Critical requirement for the membrane-proximal cytosolic tyrosine residue for CD28-mediated costimulation in vivo.
The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.
Research Institute for Biological Sciences, Science University of Tokyo, Yamazaki, Noda, Chiba, Japan.
Y
Y
Harada
M
M
Tokushima
Y
Y
Matsumoto
S
S
Ogawa
M
M
Otsuka
K
K
Hayashi
B D
BD
Weiss
C H
CH
June
R
R
Abe
D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000818:Animals; D018106:CD28 Antigens / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003433:Crosses, Genetic; D003600:Cytosol / Q000276:immunology* / Q000378:metabolism; D005110:Extracellular Space / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006087:Graft vs Host Reaction / Q000235:genetics / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007275:Injections, Intravenous; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000235:genetics; D017710:Lymphocyte Transfusion; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D018345:Mice, Knockout; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000637:transplantation; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D019076:Transgenes / Q000276:immunology; D014443:Tyrosine / Q000235:genetics / Q000276:immunology*
D018106:CD28 Antigens; D007376:Interleukin-2; D014443:Tyrosine