Murine thymic lymphomas as model tumors for T-cell studies

CELLULAR

21, 97-111 (1976)

IMMUNOLOGY

Murine T-Cell

Thymic Markers,

Lymphomas

as Model Tumors

lmmunoglobulin

and Fc-Receptors

for T-Cell

Studies

on AKR Thymomas

PETER H. KRAMMER,RONALD CITRONBAUM,~ STANLEYE.READ,~ LUCIANA

FORNI, AND ROSEMARIE LANG

The Base1 Institute

for Imnzunology,

Received

September

Basel, Szvit~crland

11,1975

Fifteen AKR thymic lymphoma lines were studied for presence or absence of Tand B-cell membrane markers, immunoglobulin (Ig), Fc- and (?-receptors. All tumors carried Thy-l antigen and mouse-specific lymphocyte antigen (MSLA), and 9 out of 14 tumors were thymus leukemia alloantigen (TL) positive. Bone-marrowderived lymphocyte antigen (MBLA) was expressed on none of these tumors. This is strong evidence that the tumor cells are malignant variants of normal T cells. The biosynthesis of Ig was shown for one T tumor. Ig on this tumor was present on the membrane of all cells. The Ig was a molecule which reacted with anti-PA antiserum. It had the size of a 10s molecule. Several of the tumors expressed Fc-receptors (FcR) for the constant portion of the -y-chain, preferentially IgGZb. One tumor was positive for Ig and FcR. C’-receptors were not found on any of the tumor lines. The results are discussed with the concept that the tumors might be a model for the study of T cells. Evidence is presented that membrane properties of the tumors are also found in normal T-cell subpopulations. It is suggested that the tumors represent the physiological variety of T cells and that they might help to solve the problem of the T-cell receptor for antigen.

INTRODUCTION Specific functions in the immune system can be attributed cells s ( 1). T cells are involved in cell-mediated immunity,

to effector T and B graft rejection and

1 Present address: School of Medicine, University of Southern California, Los Angeles, Calif. 90033. 2 Present address : Rockefeller University, New York, N.Y. 10021. 3 Abbreviations : ATC, activated T cells ; B cells, thymus-independent lymphocytes ; C, complement; CFTD, complement-fixation-test diluent ; E-rosette, erythrocyte rosette ; EAIgGaI, ORBC sensitized with serum fraction 2b; EAIgGx, ORBC sensitized with serum 224; EAIgMR, ORBC sensitized with serum fraction la; EAIgMR, ORBC sensitized with serum fraction 205 ; EACr, ORBC sensitized with serum fraction la and mouse C’ ; EACE, ORBC sensitized with serum fraction 205 and mouse C’; FCS, foetal bovine serum; FcR, Fc-receptor; FDA, fluorescein diacetate ; FITC, fluorescein isothiocyanate conjugated; GPC, guinea pig complement; Ig, immunoglobulin; Thy-1.1, @AKR alloantigen; MBLA, mouse-specific bone marrowderived lymphocyte antigen ; MLR, mixed lymphocyte reaction ; MSLA, mouse-specific lymphocyte antigen ; NRS, normal rabbit serum; ORBC, ox red blood cells; PBS, phosphate-buffered saline ; RaMIg, rabbit anti-mouse immunoglobulin IgG ; SaMIg, sheep anti-mouse immunoglobulin IgG; SaRIg, sheep anti-rabbit immunoglobulin IgG ; SRC, sheep erythrocytes ; Tc, tissue culture ; T cells, thymus-dependent lymphocytes ; TL, thymus leukemia alloantigen ; TRITC, tetramethylrhodamine isothiocyanate conjugated. 97 Copyright0 1976 by AcademicPress, Inc. All rights of reproductionin any form reserved.

