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Somatic cell genetics, immunogenetics and gene mapping
Immunology Today, vol. 4, No. 8, 1983
230 2 Paterson, P. Y. (1976) in Textbookoflmmunopathology (Miescher, P. and Miiller-Eberhard, H. J., eds), 2nd edn, pp. 179-213, Gmne and Stratton, New York 3 Ben-Nun, A. and Cohen, I. R. (1982)J. Imrnunol. 128, 1450-1457 4 Rose, N. R., Twarog, F. J. and Crowle, A. J. (1971)J lmmunol. 106, 698-704 5 Pearson, C. M. (1963)J. ChronicDis. 16, 863-874 6 Trentham, D. E., McCune, W. J., Susman, P. and David,J. R. (1980) J. Clin. Invest. 66, 1109-1117 7 Iizuka, Y. and Chang, Y. H. (1982) ArthritisRheum. 25, 1325-1332 8 Ben-Nun, A., Wekerle, H. and Cohen, I. R. (1981) Eur. J. Immunol. 11, 195-199 9 Ben-Nun, A. and Cohen, I. R. (1982)J. Immunol. 129, 303-308 10 Ben-Nun, A., Eisenstein, S. and Cohen, I. R. (1982)J. Immunol. 129, 918-919 11 Naparstek, Y., Ben-Nun, A., Holoshitz, J. et al. Eur. J. Immunol. (in press)
12 Holoshitz, J., Naparstek, Y., Ben-Nun, A. and Cohen, I. R. (1983) Science219, 56-58 13 Naparstek, Y., Holoshitz, J., Eisenstein, S. et al. (1982) Nature(London) 300, 262-263 14 Cohen, I. R., Globerson, A. and Feldman, M. (1971)J. Exp. Med. 133, 834-845 15 Orgad, S. and Cohen, I. R. (1974) Science183, 1083-1085 16 Jerne, K. (1974) Ann. Immunol. (Paris) 124C, 373-389 17 Wigzell, H. (1977) in Autoimmuni~: Genetic,Immunologicand ClinicalAspects. (Talal, N., ed.), pp. 693-707, Academic Press, New York 18 Cohen, I. R. in Advancesin InternalMedicine(Stollerman, G. H. ed.), Vol. 29, Year Book Medical Publishers, C. V. Mosby, St Louis (in press) 19 Ben-Nun, A., Wekerle, H. and Cohen, I. R. (1981)Nature(London)292, 60-61 20 Ben-Nun, A. and Cohen, I. R. (1981)Eur. J. Immunol. 11, 949-952
Somatic cell genetics, immunogenetics and gene mapping A. Tunnacliffe*, C. Jones* and P. Goodfellow* The use of immunological techniques to study genetics and the reciprocal use of genetic analysis to study immunology has proved to be a very powerful combination resulting in the definition of many systems including the major histocompatibility complex. The production of monoclonal antibodies by somatic cell hybrids (usually known as 'hybridomas') provides a more recent example of the exploitation of a genetic techniquefor the benefit of immunology. The reciprocal to the hybridoma technique is to use antibodies to analyse somatic cell hybrids. In this review Alan Tunnacliffe and colleagues describe the analysis of the human cell suoCace using somatic cell genetics in conjunction with monoclonal antibodies, and suggest that this is a powerful approach which should be applied more specifically to cells of the lymphoid system. T h e t e c h n i q u e s o f s o m a t i c cell genetics h a v e a l l o w e d a r a p i d a c c e l e r a t i o n in d e f i n i n g t h e h u m a n g e n e m a p since 1971, w h e n t h e g e n e for s o l u b l e t h y m i d i n e k i n a s e w a s a s s i g n e d to c h r o m o s o m e 171. S i n c e t h e n , s e v e r a l h u n d r e d g e n e s h a v e b e e n a s s i g n e d to specific c h r o m o s o m e s o r c h r o m o s o m a l r e g i o n s a n d o t h e r s c o n t i n u e to b e a s s i g n e d at a n i n c r e a s i n g rate 2'3. T h i s p h e n o m e n a l success is b a s e d o n t h e o b s e r v a t i o n b y W e i s s a n d G r e e n 4 in 1967 t h a t human-rodent s o m a t i c cell h y b r i d s s e g r e g a t e n o n selected h u m a n c h r o m o s o m e s . T h i s allows t h e a s s e m b l y of a panel of hybrids with various numbers and distributions o f h u m a n c h r o m o s o m e s , w h i c h c a n b e u s e d to a s s a y for a p a r t i c u l a r h u m a n g e n e o r g e n e p r o d u c t . B y correla t i n g t h e p r e s e n c e o f a g e n e w i t h the p r e s e n c e o f a particular human chromosome, chromosomal assignment of that gene can be made. T h e g e n e a s s a y is a l l - i m p o r t a n t : n o t o n l y m u s t it b e specific for t h e g e n e to b e m a p p e d , it m u s t also b e speciesspecific to d i s t i n g u i s h it f r o m its r o d e n t c o u n t e r p a r t in hyb r i d s . R e c e n t a d v a n c e s in r e c o m b i n a n t D N A t e c h n o l o g y h a v e a l l o w e d d i r e c t m a p p i n g at t h e g e n e level, p r o v i d i n g t h e a p p r o p r i a t e p r o b e is a v a i l a b l e (for r e v i e w , see R e f . 5). T h i s m e t h o d relies o n t h e s e q u e n c e d i f f e r e n c e s a n d / o r restriction fragment length polymorphisms between human and rodent genes. * Laboratory of H u m a n Molecular Genetics, Imperial Cancer Research Fund, Lincoln's Inn Fields, L o n d o n W C 2 A 3PX, UK. *Eleanor Roosevelt Institute for Cancer Research, Denver, C O . 80262, USA. © Elsevier Biomedical press 1983 0167-4919183/0000-0000151.00
A m o r e i n d i r e c t m e t h o d o f g e n e m a p p i n g involves a s s a y for a n R N A o r p r o t e i n e n c o d e d b y t h e g e n e o f interest. T h e specificity o f a n e n z y m e for its s u b s t r a t e , o r a related substance which can be calorimetrically or radioactively d e t e r m i n e d , c o u p l e d w i t h h u m a n - r o d e n t electrop h o r e t i c p o l y m o r p h i s m s w h e n h y b r i d cell e x t r a c t s a r e f r a c t i o n a t e d o n gels, h a v e e n a b l e d m a n y e n z y m e - c o d i n g g e n e s to b e m a p p e d . F o r e x a m p l e , t h e g e n e for lactate deh y d r o g e n a s e A ( L D H A ) , t h e first n o n - s e l e c t a b l e m a r k e r a s s i g n e d , is o n c h r o m o s o m e 11 s. T h e specificity o f a n a n t i b o d y for its c o g n a t e a n t i g e n h a s b e e n e x p l o i t e d as a n o t h e r m e a n s o f i n d i r e c t g e n e m a p p i n g a n d s o m e successes TABLE I. Genes for cell-surface antigens characterized by polyclonal antisera Chromosomal Location 6 7 11 12 15 17 21 X
Antigen
Refs
HLA EGF receptor Sl, $2 $3, S4 02m Interferon receptor S10 (SAX)
15 16 9-11, 13 17 18 19 20 21
Regional mapping has also been done for some of these loci using chromosome deletions or translocations and/or other genetic methods. The original reference does not always identify the antigen: for example the EGF receptor was only recently shown to be identical to the antigen described by Aden and Knowles22.
