Early Expression of a T-Cell Receptor ,8-Chain Transgene Suppresses

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 9729-9732, December 1988 Immunology

Early expression of a T-cell receptor ,8-chain transgene suppresses rearrangement of the Vy4 gene segment (transgenic mouse/T-cell receptor y6 heterodimer/allelic exclusion) HARALD VON BOEHMER*, MARC BONNEVILLEt, ISAO ISHIDAt, STEFAN RYSER*t, GLORIA LINCOLN§, RICHARD T. SMITH§, HIROYUKI KISHI*, BERNADETTE SCOTT*, PAWEL KISIELOW*, AND SUSUMU TONEGAWAt¶ tHoward Hughes Medical Institute and Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; *Basel Institute for Immunology, Basel, Switzerland; and §Department of Pathology, College of Medicine, University of Florida, Gainesville, FL 32610 Contributed by Susumu Tonegawa, September 8, 1988

ABSTRACT 18 transgenic mice have a T-cell receptor Thymocyte Preparation. Embryonic thymi were prepared 13-chain gene that is prematurely expressed on the surface of as described from time-mated females (7). Thymus lobes CD4-CD8- thymocytes and paired with an uncharacterized were carefully dissected and single-cell suspensions were non-T-cell receptor a-chain polypeptide. The rearrangement prepared. CD4-CD8- thymocytes were obtained after two ofthe T-cell receptor variable region y chain gene segment VY4, cycles oftreatment with monoclonal CD4 and CD8 antibodies a component of the y-chain gene that is rearranged and and complement as described (8). expressed preferentially on thymocytes of normal adult mice, Cell Surface Staining of Lymphoid Cells. Single-cell sus- is severely repressed in 13 transgenic mice. Consequently no 'yi pensions were incubated with CD3, CD4, CD5, and F23.1 T-cell receptor heterodimers are detectable on the surface of antibodies which were uncoupled, biotinylated, or coupled adult thymocytes or splenic T cells. These results indicate that with fluorescein isothiocyanate. Second-step reagents con- cells expressing a.3 or v(Vy4)-6 TCRs originate from a sisted of fluorescein isothiocyanate- or phycoerythrine- common precursor in which the first productive rearrange- coupled anti-mouse or -rat immunoglobulin antibodies. Cells ment of either the 13 or y locus determines the further were incubated with first- or second-step reagents for 30 min differentiation pathway into either a13 or y6 T cells. The at 40C. Cell surface staining was analyzed on a fluorescence- repression of V,,4 rearrangement by a preexisting 13-chain gene activated cell sorter and data were analyzed and plotted with may be indicative of one of several mechanisms which ensure a Consort C30 program. that y6 and a13 receptors do not as a rule appear on the surface Immunoprecipitation. TCRs were precipitated from thy- of the same cell. mocyte lysates by either the F23.1 antibody, an antiserum specific for the constant region of the a chain (Ca) domain (9) T cells expressing y8 or ap heterodimeric T-cell receptors or the anti-y-chain monoclonal antibody KN365 (anti-y) (10). (TCRs) are formed in the thymus where all four receptor loci Some lysates were cleared by incubation with anti-Ca anti- undergo rearrangement (1-4). Nonproductive, or the occa- bodies and Staphylococcus as described (6). Precipitates sional productive, y-chain rearrangement(s) are found in T were analyzed under reducing and nonreducing conditions by cells expressing the af3 TCR (ap3 T cells) and a-chain polyacryamide gel electrophoresis. rearrangement is found in T cells expressing the y3 TCR (y8 T-Cell Clones. CD8+CD4- T-cell clones from ,3 transgenic T cells) (5). This suggests that both types of T cells may mice were obtained by stimulating spleen cells from f3 originate from a common intrathymic precursor. The argu- transgenic mice with allogenic DBA/2 irradiated stimulator ment would be considerably strengthened ifit was shown that cells in vitro. T cells were after the second restimu- productive rearrangement of one of the loci would influence cloned rearrangement of other loci. A rearranged TCR 8-chain lation of interleukin 2-containing medium as described (6). transgene suppresses completely the rearrangement of Southern and Northern Blot Analyses. HindIII-digested endogenous