Immunology Today, vol. 8, No. I0, 1987 .......
A second level of regulation may be provided by the thymus, either by providing a microenvironmer~t for the accelerated expansion of cells bearing the el~ heterodimer, or by negatively regulating ~/~ cells. Since there is no increase in the frequency of CD3 +, -/a cells in congenitally athymic mice, the latter possibility seems unlikely. Rather, 0=6 cells may receive stimuli (in the form of contact with stromal elements, exposure to lymphokines, etc.) which result in accelerated proliferation of selected clon¢3 dnd negative selection of self-reactive clones with the net result of generation of the antigenspecific, MHC-restricted repertoire. The model that we h3ve described allows for the descendants of a single i;rc~cursor cell to evolve along either o; two mutually exclusive pathways of differentiation, gi~!ng rise, alternatively, to cells bearing o¢1~or Ira receptors in association with the CD3 complex. A precursor--product relationship is excluded as no cells express 71~ heterodimers or both o=l~and -~a heterodimers. Indeed, an obligate requirement for expression of two independently generated receptor complexes, each of which the result of random gene rearrangements, during the life history of a single clonal lineage, is both statistically unlikely and biologically unfavorable. On balance, current data suggest that two independent lineages with different receptors arise from a common precursor. Note added in proof. The Cx genedescribedby Chienet al.23 has been
confirmedto encodethe antigen receptor~ chain by immunoprecipitation using antisera raised to synthetic peptides corresponding to sequencesof the murine (J.P.Allison and M. Bonyhadi, unpublished) and human(A. Weiss. L Lanier,E. Lohand Y-H. Chien, unpublished)Cx genes.The propertiesof the TCR~ geneare consistentwith our model. We thank Dr Mike Brenner and Dr Drew Pardoll for providing preprints of relevantdata. Dr PardoUand his colleagueshavedescribed a similarmodel.
References
1 Kronenberg,M., Siu,G., Hood, L.E.and Shastri,N. (1986)Annu. Rev.
Immuno:~ 4, 529-591
2 Allison.J.P.and Lanier,L.L.(1987)Annu. Rev. Immunol. 5, 503-540 3 Saito. I-'. Kranz,DM., Takagaki,Y., Hayday,A.C. etal. (1984)Nature 309, 757-762 4 Hayday,A.C., Saito, H., Gillies,S.D.etal. (1985)Cell40, 259-269 5 Kranz, DM., Saito, H., Heller,M. etal. (1985) Nature313, 752-755 6 Iwamoto A., Rupp,F., Ohashi,P.S.etal. (1986)J. Exp. Med. 163, 1203-1212 7 LeFranc, M-P.and Rabbitts,T.H. (1985)Nature316, 464-466 II Raulet,D.H., Garman,R.D.,Saito,H. and Tonegawa,S.(1985) Nature 314, 103-107 g Snodgrass,H.R.,Dembic,Z., Steinmetz,M and von Boehmer,H. (1985)Nature 315, 232-233 10 Born, W., Rathbun,G., Tucker,P., Marrack, P. and Kappler,J. (1986) Science 234, 479-482 11 Pernis,B. and Axel, R.(1985)Ce1141,13-16 12 Garman,R.D.,Doherty,P.J.and Raulet,D.H.(1986)Cell45, 733-742 13 Reilly,E.B.,Kranz, D.M, Tonegawa,S. and Eisen,H.N.(1986) Nature 321,878-880 14 Lanier,L.L.and Weiss,A. (1986)Nature 324, 268-270 |$ Lew,A.M, Pardoll,D.M, Maloy,W.L. etal. (1986)Science 234, 1401-1405 16 Kuziel,W., Takashima,A., Bonyhadi,M. etal. (1987)Nature 328, 263-266 17 Borst,J., van de Griend, R.J.,van Oostveen,J.W. etal. (1987)Nature 325, 683-688 18 Brenner,MB., McLean,J., Scheft, H. et ~1.(1987)Nature325, 689-694 19 Lanier,L.L.,Federspiel,N.A., Ruitenberg,J.J.et al. (1987)J. Exp. Meal. (in press) 20 Brenner, MB., McLean,J., Dialynas,D.P.etal. (1986)Nature 322, 145-149 21 Bank,I., DePinho,R.A.,Brenner,M.B. etal. (1986)Nature322, 179-181 22 Weiss,A., Newton, M. and Crommie,D. (1986)Proc. NatlAcad. Sci. USA 83, 6998-7002 23 Chien,Y-H., Iwashima,M., Kaplan,K.B., Elliott,J.F.and Davis,M.M. (1987) Nature 327, 677-680 24 Littman, D.R.,Newton, M., ~-rommie,D. eta/. (1987)Nature326, 85-88 2S Pardoll,D., Fowlkes,B.J.,Bluestone,J.A. eta/. (1987)Nature326, 79-81
