PAPERS Flashcards
(9 cards)
Immunity Sept 2001
The Src like adaptor protein downregulates tcr on CDr+ CD8+Thymocytes and regulates positive selection Tomaz is first autho, art is last
Key Points: Sosinowski et al 2001 immunity
SLAP is expressed in DP thymocytes and responsible for regulation of TCR levels at the DP stages (specifically controls the low levels that are observed on DPs)
CD4, CD5, CD8 are also upregualted in the knockout
SLAP1 is induced by pre-TCR signaling
SLAP1 regulates positive but not negative selection
How was the SLAP1 KO made?
Described in Sosinowsky et all 2001 (immunity) Figure 1
the construction of the SLAP1 KO. 470 nt including the promotor region and first 21 nt of SLAP were replaced with a neomycin cassette. Western blot analysis reveals that the protein is largely not expressed although there is a slight signal due to an out of frame truncated neo-slap fusion (RT-PCR)
What surface markers are changed on SLAP1-deficient thymocytes
Sosinowsky et all immunity 2001
Figure 2 examines surface markers in the WT repertoire: CD3 and TCRb are markedly upregulated, while CD4, CD5, CD8, CD69 are a more moderately increased.
SLAP1 regulates the downmodulation of TCR at the DN to DP transition. How was this shown?
Even transgenic TCRs, which are expressed at unusually high levels in DN cells, are downregulated to WT TCR levels at the DP stage. Sosinowski and weiss showed that the SLAP1 deficiency prevents the downregulation of the HY and DO11.10 TCRs on DP thymocytes.
To prove that this wasn’t an artefact of uniform downregulation of a a TCR that is positively selected, they crossed the HY TCR onto the nonselecting H-2k background. Downregulation was also observed.
What causes expression of SLAP1 in thymocytes?
Pre-TCR signaling likely induces SLAP1. To distinguish between whether SLAP1 is induced by pre-TCR or ab TCR signaling, Sosinowski and weiss showed that Lck/Fyn-deficient mice (arrested at DN3) do not express SLAP1, but PMA/Iono treatment for 4 hrs (which is not long enough to promote the DN3-DP transition) induced expression of SLAP1
Zap70 deficient thymocytes express SLAP1
What are the effects of SLAP1 deficiency on selection?
DO11.10 TCR transgenic cells are selected twice as efficiently in the selecting background (H2d) in the absence of SLAP1, while the H2b (deleting) background doesn’t appear to have any differences.
Hy TCR positive selection (females, h2b) or negative selection (H2d) appear to be unaffected by SLAP1 deficiency.
JEM, February 2007
Src-Like adaptor protein is a negative regulator of T cell receptor signaling
Key Points Sosinowski, JEM, 2001
SLAP1 is expressed in thymocytes, peripheral T cells and B cells
SLAP1 expression in cell lines potently inhibits TCR signaling in a dose-dependent manner
SLAP1 expression does not affect responsiveness to PMA/Ionomycin (TCR proximal)
