8
Immunology Today, vol. 4, No. 1, 1983
associative recognition elements. Given the known inducer function of T4 + cells and suppressor function of T8 + cells, one would suspect that the former would be associated with inducer and the latter with suppressor molecules (reviewed in Ref. 23).
3 4 5 6
Testing the model Now that functional human T-cell clones are available in many laboratories and multiple monoclonal antibodies to their surface molecules exist, it should be possible to obtain more precise biochemical data regarding variability in the functionally critical T-cell surface molecules at both protein and DNA levels. Specifically, two-dimensional gel electrophoresis, peptide maps and nudeotide sequences will be critical for the characterization ofT[,, T3, T4 and T8 structures. In addition, examination of the functional effects of the purified glycoproteins themselves and their binding properties will further test the validity of the proposed model.
8
7
9 10 11 12 13 14 15 16 17
Acknowledgements This work was supported by N I H grants CA 19589 and RO1 NS 17182. Stefan Meuer is a recipient of a fellowship from the Deutsche Forschungsgemeinschaft (DFG; Me 693/1-1).
20 21
References
22
1 Benacerraf, B. and McDevitt, H. O. (1972) &'knee 175, 273 2 Zinkerrmgel, R. M. and Doherty, P. C. (1975)J. F_~. Med. 141, 1427
18 19
Schlossman, S. F. (1972) Tramp/ant. Rev. 10, 97 Zinkernagel, R. M. (1978) lmmunol. Rev. 42, 224 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495 Meuer, S. C., Schlossman, S. F. and Reinherz, E. L. (1982) Prv¢. Natl Ac.ad. Sa" U.S.A. 79, 4590 Meuer, S. C., Hussey, R. E., Hodgdon, J. C., Hercend, T., Schlossrnan, S. F. and Reinherz, E. L. (1982) S¢/ence218, 471 Reinherz, E. L., Hussey, R. E., Fitzgerald, K. A., Snow, P., Terhorst, C. and Scl'dossman, S. F. (1981) Nature(London) 294, 168 Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., Levine, H. and Scl'dossman, S. F. (1982) Cell 30, 735 Krensky, A. M., Reiss, C. S., Mier, J. W., Strominger, J. L. and Burakoff, S.J. (1982) Pr0~. NatlAcad. Sci_ U.S.A. 79, 2365 Biddison, W. E., Rao, P. E., Talle, M. A., Goldstein, G. and Shaw, S. (1982)J. Exp. Meg. 156, 1065 Ball, E. J. and Stasny, P. (1982) lmmunogenetics 16, 157 Chang, T. W., Kung, P. C., Gingras, S. P. and Goldstein, G. (1981) Proc. Natl Acad. Sci. U.S.A. 78, 1805 Reinherz, E. L., Hussey, R. E. and Schlossman, S. F. (1980) Eur. J. lmmunol. 10, 758 Bums, G. F., Boyd, A. W. and Beverley, P. C. (1982)J. lmmunol. 129, 1451 van Wauwe, F. P., DeMay, J. R. and Goossener, J. G. (1980)ft. ImmunoL 124, 2708 Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H. and Schlossman, S. F. (1980)/~'oc. NatlAtad. &'i. U.S.A. 77, 1588 Umiel, T., Daley, J. F., Bhan, A. K., Levey, R. H., Schlossman, S. F. and Reinherz, E. L. (1982)J. Immunol. 129, 1054 Wallace, L. E., Rickinmn, A. B., Rowe, M. and Epstein, M. A. (1982) Nature (London) 297, 413 Pastan, J. H. and Willingham, M. C. (1981) ,.Terence214, 504 Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Schlossman, S. F. and Reinherz, E. L.J. F__~. Med. (in press) Malisnen, B., Rebai, N., Liabeuf, A. and Mawas, C. Eur. J. Immunol. (in
press)
23 Reinherz, E. L. and Schlossman, S. F. (1980) Cell 19, 821
D N A strand breaks and differentiation Cell differentiation is the process by which genetic information is selectively expressed to produce cells with various morphologies and functions. This process is fundamentally important, not least in the immune system where co-ordinated control of progression along several branched developmental pathways is vital for correct functioning. It has been known for some time that different types of differentiated cells contain different populations of messenger R N A which are translated to produce the proteins peculiar to each type. However, many of the processes involved in the integrated changes necessary for selectively expressing genetic information during differentiation are still obscure. Recent work I has produced some intriguing results on early nuclear changes following mitogen stimulation of human peripheral blood lymphocytes, which may be relevant to a generalized mechanism for controlling the expression of genes during differentiation 1-3. Analysis of the rate of sedimentation of 'nucleoids '+ (supercoiled DNA) showed that D N A from mitogenstimulated lymphocytes contained fewer strand breaks than D N A from resting cells a. This implies that (a) circulating quiescent lymphocytes contain DN~k strand breaks; and (b) these breaks are rejoined after stimulation. This D N A ligation occurs rapidly [ 1-8h after addition ofphyto© ElsevierBiomedicalFrel119830167-4919153~$1.