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Ultrastructural distribution of surface immunoglobulin determinants on mouse lymphoid cells
Experimental
ULTRASTRUCTURAL IMMUNOGLOBULIN VIVIEN
Cell Research 12 (1972) 317-386
DISTRIBUTION
DETERMINANTS SANTER,
OF SURFACE
ON MOUSE
A. D. BANKHURST
LYMPHOID
CELLS
and G. J. V. NOSSAL
The Walter and Elba Hall Institute of Medical Research, Victoria 3050, Australia
SUMMARY The distribution of radioiodinated anti-immunoglobulin and anti-lymphocyte antibodies on the surfaces of lymphocytes from normal mouse spleen and thymus has been investigated using electron microscope radioautography. Discrete patches of label were seen on 5&60 % of spleen cells labelled with anti-light chain antibody, and on 30 % of spleen cells labelled with anti-p. Only a very small proportion of thymus cells were labelled by anti-light chain, and none bound anti-p. In contrast, anti-lymphocyte antibody labelled 90-100% of both spleen and thymus cells. This was either generalized labelling surrounding the whole cell, or heavy labelling over a uropod-like extension of the cytoplasm. The close similarity between labelling patterns with radioactively labelled antigen and immunoglobulin supports the hypothesis that the antigen receptor is immunoglobulin in nature, and the great difference in density of surface immunoglobulin between thymus and bone-marrow derived lymphocytes is once more emphasised.
The interaction of a lymphocyte with an antigen requires the presence of a specific surface receptor on the lymphocyte. Evidence from several studies in which lymphocyte activity was blocked with anti-immunoglobulin antibodies strongly suggests that this receptor is an immunoglobulin [l, 21. In support of this hypothesis, immunoglobulin determinants have been directly demonstrated on the surfaces of both non-thymus derived (B) lymphocytes [3-71 and thymus-derived (T) lymphocytes [8]. The ultrastructural characteristics of antigen-binding by lymphocytes have been described [lo], but very little work has been done on surface immunoglobulins at the electron microscope level. The present study examines surface immunoglobulins and 1 Present address: Unite de Recherches, Division d’hematologie, HBpital Cantonal, Geneva, Switzerland. 2&
721806
other surface antigens by radioautography with the electron microscope. Two experimental approaches were used: direct labelling with radioiodinated rabbit anti-mouse immunoglobulin antibody, and indirect (sandwich) labelling with rabbit anti-mouse immunoglobulin antibody followed by radioiodinated polyvalent sheep anti-rabbit globulin antibody. MATERIALS
AND METHODS
Animals For direct labelling experiments, male or female CBA mice 50-120 days old were used. For sandwich labelling experiments, male specific pathogen free C3H mice, 10-12 weeks old were used.
Preparation of cell suspensions Direct labelling: Mice were killed by exposure to ether. The spleens and thymuses were removed and teased gently through a fine stainless steel sieve into Exptl Cell Res 72
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ice-cold Eisen balanced salt solution (EBSS). The cells were washed at least four times through gradients of foetal calf serum (FCS; Commonwealth Serum Laboratories, Melbourne) [12] or bovine serum albumin (BSA: Commonwealth Serum Laboratories) to remove non-adherent surface material and cell debris. The viability of all cell suspensions was measured by trypan blue exclusion.
Sandwich labelling This was done according to a method recently described by Nossal et al. [9]. Briefly, mice were killed bv cervical dislocation. and the snleens and thymuses were removed and placed in -cold 10 % FCS in Eagle’s minimal essential medium (GIBCo, Grand Island, N.Y.) buffered with 10 mM HEPES (N-2-hydroxyethylpiperazine-N-1, 2-ethane sulphonic acid; CalBiochem, Los Angeles, Cnlif.). The spleens were teased through a fine stainless steel sieve and the clumos allowed to settle over FCS. The cell suspension was washed through 1 ml of FCS; debris and damaged cells were removed by the BSA method of Shortman et al. [13], and the remaining cells resuspended in 10 % FCS-Eagle medium-HEPES, and counted.
Preparation of antisera The preparation of antisera against mouse light chains, IgM and IgG has been previously described [ll]. Briefly, antisera were raised in rabbits using ourified mveloma nroteins. and the gammaglobulin fraction was separated by ~starch gel electrophoresis in Verona1 buffer. DH 8.2. These fractions were tested for anti-immunoglobulin activity by radioprecipitation assays [ll] and made monospecific by absorption with purified immunoglobulins coupled to polyaminopolystyrene [l 11. Labelling of cells by these purified anti-immunoglobulins could be specifically- blocked by prior absorption of the prenaration with uurified immunoglobulins 1121. Anti-mouse lymphocyte serum (ALS) was-prepared in rabbits using mouse thymocytes as previously described [14]. -Sheep a&rabbit globulin serum (SARG) was a gift from Dr Zoltan Ovary. Globulin fractions (IgG)-of these antisera were prepared by agar block electrophoresis or starch block electrophoresis in Verona1 buffer, pH 8.2.
Radioiodination of antisera The globulin fractions were labelled with ?odine according to the method of Hunter & Greenwood [15]. The specific activity of the anti-immunoglobulin IgG was 90-140 pCi/pg protein and of the sheep anti-rabbit immunoglobulin IgG was 6-10 ,&i/pg protein.
Cell labelling Direct method: Cell suspensions were incubated with 1261-anti-immunoglobulin IgG in 10% FCSExptl
Cell Res 72
Dulbecco medium for 1 h at 0°C. Generally 5 x IO6 viable cells were incubated with 5-7.5 ,ug of protein. The cells were then washed four times through FCS gradients before fixation for electron microscopy. Sandwich method: Cells were incubated for 30 min at 0°C with unlabelled anti-immunoglobulin IgG at a final dilution of 1: 100-l: 300, washed through FCS-Eagle-HEPES gradients (50 %, 75 %, 100 sb) then incubated 30 min at 0°C with rzSI-sheep-antirabbit-globulin IgG at a concentration of 5 pug/ml. They were then washed through two more gradients before fixation for electron microscopy.
Processingof cellsfor electron microscopy The cells were resuspended in FCS and spun down gently in small cellulose nitrate ultracentrifuge tubes (Beckman Instruments Inc., Palo Alto, Calif.). The supernatant was removed and cold fixative (2.5 % glutaraldehyde in sodium cacodylate-HCl buffer, pH 7.4) was layered over the cell pellet. After a total fixation time of 1 h at 4°C with changes of fixative at 20 and 40 min, the end of the tube containing the cell pellet was cut off and left in cacodylate-HCl buffer, pH 7.4 overnight at 4°C. The pellet was then post-fixed in 2 % osmic acid for 2 h, dehydrated in graded acetone solutions (30-100 %), and embedded in Araldite M (CIBA Co. Pty. Ltd, Melbourne). The cellulose nitrate tube fragment dissolved in 100 % acetone [16].
