lnt. . I Cancer: . 19, 66-76 (1977)

SOMATIC CELL HYBRIDS BETWEEN HUMAN LYMPHOMA LINES. 111. SURFACE MARKERS George KLEINI, Paul TERASAKI 2, Ron BILLING z, Ron HONIG2, Mikael JONDAL l, Anders ROSENl, Jesper 3, and Geoffrey CLEMENTS ZEUTHEN Department of Tumor Biology, Karolinska Institutet, S 10401 Stockholm 60, Sweden; a Department of Surgery, School of Medicine, UCLE, Los Angeles, Calif. 90024, USA; Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark; and Department of Pathology, University of Glasgow, Western Infirmary, Glasgow GI1 6NT, Scotland

Hybrids between t w o human lymphoma lines, Raji and Oaudi (8A) and Raji and BJAB(83) were examined for genetically determined and/or differentiationrelated surface markers. HL-A, B cell alloantigens, Fc and complement receptors, EBV receptors and comicroglobulin showed an autonomous dominant") expression i n the hybrid. This i s in contrast t o most previous studies on other differentiation markers, involving as a rule crosses between cells of different lineages, where t h e differentiated pattern usually became 6 6 eclipsed " i n the hybrid. Staining of activated complement and complement consumption tests showed intermediate o r partially suppressed expression i n the hybrids. This may be viewed i n relation t o the fact that these reactions do n o t merely depend on complement binding t o the receptor, but also on subsequent activation and binding of the activated complement. A m o r e complex interaction i s also suggested for immunoglobulin production. Surface immunoglobulin showed a suppressive o r intermediate pattern i n both hybrids, whereas intracellular kappa chain production showed an amplification in the 83 hybrid. The PZ microglobulin deficiency of the Daudi parent was corrected i n t h e Raji/Daudi hybrid. Two new HL-A specificities, A10 and BW17, appeared on t h i s hybrid which were not present on the parental lines. This suggests that the HL-A deficiency of t h e microglobulin. Daudi cell i s due t o i t s lack of

c'

BZ

In a previous paper (Clements et al., 1976) we have described the fusion of two EBV genomecarrying Burkitt lymphoma lines, Raji and Daudi, followed by the successful isolation of a hybrid line, designated 8A. The same paper also reports the establishment of the first hybrid between an EBV genome positive and a negative B cell lymphoma line, Raji and BJAB, designated " 83 ". In a subsequent paper (Klein et al., 1976) we have analyzed the Epstein-Barr virus (EBV) antigen production patterns of both hybrids. Our studies included spontaneous production, and also antigen induction by P3HR-1 virus superinfection and by IUdR treatment, respectively. The three parental lines that gave rise to these two hybrids differed in a number of surface characteristics, including surface immunoglobulin, HL-A and B cell (Ia) antigen patterns, j?% microglobulin, (B,M), Fc and complement receptors, and EBV

receptors. It was of considerable interest to test the hybrids with regard to these markers, in comparison with the parental lines, particularly since some of the markers were genetically determined and present on all cells, whereas others were differentiationdependent. As a rule, markers of the former type (e.g. HL-A) are expressed on somatic cell hybrids in an autonomous (codominant) fashion. On the other hand, Ia antigens, surface immunoglobulin, Fc, complement, and EBV receptors are differentiation-related markers, characteristic for (and in part restricted to) the B lymphocyte. With only some notable exceptions, differentiation markers tend to become " eclipsed " in somatic hybrids, only to re-emerge later after certain chromosomes, derived from the less differentiated partner, have been lost (see Ephrussi, 1970; Weiss et al., 1972. For recent reviews see Davis and Adelberg, 1973; Handmaker, 1973 ; Davidson, 1974). However, surface markers in general and B lymphocyte markers in particular have been studied only very sporadically or not at all. Moreover, the eclipse phenomenon was usually seen in hybrids derived from the fusion of cells of different lineages, in contrast to the present study where phenotypically different cells of the same lineage were hybridized. A point of special interest concerned the Daudi cell. This cell is deficient in both ,& microglobulin and HL-A expression (Raff, 1975; Zeuthen et al., submitted for publication). It is generally believed that these two deficiencies are related to each other. Since the Raji partner had a normal B2M and HL-A expression, the Raji/Daudi hybrid could be examined for possible complementation that would be expected to lead to the expression of Daudi-derived HL-A antigens. Finally, it was of interest to examine the possible implications of the recently demonstrated relationship between the EBV receptor and the B-lymphocyte-associated complement receptor (Jondal et al., 1976; Yefenof et al., 1976) in the parental lines and their hybrids. Received: June 16, 1976.

