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TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes.

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Thaker, Youg Raj 
Schneider, Helga 
Rudd, Christopher E 


The transcription factor NF-κB is needed for the induction of inflammatory responses in T-cells. Whether its activation by the antigen-receptor and CD28 is mediated by the same or different intracellular signaling pathways has been unclear. Here, using T-cells from various knock-out (Cd28(-/-), adap(-/-)) and knock-in (i.e. Cd28 Y-170F) mice in conjunction with transfected Jurkat T-cells, we show that the TCR and CD28 use distinct pathways to activate NF-κB in T-cells. Anti-CD28 ligation alone activated NF-κB in primary and Jurkat T-cells as measured by NF-κB reporter and EMSA assays. Anti-CD28 also activated NF-κB normally in primary T-cells from adap(-/-) mice, while anti-CD3 stimulation required the adaptor ADAP. Over-expression of ADAP or its binding partner SKAP1 failed to enhance anti-CD28 activation of NF-κB, while ADAP greatly increased anti-CD3 induced NF-κB activity. By contrast, CD28 activation of NF-κB depended on GRB-2 binding to CD28 as seen in CD28 deficient Jurkat T-cells reconstituted with the CD28 YMN-FM mutant, and in primary T-cells from CD28 Y170F mutant knock-in mice. CD28 associated with GRB-2, and GRB-2 siRNA impaired CD28 NF-κB activation. GRB-2 binding partner and guanine nucleotide exchange factor, VAV1, greatly enhanced anti-CD28 driven activation of NF-κB. Further, unlike in the case of anti-CD28, NF-κB activation by anti-CD3 and its cooperation with ADAP was strictly dependent on LAT expression. Overall, we provide evidence that CD28 and the TCR complex regulate NF-κB via different signaling modules of GRB-2/VAV1 and LAT/ADAP pathways respectively.



ADAP, Adaptors, GRB-2, NF-κB, VAV-1, Adaptor Proteins, Signal Transducing, Animals, CD28 Antigens, Humans, Jurkat Cells, Mice, Mice, Knockout, NF-kappa B, Receptors, Antigen, T-Cell, Signal Transduction, T-Lymphocytes

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Immunol Lett

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This work was supported by Wellcome Trust Progam Grant (PG) PKAG/504 to Principal Research Fellow (PRF) C.E. Rudd. We are grateful to Oreste Ocuto and Enzo Cerundolo from University of Oxford for providing CHC17 and 1G4 (Cd28−/−) Jurkat cells.