Electron microscoperadioautography The method used was that of Salpeter & Bachmann [17], as modified by Mitchell & Abbot [18]. Briefly, ultra-thin sections were cut on an LKB Ultrotome III ultra-microtome (LKB-Produkter AB. Stockholm) using glass knives, ~and placed on flame-polished; collodion-coated glass slides. The slides were then coated with carbon in a Dynavac evaporating unit (Dynavac Vacuum Pty. Ltd., Burwobd, Victoria) and dipped in Kodak NTE emulsion (Eastman Kodak Co., Rochester, N.Y.). The slides were allowed to dry and exposed for 3-13 weeks at 4°C with calcium sulphate as desiccant. They were developed with Dektol (Kodak (Australasia) Pty. Ltd., Coburg, Victoria), fixed with’ Amfix (May and Baker Ltd; West Footscray, Victoria), then the sections were stripped off the slides and mounted on copper 100 mesh grids or 1 x2 mm slotted discs (Mason and Morton Ltd, London). The sections were examined unstained in a Philips EM-300 electron microscope, using a 30 pm objective aperture and an accelerating voltage of 60 or 80 kV. At least four grids or discs were examined for each samnle at each radioautoaranhic exnosure time. Thus for each sample, at least 100 cells were studied. A cell was regarded as labelled if it had at least one patch of three or more grains, since background was generally extremely low. For most preparations, low magnification photomontages were made and surveys of an average of 50 cells made to assess labelling patterns and background.
Surface immunoglobulins
379
Table 2. Numbers of labelled cells
RESULTS Generally no difference was observed by electron microscope radioautography between cells labelled by the direct method and those labelled by the sandwich method. Therefore the two methods will be regarded as equivalent in the description of the results. Estimation
on Iymphoid cells
Intact lymphocytes were counted in the same photomontages as those used for table 1. Labelled cells were those with at least one patch of 3 or more grains
Specimen Spleen + anti-light chain IgG + ‘%SARGa, b Spleen + anti-light chain IgG + iasI-SARGb Spleen f Y-anti-light chain IgG Spleen + 1261-anti-p IgG Spleen + ALS IgG + ‘Y-SARG Thymus + ALS IgG + lz51-SARG
of background
No. of labelled cells
33
Total no. of cells counted
52
Percentage of cells labelled
64
31 57 54 Radioautographic background was assessed by counting grains not associated with cells 51 80 64 11 35 31 or debris in low-power photomontages of randomly-chosen areas in sections exposed 38 43 88 for a full half-life of lz51. Areas of 100 pm2 114 118 97 were counted, and the results are summarized in table 1. In general, the counts were ex- a SARG, sheep anti-rabbit globulin. tremely low; 100 pm2 corresponds to about 4 b Two different areas from the same specimen. cell areas, and so the background was from 0.25-1.5 grains/cell. Thus it was very unlikely in a diffuse pattern [lo]. The higher backthat a cell with a patch of grains was labelled non-specifically. However, cell debris is ground observed in preparations labelled with ALS IgG could be accounted for by the known to bind radioactively labelled antigen higher specific activity of the labelled antibody used. Table 1. Estimation of background Low-power photomontages of sections which had been exposed for one half-life of lasI were divided into areas equivalent to 100 pm2, and grains not associated with cells or debris were counted in each area
Specimen
No. of areas counted
Spleen + anti-light chain IgG + ‘261-SARGa, p 22 Spleen + anti-light chain IgG + =%SARGb 27 Spleen + Y-anti-light chain IgG 29 Spleen + 1261-anti-p IgG 28 Spleen + ALS IgG + lz51-SARG 25 Thymus + ALS IgG + ‘251-SARG 34
Grains/ 100 pm2 Mean and range
1.5 (O-7) 0.7 (O-4) 3.0 (o-5) 2.5 (l-5) 4.5 (2-17) 6.1 (l-9)
a SARG, sheep anti-rabbit globulin. b Two different areas of the same specimen.
Numbers of labelled cells
The same photomontages as those used to assess background were used to estimate the percentages of labelled cells in the preparations. Only intact lymphocytes were counted, and in view of the low background, cells with one or more patches of at least 3 grains were regarded as labelled. The results are summarized in table 2. Because of the very low frequency of labelling of thymus cells under the conditions used, a low-power photomontage of about 100 cells failed to reveal any labelled lymphocytes. However, an exhaustive search involving scanning of entire grids revealed moderate numbers of lightly labelled cells. No thymus cells labelled with anti+ were found, even after high magnification scanning. Exptl
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Vivien Santer et al.
1-3. Radioautographs of cells incubated with ‘251-labelled IgG specific for light chains. 1. (a, b) Serial sections of a spleen lymphocyte, showing that most patches are repeated in adjacent sections. Small patches (arrows) are sometimes not repeated, indicating the finite size of the immunoglobulin site. The specificity of very small patches of grains is shown by the presence of larger patches in the same location on the adjacent section (circles). Exposed 13 weeks; x 12 000; Fig. 2. Spleen lymphocyte, showing polar label. Exposed 13 weeks; x 13 500; Fig. 3. Thymus lymphocyte, showing a single small patch of grains at the cell membrane. Thymus cells showed a lower percentage of labelled cells than did spleen, and the cells were more lightly labelled. Exposed 8 weeks; x 12 000.
Figs Fig.
Exprl
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Surface immunoglobulins
on lymphoid cells
381
Figs 4-5. Spleen cells labelled with 1251-IgG specific against ,u chains. Fig, 4. Spleen lymphocyte, showing a pattern of labelling similar to that observed with anti-light chain. Note some grains in a small vacuole (arrow). Exposed 8 weeks; x 13 500; Fig. 5. Spleen lymphocyte, showing patches of grains clearly inside the cell, in addition to surface label. This type of labelling was less common than surface patches of grains only. Exposed 3 weeks; x18700.
