Immunology 1979 37 653
Surface immunoglobulin on cultured foetal mouse thymocytes
D. HAUSTEIN & T. E. MAN DEL The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia
Received 28 November 1978; acceptedfor publication
Summary. Organ cultures of 14-15 day foetal mouse thymus were used as a source of non-neoplastic differentiating T cells, free of contaminating B cells. Viable cells obtained from such cultured thymuses were radio-iodinated and immunoglobulins (Ig) were isolated by co-precipitation from the 1251-labelled cellsurface proteins released during 1 h of incubation at 37°. The precipitates, both reduced and unreduced, were then analysed by polyacrylamide gel electrophoresis. The unreduced material migrated in a 5% gel as a single peak with a mobility slightly faster than that of mouse IgG. After reduction, however, two peaks were obtained (in a 10% gel), one corresponding in migration to mouse light chain and the other which moved slightly faster than mouse p chain. This pattern was identical with that previously seen for both surface Ig of normal mouse thymocytes and neoplastic T lymphoma cells. Uncultured, 15 day foetal thymocytes did not produce any detectable co-precipitated cell surface material. Ig detected in these experiments was therefore produced during in vitro culture by nonneoplastic T cells in a system free of contaminating B cells and mouse serum proteins.
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December 1978
from the cell surfaces of various populations of murine T cells, including thymocytes (Marchalonis, Cone & Santer 1972; Boylston & Mowbray, 1973; Rieber & Riethmuller, 1974; Cone & Marchalonis, 1974; Haustein & Goding, 1975; Cone, 1976; Cone & Brown, 1976; Hamerling, Pickel, Mack & Master, 1976), peripheral T lymphocytes (Marchalonis & Cone, 1973; Marchalonis, Cone & Boehmer, 1974) and monoclonal, continuously cultured T lymphoma cells (Marchalonis, Cone & Atwell 1972; Boylston & Mowbray, 1974; Haustein, Marchalonis & Crumpton, 1974; Haustein, 1975; Haustein, Marchalonis & Harris, 1975; Krammer, Citronbaum, Read, Forni & Lang, 1976). Detection of Ig in solubilized surface proteins of normal T lymphocytes, however, does not necessarily establish its source since it may be cytophilically adsorbed from serum, as shown for some activated T-cell populations (Hudson & Sprent, 1976), or be derived from small numbers of contaminating B lymphocytes or plasma cells. This possibility does not, however, account for all situations as recent studies indicated that thymus lymphocytes (Haustein & Goding, 1975; Cone, 1976; Hammerling et al., 1976) and T lymphoma cells (Haustein et al., 1975) express relatively small quantities of a surface Ig which differs from serum and B cell surface Igs in physicochemical properties. Established long term cultures of monoclonal T lymphoma cells represent the only population of highly purified T cells described in the literature so far which are free from contamination by B cells, plasma cells or mouse serum proteins. There may, however,
INTRODUCTION Several groups have isolated immunoglobulin (Ig) * Correspondence: Dr. Dieter Haustein, Institut fur experimentelle Immunologie, Philipps-Universitat, Deutschhausstr. 1, D-3550 Marburg, Fed. Rep. Germany.
0019-2805/79/0700-0653 $02.00 A) 1979 Blackwell Scientific Publications
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remain some doubt whether this expression of Ig represents a characteristic property of T cells or an aberrant phenomenon related to the neoplastic state of the cells, although the latter alternative seems unlikely because the Ig of T lymphoma cells differs from that of B cells in functional (Stocker, Marchalonis & Harris, 1974; Feldmann, Boylston & Hogg, 1975) and physico-chemical (Haustein et al., 1975) properties. In order to exclude such criticism, we describe in this paper similar studies with a source of T cells which, like the T lymphoma cells model, are uncontaminated with B cells, plasma cells or mouse serum proteins and serve therefore as an ideal T-cell population for studies of T-cell 1g. The cells studied comprise thymocytes obtained from foetal thymuses taken early in gestation (14-15 days) and grown in vitro as organ cultures for 5-6 days. Thymus cultured for these periods contain newly formed Thy- 1-positive small lymphocytes but no detectable plasma cells or B cells (Mandel & Kennedy, 1978). We have used these cell populations to show that the Ig exhibited on the surface of these cultured thymocytes appears during their development in vitro since Ig cannot be detected on the surface of their uncultured 15 day foetal precursors. In addition, we describe certain physico-chemical properties of the surface Ig isolated from the cultured cells.
MATERIALS AND METHODS
Culture technique Vaginally plugged CBA mice were used as a source of age dated foetuses; the day on which a plug was noted was taken as day 0 of gestation. Thymus lobes were dissected from 14 or 15 day foetuses and any adherent connective tissue was carefully removed. The lobes were washed twice in protein-free Eisen's balanced salt solution (EBSS) and were then placed on to Nuclepore membranes (General Electric, pore size 0-8 tm). The Nuclepore was glued over 3 mm holes punched in a carrier of thin lucite which was placed over the well of an organ culture dish (Falcon Plastics, No. 3010). The medium used for culture was Dulbecco's modified Eagle's medium (DMEM), supplemented with 15% foetal calf serum (FCS) (DMEM and FCS from Commonwealth Serum Laboratories Melbourne, Australia) and containing penicillin G (50 ul/ml), streptomycin sulphate 50 pg/ml and sodium bicarbonate (3 4 g/l). The cultures were grown in a fully
humidified incubator at 37.5-38° in 10% CO2 in air. Media were changed on the third or fourth day. Preparation of cell suspensions Since mechanical disruption of the early foetal thymuses and of cultured tissue resulted in poor viability, enzyme digestion was used in order to obtain maximum numbers of viable cells. Trypsin (0o25%, Calbiochem, A Grade) in phosphate buffered saline, pH 7 3 containing 0.02% EDTA was used. Thymus lobes were washed twice in Eisen's balanced salt solution and incubated in the trypsin solution for 15-30 min at 370. Previous studies have shown that under these conditions trypsinization did not reduce the percentage of Ig positive cells in the adult mouse spleen when assayed by direct immunofluorescence with polyvalent fluorescein-labelled rabbit anti-mouse Ig (Mandel & Kennedy, 1978). Furthermore, it was found that trypsinization has no detectable effect on the expression of Thy-l or TL (Mandel & Kennedy, 1978) nor did it affect the expression of H-2 (Mandel, unpublished observation) as assayed by immunofluorescence. These results are in agreement with those of Scheid, Boyse, Carswell & Old (1972) who showed that trypsinization has no demonstrable effect on the expression of various alloantigens on epidermal cells. Enzyme action was stopped with FCS and the thymus lobes were disrupted into a single-cell suspension by gentle pipetting. The viability was always greater than 95% by dye exclusion. The cells were washed twice, counted in a haemocytometer and radio-iodinated as described below.
Assay for B-cells A critical factor in these studies was the belief that no B cells or their progeny were present in the cultured tissue. Three assays were used to search for these cells. First, electron microscopy of cell suspensions was used to look for plasma cells and plasma blasts as described elsewhere (Mandel & Kennedy, 1978). Second, direct immunofluorescence using 1:16 dilution of polyvalent FIR anti-MIg and Ploem epiillumination was used to detect surface Ig positive cells. As a positive control trypsin treated adult CBA spleen cells were used and dilutions of the antiserum down to 1:256 were used. Third, in an attempt to introduce a greater degree of sensitivity into the detection of surface Ig (Nossal, Warner, Lewis & Sprent 1972) 125I-rabbit anti-MIg was used and the cells were examined by radioautography. The antiserum was a polyvalent rabbit (R) anti-MIg and was a gift from Prof. G.
655
Thymocyte surface immunoglobulin J. V. Nossal. Conditions for autoradiography were chosen to pick up only B cells and macrophages. The relatively low exposure time used did not allow demonstration of T cell Ig.