SURFACE MARKERS IN SOMATIC HYBRIDS MATERIAL AND METHODS c 0 -4

Cell lines and hybrids

Relevant marker characteristics of the parental cell lines are listed in Table I. All lines were maintained on RPMI 1640 medium, with 10% fetal calf serum. All lines were regularly tested for mycoplasma contamination. 3 T h e production, karyotypic and isozyme marker characterization (Clements et al., 1976) and EBVinduction patterns of the hybrids (Klein et al., 1976) have been described previously.

8 + i?

L

Vl

HL-A and B cell antigen typing The cultured lines were tested against a panel of 104 monospecific HL-A alloantisera covering 31 different specificities. Recently we have found that some of the HL-A alloantisera contained antibodies directed against a newly discovered alloantigenic system present on B cells but not on T cells (Ting et af., 1976). These non-HL-A antibodies reacted with the cultured B cell lines such as those described here. In this report, reactions of typing sera which are due to B antigens have been used as phenotypic markers in a similar way to the HL-A specificities. The B cell antigen reactions were particularly useful in the hybridization studies with Daudi because this line does not express HL-A antigens but it does express B cell antigens. Although some of the typing sera used contain both HL-A and B cell specificities, the reactions could be distinguished by the use of several antisera against each HL-A specificity. For example, Raji was considered to express HL-A A3 because three different HL-A sera of this specificity reacted with Raji. When only one or two sera out of a group of HL-A sera reacted they were considered to be B antigen reactions. A reaction was considered positive when more than 80% of the cells were killed by microcytotoxicity using rabbit complement (Terasaki and McClelland, 1964).

+t t

+ +t

.5

d

D

B

Surface immunoglobulin

Surface Ig was assessed by direct membrane immunofluorescence and quantitated by absorption of fluorescence and cytotoxicity. For direct fluorescence, FITC-conjugated anti-IgM, gamma, kappa and lambda chain reagents were used against living target cells, at a dilution of 1:15 (Hyland Laboratories, Los Angeles, Calif.). IgM and kappa were quantitated by absorption using Daudi cells as the anti-IgM or the anti-kappa target in fluorescence and cytotoxicity tests. For fluorescence, the direct FITCconjugated reagent was titrated against Daudi and a conjugate dilution was chosen at a point where the

C

.B

8

5 III

68

KLEIN ET AL.

number of stained cells started to fall but with 7080% of the cells still stained. For absorption, twofold cell number dilutions ranging from 0.5 x lo6 to 12 x los were suspended in 20 pl BSS, and incubated with an equal amount of the desired reagent dilution at 37" C for 45min with gentle agitation. Subsequently, the cells were pelleted and supernatants removed and tested against the Daudi target in the usual way. Known surface IgM kappa-positive and -negative cells were always processed in parallel as controls. For the cytotoxic absorption experiments, cell numbers ranged between 1.25 x lo5and 4 x lo6. Sera were tested against Daudi cells in the conventional, complement-dependent cytotoxicity assay. Guineapig complement was used, lacking significant cytotoxicity against the Daudi target cell.