Cells labelled with anti-light chain antibody Fifty to 60 % of spleencellswere labelled (table 2). Most of these had grains in discrete patches at or just inside the cell membrane, closely resembling the distribution of radioactively labelled antigen on antigen-binding lymphocytes [lo]. As was observed with labelled antigen, dead cells and debris showed diffuse labelling. Patches were found at the same site in serial sections (fig. la, b). Small cell processeswere frequently labelled. Clumps of grains were also sometimesseenapparently just inside the cell; this could have been the result of invagination of a labelled portion of cell membrane, or, indeed, of endocytosis (fig. 5). A typical cell labelled with antilight chain antibody is shown in fig. 1. Most of the labelled cells were small or medium lymphocytes. A rare plasma cell had
patches of surface label; however, in general these were more lightly labelled than lymphocytes, with fewer and smaller patches. In some cases,labelled lymphocytes showed an asymmetrical (polar) distribution of grains, with 30-50 % of the cell surface heavily labelled and the rest virtually unlabelled (fig. 2). In contrast to spleen cells, thymus cells were rarely labelled (see above). The grains on these were again in patches at the cell membrane, but very few cells had more than a single patch of grains and in general the patches appeared smaller than those found on splenic lymphocytes (fig. 3). As with spleen cells, thymus cells were labelled in serial sections, but the same site was labelled in no more than three consecutive sections, compared with often three to six serial sections Exptl
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Vivien Santer et al.
Fig. 6. Spleen cells labelled with Y-IgG specific against light chains. Grains are in patches on the surfaces of lymphocytes. A plasma cell and debris in the field are lightly labelled, but the grains are not in patches. Red cells are unlabelled. Exposed 13 weeks; x 5 700; Fig. 7. Thymus cells labelled with anti-lymphocyte IgG. Label is more scattered than with anti-immunoglobulin labelling. One cell shows labelling of the uropod (arrow). Exposed 9 weeks; x 6 700.
Expti
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Surface immunoglobulirls
on lymphoid cells
383
ii Figs 8-9. Cells lsbelled with anti-lymphocyte IgG. Fig. 8. Typical spleen lymphocyte, showing the more scattered distribution of grains. Note that small cell processes are labelled (auvow). Exposed 5 weeks; x 14 700; Fig. 9. Thymus lymphocytes, each with a preferentially-labelled uropod-like extension of the cytoplasm containing the Golgi region. Many single cells showing a similar pattern of labelling were also seen. Exposed 5 weeks; x 8 000.
of spleen cells. No thymus cells with an asymmetrical, polar distribution of grains were seen.
Cells labelled with anti-,u chain antibody Spleen cells labelled with anti-,u IgG presented a similar appearance to those labelled with anti-light chain antibody. About 30 % of cells were labelled (table 1). Again, the label was usually in patches at the cell membrane or on small processes,with occasional cells having label apparently just inside. A typical cell labelled with anti-p is shown in fig. 4. Most of the labelled cells were small and medium lymphocytes, but plasma cells occasionally had grains at the cell membrane. No thymus cells were labelled with anti-p at the concentrations used in these experiments.
One unusual feature of the spleencell preparations incubated after both anti-light chain and anti+ was the presence of a significant number of cells after (at least 15 % of all labelled cells) grains which were clearly well inside the cell. This label was seen in serial sections: an example is shown in fig. 5. Possible explanations for this phenomenon will be explored in the discussion.
Cells labelled with anti-lymphocyte
antibody
Spleenand thymus cellslabelled with ALS IgG were examined to compare the distribution of immunoglobulin determinants with that of other antigens on the lymphocyte surface. Virtually all lymphoid cells from both spleen and thymus were labelled, even after short radioautographic exposure times, and at 9 weeks exposure, 88 % of spleen cells and Exptl Cell Res 72
384
Vivien Santer et al.
97 % of thymus cells were labelled. In the sandwich preparations, red cells quite frequently had light label scattered around the membrane, possibly because of the high concentration of ALS IgG used, and inadequacy of absorption with erythrocytes. If this were taken as the background level of labelling, some lymphoid cells with light accumulations of grains could not be regarded as specifically labelled. Only heavily labelled cells are discussed here. Occasionally plasma cells were labelled, but eosinophils and macrophages were not. Grains were found predominantly at the cell membrane, often on small cell processes, but their distribution differed from that seen with anti-immunoglobulins. This is shown in the low power photographs in fig. 6, showing spleen cells labelled with anti-light chain, and fig. 7, showing spleen cells labelled with ALS IgG. With ALS IgG, the label was usually scattered along the membrane (fig. 8) rather than in discrete patches, although these also were observed on some cells. A polar distribution of label was more common than on cells labelled with anti-light chain antibody (fig. 9). This polar label was maintained in serial sections. A remarkable feature of ALS IgG-labelled preparations was that many cells from both spleen and thymus showed a large projection of the cytoplasm, usually containing the Golgi region, which was frequently preferentially labelled. An example of this is shown in fig. 9. This type of labelling was not seen on cells incubated with anti-immunoglobulins. DISCUSSION Surface immunoglobulins were identified in discrete patches on the surfaces of lymphocytes. Investigations with labelled antigens have also identified binding sites distributed in discrete patches [lo, 201. Such a similarity Exptl
Cell Res 72
between the distributions of surface immunoglobulins and antigen-binding sites lends morphological support to the hypothesis that the antigen receptor is immunoglobulin in nature. The present results confirm the findings of Bosman & Feldman [21] who observed a patchy distribution of label on rat lymph node cells binding iz51-anti-p and anti-y antibodies; however, they found the same distribution of label on both lymphocytes and plasma cells. Using surface IgM-reactive cell lines of human neoplastic lymphoblasts, Hammond [22] showed a discontinuous distribution of ferritin-conjugated anti-p and anti-x chain. Jones et al. reported a patchy distribution of anti-allotype antibody bound by rabbit lymphocytes [23], and Biberfeld et al. [24] found that human lymphocytes cultured with or without phytohaemagglutinin bound ferritin-conjugated anti-x or anti-A in discontinuous areas of varying size on the cell membrane. No cells reacted with anti-y or anti-p, in contrast to our findings. The uropods of phytohaemagglutinin-transformed cells were preferentially labelled with anti-x or anti-& while with ALS both normal and transformed cells showed an even distribution of label over a large area of the surface. Thus in general the findings of the present study are in agreement with other results, although there may be some species variation. The relatively light labelling observed with anti-light chain antibody on thymus cells deserves comment. Previous electron microscope studies on surface immunoglobulins have been performed with mixed B and T cell populations [20-241. The present study provides the first electron microscope study of immunoglobulins on the surfaces of T cells. The results support the findings of Bankhurst & Warner, who have recently shown, using light microscope radioautography, that immunoglobulins are present on the surfaces