genes for the variable region of the f chain (Vo) DNAs were electrophoresed into a 0.7% agarose gel, blotted (6). Here we show that the same transgene suppresses the onto a nitrocellulose filter, and hybridized with random- rearrangement and expression of endogenous y-chain genes primed probes as described (8). The Jy1 probe for the joining (y gene). (J) region of the y-chain gene was a Sty I-HindIII fragment from a V44-Jyl genomic DNA where V.4 is a locus for the variable region of the y chain and was comprised of Jy, and MATERIALS AND METHODS a part of the flanking intron (11). Assignments of bands to Mice. The transgenic founder mice 95 and 93 contain 2 and germ-line or rearranged genes was done as described (12). 20 copies, respectively, of the V,82 gene segment that codes Formaldehyde-treated RNAs were electrophoresed into 1% for an amino acid sequence which can be identified by the agarose/formaldehyde gel, blotted onto nylon membranes, monoclonal antibody F23.1. The transgenic mice were back- and hybridized to random-primed probes as described (8). crossed to C57L mice lacking the V,8.2 gene family. Trans- The Vy2 probe is a Pst I-Ava I fragment from pHDS203 (13). genic and nontransgenic mice were identified by Southern blot hybridization ofDNA isolated from the tail of the mouse Abbreviations: TCR, T-cell receptor; V, J, and C, variable, joining, (6). In this report we have analyzed transgenic and nontrans- and constant regions of the TCR genes, respectively; Vp,Vy, etc., V genic littermates as well as normal C57BL/6 mice. region ofthe f3 chain, ychain, etc., respectively; C, C,, etc., constant region of the a chain, y chain, etc., respectively; Jy, J region of the y chain. The publication costs of this article were defrayed in part by page charge tPresent address: Central Research Unit, Hoffmann-La Roche, payment. This article must therefore be hereby marked "advertisement" Basel, Switzerland in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 9729 Downloaded by guest on September 25, 2021 9730 Immunology: von Boehmer et al. Proc. Natl. Acad. Sci. USA 85 (1988) RESULTS Surface Markers on Thymocyte Subsets. In Fig. 1A we show that P transgenic mice and nontransgenic littermates contain almost identical populations of thymocytes defined by CD4 and CD8 surface antigens. In addition the CD3 complex is expressed to a similar extent by thymocytes from transgenic 100 ' 102 ' 1'04 and nontransgenic mice: most cells express low levels and Fluorescence intensity -20% of the cells expresses higher levels of CD3 molecules. The obvious difference is that practically all transgenic thy- FIG. 2. Staining ofthymocytes (solid line) from /3 transgenic mice mocytes express the transgenic /8 chain on the cells surface: from day 15 of gestation by F23.1 antibodies (broken line, unstained again the majority expresses lower levels while -20% ofcells, cells). representing the single positive CD4+CD8- and CD4-CD8' and normal mice could be compared. As shown in Fig. 1B the thymocytes (14), shows higher levels of receptor. majority of double-negative cells from 83 transgenic mice We have prepared double-negative cells by cytotoxic expresses low levels of CD3 and, unlike cells from C57BL/6, treatment of thymocytes from /3 transgenic as well as low levels (comparable to that of CD4+CD8' thymocytes) of C57BL/6 mice with anti-CD4 and -CD8 antibodies. We used the transgenic /3 chain (detected by F23.1) on the cell surface. C57BL/6 mice because a significant fraction of their T cells Staining of these cells with CD5 antibodies shows that they expresses TCRs in part encoded by the V188 gene family so are CD5 dull (Fig. 1C). Because CD5-dull CD4-CD8- thy- that intensity ofexpression of VP8 gene products in transgenic mocytes contain the precursors for all other thymocyte subpopulations (15), these experiments suggested an "early" Aj J I expression of a receptor containing the TCR p chain on cells which usually do not express TCRs containing 3 chain (16, 17). This was verified by the analysis of thymocytes from 14- and 15-day-old embryos: the transgenic 8 chain could be detected on the surface of ;10% of thymocytes from 14- day-old embryos