T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells.
To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.
Center for Molecular Medicine, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, N-0318 Oslo, Norway.
Maria Elisabeth
ME
Kalland
Nikolaus Günter
NG
Oberprieler
Torkel
T
Vang
Kjetil
K
Taskén
Knut Martin
KM
Torgersen
D018801:CD2 Antigens / Q000378:metabolism / Q000502:physiology*; D018106:CD28 Antigens / Q000378:metabolism / Q000502:physiology*; D002469:Cell Separation; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D020935:MAP Kinase Signaling System / Q000276:immunology*; D016328:NF-kappa B / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000276:immunology*; D038601:Ribosomal Protein S6 / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism
D018801:CD2 Antigens; D018106:CD28 Antigens; D016328:NF-kappa B; D038601:Ribosomal Protein S6