Surface immunoglobulins of normal and antigen-activated thymus cells but that the label is much lighter than on the B cell [8]. The present investigation verifies this difference in the amount of immunoglobulin displayed on the B cell compared to the T cell. In many of the specimens, particularly those incubated with ALS IgG, some cells were attached to one another; these were labelled predominantly at the interface between the cells (e.g. fig. 6). This may be because attachment between cells is strongest at the active region, which is also a site often preferentially labelled by anti-immunoglobulin reagents and by ALS. Alternatively, the cells could be held together by the labelling antibody itself, which is divalent. This is particularly likely for ALS IgG, which is a known agglutinator of lymphocytes. It was noted that about 15 % of cells labelled with anti-light chain and anti-p antibodies contained intracellular label, sometimes at the Golgi region. Such cells usually appeared undamaged, and it is possible that this intracellular labelling was the result of endocytosis of the anti-immunoglobulin by the lymphocyte. Although the cells were maintained at 0°C during the series of labelling reactions and washes, a period of several hours elapsed between killing of the mouse and final fixation of the cells. In addition, no metabolic inhibitors were present in any of the media used, and in some experiments, bench centrifuges were used. Thus, although cell metabolism was decreased by the low temperature, over such a period of time some pinocytosis probably occurred. There is recent evidence that the attachment of bivalent antibody to the lymphocyte surface promotes changes including assumption of a polar distribution of the attached reagent, and subsequent endocytosis of this reagent [19]. This question is being explored further, using (a) brief labelling periods in the cold followed
on lymphoid cells
385
by immediate fixation; (b) brief culture of cells at 37°C after labelling. All the anti-immunoglobulin reagents used in these experiments were extensively absorbed to ensure specificity for the target immunoglobulin [9]. The sheep anti-rabbit globulin used in the sandwich assay was absorbed with normal mouse y-globulin on polyaminopolystyrene or with washed mouse erythrocytes, and rabbit antisera were absorbed twice with mouse erythrocytes or up to 8 times with mouse thymocytes. Thus antimembrane activity of the antibodies could be disregarded as a cause of anti-immunoglobulin binding. With anti-immunoglobulin reagents, non-lymphoid cells generally were completely unlabelled. Some plasma cells were labelled, probably because of antibody at the point of release by the cell. Preliminary results support the view that plasma cells may bind anti-immunoglobulins (Santer, unpublished work). However, with ALS IgG the background level of radioactivity was somewhat higher (table l), and some erythrocytes were very lightly labelled. This may have been because the concentration of the antibody and specific activity of the 12jIsheep anti-rabbit globulin in the ALS experiments were considerably higher than for the anti-immunoglobulin experiments. Absorption of the ALS IgG with cells other than erythrocytes (e.g. mouse kidney) might decrease this non-specific labelling. The diffuse pattern of surface labelling observed with ALS suggests that not all surface antigens of the lymphocyte had the same discrete localization noted with antigen and anti-immunoglobulin labelling. The distribution of H-2, 0, and TL antigens on mouse lymphocytes described by Aoki et al. [25] supports this idea of a generalized distribution of surface antigens other than immunoglobulins. Similarly, Biberfeld et al. reported recently that ALS labels large areas Exptl Cell Res 72
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Vivien Santer et al.
of the membranes of human lymphocytes [23]. It should be pointed out, however, that a higher concentration of anti-immunoglobulin might progressively blur the patchy nature of the distribution of immunoglobulins, which may be related to the conditions of the reaction. However, in our experiments prolonged exposure (13 weeks) of anti-immunoglobulin labelled specimens still gave cells with a patchy distribution of grains and some parts of the membrane still completely unlabelled. The patchy distribution of antigen and anti-immunoglobulin labelling supports the hypothesis that the antigen receptor is an immunoglobulin. Such a view fits in with the results obtained with functional blocking of lymphocyte activity with anti-immunoglobulins. The authors thank Mr John Pye for performing iodinations. Vivien Santer wishes to thank Mrs Judith Mitchell for valuable discussions. This work was supported by grants from the National Health and Medical Research Council and the Australian Research Grants Commission, Canberra and USPHS Al-0-3958. This is publication number 1602 from the Walter and Eliza Hall Institute. V. S. is the recipient of an Australian Commonwealth Postgraduate Research Award. A. D. B. is a Cleveland Fellow of the Royal Melbourne Hospital.
biology (ed M Hanna) vol. 1. Plenum Press, New York. In press. 3. Coombs, R R A, Feinstein, A & Wilson, A B, Lancet II (1969) 1157. 4. Raff, M C, Sternberg, M & Taylor, R B, Nature 225 (1970) 553. 5. Pernis, B, Forni, L & Amante, L, J exptl med 132 (1970) 1001. 6. Rabellino, E, Colon, S, Grey, H M & Unanue, E R, J exptl Led 133 (i971) i56. 7. Bankhurst, A D & Warner, N L. In preparation. 8. Bankhurst, A D, Warner, N L & Sprent, J, J exptl med 134 (1971) 1005. 9. Nossal, G J V,‘Warner, N L, Lewis, H & Sprent, J, J exptl med. In press. 10. Mandel, T, Byrt, P & Ada G L, Exptl cell res 58 (1969) 179. 11. Herzenberg, L & Warner, N L, Regulation of the antibody response (ed B Cinader). Charles C Thomas, Springfield, Ill. (1968). 12. Bankhurst,.A D & Warner, N L, J immunol 107 (1971) 368. 13. Shortman, K D, Williams, N & Adams, P, J immunol methods. In press. 14. Levey, R H & Medawar, P B, Ann NY acad sci 129 (1956) 164. 15. Hunter, W M & Greenwood, F C, Nature 194 (1962) 495. 16. Mandel, T. Personal communication. 17. Salneter. M M & Bachmann. L J cell biol 22 (1964) 469. 18. Mitchell, J & Abbot, A, Nature 208 (1965) 500. 19. Taylor, R B, Duffus, W P H, Raff, M C & de Petris, S, Nature new biol 233 (1971) 225. 20. Bosman, C, Feldman, J D & Pick, E, J exptl med 129 (1969) 1029. 21. Bosman, C & Feldman, J D, Lab invest 22 (1970) 309. 22. Hammond, E, Exptl cell res 59 (1970) 359. 23. Jones, G, Marcuson, E C & Roitt, I M, Nature 227 (1970) 1051. 24. Biberfeld, P, Biberfeld, G & Perlmann, P, Exptl cell res 66 (1971) 177. 25. Aoki, T, Hammerling, U de Harven, E, Boyse, E A & Old, L J, J exptl med 130 (1969) 979.
