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A point mutation in CD28 distinguishes proliferative signals from survival signals

Nature Immunology volume 2, pages 325–332 (2001)Cite this article

Abstract

Upon interaction with its ligand, B7, CD28 becomes phosphorylated on tyrosines. One tyrosine in particular (Y170 in mouse CD28, Y173 in human CD28) has received much attention. This is because it permits CD28 to recruit SH2-containing signaling molecules, including phosphoinositide 3 kinase, Grb2 and Gads. Using mice we employed a transgenic approach to express a tyrosine→phenylalanine mutant form of CD28 that uncouples these SH2-mediated interactions from CD28. The CD28 mutant is unable to up-regulate expression of the prosurvival protein Bcl-xL, rendering the T cells more susceptible to radiation-induced death. Nonetheless, this mutated form of CD28 still prevents the induction of anergy and promotes T cell proliferation, interleukin 2 secretion and B cell help. Thus, we describe a single point mutation within the CD28 cytoplasmic domain that uncouples signals required for proliferation and survival.

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Figure 1: Generation of CD28 transgenic mice.
Figure 2: CD28 Y170F Tg-1 is unable to bind to the p85 subunit of PI3K or couple to the PI3K target PKB.
Figure 3: CD28-dependent proliferation of Y170F mutant lymph node cells.
Figure 4: CD28 Y170F responds normally to peptide antigen.
Figure 5: CD28 Y170F prevents the induction of anergy.
Figure 6: The YMNM motif is required for CD28-dependent up-regulation of Bcl-xL and for CD28-promotion of radiation resistance.
Figure 7: The Y170F mutant of CD28 supports an antigen-specific humoral immune response.

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Acknowledgements

We thank J. Ellis and A. Huang at the Hospital for Sick Children Transgenic Facility, Toronto for micro-injecting DNA and A. Veillette, S. Ilangumaran and B. Vanhaesebroeck for advice and for reviewing the manuscript.. Supported by grants from the Arthritis Society of Canada and the Medical Research Council (to R. R.) and the National Cancer Institute (to P. S. O.).

Author information

Author notes

  1. Klaus Okkenhaug

    Present address: Ludwig Institute for Cancer Research, 91 Riding House St., London, W1W 7BS, UK

  2. Klaus Okkenhaug and Linda Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology, Toronto, M5S 1A2, Ontario, Canada

    Klaus Okkenhaug, Linda Wu, Tak W. Mak, Pamela S. Ohashi & Robert Rottapel

  2. Ontario Cancer Institute and Princess Margaret Hospital, 610 University Ave, Ontario, M5G 2M9, Canada

    Klaus Okkenhaug, Linda Wu, Kristine M. Garza, Jose La Rose, Tak W. Mak, Pamela S. Ohashi & Robert Rottapel

  3. Amgen Institute, Toronto, Ontario, Canada

    Wilson Khoo & Tak W. Mak

  4. Institute for Experimental Immunology, University of Zürich, Zürich, 8091, Switzerland

    Bernhard Odermatt

  5. Department of Medical Biophysics, Toronto, M5S 1A2, Ontario, Canada

    Tak W. Mak & Robert Rottapel

  6. Medicine University of Toronto, Toronto, M5S 1A2, Ontario, Canada

    Robert Rottapel

  7. St. Michaels Hospital, 80 Bond St., Toronto, M5B 1W8, Ontario, Canada

    Robert Rottapel

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Correspondence to Robert Rottapel.

Supplementary information

Web Figure 1.

The YMNM motif is required for CD28-dependent up-regulation of Bcl-xL. Lymph node cells from C57BL/6, CD28-/-, WT Tg and Y170F Tg –1 were left unstimulated or stimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 6, 10, 22, 34 and 46 h. Cells were collected, washed in PBS and lysed. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes and probed with anti–Bcl-xL and anti-actin. (JPG 39 kb)

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Okkenhaug, K., Wu, L., Garza, K. et al. A point mutation in CD28 distinguishes proliferative signals from survival signals. Nat Immunol 2, 325–332 (2001). https://doi.org/10.1038/86327

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