Deletion of the CD4 silencer element supports a stochastic mechanism of thymocyte lineage commitment


The mechanism of T cell lineage commitment remains controversial; to examine it we deleted the CD4-silencer element in the germ line of a mouse using a combination of gene targeting and Cre/LoxP-mediated recombination. We found that these mice were unable to extinguish CD4 expression either in immature thymocytes or mature CD8+ cytotoxic T cells (CTLs), which resulted in the development of major histocompatibility complex class II–restricted double-positive CTLs in the periphery. This finding strongly supports a stochastic over an instructive mechanism of coreceptor down-regulation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Gene-targeting at the CD4 locus.
Figure 2: Expression of CD4 on lymph node T cells.
Figure 3: Mature DP thymocytes in CD4Δsil mice.
Figure 4: Mature DP T cells in CD4Δsil mice.
Figure 5: MHC class II–restriction of DP cells.
Figure 6: Mature DP thymocytes and lymph node T cells in CD4Δsil mice that lacked MHC class I.


  1. 1

    Jorgensen, J. L., Reay, P. A., Ehrich, E. W. & Davis, M. M. Molecular components of T-cell recognition. Annu. Rev. Immunol. 10, 835–873 (1992).

  2. 2

    Robey, E. & Fowlkes, B. J. Selective events in T cell development. Annu. Rev. Immunol. 12, 675–705 (1994).

  3. 3

    Marrack, P. & Kappler, J. Positive selection of thymocytes bearing αβ T cell receptors. Curr. Opin. Immunol. 9, 250–255 (1997).

  4. 4

    von Boehmer, H. & Kisielow, P. Lymphocyte lineage commitment: instruction versus selection. Cell 73, 207–208 (1993).

  5. 5

    Davis, C. B. & Littman, D. R. Thymocyte lineage commitment: is it instructed or stochastic? Curr. Opin. Immunol. 6, 266–272 (1994).

  6. 6

    Chan, S. H., Benoist, C. & Mathis, D. A challenge to the instructive model of positive selection. Immunol. Rev. 135, 119–131 (1993).

  7. 7

    Chan, S. H., Cosgrove, D., Waltzinger, C., Benoist, C. & Mathis, D. Another view of the selective model of thymocyte selection. Cell 73, 225–236 (1993).

  8. 8

    van Meerwijk, J. P. & Germain, R. N. Development of mature CD8+ thymocytes: selection rather than instruction? Science 261, 911–915 (1993).

  9. 9

    Itano, A., Kioussis, D. & Robey, E. Stochastic component to development of class I major histocompatibility complex-specific T cells. Proc. Natl Acad. Sci. USA 91, 220–224 (1994).

  10. 10

    Robey, E., Itano, A., Fanslow, W. C. & Fowlkes, B. J. Constitutive CD8 expression allows inefficient maturation of CD4+ helper T cells in class II major histocompatibility complex mutant mice. J. Exp. Med. 179, 1997–2004 (1994).

  11. 11

    Baron, A., Hafen, K. & von Boehmer, H. A human CD4 transgene rescues CD4CD8+ cells in β 2-microglobulin-deficient mice. Eur. J. Immunol. 24, 1933–1936 (1994).

  12. 12

    Corbella, P. et al. Functional commitment to helper T cell lineage precedes positive selection and is independent of T cell receptor MHC specificity. Immunity 1, 269–276 (1994).

  13. 13

    Paterson, R. K. et al. Thymic development in human CD4 transgenic mice. Positive selection occurs after commitment to the CD8 lineage. J. Immunol. 153, 3491–3503 (1994).

  14. 14

    von Boehmer, H. The selection of the αβ heterodimeric T cell receptor for antigen. Immunol. Today 7, 333–336 (1986).

  15. 15

    Teh, H. S. et al. Thymic major histocompatibility complex antigens and the αβ T-cell receptor determine the CD4/CD8 phenotype of T cells. Nature 335, 229–233 (1988).

  16. 16

    Robey, E. A. et al. Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage. Cell 64, 99–107 (1991).

  17. 17

    Borgulya, P., Kishi, H., Muller, U., Kirberg, J. & von, B. H. Development of the CD4 and CD8 lineage of T cells: instruction versus selection. EMBO J. 10, 913–918 (1991).

  18. 18

    Davis, C. B., Killeen, N., Crooks, M. E., Raulet, D. & Littman, D. R. Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes. Cell 73, 237–247 (1993).

  19. 19

    Chan, S. H., Waltzinger, C., Baron, A., Benoist, C. & Mathis, D. Role of coreceptors in positive selection and lineage commitment. EMBO J. 13, 4482–4489 (1994).

  20. 20

    Crump, A. L., Grusby, M. J., Glimcher, L. H. & Cantor, H. Thymocyte development in major histocompatibility complex-deficient mice: evidence for stochastic commitment to the CD4 and CD8 lineages. Proc. Natl Acad. Sci. USA 90, 10739–10743 (1993).