REFERENCES 1. Warner, N L, Byrt, P & Ada, G L, Nature 226 (1970) 942. 2. Warner, N L, Contemporary topics in immuno-
Exptl
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Received September 21, 1971 Revised version received November 29, 1971
ULTRASTRUCTURAL IMMUNOGLOBULIN VIVIEN
Cell Research 12 (1972) 317-386
DISTRIBUTION
DETERMINANTS SANTER,
OF SURFACE
ON MOUSE
A. D. BANKHURST
LYMPHOID
CELLS
and G. J. V. NOSSAL
The Walter and Elba Hall Institute of Medical Research, Victoria 3050, Australia
SUMMARY The distribution of radioiodinated anti-immunoglobulin and anti-lymphocyte antibodies on the surfaces of lymphocytes from normal mouse spleen and thymus has been investigated using electron microscope radioautography. Discrete patches of label were seen on 5&60 % of spleen cells labelled with anti-light chain antibody, and on 30 % of spleen cells labelled with anti-p. Only a very small proportion of thymus cells were labelled by anti-light chain, and none bound anti-p. In contrast, anti-lymphocyte antibody labelled 90-100% of both spleen and thymus cells. This was either generalized labelling surrounding the whole cell, or heavy labelling over a uropod-like extension of the cytoplasm. The close similarity between labelling patterns with radioactively labelled antigen and immunoglobulin supports the hypothesis that the antigen receptor is immunoglobulin in nature, and the great difference in density of surface immunoglobulin between thymus and bone-marrow derived lymphocytes is once more emphasised.
The interaction of a lymphocyte with an antigen requires the presence of a specific surface receptor on the lymphocyte. Evidence from several studies in which lymphocyte activity was blocked with anti-immunoglobulin antibodies strongly suggests that this receptor is an immunoglobulin [l, 21. In support of this hypothesis, immunoglobulin determinants have been directly demonstrated on the surfaces of both non-thymus derived (B) lymphocytes [3-71 and thymus-derived (T) lymphocytes [8]. The ultrastructural characteristics of antigen-binding by lymphocytes have been described [lo], but very little work has been done on surface immunoglobulins at the electron microscope level. The present study examines surface immunoglobulins and 1 Present address: Unite de Recherches, Division d’hematologie, HBpital Cantonal, Geneva, Switzerland. 2&
721806
other surface antigens by radioautography with the electron microscope. Two experimental approaches were used: direct labelling with radioiodinated rabbit anti-mouse immunoglobulin antibody, and indirect (sandwich) labelling with rabbit anti-mouse immunoglobulin antibody followed by radioiodinated polyvalent sheep anti-rabbit globulin antibody. MATERIALS
AND METHODS
Animals For direct labelling experiments, male or female CBA mice 50-120 days old were used. For sandwich labelling experiments, male specific pathogen free C3H mice, 10-12 weeks old were used.
Preparation of cell suspensions Direct labelling: Mice were killed by exposure to ether. The spleens and thymuses were removed and teased gently through a fine stainless steel sieve into Exptl Cell Res 72
378
Vivien Santer et al.
ice-cold Eisen balanced salt solution (EBSS). The cells were washed at least four times through gradients of foetal calf serum (FCS; Commonwealth Serum Laboratories, Melbourne) [12] or bovine serum albumin (BSA: Commonwealth Serum Laboratories) to remove non-adherent surface material and cell debris. The viability of all cell suspensions was measured by trypan blue exclusion.
Sandwich labelling This was done according to a method recently described by Nossal et al. [9]. Briefly, mice were killed bv cervical dislocation. and the snleens and thymuses were removed and placed in -cold 10 % FCS in Eagle’s minimal essential medium (GIBCo, Grand Island, N.Y.) buffered with 10 mM HEPES (N-2-hydroxyethylpiperazine-N-1, 2-ethane sulphonic acid; CalBiochem, Los Angeles, Cnlif.). The spleens were teased through a fine stainless steel sieve and the clumos allowed to settle over FCS. The cell suspension was washed through 1 ml of FCS; debris and damaged cells were removed by the BSA method of Shortman et al. [13], and the remaining cells resuspended in 10 % FCS-Eagle medium-HEPES, and counted.
Preparation of antisera The preparation of antisera against mouse light chains, IgM and IgG has been previously described [ll]. Briefly, antisera were raised in rabbits using ourified mveloma nroteins. and the gammaglobulin fraction was separated by ~starch gel electrophoresis in Verona1 buffer. DH 8.2. These fractions were tested for anti-immunoglobulin activity by radioprecipitation assays [ll] and made monospecific by absorption with purified immunoglobulins coupled to polyaminopolystyrene [l 11. Labelling of cells by these purified anti-immunoglobulins could be specifically- blocked by prior absorption of the prenaration with uurified immunoglobulins 1121. Anti-mouse lymphocyte serum (ALS) was-prepared in rabbits using mouse thymocytes as previously described [14]. -Sheep a&rabbit globulin serum (SARG) was a gift from Dr Zoltan Ovary. Globulin fractions (IgG)-of these antisera were prepared by agar block electrophoresis or starch block electrophoresis in Verona1 buffer, pH 8.2.
Radioiodination of antisera The globulin fractions were labelled with ?odine according to the method of Hunter & Greenwood [15]. The specific activity of the anti-immunoglobulin IgG was 90-140 pCi/pg protein and of the sheep anti-rabbit immunoglobulin IgG was 6-10 ,&i/pg protein.
Cell labelling Direct method: Cell suspensions were incubated with 1261-anti-immunoglobulin IgG in 10% FCSExptl
Cell Res 72
Dulbecco medium for 1 h at 0°C. Generally 5 x IO6 viable cells were incubated with 5-7.5 ,ug of protein. The cells were then washed four times through FCS gradients before fixation for electron microscopy. Sandwich method: Cells were incubated for 30 min at 0°C with unlabelled anti-immunoglobulin IgG at a final dilution of 1: 100-l: 300, washed through FCS-Eagle-HEPES gradients (50 %, 75 %, 100 sb) then incubated 30 min at 0°C with rzSI-sheep-antirabbit-globulin IgG at a concentration of 5 pug/ml. They were then washed through two more gradients before fixation for electron microscopy.