231
Immunology Today, vol. 4, No. 8, 1983 _ 1 -
Sl,S3,S4 W6/34, F10.44.2
S2 TRAI.IO, 4D12
Fig. l. A summary of cell surface antigens whose controlling loci have been mapped to human chromosome 11.
with both polyclonal and monoclonal antibodies will be outlined in this review. The presence of human-specific antigens on h u m a n mouse hybrids was noted in the pioneering experiments of Weiss and Green (1967) 4, Nabholz et al. (1969) 7 and Kano et al. (1969) 8. In all cases, polyclonal antisera raised against h u m a n cells in rabbits and absorbed against mouse cells to remove heterophilic antibodies showed reactivity with human-mouse hybrids, demonstrating the presence of h u m a n antigens on the hybrid cell surface. In addition, Nabholz et al. suggested that human-specific antigens might be coded for on the same chromosome as one of the L D H l o c i . In 1971, Puck et al. 9 found synteny between the genes for h u m a n 'lethal antigens' (AL) and LDHA in human-Chinese-hamster hybrids, and when LDHA was assigned to chromosome 116 this simultaneously assigned the Au system to that chromosome. Puck, Jones, Kao and colleagues have since gone on further to split Ac into at least three distinct antigenic identities - a~, a2 and a~, subsequently renamed S1, $2 and $3, all encoded by chromosome 11. These antigens can be distinguished by various antisera which are assayed by their cytotoxicity, in the presence of complement, towards cells carrying cognate antigens; hence their name 'lethal antigens'. For example, rabbits immunized with HeLa cells make antibodies against both S1 and $2 but immunization with h u m a n red blood cells (RBC) stimulates production of antibodies against S1 only, producing an anti-S1 serum. Thus if anti-HeLa sera are absorbed with RBC, $1 activity is removed, leaving an anti-S2 serum ~°. The $3 component was identified after a human-Chinese-hamster hybrid, containing only chromosome 11 as its h u m a n genetic material, was mutagenized and selected for resistance to killing by both anti-S1 and anti-S2 sera. This leads to $1$2- mutants". However, antisera prepared against human lymphoblasts in horses still kill these mutants, indicating the presence of another 'lethal antigen', $3. Hyperimmunization of rabbits or standard immunization of sheep with RBC also produces anti-S3 activity. By further use of specific antisera in conjunction with hybrids with deleted chromosomes 11, it was possible to locate the position of the AL genes more accurately'2 - $1 and $3 to 1lpter-pl3 and $2 to 1lq13-qter (Fig. 1).
A different approach was taken by Buck and Bodmer j' who used whole cells of human-mouse hybrids with few human chromosomes to immunize a strain of mice from which the mouse parent of the hybrids was derived. In theory, this should give rise to antisera specific for human cell-surface antigens, but in practice a strong response to C-type virus tumour antigens is seen. Substrain dii~ ferences between the progenitor of the mouse cell line and the immunized mouse may also be responsible tbr antimouse activity in the antiserum. However, after absorption with mouse cells, a human-specific antiserum can be isolated. Using a hybrid with h u m a n chromosomes X, 11 and 13 only, an antiserum was produced which was shown to recognize an antigen or antigens coded for by h u m a n chromosome 11~. This was named SA-1 (species antigen 1) and subsequently renamed $4. Further study ~ showed the $4 gene to be carried on the short arm of chromosome 11. These studies illustrate the possibilities tbr mapping genes for cell-surface antigens using conventional antisera. Other chromosomes have been shown to carry genes for cell-surface antigens by these methods and these are listed in Table 1. It can be seen that at least a third of the h u m a n chromosomes carry genes which control the expression of cell-surface antigens. Indeed, there is a reasonable probability that all the human chromosomes will contain at least one such gene. It was inevitable that, after Kohler and Mi]stein introduced hybridoma technology in 19752'~, monoclonal antibodies would begin to be used in the genetic analysis of cell-surface antigens (for reviews of hybridoma lechnology, see Ref. 24). The advantages ofmonoclonal over polyclonal antisera have often been stressed, but in the context of gene mapping these include: (1) standardization of reagents; (2) reproducibility; (3) unlimited availability of reagents; and (4) immunochemicai purity ,~! reagents. Coupled with such techniques as the indire~, radioimmunoassay (IRIA) 2~ or EI~ISA, a rapid aualvsi~ of antigenic determinants on the cell surt~tce is possible. By immunizing mice with human tonsil leucocvt~ s ai~d subsequent hybridoma tusions, Barnstable eta/. { 1978) were able to produce, arnongst others, monoclonal antibodies against HLA-A, -B, -C (W6/32) and an antige~ encoded by a gene on the short arm of chromosome I 1 (W6/34). These antibodies probably correspond to some component of the polyclonal antisera directed against these determinants. It would seem likely that monoclonal antibodies corresponding to all the polyclonal antisera of Table I could be made and that other, so far unidentified, chromosomal antigenic markers might be brought to light. This has been attempted in our own and other laboratories either by screening existing monoclonal antibodies against a hybrid panel or by producing new monoclonal antibodies after the method of Buck and Bodmer ~:~. Table II outlines the current situation. Comparison with Table I shows that all polyclonal antisera, with the exception of that against the interferon receptor, now have monoelonal antibody counterparts, in some cases recognizing the same antigen (for example HLA-A, -B, -C;/J2m; EGFR). In addition, we have extended the panel of antibodies to cover chromosomes 3 and Y and in some cases have characterized several distinct antigens encoded
Immunology Today, vol. 4, No. 8, 1983
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TABLE II. Genes for cell-surfaceantigens identified by monoclonal antibodies Chromosomal Gene location
Antibody
Antigen
Refs
Transferrin receptor HLA-A, -B, -C Epidermal growth factor receptor Glycolipid 80K glycoprotein 100K protein
27
3
TFRC
OKT9
6
HLA-A,
W6/32
-B, -C 7
EGFR
EGFR1
11
11
MIC1 a MIC4
W6134 F10.44.2
11
MIC9
4D12
11
MIC8
TRA1.10
80K, 40K protein
12 15
MIC3 B2M
602 BBM1
15
MIC7
28
21K protein 02 microglobulin 95K protein
17 X X Y
MIC6 MIC2 MIC5 MIC2Y
H207 12E7 RI 12E7
26 28 26 29 A. Tunnacliffe, unpublished observations P. Andrews, unpublished observations 30 31
C. Blaineau et aL, unpublished observations 125K protein 32 34K protein 33 34 34K protein 33
aThe designation M I C No. is a provisional local name for genes mapped by
monoclonal antibodies at ICRF, a nomenclature agreed upon at the Sixth International H u m a n Gene Mapping Workshop, Oslo, 1981.