{KI
haemaglutinin (PHA)] and represents one of the earliest nuclear responses to stimulation to be detected (the reported acetylation and phosphorylation of nuclear proteins may be attributable to changes in the permeability of the plasma membrane to the added radiochemicalsS). The enzyme ADP-ribosyl transferase (ADPRT) was implicated in the DNA rejoining process by showing that competitive inhibitors of the enzyme slowed the ligation and prevented lymphocyte activation 1. A D P R T is known to regulate D N A repair by enhancing the activity of D N A ligase II (Ref. 6). Inhibitors of A D P R T were only effective early during lymphocyte activation; they did not prevent subsequent cell proliferation if added later'. This suggests that similar chemicals may be of eventual clinical use in suppressing newly initiated immune responses whilst allowing established ones to continue (cf. use of cyclosporin A in transplantation managemend). The inhibitors presently available affect differentiation in general ~a and so may not be specific enough for use as drugs. The presence of breaks in the D N A strands of resting lymphocytes may explain several previously puzzling observations. One example is the extreme susceptibility of circulating lymphocytes to radiation damage, which decreases after P H A stimulation s. The authors of a recent report on the blocking of a mitogenic response by U V ir-
Immunology Today, vol. 4, No. 1, 1983
radiation only if administered early during activation9 would probably have been less puzzled if they had been aware of the DNA breaks. These breaks may also be relevant when considering cases of generalized defects in DNA repair which are clinically associated with impaired immune function - e.g. ataxia telangiectasia and Fanconi' s anaemia 1°. The rejoining of DNA breaks during lymphocyte activation appears to be part of a general differentiation mechanism because similar observations have been made with chick-embryo myoblasts2's and the protozoan parasite T~ypanosoma cruzi (G. T. Williams, unpublished observations). Single-strand breaks appear in DNA during the transition from myoblasts to myotubes~.S; this suggests that circulating quiescent lymphocytes are held part-way through a differentiation process, awaiting a stimulatory signal before rejoining their DNA breaks and continuing their differentiation. Farzaneh et aL 2's first proposed that breaking and rejoining of DNA breaks, regulated by ADPRT, may be a ubiquitous mechanism for coherently altering gene expression during differentiation. The transient breaks could be involved in changing the degree of supercoiling in specific parts of the DNA, allowing new genes to be transcribed *','2. Alternatively, they may be indicative of a rearrangement of genetic material. Such rearrangements in eukaryotes have a long history beginning with McClintock's work on maize ~3. Since then, they have been detected in yeast, SchizophyUum commune, Trypanosoma brucei, Drosphila and, of course, the selection of
9
antibody specificity and immunoglobulin class switching in mammals. This latest work l-s, together with some recent evidence of variation in DNA primary structure between different tissues of an individual '4, suggests that such rearrangement of genetic material may be much more widespread than previously imagined. ALANJOHNSTONE GWYN WILLIAMS Departmentof Immunology, St George'sHospitalMedicalSchool, London SW17 ORE, U.K.