Processingof cellsfor electron microscopy The cells were resuspended in FCS and spun down gently in small cellulose nitrate ultracentrifuge tubes (Beckman Instruments Inc., Palo Alto, Calif.). The supernatant was removed and cold fixative (2.5 % glutaraldehyde in sodium cacodylate-HCl buffer, pH 7.4) was layered over the cell pellet. After a total fixation time of 1 h at 4°C with changes of fixative at 20 and 40 min, the end of the tube containing the cell pellet was cut off and left in cacodylate-HCl buffer, pH 7.4 overnight at 4°C. The pellet was then post-fixed in 2 % osmic acid for 2 h, dehydrated in graded acetone solutions (30-100 %), and embedded in Araldite M (CIBA Co. Pty. Ltd, Melbourne). The cellulose nitrate tube fragment dissolved in 100 % acetone [16].
Electron microscoperadioautography The method used was that of Salpeter & Bachmann [17], as modified by Mitchell & Abbot [18]. Briefly, ultra-thin sections were cut on an LKB Ultrotome III ultra-microtome (LKB-Produkter AB. Stockholm) using glass knives, ~and placed on flame-polished; collodion-coated glass slides. The slides were then coated with carbon in a Dynavac evaporating unit (Dynavac Vacuum Pty. Ltd., Burwobd, Victoria) and dipped in Kodak NTE emulsion (Eastman Kodak Co., Rochester, N.Y.). The slides were allowed to dry and exposed for 3-13 weeks at 4°C with calcium sulphate as desiccant. They were developed with Dektol (Kodak (Australasia) Pty. Ltd., Coburg, Victoria), fixed with’ Amfix (May and Baker Ltd; West Footscray, Victoria), then the sections were stripped off the slides and mounted on copper 100 mesh grids or 1 x2 mm slotted discs (Mason and Morton Ltd, London). The sections were examined unstained in a Philips EM-300 electron microscope, using a 30 pm objective aperture and an accelerating voltage of 60 or 80 kV. At least four grids or discs were examined for each samnle at each radioautoaranhic exnosure time. Thus for each sample, at least 100 cells were studied. A cell was regarded as labelled if it had at least one patch of three or more grains, since background was generally extremely low. For most preparations, low magnification photomontages were made and surveys of an average of 50 cells made to assess labelling patterns and background.
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Table 2. Numbers of labelled cells
RESULTS Generally no difference was observed by electron microscope radioautography between cells labelled by the direct method and those labelled by the sandwich method. Therefore the two methods will be regarded as equivalent in the description of the results. Estimation
on Iymphoid cells
Intact lymphocytes were counted in the same photomontages as those used for table 1. Labelled cells were those with at least one patch of 3 or more grains
Specimen Spleen + anti-light chain IgG + ‘%SARGa, b Spleen + anti-light chain IgG + iasI-SARGb Spleen f Y-anti-light chain IgG Spleen + 1261-anti-p IgG Spleen + ALS IgG + ‘Y-SARG Thymus + ALS IgG + lz51-SARG
of background
No. of labelled cells
33
Total no. of cells counted
52
Percentage of cells labelled
64
31 57 54 Radioautographic background was assessed by counting grains not associated with cells 51 80 64 11 35 31 or debris in low-power photomontages of randomly-chosen areas in sections exposed 38 43 88 for a full half-life of lz51. Areas of 100 pm2 114 118 97 were counted, and the results are summarized in table 1. In general, the counts were ex- a SARG, sheep anti-rabbit globulin. tremely low; 100 pm2 corresponds to about 4 b Two different areas from the same specimen. cell areas, and so the background was from 0.25-1.5 grains/cell. Thus it was very unlikely in a diffuse pattern [lo]. The higher backthat a cell with a patch of grains was labelled non-specifically. However, cell debris is ground observed in preparations labelled with ALS IgG could be accounted for by the known to bind radioactively labelled antigen higher specific activity of the labelled antibody used. Table 1. Estimation of background Low-power photomontages of sections which had been exposed for one half-life of lasI were divided into areas equivalent to 100 pm2, and grains not associated with cells or debris were counted in each area
Specimen
No. of areas counted
Spleen + anti-light chain IgG + ‘261-SARGa, p 22 Spleen + anti-light chain IgG + =%SARGb 27 Spleen + Y-anti-light chain IgG 29 Spleen + 1261-anti-p IgG 28 Spleen + ALS IgG + lz51-SARG 25 Thymus + ALS IgG + ‘251-SARG 34
Grains/ 100 pm2 Mean and range
1.5 (O-7) 0.7 (O-4) 3.0 (o-5) 2.5 (l-5) 4.5 (2-17) 6.1 (l-9)
a SARG, sheep anti-rabbit globulin. b Two different areas of the same specimen.
Numbers of labelled cells
The same photomontages as those used to assess background were used to estimate the percentages of labelled cells in the preparations. Only intact lymphocytes were counted, and in view of the low background, cells with one or more patches of at least 3 grains were regarded as labelled. The results are summarized in table 2. Because of the very low frequency of labelling of thymus cells under the conditions used, a low-power photomontage of about 100 cells failed to reveal any labelled lymphocytes. However, an exhaustive search involving scanning of entire grids revealed moderate numbers of lightly labelled cells. No thymus cells labelled with anti+ were found, even after high magnification scanning. Exptl
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1-3. Radioautographs of cells incubated with ‘251-labelled IgG specific for light chains. 1. (a, b) Serial sections of a spleen lymphocyte, showing that most patches are repeated in adjacent sections. Small patches (arrows) are sometimes not repeated, indicating the finite size of the immunoglobulin site. The specificity of very small patches of grains is shown by the presence of larger patches in the same location on the adjacent section (circles). Exposed 13 weeks; x 12 000; Fig. 2. Spleen lymphocyte, showing polar label. Exposed 13 weeks; x 13 500; Fig. 3. Thymus lymphocyte, showing a single small patch of grains at the cell membrane. Thymus cells showed a lower percentage of labelled cells than did spleen, and the cells were more lightly labelled. Exposed 8 weeks; x 12 000.
Figs Fig.
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Surface immunoglobulins
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Figs 4-5. Spleen cells labelled with 1251-IgG specific against ,u chains. Fig, 4. Spleen lymphocyte, showing a pattern of labelling similar to that observed with anti-light chain. Note some grains in a small vacuole (arrow). Exposed 8 weeks; x 13 500; Fig. 5. Spleen lymphocyte, showing patches of grains clearly inside the cell, in addition to surface label. This type of labelling was less common than surface patches of grains only. Exposed 3 weeks; x18700.