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“helper effect” in response to certain antigens while B cells are directly responsible for antibody production. Thymus and thymus-derived T cells carry Thy-I (8) as well as MSLA membrane antigens (2, 3). In some mouse strains T cells acquire TL antigens during differentiation in the thymus which are subsequently lost when the cells leave the thymic gland (4). B cells have been characterized by MBLA antigen (5), easily detectable immunoglobulin as antigen receptor (fj), a receptor for the Fc-fragment of antibody, particularly when presented as antigenantibody complex (7), and a receptor for the C’3 component of complement (8). A great deal of our present knowledge about B cells, and plasma cells, especially structure, synthesis, assembly, and secretion of their immunoglobulin molecules, comes from work with myelomas and homogeneous B-cell tumors (9). Furthermore, by detection of immunoglobulin, B cells can be characterized on a single-cell level (10). Several controversies have arisen due to the fact that a single-cell assay for T cells is not available. The demonstration of immunoglobulin on T cells is still a matter of discussion (11-16) as well as the presence or absence of other membrane receptors like Fc- and C-receptors and their function (17). Equally controversial is the concept of subsets within the T- and B-cell lineage, their function and the question whether these are stages of differentiation within stem cell-derived clones (18, 19). For reasons similar to those for which myelomas are chosen to represent certain features of differentiated B cells and plasma cells, we wanted to assess whether Gross virus-induced lymphomas spontaneously arising in thymuses of aging AKR mice (20, 21) might serve as a T-cell model. This paper describes the presence of T-cell markers on thymic lymphomas while certain B-cell markers are absent. The finding of immunoglobulin and FcR on some T tumors allows a classification of tumors in groups that might eventually represent T-cell subsets. MATERIALS

AND METHODS

Aninzals. Four- to six-week-old male AKR/J mice were obtained from the Jackson Laboratory, Bar Harbour, Me., or from our own colony at Fiillingsdorf, Basel. Tumo~.s. Spontaneous thymic lymphomas detected in aging male AKR/J mice were passaged every 1-2 weeks by serial subcutaneous passage in two to five syngeneic male mice. One tumor (2590) initially grew in spleen and lymph nodes. This tumor, after several passages, localized to the site of subcutaneous injec,tion. Cell suspensions. Except for tumor 2590 (see below), exclusively local tumors were used for assay. Under aseptic technique tumors were dissected avoiding the local lymph node as well as necrotic tumor areas. Single-cell suspensions were prepared by teasing the tissue through a 60-mesh stainless-steel screen into cold Tc Medium 199 (Difco Laboratories) plus 10% FCS (Flow Laboratories) and gently pipetting the cells through a l-ml pipet. Cells were washed three times in cold medium and clumps removed by letting the cell suspension settle for 5 min. Viability in $the supernatant fluid was determined either by trypan blue dye exclusion or by fluorochromasia with FDA (Sigma) (22). Both methods gave essentially the same results. Viability was usually between 85 and 95%. Antiserum and fluorescent conjugates. C3H anti-Thy-l.1 serum (gift from Dr. G. Roelants) was raised by the method of Raff (2). Anti-TL an,tiserum (gift from Dr.

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T-CELL

TUMORS

99

J. R. Little) 4 was prepared by injecting TL+ leukaemic cells (A%1 with the TL specificities 1, 2 and 3) from A/J mice into the congenic strain A-TL-. Rabbit anti-MSLA and rabbit anti-MBLA (anti-B-cell serum) ; gift from Dr. A. Matter) were prepared as described elsewhere (23). The Ig fraction of rabbit anti-MBLA was made and absorbed according to Niederhuber (24). Before use in was indirect immunofluorescence on AKR/ J-d erived lymphoma lines, anti-MBLA repeatedly further absorbed on pooled thymus cells from &week-old AKR/J mice. After immunoelectrophoresis the serum fraction (3 mg of protein/ml) gave one line in the IgG region with sheep anti-total rabbit serum. In indirect immunofluorescence, no staining was observed on AKR/J thymus cells. RaMIg was raised as described before (25). SaMIg was prepared by injecting a sheep in the same manner as described (25) for RaMIg. SaRIg was raised in sheep by injection of rabbit Cohn FII (Mann Research Laboratories, New York) ; this antiserum did not stain mouse cells. All antisera as well as their fluorescent conjugates (see below) were submitted to 100,OOOgcentrifugation for 1 hr to remove all aggregates. Conjugation of antisera and staining of cells. The antisera were conjugated with fluorochromes by the method of Cebra and Goldstein (26) as modified by Amante and Giuriani (27). Immunofluorescent staining of cells was performed as described (25, 28), Conditions for spot and cap formation on lymphocytes, as well as their inhibition, were used according to published methods (29, 30). Microscopy. Cell preparations were examined with a Leitz Ortholux microscope (E. Leitz, GMBH, Wetzlar, Germany) as described before (31). Between 500 and 1000 cells were usually examined. Photographs were recorded on Kodak TRI-X Pan 27 DIN film. Biosynthesis of immunoglobulin. Cells from local subcutaneous tumors were used. For tumor 2590 splenic tumor cells and cells from the local subcutaneous tumor, after the tumor had localized to the site of injection, were used. Splenic tumor cells were isolated by the velocity sedimentation method of Miller and Phillips (32). Labelling of cells with L-4, [5-3H]leucine (50 Ci/nmol, 66 &i/ml, Batch 40, The Radiochemical Centre, Amersham, U.K.), separation of the labeled cells from their supernatant medium, dialysis of the supernatam media, determinations of total radioactivity incorporation into macromolecular, nondialysable material and into serologically precipitable material have been described elsewhere (33). Serological precipitations were carried out by reacting soluble complexes of supernatant media with excess rabbit (anti-mouse Ig) antiserum with pig (antirabbit Ig) globulin (gift from Dr. F. Franek, Prague) at equivalence point (sandwich technique). Values obtained with specific (ami-mouse Ig) precipitations were corrected by subtracting values obtained with nonspecific (anti-Escherichia coli ,&galactosidase) precipitations. All serological determinations were carried out at least in duplicate. The IgM-specific amisera were raised in rabbits by repeated injections of 1 mg each of purified (34) extracellular 19s MOPC 104E IgM (A, r). The specificity of this antiserum has been described (35). The size of the radioactive specific precipitations obtained from secreted material was determined by polyacrylamide-gel electrophoresis. Composi,te gels (36, 37) of 2.5% crosslinked polyacrylamide in 0.5% agarose were used for the dissociated

