Monday, August 21, 2006

Immunity: New T cell Regulatory Proteins

Anonymous writes "Chu and colleagues found 33 proteins that decreased T cell activation induced by triggering the T cell antigen receptor (TCR). They screened for increased CD69, a marker of T activation, induced by antibodies to the TCR in Jurkat cells transduced with a retroviral expression library. Although not exhaustive, the screen identified many truncation mutants of expected (Lck, ZAP70, SHP-1, etc.) and surprising (an integrin, Grb7, etc.) proteins. One new gene was identified, a suspected ubiquitin ligase they named TRAC-1 that previously was only an EST. The proteins’ functions were confirmed in primary T cells. As the authors note, the post genome advances will come from assigning functions to proteins. Their approach may help identify proteins in complex signaling systems. PubMed
J. Biol." (Originally posted on October 2, 2003)
Systematic identification of regulatory proteins critical for T-cell activation
Peter Chu, Jorge Pardo, Haoran Zhao, Connie C Li, Erlina Pali, Mary M Shen, Kunbin Qu, Simon X Yu, Betty CB Huang, Peiwen Yu, Esteban S Masuda1, Susan M Molineaux, Frank Kolbinger, Gregorio Aversa, Jan de Vries, Donald G Payan and X Charlene Liao
Journal of Biology 2003 2:21


Anonymous said...

Useful Winnowing Step

A key technical advantage was their use of a tetracylin-regulated ('Tet off') retroviral expression construct. The promoter controlling expression of the inserted cDNA contained a Tet-regulated element (TRE, Fig 1).

Tet regulation allowed them to quickly check the candidate T cell clones for artifactual (epigenetic) CD69 inhibition. If adding tetracyclin (thereby turning off expression of the transduced protein) did not restore CD69 induction, then the transduced protein was not responsible for the inhibition and the investigators could discard the clone. More than half the transductants showing decreased CD69 induction were NOT rescued by Tet treatment (Fig 2c), so this check saved significant effort by allowing them to focus on analyzing the 1,323 clones that passed this test out of 2,828 clones obtained initially (page 6).

Anonymous said...

The most frequent clone they obtained (46 times) was a T cell receptor beta chain (TCRb, Table 1), which replaced the endogenous TCRb. The altered TCR was no longer triggered by the antibody, so CD69 was not increased. Tet turned off the new beta chain, allowing the old TCR to be expressed.

Any ideas why they didn't find TCR alpha clones? Is this chain expressed at higher levels (making it less easily competed by the transduced gene) or is it much more conserved (so that the antibody could still bind)?

What does not kill me makes me more susceptible to the next infection.