by the same chromosome, in particular four different antigens encoded by chromosome 11. The question arises as to the nature of the four antigens whose expression is controlled by chromosome 11 and their possible relationships to the AL system defined by Puck et al. with polyclonal antisera. Collaborative research between our laboratories has shown that in a series of hybrids with deleted human chromosomes 11, and in HeLa mutants, W6/34 antigen is indistinguishable from S 1 (unpublished observations). Furthermore, the S 1 antigenic determinant is found on a macroglycolipid isolated from human RBC ~5. The antigen defined by monoclonal antibody W6/34 is also found on a glycolipid26 and thus may be identical or closely related to S1. W6/34 and F10.44.2 antigens are biochemieally and genetically distinct (Ref. 29; G. Banting, P. N. Goodfellow, G. Lenoir et al., unpublished observations) and preliminary evidence suggests a relationship between F10.44.2 antigen and $3. The antigens recognized by monoclonal antibodies 4D12 and T R A I . 1 0 are genetically distinct (our own unpublished observations) although both probably map to the long arm of chromosome 11 and are absent from RBC and lymphocytes. These antigens may be related to $2 and this is currently under investigation. The present situation is summarized in Fig. 1. We hope to extend the panel of monoclonal antibodies to cover the whole range of human chromosomes as this would provide a valuable set of reagents for somatic cell genetics. Possible uses are: (1) Rapid evaluation of the human chromosome content of human-rodent hybrids with relatively small numbers of cells. At present, karyotypic and/or isozyme analysis is mandatory and is labour- and time-intensive. (2) Manipulation of hybrid chromosome content. Selec-
tion for the presence or absence of a particular chromosome in a hybrid is possible by use of the appropriate antibody and flow cytofluorimetry on the fluorescenceactivated cell sorter (FACS) (for example, see Ref. 29). (3) The FACS also allows rapid syntenic and linkage analysis in a single population of cells between an antigencoding gene and any other gene. For example, Goodfellow et al., 198229, were able to show segregation of L D H A activity and W6/34 and F10.44.2 antigens in a mass population of a hybrid segregating chromosomes 11 and 15. The characterization of monoclonal antibodies against cell-surface determinants often leads to other interesting lines of enquiry. An example is 12E7, an antibody made against human T - A L L cell membranes 36 and which recognizes an antigen found generally on human tissues (except spermatozoa) although at highest levels on cortical thymocytes. Somatic cell genetic analysis has indicated the presence of homologous loci for the cell surface expression of 12E7 antigen on both the X and Y chromosomes (Ref. 33; P. Goodfellow, G. Banting, D. Sheer, unpublished observations). This is the first demonstration of an active structural gene on the human Y chromosome. Intriguingly, the X-specific location is at the very tip of the short arm (Xp2.23), a region thought to pair with the Y chromosome during meiosis 37. This region is also believed to escape X inactivation in females 3a-~°. The 12E7 antibody may prove a useful tool for investigating these phenomena and sex-chromosome evolution. Most of the antigens studied so far by this technique have a wide tissue distribution. However, by using the appropriate mouse and human parental cells, tissuespecific antigens can be studied (for example, see Ref. 41). Extension of this approach to study antigenic markers of human lymphoid cells will be a fruitful area for further research. Indeed recently, using hybrids between cALL lymphoblasts and mouse myeloma, F. Katz and others (unpublished observations) have succeeded in mapping the gene(s) responsible for OKT10 antigen expression to human chromosome 4. In summary, the techniques of somatic cell genetics, used in conjunction with polyclonal and monoclonal antisera, have allowed chromosomal assignment and genetic analysis of the genes controlling expression of several human cell-surface antigens. In particular, monoclonal antibodies to specific antigenic chromosome markers have provided us with powerful tools for hybrid manipulation and characterization.
