"°
`
`IJETFERS T0 NATURE
`
`Table 3 Expression of Lyt 1.1 and Lyt 2.1 antigens by FACS analysis
`
`Control mice
`VAA-fed mice
`Student’s t-test
`
`‘/9 Lymph node cells expressing
`Lyt 1.1
`Lyt 2.1
`83.6:t1.1
`24.4: 1.4
`90.1 :0.6
`24.2:l:O.7
`P < 0.001
`Non significant
`difference
`
`Transfer of a cloned immunoglobulin
`light-chain gene to
`mutant hybridoma cells
`restores specific antibody production
`
`NATURE VOL. 302 24 MARCH 1983
`
`Immunofluorescently stained CBA lymph node cells were analysed
`using FACS—II (Becton-Dickinson) as described previouslyiom, but
`with the argon ion laser set at 300 mW, 488 nm and the photomultiplier
`tube (EMI 9524A) at 750 V with a fluorescence gain of 8 and a light
`scatter gain of 4. Data for each individual mouse were derived from
`the analysis of 5 X 10" lymph node cells. The fluorescein isothiocyanate-
`coupled rabbit anti-mouse immunoglobulin (obtained from Nordic) was
`selected for its binding to viable cells expressing immunoglobulin at
`the cell surface while producing negligible staining of other cells”. The
`values in the table were calculated after subtracting the count for
`immunoglobulin-positive cells and represent the mean is.e. calculated
`from seven determinations. These results have been repeated in two
`subsequent experiments.
`
`Our data are consistent with recent studies of Loveland and
`McKenzieZ°'“ who have clearly shown that Lyt 1* T cells alone
`(depleted of Lyt 2* T cells) were sufficient to restore immune
`responses generated against H-2 and non-H-2 alloantigens in
`ATXBM (adult thymectomized, irradiated and bone-marrow
`reconstituted) mice. Our data also corroborate their view“ that
`Lyt 1* cells have a central role in allograft responses. In either
`case, vitamin A acetate seems to be unique in that it enhances
`immune responses against an allograft when given by mouth
`and the mechanism of this action will require much further
`attention.
`
`In summary, these findings support the hypothesis” that the
`anti-cancer action of vitamin A acetate "'22 is mediated through
`an immunological process and we have shown that it acts by
`increasing the representation of the Lyt 1+2‘ phenotype in the
`T-cell population.
`We thank Dr G. L. Asherson for the gift of the F(ab’)2 rabbit
`anti-mouse immunoglobulin and Prof. I. F. C. McKenzie for
`the monoclonal antibodies. We thank Drs E. Simpson
`and B. E. Loveland for discussions and Mrs Pat McFarlane
`for preparation of the manuscript. This work was supported by
`the CRC and MRC.
`Received 1 December 1982; accepted 21 January 1983.
`1. Moore, T. in The Vitamins (eds Sehrell, W. H. & Harris, R. S.) 245-266 (Academic, New
`York, 1967).
`2. Goodman, D. S. Fedu Proc. 38. 2501 -2503 (1979).
`3. Bollag, W. in Retinoids. Advances in Basic Research and Therapy (eds Orfanos, C. E. et
`al.) 5-11 (Springer, Berlin, 1981).
`.‘e
`Spam, M. B. in Retinnidr, Adtmnrrr in Basic Rexearrh and Therapy (eds Orfanos. C. E.
`pt al.) 73-76 (Springer. Berlin, 1981).
`5. Pawson. B. A. Ann. N. Y. Acad. Sci. 359. 1-8 (19811.
`6. Dresser, D. W. Nature 217. 527-529 (1968).
`7. Floersheim, G. L. & Bollag, W. Transplantation 15, 564-567 (1972).
`X. Jurin. M. & Tannock, I. F. Immunology 23, 283-287 (1972).
`9. Cohen, B. E. & Cohen, 1. K. J. Immun. 111, 1376-1380 (1973).
`10. Blalock, .1. E. & Gifford, G. E. Proc. mun. Acad. SCI. U.S.A. 74, 5382-5386 (1977).
`11. Dennert, G. & Lotan, R. Eur. J. Immun. 8, 23-29 (1978).