References 1 Johnstone, A. P. and Williams, G. T. (1982) Nature(London) 300, 368-370 2 Farzaneh, F., Shall, S. and Zalin, R. (1980) in Novel ADP-Ribosylations of Regulato~ Enzymes ($mulson, M. and Sugimura, T., eds), pp. 217-225, Elsevier, Amsterdam 3 Farzaneh, F., Zalin, R., BriU, D. and Shall, S. (1982) Nature(London) 300, 362-366 4 Cook, P. R. and Brazen, I. A. (1976)J. Cell Sci. 22, 287-302 5 Ling, N. R. and Kay, J. E. (1975) Lymphocytestimulation, 2nd edn, NorthHolland, Amsterdam 6 Creissen, D. and Shall, 8. (1982) Nature(London) 296, 271-272 7 Green, C. J. (1982) Immunol. Today 3, 121-123 8 Schrek, R. and Stefani, S. (1964)J. Nail C~mctrlast. 32, 507-521 9 Castellanos, G., Owens, T., Rudd, C., Bladon, T., Setterfield, G. and Kaplan, J. G. (1982) Can. J. Biochon. 60, 854-860 10 Cleaver, J. E. (1978) in Birth Deficts (Litt/e£eld, J. w. and de Groucy, J., eds), pp. 85-100, Excerpta Medica, Amsterdam 11 Akrigg, A. and Cook, P. R. (1980) Nu¢l. Ao~. Rts. 8, 845-854 12 MacDermott, A. (1982) New 3¢/. 95, 228-231 13 McClintock, B. (1956) ColdSt~q'ngHarborSymp. Quant. Biol. 21,197-216 14 Calabretta, B., Robberson, D. C., Barrera-Saldana, H. A., Lambrou, T. P. and Sannders, G. F. (1982) Nature (London) 296, 219-225
Immunology Today, vol. 4, No. 1, 1983
associative recognition elements. Given the known inducer function of T4 + cells and suppressor function of T8 + cells, one would suspect that the former would be associated with inducer and the latter with suppressor molecules (reviewed in Ref. 23).
3 4 5 6
Testing the model Now that functional human T-cell clones are available in many laboratories and multiple monoclonal antibodies to their surface molecules exist, it should be possible to obtain more precise biochemical data regarding variability in the functionally critical T-cell surface molecules at both protein and DNA levels. Specifically, two-dimensional gel electrophoresis, peptide maps and nudeotide sequences will be critical for the characterization ofT[,, T3, T4 and T8 structures. In addition, examination of the functional effects of the purified glycoproteins themselves and their binding properties will further test the validity of the proposed model.
8
7
9 10 11 12 13 14 15 16 17
Acknowledgements This work was supported by N I H grants CA 19589 and RO1 NS 17182. Stefan Meuer is a recipient of a fellowship from the Deutsche Forschungsgemeinschaft (DFG; Me 693/1-1).