The treatmentof adenosinedeaminase deficiency A'!eviation of the desperate prognosis for infants with severe combined immunodeficiency (SClD) has been a major challenge for clinical medicine. It has encouraged innovative approaches to therapy which have had consequences far beyond the treatment of this very rare disorder - bone marrow transplantation with an HLAcompatible sibling as donor was first used two decades ago as treatment for an infant with SClD. Recently, several reports describing equally innovative approaches to treatment have appeared with the potential for even greater impact on clinical medicine. Although the biochemical basis of most cases of SCID is unknown, about a quarter are caused by deficiency of adenosine deaminase (ADA). ADA is a key enzyme of purine metabolism that catalyses the deamination of adenosine and deoxyadenosine. Although the precise mechanism by which ADA deficiency leads to iraCellular Immunology Section, Metabolism Branch, National Cancer 296
Institute, National Institutes of Health, Bethesda, MD 20892, USA
R. MichaelBlaese,DonaldB. Kohnand RobertC. Moen munodeficiency remains controversial, there is fairly general agreement that the accumulation of deoxyadenosine and its phosphorylated derivatives (particularly dATP) in the lymphoid tissues is significant in inhibiting lymphocyte development and function. Although ADA- SCID is curable by transplantation with HLA-matched bone marrow, a suitable donor is available for only a minority of patients and this has led to the search for other effective forms of treatment. As a result of deficiency of ADA enzymatic activity, patients have elevated concentrations of deoxyadenosine in their cells, plasma and urine as well as extraordinarily high levels of dATP in their lymphocytes and erythrocytes. Since the intracellular metabolites are effectively in equilibrium with the plasma, removal of plasma deoxyadenosine results in corresponding decreases in the (~ 1987,ElseviePubl r ications,Cambridge0167- 4919/87,'$02.00
Immunology Today, vol. 8, No. 10, 1987
intracellular metabolite concentrations. Normal erythrocytes contain ADA, and transfusions of these normal cells (and thus this enzyme) to ADA-deficient children partially correct the metabolic abnormalities seen in their plasma, urine and blood cells1. Several ADA-deficient patients have been treated with periodic transfusions of irradiated normal erythrocytes and some, but not all, have detectably altered immune ~unctiGi,--s. Increases in absolute lymphocyte count, T-c~!l numbers and proliferative responses to mitogens have occurred in about half the reported cases. Antigen-specific responses, however, have not usually been seen, and in several patients the initial mitogen response and increase in lymphocyte count have not been maintained. At best, the effect of erythrocyte transfusions has been modest. In an attempt to increase the amount of ADA administered and to avoid the risks associated with prolonged transfusion therapy, Hershfield and colleagues 6 have recently reported a novel approach to enzyme replacement therapy. They treated two patients suffering from ADA- SClD with bovine intestinal ADA conjugated to polyethylene glycol (PEG-ADA). PEG conjugation inactivates the antigenicity of various protein antigens7 and is effective in diminishing the immunogenicity of bovine ADA in mice. Importantly, PEG conjugation prolonged the half-life of bovine ADA in plasma from a few minutes to 24 hours8. With weekly intramuscular injections, total blood ADA concentrations two to three times higher than normal were achieved along with almost complete reversal of the principal biochemical consequences of •ADA deficiency. Lymphocyte numbers and mitogen responses improved and the patients gained weight and were free from infection. These early results are very encouraging and suggest that PEG-conjugated enzymes may be useful in a number of metabolic disorders in addition to ADA deficiency and the similar immunodeficiency disease caused by purine nucleoside phosphorylase (PNP) deficiency. Before final judgment, a Ionaer follnw un will hp rpnHirprl tn d~t~rmin~ if th~ resl~onse- to- I~E~G-AD~.