal rabbit serum for the anti- Stzary serum were negative in each experiment u Standard deviation was determined for the normal lymphocytes only

cluded controls with no rma l rabbi t se rum as the first an t ibody. N o posit ive cells were detected.

The ant i -S~zary se rum was further absorbed five times, with 5 x 108 S tzary- r ich cells/ml (> 70% S tza ry

cells) to see whether the cross-react ivi ty against no rma l

cells could be removed by the S~zary cells. Table 3 shows

the effects of such absorpt ion. There were 7 .25% norma l peripheral b lood lymphocytes react ing with an t i -S tza ry serum. Fol lowing absorp t ion with S t z a r y cells this de- clined to 0.75%.

Tab le 4 shows the results obta ined with thymocy tes and lymphocy tes obta ined from bone mar row. T h y m o - cytes failed to show fluorescence, while the bone m a r r o w lymphocy tes conta ined a high percentage (34 + 13.9) of

cells bear ing surface markers tha t reacted with an t i -a serum. Aga in , the control assays with absorbed no rma l rabbi t se rum were negat ive for immunof luorescence .

Double Labeling. The double labeling for Ig and a ant igen showed that the no rma l peripheral b lood lymphocytes bear ing Ig did no t react with anti-o- serum. The propor-

t ions of no rma l lymphocy tes (from two individuals) show- ing positive immunof luoreseence with rhodamine -con ju - gated an t i se rum (Ig bear ing cells) were 11% and 8% (average 9.5%). The a-posi t ive cells in the same two individuals a m o u n t e d to 7% and 6% (average 6.5%). The two popula t ions of cells were dist inct f rom each other.

228 A. Khan et al.: A Unique Antigen (Sigma) on S~zary Cells

Fig. 1. Photomicrograph of S6zary cells following indirect immunofluorescence assay (× 550). The anti-S~zary serum was used as the first antibody. These cells are positive for immunofluorescence

Fig. 2. Photomicrograph of a normal lymphocyte positive for indirect immunofluorescence with the anti-S~zary serum (x 550)

Table 3. Percentage of normal peripheral lymphocytes reacting with anti-a serum before and after absorption with S6zary cells

Donor Immunofluorescence with

Anti-c~ serum Absorbed anti-a serum

1 6 0 2 10 0 3 7 3 4 6 0

Mean 7.25 Mean 0.75

Table 4. Percentage of thymus and bone marrow lymphocytes reacting with the antiserum

Tissue No. tested Mean ± SD

Thymus 4 0.5 ± 0.58 Bone marrow 5 34.6 ± 13.9

D i s c u s s i o n

Circulating neoplastic cells in S6zary syndrome bear T- cell markers. Both helper and suppressor cell functions have been ascribed to these cells [2, 8]. The S~zary cells can lose E-rosette-forming ability during the evolution of the disease [22]. Cells for preparing antiserum were ob- tained from a patient who had a high percentage ofnon-E- rosetting cells that were morphological ly S~zary cells. Only 14% of his cells formed E-rosettes and 5% formed EAC-roset tes . The purpose of purification was to obtain a uniform populat ion of Sbzary cells. This was accom- plished by excluding phagocyt ic cells and those cells that formed E-rosettes and EAC-roset tes . The antiserum was prepared against a pure (nonrosetting) preparat ion of S6zary cells. I t was made specific by absorpt ion with C L L cells (B lymphocytes) and normal peripheral blood lym- phocytes, which would have a high percentage of T cells. The antiserum, which was initially cytotoxic to normal and C L L lymphocytes , lost cytotoxici ty towards these

A. Khan et al.: A Unique Antigen (Sigma) on S6zary Cells

cells following absorption. However, the antiserum main- tained cytotoxieity towards purified S~zary cells.

It is important to know whether S~zary cells possess any unique marker on their surfaee. This information should be helpful not only in elucidating the mechanisms of basic immunology, but also in understanding this dis- ease. Extensive absorption with normal and CLL cells rendered the antiserum highly reactive for S~zary cells. The lack of activity of this antiserum against T cells was demonstrated by the lack of its reactivity against thy- mocytes and E-rosette-positive acute lymphocytic leu- kemia cells (T cells). Both CL L patients had 8% a-pos- itive cells. One of these patients had only 2% E-rosette- forming cells, again suggesting that the antiserum reacted against cells other than the ones forming E-rosettes.

The double immunofluorescenee study was per- formed to investigate the relationship of B cell markers and g antigen. Rhodamine-labeled antiserum was used to detect Ig markers on B cells (direct immunofluorescence) and anti-g serum (indirect immunofluorescence), to iden- tify the g antigen-bearing ceils. Ig-bearing cells did not react with anti-g serum, showing that B cells do not carry g antigen. Similarly, the majority of CL L cells (B cells) failed to react with the antiserum. These observations suggest that the normal lymphocyte, which carries g antigen, is different from the typical T or B cell. The immunofluorescence assay showed that the antiserum reacted against a marker on the purified S~zary cells. Demonstration of an increased number of g antigen- positive cells may serve as an additional diagnostic cri- terion, along with the previously delineated features of S6zary syndrome [7, 14, 16, 18, 21]. The antiserum reacted with a small number of cells obtained from normal individuals and C L L patients in spite of absorption with C L L and normal lymphocytes. This can be explained on the basis that the number of g-positive cells in CLL and normal lymphocytes was very small and was not enough to absorb significant amount of antibody. Further ab- sorption of the antiserum with S~zary cells removed reac- tivity against normal lymphocytes (Table 3). This sug- gested that the antiserum was detecting a common antigen on S6zary cells and 5.7% of the normal peripheral blood lymphocytes. This marker was designated as sigma (g) antigen [ 11].

It is important to resolve whether the g antigen is related to neoplastic change. Our data suggest that a small number (5.7%) of normal peripheral blood lymphocytes possess g antigen. It is, therefore, likely that cells bearing this unique antigen are normally present in small numbers in the normal peripheral blood. The S6zary cells may represent proliferation and malignant transformation of g-positive cells. The source of these cells may be the bone marrow, since a high percentage of g-positive ceils was seen in this organ. The presence of g cells in the bone marrow, an organ that is spared by the disease, needs

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explanation. However, there are examples of similar spar- ing of bone marrow in some patients with B-cell ma- lignancies ( lymphoma may be limited to lymph nodes, spleen, or other organs without involvement of bone mar- row). Future work should elucidate this point.

Acknowledgements. We gratefully acknowledge the technical assis- tance of Janet Bailey, Patty Parker, Donna Corbett, Mary Terry, Rose Vilarreal, and Odessa Madison. This work was supported in part by Medical Research Inc. of Dallas.

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A. Khan et al.: A Unique Antigen (Sigma) on S~zary Cells

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Recdved June 22, 1979/Accepted February 14, 1980