References 1 Miller, O. J., Allderdice, P. W. and Miller, D. A. (1971) Science 173, 244-245 2 McKusick, V. A. and Ruddle, F. H. (1977) Science 196, 390-405 3 Proceedings of the Sixth International Workshop on Human Gene Mapping, Oslo, 1981. Birth Defects: On'g~nalArticleSeries 18 2; and Cytogenet. Cell C,enet. 32 (1982) 4 Weiss, M. C. and Green, H. (1967) Pr0c. Natl Acad. Sci. USA 58, 1104-1111 5 Ruddle, F. H. (1981)Nature(London)294, 115-120 6 Boone, C., Chen, T. R. and Ruddle, F. H. (1972)Proc. NatlAcad. &i. USA 69, 510-514 7 Nabholz, M., Miggiano, V. and Bodmer, W. F. (1969) Nature (London) 223, 358--363
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8 Kano, K., Baranska, W., Knowles, B. B. et al. (1969)J. Immunol. 103, 1050-1060 9 Puck, T. T., Wuthier, P., Jones, C. and Kao, F. (1971) Proc. NatlAcad. Sci. USA 68, 3102 3106 10 Jones, C., Wuthier, P. and Puck, T. T. (1975) Somatic Cell Go~t. 1, 235-246 11 Jones, C., and Puck, T. T. (1977) Somatic Cell Genet. 3,407-420 12 Kao, F.-T., Jones, C. and Puck, T. T. (1977) Somatic Cell Genet. 3, 421-429 13 Buck, D. W. and Bodmer, W. F. (1974) Birth Defects: OriginalArticle Series 11 3, 87-89 14 Buck, D. W., Bodmer, W. F., Bobrow, M. and Francke, U. (1976) Cytogenet. Cell Genet. 16, 97-98 15 van Someren, H., Westerveld, A., Hagemeijer, A. et al. (1974)Proc. Natl Acad. Sci. USA 71,962-965 16 Aden, D. P. and Knowles, B. B. (1976) Immunogenetics 3, 209-221 17 Seravalli, E., Schwab, R., Penvis, B. and Siniscalco, M. (I978) Cytogenet. Cell Genet. 22, 260-264 18 Goodfellow, P. N., Jones, E. A., van Heyningen, V. et al. (1975). Nature (London) 254, 267-269 19 Cicurel, L. and Croce, C. M. (1977)J. Irnrnunol. t18 1951-1956 20 Revel, M., Bash, D. and Ruddle, F. H. (1976) Nature (London) 260, 139-141 21 Buck, D. W. and Bodmer, W. F. (1976) Birth Defects: OrtginalArticle Series 12 7, 376-377 22 Carlin, C. R. and Knowles, B. B. (1982) Proc. Natl Acad. Sci USA 79, 5026--5030 23 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 24 McMichael, A . J . and Fabre, J. w . (1982) MonodonalAntibodies in Clinical Medicine, Academic Press, New York 25 Williams, A. F. (1977) Contemp. Top. Mol. lmmunoL 6, 83-116
26 Barnstable, C. J., Bodmer, W. F., Brown, G. et al. (1978) Cell 14, 9-20 27 Goodfellow, P. N., Banting, G., Sutherland, R. et al. (1982) Somatic Cell Genet. 8, 197-206 28 Goodfellow, P. N., Banting, G., Waterfield, M. and Ozanne, B. (1981) in Proceedings of the Sixth International Workshop on Human Gene Mapping, Oslo, 1981. Birth Defects: Onginal Article Series 18 2; and Cytogenet. Cell C,enet. 32 (1982) 29 Goodfellow, P. N., Banting, G., Tunnacliffe, A. et al. (1982) Eur. J. ImmunoL 12, 659-663 30 Andrews, P. W., Knowles, B. B. and Goodfellow, P. N. (1981) Somatic Cell Genet. 7, 435-443 31 Brodsky, F. M., Bodmer, W. F. and Parham, P. (1979) Eur. J. ImmunoL 9, 536-545 32 Yan, B., Sheer, D., Hinrns, L. etal. (1982)Ann. Hum. C,enet. 46,337-347 33 GoodfeUow, P., Banting, G., Sheer, D. et al. (1983) Nature (London) 6, 777-787; 302, 346-349 34 Hope, R. M., Goodfellow, P. N., Solomon, E. and Bodmer, W. F. (1982) Cytogenet. Cell Genet. 33, 204-212 35 Jones, C., Moore, E. E. and Lehman, D. W. (1979) Proc. NatlAcad. Sci. USA 76, 6491-6495 36 Levy, R., Dilley, J., Fox, R. I. and Warnke, R. (1979)Proc. NatlAcad. Sci. USA 76, 6552-6556 37 Pearson, P. C. and Bobrow, M. (1970) Nature (London) 226, 959-961 38 Race, R. and Sanger, R. (1975) Blood Groups in Man, 6th Edn, Blackwell Scientific, Oxford 39 Shapiro, C. J., Mohandas, T., Weiss, R. and Romeo, G. (1979) Science 204, 1224 1226 40 Muller, C. R., Migl, B., Traupe, H. and Ropers, H. H. (1980) Hum. Genet. 54, 197-199 41 Quinn, C. A., Goodfellow, P. N., Povey, S. and Walsh, F. S. (1981)Proc. Natl Acad. Sci. USA 78, 5031-5035
Immunoregulatory T-cell defects Raif S. Geha and Fred S. Rosen In normal circumstanceshomeostatic balance is maintained between T-cell help and suppression. In this review Raif Geha and Fred Rosen discuss examples of human diseases which involve excess or deficiency of T-cell help or T-ceU suppression - diseases which have contributedgreatly to an understanding of how T cells regulate the human immune response.
T lymphocytes play a major role in the regulation of the immune response. Helper T cells recognize antigen processed by macrophages in association with determinants encoded in the mouse I-A or human HLA-D regions. They are stimulated to proliferate and to induce directly or via the secretion of soluble mediators other lymphoid cells to differentiate into effector cells. Conversely, suppressor T cells appear to recognize antigen directly, or perhaps in the mouse in the context of determinants encoded in the I-J region, after which they are activated to suppress the immune response. The targets of T-cell regulation include B cells, other T cells, natural killer cells and non-lymphoid cells such as erythroid cell precursors. The availability of monoclonal antibodies to human T-cell surface antigens and of antigen-specific human T-cell clones have led to a better understanding of the relationship between surface markers and T-cell function. It appears that the 20K T3 antigen expressed by all mature T lymphocytes is closely associated with the T-cell receptor for antigen which recognizes antigen X plus a polymorphic M H C gene product, whereas the 62K T4 Divisions of Allergy and Immunology, The Children's Hospital and the Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
antigen and the 76 K T8 antigen serve as associative recognition structures which bind to a monomorphic portion of class-II and class-I M H C gene products, respectively 1. Thus cytotoxic T-cell clones express either T4 or T8 antigen, depending on their restriction for recognition of class-II or class-I antigens 2. However, in bulk populations of T cells, helper function resides mostly in T4-positive cells, whereas suppressor and cytotoxic functions reside predominantly in T8-positive cells 3. For all functional T-cell subsets activation is accompanied by the expression of new surface markers which include Ia/HLA-DR antigens, and receptors for interleukin 2 (IL-2) and for transferrin. Diseases with failure of T-ceU help Transient hypogammaglobulinemia of infancy is characterized by recurrent respiratory infections. These patients have low levels of IgG which often return rather abruptly to normal by age 4 years. Affected infants have normal numbers of B cells and normal intrinsic B-cell function: their peripheral blood lymphocytes (PBL) generate a normal number of IgG- and IgM-plaqueforming cells following activation by the T-cell-independent polyclonal B-cell activator Epstein-Barr virus (EBV). Their T cells, however, are deficient in the capacity to generate help for immunoglobulin (Ig) synthesis by
