Europ. J. Cancer Vol. 10, pp. 517-521. Pergamon Press 1974. Printed in Great Britain.

Redistribution of Immunoglobulin Determinants on Human Lymphocytes in Lymphoproliferative Disorders*

CHRISTOPH HUBER, GERT MICHLMAYR, HERBERT BRAUNSTEINER and HEINZ HUBER

Department of Medicine, University of Innsbruck, Austria

Abstract--Us/rig 12 si. or FITC-labeUed anti-immunoglobulin antisera we studied the redistribution of surface Ig-anti-Ig complexes on the surface of human normal and neoplastic B lymphoe.ytes, obtained from patients with chronic lymphocytic leukaemia and lymphosarkoma. The results were compared with those obtained from murine spleen cells. Whereas the majority of mouse lymphocytes exhibited cap formation under appro- priate conditions, this staining pattern was only demonstrable on a very small, but distinct percentage of normal human lymphocytes and lymphosarcoma cells. In contrast typical cap formation was not observed on leukaemic blood lymphocytes. These findings are discussed with respect to the funotional impairment of leukaemic lymphotytes.

I N T R O D U C T I O N

ON MURINE B-cells the redistribution of surface immunoglobulin (Ig) molecules, after reaction with labelled antibodies, has been studied extensively by several groups [1-6].

Under appropriate conditions crosslinking of surface molecules by bivalent antibodies resulted in "cap formation" at distinct areas overlying the Golgi field. On human peripheral blood lymphocytes a different spacing of Ig- determinants on the cell surface has been reported [7], which makes these cells less capable of cap formation. Alterations of the membrane structure of lymphocytes in chronic leukaemia (CLL) were postulated by various investigators [8, 9, 10, 11, 12], but the fate of Ig-determinants after reaction with antibodies has not yet been reported in detail. We studied the redistribution of Ig- anti Ig complexes on the surface of lymphocytes of patients with lymphoproliferative disorders. Results were

Accepted 28 March 1974 *This work was supported by the Austrian funds "Zur F6rderung der wissenschaftlichen Forschung".

517

compared with those obtained from normal human and mouse lymphocytes.

M E T H O D S

Column separated normal blood lympho- cytes, blood cells from patients with chronic lymphocytic leukaemia (CLL) and lymph node suspensions from patients with lymphosarcoma, showing surface Ig of the #k-type on the majority of cells, as well as murine spleen cells were tested. 2 x 106 lymphocytes and labelled anti-Ig antibody, resuspended in a total volume of 100 #1 tissue culture medium TC 199, were incubated at 4°C for 30 min. At the same time, cells were incubated with the same amount of unlabelled rabbit antibody; they were washed and then stained in the cold with labelled anti-rabbit Ig antibody in excess. A rabbit anti- mouse k-chain antiserum (RAM- K, 13,), 12si- labelled anti- human k- or #-chain antisera (RAH-k, RAH-#, [14]) and a l zsI- or FITC- labelled swine anti-rabbit Ig antiserum (SARIg, Fa. Dakopats, Denmark) were used. For optimal capping conditions various amounts of antibodies including con-

518 Christoph Huber, Gert Michlmayr, Herbert Braunsreiner and Heinz Huber

centrations with marked antibody excess ([14], see also Fig. 1) were employed. After 4 washings in the cold cells were resuspended in 2"0 ml TC 199 supplemented with 10% fetal calf serum and incubated at 37°C. After various intervals of incubation, cells were harvested and examifled for surface Ig distribution by autoradiography [14] or immunofluorescence

20

Cc I 15

I0

u

~ 5

I I I I i l l ] I ! , l 0-5 1.0 5"0

12'~1 - RAH - k (/.¢cj)

Fig. 1. Correlation between the dose of x a sI-RAH-k anti- body and the percentage of labelled normal human blood lymphocytes. Data of two controls are given. With this anti- serum the mean percentage of stained normal blood lympho-

cytes was 18~ [14].

[13]. Cap formation was considered, if staining was confined to less than 1 ]4 of the cell surface [5]. Cap formation was a rare event on human lymphocytes. Intermediate stages between caps and diffuse staining, however, were often observed, but only under conditions favouring cap formation and were classified as follows (see also Fig. 2): less than 1[4 of the cell surface stained--typical caps 1/4-1/2 of the cell surface stained--incomplete caps more than 1/2 of the cell surface stained, with pre- ferential staining over one pole--intermediate forms--diffuse staining.

RESULTS

Treatment of a constant number of blood lymphocytes with increasing amounts of the labelled antibody resulted in an increase of staining intensity and percentage of labelled cells, until a saturation point was reached (Fig. 1). Higher doses of antibody d id not further increase the percentages of stained cells. Crosslinking of the majority of surface Ig molecules appeared necessary for optimal cap formation. Amounts of antibody 2-4 times those required for obtaining saturation doses

2) so f

6 0

4O

20

I0 60 120 180

Time of incubof ion of 37°C, m i n

Fig. 2. Redistribution of Ig-determinants on human normal and neoplastic lymphocytes [] [] normal blood lymphocytes [] [] leukuemic blood lymphocytes [] lymphosarcoma cells, obtained from lymph node

biopsies [] [] [] direct staining technique [] [] sandwich technique 1. "diffusely stained cells" 2. "intermediate forms" 3. "incomplete caps'" 4. "typical caps" (for details of definition see under

methods) 5. P values versus normal controls> 0.05.