`12. Dennert, G.. Crowley, C., Kouba, J. & Lotan, R. J. natn. Cancer Inst. 62, 89-94 (1979).
`13. Goldfarb, R. H. & Herberman, R. B. J. Imrnun. 126. 2129-2135 (1981).
`14. Abb, 1., Abb, H. & Deinhardt. F. Imrnunopharmacology 4, 303-310 (1982).
`15
`. Sporn, M. B. & Newton, D. L. Fedn Proc. 38, 2528-2534 (1979).
`16
`Bollag, W. & Matter. A. Ann. N. Y. Acad. Sci. 359, 9-23 (1981).
`17
`. Medawar, P. B. & Hunt, R. Immunology 42, 349-353 (1981).
`18
`. Medawar, P. B.. Hunt. R. & Martin, 1. Pros. R. Soc. Land. B206, 265-280 (1979).
`19
`. Malkovsky, M. et al. J. lmmun. 130, 785-790 I1983).
`20
`. Loveland, B. E., Hogarth, P. M., Ceredig, Rh. & McKenzie, l.F.C. J. exp. Med. 153,
`1044-1057 (1981).
`21. Loveland, B. E. & McKenzie. I. F. C. Transplantation 33, 217-221 (1982).
`22. Moon, R. C.. Grubbs, C. J. & Sporn, M. B. Cancer Res. 36, 2626-2630 (1976).
`23. Malkovsky, M., Asherson, C. L., Stockinger, B. & Watkins, M. C. Nature 300, 652-655
`(1982).
`24. Hughes, W. L. et al. Fedn Proc. 23, 6411-648 (1964).
`25. Julius. M. H., Simpson. E. & Herzenbetg, L. A. Eur. J. Immun. 3, 645-649 (1973).
`26. Mage, M. G., McHugh, L. L. & Ruthslein, T. L. J. Immun. Meth. 15, 47-56 (1977).
`27. Mags, M. et al. Eur. J. Immun. 11, 228-235 (1981).
`28. Zembala, M. A., Asherson, G. L., James, B. M. B.. Stein, V. E. & Watkins, M. C. J.
`Irnmun. 129, 1823-1829 (1982).
`29. Gorini, G., Medgyesi, G. A. & Doria, G. J. Immun. 103, 1132-1142 (1969).
`30. Abehsira, 0., Edwards, A & Simpson, E. Eur. J. Immun. 11, 275-281 (1981).
`31. Simon, M. M. et al. Fur. I. Immun. 11. 246-2501191111.
`
`0028-0836/83/120340-03$01.(l(l
`
`Atsuo Ochi, Robert G. Hawley,
`Marc J. Shulman"‘ & Nobumichi Hozumi
`
`Ontario Cancer Institute and Department of Medical Biophysics,
`University of Toronto, 500 Sherbourne Street, Toronto,
`Canada M4X 1K9
`* Rheumatic Disease Unit, Wellesley Hospital, and
`Department of Medical Biophysics, University of Toronto,
`Toronto, Canada M4Y 113
`
`The expression of immunoglobulin (lg) genes is regulated at
`several
`levels. For example, although K-chain production
`requires a DNA rearrangement that juxtaposes variable and
`joining segments‘, this rearrangement is not sufficient for K-
`chain gene expression; that is, some cell types do not permit
`immunoglobulin production“". The mechanisms responsible
`for the regulation of the expression of rearranged immuno-
`globulin genes are poorly understood. The technique of modify-
`ing cloned genes in vitro and transferring the modified genes
`to cells in culture provides a tool for identifying the structural
`features required for gene expression. To analyse immuno-
`globulin genes in this manner, however, it is first necessary to
`use, as recipients, cells that normally permit immunoglobulin
`production. We report here that a cloned K-chain gene is
`expressed in immunoglobulin-producing hybridoma cells. Fur-
`thermore, the product of the transferred K -chain gene is capable
`of restoring specific antibody production to the transformed
`cells.