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serological precipitations for size analysis of the whole molecule. The gel procedure was adapted to the soluble gel procedure of Choules and Zimm (38). Test for Fc- and C-receptors. A rosette test with nonagglutjnable bovine erythrocytes (ORBC) as indicator cells (39) was used to detect FcR (EA-rosettes) and C-receptors (EAC rosettes) on lymphojd and tumor cells. Rabbit (gift from Dr. M. Ferrarini, Genova) as well as mouse (Balb/c M) ad-ORBC antisera were used. Rabbit anti-ORBC antiserum 224 (rich in IgG), rabbit anti-ORBC antiserum 205 (purified IgM fraction at a djlution of 1: 50 in CFTD) (40, 41), Balb/cM anti-ORBC antiserum fraction la (Sephadex G-200 column-purified IgM) and 2b (Sephadex G-200 column-purified IgG) were used for sensitization of indicator cells. Neither fraction la nor 2b showed agglutination of ORBC while the hemolytic titer (with GPC diluted 1: 10) was 1: 32 for la and 1: 64 for 2b. For sensitization of ORBC, la and 2b were both used jn a dilution of 1: 16 ; la was diluted in PBS and 2b in CFTD (40). Preparation of sensitized red cells for EA-rosettes. A slightly modified method described by Cohen et al. (41) was used. Equal volumes of a 2% ORBC suspensions in PBS and either rabbit or mouse anti-ORBC antiserum (serum 224 or serum fraction 2a) in dilutions indicated above were incubated for 0.5 hr at room temperature. Following incubation, the red cells were washed twice in PBS and resuspended to a 0.5% suspension in Tc medium 199 plus 10% FCS. Preparation of sensitized red cells for EAC-rosettes. A modified technique described by Nussenzweig and Say, was used (42). ORBC, thoroughly washed in CFTD were sensitized as above with ,the indicated dilutions of either serum fraction 205 or la and washed twice in CFTD. The EA complex was then treated with mouse complement, previously absorbed at 4°C with an equal volume of washed, packed ORBC and diluted 1: 2 in CFTD, washed twice in CFTD and adjusted to a concentration of 0.5% in Medium 199 plus 10% FCS. Rosette test. A 20-,uI portion of the 0.5% suspension of sensitized red cells was added to 20 ~1 of cell suspension (3 x lo6 cells/ml) in a small plastic tube (Luckhan Ltd., LPZ). Tubes were centrifuged at 4009 for 5 min at 4°C. EAC-rosettes were gently resuspended with a Pasteur pjpet directly, EA-rosettes after 0.5 hr at 0°C. Rosettes were examined under the microscope on a siliconized slide with a siliconjzed coverslip overlaid and wax-sealed at the edges. Every test was done in triplicate and *the percentage of rosettes determined from 300 cells counted per slide. Cells showing more than 4 adherent erythrocytes were counted as positive. Every test included E-rosettes as simultaneous controls for EAIgG- and EAIgMrosettes as controls for EAC-rosettes. Both controls were always negative. To facilitate counting, toluidine blue dye was added to the slides to stain the nucleated cells, and rosettes were examined under the light microscope. Fluorochromasia with FDA (22) allowed assessment of the percentage of live cells giving rosettes. Examination under appropriate conditions for selective visualization of Auorescein with a low transmitted-light background made it possible to see both live fluorescent nucleated cells and rosetting erythrocytes at the same time. Both counting markers did not reveal any considerable difference in the percentage of resetting cells. I&&&n of Fc-rosettes. For inhibition of EA-rosette formation, purified myeloma proteins (gift from Drs. G. Torrigiani and R. Pink) at final concentration of 500 pi/ml were employed. These were added to the cell suspensions at the same time as the IgG-coated ORBC.