Redistribution of Immunoglobulin Determinants on Human Lymphocytes 519

Table 1. Redistribution of Ig-determinants : comparison of normal human and mouse lymphocytes with cells obtained from patients with chronic lymphocytic leukaemia and lymphosarcomas

I

Percentage of polar label led cells* (mean values)

Cell source Antisera Exper iments T i m e of incubat ion at 37°C (rain) No. 0 10 60 120 180

Mouse spleen cells R A M - k + F I T C - S A R I g 5 < 1 (-) 88(12) - - t - - - - H u m a n normal blood 12SI=RAH-t~ 5 0 ( < 1 ) 1(16) - - - - - -

lymphocytes x 2 S I - R A H - k 6 0 ( < 1) 2(15) 3(40) 7(51) 8(62) R A H - k + 1 2 s I - S A R I g 6 0 ( < 1) 2(23) 6(43) 16(59) 21(67)

H u m a n leukaemic blood 12SI-RAH-/~ 8 0 ( < 1) 0(3) - - - - - - lymphocytes 12 S I - R A H = k 5 0 ( < 1) 0(3) 0(7) 0(5) 0(8)

R A H - k + ~2 s I - S A R I g 5 0 ( < l) 0(8) 0(7) 0(9) 0(10) H u m a n lymphosarcoma 12 5 I - R A H - ~ 5 - - 5(25) - - - - - -

lymph node cells x 2 S I - R A H - k 5 - - 4(31) - - - - - - / i

*Numbers of typical cap forming cells and in brackets percentages of cells with " incomple te caps" (for definition see methods) are given.

t N o t done.

were therefore employed during our subsequent studies.

At 4°C normal and pathological human, as well as murine lymphoid,cells, showed a homo- genous distribution of label. After a short incubation at 37°C the majority of mouse spleen cells showed typical c a p formation. After 10 min of incubation approximately 90 7o of murine B-cells formed caps (Table 1). Polar distribution of the label defined as caps was rarely seen on human normal blood lymphocytes after this short time of incubation (Table 1 and Fig. 2). A considerable higher percentage of human blood tymphocytes (bet- ween 12 and 16 70) showed a partial redistribu- tion of label ("incomplete cap formation"). During prolonged incubation intervals at 37°C an increasing percentage of cells was observed with this staining pattern and labelling intensity decreased. Nevertheless, the marked differences in the redistribution of the label between murine and human lymphocytes remained even after an incubation time of 180 min.

Staining of leukaemic lymphocytes with anti-Ig antisera and the redistribution of label differed from normal human blood lympho- cytes. The density of both /~- and k-determi- nants was markedly decreased in 4 out of 8 CLL patients. Polar redistribution of the stain was a very rare finding even after long incuba- tion, and labelling intensity decreased more slowly. Typical caps were not observed and the percentages of leukaemic lymphocytes with "incomplete caps" were considerably lower in comparison to normal blood lymphocytes.

The formation of uropods, which carried usually the whole label, was frequent on murine spleen cells; they were occasionally seen on normal human lymphocytes and not

observed on leukaemic cells. In contrast, lymphosarkoma cells obtained from biopsies showed a normal or increased staining after incubation with anti-Ig antisera [14]. The percentage of lymphosarcoma cells with polar staining pattern was comparable or somewhat higher in comparison to normal blood lympho- cytes.

DISCUSSION

Cap formation depends on various factors including density and spatial distribution of surface molecules [1-7]. The redistribution of Ig-anti Ig complexes after incubation of normal human lymphocytes was strikingly less pro- nounced in comparison to murine lymphocytes. This may be caused by the different distribution of surface immunoglobulins in mice and man [7]. Nevertheless, on normal human lympho- cytes a small, but distinct percentage of cells showed a polar distribution of label, which increased with prolonged incubation. Beside the process of cap formation various cellular functions as endocytosis with subsequent enrich- ment of Ig-anti Ig complexes at distinct intra- cellular areas, as well as partial shedding of the label may cause a polar staining pattern. Whereas endocytosis and/or shedding of label are presumably not responsible for the redistri- bution observed within a few minutes, these processes may influence the staining pattern after longer incubation periods.

On leukaemic lymphocytes even a partial redistribution of the stain was rarely observed. The density of surface Ig molecules was reduced in some CLL patients [17, 14]. However, in patients with lymphocytes exhibiting high concentrations of anti-Ig binding sites the

520 Christoph Huber, Gert Middmayr, Herbert Braunsteiner and Heinz Huber

redistribution of label was markedly impaired. Moreover, a wide range of antibody concentra- tions as well as sandwich techniques were employed for facilitating lattice formation, but the redistribution of the label was not improved. B-lymphocytes in our patients with lympho- sarcomas exhibited membrane pattens com- parable to normal controls. Marked differences in staining density and membrane redistribu- tion of lymphosarcoma ceils and CLL lympho- cytes were observed.

The significance of cap formation has not yet been established in detail. Its role in the early steps of lymphocyte transformation has been considered, but yet not conclusively demonstrated [4, 5, 15]. Membrane fluidity

as assessed by the capacity for cap formation may be related to cell motility [4, 5, 6, 16]. An impaired recirculation of leukaemic lym- phocytes has been reported [17], and in this respect our finding of an impaired redistribution of Ig-determinants deserves particular interest.

We hope that further studies on membrane dynamics and structure will enable a better understanding of the functional impairment of leukaemic cells.

Acknowledgement--We are indebted to Prof. W. Sachsenmaicr, head of the Institute of Biochemistry and Cancer Research of the University of Innsbruck for critical correction of our manuscript and to Mrs. A.-M. F6dinger and Mrs. K. Krenkel for skilful technical assistance.

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Redistribution of lmmunoglobulin Determinants on Human Lymphocytes 521

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