`
`The hybridoma Sp603 produces IgM(K) specific for the hap-
`ten 2,4,6-trinitrophenyl (TNP)5. The rearranged gene encoding
`the TNP-specific K chain (Kmp) has been cloned . As recipient
`cells, we used the mutant cell line igk-14 which was derived
`from Sp603 and does not produce the K'nvp chain. As shown
`in Fig. 3, the Km,» gene is apparently deleted from the igk—14
`cell line. Because the igk—14 cells still produce the TNP-specific
`pt heavy chain, it would be expected that the expression of the
`Km; gene in these cells would restore the production of TNP-
`specific IgM. To select for cells that have taken up the K-mp
`gene, the cloned Kmp gene, designated TK1, was inserted into
`the plasmid pSV2-neo, which contains a bacterial phos-
`photransferase gene (neo) and confers resistance to the amino-
`glycoside antibiotic G418 (ref. 7). In the present study, we have
`used two recombinant plasmids: in the plasmid pT-TK1, TK1
`was inserted so that the direction of transcription would be in
`the same direction as transcription initiated from the SV40
`early promoter;
`in pR-TK1,
`the orientation of the gene is
`reversed (Fig. 1).
`To transfer the DNA, bacteria harbouring the pT-TK1 and
`pR-TK1 plasmids were converted to protoplasts and fused with
`igk-14, after the method of Schaffnerg. G418-resistant transfor-
`mants of igk-14 were then selected as described in the legend
`to Table 1. The frequency of G418-resistant cells ranged from
`5 X 10"’ to 10-5 per input igk-14 cell, and was comparable for
`the pT-TK1, pR-TK1 and pSV2-neo plasmids. The resistant
`cells were then tested for the ability to make TNP-specific
`plaques. By this criterion about 10% of the G418-resistant
`transformants from pT-TK1 and about 30% of the transfor-
`mants from pR—TK1 made TNP-specific IgM. By contrast, of
`30 G418-resistant transformants from the pSV2-neo vector
`alone, none made TNP-specific plaques.
`Representative transformants were selected, cloned by limit-
`ing dilution and studied further. The production of the K’[‘Np
`chain has been assayed by TNP-specific haemagglutination and
`© 1983 Macmillan Journals Ltd
`
`Merck Ex. 1018, pg 659
`
`Merck Ex. 1018, pg 659
`
`
`
`IJETFERS T0 NATURE
`
`Table 3 Expression of Lyt 1.1 and Lyt 2.1 antigens by FACS analysis
`
`Control mice
`VAA-fed mice
`Student’s t-test
`
`‘/9 Lymph node cells expressing
`Lyt 1.1
`Lyt 2.1
`83.6:t1.1
`24.4: 1.4
`90.1 :0.6
`24.2:l:O.7
`P < 0.001
`Non significant
`difference
`
`Transfer of a cloned immunoglobulin
`light-chain gene to
`mutant hybridoma cells
`restores specific antibody production
`
`NATURE VOL. 302 24 MARCH 1983
`
`Immunofluorescently stained CBA lymph node cells were analysed
`using FACS—II (Becton-Dickinson) as described previouslyiom, but
`with the argon ion laser set at 300 mW, 488 nm and the photomultiplier
`tube (EMI 9524A) at 750 V with a fluorescence gain of 8 and a light
`scatter gain of 4. Data for each individual mouse were derived from
`the analysis of 5 X 10" lymph node cells. The fluorescein isothiocyanate-
`coupled rabbit anti-mouse immunoglobulin (obtained from Nordic) was
`selected for its binding to viable cells expressing immunoglobulin at
`the cell surface while producing negligible staining of other cells”. The
`values in the table were calculated after subtracting the count for
`immunoglobulin-positive cells and represent the mean is.e. calculated
`from seven determinations. These results have been repeated in two
`subsequent experiments.
`
`Our data are consistent with recent studies of Loveland and
`McKenzieZ°'“ who have clearly shown that Lyt 1* T cells alone
`(depleted of Lyt 2* T cells) were sufficient to restore immune
`responses generated against H-2 and non-H-2 alloantigens in
`ATXBM (adult thymectomized, irradiated and bone-marrow
`reconstituted) mice. Our data also corroborate their view“ that
`Lyt 1* cells have a central role in allograft responses. In either
`case, vitamin A acetate seems to be unique in that it enhances
`immune responses against an allograft when given by mouth
`and the mechanism of this action will require much further
`attention.