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101

TUMORS

RESULTS Fifteen AKR/J lymphoma lines of thymic origin were examined for known membrane antigens that might relate them to T cells, namely for Thy-l.1 (formerly BAKR) and MSLA. T Membrane Markers Table 1 shows that all 15 lines are Thy-l.1 as well as MSLA positive. Thy-l .l. Thy-l.1 antigen was detected with a sandwich immunofluorescence technique using C3H anti-Thy-l.1 serum in a dilution of 1 : 80 and FITC-SaMIg. All tumor cells of all 15 lines were brightly positive. Controls with normal mouse serum gave no fluorescent staining. In order to exclude that FITC-SaMIg might have stained either mouse Ig picked up unspecifically from the serum of the tumor host in viva or Ig synthesized by the tumors themselves (for tumor 2.590, see Relationshi! of Ig and FcR on Twnor 2590) instead of the anti-Thy-l.1 serum, tumors were also incubated with TRITCRaMIg under capping conditions before staining with anti-Thy-l.1 serum plus FITC-SaMIg. Thus, Ig could be kept in a red cap while green fluorescence for Thy-l.1 antigen was distributed all around the cell membrane. This staining pattern was obtained only for the tumors D, H, and 2590. In the case of tumors D and H, red caps were quite faint whereas, for 2590, they showed much stronger fluorescence. In all three cases, green fluorescence for Thy-l.1 was strong. It will be pointed out below that only tumor 2590 synthesized Ig while tumors D and H TABLE MEMBRANE

Tumor line

MARKERS

ON 15

Thy 1.1 (8) antigen by indirect fluorescence with C3H antiThy-l.1 antiserum + FITC-SaMIg

A B C D E F G J”

2590 I 111 IV V 1X o For explanation b For explanation

‘&YMIc

LYMPHOMAS

MSLA antigen by indirect fluorescence with rabbit antiMSLA antiserum + TRITC-SaRIg

+ + + +” + + + +a + f” + + + + + of positivity of negativity

+ + + + + + + + + + + + + + + see text. see text.

1 SPONTANEOUSLY

ARISEN

TL antigens by indirect immunofluorescence with antiTL1.2.3 antiserum + FITC-SaMIg +

IN %x.&w

M BLA indirect with MBLA

AKR/J

antigen by fluorescence rabbit anti+ TRITCSaRIg -

+

-

+

-

+

-b

+

-

+

-

+ -

-b

+ -

-b

-

-

+ -

-

+ -I-

-

102

KRAMMER

ET

AL.