230 2 Paterson, P. Y. (1976) in Textbookoflmmunopathology (Miescher, P. and Miiller-Eberhard, H. J., eds), 2nd edn, pp. 179-213, Gmne and Stratton, New York 3 Ben-Nun, A. and Cohen, I. R. (1982)J. Imrnunol. 128, 1450-1457 4 Rose, N. R., Twarog, F. J. and Crowle, A. J. (1971)J lmmunol. 106, 698-704 5 Pearson, C. M. (1963)J. ChronicDis. 16, 863-874 6 Trentham, D. E., McCune, W. J., Susman, P. and David,J. R. (1980) J. Clin. Invest. 66, 1109-1117 7 Iizuka, Y. and Chang, Y. H. (1982) ArthritisRheum. 25, 1325-1332 8 Ben-Nun, A., Wekerle, H. and Cohen, I. R. (1981) Eur. J. Immunol. 11, 195-199 9 Ben-Nun, A. and Cohen, I. R. (1982)J. Immunol. 129, 303-308 10 Ben-Nun, A., Eisenstein, S. and Cohen, I. R. (1982)J. Immunol. 129, 918-919 11 Naparstek, Y., Ben-Nun, A., Holoshitz, J. et al. Eur. J. Immunol. (in press)
12 Holoshitz, J., Naparstek, Y., Ben-Nun, A. and Cohen, I. R. (1983) Science219, 56-58 13 Naparstek, Y., Holoshitz, J., Eisenstein, S. et al. (1982) Nature(London) 300, 262-263 14 Cohen, I. R., Globerson, A. and Feldman, M. (1971)J. Exp. Med. 133, 834-845 15 Orgad, S. and Cohen, I. R. (1974) Science183, 1083-1085 16 Jerne, K. (1974) Ann. Immunol. (Paris) 124C, 373-389 17 Wigzell, H. (1977) in Autoimmuni~: Genetic,Immunologicand ClinicalAspects. (Talal, N., ed.), pp. 693-707, Academic Press, New York 18 Cohen, I. R. in Advancesin InternalMedicine(Stollerman, G. H. ed.), Vol. 29, Year Book Medical Publishers, C. V. Mosby, St Louis (in press) 19 Ben-Nun, A., Wekerle, H. and Cohen, I. R. (1981)Nature(London)292, 60-61 20 Ben-Nun, A. and Cohen, I. R. (1981)Eur. J. Immunol. 11, 949-952
Somatic cell genetics, immunogenetics and gene mapping A. Tunnacliffe*, C. Jones* and P. Goodfellow* The use of immunological techniques to study genetics and the reciprocal use of genetic analysis to study immunology has proved to be a very powerful combination resulting in the definition of many systems including the major histocompatibility complex. The production of monoclonal antibodies by somatic cell hybrids (usually known as 'hybridomas') provides a more recent example of the exploitation of a genetic techniquefor the benefit of immunology. The reciprocal to the hybridoma technique is to use antibodies to analyse somatic cell hybrids. In this review Alan Tunnacliffe and colleagues describe the analysis of the human cell suoCace using somatic cell genetics in conjunction with monoclonal antibodies, and suggest that this is a powerful approach which should be applied more specifically to cells of the lymphoid system. T h e t e c h n i q u e s o f s o m a t i c cell genetics h a v e a l l o w e d a r a p i d a c c e l e r a t i o n in d e f i n i n g t h e h u m a n g e n e m a p since 1971, w h e n t h e g e n e for s o l u b l e t h y m i d i n e k i n a s e w a s a s s i g n e d to c h r o m o s o m e 171. S i n c e t h e n , s e v e r a l h u n d r e d g e n e s h a v e b e e n a s s i g n e d to specific c h r o m o s o m e s o r c h r o m o s o m a l r e g i o n s a n d o t h e r s c o n t i n u e to b e a s s i g n e d at a n i n c r e a s i n g rate 2'3. T h i s p h e n o m e n a l success is b a s e d o n t h e o b s e r v a t i o n b y W e i s s a n d G r e e n 4 in 1967 t h a t human-rodent s o m a t i c cell h y b r i d s s e g r e g a t e n o n selected h u m a n c h r o m o s o m e s . T h i s allows t h e a s s e m b l y of a panel of hybrids with various numbers and distributions o f h u m a n c h r o m o s o m e s , w h i c h c a n b e u s e d to a s s a y for a p a r t i c u l a r h u m a n g e n e o r g e n e p r o d u c t . B y correla t i n g t h e p r e s e n c e o f a g e n e w i t h the p r e s e n c e o f a particular human chromosome, chromosomal assignment of that gene can be made. T h e g e n e a s s a y is a l l - i m p o r t a n t : n o t o n l y m u s t it b e specific for t h e g e n e to b e m a p p e d , it m u s t also b e speciesspecific to d i s t i n g u i s h it f r o m its r o d e n t c o u n t e r p a r t in hyb r i d s . R e c e n t a d v a n c e s in r e c o m b i n a n t D N A t e c h n o l o g y h a v e a l l o w e d d i r e c t m a p p i n g at t h e g e n e level, p r o v i d i n g t h e a p p r o p r i a t e p r o b e is a v a i l a b l e (for r e v i e w , see R e f . 5). T h i s m e t h o d relies o n t h e s e q u e n c e d i f f e r e n c e s a n d / o r restriction fragment length polymorphisms between human and rodent genes. * Laboratory of H u m a n Molecular Genetics, Imperial Cancer Research Fund, Lincoln's Inn Fields, L o n d o n W C 2 A 3PX, UK. *Eleanor Roosevelt Institute for Cancer Research, Denver, C O . 80262, USA. © Elsevier Biomedical press 1983 0167-4919183/0000-0000151.00
A m o r e i n d i r e c t m e t h o d o f g e n e m a p p i n g involves a s s a y for a n R N A o r p r o t e i n e n c o d e d b y t h e g e n e o f interest. T h e specificity o f a n e n z y m e for its s u b s t r a t e , o r a related substance which can be calorimetrically or radioactively d e t e r m i n e d , c o u p l e d w i t h h u m a n - r o d e n t electrop h o r e t i c p o l y m o r p h i s m s w h e n h y b r i d cell e x t r a c t s a r e f r a c t i o n a t e d o n gels, h a v e e n a b l e d m a n y e n z y m e - c o d i n g g e n e s to b e m a p p e d . F o r e x a m p l e , t h e g e n e for lactate deh y d r o g e n a s e A ( L D H A ) , t h e first n o n - s e l e c t a b l e m a r k e r a s s i g n e d , is o n c h r o m o s o m e 11 s. T h e specificity o f a n a n t i b o d y for its c o g n a t e a n t i g e n h a s b e e n e x p l o i t e d as a n o t h e r m e a n s o f i n d i r e c t g e n e m a p p i n g a n d s o m e successes TABLE I. Genes for cell-surface antigens characterized by polyclonal antisera Chromosomal Location 6 7 11 12 15 17 21 X
Antigen
Refs
HLA EGF receptor Sl, $2 $3, S4 02m Interferon receptor S10 (SAX)
15 16 9-11, 13 17 18 19 20 21
Regional mapping has also been done for some of these loci using chromosome deletions or translocations and/or other genetic methods. The original reference does not always identify the antigen: for example the EGF receptor was only recently shown to be identical to the antigen described by Aden and Knowles22.