p2 microglobulin p2M was visualized in an indirect test, using an 1:lo0 diluted rabbit anti+, microglobulin reagent (Dakopatts, Denmark), followed by a 1:20 diluted goat anti-rabbit immunoglobulin conjugate (Hyland Laboratories). Fluorescence was read in a Leitz Ortholux fluorescence microscope, at oil immersion. p2M was also quantitated in fluorescence and cytotoxic absorption tests, performed in the same way as described above for surface immunoglobulin but with Raji as the target cell. The &M-deficient Daudi was used as the negative absorption control. Radioimmunoassay for surface immunoglobulin and p2 microglobulin The radioimmunoassay for surface-localized kappa chains was performed as described elsewhere (Rosen et al., submitted for publication). Five or ten million cells were counted in a Biirker chamber, washed twice in BSS and detergent-extracted in 0.2 mi, 20 mM Tris HCI buffer containing 100 mM NaCI, 1 mM EDTA and 0.4%Triton X-100. This procedure extracts all membrane-bound Ig and also releases intracellular Ig (Jensenius, 1976). According to Grundke-Iqbal and Uhr (1974) and Nilsson et al. (1974) established human lymphoid lines contain only small amounts of intracellular Ig, if any. Most Burkitt lymphoma derived lines fail to secrete Ig, Tenpl of extract were tested in duplicate or triplicate tubes at different dilutions in a doubleantibody radioimmunoassay. Purified kappa or lambda light chains (from Bence-Jones proteinuria patients) were labelled with lS6I.Rabbit anti kappa/ lambda (Daco, Copenhagen) IgG diluted 1:3,000 gave 50% binding of the labelled protein. The Igcontaining cell extracts were compared to a purified standard reagent (kappa/lambda) for their ability to inhibit the reaction after precipitation by a second antibody, sheep anti-rabbit gammaglobulin, followed

by three washes, centrifugation of the precipitates and counting in a gamma counter. pz microglobulin was quantified by a solid phase radioimmunoassay, (Evrin et al., 1971). The first antibody was coupled to cyanogen-bromideactivated Sephadex. The commercially available test was obtained from Pharmacia Diagnostics, Uppsala, Sweden. The cells were extracted in the same manner as for immunoglobulins and the extracts were tested in duplicate at dilutions I :2, 1 :25, 150. Fc and complement receptor tests

These were carried out by two methods: EA and EAC rosettes and membrane fluorescence. The rosette tests were performed as described elsewhere (Jondal, 1974). Fc receptors were scored either by the adsorption of aggregated IgG, as described by Dickler and Kunkel (1972) or by rosette formation with IgG sensitized sheep erythrocytes (SRBC) (Jondal, 1974), called EA. C3b (EAC,) and C3d (EAC,) receptors were detected by rosettte formation with EAC cells sensitized with TgM and human and mouse complement respectively (Jondal, 1974). Fluorescence tests for the Fc and complement receptors were performed as described previously (Yefenof et al., 1976). For the Fc receptor test, the cells were exposed to heat-aggregated human gammaglobulin (HAG) for 30 min at 37" C, followed by washing and staining with an anti-Fc (gamma) reagent, at a dilution of 1:lO. The complement receptor was visualized by exposing the cells to fresh EBV-negative human serum, diluted 1 :20, followed by an FITC-conjugated goat anti-human ,!?lc/fila reagent at a dilution of 1 :40 (Hyland Laboratories). It is known that C3 binds to the complement receptor of B-lymphoid lines and becomes activated via the indirect pathway (Okada and Baba, 1974). Complement consumption tests The complement binding capacity of the different lines was tested in a quantitative complement consumption test. The hemolytic system consisted of 1.25% sensitized SRBC, washed three times in gelatin veronal buffer (GVB) with calcium and magnesium. The cells of the different lines were washed twice with GVB. The complement consumption test was carried out in precipitin tubes, with different cell numbers. The cells were incubated with 100 p1 at 37" C for 30 min, and shaken every 10 min. After centrifugation, 25 pl was removed and tested for hemolytic activity in microplates (incubation for 1 h at 37" C).

EB V receptors Receptors were assessed by two tests. Qualitative tests were performed by adsorbing virus-containing supernatants of the P3HR-1 line to live cells and

69

SURFACE MARKERS IN SOMATIC HYBRIDS

TYPING REACTIONS OF RAJI-ATG, DAUDI, AND BA HYBRID

Line

3,

)(In, 35)

Raji-ATG Specific Reaction Daudi Specific Reaction

Raji-ATG and Daudi Reactions

H New Hybrid Reactions

FIGURE 1

Typing reactions of Raji, Daudi and 8A hybrid.

demonstrating the attached, EBV-MA-containing material by direct fluorescence as described previously (Klein et al., 1972a). A quantitative test was performed by the method of Sairenji and Hinuma (1973): 1:30 diluted EBV containing P3HR-1 cell supernatants commonly capable of inducing 21% EApositive cells in lo6 Raji target cells after 48 h, were incubated with lo6 and lo7 test cells, respectively. The total reaction mixture of 1 ml was incubated at 4" C overnight, centrifuged, and added to 0.5 x lo6 Raji target cells. Unabsorbed virus supernatants were used as parallel controls. After incubation at 37" C for 1 h, the cells were pelleted, suspended in tissue culture medium with 8 % FCS and cultivated for 2 days. Subsequently, the cells were harvested, acetone-fixed smears were prepared and EA-positive cells were stained with a direct EA+VCA+FITCconjugate (F,-Ndungu, 1 :80).