Cells labelled with anti-light chain antibody Fifty to 60 % of spleencellswere labelled (table 2). Most of these had grains in discrete patches at or just inside the cell membrane, closely resembling the distribution of radioactively labelled antigen on antigen-binding lymphocytes [lo]. As was observed with labelled antigen, dead cells and debris showed diffuse labelling. Patches were found at the same site in serial sections (fig. la, b). Small cell processeswere frequently labelled. Clumps of grains were also sometimesseenapparently just inside the cell; this could have been the result of invagination of a labelled portion of cell membrane, or, indeed, of endocytosis (fig. 5). A typical cell labelled with antilight chain antibody is shown in fig. 1. Most of the labelled cells were small or medium lymphocytes. A rare plasma cell had
patches of surface label; however, in general these were more lightly labelled than lymphocytes, with fewer and smaller patches. In some cases,labelled lymphocytes showed an asymmetrical (polar) distribution of grains, with 30-50 % of the cell surface heavily labelled and the rest virtually unlabelled (fig. 2). In contrast to spleen cells, thymus cells were rarely labelled (see above). The grains on these were again in patches at the cell membrane, but very few cells had more than a single patch of grains and in general the patches appeared smaller than those found on splenic lymphocytes (fig. 3). As with spleen cells, thymus cells were labelled in serial sections, but the same site was labelled in no more than three consecutive sections, compared with often three to six serial sections Exptl
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Fig. 6. Spleen cells labelled with Y-IgG specific against light chains. Grains are in patches on the surfaces of lymphocytes. A plasma cell and debris in the field are lightly labelled, but the grains are not in patches. Red cells are unlabelled. Exposed 13 weeks; x 5 700; Fig. 7. Thymus cells labelled with anti-lymphocyte IgG. Label is more scattered than with anti-immunoglobulin labelling. One cell shows labelling of the uropod (arrow). Exposed 9 weeks; x 6 700.
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ii Figs 8-9. Cells lsbelled with anti-lymphocyte IgG. Fig. 8. Typical spleen lymphocyte, showing the more scattered distribution of grains. Note that small cell processes are labelled (auvow). Exposed 5 weeks; x 14 700; Fig. 9. Thymus lymphocytes, each with a preferentially-labelled uropod-like extension of the cytoplasm containing the Golgi region. Many single cells showing a similar pattern of labelling were also seen. Exposed 5 weeks; x 8 000.
of spleen cells. No thymus cells with an asymmetrical, polar distribution of grains were seen.
Cells labelled with anti-,u chain antibody Spleen cells labelled with anti-,u IgG presented a similar appearance to those labelled with anti-light chain antibody. About 30 % of cells were labelled (table 1). Again, the label was usually in patches at the cell membrane or on small processes,with occasional cells having label apparently just inside. A typical cell labelled with anti-p is shown in fig. 4. Most of the labelled cells were small and medium lymphocytes, but plasma cells occasionally had grains at the cell membrane. No thymus cells were labelled with anti-p at the concentrations used in these experiments.
One unusual feature of the spleencell preparations incubated after both anti-light chain and anti+ was the presence of a significant number of cells after (at least 15 % of all labelled cells) grains which were clearly well inside the cell. This label was seen in serial sections: an example is shown in fig. 5. Possible explanations for this phenomenon will be explored in the discussion.
Cells labelled with anti-lymphocyte
antibody
Spleenand thymus cellslabelled with ALS IgG were examined to compare the distribution of immunoglobulin determinants with that of other antigens on the lymphocyte surface. Virtually all lymphoid cells from both spleen and thymus were labelled, even after short radioautographic exposure times, and at 9 weeks exposure, 88 % of spleen cells and Exptl Cell Res 72
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97 % of thymus cells were labelled. In the sandwich preparations, red cells quite frequently had light label scattered around the membrane, possibly because of the high concentration of ALS IgG used, and inadequacy of absorption with erythrocytes. If this were taken as the background level of labelling, some lymphoid cells with light accumulations of grains could not be regarded as specifically labelled. Only heavily labelled cells are discussed here. Occasionally plasma cells were labelled, but eosinophils and macrophages were not. Grains were found predominantly at the cell membrane, often on small cell processes, but their distribution differed from that seen with anti-immunoglobulins. This is shown in the low power photographs in fig. 6, showing spleen cells labelled with anti-light chain, and fig. 7, showing spleen cells labelled with ALS IgG. With ALS IgG, the label was usually scattered along the membrane (fig. 8) rather than in discrete patches, although these also were observed on some cells. A polar distribution of label was more common than on cells labelled with anti-light chain antibody (fig. 9). This polar label was maintained in serial sections. A remarkable feature of ALS IgG-labelled preparations was that many cells from both spleen and thymus showed a large projection of the cytoplasm, usually containing the Golgi region, which was frequently preferentially labelled. An example of this is shown in fig. 9. This type of labelling was not seen on cells incubated with anti-immunoglobulins. DISCUSSION Surface immunoglobulins were identified in discrete patches on the surfaces of lymphocytes. Investigations with labelled antigens have also identified binding sites distributed in discrete patches [lo, 201. Such a similarity Exptl
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between the distributions of surface immunoglobulins and antigen-binding sites lends morphological support to the hypothesis that the antigen receptor is immunoglobulin in nature. The present results confirm the findings of Bosman & Feldman [21] who observed a patchy distribution of label on rat lymph node cells binding iz51-anti-p and anti-y antibodies; however, they found the same distribution of label on both lymphocytes and plasma cells. Using surface IgM-reactive cell lines of human neoplastic lymphoblasts, Hammond [22] showed a discontinuous distribution of ferritin-conjugated anti-p and anti-x chain. Jones et al. reported a patchy distribution of anti-allotype antibody bound by rabbit lymphocytes [23], and Biberfeld et al. [24] found that human lymphocytes cultured with or without phytohaemagglutinin bound ferritin-conjugated anti-x or anti-A in discontinuous areas of varying size on the cell membrane. No cells reacted with anti-y or anti-p, in contrast to our findings. The uropods of phytohaemagglutinin-transformed cells were preferentially labelled with anti-x or anti-& while with ALS both normal and transformed cells showed an even distribution of label over a large area of the surface. Thus in general the findings of the present study are in agreement with other results, although there may be some species variation. The relatively light labelling observed with anti-light chain antibody on thymus cells deserves comment. Previous electron microscope studies on surface immunoglobulins have been performed with mixed B and T cell populations [20-241. The present study provides the first electron microscope study of immunoglobulins on the surfaces of T cells. The results support the findings of Bankhurst & Warner, who have recently shown, using light microscope radioautography, that immunoglobulins are present on the surfaces