Cell surface radio-iodination The foetal thymocytes were washed twice before iodination in phosphate buffered saline, pH 7 3, containing 0 05 M sodium phosphate-0- 15 M NaCl (PBS). The cells were iodinated without losing viability (> 95% viable) by a modification (Haustein, 1975) of the lactoperoxidase catalysed reaction (Marchalonis et al., 1971). The conditions used were as follows. To each 107 cells was added 20 p1 of 0-05 M'251-iodide (100 mCi/ml Radiochemical Centre, Amersham, England) followed by three additions of 10 p1 of lactoperoxidase (0 25 mg/ml) and four additions of 10 p1 of hydrogen peroxide (0 03%). In contrast to previous studies (Marchalonis et al., 1971) no carrier 1271 was added during the iodination of cultured foetal thymocytes. The iodinated cells were washed once with PBS (1 5 ml per 107 cells). About 40% of the added iodide was associated with the washed cells and between 1 and 3 X 107 cells were used in each experiment.
Solubilization of radio-iodinated cell surface proteins lodinated cells were suspended in DMEM containing 10% FCS (0 7 ml of medium per 107 cells) and incubated in a 35 mm diameter sterile plastic dish (Falcon Plastics) for 1 h at 370 in a humidified incubator in a 10% CO2 in air. After 1 h incubation the viability ofthe cells was generally never less than 5% below that of the cells before incubation and the radioactivity released by the cells into the medium was 90 95% of the originally cell-associated radioactivity. The cells (incubated for 1 h) were spun out at 300 g for 5 min at 40 and the supernatant was centrifuged again under the same conditions in order to remove small numbers of remaining cells and was then dialysed against three changes of 2 L of PBS at 40 for 4 h. About 5% of the solubilized radioactive material was non-dialysable. Immunological co-precipitation Solubilized 1251-labelled cell-surface proteins were centrifuged at 12,000 g for 20 min in order to remove aggregates. Ig was isolated by specific co-precipitation with mouse IgM (MIgM) and anti-MIgM as described in detail elsewhere (Haustein et al., 1975). Control precipitates consisted of fowl IgG (FIgG) and antiFIgG. Antisera against MIgM and FIgG were made by immunizing rabbits with these immunoglobulins
(Haustein et al., 1975). As determined by a radioimmunoassay the anti-MIgM antiserum was predominantly specific for mouse p chain and showed only minute activity against light chains. A detailed description of the characterization of this antiserum was given previously (Haustein et al., 1975). Precipitations were carried out routinely with 200 p1 aliquots of solubilized cell-surface proteins. Details of the precipitation procedure are described elsewhere (Haustein et al., 1975). Disc electrophoresis in polyacrylamide gel Precipitated materials were analysed by polyacrylamide gel electrophoresis (PAGE) in a discontinuous buffer system according to the method of Laemmli (Laemmli, 1970), as described previously (Haustein et al., 1975).
RESULTS Thymuses of 14-15 day foetal CBA mice contain between 3 x 104 and 5 x 104 cells per lobe of which about 50% are lymphoid precursors (Mandel & Kennedy, 1978). During the 5-6 day culture period proliferation of these cells occurs and typical small lymphocytes are produced. By day 5-6 virtually all the lymphoid cells are typical small lymphocytes and these account for up to 9000 of the total cells in a cultured thymus lobe. The characteristics of these cells are described in detail elsewhere (Mandel & Kennedy, 1978). B lymphocytes or their progeny (plasmablasts and plasma cells) were not detected either by direct immunofluorescence with FIR anti-MIg or by radioautography with '25IR anti-MIg, or as plasma cells and plasmablasts by electron microscopy. It should be stressed that, under identical conditions for cell preparation and immunofluorescence, Ig positive cells in CBA spleen were readily detected even at antiserum dilutions far higher (1:128 and 1:256) than those used to study thymocytes for surface Ig. Furthermore the sensitive technique of radioautography also failed to detect Ig positive lymphocytes. Surface proteins of both uncultured cells and ofcells cultured for 5-6 days were radio-iodinated and subsequently solubilized by metabolic release. After dialysis and centrifugation of the solubilized surface proteins, Ig was isolated by immunological coprecipitation. As shown in Table 1, in the case of cells of uncultured thymuses only a minute difference was present between the radioactivity associated with the specific
D. Haustein & T. E. Mandel
656
Table 1. Results of a typical experiment demonstrating the isolation of radio-iodinated immunoglobulin by metabolic release from 1251-labelled foetal thymocytes
Radioactivity* precipitated by Percentage of non-dialysable Radioactivity* material specifically non-dialysable MIgM+anti-MIgM FIgG+anti-FIgG precipitated as Ig (c.p.m. x 10-4) (c.p.m. x 10-4) Foetal thymocytes (c.p.m. x l0-4)
Uncultured Cultured for 5 days
1029 1289
9 0+0 7 21 9+1.9
8 1+0 6 8 4+0-7
0.1 10
* Radioactivity (1251 c.p.m.) is normalized for 107 cells. Results represent the arithmetic mean + SE of at least five replicates.
precipitation system (MIgM+anti-MIgM) and the radioactivity associated with the control precipitation system (FIgG + anti-FIgG). By contrast a highly significant difference between the specific and control precipitations was obtained when surface proteins of cells of cultured thymuses were examined. 100% of non-dialysable material was specifically precipitated as Ig (radioactivity of control precipitate was subtracted) in the case of cells of cultured thymuses whereas less than 0-1% of non-dialysable material from cell of uncultured organs was specifically precipitated. We made an attempt to calculate the number of molecules of surface Ig per cell of cultured thymuses. The formula for the calculation is given elsewhere (Haustein et al., 1975). The calculation is based on the assumption that all the thymocytes express the same amount of surface Ig and that the counts precipitated as Ig represent a true measure of mass contribution of this protein relative to other components of the cell membrane. The calculated number of molecules of surface Ig per foetal thymocyte was 3 4 x 104 molecules per cell. This number was calculated on the basis of the precipitation data given in Table I (radioactivity precipitated by the specific minus radioactivity precipitated by the non-specific precipitation system). There was the possibility that a protein of FCS in which thymuses were cultured could be bound to the thymus cells during the 5-6 day culture and the mouse Ig-specific antiserum could have cross-reactivity for this protein. In order to exclude this possibility coprecipitation studies were performed with 1251-labelled FCS which failed to show any binding of radioiodinated FCS components by the MIgM/anti-MIgM system. Precipitated '251-labelled proteins were analysed by polyacrylamide gel electrophoresis (PAGE) in the pre-
sence of sodium dodecylsulphate (SDS). Although there was a small difference between material of cellsurface proteins from cells of uncultured thymuses bound to the specific precipitates and material bound to the control precipitates this did not resolve into discrete peaks but gave a low background over the length of the gel when analysed by PAGE. By contrast radioactive material specifically precipitated from solubilized 1251-labelled surface proteins of cells from thymuses cultured for 5-6 days resolved in one sharp peak of radioactivity when analysed in a 5% gel (Fig. la). Control precipitates gave a low background over the length of the gel. The electrophoretic mobility of the intact surface Ig of cultured foetal thymocytes was distinctly faster than that of the mouse IgG standard (Fig. I a). An attempt was made to elute this peak from the 5% gel in order to reduce the eluted material with mercaptoethanol and analyse it on a 10% gel. The amount of radioactivity eluted from the gel, however, was not enough to re-electrophorese the material on a 10% gel. In this connection it should be mentioned that the amount of protein related to the amount of radioactivity was very low compared with earlier experiments performed with thymocytes of adult mice (Haustein & Goding, 1975) or T lymphoma cells (Haustein et al., 1975). The total number of cells per experiment was usually not higher than 2 x 107 cells. Approximately eighty thymus lobes were needed to obtain this number of cells which was about the limit of organ cultures we were able to handle. When aliquots of specifically precipitated material were reduced with mercaptoethanol to cleave interchain disulphide bonds and were then analysed in a 10% gel, the specifically precipitated material resolved into three descrete peaks (Fig. 1 b): one corresponding exactly to the position of light chain of a mouse Ig standard, one with an electrophoretic mobility
Thymocyte surface immunoglobulin
657
1975). Ig isolated from such cell lysates gave similar patterns to those described above for metabolically released material. These patterns, however, showed a high background of radioactivity and were not as reproducible as those obtained for Ig solubilized by metabolic release. DISCUSSION E
0
n
0
0-50
0'25
'e00
0'75
Relative mobility
Figure 1. Analysis by polyacrylamide gel electrophoresis in
SDS-containing 1251-labelled
buffers
surface
Ig
of
intact
of cells
(a)
and
of thymuses
reduced which
(b) were
removed from 14 day old foetal CBA mice and then grown in vitro as organ cultures for 6 days (a, 5% gel; b, 10% gel). Ig was isolated by specific co-precipitation from
21
sIlabelled
cell surface proteins which were solubilized by metabolic release.