and of the majority of thymocytes from 15-day-old embryos (Fig. 2). Surface Expression of f3 Chains Without TCR a Chains. To analyze the chain composition of the receptor expressed on CD4-CD8- thymocytes, we precipitated lysates from these cells with either F23.1 or Ca antibodies before and after preclearing of the lysates with Ca antibodies. In control experiments, the lysates from the ap-TCR-expressing B6.2.16 clone were used. Fig. 3 shows that F23.1 antibodies precipitated a disulfide-linked dimer from the CD4-CD8- B6 P.TG thymocytes of p transgenic mice as well as the B6.2.16 clone. In contrast Ca antibodies required a similar dimer from the B D B6.2.16 clone but not from the CD4-CD8- thymocytes. 0D3 CD3 Preclearing of the lysates with C, antibodies removed the dimers precipitated by both the F23.1 as well as the C, antibody from the lysates of B6.2.16 clone but did not affect the precipitate obtained with the F23.1 antibody and the lysates of CD4-CD8- thymocytes from /3 transgenic mice. This indicates that the transgenic P chain expressed on the surface of CD4-CD8- thymocytes is not paired with a TCR F23.1 F23.1 a chain. A B CD48 cells B6.2.16 CD48 cells B6.2.16 A B C D E A B C D E A B C D E A B C D E kd kd cD5 iD5 97- & 97- 68- *o 68-

- 4 3- | .: 43- N iM s-. 100 102 10 100 102 104 Fluorescence intensity FIG. 1. (A) Staining of thymocytes from ,/ transgenic mice (solid FIG. 3. Association of TCR p chains with molecules other than line) and from nontransgenic littermates (broken lines) by CD3 and TCR a chains on CD4-CD8- thymocytes in ,/ transgenic mice. TCRs F23.1 (Upper) and CD4 and CD8 antibodies (Lower). (B) Staining of were precipitated before (lanes A and C) or after (lanes B and D) CD4-CD8- thymocytes from normal C57BL/6 (B6) and ,8 transgenic preclearing with anti-Ca antibody from CD4-CD8- thymocytes or (13.TG) mice with CD3 and F23.1 antibodies (broken lines, unstained B6.2.16 cells and analyzed by NaDodSO4/PAGE under reducing (A) controls). (C) Staining of CD4-CD8- thymocytes from normal or nonreducing (B) conditions. Lanes: A and B, immunoprecipitates C57BL/6 (B6) and /3 transgenic (,3.TG) with CD5 antibodies (broken by anti-Ca; C and D, immunoprecipitates by F23.1; E, precipitates lines, unstained controls). without antibody. Downloaded by guest on September 25, 2021 Immunology: von Boehmer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9731 Lack of y8 on Transgenic Thymocytes. The 'yS TCR is Host normally expressed on about 5% of CD5-dull CD4-CD8- A B/6 PTG B 3TG thymocytes in adult mice (18). Since in /3 transgenic mice the A B C D A B C D developmental timing of the TCR 13-chain expression is abnormal and the vast majority of this thymocyte subpop- J2C2a~ ulation express a TCR containing the P transgene, the J3C3,-V$0*z k W 18S- question arose as to the effect of the /3 transgene on the I'I generation of y8-TCR thymocyte population. This was analyzed by immunoprecipitation of lysates prepared from V2J2- 10 surface-labeled total CD4-CD8- thymocyte populations with VY2 the anti-y monoclonal antibody KN365. Lysates from nontransgenic littermates (Fig. 4a, lanes A and B, and Fig. 4b, lane B) and the control 'y8 T-cell hybridoma KN6 (Fig. 4a, lanes E, and Fig. 4b, lane A) gave characteristic vy het- V4J1- * 18S-,1 erodimers while those from transgenic mice exhibited no detectable Vy signals (Fig. 4a, lanes C and D, and Fig. 4b, lane C). As expected, ample a,8 signals were obtained when cell Jyl lysates precleared with anti-y antibody were subsequently Actin immunoprecipitated with a purified anti-a antiserum (Fig. 4b, FIG. 5. y gene rearrangement and expression in (3 transgenic lanes D and E). mice. (A) Analysis of y gene rearrangement in j3 transgenic mice. y Gene Rearrangement and Expression in Transgenic Mice. HindIll-digested DNA from thymocytes (lanes A and C) and sple- Is the lack of 'y5 TCR on transgenic thymocytes due to nocytes (lanes B and D) of C57BL/6 and /8 transgenic mice were repression of gene rearrangement? We investigated this electrophoresed on agarose gel, transferred onto a nylon filter, and question by Southern blot analysis of DNA isolated from hybridized with the Jyj probe. This