- is-~n~ai'ntained-ancl"'wl~ether antigen-specific immune responsiveness develops in these patients. In a different approach to enzyme replacement in ADA deficiency, Palmer and associates9 have recently presented in-vitro evidence that skin fibroblasts from ADASCID patients, when transduced with a retroviral vector containing the gene for human ADA, will produce up to 12 times the amount of enzyme produced by normal fibroblasts. These gene-treated fibroblasts could rapidly metabolize exogenous adenosine and deoxyadenosine in tissue culture. The authors speculate that sufficient autologous gene-treated fibroblasts could be grown and transplanted back into the patients to influence the total body deoxyadenosine pool and consequently correct the immunodeficiency. However, there are no reports that in intact animals nontransformed gene-treated fibroblasts can produce sufficient enzyme for long enough to have a beneficial effect, and many potential barriers exist to the success of this approach for ADA deficiency. Nevertheless, the use of autologous fibroblasts for gene therapy, perhaps in combination with cultured epidermal keratinocytes, could be uniquely valuable for disorders where such relatively high levels of enzyme production are not required. A patch of genetically altered skin producing a polypeptide hormone could potentially be used to treat endocrine defects. The concentration of the gene prod-
uct delivered could be coarsely controlled by varying the size of the skin patch. Further, the addition of regulatory sequences responsive to light (e.g. from melanocytes or plants) to such ~. hormone construct might permit fine regulation of thP production of, for example, insulin by an externally ap!Jiied llgi~t source. The ultimate goal of gene therapy is the insertion of a functional aene directly into defective cells and their precursors in a patient, thereby curing the disease at the most fundamental level. ADA deficiency would be particularly well suited for this type of gene therapy lo. Since ADA-deficiency SCID can be cured by Hl.~,-matched bone marrow transplantation, the use of a gene-treated autologous bone marrow transplant should be sufficient to correct all clinically significant features of the disease. The fact that Hershfield's patients showed positive responses to increased enzyme levels suggests that lymphoid precursor cells are not irreversibly inactivated as a consequence of ADA deficiency and therefore should be available for gene correction. All the metabolic abnormalities of T-cell lines from ADA- SCID patients can be efficiently corrected by retroviral-mediated transfer of the ADA gene in vitro 11. Very recently, Kantoff has reported initial studies of a retroviral ADA gene delivery system in autologous bone marrow transplantation in a primate 12. Lethally irradiated donor monkeys were infused with autologous bone marrow which had been treated with a retroviral vector containing genes for both human ADA and bacterial neomycin resi~t,~nce. Low levels of expression of both human ADA and neomycin resistance genes wer~ detected in four out of five reconstituted animals. However, expression of the transferred genes persisted for only a few months suggesting that a relatively committed stem cell population was being infected with the vector. As we learn more about the factors critical for efficient gene transfer and expression, as well as those important for targeting the retrovirus to the most appropriate precursor cell population, therapeutic tool with benefits ranging far beyond the treatment of this rare immunodeficiency disease. References
1 Polmar,S.H., Stern, R.C., Schwartz,A.L., etal. (1976)New Engl. J. Med. 295, 1337-1343
2 Rubinstein,A., Hirshhorn,R., Sicklick, M. and Murphy, R.A. (1979) New Engl. J. Med. 300, 387-392 3 Ziegler,J.B., Lee,C.H., Van der Weyden, M.B., Bagnara,A.S. and Beveridge,J. (1980)Arch. Dis. Childhood 55, 452-457 4 Hutton, JJ., Wiginton, D.A., Coleman, M.S., Fuller,S.A., Limouze, S. and Lampkin,B.C. (1981)J. Clin. Invest. 68, 413-421 5 Davies,E.G., Levinsky,R.J.,Webster, D.R., Simmonds,H.A. and Perrett, D. (1982) Clin. Exp. Irnrnunol. 50, 303-310 6 Hershfield,M.S., Buckley,R.H.Greenberg, M.L. etal. (1987) New Engl. J. Med. 3 ! 6, 589-596 7 Abuchowski,A., van Es,T., Palczuk,N.C. and Davis,F.F. (1977) J. Biol. Chem. 252, 3578-3581 8 Davis,S., Abuchowski,A., Park,Y.K. and Davis,F.F.(1981) Clin. Exp. Imrnunol. 46, 649-652 g Palmer,T.D., Hock, R.A., Osborne,W.R.A. and Miller, A.D. (1987) Proc Natl Acad. Sci. USA 84, 105~-1059 10 Anderson,W.F. (1984)Science226, 401-409 11 Kantoff, P.W., Kohn, D.B., Mitsuya, H. etal. (1986)Proc. Natl Acad. Sci. USA 83, 6563-6567 12 Kantoff, P.W., Gillio, A.P., McLachlin,J.R.etal. (1987) J. Exp. Med. 166, 219-234