have very strong FcR which also pick up the TRITC-RaMIg in an unspecific way and thus give a faint staining. FITC-SaMIg alone was never observed to be picked UP unspecifically. Thus, under strict observation of experimental conditions all 1.5 tumors could be shown to be Thy-l.1 positive. MSLA. For MSLA antigen as well, a sandwich technique for fluorescence with rabbit anti-MSLA antiserum and TRITC-SaRIg was used. Controls included NRS plus TRI’K-SaRIg. On thymus, lymph node and tumor cells, controls in appropriate dilutions were completely negative while specific fluorescence was recorded medium to strong. With the rabbit anti-MSLA antiserum, 98% of AKR/J thymus and 507% of lymph node cells were positive (Thy-l.1 on lymph node cells, 55% ; Ig on lymph node cells, about 30%). All cells of all 15 tumors equally showed positive membrane fluorescence. TL. TL-antigens: Mice of the AKR/J strain do not exhibit any of the thymus confined differentiation antigens TL 1, 2, 3 whereas leukemias of that strain can be either TL+ or TL-. On leukemic cells TL 1, 2 and 4 are expressed (4). The expression of TL might be a further indication for the T-cell origin of the tumors. For detection of TL antigens we used an anti-TL antiserum with specificities against TL1, 2, 3. The same technique for detection with membrane immunoffuorescence as for Thy-l.1 was chosen. Table 1 shows that 11 out of 15 leukemias were TL’. Cells of these 11 tumors were all brightly positive. Anti-TL antiserum was diluted 1: 100, a concentration 102-lo3 times higher than required to get cell lysis by C’.4 Leukemias were recorded TL- when no staining could be observed with an anti-TL antiserum diluted 1: 50. B Membrane Marker MBLA MBLA antigen is supposed to be a membrane marker for B cells (5). For detection of MBLA the same sandwich technique of fluorescence as for MSLA was employed. In order to validate negative results, lymph node cells from young AKR/J mice were included as positive controls. Rabbit anti-MBLA was negative on AKR/J thymus cells (dilution 1: 5) and stained ZO-30% of AKR/J lymph node cells (dilution 1: 5 and 1 : 10) with a medium to strong fluorescence (Ig on lymph node cells, approximately 30%). Spleen cells from “nude” thymusless mice on Balb/c background were positive up to 55% and lymph node cells up to 70% (dilution 1 : 10). The 1 : 10 dilution which gave positive results on all cells indicated above did not stain any tumor except tumors D, H, and 2590 (Table 1) . As tumors D, H, and 2590 were also positive with the NRS controls in the same dilution, positivity was thought to be due to unspecific uptake of the antiserum by strong FcR (see below). This proved to be true because when FcR were capped away in a red cap by NRS plus TRITC-SaRIg, all cells that showed complete capping of their FcR were negative for green fluorescence with anti-MBLA ph FITC SaRIg whereas controls with anti-MSLA were still positive. In conclusion, all 15 lymphomas do not express MBLA (B) antigen on the membrane. Ivnwaunoglobulin of Thyndc Lynzphomas The presence of membrane Ig on ,thymic lymphomas was repeatedly checked with antiserum of known specificity against mouse Ig. FITC-SaMIg, TRITC4 Loor, E., Block, N., Little, J. R., Dynamics cells. Cell. Zmmunol. 17, 351, 1975.

of the TL

antigens on thyrnus and leukemia

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T-CELL

TABLE IMM~NOGLOBULINS

Membrane

Tumor line FITC-SaMIg

2 THYMICLYMPNOMAS

Biosynthesis

Ig by fluorescence

TRITC-RaMIg

TRITC-RaMIg + TRITC-SaRIg

Secreted Ig

-

+

+++” -

-

+

++-t -

-

++

++

+++ -

+ -

-

A B C D E F G J” 2590 I III IV V IX

OF

103

TUMORS

0 Plus signs indicate

intensity

of fluorescence.

RaMIg and TRITC-RaMIg plus TRITC-SaRIg in a sandwich technique were used (Table 2). Table 2 shows that all tumors were negative except D, H, and 2590, among which 2590 was the only one staining rather strongly with FITC-SaMIg alone. Also red fluorescence with TRITC-RaMIg was much brighter on this tumor than on D and H. As NRS plus TRITC-RaMIg gave the same staining pattern on D and H and FITC-SaMIg did not show nonspecific staining via FcR, evidence was rather suggestive that only on 2590 mouse was Ig specifically detected. Capping and resynthesis of Ig on tamor 2590. To investigate further the question whether Ig of tumor 2590 was actually synthesized by the tumor itself, membrane Ig was submitted to capping and resynthesis as described in Materials and Methods. After 6 and 18 hr in culture, no restaining with TRITC-SaRIg of unlabeled RaMIg used for capping could be observed, whereas resynthesis of mouse Ig could be shown by bright staining with TRITC-RaMIg (Fig. 1). Support of data obtained by fluorescence came from results of biosynthetic studies. Biosynthetic studies. All tumors were surveyed for synthesis of Ig by precipitation of 4-hr pulsed cultures as described in Materials and Methods. As expected, tumor 2590 was found to have synthesis of Ig. Ig of tumor 2590 was subjected to size analysis on polyacrylamide-gel electrophoresis. Dissociated precipitates of secreted Ig from tumor 2590 are shown in Fig. 2. The molecule migrates in about the 10s position. Positions of 7s and 19s IgM were used as external standards. Fc and C-Receptors Fc- and C-receptors on tumor and pooled lymph node cells were detected as described under Materials and Methods. Normal AKR/J lymph node cells were

104

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FIG. 1. (A), Phase contrast picture of T-cell tumor 2.590 in culture. (B), Same cells as in (A). Ig on the tumor cell membrane capped. (C), Phase contrast picture of T-cell tumor 2590 in culture 6 hr after capping of Ig. (D), Resynthesis of Ig. Same cells as in (C).

always included in the test as positive controls and results on counted only when the percentage of rosettes for lymph node range indicated in Table 3. Generally, five to eight ORBC were attached to lymph node cells were surrounded by considerably more indicator cells. rosettes were counted immediately, keeping them at 4°C overnight results.

tumor cells were cells was in the cells while tumor Even though all did not alter the

.