`
`In summary, these findings support the hypothesis” that the
`anti-cancer action of vitamin A acetate "'22 is mediated through
`an immunological process and we have shown that it acts by
`increasing the representation of the Lyt 1+2‘ phenotype in the
`T-cell population.
`We thank Dr G. L. Asherson for the gift of the F(ab’)2 rabbit
`anti-mouse immunoglobulin and Prof. I. F. C. McKenzie for
`the monoclonal antibodies. We thank Drs E. Simpson
`and B. E. Loveland for discussions and Mrs Pat McFarlane
`for preparation of the manuscript. This work was supported by
`the CRC and MRC.
`Received 1 December 1982; accepted 21 January 1983.
`1. Moore, T. in The Vitamins (eds Sehrell, W. H. & Harris, R. S.) 245-266 (Academic, New
`York, 1967).
`2. Goodman, D. S. Fedu Proc. 38. 2501 -2503 (1979).
`3. Bollag, W. in Retinoids. Advances in Basic Research and Therapy (eds Orfanos, C. E. et
`al.) 5-11 (Springer, Berlin, 1981).
`.‘e
`Spam, M. B. in Retinnidr, Adtmnrrr in Basic Rexearrh and Therapy (eds Orfanos. C. E.
`pt al.) 73-76 (Springer. Berlin, 1981).
`5. Pawson. B. A. Ann. N. Y. Acad. Sci. 359. 1-8 (19811.
`6. Dresser, D. W. Nature 217. 527-529 (1968).
`7. Floersheim, G. L. & Bollag, W. Transplantation 15, 564-567 (1972).
`X. Jurin. M. & Tannock, I. F. Immunology 23, 283-287 (1972).
`9. Cohen, B. E. & Cohen, 1. K. J. Immun. 111, 1376-1380 (1973).
`10. Blalock, .1. E. & Gifford, G. E. Proc. mun. Acad. SCI. U.S.A. 74, 5382-5386 (1977).
`11. Dennert, G. & Lotan, R. Eur. J. Immun. 8, 23-29 (1978).
`12. Dennert, G.. Crowley, C., Kouba, J. & Lotan, R. J. natn. Cancer Inst. 62, 89-94 (1979).
`13. Goldfarb, R. H. & Herberman, R. B. J. Imrnun. 126. 2129-2135 (1981).
`14. Abb, 1., Abb, H. & Deinhardt. F. Imrnunopharmacology 4, 303-310 (1982).
`15
`. Sporn, M. B. & Newton, D. L. Fedn Proc. 38, 2528-2534 (1979).
`16
`Bollag, W. & Matter. A. Ann. N. Y. Acad. Sci. 359, 9-23 (1981).
`17
`. Medawar, P. B. & Hunt, R. Immunology 42, 349-353 (1981).
`18
`. Medawar, P. B.. Hunt. R. & Martin, 1. Pros. R. Soc. Land. B206, 265-280 (1979).
`19
`. Malkovsky, M. et al. J. lmmun. 130, 785-790 I1983).
`20
`. Loveland, B. E., Hogarth, P. M., Ceredig, Rh. & McKenzie, l.F.C. J. exp. Med. 153,
`1044-1057 (1981).
`21. Loveland, B. E. & McKenzie. I. F. C. Transplantation 33, 217-221 (1982).
`22. Moon, R. C.. Grubbs, C. J. & Sporn, M. B. Cancer Res. 36, 2626-2630 (1976).
`23. Malkovsky, M., Asherson, C. L., Stockinger, B. & Watkins, M. C. Nature 300, 652-655
`(1982).
`24. Hughes, W. L. et al. Fedn Proc. 23, 6411-648 (1964).
`25. Julius. M. H., Simpson. E. & Herzenbetg, L. A. Eur. J. Immun. 3, 645-649 (1973).
`26. Mage, M. G., McHugh, L. L. & Ruthslein, T. L. J. Immun. Meth. 15, 47-56 (1977).
`27. Mags, M. et al. Eur. J. Immun. 11, 228-235 (1981).
`28. Zembala, M. A., Asherson, G. L., James, B. M. B.. Stein, V. E. & Watkins, M. C. J.
`Irnmun. 129, 1823-1829 (1982).
`29. Gorini, G., Medgyesi, G. A. & Doria, G. J. Immun. 103, 1132-1142 (1969).