*,
specific precipitates; o, control precipitates.
p,
y
and L refer to positions of standard mouse Ig chains; MIgG refers to the position of intact mouse IgG standard.
slightly, but reproducibly, faster than that of p-chain
of a mouse serum
IgM
standard and one peak with an
electrophoretic mobility of 0 45 which was also present when the control precipitate was analysed (Fig.
1lb).
The electrophoretic mobility of the latter peak
was clearly distinct from that of y-chain of a mouse IgG standard and corresponded to a molecular weight
of 40,000-45,000. This peak is not an artifact depending on the amount of protein applied to the gels as shown by experiments in which various amounts of radioactivity
were loaded on gels.
Furthermore,
it
should be stressed again that in these experiments the amount of protein related to the amount of radioactivity is extremely low (see Material and Methods). Radio-iodinated surface proteins of cells from thymuses cultured for 5-6
days were also solubilized by
extraction with 1% Nonidet P40/6 M urea (Haustein,
A major controversy in contemporary immunology is whether or not T cells produce Ig. The objections to previous studies that demonstrated the presence of Ig in populations of T cells are three-fold. First, it was usually impossible to categorically exclude contaminating B cell or plasma cells. Second, the presence of mouse serum 1g, possibly adsorbed on the T-cell surface, was difficult to exclude. Third, when cloned T lymphoma cells were used, there is a remote possibility that these cells may express aberrant depressed genetic information coding for 1g. We believe that we have overcome these objections by using T cells differentiating in vitro from their progenitor cells which are present in the early foetal mouse thymus. The foetal thymuses were obtained from animals at a stage of gestation well before any B cells could be demonstrated anywhere in the foetus (Nossal & Pike, 1973; Spear, Wang, Lukishauser & Edelmann, 1973). In addition, we could not demonstrate Ig-positive cells either in the starting population by direct immunofluorescence, during the 6 day culture period by direct immunofluorescence or by radioautography (Mandel & Kennedy, 1978) or as plasma cells by electron microscopy (Mandel & Kennedy, 1978). Furthermore we could not demonstrate Ig on the 15 day foetal thymocytes by surface iodination although it was regularly detected after growing these cells in vitro for 5-6 days. Since the thymuses were not exposed to murine serum after their isolation from the pregnant uterus and were grown for 5-6 days in mouse Ig-free foetal calf serum, the Ig detected must have been produced by the cells themselves and could not be due to cytophillic adsorption. The finding that surface Ig of cells of 5-6 day cultured thymuses was not detected by the very sensitive autoradiographic technique but by solubilization of radio-iodinated surface proteins and subsequent coprecipitation can be explained as follows. Employing radio-iodinated anti-Ig antisera within the autoradiographic technique we may not be recognizing determinants of the T-cell Ig with which the antiserum
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D. Haustein & T. E. Mandel
would react because these determinants are buried in the T-cell membrane (Marchalonis & Cone, 1973). Another possibility is that the avidity of binding is so low that the radiolabelled material is lost during processing. By contrast as a result of the solubilization of radio-iodinated surface protein all determinants of the thymocyte surface Ig are accessible for the anti-MIgM antibodies and hence this technique may be more sensitive in detecting T-cell Ig than autoradiography. With regard to the demonstration of Ig on thymocytes of foetal mice by immunofluorescence we would like to mention the following recent finding. As shown by Szenberg, Marchalonis & Warner (1977) chicken antibodies produced against the F(ab')2 fragment of normal mouse IgG give strong indirect fluorescence with murine T cells and cultured T lymphoma cells. Using such chicken antibodies thymocytes of foetal mice cultured for 6 days (then dispersed by trypsinization) also show strong fluorescence (Mandel & Szenberg, unpublished observation). The evidence that the lymphocytes which differentiate in the cultured foetal thymus are not transformed into neoplastic cells comes from our previous studies in which we grafted cultured foetal thymuses under the kidney capsules of syngeneic mice. These grafts developed into apparently histologically normal thymuses and no such grafted mice showed any evidence of either local or disseminated lymphoma (Mandel & Russell, 1971). The PAGE analysis of the unreduced material obtained from the surfaces of the cultured foetal thymocytes showed that it migrated in a 5% gel as a single peak with a mobility slightly faster than that of a mouse IgG standard. It therefore differed from the Ig obtained previously from adult thymocytes (Cone & Marchalonis, 1974; Marchalonis & Cone, 1973) and from T lymphoma cells (Boylston & Mowbray, 1974; Haustein et al., 1975) which migrated slower than the IgG standard. Recently, however, Cone & Brown (1976) found that Ig exist on the surface of thymocytes of adult mice as molecules whose heavy and light chains are not linked by interchain disulphide bonds. Furthermore, Moseley, Marchalonis, Harris & Pye (1977) isolated Ig from medium in which T lymphoma cells had been grown and showed that the intact Ig had an electrophoretic mobility comparable to that of IgG. These authors proposed that the released molecule consists of a pair of heavy chains linked by disulphide bonds and light chains non-covalently bound to the heavy chains. The different mobilities of these intact T-cell surface Igs compared with those obtained in
earlier experiments may be most probably explained by the use of different solubilization techniques. The electrophoretic mobility of the intact Ig of foetal mice is only slightly faster than the mobility of the IgG standard and therefore similar to the mobility of intact Ig of T lymphoma cells which Moseley and co-workers (1977) isolated from culture fluid. Preliminary experiments by which the intact surface Ig of cells of cultured foetal thymuses was analysed in a lO% gel have shown that two peaks can be identified: one with a mobility corresponding to a molecular weight of 40,000-45,000 which was also present in the control precipitate (see above) and the other with a mobility corresponding to a light chain monomer of an IgG standard. The mobility of the last peak indicates that the light chain does not exist as disulphide-linked light chain dimer. We assume therefore that the surface Ig of thymocytes of foetal mice consists also of two disulphide linked heavy chains and two light chains non-covalently bound to the heavy chains. After reduction of the co-precipitated material and PAGE analysis of the products obtained in a I00% gel a light chain and a heavy chain peak was observed. The light chain corresponded in its mobility with the mouse light chain standard whereas the heavy chain peak moved slightly faster than the p chain standard. Furthermore, a third peak with a mobility of 0-45 was also present. This compound probably represents a molecule with an affinity for antigen-antibody complexes (Haustein et al., 1975; Premkumar, Potter, Singer & Sklar, 1975) and has also been identified in analyses of surface Ig from splenocytes (Haustein & Goding, 1975) adult thymocytes (Haustein & Goding, 1975; Cone, 1975) and T lymphoma cells (Haustein et al., 1975). The experiments mentioned above by which intact surface Ig of cells of cultured foetal thymuses was analysed in a I0% gel show that this component is non-covalently linked to surface Ig. In a 5% gel this component migrates together with the dye-marker where an appropriate amount of radioactivity was routinely found. Similar results were obtained when intact surface Ig of thymocytes of adult mice and T lymphoma cells were analysed in a 10% gel (Haustein, unpublished observation). Recently Bourgois, Abney & Parkhouse (1977) reported that Fc-receptor material isolated by certain antigen-antibody complexes contains fragments of approximately the size of polypeptide chains of IgM when no protease inhibitor was added to the detergent extract. Moreover, these authors argue that this material may have been mistakenly identified as Ig on
Thymocyte surface immunoglobulin T cells. We should like to state clearly, however, that the data we present in this paper cannot be so explained by the finding of these authors for at least two reasons. First, these authors use different extraction conditions which we previously showed not to be adequate for T-cell Ig (Haustein, 1975). Secondly, we used appropriate control precipitates expressing the Fc-region of rabbit IgG. Employing metabolically released material the FIgG/anti-FIgG control and other controls we used (e.g. haemocyanin/antihaemocyanin; ovalbumin/anti-ovalbumin) did not yield the Fc-receptor fragments Bourgouis and coworkers found, but only the peak corresponding to a molecular weight of 40,000-45,000. Finally we would like to stress that the Ig obtained from all T-cell sources, including cultured foetal thymocytes, adult thymocytes (Haustein & Goding, 1975; Cone, 1976) and T lymphoma cells (Haustein et al., 1975), all show a single heavy chain peak in contrast to the two heavy chain peaks present in the reduced Ig from B cells (Haustein & Goding, 1975; Vitetta & Uhr, 1975; Abney & Parkhouse, 1974). The mobility of the T Ig heavy chain is distinct from both B heavy chains but it can be precipitated by anti-IgM antiserum. The fact that the heavy chains of the Ig from all T-cell sources so far studied show identical mobility suggests that all of these cells carry a similar molecule on their surface and that this molecule differs at least in heavy chain properties from the Ig found on B cells. This lends further weight to the argument that the Ig we have isolated from the cultured foetal thymocyte is indeed a product of the cells and not a cytophilically adsorbed molecule.