probe hybridizes to y,,j, Jy2, and Jy3 gene segments (12). J2C2, J3C3, and JC,, germ-line position; V2J2 thymocyte populations as well as from individual T-cell and V4J1, rearranged position. A strain polymorphism explains the clones prepared from the ,/ transgenic mice. As shown in Fig. different migration patterns ofJ2C2 germ-line bands of C57BL/6 and 5A, V4-J, t-chain rearrangement, which is primarily respon- (3 transgenic mice. (B) Northern blot analysis of thymocyte and sible for the surface expression of vy TCRs on adult thymo- splenocyte RNA from 13 transgenic mice. Total RNAs from spleno- cytes and splenic T cells in normal mice (refs. 8, 17, 18, and cytes (lanes A and C) and thymocytes (lanes B and D) of 8 transgenic Y. Takagaki, N. Nakanishi, I.I., 0. Kenagawa, and S.T., mice and control littermates were electrophoresed on agarose gel, unpublished observation), is severely hampered in transgenic blotted onto nylon filters, and hybridized with Vy2 and actin probes. mice (lanes C and D). By contrast the V2-2 rearrangement the fifth one (Fig. 5A) and the transcription ofthe resulting V2J2C2 y-chain (four out offive T-cell clones were examined but gene (Fig. 5B) are only moderately affected by the presence was not). ofthe ,3 transgene. The actin probe serves as a control for the amount of total RNA analyzed (Fig. 5B). DISCUSSION In further experiments a,8-TCR-bearing T-cell clones from The results reported here indicate that the productive re- /3 transgenic mice were obtained by stimulation with alloge- arrangement of a TCR /-chain gene can lead to the surface neic x-irradiated DBA/2 spleen cells and cloned in interleu- expression of that ,/ gene without concomitant expression of kin 2-containing medium (6). Analyses ofthe y gene loci in at a TCR a chain. At present we do not know whether this /3 least four offive T-cell clones are in excellent agreement with chain is paired with another polypeptide chain or expressed the population analysis described above. Thus, unlike most as a homodimer. Ifit is expressed as a heterodimer, it is most ap T-cell clones prepared from normal mice (5), all ofthe five likely not paired with TCR y chain because anti-y antibodies T-cell clones from ,B-transgenic mice had Jy, in the germ-line fail to precipitate a heterodimer from CD4-CD8- thymo- configuration while, as in most "normal" T-ceil clones, J.2 cytes. Pairing with a 6 chain is also unlikely because most was rearranged in all clones derived from transgenic mice known mouse 8 chains migrate slower than /3 chains (refs. 8, 10, 17, 18, and Y. Takagaki, N. Nakanishi, I.I., 0. Kena- a B/6 PTG b antiy anti a gawa, and S.T., unpublished observations) while the precip- A B C D E A B C D E itation with F23.1 antibodies did not show a band of higher molecular weight than that of the /3 chain (Fig. 3). It is fs, possible that a new form ofa TCR is present in nontransgenic * re but has escaped detection because of the very similar r- molecular masses of both chains to the masses of the a and /3 chains. However, the possibility that the transgenic /3 chain pairs with a rare or unknown 6 chain cannot be ruled out. TCR FIG. 4. Analysis of yS- and a3-TCR proteins expression on Whatever the form of the surface receptor containing transgenic thymocytes. (a) Cell lysates from total (lanes A and C) or ,8 chains, the results reported here suggest that the productive CD4-CD8- (lanes B and D) thymocytes were immunoprecipitated rearrangement of the TCR P-chain gene and possibly its with the anti-y monoclonal antibody KN365 and analyzed by expression on the cell surface are events that have pleiotropic NaDodSO4/PAGE under reducing conditions. Lanes: A and B, effects on the development ofboth a,3 and Vy8T cells: not only C57BL/6; C and D, 18 transgenic; E, yS-expressing hybridoma. (b) is the productive rearrangement of endogenous V3 genes Lysates made from total thymocytes were first immunoprecipitated completely suppressed (6) but also the rearrangement of with protein A-Sepharose beads coated with the anti--y antibody certain y gene segments (V4), but not other y gene segments KN365. The unprecipitated material was then subjected to a second is completely