RESULTS

HL-A and B-cell antigens The parental and hybrid lines were typed for HL-A and B lymphocyte alloantigens. Figures 1 and 2 show the positive reactions obtained from the panel of typing sera used. The 8A hybrid expresses the HL-A and B antigens of parental Raji ATG line and also the B antigens of Daudi parental line (Fig. 1). Only Daudi B cell antigens can be considered because the Daudi line lacks HL-A antigens. However, besides expressing the HL-A antigens of Raji, i.e., (3,-) (18, 35) 8A also expressed two new HL-A antigens, A10 and BW17, which were not expressed by either parental line. 8A also shows two new B cell reactions not seen on either of the parental lines. The hybrid characteristics of the 83 hybrid of Raji and BJAB are clearly seen from typing reactions

TYPING REACTIONS OF RAJI-ATG, BJAB, AND 83 HYBRID

Raji-ATG Specific Reaction Raji-ATG and BJAB Specific Reactions BJAB Specific Reaction

FIGURE 2 Typing reactions of Raji, BJAB and 83 hybrid.

70

KLEIN ET AL.

(Fig. 2). 83 expresses HL-A antigens (3, 1, 2, 10) of the first HL-A locus and (13, 35, 18) from the second locus: (3,lO) (18,35) came from the Raji parental lines and (1,2) (13,35) from the BJAB parental lines. The B reactions from both parents are expressed in the 83 hybrid line, clearly showing that it expresses antigenic information from both parental lines. Surface immunoglobulin Table I1 summarizes the results of the surface immunoglobulin tests. Each figure is based on at least three tests. The Raji ATG parent contains only a minimal amount of immunoglobulin (trace quantities of kappa and some mu-chains (Roskn et al., submitted for publication). The two other partner cells that were fused with Raji, Daudi and BJAB, carry a relatively high concentration of surface-associated IgM kappa. Cytotoxic and fluorescence absorption tests for mu and kappa chains concurred in showing that their surface-exposed immunoglobulin concentration is virtually identical. The radioimmunoassay for kappa chains that measures both surface-exposed and intracellular moieties gave similar results (18.5 vs 19.6 x lo4 molecules per cell). In spite of the similarity of the two parental combinations, the surface immunoglobulin expression of the two hybrids was quite different. The Raji/Daudi hybrid 8A had a much lower expression of both mu and kappa than Daudi, although it was slightly higher than Raji. According to the radioimmunoassay for kappa, it contained 4.12 x lo4 molecules per cell, compared to 2.5 in Raji and 18.5 in Daudi. In the absorption tests, it showed a slightly higher absorptive capacity than Raji but was clearly inferior to Daudi. Its surface-associated kappa expression appeared to be relatively more suppressed compared to Daudi than its expression of mu chains. In direct membrane fluorescence, the 83 hybrid gave a brilliant IgM-kappa reaction, fully comparable to the parental BJAB and much superior to 8A. The radioimmunoassay showed a doubling of kappa chains (surface and intracellular), compared to BJAB. Absorption tests for surface-expressed kappa chains, tested by cytotoxicity or fluorescence, were less impressive but they nevertheless showed that 83 carried a larger number of surface-associated kappa molecules than Raji, but less than BJAB. Similar findings were made with regard to surface-associated mu chain expression, with only minor differences as shown in Table 11. Figure 3 illustrates a representative IgM-cytotoxicity absorption test. The good and approximately equal absorptive capacity of Daudi and BJAB contrasts with the low and approxiamtely equal absorptive capacity of 8A and Raji, and the intermediate absorptive capacity of the 83 hybrid.

n

1 3

$1

2

A

&

A

I

I

d 0

-b 2 .-

.Y 0

I

B U

z,

I

"dr"