ACKNOWLEDGMENTS The authors are grateful for the assistance of Drs J. J. Marchalonis and N. L. Warner in the preparation of this paper, and for the excellent technical help of Ms G. Marton and Ms M. M. Kennedy. This project was supported by postdoctoral fellowship of the Max Planck Society of Germany to D. H., and by grants from the National Health and Medical Research Foundation of Australia, by grant No. 11-2147 from the Volkswagen Foundation, Germany and by USPHS Grant No. CA-20085 This is publication No. 2218 from the Walter and Eliza Hall Institute of Medical Research.
659 REFERENCES
ABNEY E.R. & PARKHOUSE R.M.E. (1974) Candidate for immunoglobulin D present on murine B lymphocytes. Nature (London.) 252, 600. BoURGoIs A. ABNEY E.R. & PARKHOUSE R.M.E. (1977) Structure of mouse Fc receptor. Europ. J. Immunol. 7, 691. BOYLSTON A.W. & MOWBRAY J.F. (1973) T and B Lymphocytes: Origin, properties and Roles in Immune Responses (Ed. by M.E. Greaves, J.J.T. Owen, and M.C. Rafl). 1st edn, p. 116. Exerpta Medica, Amsterdam. BOYLSTON A.W. & MOWBRAY J.F. (1974) Surface immunoglobulin of a mouse T-cell lymphoma. Immunology, 27, 855. CONE R.E. (1976) Factors influencing the isolation of membrane immunoglobulins from T and B lymphocytes. I. Detergent effects and iodination conditions. J. Immunol. 116, 847. CoNE R.E. & BROWN W.C. (1976) Isolation of membrane associated immunoglobulins from T lymphocytes by nonionic detergents. Immunochemistry, 13, 571. CONE R.E. & MARCHALONIS J.J. (1974) Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes: selective isolation of immunoglobulin and the theta antigen by non-ionic detergents. Biochem. J. 140, 345. FELDMANN M., BOYLSTON A. & HOGG N.M. (1975) Immunological effects of IgT synthesized by theta-positive cell lines. Europ. J. Immunol. 5,429. HAMMERLING U., PICKEL H.J., MACK C. & MASTER P. (1976) Immunochemical study of surface associated immunoglobulin of murine thymocytes. Immunochemistry, 13, 533. HAUSTEIN D., MARCHALONIS J.J. & CRUMPTON M.J. (1974) Immunoglobulin of T lymphoma cells is an integral membrane protein. Nature (Lond.) 252, 602. HAUSTEIN D. (1975) Effective radioiodination by lactoperoxidase and solubilisation of cell surface proteins of cultured murine T lymphoma cells. J. immunol. Meth. 7, 25. HAUSTEIN D., MARCHALONIS J.J. & HARRIS A.W. (1975) Immunoglobulin of T-lymphoma cells. Biosynthesis, surface representation and partial characterization. Biochemistry, 14, 1826. HAUSTEIN D. & GODING J.W. (1975) Surface immunoglobulin heavy chains of murine splenocytes and thymocytes are different. Biochem. Biophys. Res. Commun. 65,483. HUDSON L. & SPRENT J. (1976) Specific adsorption of IgM antibody onto H-2-activated mouse T lymphocytes. J. exp. Med. 143,444. KRAMMER P.H., CITRONBAUM R., READ ST E., FORNI L. & LANG R. (1976) Murine thymic lymphomas as model tumors for T-cell studies. T-cell markers, immunoglobulin and Fc-receptors on AKR thymomas. Cell. Immunol. 21,97. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680. MANDEL T.E. & RUSSELL P.J. (1971) Differentiation of foetal mouse thymus. Ultrastructure of organ cultures and of subcapsular grafts. Immunology, 21, 659. MANDEL T.E. & KENNEDY M.M. (1978) The differentiation
660
D. Haustein & T. E. Mandel
of murine thymocytes in vivo and in vitro. Immunology, 35, 317. MARCHALONIS J.J., CONE R.E. & SANTER V. (1971) Enzymic iodination. A probe for accessible surface proteins of normal and neoplastic lymphocytes. Biochem. J. 124,921. MARCHALONIS J.J., CONE R.E. & ATWELL J.L. (1972) Isolation and partial characterization of lymphocyte surface immunoglobulin. J. exp. Med. 135, 956. MARCHALONIS J.J. & CONE R.E. (1973) Biochemical and biological characteristics of lymphocyte surface immunoglobulin. Transplant. Rev. 14, 3. MARCHALONIS J.J., CONE R.E. & BOEHMER H. (1974) Surface immunoglobulins of peripheral thymus-derived lymphocytes. Immunochemistry, 11, 271. MOSELEY J.M., MARCHALONIS J.J., HARRIS A.W. & PYE J. (1977) Molecular properties of T lymphoma immunoglobulin. I. Serological and general physico-chemical properties. J. Immunogenet. 4, 233. NOSSAL G.J.V., WARNER N.L., LEWIS M. & SPRENT J. (1972) Quantitative features of a sandwich radioimmunolabelling technique for lymphocyte surface receptors. J. exp. Med. 135, 405. NOSSAL G.J.V. & PIKE B.L. (1973) Studies on the differentiation of B lymphocytes in the mouse. Immunology, 25, 33.
PREMKUMAR E., POTTER M., SINGER P.L. & SKLAR M.D. (1975) Synthesis, surface deposition, and secretion of
immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines. Cell, 6, 149. RIEBER E.P. & RIETHMULLER G. (1974) Surface immunoglobulin on thymus cells. 1. Release of heterologous antiIg-antibodies from thymus cells as an immunogenic complex. Z. Immun. Forsch. 147, 276. SCHEID M., BoYSE E.A., CARSWELL E.A. & OLD L.J. (1972) Serologically demonstrable alloantigens of mouse epidermal cells. J. exp. Med. 135, 938. SPEAR P.G., WANG A., RUTISHAUSER U. & EDELMANN G.M.
(1973) Characterization of splenic lymphoid cells in fetal and newborn mice. J. exp. Med. 138, 557. STOCKER J.W., MARCHALONIS J.J. & HARRIS A.W. (1974) Inhibition of a T-cell dependent immune response in Nitro by thymoma cell immunoglobulin. J. exp. Med. 139, 785. SZENBERG A., MARCHALONIS J.J. & WARNER N.L. (1977) Direct demonstration of murine thymus-dependent cell surface endogenous immunoglobulin. Proc. natn. Acad. Sci. (U.S.A.), 74, 2113. VITETTA E.S. & UHR J.W. (1975) Immunoglobulin-receptors revisited. A model for the differentiation of bone marrowderived lymphocytes is described. Science, 189, 964.