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Immunology Today, vol. 4, No. 8, 1983 _ 1 -
Sl,S3,S4 W6/34, F10.44.2
S2 TRAI.IO, 4D12
Fig. l. A summary of cell surface antigens whose controlling loci have been mapped to human chromosome 11.
with both polyclonal and monoclonal antibodies will be outlined in this review. The presence of human-specific antigens on h u m a n mouse hybrids was noted in the pioneering experiments of Weiss and Green (1967) 4, Nabholz et al. (1969) 7 and Kano et al. (1969) 8. In all cases, polyclonal antisera raised against h u m a n cells in rabbits and absorbed against mouse cells to remove heterophilic antibodies showed reactivity with human-mouse hybrids, demonstrating the presence of h u m a n antigens on the hybrid cell surface. In addition, Nabholz et al. suggested that human-specific antigens might be coded for on the same chromosome as one of the L D H l o c i . In 1971, Puck et al. 9 found synteny between the genes for h u m a n 'lethal antigens' (AL) and LDHA in human-Chinese-hamster hybrids, and when LDHA was assigned to chromosome 116 this simultaneously assigned the Au system to that chromosome. Puck, Jones, Kao and colleagues have since gone on further to split Ac into at least three distinct antigenic identities - a~, a2 and a~, subsequently renamed S1, $2 and $3, all encoded by chromosome 11. These antigens can be distinguished by various antisera which are assayed by their cytotoxicity, in the presence of complement, towards cells carrying cognate antigens; hence their name 'lethal antigens'. For example, rabbits immunized with HeLa cells make antibodies against both S1 and $2 but immunization with h u m a n red blood cells (RBC) stimulates production of antibodies against S1 only, producing an anti-S1 serum. Thus if anti-HeLa sera are absorbed with RBC, $1 activity is removed, leaving an anti-S2 serum ~°. The $3 component was identified after a human-Chinese-hamster hybrid, containing only chromosome 11 as its h u m a n genetic material, was mutagenized and selected for resistance to killing by both anti-S1 and anti-S2 sera. This leads to $1$2- mutants". However, antisera prepared against human lymphoblasts in horses still kill these mutants, indicating the presence of another 'lethal antigen', $3. Hyperimmunization of rabbits or standard immunization of sheep with RBC also produces anti-S3 activity. By further use of specific antisera in conjunction with hybrids with deleted chromosomes 11, it was possible to locate the position of the AL genes more accurately'2 - $1 and $3 to 1lpter-pl3 and $2 to 1lq13-qter (Fig. 1).
A different approach was taken by Buck and Bodmer j' who used whole cells of human-mouse hybrids with few human chromosomes to immunize a strain of mice from which the mouse parent of the hybrids was derived. In theory, this should give rise to antisera specific for human cell-surface antigens, but in practice a strong response to C-type virus tumour antigens is seen. Substrain dii~ ferences between the progenitor of the mouse cell line and the immunized mouse may also be responsible tbr antimouse activity in the antiserum. However, after absorption with mouse cells, a human-specific antiserum can be isolated. Using a hybrid with h u m a n chromosomes X, 11 and 13 only, an antiserum was produced which was shown to recognize an antigen or antigens coded for by h u m a n chromosome 11~. This was named SA-1 (species antigen 1) and subsequently renamed $4. Further study ~ showed the $4 gene to be carried on the short arm of chromosome 11. These studies illustrate the possibilities tbr mapping genes for cell-surface antigens using conventional antisera. Other chromosomes have been shown to carry genes for cell-surface antigens by these methods and these are listed in Table 1. It can be seen that at least a third of the h u m a n chromosomes carry genes which control the expression of cell-surface antigens. Indeed, there is a reasonable probability that all the human chromosomes will contain at least one such gene. It was inevitable that, after Kohler and Mi]stein introduced hybridoma technology in 19752'~, monoclonal antibodies would begin to be used in the genetic analysis of cell-surface antigens (for reviews of hybridoma lechnology, see Ref. 24). The advantages ofmonoclonal over polyclonal antisera have often been stressed, but in the context of gene mapping these include: (1) standardization of reagents; (2) reproducibility; (3) unlimited availability of reagents; and (4) immunochemicai purity ,~! reagents. Coupled with such techniques as the indire~, radioimmunoassay (IRIA) 2~ or EI~ISA, a rapid aualvsi~ of antigenic determinants on the cell surt~tce is possible. By immunizing mice with human tonsil leucocvt~ s ai~d subsequent hybridoma tusions, Barnstable eta/. { 1978) were able to produce, arnongst others, monoclonal antibodies against HLA-A, -B, -C (W6/32) and an antige~ encoded by a gene on the short arm of chromosome I 1 (W6/34). These antibodies probably correspond to some component of the polyclonal antisera directed against these determinants. It would seem likely that monoclonal antibodies corresponding to all the polyclonal antisera of Table I could be made and that other, so far unidentified, chromosomal antigenic markers might be brought to light. This has been attempted in our own and other laboratories either by screening existing monoclonal antibodies against a hybrid panel or by producing new monoclonal antibodies after the method of Buck and Bodmer ~:~. Table II outlines the current situation. Comparison with Table I shows that all polyclonal antisera, with the exception of that against the interferon receptor, now have monoelonal antibody counterparts, in some cases recognizing the same antigen (for example HLA-A, -B, -C;/J2m; EGFR). In addition, we have extended the panel of antibodies to cover chromosomes 3 and Y and in some cases have characterized several distinct antigens encoded
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TABLE II. Genes for cell-surfaceantigens identified by monoclonal antibodies Chromosomal Gene location
Antibody
Antigen
Refs
Transferrin receptor HLA-A, -B, -C Epidermal growth factor receptor Glycolipid 80K glycoprotein 100K protein
27
3
TFRC
OKT9
6
HLA-A,
W6/32
-B, -C 7
EGFR
EGFR1
11
11
MIC1 a MIC4
W6134 F10.44.2
11
MIC9
4D12
11
MIC8
TRA1.10
80K, 40K protein
12 15
MIC3 B2M
602 BBM1
15
MIC7
28
21K protein 02 microglobulin 95K protein
17 X X Y
MIC6 MIC2 MIC5 MIC2Y
H207 12E7 RI 12E7
26 28 26 29 A. Tunnacliffe, unpublished observations P. Andrews, unpublished observations 30 31
C. Blaineau et aL, unpublished observations 125K protein 32 34K protein 33 34 34K protein 33
aThe designation M I C No. is a provisional local name for genes mapped by
monoclonal antibodies at ICRF, a nomenclature agreed upon at the Sixth International H u m a n Gene Mapping Workshop, Oslo, 1981.