I

I

P m

4

x

9

.a

a"

I

I

71

SURFACE MARKERS IN SOMATIC HYBRIDS

1

1.0 T e s t s y s t e m a n t i I g M - D o u d i

1

-

-8 X 0)

06-

--- - -

FIGURE 3 Quantitative absorption test. Cytotoxicity assay was carried out with an anti-IgM serum and Daudi target cells. The logarithmic number of cells used for absorption is plotted against the cytotoxic index. The dotted line shows the cytotoxicity of unabsorbed serum. 0, Daudi; X , Raji; 0 , BJAB; A , 8A (Raji/Daudi); 0 , 83 (Raji/BJAB).

-

U

.-c

.-0 .4-

-

X

0 c 0 c

-

*2

-

)r

0

P

0

0

Figure 4. Raji target cells were used, exposed to the 1 :120 diluted anti+, microglobulin reagent, in the presence of complement. Daudi lacked all detectable Bz microglobulin expression whereas Raji absorbed

BJAB is somewhat higher than Raji and the Raji/ BJAB hybrid resembles the Raji parent. In contrast to the full p2 microglobulin expression of the Raji/Daudi hybrid, it is noteworthy that the " missing " HL-A specificities of the Daudi cell were not re-established. As already shown in Figure 1, no

1.0-

-

+

o,,

.8-

Log

a

X

number

g

of

for absorption

~

~

hybrid, o, ~ ~ ~ x,d ~ i ~ ; j . i, ;BJAB; =, Daudi).

8,

~

(Raji/

~

72

KLEIN ET AL. TABLE 111

SURFACE-ASSOCIATED Pa MICROGLOBULIN EXPRESSION, MEASURED BY ABSORPTION OF DIRECT FLUORESCENCE, A N D TOTAL 8%MICROGLOBULIN, DETERMINED BY THE RADIOIMMUNOMETRIC METHOD

Cell

'

Number of cells Total B,M (radioimmunometric e x ~ ~ ~ ~ ~ ~ ~ ~ ' s M assay) fluorescence

Raji Daudi

17.0&3.1

0

No absorption

8A (Raji/Daudi)

12.7rt2.1 23.6h2.0 17.9rt5.6

up to 1 2 x 106 5 X 1O5-1OS 5 X 106-108 106-2X106

BJAB 83 (Raji/BJAB)

5 x 105-106

Id

sQ*

l:

g2 W

ng &M/10@cells. Mean value of 4-6 tests. - a Cell numbers refer to a reaction mixture with 50 pl anti &M reagent, tested by indirect membrane fluorescence against Raji target cells as described under " Methods Three independent tests were performed with each cell.

".

that 8A contains at least one Daudi-derived chromosome 6. If the deficient HL-A expression of Daudi were merely due t o its & M deficiency, a complementation effect might have been expected in the hybrid, with the expression of new, Daudi-derived HL-A specificities.

P

Fc and complement receptors

Separate rosette tests were performed for Fc (EA), C3b ( E A C B ) and Cd receptors (EACD and EAC,,,, C, respectively). Fc and C' receptor-dependent reactions were also examined by an independent method, membrane fluorescence. As described elsewhere (Yefenof et al., 1976) the Fc receptor was stained by exposing the cells to aggregated human IgG (prepared from an EBV-negative serum), followed by staining with a goat anti-Fc (gamma) reagent that gives no background staining with any of the cell lines or hybrids used in the present study. To stain the complement receptor, the alternate pathway of complement activation was used. This was based on the observation of Okada and Baba (1974) that C' receptor-positive human lymphoid cell lines activate the alternative pathway. Similar results were recently reported by Simonian et al. (1976) in relation to normal human lymphocytes and by Budzko et al. (1976) for additional lymphoid lines. The staining reaction involves exposure of the target cells to human complement from EBV-negative donors, followed by an FITC or TRITC-conjugated anti-& reagent. We have previously found a good correlation between this staining reaction and the EAC rosette test (Yefenof et al., 1976). Table IV shows the results of Fc, complement and EBV-receptor tests, together with the quantitative complement consumption and EBV absorption tests.