Surface immunoglobulin on cultured foetal mouse thymocytes
D. HAUSTEIN & T. E. MAN DEL The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia
Received 28 November 1978; acceptedfor publication
Summary. Organ cultures of 14-15 day foetal mouse thymus were used as a source of non-neoplastic differentiating T cells, free of contaminating B cells. Viable cells obtained from such cultured thymuses were radio-iodinated and immunoglobulins (Ig) were isolated by co-precipitation from the 1251-labelled cellsurface proteins released during 1 h of incubation at 37°. The precipitates, both reduced and unreduced, were then analysed by polyacrylamide gel electrophoresis. The unreduced material migrated in a 5% gel as a single peak with a mobility slightly faster than that of mouse IgG. After reduction, however, two peaks were obtained (in a 10% gel), one corresponding in migration to mouse light chain and the other which moved slightly faster than mouse p chain. This pattern was identical with that previously seen for both surface Ig of normal mouse thymocytes and neoplastic T lymphoma cells. Uncultured, 15 day foetal thymocytes did not produce any detectable co-precipitated cell surface material. Ig detected in these experiments was therefore produced during in vitro culture by nonneoplastic T cells in a system free of contaminating B cells and mouse serum proteins.
11
December 1978
from the cell surfaces of various populations of murine T cells, including thymocytes (Marchalonis, Cone & Santer 1972; Boylston & Mowbray, 1973; Rieber & Riethmuller, 1974; Cone & Marchalonis, 1974; Haustein & Goding, 1975; Cone, 1976; Cone & Brown, 1976; Hamerling, Pickel, Mack & Master, 1976), peripheral T lymphocytes (Marchalonis & Cone, 1973; Marchalonis, Cone & Boehmer, 1974) and monoclonal, continuously cultured T lymphoma cells (Marchalonis, Cone & Atwell 1972; Boylston & Mowbray, 1974; Haustein, Marchalonis & Crumpton, 1974; Haustein, 1975; Haustein, Marchalonis & Harris, 1975; Krammer, Citronbaum, Read, Forni & Lang, 1976). Detection of Ig in solubilized surface proteins of normal T lymphocytes, however, does not necessarily establish its source since it may be cytophilically adsorbed from serum, as shown for some activated T-cell populations (Hudson & Sprent, 1976), or be derived from small numbers of contaminating B lymphocytes or plasma cells. This possibility does not, however, account for all situations as recent studies indicated that thymus lymphocytes (Haustein & Goding, 1975; Cone, 1976; Hammerling et al., 1976) and T lymphoma cells (Haustein et al., 1975) express relatively small quantities of a surface Ig which differs from serum and B cell surface Igs in physicochemical properties. Established long term cultures of monoclonal T lymphoma cells represent the only population of highly purified T cells described in the literature so far which are free from contamination by B cells, plasma cells or mouse serum proteins. There may, however,
INTRODUCTION Several groups have isolated immunoglobulin (Ig) * Correspondence: Dr. Dieter Haustein, Institut fur experimentelle Immunologie, Philipps-Universitat, Deutschhausstr. 1, D-3550 Marburg, Fed. Rep. Germany.
0019-2805/79/0700-0653 $02.00 A) 1979 Blackwell Scientific Publications
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remain some doubt whether this expression of Ig represents a characteristic property of T cells or an aberrant phenomenon related to the neoplastic state of the cells, although the latter alternative seems unlikely because the Ig of T lymphoma cells differs from that of B cells in functional (Stocker, Marchalonis & Harris, 1974; Feldmann, Boylston & Hogg, 1975) and physico-chemical (Haustein et al., 1975) properties. In order to exclude such criticism, we describe in this paper similar studies with a source of T cells which, like the T lymphoma cells model, are uncontaminated with B cells, plasma cells or mouse serum proteins and serve therefore as an ideal T-cell population for studies of T-cell 1g. The cells studied comprise thymocytes obtained from foetal thymuses taken early in gestation (14-15 days) and grown in vitro as organ cultures for 5-6 days. Thymus cultured for these periods contain newly formed Thy- 1-positive small lymphocytes but no detectable plasma cells or B cells (Mandel & Kennedy, 1978). We have used these cell populations to show that the Ig exhibited on the surface of these cultured thymocytes appears during their development in vitro since Ig cannot be detected on the surface of their uncultured 15 day foetal precursors. In addition, we describe certain physico-chemical properties of the surface Ig isolated from the cultured cells.
MATERIALS AND METHODS
Culture technique Vaginally plugged CBA mice were used as a source of age dated foetuses; the day on which a plug was noted was taken as day 0 of gestation. Thymus lobes were dissected from 14 or 15 day foetuses and any adherent connective tissue was carefully removed. The lobes were washed twice in protein-free Eisen's balanced salt solution (EBSS) and were then placed on to Nuclepore membranes (General Electric, pore size 0-8 tm). The Nuclepore was glued over 3 mm holes punched in a carrier of thin lucite which was placed over the well of an organ culture dish (Falcon Plastics, No. 3010). The medium used for culture was Dulbecco's modified Eagle's medium (DMEM), supplemented with 15% foetal calf serum (FCS) (DMEM and FCS from Commonwealth Serum Laboratories Melbourne, Australia) and containing penicillin G (50 ul/ml), streptomycin sulphate 50 pg/ml and sodium bicarbonate (3 4 g/l). The cultures were grown in a fully
humidified incubator at 37.5-38° in 10% CO2 in air. Media were changed on the third or fourth day. Preparation of cell suspensions Since mechanical disruption of the early foetal thymuses and of cultured tissue resulted in poor viability, enzyme digestion was used in order to obtain maximum numbers of viable cells. Trypsin (0o25%, Calbiochem, A Grade) in phosphate buffered saline, pH 7 3 containing 0.02% EDTA was used. Thymus lobes were washed twice in Eisen's balanced salt solution and incubated in the trypsin solution for 15-30 min at 370. Previous studies have shown that under these conditions trypsinization did not reduce the percentage of Ig positive cells in the adult mouse spleen when assayed by direct immunofluorescence with polyvalent fluorescein-labelled rabbit anti-mouse Ig (Mandel & Kennedy, 1978). Furthermore, it was found that trypsinization has no detectable effect on the expression of Thy-l or TL (Mandel & Kennedy, 1978) nor did it affect the expression of H-2 (Mandel, unpublished observation) as assayed by immunofluorescence. These results are in agreement with those of Scheid, Boyse, Carswell & Old (1972) who showed that trypsinization has no demonstrable effect on the expression of various alloantigens on epidermal cells. Enzyme action was stopped with FCS and the thymus lobes were disrupted into a single-cell suspension by gentle pipetting. The viability was always greater than 95% by dye exclusion. The cells were washed twice, counted in a haemocytometer and radio-iodinated as described below.
Assay for B-cells A critical factor in these studies was the belief that no B cells or their progeny were present in the cultured tissue. Three assays were used to search for these cells. First, electron microscopy of cell suspensions was used to look for plasma cells and plasma blasts as described elsewhere (Mandel & Kennedy, 1978). Second, direct immunofluorescence using 1:16 dilution of polyvalent FIR anti-MIg and Ploem epiillumination was used to detect surface Ig positive cells. As a positive control trypsin treated adult CBA spleen cells were used and dilutions of the antiserum down to 1:256 were used. Third, in an attempt to introduce a greater degree of sensitivity into the detection of surface Ig (Nossal, Warner, Lewis & Sprent 1972) 125I-rabbit anti-MIg was used and the cells were examined by radioautography. The antiserum was a polyvalent rabbit (R) anti-MIg and was a gift from Prof. G.
655
Thymocyte surface immunoglobulin J. V. Nossal. Conditions for autoradiography were chosen to pick up only B cells and macrophages. The relatively low exposure time used did not allow demonstration of T cell Ig.