by the same chromosome, in particular four different antigens encoded by chromosome 11. The question arises as to the nature of the four antigens whose expression is controlled by chromosome 11 and their possible relationships to the AL system defined by Puck et al. with polyclonal antisera. Collaborative research between our laboratories has shown that in a series of hybrids with deleted human chromosomes 11, and in HeLa mutants, W6/34 antigen is indistinguishable from S 1 (unpublished observations). Furthermore, the S 1 antigenic determinant is found on a macroglycolipid isolated from human RBC ~5. The antigen defined by monoclonal antibody W6/34 is also found on a glycolipid26 and thus may be identical or closely related to S1. W6/34 and F10.44.2 antigens are biochemieally and genetically distinct (Ref. 29; G. Banting, P. N. Goodfellow, G. Lenoir et al., unpublished observations) and preliminary evidence suggests a relationship between F10.44.2 antigen and $3. The antigens recognized by monoclonal antibodies 4D12 and T R A I . 1 0 are genetically distinct (our own unpublished observations) although both probably map to the long arm of chromosome 11 and are absent from RBC and lymphocytes. These antigens may be related to $2 and this is currently under investigation. The present situation is summarized in Fig. 1. We hope to extend the panel of monoclonal antibodies to cover the whole range of human chromosomes as this would provide a valuable set of reagents for somatic cell genetics. Possible uses are: (1) Rapid evaluation of the human chromosome content of human-rodent hybrids with relatively small numbers of cells. At present, karyotypic and/or isozyme analysis is mandatory and is labour- and time-intensive. (2) Manipulation of hybrid chromosome content. Selec-
tion for the presence or absence of a particular chromosome in a hybrid is possible by use of the appropriate antibody and flow cytofluorimetry on the fluorescenceactivated cell sorter (FACS) (for example, see Ref. 29). (3) The FACS also allows rapid syntenic and linkage analysis in a single population of cells between an antigencoding gene and any other gene. For example, Goodfellow et al., 198229, were able to show segregation of L D H A activity and W6/34 and F10.44.2 antigens in a mass population of a hybrid segregating chromosomes 11 and 15. The characterization of monoclonal antibodies against cell-surface determinants often leads to other interesting lines of enquiry. An example is 12E7, an antibody made against human T - A L L cell membranes 36 and which recognizes an antigen found generally on human tissues (except spermatozoa) although at highest levels on cortical thymocytes. Somatic cell genetic analysis has indicated the presence of homologous loci for the cell surface expression of 12E7 antigen on both the X and Y chromosomes (Ref. 33; P. Goodfellow, G. Banting, D. Sheer, unpublished observations). This is the first demonstration of an active structural gene on the human Y chromosome. Intriguingly, the X-specific location is at the very tip of the short arm (Xp2.23), a region thought to pair with the Y chromosome during meiosis 37. This region is also believed to escape X inactivation in females 3a-~°. The 12E7 antibody may prove a useful tool for investigating these phenomena and sex-chromosome evolution. Most of the antigens studied so far by this technique have a wide tissue distribution. However, by using the appropriate mouse and human parental cells, tissuespecific antigens can be studied (for example, see Ref. 41). Extension of this approach to study antigenic markers of human lymphoid cells will be a fruitful area for further research. Indeed recently, using hybrids between cALL lymphoblasts and mouse myeloma, F. Katz and others (unpublished observations) have succeeded in mapping the gene(s) responsible for OKT10 antigen expression to human chromosome 4. In summary, the techniques of somatic cell genetics, used in conjunction with polyclonal and monoclonal antisera, have allowed chromosomal assignment and genetic analysis of the genes controlling expression of several human cell-surface antigens. In particular, monoclonal antibodies to specific antigenic chromosome markers have provided us with powerful tools for hybrid manipulation and characterization.