297
A second level of regulation may be provided by the thymus, either by providing a microenvironmer~t for the accelerated expansion of cells bearing the el~ heterodimer, or by negatively regulating ~/~ cells. Since there is no increase in the frequency of CD3 +, -/a cells in congenitally athymic mice, the latter possibility seems unlikely. Rather, 0=6 cells may receive stimuli (in the form of contact with stromal elements, exposure to lymphokines, etc.) which result in accelerated proliferation of selected clon¢3 dnd negative selection of self-reactive clones with the net result of generation of the antigenspecific, MHC-restricted repertoire. The model that we h3ve described allows for the descendants of a single i;rc~cursor cell to evolve along either o; two mutually exclusive pathways of differentiation, gi~!ng rise, alternatively, to cells bearing o¢1~or Ira receptors in association with the CD3 complex. A precursor--product relationship is excluded as no cells express 71~ heterodimers or both o=l~and -~a heterodimers. Indeed, an obligate requirement for expression of two independently generated receptor complexes, each of which the result of random gene rearrangements, during the life history of a single clonal lineage, is both statistically unlikely and biologically unfavorable. On balance, current data suggest that two independent lineages with different receptors arise from a common precursor. Note added in proof. The Cx genedescribedby Chienet al.23 has been
confirmedto encodethe antigen receptor~ chain by immunoprecipitation using antisera raised to synthetic peptides corresponding to sequencesof the murine (J.P.Allison and M. Bonyhadi, unpublished) and human(A. Weiss. L Lanier,E. Lohand Y-H. Chien, unpublished)Cx genes.The propertiesof the TCR~ geneare consistentwith our model. We thank Dr Mike Brenner and Dr Drew Pardoll for providing preprints of relevantdata. Dr PardoUand his colleagueshavedescribed a similarmodel.
References
1 Kronenberg,M., Siu,G., Hood, L.E.and Shastri,N. (1986)Annu. Rev.
Immuno:~ 4, 529-591
2 Allison.J.P.and Lanier,L.L.(1987)Annu. Rev. Immunol. 5, 503-540 3 Saito. I-'. Kranz,DM., Takagaki,Y., Hayday,A.C. etal. (1984)Nature 309, 757-762 4 Hayday,A.C., Saito, H., Gillies,S.D.etal. (1985)Cell40, 259-269 5 Kranz, DM., Saito, H., Heller,M. etal. (1985) Nature313, 752-755 6 Iwamoto A., Rupp,F., Ohashi,P.S.etal. (1986)J. Exp. Med. 163, 1203-1212 7 LeFranc, M-P.and Rabbitts,T.H. (1985)Nature316, 464-466 II Raulet,D.H., Garman,R.D.,Saito,H. and Tonegawa,S.(1985) Nature 314, 103-107 g Snodgrass,H.R.,Dembic,Z., Steinmetz,M and von Boehmer,H. (1985)Nature 315, 232-233 10 Born, W., Rathbun,G., Tucker,P., Marrack, P. and Kappler,J. (1986) Science 234, 479-482 11 Pernis,B. and Axel, R.(1985)Ce1141,13-16 12 Garman,R.D.,Doherty,P.J.and Raulet,D.H.(1986)Cell45, 733-742 13 Reilly,E.B.,Kranz, D.M, Tonegawa,S. and Eisen,H.N.(1986) Nature 321,878-880 14 Lanier,L.L.and Weiss,A. (1986)Nature 324, 268-270 |$ Lew,A.M, Pardoll,D.M, Maloy,W.L. etal. (1986)Science 234, 1401-1405 16 Kuziel,W., Takashima,A., Bonyhadi,M. etal. (1987)Nature 328, 263-266 17 Borst,J., van de Griend, R.J.,van Oostveen,J.W. etal. (1987)Nature 325, 683-688 18 Brenner,MB., McLean,J., Scheft, H. et ~1.(1987)Nature325, 689-694 19 Lanier,L.L.,Federspiel,N.A., Ruitenberg,J.J.et al. (1987)J. Exp. Meal. (in press) 20 Brenner, MB., McLean,J., Dialynas,D.P.etal. (1986)Nature 322, 145-149 21 Bank,I., DePinho,R.A.,Brenner,M.B. etal. (1986)Nature322, 179-181 22 Weiss,A., Newton, M. and Crommie,D. (1986)Proc. NatlAcad. Sci. USA 83, 6998-7002 23 Chien,Y-H., Iwashima,M., Kaplan,K.B., Elliott,J.F.and Davis,M.M. (1987) Nature 327, 677-680 24 Littman, D.R.,Newton, M., ~-rommie,D. eta/. (1987)Nature326, 85-88 2S Pardoll,D., Fowlkes,B.J.,Bluestone,J.A. eta/. (1987)Nature326, 79-81