Cell surface radio-iodination The foetal thymocytes were washed twice before iodination in phosphate buffered saline, pH 7 3, containing 0 05 M sodium phosphate-0- 15 M NaCl (PBS). The cells were iodinated without losing viability (> 95% viable) by a modification (Haustein, 1975) of the lactoperoxidase catalysed reaction (Marchalonis et al., 1971). The conditions used were as follows. To each 107 cells was added 20 p1 of 0-05 M'251-iodide (100 mCi/ml Radiochemical Centre, Amersham, England) followed by three additions of 10 p1 of lactoperoxidase (0 25 mg/ml) and four additions of 10 p1 of hydrogen peroxide (0 03%). In contrast to previous studies (Marchalonis et al., 1971) no carrier 1271 was added during the iodination of cultured foetal thymocytes. The iodinated cells were washed once with PBS (1 5 ml per 107 cells). About 40% of the added iodide was associated with the washed cells and between 1 and 3 X 107 cells were used in each experiment.
Solubilization of radio-iodinated cell surface proteins lodinated cells were suspended in DMEM containing 10% FCS (0 7 ml of medium per 107 cells) and incubated in a 35 mm diameter sterile plastic dish (Falcon Plastics) for 1 h at 370 in a humidified incubator in a 10% CO2 in air. After 1 h incubation the viability ofthe cells was generally never less than 5% below that of the cells before incubation and the radioactivity released by the cells into the medium was 90 95% of the originally cell-associated radioactivity. The cells (incubated for 1 h) were spun out at 300 g for 5 min at 40 and the supernatant was centrifuged again under the same conditions in order to remove small numbers of remaining cells and was then dialysed against three changes of 2 L of PBS at 40 for 4 h. About 5% of the solubilized radioactive material was non-dialysable. Immunological co-precipitation Solubilized 1251-labelled cell-surface proteins were centrifuged at 12,000 g for 20 min in order to remove aggregates. Ig was isolated by specific co-precipitation with mouse IgM (MIgM) and anti-MIgM as described in detail elsewhere (Haustein et al., 1975). Control precipitates consisted of fowl IgG (FIgG) and antiFIgG. Antisera against MIgM and FIgG were made by immunizing rabbits with these immunoglobulins
(Haustein et al., 1975). As determined by a radioimmunoassay the anti-MIgM antiserum was predominantly specific for mouse p chain and showed only minute activity against light chains. A detailed description of the characterization of this antiserum was given previously (Haustein et al., 1975). Precipitations were carried out routinely with 200 p1 aliquots of solubilized cell-surface proteins. Details of the precipitation procedure are described elsewhere (Haustein et al., 1975). Disc electrophoresis in polyacrylamide gel Precipitated materials were analysed by polyacrylamide gel electrophoresis (PAGE) in a discontinuous buffer system according to the method of Laemmli (Laemmli, 1970), as described previously (Haustein et al., 1975).
RESULTS Thymuses of 14-15 day foetal CBA mice contain between 3 x 104 and 5 x 104 cells per lobe of which about 50% are lymphoid precursors (Mandel & Kennedy, 1978). During the 5-6 day culture period proliferation of these cells occurs and typical small lymphocytes are produced. By day 5-6 virtually all the lymphoid cells are typical small lymphocytes and these account for up to 9000 of the total cells in a cultured thymus lobe. The characteristics of these cells are described in detail elsewhere (Mandel & Kennedy, 1978). B lymphocytes or their progeny (plasmablasts and plasma cells) were not detected either by direct immunofluorescence with FIR anti-MIg or by radioautography with '25IR anti-MIg, or as plasma cells and plasmablasts by electron microscopy. It should be stressed that, under identical conditions for cell preparation and immunofluorescence, Ig positive cells in CBA spleen were readily detected even at antiserum dilutions far higher (1:128 and 1:256) than those used to study thymocytes for surface Ig. Furthermore the sensitive technique of radioautography also failed to detect Ig positive lymphocytes. Surface proteins of both uncultured cells and ofcells cultured for 5-6 days were radio-iodinated and subsequently solubilized by metabolic release. After dialysis and centrifugation of the solubilized surface proteins, Ig was isolated by immunological coprecipitation. As shown in Table 1, in the case of cells of uncultured thymuses only a minute difference was present between the radioactivity associated with the specific
D. Haustein & T. E. Mandel
656
Table 1. Results of a typical experiment demonstrating the isolation of radio-iodinated immunoglobulin by metabolic release from 1251-labelled foetal thymocytes
Radioactivity* precipitated by Percentage of non-dialysable Radioactivity* material specifically non-dialysable MIgM+anti-MIgM FIgG+anti-FIgG precipitated as Ig (c.p.m. x 10-4) (c.p.m. x 10-4) Foetal thymocytes (c.p.m. x l0-4)
Uncultured Cultured for 5 days
1029 1289
9 0+0 7 21 9+1.9
8 1+0 6 8 4+0-7
0.1 10
* Radioactivity (1251 c.p.m.) is normalized for 107 cells. Results represent the arithmetic mean + SE of at least five replicates.
precipitation system (MIgM+anti-MIgM) and the radioactivity associated with the control precipitation system (FIgG + anti-FIgG). By contrast a highly significant difference between the specific and control precipitations was obtained when surface proteins of cells of cultured thymuses were examined. 100% of non-dialysable material was specifically precipitated as Ig (radioactivity of control precipitate was subtracted) in the case of cells of cultured thymuses whereas less than 0-1% of non-dialysable material from cell of uncultured organs was specifically precipitated. We made an attempt to calculate the number of molecules of surface Ig per cell of cultured thymuses. The formula for the calculation is given elsewhere (Haustein et al., 1975). The calculation is based on the assumption that all the thymocytes express the same amount of surface Ig and that the counts precipitated as Ig represent a true measure of mass contribution of this protein relative to other components of the cell membrane. The calculated number of molecules of surface Ig per foetal thymocyte was 3 4 x 104 molecules per cell. This number was calculated on the basis of the precipitation data given in Table I (radioactivity precipitated by the specific minus radioactivity precipitated by the non-specific precipitation system). There was the possibility that a protein of FCS in which thymuses were cultured could be bound to the thymus cells during the 5-6 day culture and the mouse Ig-specific antiserum could have cross-reactivity for this protein. In order to exclude this possibility coprecipitation studies were performed with 1251-labelled FCS which failed to show any binding of radioiodinated FCS components by the MIgM/anti-MIgM system. Precipitated '251-labelled proteins were analysed by polyacrylamide gel electrophoresis (PAGE) in the pre-
sence of sodium dodecylsulphate (SDS). Although there was a small difference between material of cellsurface proteins from cells of uncultured thymuses bound to the specific precipitates and material bound to the control precipitates this did not resolve into discrete peaks but gave a low background over the length of the gel when analysed by PAGE. By contrast radioactive material specifically precipitated from solubilized 1251-labelled surface proteins of cells from thymuses cultured for 5-6 days resolved in one sharp peak of radioactivity when analysed in a 5% gel (Fig. la). Control precipitates gave a low background over the length of the gel. The electrophoretic mobility of the intact surface Ig of cultured foetal thymocytes was distinctly faster than that of the mouse IgG standard (Fig. I a). An attempt was made to elute this peak from the 5% gel in order to reduce the eluted material with mercaptoethanol and analyse it on a 10% gel. The amount of radioactivity eluted from the gel, however, was not enough to re-electrophorese the material on a 10% gel. In this connection it should be mentioned that the amount of protein related to the amount of radioactivity was very low compared with earlier experiments performed with thymocytes of adult mice (Haustein & Goding, 1975) or T lymphoma cells (Haustein et al., 1975). The total number of cells per experiment was usually not higher than 2 x 107 cells. Approximately eighty thymus lobes were needed to obtain this number of cells which was about the limit of organ cultures we were able to handle. When aliquots of specifically precipitated material were reduced with mercaptoethanol to cleave interchain disulphide bonds and were then analysed in a 10% gel, the specifically precipitated material resolved into three descrete peaks (Fig. 1 b): one corresponding exactly to the position of light chain of a mouse Ig standard, one with an electrophoretic mobility
Thymocyte surface immunoglobulin
657
1975). Ig isolated from such cell lysates gave similar patterns to those described above for metabolically released material. These patterns, however, showed a high background of radioactivity and were not as reproducible as those obtained for Ig solubilized by metabolic release. DISCUSSION E
0
n
0
0-50
0'25
'e00
0'75
Relative mobility
Figure 1. Analysis by polyacrylamide gel electrophoresis in
SDS-containing 1251-labelled
buffers
surface
Ig
of
intact
of cells
(a)
and
of thymuses
reduced which
(b) were
removed from 14 day old foetal CBA mice and then grown in vitro as organ cultures for 6 days (a, 5% gel; b, 10% gel). Ig was isolated by specific co-precipitation from
21
sIlabelled
cell surface proteins which were solubilized by metabolic release.