References 1 Miller, O. J., Allderdice, P. W. and Miller, D. A. (1971) Science 173, 244-245 2 McKusick, V. A. and Ruddle, F. H. (1977) Science 196, 390-405 3 Proceedings of the Sixth International Workshop on Human Gene Mapping, Oslo, 1981. Birth Defects: On'g~nalArticleSeries 18 2; and Cytogenet. Cell C,enet. 32 (1982) 4 Weiss, M. C. and Green, H. (1967) Pr0c. Natl Acad. Sci. USA 58, 1104-1111 5 Ruddle, F. H. (1981)Nature(London)294, 115-120 6 Boone, C., Chen, T. R. and Ruddle, F. H. (1972)Proc. NatlAcad. &i. USA 69, 510-514 7 Nabholz, M., Miggiano, V. and Bodmer, W. F. (1969) Nature (London) 223, 358--363
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8 Kano, K., Baranska, W., Knowles, B. B. et al. (1969)J. Immunol. 103, 1050-1060 9 Puck, T. T., Wuthier, P., Jones, C. and Kao, F. (1971) Proc. NatlAcad. Sci. USA 68, 3102 3106 10 Jones, C., Wuthier, P. and Puck, T. T. (1975) Somatic Cell Go~t. 1, 235-246 11 Jones, C., and Puck, T. T. (1977) Somatic Cell Genet. 3,407-420 12 Kao, F.-T., Jones, C. and Puck, T. T. (1977) Somatic Cell Genet. 3, 421-429 13 Buck, D. W. and Bodmer, W. F. (1974) Birth Defects: OriginalArticle Series 11 3, 87-89 14 Buck, D. W., Bodmer, W. F., Bobrow, M. and Francke, U. (1976) Cytogenet. Cell Genet. 16, 97-98 15 van Someren, H., Westerveld, A., Hagemeijer, A. et al. (1974)Proc. Natl Acad. Sci. USA 71,962-965 16 Aden, D. P. and Knowles, B. B. (1976) Immunogenetics 3, 209-221 17 Seravalli, E., Schwab, R., Penvis, B. and Siniscalco, M. (I978) Cytogenet. Cell Genet. 22, 260-264 18 Goodfellow, P. N., Jones, E. A., van Heyningen, V. et al. (1975). Nature (London) 254, 267-269 19 Cicurel, L. and Croce, C. M. (1977)J. Irnrnunol. t18 1951-1956 20 Revel, M., Bash, D. and Ruddle, F. H. (1976) Nature (London) 260, 139-141 21 Buck, D. W. and Bodmer, W. F. (1976) Birth Defects: OrtginalArticle Series 12 7, 376-377 22 Carlin, C. R. and Knowles, B. B. (1982) Proc. Natl Acad. Sci USA 79, 5026--5030 23 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 24 McMichael, A . J . and Fabre, J. w . (1982) MonodonalAntibodies in Clinical Medicine, Academic Press, New York 25 Williams, A. F. (1977) Contemp. Top. Mol. lmmunoL 6, 83-116
26 Barnstable, C. J., Bodmer, W. F., Brown, G. et al. (1978) Cell 14, 9-20 27 Goodfellow, P. N., Banting, G., Sutherland, R. et al. (1982) Somatic Cell Genet. 8, 197-206 28 Goodfellow, P. N., Banting, G., Waterfield, M. and Ozanne, B. (1981) in Proceedings of the Sixth International Workshop on Human Gene Mapping, Oslo, 1981. Birth Defects: Onginal Article Series 18 2; and Cytogenet. Cell C,enet. 32 (1982) 29 Goodfellow, P. N., Banting, G., Tunnacliffe, A. et al. (1982) Eur. J. ImmunoL 12, 659-663 30 Andrews, P. W., Knowles, B. B. and Goodfellow, P. N. (1981) Somatic Cell Genet. 7, 435-443 31 Brodsky, F. M., Bodmer, W. F. and Parham, P. (1979) Eur. J. ImmunoL 9, 536-545 32 Yan, B., Sheer, D., Hinrns, L. etal. (1982)Ann. Hum. C,enet. 46,337-347 33 GoodfeUow, P., Banting, G., Sheer, D. et al. (1983) Nature (London) 6, 777-787; 302, 346-349 34 Hope, R. M., Goodfellow, P. N., Solomon, E. and Bodmer, W. F. (1982) Cytogenet. Cell Genet. 33, 204-212 35 Jones, C., Moore, E. E. and Lehman, D. W. (1979) Proc. NatlAcad. Sci. USA 76, 6491-6495 36 Levy, R., Dilley, J., Fox, R. I. and Warnke, R. (1979)Proc. NatlAcad. Sci. USA 76, 6552-6556 37 Pearson, P. C. and Bobrow, M. (1970) Nature (London) 226, 959-961 38 Race, R. and Sanger, R. (1975) Blood Groups in Man, 6th Edn, Blackwell Scientific, Oxford 39 Shapiro, C. J., Mohandas, T., Weiss, R. and Romeo, G. (1979) Science 204, 1224 1226 40 Muller, C. R., Migl, B., Traupe, H. and Ropers, H. H. (1980) Hum. Genet. 54, 197-199 41 Quinn, C. A., Goodfellow, P. N., Povey, S. and Walsh, F. S. (1981)Proc. Natl Acad. Sci. USA 78, 5031-5035
Immunoregulatory T-cell defects Raif S. Geha and Fred S. Rosen In normal circumstanceshomeostatic balance is maintained between T-cell help and suppression. In this review Raif Geha and Fred Rosen discuss examples of human diseases which involve excess or deficiency of T-cell help or T-ceU suppression - diseases which have contributedgreatly to an understanding of how T cells regulate the human immune response.
T lymphocytes play a major role in the regulation of the immune response. Helper T cells recognize antigen processed by macrophages in association with determinants encoded in the mouse I-A or human HLA-D regions. They are stimulated to proliferate and to induce directly or via the secretion of soluble mediators other lymphoid cells to differentiate into effector cells. Conversely, suppressor T cells appear to recognize antigen directly, or perhaps in the mouse in the context of determinants encoded in the I-J region, after which they are activated to suppress the immune response. The targets of T-cell regulation include B cells, other T cells, natural killer cells and non-lymphoid cells such as erythroid cell precursors. The availability of monoclonal antibodies to human T-cell surface antigens and of antigen-specific human T-cell clones have led to a better understanding of the relationship between surface markers and T-cell function. It appears that the 20K T3 antigen expressed by all mature T lymphocytes is closely associated with the T-cell receptor for antigen which recognizes antigen X plus a polymorphic M H C gene product, whereas the 62K T4 Divisions of Allergy and Immunology, The Children's Hospital and the Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
antigen and the 76 K T8 antigen serve as associative recognition structures which bind to a monomorphic portion of class-II and class-I M H C gene products, respectively 1. Thus cytotoxic T-cell clones express either T4 or T8 antigen, depending on their restriction for recognition of class-II or class-I antigens 2. However, in bulk populations of T cells, helper function resides mostly in T4-positive cells, whereas suppressor and cytotoxic functions reside predominantly in T8-positive cells 3. For all functional T-cell subsets activation is accompanied by the expression of new surface markers which include Ia/HLA-DR antigens, and receptors for interleukin 2 (IL-2) and for transferrin. Diseases with failure of T-ceU help Transient hypogammaglobulinemia of infancy is characterized by recurrent respiratory infections. These patients have low levels of IgG which often return rather abruptly to normal by age 4 years. Affected infants have normal numbers of B cells and normal intrinsic B-cell function: their peripheral blood lymphocytes (PBL) generate a normal number of IgG- and IgM-plaqueforming cells following activation by the T-cell-independent polyclonal B-cell activator Epstein-Barr virus (EBV). Their T cells, however, are deficient in the capacity to generate help for immunoglobulin (Ig) synthesis by