`30. Abehsira, 0., Edwards, A & Simpson, E. Eur. J. Immun. 11, 275-281 (1981).
`31. Simon, M. M. et al. Fur. I. Immun. 11. 246-2501191111.
`
`0028-0836/83/120340-03$01.(l(l
`
`Atsuo Ochi, Robert G. Hawley,
`Marc J. Shulman"‘ & Nobumichi Hozumi
`
`Ontario Cancer Institute and Department of Medical Biophysics,
`University of Toronto, 500 Sherbourne Street, Toronto,
`Canada M4X 1K9
`* Rheumatic Disease Unit, Wellesley Hospital, and
`Department of Medical Biophysics, University of Toronto,
`Toronto, Canada M4Y 113
`
`The expression of immunoglobulin (lg) genes is regulated at
`several
`levels. For example, although K-chain production
`requires a DNA rearrangement that juxtaposes variable and
`joining segments‘, this rearrangement is not sufficient for K-
`chain gene expression; that is, some cell types do not permit
`immunoglobulin production“". The mechanisms responsible
`for the regulation of the expression of rearranged immuno-
`globulin genes are poorly understood. The technique of modify-
`ing cloned genes in vitro and transferring the modified genes
`to cells in culture provides a tool for identifying the structural
`features required for gene expression. To analyse immuno-
`globulin genes in this manner, however, it is first necessary to
`use, as recipients, cells that normally permit immunoglobulin
`production. We report here that a cloned K-chain gene is
`expressed in immunoglobulin-producing hybridoma cells. Fur-
`thermore, the product of the transferred K -chain gene is capable
`of restoring specific antibody production to the transformed
`cells.
`
`The hybridoma Sp603 produces IgM(K) specific for the hap-
`ten 2,4,6-trinitrophenyl (TNP)5. The rearranged gene encoding
`the TNP-specific K chain (Kmp) has been cloned . As recipient
`cells, we used the mutant cell line igk-14 which was derived
`from Sp603 and does not produce the K'nvp chain. As shown
`in Fig. 3, the Km,» gene is apparently deleted from the igk—14
`cell line. Because the igk—14 cells still produce the TNP-specific
`pt heavy chain, it would be expected that the expression of the
`Km; gene in these cells would restore the production of TNP-
`specific IgM. To select for cells that have taken up the K-mp
`gene, the cloned Kmp gene, designated TK1, was inserted into
`the plasmid pSV2-neo, which contains a bacterial phos-
`photransferase gene (neo) and confers resistance to the amino-
`glycoside antibiotic G418 (ref. 7). In the present study, we have
`used two recombinant plasmids: in the plasmid pT-TK1, TK1
`was inserted so that the direction of transcription would be in
`the same direction as transcription initiated from the SV40
`early promoter;
`in pR-TK1,
`the orientation of the gene is
`reversed (Fig. 1).
`To transfer the DNA, bacteria harbouring the pT-TK1 and
`pR-TK1 plasmids were converted to protoplasts and fused with
`igk-14, after the method of Schaffnerg. G418-resistant transfor-
`mants of igk-14 were then selected as described in the legend
`to Table 1. The frequency of G418-resistant cells ranged from
`5 X 10"’ to 10-5 per input igk-14 cell, and was comparable for
`the pT-TK1, pR-TK1 and pSV2-neo plasmids. The resistant
`cells were then tested for the ability to make TNP-specific
`plaques. By this criterion about 10% of the G418-resistant
`transformants from pT-TK1 and about 30% of the transfor-
`mants from pR—TK1 made TNP-specific IgM. By contrast, of
`30 G418-resistant transformants from the pSV2-neo vector
`alone, none made TNP-specific plaques.
`Representative transformants were selected, cloned by limit-
`ing dilution and studied further. The production of the K’[‘Np
`chain has been assayed by TNP-specific haemagglutination and
`© 1983 Macmillan Journals Ltd
`
`Merck Ex. 1018, pg 659
`
`Merck Ex. 1018, pg 659
`
`
`
`
`
`© Nature Publishing Group1983
`
`Merck Ex. 1018, pg 660
`
`
`
`
`
`© Nature Publishing Group1983
`
`Merck Ex. 1018, pg 661
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