& a

++ + + 8 8 + + ++:: + + + + g> 3>

73

SURFACE MARKERS IN SOMATIC HYBRIDS

Fc receptors that are virtually absent on the Raji parent cell but strongly exposed on Daudi, were fully expressed on the 8A Raji/Daudi hybrid, both by immunofluorescence and by EA-rosetting. The BJAB line shows a relatively weak Fc receptor expression, compared to Daudi, although it is clearly positive, in contrast to the negative Raji. The RajilBJAB hybrid resembled the BJAB parent. Thus it appears that Fc expression was " dominant " over non-expression and its level corresponded approximately t o the level of the expressor parent. As far as the complement receptor is concerned, an interesting discordance was obtained between EACrosetting and the immunofluorescence reaction. The former measures binding to the complement receptor whereas the latter depends on binding and subsequent activation via the alternative pathway. In the rosette test, expression was dominant over non-expression, as for the Fc receptor. Thus, Raji is 100% EACB positive, whereas Daudi is virtually negative. 8A resembled Raji in its 100% positivity. The same was found with BJAB (also negative for EAC,) as compared t o the high expressor 83 Raji/ BJAB hybrid. The EACD receptor was highly expressed on all lines and there were no significant differences in the rosette tests, although both BJAB and the 83 hybrid formed somewhat less rosettes than the other cells. Immunofluorescence staining of bound complement was very weak in BJAB, compared to Raji, and the 83 Raji/BJAB hybrid resembled BJAB. In the Raji-Daudi combination, the differences between the parental cells were too small for visual evaluation and it was therefore not possible to draw any conclusions about the behavior of the hybrid. In the quantitative complement consumption test, 2.7 times more Daudi cells than Raji cells were required to obtain 50% inhibition. For the 8A Raji/ Daudi hybrid, the figure was 2.1. BJAB consumed less complement than any of the other lines tested; 5.8

times more BJAB cells were required, compared to Raji, to obtain the same inhibition. The 83 Raji/ BJAB hybrid was intermediate, at a 2.6-fold excess over Raji. The three complement tests thus showed three different patterns : dominant expressivity in the rosette test, suppression to the less expressive level in staining and intermediate values in C' consumption. Since all three tests measure different C'dependent phenomena, this is not too surprising. Quantitative measurements of the EBV receptor by the Hinuma test gave interesting and, within themselves, consistent results. Table IV shows the absorptive activity of lo* and lo7cells of the various lines, towards a standard P3HR-1 virus suspension, in the Raji-EA induction test. One million Daudi cells had a somewhat lower absorptive capacity than Raji. The 8A hybrid resembled Raji. The same pattern was even clearer in the other combination. BJAB had a much lower absorptive capacity than Raji (and also lower than Daudi) but the 83 hybrid resembled the more expressive Raji parent, almost precisely. In the direct EBV-receptor staining test, there was no visible difference between Raji and Daudi and the 8A hybrid could therefore not be evaluated. However, BJAB showed a clearly reduced staining, in agreement with the absorption test. The 83 hybrid again showed a strong staining. This is also entirely in line with the absorption test. A clear relationship was found between the EBV receptor and the complement receptor of the human B lymphocyte (Jondal et al., 1976; Yefenof et al., 1976). This relationship is also seen in the present study, reflected by the good agreement between the EACmoUserosette tests (the same as used in the previous study) and the quantitative and qualitative EBV receptor tests. A schematic summary of the surface receptov tests is given in Table V.

TABLE V SCHEMATIC SUMMARY OF SURFACE RECEPTOR TESTS * ~~~

Surface Ig (mu/kappa) &-microglobulin Fc receptors EACmouserosettes EACBrosettes Complement staining Complement consumption EBV receptor staining EBV absorption test

Raji

Daudi

8A hybrid

-*

+++ ++ * +++ +++ Medium * ++ Medium

+++ ++ +++ +++ +++ Medium +++ High

+++ * +++ +++ * +++ High +++ High

f

Raji

-

+++ +++ * +++ * +++ High +++ * High *

BJAB

83 hybrid

+++* +++ ++* f

+-++ +++ ++ +++ +++

* Symbols marked with an asterisk designate the parental characteristic that is most prevalent in the hybrid.