*,
specific precipitates; o, control precipitates.
p,
y
and L refer to positions of standard mouse Ig chains; MIgG refers to the position of intact mouse IgG standard.
slightly, but reproducibly, faster than that of p-chain
of a mouse serum
IgM
standard and one peak with an
electrophoretic mobility of 0 45 which was also present when the control precipitate was analysed (Fig.
1lb).
The electrophoretic mobility of the latter peak
was clearly distinct from that of y-chain of a mouse IgG standard and corresponded to a molecular weight
of 40,000-45,000. This peak is not an artifact depending on the amount of protein applied to the gels as shown by experiments in which various amounts of radioactivity
were loaded on gels.
Furthermore,
it
should be stressed again that in these experiments the amount of protein related to the amount of radioactivity is extremely low (see Material and Methods). Radio-iodinated surface proteins of cells from thymuses cultured for 5-6
days were also solubilized by
extraction with 1% Nonidet P40/6 M urea (Haustein,
A major controversy in contemporary immunology is whether or not T cells produce Ig. The objections to previous studies that demonstrated the presence of Ig in populations of T cells are three-fold. First, it was usually impossible to categorically exclude contaminating B cell or plasma cells. Second, the presence of mouse serum 1g, possibly adsorbed on the T-cell surface, was difficult to exclude. Third, when cloned T lymphoma cells were used, there is a remote possibility that these cells may express aberrant depressed genetic information coding for 1g. We believe that we have overcome these objections by using T cells differentiating in vitro from their progenitor cells which are present in the early foetal mouse thymus. The foetal thymuses were obtained from animals at a stage of gestation well before any B cells could be demonstrated anywhere in the foetus (Nossal & Pike, 1973; Spear, Wang, Lukishauser & Edelmann, 1973). In addition, we could not demonstrate Ig-positive cells either in the starting population by direct immunofluorescence, during the 6 day culture period by direct immunofluorescence or by radioautography (Mandel & Kennedy, 1978) or as plasma cells by electron microscopy (Mandel & Kennedy, 1978). Furthermore we could not demonstrate Ig on the 15 day foetal thymocytes by surface iodination although it was regularly detected after growing these cells in vitro for 5-6 days. Since the thymuses were not exposed to murine serum after their isolation from the pregnant uterus and were grown for 5-6 days in mouse Ig-free foetal calf serum, the Ig detected must have been produced by the cells themselves and could not be due to cytophillic adsorption. The finding that surface Ig of cells of 5-6 day cultured thymuses was not detected by the very sensitive autoradiographic technique but by solubilization of radio-iodinated surface proteins and subsequent coprecipitation can be explained as follows. Employing radio-iodinated anti-Ig antisera within the autoradiographic technique we may not be recognizing determinants of the T-cell Ig with which the antiserum
658
D. Haustein & T. E. Mandel
would react because these determinants are buried in the T-cell membrane (Marchalonis & Cone, 1973). Another possibility is that the avidity of binding is so low that the radiolabelled material is lost during processing. By contrast as a result of the solubilization of radio-iodinated surface protein all determinants of the thymocyte surface Ig are accessible for the anti-MIgM antibodies and hence this technique may be more sensitive in detecting T-cell Ig than autoradiography. With regard to the demonstration of Ig on thymocytes of foetal mice by immunofluorescence we would like to mention the following recent finding. As shown by Szenberg, Marchalonis & Warner (1977) chicken antibodies produced against the F(ab')2 fragment of normal mouse IgG give strong indirect fluorescence with murine T cells and cultured T lymphoma cells. Using such chicken antibodies thymocytes of foetal mice cultured for 6 days (then dispersed by trypsinization) also show strong fluorescence (Mandel & Szenberg, unpublished observation). The evidence that the lymphocytes which differentiate in the cultured foetal thymus are not transformed into neoplastic cells comes from our previous studies in which we grafted cultured foetal thymuses under the kidney capsules of syngeneic mice. These grafts developed into apparently histologically normal thymuses and no such grafted mice showed any evidence of either local or disseminated lymphoma (Mandel & Russell, 1971). The PAGE analysis of the unreduced material obtained from the surfaces of the cultured foetal thymocytes showed that it migrated in a 5% gel as a single peak with a mobility slightly faster than that of a mouse IgG standard. It therefore differed from the Ig obtained previously from adult thymocytes (Cone & Marchalonis, 1974; Marchalonis & Cone, 1973) and from T lymphoma cells (Boylston & Mowbray, 1974; Haustein et al., 1975) which migrated slower than the IgG standard. Recently, however, Cone & Brown (1976) found that Ig exist on the surface of thymocytes of adult mice as molecules whose heavy and light chains are not linked by interchain disulphide bonds. Furthermore, Moseley, Marchalonis, Harris & Pye (1977) isolated Ig from medium in which T lymphoma cells had been grown and showed that the intact Ig had an electrophoretic mobility comparable to that of IgG. These authors proposed that the released molecule consists of a pair of heavy chains linked by disulphide bonds and light chains non-covalently bound to the heavy chains. The different mobilities of these intact T-cell surface Igs compared with those obtained in
earlier experiments may be most probably explained by the use of different solubilization techniques. The electrophoretic mobility of the intact Ig of foetal mice is only slightly faster than the mobility of the IgG standard and therefore similar to the mobility of intact Ig of T lymphoma cells which Moseley and co-workers (1977) isolated from culture fluid. Preliminary experiments by which the intact surface Ig of cells of cultured foetal thymuses was analysed in a lO% gel have shown that two peaks can be identified: one with a mobility corresponding to a molecular weight of 40,000-45,000 which was also present in the control precipitate (see above) and the other with a mobility corresponding to a light chain monomer of an IgG standard. The mobility of the last peak indicates that the light chain does not exist as disulphide-linked light chain dimer. We assume therefore that the surface Ig of thymocytes of foetal mice consists also of two disulphide linked heavy chains and two light chains non-covalently bound to the heavy chains. After reduction of the co-precipitated material and PAGE analysis of the products obtained in a I00% gel a light chain and a heavy chain peak was observed. The light chain corresponded in its mobility with the mouse light chain standard whereas the heavy chain peak moved slightly faster than the p chain standard. Furthermore, a third peak with a mobility of 0-45 was also present. This compound probably represents a molecule with an affinity for antigen-antibody complexes (Haustein et al., 1975; Premkumar, Potter, Singer & Sklar, 1975) and has also been identified in analyses of surface Ig from splenocytes (Haustein & Goding, 1975) adult thymocytes (Haustein & Goding, 1975; Cone, 1975) and T lymphoma cells (Haustein et al., 1975). The experiments mentioned above by which intact surface Ig of cells of cultured foetal thymuses was analysed in a I0% gel show that this component is non-covalently linked to surface Ig. In a 5% gel this component migrates together with the dye-marker where an appropriate amount of radioactivity was routinely found. Similar results were obtained when intact surface Ig of thymocytes of adult mice and T lymphoma cells were analysed in a 10% gel (Haustein, unpublished observation). Recently Bourgois, Abney & Parkhouse (1977) reported that Fc-receptor material isolated by certain antigen-antibody complexes contains fragments of approximately the size of polypeptide chains of IgM when no protease inhibitor was added to the detergent extract. Moreover, these authors argue that this material may have been mistakenly identified as Ig on
Thymocyte surface immunoglobulin T cells. We should like to state clearly, however, that the data we present in this paper cannot be so explained by the finding of these authors for at least two reasons. First, these authors use different extraction conditions which we previously showed not to be adequate for T-cell Ig (Haustein, 1975). Secondly, we used appropriate control precipitates expressing the Fc-region of rabbit IgG. Employing metabolically released material the FIgG/anti-FIgG control and other controls we used (e.g. haemocyanin/antihaemocyanin; ovalbumin/anti-ovalbumin) did not yield the Fc-receptor fragments Bourgouis and coworkers found, but only the peak corresponding to a molecular weight of 40,000-45,000. Finally we would like to stress that the Ig obtained from all T-cell sources, including cultured foetal thymocytes, adult thymocytes (Haustein & Goding, 1975; Cone, 1976) and T lymphoma cells (Haustein et al., 1975), all show a single heavy chain peak in contrast to the two heavy chain peaks present in the reduced Ig from B cells (Haustein & Goding, 1975; Vitetta & Uhr, 1975; Abney & Parkhouse, 1974). The mobility of the T Ig heavy chain is distinct from both B heavy chains but it can be precipitated by anti-IgM antiserum. The fact that the heavy chains of the Ig from all T-cell sources so far studied show identical mobility suggests that all of these cells carry a similar molecule on their surface and that this molecule differs at least in heavy chain properties from the Ig found on B cells. This lends further weight to the argument that the Ig we have isolated from the cultured foetal thymocyte is indeed a product of the cells and not a cytophilically adsorbed molecule.