  21. 21

    Kydd, R., Lundberg, K., Vremec, D., Harris, A. W. & Shortman, K. Intermediate steps in thymic positive selection. Generation of CD48+ T cells in culture from CD4+8+, CD4int8+, and CD4+8int thymocytes with up-regulated levels of TCR- CD3. J. Immunol. 155, 3806–3814 (1995).

  22. 22

    Lundberg, K., Heath, W., Kontgen, F., Carbone, F. R. & Shortman, K. Intermediate steps in positive selection: differentiation of CD4+8int TCRint thymocytes into CD48+TCRhi thymocytes. J. Exp. Med. 181, 1643–1651 (1995).

  23. 23

    Suzuki, H., Punt, J. A., Granger, L. G. & Singer, A. Asymmetric signaling requirements for thymocyte commitment to the CD4+ versus CD8+ T cell lineages: a new perspective on thymic commitment and selection. Immunity 2, 413–425 (1995).

  24. 24

    Lucas, B. & Germain, R. N. Unexpectedly complex regulation of CD4/CD8 coreceptor expression supports a revised model for CD4+CD8+ thymocyte differentiation. Immunity 5, 461–477 (1996).

  25. 25

    Benveniste, P., Knowles, G. & Cohen, A. CD8/CD4 lineage commitment occurs by an instructional/default process followed by positive selection. Eur. J. Immunol. 26, 461–471 (1996).

  26. 26

    Yasutomo, K., Doyle, C., Miele, L. & Germain, R. N. The duration of antigen receptor signaling determines CD4+ versus CD8+ T-cell lineage fate. Nature 404, 506–510 (2000).

  27. 27

    Hernandez-Hoyos, G., Sohn, S. J., Rothenberg, E. V. & Alberola-Ila, J. Lck activity controls CD4/CD8 T cell lineage commitment. Immunity 12, 313–322 (2000).

  28. 28

    Legname, G. et al. Inducible expression of a p56Lck transgene reveals a central role for Lck in the differentiation of CD4 SP thymocytes. Immunity 12, 537–546 (2000).

  29. 29

    Brugnera, E. et al. Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Immunity 13, 59–71 (2000).

  30. 30

    Sternberg, N. & Hamilton, D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J. Mol. Biol. 150, 467–486 (1981).

  31. 31

    Sawada, S., Scarborough, J. D., Killeen, N. & Littman, D. R. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell 77, 917–929 (1994).

  32. 32

    Siu, G., Wurster, A. L., Duncan, D. D., Soliman, T. M. & Hedrick, S. M. A transcriptional silencer controls the developmental expression of the CD4 gene. EMBO J. 13, 3570–3579 (1994).

  33. 33

    McCready, P. M., Hansen, R. K., Burke, S. L. & Sands, J. F. Multiple negative and positive cis-acting elements control the expression of the murine CD4 gene. Biochim. Biophys. Acta 1351, 181–191 (1997)

  34. 34

    Rushton, J. J., Zorich, G. P., Stolc, V. and Neudorf, S. M. Characterization of a promoter within the first intron of the human CD4 gene. Eur. J. Biochem. 245, 768–773 (1997)

  35. 35

    Liao, N. S., Maltzman, J. & Raulet, D. H. Positive selection determines T cell receptor Vβ14 gene usage by CD8+ T cells. J. Exp. Med. 170, 135–143 (1989).

  36. 36

    Jameson, S. C., Kaye, J. & Gascoigne, N. R. A T cell receptor Vα region selectively expressed in CD4+ cells. J. Immunol. 145, 1324–1331 (1990).

  37. 37

    Oxenius, A. et al. Presentation of endogenous viral proteins in association with major histocompatibility complex class II: on the role of intracellular compartmentalization, invariant chain and the TAP transporter system. Eur. J. Immunol. 25, 3402–3411 (1995)

  38. 38

    Bosselut, R., Feigenbaum, L., Sharrow, S. O. and Singer, A. Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes. Immunity 14, 483–494 (2001).

Download references


We thank H. Waldmann, R. Lechler and R. Zinkernagel for advice and helpful discussions; J. Rossant for the D3 embryonic stem cell line; K. Rajewski for the Cre-Lox cassettes; and R. Sumner and C. Hetherington for help with some blastocyst injections. Supported by the Croucher Foundation (R. L) and the Wellcome Trust (K. T and A. R).

Author information

Correspondence to Amin Rahemtulla.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Leung, R., Thomson, K., Gallimore, A. et al. Deletion of the CD4 silencer element supports a stochastic mechanism of thymocyte lineage commitment. Nat Immunol 2, 1167–1173 (2001) doi:10.1038/ni733

Download citation

Further reading