Table 1.
The identity of cDNAs identified from positive interactors.
Table 2.
CD3δ interacts with TCR subunits in S. cerevisiae.
Figure 1.
HEK293T cells were transfected with plasmids encoding CD81 protein in the presence (lane 4) or absence of CD3δ (lanes 3). Top row: Lysates from untransfected HEK293T cells (lane 1) or transfected cells (lanes 2–4) were immunoprecipitated with anti-Myc antibodies and immunoblotted with anti-HA antibodies. Anti-HA immunoblot shows the presence of a 26 kD band corresponding to the CD81 protein co-immunoprecipitated with Myc-CD3δ protein. Middle row: anti-HA immunoblot of lysates demonstrates the expression of CD81 specifically in cells transfected with this construct (lane 3–4). Bottom row: anti-Myc immunoblot of lysates demonstrates the expression of CD3δ in lanes 2 and 4.
Figure 2.
CD81shRNAs knock down CD81 protein expression and surface CD81 expression.
(a) HA-CD81 transfected HEK293T cells with or without shRNA expression were solubilized in Triton X-100 detergent and resolved by reducing SDS-PAGE and blotted with anti-HA Ab. Lysates from HEK293T cells expressing HA-CD81 alone (lane1), untransfected cells (lane 2), cells expressing HA-CD81 plus empty pLMP shRNA expression construct (lane 3) and HA-CD81 plus sh1CD81 or sh2CD81 expressing pLMP constructs (lanes 4 and 5 respectively) were blotted with anti-HA Ab revealing a 26 KDa HA tagged CD81 band. Equal loading was confirmed by blotting the same membrane with anti-calnexin Ab revealing a 90 KDa calnexin band. (b) NIH3T3 cells infected with retrovirus coding shRNA against EGFP (shEGFP) (heavy dotted histogram), empty pLMP shRNA expression construct (solid histogram) or sh1CD81 expressing pLMP construct (tinted histogram) were stained with anti-CD81 biotin followed by streptavidin-PE. Histograms show surface CD81 levels of the cells. The relative mean fluorescence intensity (MFI) normalized to CD81 expression on empty LMP transfected NIH3T3 cells (set to 100) is plotted as a bar graph.
Figure 3.
Stable expression of sh1CD81 downregulates surface CD81 expression and increases TCR mediated activation.
(a) GFP expression before and after puromycin selection in VL3.3M2 cell lines transfected with pLMP encoding CD81 shRNA (sh1CD81) shows the percentage of cells expressing CD81 shRNA. Shaded histograms show fluorescensce of untransfected VL3.3M2 cells and black lined histograms show GFP expression in transfected and antibiotic selected cells. (b) CD81 shRNAs decrease surface CD81 expression in stably transfected VL3.3M2 cells. Relative MFI of CD81 expression on the surface of VL3.3M2 cells that are untransfected (U), or stably transfected with empty pLMP constructs (LMP) or with pLMP-sh1CD81 constructs (sh1CD81) or single cell cloned stable sh1CD81 expressing clones (clone1 and clone2) was determined by flow cytometry and plotted as bar graphs. Surface CD81 expression of untransfected VL3.3M2 cells was set to 100. (c) Relative MFI of surface CD69 expression on VL3.3M2 cells stimulated with plate bound anti-TCRß (1 µg) and anti-CD4 (1 µg). Surface CD69 expression of empty pLMP transfected VL3.3M2 cells was set to 100.
Figure 4.
Inreased signaling intensity in CD81−/− thymocytes.
(a) Deficiency of CD81 or CD9 does not affect T lymphocyte development and thymic cellularity. Thymocytes from B10, CD81−/− and CD9−/− mice were analyzed by multicolor flow cytometry. CD4 vs CD8 contour plots of thymocytes (top row) and lymph node cells (bottom row) are shown. (b) CD81 deficiency increases in vivo signaling intensity in DP thymocytes. Single parameter histograms of TCRß, CD5 and CD69 expression on electronically gated CD4+CD8+ DP thymocytes from B10 (dashed line), CD81−/− (black line) and CD9−/− (grey line) are shown. The electronic gate used is depicted as a box in the top panels in (a).
Figure 5.
CD81 deficiency increases in vitro TCR signaling intensity in CD4+ lymph node T cells.
(a) Deficiency of CD81 results in more cells that upregulate CD69 upon TCR signaling. Purified LN T cells from B10 or CD81−/− mice were assessed for anti-TCR+anti-CD4 induced up-regulation of CD69 expression. Left panel: After overnight stimulation with plate bound anti-TCR antibodies, electronically gated CD81−/− CD4 LN T cells (filled circles) upregulated dramatically more CD69 compared to WT B10 CD4 LN T cells (open circles). Right panel: Stimulation with anti-TCR did not result in a difference between CD81−/− and WT B10 CD8 LN T cells. (b) Frequency of proliferated (>1 cell division) cells after stimulation of CFSE-labelled purified LN CD4+ cells from B6 and CD81−/− mice. Plots of CFSE dye dilution in CD4 T cells stimulated with plate bound anti-CD3 antibodies indicate the frequency of cells with >1 division. Left plot shows proliferation after 40 hr of stimulation and right plot shows proliferation after 72 hr of stimulation. At least two mice in each group were analyzed.