-

&*

Low

+

Low

*

Medium

+++ High

14

KLEIN ET AL. DISCUSSION

In addition to the Raji/Namalwa hybrids described by Nyormoi et al. (1973), and Clements et al, (1976), the Raji/Daudi and Raji/BJAB hybrids represent the only known hybrid combinations between human lymphoma cells of B-cell derivation. In view of the fact that the parental cells differed in the expression of several important surface markers and the added advantage that the hybrids maintained a nearly complete hybrid chromosome complement, as we have shown elsewhere (Clements et al., 1976), it was of interest to test their surface marker expression. While the picture is complex, it reveals a number of novel and interesting features. The HL-A and B cell antigen typing results clearly show that the hybrid lines express transplantation antigens from both parental lines. The 83 hybrid expresses the four HL-A specificities found on Raji and the four found on BJAB. This gives the 83 hybrid 7 HL-A antigens (HL-A BW35 is common to both parental lines). Normal diploid lymphocytes can express only a maximum of four HL-A antigens; therefore, this represents further phenotypic evidence of the hybrid karyotype in the 83 line. The B cell antigens have only recently been detected serologically and in this study only reactions to individual B cell antisera are shown rather than to B cell groups. However, B cell reactions are proving to be very useful phenotypic markers for cultured B cell lines. The B cell antigens detzcted are thought to be the human analogue of the mouse Ia antigen (Bach et al., 1976). The genetic locus for these antigens appears to be closely linked to the HL-A locus which in man is found on chromosome 6 (Mann et al., 1975). It is interesting that the Daudi cell line expresses Ia-like B antigens and therefore has chromosome 6, but does not express HL-A antigens which therefore must be repressed in some way. The presence of a Daudi-derived chromosome 6 has been confirmed independently by isozyme studies (Clements et al., 1976). The fact that the 8A hybrid has two “ new ” HLA antigens, A10 and BW17, which are not expressed by either the Raji or Daudi parental lines, suggests that Raji may have supp!ied the j3% microglobulin information necessary for the expression of “ repressed ” Daudi HLA antigens. Thus it appears that the lack of B2 microglobu!in information in the Daudi cell line might be responsible for its lack of expression of microglobulin HLA antigens which are linked to 8% on the cell surface (Nilsson et al., 1974). In support of this conclusion is the observation that Daudi appears to carry a chromosomal aberration in chromosome 15 (Zeuthen et al., submitted for publication) which carries the & microglobulin structural gene (Goodfellow et al., 1975).

The surface immunoglobulin patterns may be considered in relation t o our previous study on somatic cell hybrids derived from the fusion of the mu-kappa producing Raji and the mu-lambda Namalwa cell (RosCn et al., submitted for publication). Although different hybrid clones varied in their immunoglobulin expression, in some both light chains (and also the heavy chains) were expressed, at levels comparable to those of the corresponding parent of the strain. This suggested a certain autonomous regulation of light chain production in the parental cells, maintained in the hybrid. The present hybrids showed a different picture. In Raji/Daudi, there was a clear suppression of the high surface immunoglobulin expression, characteristic for Daudi; the pattern appeared to be imposed by the Raji parent. In the Raji/BJAB this was not the case. Intracellular kappa chain production, as determined by the radioimmunoassay, exceeded the high-producer BJAB parent, suggesting some kind of an amplification or complementation phenomenon. Surface-associated kappa was also higher than in the low-producer Raji but not as high as in BJAB. Surface-associated mu chains showed a similar, intermediate pattern. It is interesting to note that the Fc and complement receptors behaved, like BZ microglobulin and HL-A, as autonomously expressed, codominant markers. This resembles the usual behavior of e.g. isoantigens, isozymes, and other genetically determined characteristics in somatic cell hybrids (see e.g. Klein et al., 19726; Hyman and Stallings, submitted for publication). It differs from the usual behavior of differentiated characteristics in somatic hybrid cells that tend to be eclipsed ”, i. e. fail to be expressed in the somatic hybrid, but may become re-expressed at a later stage, after chromosomes have been lost (Davis and Adelberg, 1973; Handmaker, 1973; Davidson, 1974). However, it must be noted that the eclipse phenomenon occurs in hybrids between cells of different lineages, as a rule, rather than between cells of the same lineage, as used in the present study. Interesting differences were noted between the results of the complement-related tests, EAC rosetting, staining of activated complement and complement consumption. While the rosette tests, based on the binding of the appropriate EAC complexes to the corresponding receptor, showed a dominant pattern in the hybrids, corresponding to the more highly expressing parent, the two other tests showed an intermediate or suppressed pattern in the hybrid. Since these latter tests involve not only simple binding but activation and binding of the activated complement as well, more complex regulatory circuits may be expected to work in the hybrid than in relation t o the simple surface receptor