The treatmentof adenosinedeaminase deficiency A'!eviation of the desperate prognosis for infants with severe combined immunodeficiency (SClD) has been a major challenge for clinical medicine. It has encouraged innovative approaches to therapy which have had consequences far beyond the treatment of this very rare disorder - bone marrow transplantation with an HLAcompatible sibling as donor was first used two decades ago as treatment for an infant with SClD. Recently, several reports describing equally innovative approaches to treatment have appeared with the potential for even greater impact on clinical medicine. Although the biochemical basis of most cases of SCID is unknown, about a quarter are caused by deficiency of adenosine deaminase (ADA). ADA is a key enzyme of purine metabolism that catalyses the deamination of adenosine and deoxyadenosine. Although the precise mechanism by which ADA deficiency leads to iraCellular Immunology Section, Metabolism Branch, National Cancer 296
Institute, National Institutes of Health, Bethesda, MD 20892, USA
R. MichaelBlaese,DonaldB. Kohnand RobertC. Moen munodeficiency remains controversial, there is fairly general agreement that the accumulation of deoxyadenosine and its phosphorylated derivatives (particularly dATP) in the lymphoid tissues is significant in inhibiting lymphocyte development and function. Although ADA- SCID is curable by transplantation with HLA-matched bone marrow, a suitable donor is available for only a minority of patients and this has led to the search for other effective forms of treatment. As a result of deficiency of ADA enzymatic activity, patients have elevated concentrations of deoxyadenosine in their cells, plasma and urine as well as extraordinarily high levels of dATP in their lymphocytes and erythrocytes. Since the intracellular metabolites are effectively in equilibrium with the plasma, removal of plasma deoxyadenosine results in corresponding decreases in the (~ 1987,ElseviePubl r ications,Cambridge0167- 4919/87,'$02.00
Immunology Today, vol. 8, No. 10, 1987
intracellular metabolite concentrations. Normal erythrocytes contain ADA, and transfusions of these normal cells (and thus this enzyme) to ADA-deficient children partially correct the metabolic abnormalities seen in their plasma, urine and blood cells1. Several ADA-deficient patients have been treated with periodic transfusions of irradiated normal erythrocytes and some, but not all, have detectably altered immune ~unctiGi,--s. Increases in absolute lymphocyte count, T-c~!l numbers and proliferative responses to mitogens have occurred in about half the reported cases. Antigen-specific responses, however, have not usually been seen, and in several patients the initial mitogen response and increase in lymphocyte count have not been maintained. At best, the effect of erythrocyte transfusions has been modest. In an attempt to increase the amount of ADA administered and to avoid the risks associated with prolonged transfusion therapy, Hershfield and colleagues 6 have recently reported a novel approach to enzyme replacement therapy. They treated two patients suffering from ADA- SClD with bovine intestinal ADA conjugated to polyethylene glycol (PEG-ADA). PEG conjugation inactivates the antigenicity of various protein antigens7 and is effective in diminishing the immunogenicity of bovine ADA in mice. Importantly, PEG conjugation prolonged the half-life of bovine ADA in plasma from a few minutes to 24 hours8. With weekly intramuscular injections, total blood ADA concentrations two to three times higher than normal were achieved along with almost complete reversal of the principal biochemical consequences of •ADA deficiency. Lymphocyte numbers and mitogen responses improved and the patients gained weight and were free from infection. These early results are very encouraging and suggest that PEG-conjugated enzymes may be useful in a number of metabolic disorders in addition to ADA deficiency and the similar immunodeficiency disease caused by purine nucleoside phosphorylase (PNP) deficiency. Before final judgment, a Ionaer follnw un will hp rpnHirprl tn d~t~rmin~ if th~ resl~onse- to- I~E~G-AD~.