20 21
References
22
1 Benacerraf, B. and McDevitt, H. O. (1972) &'knee 175, 273 2 Zinkerrmgel, R. M. and Doherty, P. C. (1975)J. F_~. Med. 141, 1427
18 19
Schlossman, S. F. (1972) Tramp/ant. Rev. 10, 97 Zinkernagel, R. M. (1978) lmmunol. Rev. 42, 224 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495 Meuer, S. C., Schlossman, S. F. and Reinherz, E. L. (1982) Prv¢. Natl Ac.ad. Sa" U.S.A. 79, 4590 Meuer, S. C., Hussey, R. E., Hodgdon, J. C., Hercend, T., Schlossrnan, S. F. and Reinherz, E. L. (1982) S¢/ence218, 471 Reinherz, E. L., Hussey, R. E., Fitzgerald, K. A., Snow, P., Terhorst, C. and Scl'dossman, S. F. (1981) Nature(London) 294, 168 Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., Levine, H. and Scl'dossman, S. F. (1982) Cell 30, 735 Krensky, A. M., Reiss, C. S., Mier, J. W., Strominger, J. L. and Burakoff, S.J. (1982) Pr0~. NatlAcad. Sci_ U.S.A. 79, 2365 Biddison, W. E., Rao, P. E., Talle, M. A., Goldstein, G. and Shaw, S. (1982)J. Exp. Meg. 156, 1065 Ball, E. J. and Stasny, P. (1982) lmmunogenetics 16, 157 Chang, T. W., Kung, P. C., Gingras, S. P. and Goldstein, G. (1981) Proc. Natl Acad. Sci. U.S.A. 78, 1805 Reinherz, E. L., Hussey, R. E. and Schlossman, S. F. (1980) Eur. J. lmmunol. 10, 758 Bums, G. F., Boyd, A. W. and Beverley, P. C. (1982)J. lmmunol. 129, 1451 van Wauwe, F. P., DeMay, J. R. and Goossener, J. G. (1980)ft. ImmunoL 124, 2708 Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H. and Schlossman, S. F. (1980)/~'oc. NatlAtad. &'i. U.S.A. 77, 1588 Umiel, T., Daley, J. F., Bhan, A. K., Levey, R. H., Schlossman, S. F. and Reinherz, E. L. (1982)J. Immunol. 129, 1054 Wallace, L. E., Rickinmn, A. B., Rowe, M. and Epstein, M. A. (1982) Nature (London) 297, 413 Pastan, J. H. and Willingham, M. C. (1981) ,.Terence214, 504 Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Schlossman, S. F. and Reinherz, E. L.J. F__~. Med. (in press) Malisnen, B., Rebai, N., Liabeuf, A. and Mawas, C. Eur. J. Immunol. (in
press)
23 Reinherz, E. L. and Schlossman, S. F. (1980) Cell 19, 821
D N A strand breaks and differentiation Cell differentiation is the process by which genetic information is selectively expressed to produce cells with various morphologies and functions. This process is fundamentally important, not least in the immune system where co-ordinated control of progression along several branched developmental pathways is vital for correct functioning. It has been known for some time that different types of differentiated cells contain different populations of messenger R N A which are translated to produce the proteins peculiar to each type. However, many of the processes involved in the integrated changes necessary for selectively expressing genetic information during differentiation are still obscure. Recent work I has produced some intriguing results on early nuclear changes following mitogen stimulation of human peripheral blood lymphocytes, which may be relevant to a generalized mechanism for controlling the expression of genes during differentiation 1-3. Analysis of the rate of sedimentation of 'nucleoids '+ (supercoiled DNA) showed that D N A from mitogenstimulated lymphocytes contained fewer strand breaks than D N A from resting cells a. This implies that (a) circulating quiescent lymphocytes contain DN~k strand breaks; and (b) these breaks are rejoined after stimulation. This D N A ligation occurs rapidly [ 1-8h after addition ofphyto© ElsevierBiomedicalFrel119830167-4919153~$1.{KI