ACKNOWLEDGMENTS The authors are grateful for the assistance of Drs J. J. Marchalonis and N. L. Warner in the preparation of this paper, and for the excellent technical help of Ms G. Marton and Ms M. M. Kennedy. This project was supported by postdoctoral fellowship of the Max Planck Society of Germany to D. H., and by grants from the National Health and Medical Research Foundation of Australia, by grant No. 11-2147 from the Volkswagen Foundation, Germany and by USPHS Grant No. CA-20085 This is publication No. 2218 from the Walter and Eliza Hall Institute of Medical Research.
659 REFERENCES
ABNEY E.R. & PARKHOUSE R.M.E. (1974) Candidate for immunoglobulin D present on murine B lymphocytes. Nature (London.) 252, 600. BoURGoIs A. ABNEY E.R. & PARKHOUSE R.M.E. (1977) Structure of mouse Fc receptor. Europ. J. Immunol. 7, 691. BOYLSTON A.W. & MOWBRAY J.F. (1973) T and B Lymphocytes: Origin, properties and Roles in Immune Responses (Ed. by M.E. Greaves, J.J.T. Owen, and M.C. Rafl). 1st edn, p. 116. Exerpta Medica, Amsterdam. BOYLSTON A.W. & MOWBRAY J.F. (1974) Surface immunoglobulin of a mouse T-cell lymphoma. Immunology, 27, 855. CONE R.E. (1976) Factors influencing the isolation of membrane immunoglobulins from T and B lymphocytes. I. Detergent effects and iodination conditions. J. Immunol. 116, 847. CoNE R.E. & BROWN W.C. (1976) Isolation of membrane associated immunoglobulins from T lymphocytes by nonionic detergents. Immunochemistry, 13, 571. CONE R.E. & MARCHALONIS J.J. (1974) Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes: selective isolation of immunoglobulin and the theta antigen by non-ionic detergents. Biochem. J. 140, 345. FELDMANN M., BOYLSTON A. & HOGG N.M. (1975) Immunological effects of IgT synthesized by theta-positive cell lines. Europ. J. Immunol. 5,429. HAMMERLING U., PICKEL H.J., MACK C. & MASTER P. (1976) Immunochemical study of surface associated immunoglobulin of murine thymocytes. Immunochemistry, 13, 533. HAUSTEIN D., MARCHALONIS J.J. & CRUMPTON M.J. (1974) Immunoglobulin of T lymphoma cells is an integral membrane protein. Nature (Lond.) 252, 602. HAUSTEIN D. (1975) Effective radioiodination by lactoperoxidase and solubilisation of cell surface proteins of cultured murine T lymphoma cells. J. immunol. Meth. 7, 25. HAUSTEIN D., MARCHALONIS J.J. & HARRIS A.W. (1975) Immunoglobulin of T-lymphoma cells. Biosynthesis, surface representation and partial characterization. Biochemistry, 14, 1826. HAUSTEIN D. & GODING J.W. (1975) Surface immunoglobulin heavy chains of murine splenocytes and thymocytes are different. Biochem. Biophys. Res. Commun. 65,483. HUDSON L. & SPRENT J. (1976) Specific adsorption of IgM antibody onto H-2-activated mouse T lymphocytes. J. exp. Med. 143,444. KRAMMER P.H., CITRONBAUM R., READ ST E., FORNI L. & LANG R. (1976) Murine thymic lymphomas as model tumors for T-cell studies. T-cell markers, immunoglobulin and Fc-receptors on AKR thymomas. Cell. Immunol. 21,97. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680. MANDEL T.E. & RUSSELL P.J. (1971) Differentiation of foetal mouse thymus. Ultrastructure of organ cultures and of subcapsular grafts. Immunology, 21, 659. MANDEL T.E. & KENNEDY M.M. (1978) The differentiation
660
D. Haustein & T. E. Mandel
of murine thymocytes in vivo and in vitro. Immunology, 35, 317. MARCHALONIS J.J., CONE R.E. & SANTER V. (1971) Enzymic iodination. A probe for accessible surface proteins of normal and neoplastic lymphocytes. Biochem. J. 124,921. MARCHALONIS J.J., CONE R.E. & ATWELL J.L. (1972) Isolation and partial characterization of lymphocyte surface immunoglobulin. J. exp. Med. 135, 956. MARCHALONIS J.J. & CONE R.E. (1973) Biochemical and biological characteristics of lymphocyte surface immunoglobulin. Transplant. Rev. 14, 3. MARCHALONIS J.J., CONE R.E. & BOEHMER H. (1974) Surface immunoglobulins of peripheral thymus-derived lymphocytes. Immunochemistry, 11, 271. MOSELEY J.M., MARCHALONIS J.J., HARRIS A.W. & PYE J. (1977) Molecular properties of T lymphoma immunoglobulin. I. Serological and general physico-chemical properties. J. Immunogenet. 4, 233. NOSSAL G.J.V., WARNER N.L., LEWIS M. & SPRENT J. (1972) Quantitative features of a sandwich radioimmunolabelling technique for lymphocyte surface receptors. J. exp. Med. 135, 405. NOSSAL G.J.V. & PIKE B.L. (1973) Studies on the differentiation of B lymphocytes in the mouse. Immunology, 25, 33.
PREMKUMAR E., POTTER M., SINGER P.L. & SKLAR M.D. (1975) Synthesis, surface deposition, and secretion of
immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines. Cell, 6, 149. RIEBER E.P. & RIETHMULLER G. (1974) Surface immunoglobulin on thymus cells. 1. Release of heterologous antiIg-antibodies from thymus cells as an immunogenic complex. Z. Immun. Forsch. 147, 276. SCHEID M., BoYSE E.A., CARSWELL E.A. & OLD L.J. (1972) Serologically demonstrable alloantigens of mouse epidermal cells. J. exp. Med. 135, 938. SPEAR P.G., WANG A., RUTISHAUSER U. & EDELMANN G.M.
(1973) Characterization of splenic lymphoid cells in fetal and newborn mice. J. exp. Med. 138, 557. STOCKER J.W., MARCHALONIS J.J. & HARRIS A.W. (1974) Inhibition of a T-cell dependent immune response in Nitro by thymoma cell immunoglobulin. J. exp. Med. 139, 785. SZENBERG A., MARCHALONIS J.J. & WARNER N.L. (1977) Direct demonstration of murine thymus-dependent cell surface endogenous immunoglobulin. Proc. natn. Acad. Sci. (U.S.A.), 74, 2113. VITETTA E.S. & UHR J.W. (1975) Immunoglobulin-receptors revisited. A model for the differentiation of bone marrowderived lymphocytes is described. Science, 189, 964.