- is-~n~ai'ntained-ancl"'wl~ether antigen-specific immune responsiveness develops in these patients. In a different approach to enzyme replacement in ADA deficiency, Palmer and associates9 have recently presented in-vitro evidence that skin fibroblasts from ADASCID patients, when transduced with a retroviral vector containing the gene for human ADA, will produce up to 12 times the amount of enzyme produced by normal fibroblasts. These gene-treated fibroblasts could rapidly metabolize exogenous adenosine and deoxyadenosine in tissue culture. The authors speculate that sufficient autologous gene-treated fibroblasts could be grown and transplanted back into the patients to influence the total body deoxyadenosine pool and consequently correct the immunodeficiency. However, there are no reports that in intact animals nontransformed gene-treated fibroblasts can produce sufficient enzyme for long enough to have a beneficial effect, and many potential barriers exist to the success of this approach for ADA deficiency. Nevertheless, the use of autologous fibroblasts for gene therapy, perhaps in combination with cultured epidermal keratinocytes, could be uniquely valuable for disorders where such relatively high levels of enzyme production are not required. A patch of genetically altered skin producing a polypeptide hormone could potentially be used to treat endocrine defects. The concentration of the gene prod-
uct delivered could be coarsely controlled by varying the size of the skin patch. Further, the addition of regulatory sequences responsive to light (e.g. from melanocytes or plants) to such ~. hormone construct might permit fine regulation of thP production of, for example, insulin by an externally ap!Jiied llgi~t source. The ultimate goal of gene therapy is the insertion of a functional aene directly into defective cells and their precursors in a patient, thereby curing the disease at the most fundamental level. ADA deficiency would be particularly well suited for this type of gene therapy lo. Since ADA-deficiency SCID can be cured by Hl.~,-matched bone marrow transplantation, the use of a gene-treated autologous bone marrow transplant should be sufficient to correct all clinically significant features of the disease. The fact that Hershfield's patients showed positive responses to increased enzyme levels suggests that lymphoid precursor cells are not irreversibly inactivated as a consequence of ADA deficiency and therefore should be available for gene correction. All the metabolic abnormalities of T-cell lines from ADA- SCID patients can be efficiently corrected by retroviral-mediated transfer of the ADA gene in vitro 11. Very recently, Kantoff has reported initial studies of a retroviral ADA gene delivery system in autologous bone marrow transplantation in a primate 12. Lethally irradiated donor monkeys were infused with autologous bone marrow which had been treated with a retroviral vector containing genes for both human ADA and bacterial neomycin resi~t,~nce. Low levels of expression of both human ADA and neomycin resistance genes wer~ detected in four out of five reconstituted animals. However, expression of the transferred genes persisted for only a few months suggesting that a relatively committed stem cell population was being infected with the vector. As we learn more about the factors critical for efficient gene transfer and expression, as well as those important for targeting the retrovirus to the most appropriate precursor cell population, therapeutic tool with benefits ranging far beyond the treatment of this rare immunodeficiency disease. References
1 Polmar,S.H., Stern, R.C., Schwartz,A.L., etal. (1976)New Engl. J. Med. 295, 1337-1343
2 Rubinstein,A., Hirshhorn,R., Sicklick, M. and Murphy, R.A. (1979) New Engl. J. Med. 300, 387-392 3 Ziegler,J.B., Lee,C.H., Van der Weyden, M.B., Bagnara,A.S. and Beveridge,J. (1980)Arch. Dis. Childhood 55, 452-457 4 Hutton, JJ., Wiginton, D.A., Coleman, M.S., Fuller,S.A., Limouze, S. and Lampkin,B.C. (1981)J. Clin. Invest. 68, 413-421 5 Davies,E.G., Levinsky,R.J.,Webster, D.R., Simmonds,H.A. and Perrett, D. (1982) Clin. Exp. Irnrnunol. 50, 303-310 6 Hershfield,M.S., Buckley,R.H.Greenberg, M.L. etal. (1987) New Engl. J. Med. 3 ! 6, 589-596 7 Abuchowski,A., van Es,T., Palczuk,N.C. and Davis,F.F. (1977) J. Biol. Chem. 252, 3578-3581 8 Davis,S., Abuchowski,A., Park,Y.K. and Davis,F.F.(1981) Clin. Exp. Imrnunol. 46, 649-652 g Palmer,T.D., Hock, R.A., Osborne,W.R.A. and Miller, A.D. (1987) Proc Natl Acad. Sci. USA 84, 105~-1059 10 Anderson,W.F. (1984)Science226, 401-409 11 Kantoff, P.W., Kohn, D.B., Mitsuya, H. etal. (1986)Proc. Natl Acad. Sci. USA 83, 6563-6567 12 Kantoff, P.W., Gillio, A.P., McLachlin,J.R.etal. (1987) J. Exp. Med. 166, 219-234
297