haemaglutinin (PHA)] and represents one of the earliest nuclear responses to stimulation to be detected (the reported acetylation and phosphorylation of nuclear proteins may be attributable to changes in the permeability of the plasma membrane to the added radiochemicalsS). The enzyme ADP-ribosyl transferase (ADPRT) was implicated in the DNA rejoining process by showing that competitive inhibitors of the enzyme slowed the ligation and prevented lymphocyte activation 1. A D P R T is known to regulate D N A repair by enhancing the activity of D N A ligase II (Ref. 6). Inhibitors of A D P R T were only effective early during lymphocyte activation; they did not prevent subsequent cell proliferation if added later'. This suggests that similar chemicals may be of eventual clinical use in suppressing newly initiated immune responses whilst allowing established ones to continue (cf. use of cyclosporin A in transplantation managemend). The inhibitors presently available affect differentiation in general ~a and so may not be specific enough for use as drugs. The presence of breaks in the D N A strands of resting lymphocytes may explain several previously puzzling observations. One example is the extreme susceptibility of circulating lymphocytes to radiation damage, which decreases after P H A stimulation s. The authors of a recent report on the blocking of a mitogenic response by U V ir-
Immunology Today, vol. 4, No. 1, 1983
radiation only if administered early during activation9 would probably have been less puzzled if they had been aware of the DNA breaks. These breaks may also be relevant when considering cases of generalized defects in DNA repair which are clinically associated with impaired immune function - e.g. ataxia telangiectasia and Fanconi' s anaemia 1°. The rejoining of DNA breaks during lymphocyte activation appears to be part of a general differentiation mechanism because similar observations have been made with chick-embryo myoblasts2's and the protozoan parasite T~ypanosoma cruzi (G. T. Williams, unpublished observations). Single-strand breaks appear in DNA during the transition from myoblasts to myotubes~.S; this suggests that circulating quiescent lymphocytes are held part-way through a differentiation process, awaiting a stimulatory signal before rejoining their DNA breaks and continuing their differentiation. Farzaneh et aL 2's first proposed that breaking and rejoining of DNA breaks, regulated by ADPRT, may be a ubiquitous mechanism for coherently altering gene expression during differentiation. The transient breaks could be involved in changing the degree of supercoiling in specific parts of the DNA, allowing new genes to be transcribed *','2. Alternatively, they may be indicative of a rearrangement of genetic material. Such rearrangements in eukaryotes have a long history beginning with McClintock's work on maize ~3. Since then, they have been detected in yeast, SchizophyUum commune, Trypanosoma brucei, Drosphila and, of course, the selection of
9
antibody specificity and immunoglobulin class switching in mammals. This latest work l-s, together with some recent evidence of variation in DNA primary structure between different tissues of an individual '4, suggests that such rearrangement of genetic material may be much more widespread than previously imagined. ALANJOHNSTONE GWYN WILLIAMS Departmentof Immunology, St George'sHospitalMedicalSchool, London SW17 ORE, U.K.
References 1 Johnstone, A. P. and Williams, G. T. (1982) Nature(London) 300, 368-370 2 Farzaneh, F., Shall, S. and Zalin, R. (1980) in Novel ADP-Ribosylations of Regulato~ Enzymes ($mulson, M. and Sugimura, T., eds), pp. 217-225, Elsevier, Amsterdam 3 Farzaneh, F., Zalin, R., BriU, D. and Shall, S. (1982) Nature(London) 300, 362-366 4 Cook, P. R. and Brazen, I. A. (1976)J. Cell Sci. 22, 287-302 5 Ling, N. R. and Kay, J. E. (1975) Lymphocytestimulation, 2nd edn, NorthHolland, Amsterdam 6 Creissen, D. and Shall, 8. (1982) Nature(London) 296, 271-272 7 Green, C. J. (1982) Immunol. Today 3, 121-123 8 Schrek, R. and Stefani, S. (1964)J. Nail C~mctrlast. 32, 507-521 9 Castellanos, G., Owens, T., Rudd, C., Bladon, T., Setterfield, G. and Kaplan, J. G. (1982) Can. J. Biochon. 60, 854-860 10 Cleaver, J. E. (1978) in Birth Deficts (Litt/e£eld, J. w. and de Groucy, J., eds), pp. 85-100, Excerpta Medica, Amsterdam 11 Akrigg, A. and Cook, P. R. (1980) Nu¢l. Ao~. Rts. 8, 845-854 12 MacDermott, A. (1982) New 3¢/. 95, 228-231 13 McClintock, B. (1956) ColdSt~q'ngHarborSymp. Quant. Biol. 21,197-216 14 Calabretta, B., Robberson, D. C., Barrera-Saldana, H. A., Lambrou, T. P. and Sannders, G. F. (1982) Nature (London) 296, 219-225