4-

5

10

15

20

25

50

35

40

4.5

GEL SLICE NUMBER 2. Polyacrylamide-gel electrophoresis of secreted Ig from tumor 2,590; 19s and 7S represent positions of external standards, FIG.

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TABLE

105

TUMORS

3

PERCENTAGE RANGE OF POOLED LYMPH NODE CELLS (AKR/J) Fc- and C’-RECEPTORS (CONTROL TESTS)

Rosette type

Rosettes (range, 70)

E EA IgGaa EA IgGa EA IgMnr EA IgMa EACM EACa

12-24 19-33 11-26 10-21

Control Fc-receptor Control C’receptor

FOR

Results in Table 3 are shown as mean percentage of tests in triplicate; variation among triplicates was essentially low. Assessing viability with FDA treatment and counting rosettes and viable cells at the same time revealed that rosettes were almost exclusively formed by viable cells. Table 3 summarizes the results obtained on lymph node cells. Table 4 shows the results for all 15 tumor lines. It is evident that E-rosette and EAIgM-rosette controls as well as EAC-rosettes (for C-receptors) were negative ; low percentages for EA&-rosettes on tumors C, I, V and IX were probably due to very low host cell infiltration. The number of cells expressing FcR ( EAIgG-rosettes) varied considerably from tumor line to tumor line (from negative in tumor III to 80% in tumor D). Tumors D, H, and 2590 were repeatedly assayed for FcR and constantly gave high percentages of positive cells. Percentages of Fc-rosettes with mouse- and TABLE

4

PERCENTAGE OF Fc- AND C-RECEPTOR-BEARING CELLS ON THYMIC LYMPHOMAS AS DETECTED BY ROSETTE FORMATION WITH ORBC AS INDICATOR CELLS

Tumor line

Control E

Fc-receptor EAIgGnr

A B C D E F G J” 2590 I III IV V IX

-

3 10 1 69 7 10 1 41 24 56 12 22 15

EAIgGa

Control

C’-receptor EACH

EACR

-

-

-

-

-

EAIgMar EAIgMR

7 23 3 80 4 14 3 39 29 48 3

-

5 37 24

--

-

1 -

3

-

3 2

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TABLE Fc-RECEPTOR-BEARING

AL.

5

CELLS ON CULTURED MOUSE LYMPHOMA LINES FTC AND 2590~0

Rosette type

Control Fc-receptor

E EA IgGx EA IgGR

Rosettes (%) FTC

2590~~

-

73 92

2 7

rabbit-IgG were always in the same range. These results were also confirmed in immunofluorescence where only these three tumors gave detectable strong staining with TRITC-rabbit IgG and TRITC-SaRIg. Rabbit IgG bound strongly and could not be removed by five washes. Tumors F and 2590 with a low and a high number of FcR-bearing cells, respectively, were adapted to tissue culture by repeated passage over fibroblast monolayers until they both grew in suspension. After 6 weeks in suspension culture both tumors were tested again for FcR (Table 5). Table 5 shows that rosette counts of these cultured lines do not differ much from the in Z&JOtumors. Results for FcR on tumor FTC indicate that low numbers of cells with FcR on the in viva tumors were probably not entirely due to infiltration of local tumors by host cells. Specificity of Fc-rosettes. Specificity of Fc-rosettes on tumors D and 2590 was analysed by inhibition of rosette formation with purified myeloma proteins of differem subclasses. Table 6 shows that only with IgG2b considerable inhibition of Fc-rosettes could be achieved. Relationship

of Ig and FcR on Tumor 2590

Tumor 2590 was shown to be positive for Ig as well as for FcR. It was claimed (17) that both receptors on lymphoid cells would be functionally linked with respect to antigen binding. Therefore, we were interested in learning whether FcR could still be detected after capping of Ig. Tumor cells from tumor 2590 were incubated with RaMIg for 30 min at 0°C then put at 37°C for 30 min to allow capping, and finally assayed for Fc-rosettes (EAIgGa). Controls involved Fcrosettes without capping. TABLE INHIBITION

6

OF Fc-ROSETTE FORMATION OF Two THYMIC LYMPHOMAS, D AND 2590, WITH PURIFIED MYELOMA PROTEINS

Myeloma

Ig class

MOPC 104 MOPC 315 RPC 23 SS 63 MOPC 141

kM kA IgGl IgG2a IgG2b

Inhibition of Fc-rosettes (%) Tumor D Tumor 2590 2.3 64.6

4.2 0.7 72.5

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TABLE

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7

FcR ON TUMOR 2,590 BEFORE AND AFTER CAPPING OF Ig Rosettes

Rosette test

Control Fc-receptor

E EA IgGR

(%)