Surface immunoglobulins of normal and antigen-activated thymus cells but that the label is much lighter than on the B cell [8]. The present investigation verifies this difference in the amount of immunoglobulin displayed on the B cell compared to the T cell. In many of the specimens, particularly those incubated with ALS IgG, some cells were attached to one another; these were labelled predominantly at the interface between the cells (e.g. fig. 6). This may be because attachment between cells is strongest at the active region, which is also a site often preferentially labelled by anti-immunoglobulin reagents and by ALS. Alternatively, the cells could be held together by the labelling antibody itself, which is divalent. This is particularly likely for ALS IgG, which is a known agglutinator of lymphocytes. It was noted that about 15 % of cells labelled with anti-light chain and anti-p antibodies contained intracellular label, sometimes at the Golgi region. Such cells usually appeared undamaged, and it is possible that this intracellular labelling was the result of endocytosis of the anti-immunoglobulin by the lymphocyte. Although the cells were maintained at 0°C during the series of labelling reactions and washes, a period of several hours elapsed between killing of the mouse and final fixation of the cells. In addition, no metabolic inhibitors were present in any of the media used, and in some experiments, bench centrifuges were used. Thus, although cell metabolism was decreased by the low temperature, over such a period of time some pinocytosis probably occurred. There is recent evidence that the attachment of bivalent antibody to the lymphocyte surface promotes changes including assumption of a polar distribution of the attached reagent, and subsequent endocytosis of this reagent [19]. This question is being explored further, using (a) brief labelling periods in the cold followed
on lymphoid cells
385
by immediate fixation; (b) brief culture of cells at 37°C after labelling. All the anti-immunoglobulin reagents used in these experiments were extensively absorbed to ensure specificity for the target immunoglobulin [9]. The sheep anti-rabbit globulin used in the sandwich assay was absorbed with normal mouse y-globulin on polyaminopolystyrene or with washed mouse erythrocytes, and rabbit antisera were absorbed twice with mouse erythrocytes or up to 8 times with mouse thymocytes. Thus antimembrane activity of the antibodies could be disregarded as a cause of anti-immunoglobulin binding. With anti-immunoglobulin reagents, non-lymphoid cells generally were completely unlabelled. Some plasma cells were labelled, probably because of antibody at the point of release by the cell. Preliminary results support the view that plasma cells may bind anti-immunoglobulins (Santer, unpublished work). However, with ALS IgG the background level of radioactivity was somewhat higher (table l), and some erythrocytes were very lightly labelled. This may have been because the concentration of the antibody and specific activity of the 12jIsheep anti-rabbit globulin in the ALS experiments were considerably higher than for the anti-immunoglobulin experiments. Absorption of the ALS IgG with cells other than erythrocytes (e.g. mouse kidney) might decrease this non-specific labelling. The diffuse pattern of surface labelling observed with ALS suggests that not all surface antigens of the lymphocyte had the same discrete localization noted with antigen and anti-immunoglobulin labelling. The distribution of H-2, 0, and TL antigens on mouse lymphocytes described by Aoki et al. [25] supports this idea of a generalized distribution of surface antigens other than immunoglobulins. Similarly, Biberfeld et al. reported recently that ALS labels large areas Exptl Cell Res 72
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of the membranes of human lymphocytes [23]. It should be pointed out, however, that a higher concentration of anti-immunoglobulin might progressively blur the patchy nature of the distribution of immunoglobulins, which may be related to the conditions of the reaction. However, in our experiments prolonged exposure (13 weeks) of anti-immunoglobulin labelled specimens still gave cells with a patchy distribution of grains and some parts of the membrane still completely unlabelled. The patchy distribution of antigen and anti-immunoglobulin labelling supports the hypothesis that the antigen receptor is an immunoglobulin. Such a view fits in with the results obtained with functional blocking of lymphocyte activity with anti-immunoglobulins. The authors thank Mr John Pye for performing iodinations. Vivien Santer wishes to thank Mrs Judith Mitchell for valuable discussions. This work was supported by grants from the National Health and Medical Research Council and the Australian Research Grants Commission, Canberra and USPHS Al-0-3958. This is publication number 1602 from the Walter and Eliza Hall Institute. V. S. is the recipient of an Australian Commonwealth Postgraduate Research Award. A. D. B. is a Cleveland Fellow of the Royal Melbourne Hospital.
biology (ed M Hanna) vol. 1. Plenum Press, New York. In press. 3. Coombs, R R A, Feinstein, A & Wilson, A B, Lancet II (1969) 1157. 4. Raff, M C, Sternberg, M & Taylor, R B, Nature 225 (1970) 553. 5. Pernis, B, Forni, L & Amante, L, J exptl med 132 (1970) 1001. 6. Rabellino, E, Colon, S, Grey, H M & Unanue, E R, J exptl Led 133 (i971) i56. 7. Bankhurst, A D & Warner, N L. In preparation. 8. Bankhurst, A D, Warner, N L & Sprent, J, J exptl med 134 (1971) 1005. 9. Nossal, G J V,‘Warner, N L, Lewis, H & Sprent, J, J exptl med. In press. 10. Mandel, T, Byrt, P & Ada G L, Exptl cell res 58 (1969) 179. 11. Herzenberg, L & Warner, N L, Regulation of the antibody response (ed B Cinader). Charles C Thomas, Springfield, Ill. (1968). 12. Bankhurst,.A D & Warner, N L, J immunol 107 (1971) 368. 13. Shortman, K D, Williams, N & Adams, P, J immunol methods. In press. 14. Levey, R H & Medawar, P B, Ann NY acad sci 129 (1956) 164. 15. Hunter, W M & Greenwood, F C, Nature 194 (1962) 495. 16. Mandel, T. Personal communication. 17. Salneter. M M & Bachmann. L J cell biol 22 (1964) 469. 18. Mitchell, J & Abbot, A, Nature 208 (1965) 500. 19. Taylor, R B, Duffus, W P H, Raff, M C & de Petris, S, Nature new biol 233 (1971) 225. 20. Bosman, C, Feldman, J D & Pick, E, J exptl med 129 (1969) 1029. 21. Bosman, C & Feldman, J D, Lab invest 22 (1970) 309. 22. Hammond, E, Exptl cell res 59 (1970) 359. 23. Jones, G, Marcuson, E C & Roitt, I M, Nature 227 (1970) 1051. 24. Biberfeld, P, Biberfeld, G & Perlmann, P, Exptl cell res 66 (1971) 177. 25. Aoki, T, Hammerling, U de Harven, E, Boyse, E A & Old, L J, J exptl med 130 (1969) 979.
REFERENCES 1. Warner, N L, Byrt, P & Ada, G L, Nature 226 (1970) 942. 2. Warner, N L, Contemporary topics in immuno-
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Received September 21, 1971 Revised version received November 29, 1971