Small molecule inhibition of Csk alters affinity recognition by T cells
- University of California, San Francisco, United States
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- University of basel, Switzerland
Figures
Figure 1 with 1 supplement
Inhibiting Csk increases the magnitude of ligand-induced TCR signaling and reduces the threshold for TCR activation.
(A) Purified CskAS (AS) or wildtype (WT) CD4+ T cells stimulated for 2 min with 1 μg/ml, 5 μg/ml or 10 μg/ml anti-CD3ε antibody in the presence of DMSO or 10 μM 3-IB-PP1 were analyzed by …
Figure 1—figure supplement 1
Inhibiting Csk during TCR stimulation prolongs TCR signals.
(A) Purified CskAS CD4+ T cells stimulated for 2, 5 or 10 min with anti-CD3ε antibody in the presence of DMSO or 10 μM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylated ZAP-70, LAT and …
Figure 2 with 1 supplement
Inhibiting Csk reduces the threshold for TCR activation and prolongs signaling induced by pMHC engagement.
(A) Purified CskAS;OTII CD4+ T cells stimulated for 3 min with 5 μg/ml bead-bound control pMHC tetramer or 5 μg/ml, 2.5 μg/ml or 1.25 μg/ml bead-bound OVA pMHC tetramer in the presence of DMSO or 10 …
Figure 2—figure supplement 1
Csk inhibition shifts the threshold for cellular proliferation in response to anti-CD3 or p-MHC tetramer stimulation.
(A) Purified CD4+ T cells from CskAS mice were loaded with CFSE and stimulated with 3 μg/ml, 1 μg/ml or 0.3 μg/ml plate-bound anti-CD3ε and 1 μg/ml anti-CD28 for 72 hr in the presence or absence of …
Figure 3 with 1 supplement
Weak Csk inhibition and Lck activation potentiate agonist signaling.
(A) CskAS CD8+ T cells were rested and stimulated with indicated doses of 3-IB-PP1 for 3 min, lysed, and immunoblotted for activating (pY394) and inhibitory (pY505) site Lck phosphorylation. Below …
Figure 3—figure supplement 1
Comparison of Csk inhibition and Lck activation in CD4+ vs CD8+ T cells.
(A) CskAS CD4+ and CskAS CD8+ T cells were rested and stimulated with indicated doses of 3-IB-PP1 or DMSO for 2 min and then lysed. Lysates were immunoblotted for activating (pY394) and inhibitory …
Figure 4 with 1 supplement
Csk inhibition potentiates response to weak agonists.
(A) CskAS;OTI naive CD8+ T cells were stimulated with MitoC-treated WT splenocytes, preloaded with indicated peptides, in presence of DMSO or 5 μM 3-IB-PP1 for 4 hr and CD69 upregulation was …
Figure 4—figure supplement 1
TCR Valpha2 downregulation in cells in Figure 4A.
Only OVA triggers peptide-dependent TCR downregulation. 5 μM 3-IB-PP1 induces ligand-independent downregulation of TCR, as seen in VSV peptide. In the presence of CskAS inhibition, all the other …