SURFACE MARKERS IN SOMATIC HYBRIDS

expression. As we have illustrated before in another study (Klein et al., 1973), the power of the hybrid analysis lies in the fact that it dissociates seemingly interdependent properties and points the way towards further analyses. The EBV binding pattern was measured by two independent tests, staining of attached EBV (MA) material (Klein et al., 1972a) and by the absorption of biologically active EBV from virus-containing supernatants (Sairenji and Hinuma, 1973). Both tests concurred in showing a dominant expression in the hybrids, like the other surface receptors studied. We have previously found a close relationship, if not identity, between the EBV and the complement receptors of the human B lymphocyte (Jondal et al., 1976; Yefenof et al., 1976). They were present or absent on the same cell lines, moved together in cocapping experiments (in contrast to other surface receptor combinations) and could be cross-blocked by appropriate reagents.

75

In the present study, there was a clear correspondence between the behavior of the complement receptor, as detected by mouse complement (EAC,,,,,, the same as used in the study of Jondal et al., 1976), and the EBV receptor, in line with our previous findings. ACKNOWLEDGEMENTS

This work was supported by Contract No. NO1 CP 33316 within the Virus Cancer Program of the National Cancer Institute, by Public Health Service Research Grant No. 5R01 CA 14054-02 and the Swedish Cancer Society. The work of J. Zeuthen was supported by grant No. 521/18 from the Danish Natural Science Research Council. G. B. Clements was supported by a British Medical Research Council Travelling Fellowship and subsequently a Fellowship in Cancer Immunology (Cancer Research Institute Inc.). We wish to express our thanks to Dr. M. Fellous (Institut de Recherches sur les Maladies du Sang, Paris) for initially bringing our attention to the new HL-A specificities on the 8A hybrid.

CELLULES SOMATIQUES HYBRIDES PROVENANT DE LIGNCES DE LYMPHOME HUMAIN 111. MARQUEURS DE L A SURFACE Les hybrides entre deux lignees de lymphome humain, Raji e t Daudi (8A) e t Raji e t BJAB (83). ont 6th examines du point de vue des marqueurs de l a surface gbnetiquement dbtermines et/ou lies P la differenciation. L’expression des antighes HL-A, des alloantig&nes des cellules B, des recepteurs pour la fraction Fc e t l e complbment, der recepteurs de I’EBV e t de la microglobuline /?z est autonome (“ co-dominante ”) chez I’hybride. Ces observations sont en contradiction avec la plupart des etudes prbcedentes concernant d’autres marqueurs de l a diffbrenciation, qui 6taient gbnbralement ‘‘ Cclipsbs ” chez I’hybride; iI convient cependant de noter qu’il s’agissait d’hybrides entre cellules de lignees diffbrentes. La coloration du compl6ment activb e t les tests de fixation du complement o n t mis en evidence une expression intermediaire ou partiellement supprimhe chez les hybrides. II faut peut-6tre y voir u n rapport avec l e fait qua ces reactions ne dkpendent pas uniquement de l a liaison du complement au rbcepteur, mais aursi de I’activation e t de l a liaison du complement active. O n peut 6galement penser B une interaction plus complexe en ce qui concerne l a production d’immunoglobuline. L’expresnion de I’immunoglobuline de l a surface est supprimee ou intermediaire chez les deux hybrides, tandis que l a production intracellulaire de l a chafne kappa est amplifiCe chez I’hybride 83. La dbficience en microglobuline 82 chez le parent Daudi est corrigee chez I’hybride Raji/Daudi. Deux nouveaux antighnes HL-A, A L O e t BW17, sont apparus sur cet hybride alors qu’on ne les a pas d6celCs sur les lignees parentales. On peut en d4duire que la dbficience en H L - A de l a cellule Daudi est due B I’absence de microglobuline PZ.

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