Without capping of Ig (control)

Capping of Ig

-

-

48

51

As can be seen from Table 7, conditions that cap Ig on lymphoid affect the number of tumor cells positive for FcR by rosetting.

cells did not

DISCUSSION Data presented in this paper establish the presence of T-cell membrane markers Thy-l.1 (formerly BAKR) and MSLA on 15 AKR thymic lymphoma lines, MBLA as a marker for B cells is expressed on none of these lines. TL antigens are found in 11 out of 15 tumors. In addition, one tumor actively synthesizes Ig. Several thymoma lines express receptors for the constant portion of the y-chain (FcR) while no C-receptors are present in any of the lines. The presence of “T- and B-membrane antigens” seems to be mutually exclusive (43, 44). Thy-l and MSLA have so far always been found associated with T lymphocytes and TL antigens are only present on thymic T cells. MBLA, on the other hand, has only been found on B cells, and none of these antigens seems to be present on stem cells (45). Thy-l and MSLA are not expressed on myelomas (44) whereas MBLA can be detected (43). The presence or absence of these anti.gens on lymphoid tumor cells is therefore probably a good indication from which cell of the lymphoid compartment the malignant cell has arisen (44). Other groups have shown that Thy-l (46-49) MSLA (3) and TL (4) appear on the cell membrane of certain murine leukemias. Thymic origin of the tumors presented in this study, simultaneous presence of those independent “T markers,” and absence of a “B-marker” speak in favour of the assumption that these tumors and AKR thymomas in general are T-cell derived. As TL is normally not expressed on AKR ,thymocytes, the absence of TL on four thymomas does not contradict this statement. The fact that “normal” T-cell membrane antigens are preserved on T-cell-derived malignant cells makes it likely that other T-cell membrane properties can be found on T-cell tumors. It is still an unsolved problem whether Ig is present on the T-cell membrane and can function as an antigen receptor ( 11-16, 23, 50, 51, 53, 54). Therefore, it is of considerable importance that Ig can be present on lymphomas which otherwise kept membrane markers for T cells. Harris et al. (48), Haustein et d. (55) and Boylston (56) reported the presence of Ig on a Thy-l+ cultured mouse lymphoma. We here describe 1 (tumor 2590) out of 15 tumors which actively synthesizes Ig and exposes it on the membrane of all cells.

108 The qwtion

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arises whether Ig expression on T tumors, as on tumor 2590, is and aberrant synthesis of an Ig molecule more easily accessible to ordinary detection methods liks immunofluorescence or whether those Igi- tumors represent a small subpopulation of normal T cells. In this regard Greaves and Hogg (51) have suggested that T cells might only expose an immunoglobulin receptor molecule when stimulated by antigen. The Ig molecule of tumor 2590 is bigger than 7S, a finding that is in concurrence with the work of Boylston and Mobray (56) using T-cell lymphoma El4 but that differs from IgM for T-cell lymphoma WEH122 as described by Haustein et a,?. (55). Tumor 2590 secretes a 10s molecule precipitable with an anti-d antiserum. B-cell or plasma cell contamination cannot account for that finding as 7-8 and 19s IgM and 7S IgG would have been expected in that case. Apart from the synthesis of Ig by a thymic lymphoma, the detection of FcR on some T tumors, might be of relevance for further characterization of the T-cell membrane. FcR have been demonstrated on macrophages (57), neutrophils (58) and B cells (7). Especially FcR on B-cells have gained some importance in differentiating and separating B cells from other cells of the lymphocyte compartment (59). Harris et al. (48) and Grey et ~2. (60) described one Thy-l + lymphoma with FcR while Shevach et al. (61) and Ramasamy et al. (62) could not find FcR on Thy-l + lymphomas. The failure to detect FcR+ lines could be due to the use of a relatively less sensitive assay system (61) or to the selection of tumors tested (62). We used a rosetting system with antibody- or antibody-complement-coated ORBC a indicator cells. Specially selected, nonagglutinable ORBC (39) can be heavily coated with antibody and provide a sensitive tool for the detection of Fcand C’-receptors. Nine out of 15 tumor lines showed different numbers of cells which expressed FcR for mouse or heterologous rabbit IgG. EAIgM-rosettes, however, were never obtained. Rosette inhibition studies with purified myeloma proteins of different. subclasses revealed preferential binding for IgG2b and not IgM, IgA, IgGl and IgG2a. Fc binding on T tumors was rather strong, as shown by preservation of Fc-rosettes overnight in the cold and by the failure to remove bound IgG by several washes in the immunofluorescence test. Whether the subclass specificity of the EcR is only confined to IgG2b has to be checked with more monoclonal immunoglobulins from different myelomas because Harris et al. (48) also found specificity for IgGl and IgG2a in one FcR+ T-tumor line. The possibility that FcR have been mimicked by rheumatoid factor-like activity is excluded by the failure to detect any antibody on the cell membrane by fluorescence except on tumor 2590. It has been suggested that FcR have a function in strengthening the bond between antigen-antibody complex and receptor-antibody on the membrane (17). Antibody would then attach to the FcR via its free Fc portion. Such B model would require cocapping of Ig and FcR in the plane of the membrane. Tumor 2590 is a good model to check this hypothesis because Ig and FcR occur together on the membrane. The number of EC-rosettes on this tumor stayed the same whether Ig was capped or not, which shows that the FcR function is not impaired when anti-Ig