suppressed as shown here. One may argue immunoprecipitation with a purified anti-Ca antiserum. Immunopre- (V2), cipitates from the first (lanes B and C) and the second (lanes D and that the negative effect of the /3-chain gene on the rearrange- E) reactions were analyzed by NaDodSO4/PAGE under reducing ment of V1 and Vy genes may be executed indirectly by conditions. Lanes: A, hybridoma KN6; B and D, C57BL/6 thymo- accelerated a-chain gene expression leading to an a/3 TCR cytes; C and E, thymocytes from the 13 transgenic mouse. which shuts off all further rearrangement. Such a feedback Downloaded by guest on September 25, 2021 9732 Immunology: von Boehmer et al. Proc. Natl. Acad. Sci. USA 85 (1988) mechanism by a surface-expressed receptor is supported by 7. Kisielow, P., Leiserson, W. & von Boehmer, H. (1984) J. experiments with the immunoglobulin gene system (19, 20). Immunol. 133, 1117-1123. Regardless of whether the negative feedback occurs di- 8. Ito, K., Bonneville, M., Takagaki, Y., Nakanishi, N., Kana- rectly or indirectly the suppression of rearrangement seems gawa, O., Krecko, E. & Tonegawa, S. (1988) Proc. Natl. Acad. to be mediated by a mechanism which has its target in all Sci. USA, in press. Vp 9. Traunecker, A., Dolder, B. & Karjalainen, K. (1986) Eur. J. genes but only some V1 genes. Thus, as postulated for Immunol. 16, 851-854. immunoglobulin genes, the accessibility of certain gene 10. Maeda, K., Nakanishi, N., Rogers, B. L., Haser, W. G., segments for the recombinase rather than the acting of the Shitara, K., Yoshida, H., Takagaki, Y., Augustin, A. A. & recombinase itself may be a crucial event in allelic and Tonegawa, S. (1987) Proc. Natl. Acad. Sci. USA 84, 6536- nonallelic exclusion of TCR genes (21). 6540. The fact that a productively rearranged ,8 gene suppresses 11. Hayday, A. C., Saito, H., Gillies, S. D., Kranz, D. M., Tan- rearrangement of a nonallelic V.4 locus indicates that both ap igawa, G., Eisen, H. N. & Tonegawa, S. (1985) Cell 40, 259- and y(V4)-8 cells originate from a common precursor in which 269. the first productive rearrangement of either the /3 or 'y locus 12. Pelkonen, J., Traunecker, A. & Kardalainen, K. (1987) EMBO determines the further differentiation pathway into either a/3 J. 6, 1941. 13. Saito, H., Kranz, D. M., Takagaki, Y., Hayday, A. C., Eisen, or y8 T cells. The observations reported here may be H. N. & Tonegawa, S. (1984) Nature (London) 309, 757-762. indicative of one of several mechanisms which ensure that as 14. Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M. & a rule y5 and af3 receptors do not appear on the surface ofthe von Boehmer, H. (1988) Nature (London) 333, 742-748. same cell. 15. Fowlkes, B. J., Edison, L., Mathieson, B. J. & Chused, T. M. (1985) J. Exp. Med. 162, 802-822. 1. Snodgrass, H. R., Kisielow, P., Kiefer, M., Steinmetz, M. & 16. Cristanti, A., Celantoni, A., Snodgrass, R. & von Boehmer, H. von Boehmer, H. (1985) Nature (London) 313, 592-595. (1986) EMBO J. 5, 2837-2842. 2. Raulet, D. H., Garman, R. D., Saito, H. & Tonegawa, S. (1985) 17. Bluestone, J. A., Pardoll, D., Sharrow, S. 0. & Fowlkes, B. J. Nature (London) 314, 103-107. (1987) Nature (London) 326, 82-85. 3. Born, W., Yaguc, J., Palmer, E., Kappler, J. & Marrack, P. 18. Lew, A. M., Pardoll, D. M., Maloy, W. L., Fowlkes, B. J., (1985) Proc. Natl. Acad. Sci. USA 82, 531-535. Kruisbeek, A., Cheng, S.-F., Germain, R. N., Bluestone, 4. Chien, Y.-h., Iwashima, M., Wettstein, D. A., Kaplan, K. B., J. A., Schwartz, R. H. & Coligan, J. E. (1986) Science 234, Elliott, J. F., Born, W. & Davis, M. M. (1987) Nature (London) 1401-1405. 330, 722-727. 19. Alt, F. W., Rosenberg, N., Enea, V., Seiden, E. & Baltimore, 5. Heilig, J. S. & Tonegawa, S. (1987) Proc. Natl. Acad. Sci. USA D. (1980) Cell 21, 1-12. 84, 8070-8074. 20. Ritchie, K. A., Brinster, R. L. & Storb, U. (1984) Nature 6. Uematsu, Y., Ryser, S., Dembid, Z., Borgulya, P., Krimpen- (London) 312, 517-521. fort, P., Berns, A., von Boehmer, H. & Steinmetz, M. (1988) 21. Alt, F. W., Blackwell, T. K., DePinho, R. A., Reth, M. G. & Cell 52, 831-841. Yancopoulos, G. D. (1986) Immunol. Rev. 89, 5-30. Downloaded by guest on September 25, 2021