due to malignant transformation

MURINE

T-CELL

TABLE

109

TUMORS

8

DIFFERENT MEMBRANE PROPERTIESOF T Tu,~oas Group

I II III

Number of tumors

5 9 1

Thy-l @I

+ + +

C’-receptors

MBLA

Membrane Ig by fluorescence

Ig by Biosynthesis

Fc-receptors

+ +

-

-

-

-

-

+

-

+

+

+ +

-

MSLA

____---

antibody binds to the membrane Ig. To see whether the FcR actually cocaps with the Ig as on B cells (68) requires further studies with a fluorescent technique. Whereas T-cell membrane antigens, Ig and FcR were shown to be on T-cell lymphomas, we did not find a complement-receptor-positive T-celI line. This is in accord with reports from other groups that (Z-receptors are only on B cells (8). As to the membrane properties of T tumors described in this paper, differences emerge concerning Ig and FcR. The results of these tests allow us to divide T lymphomas into groups (Table 8). It is conceivable that the differences between T tumors may reflect the physiological variety of T cells. Even though FcR have been found on a variety of other tumors including malignant human tissue of various types (41, 63), their detection on tumors that have otherwise retained T characteristics made it worthwhile to compare our findings with results on normal T-cell subpopulations. Fc- and C’-receptors are absent from small resting T cells. In contrast to ‘this, T cells activated in V&O by H-2 determinants (ATC) carry FcR (64, P. Krammer, L. Hudson, and J. Sprent, I. -hp. Med., in press. Further experiments are needed to elaborate the function of FcR on T cells, whether FcR have something to do with cell homing, antigen concentration and presentation via unspecific uptake of antigen-antibody complexes, or cell to cell cooperation (17). Experiments by Webb et al. (65) showed that cells from agammaglobulinaemic chickens which should be devoid of B cells can only specifically rosette with SRC when they absorb anti-SRC antibody. This study, however, has not conclusively shown that these cells are T cells. In addition, it has recently been demonstrated that FcR show a close association with Ia antigens at least on the B-cell membrane (66). Whether Ia antigens are also on T cells and whether these might also be associated with the FcR remains to be established, It has been pointed out that membrane properties of resting and activated T cells are represented by certain T tumors. Experiments by Loor and Roelants (67) suggest that tumor 2590 (Thy-l+, Ig+) also has its normal counterpart. T-cell differentiation studies in the nude and normal mouse reveal the existence of a population of double positive cells, (Thy-l+, Ig+), which ranges from about 2 to 10%. It might be reasoned that tumor 2590 is arrested on this differentiation pathway of T cells. Evidence presented here points out that T tumors are a helpful tool as model tumors for the study of the T cell. Further work will show whether those tumors have also retained functions of normal T cells, e.g., in analogy to myelomas with secreted antibody of defined specificity.

110

KRAMMER

ET

AL.

ACKNOWLEDGMENTS The authors wish to thank Dr. B. Pernis for discussion of the results, Dr. D. Collavo for having provided tumor 2590, Drs. M. Ferrarini, J. R. Little, A. Matter, R. Pink, G. Roelants and G. Torrigiani for antisera and myeloma proteins, and Dr. H. Ginsburg for his help with the cultures. We are very grateful for the excellent technical assistance of Misses B. Miller and K. Burvall and for help in preparation of the manuscript by Misses M. Watton, P. Hamilton and M. McCarroll.

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