Recombinant Rat Anti-CD8 Antibody (YTS156.7) (CAT#: PABX-035)

Figure 1 Ca²⁺ mobilization in OT-I RAG-splenocytes treated with H-2Kb tetramers loaded with the OVA peptide (H-2Kb/OVA) after pre-staining with mAbs 53.6.7 (red line), YTS105.18 (purple line) or YTS156.7.7 (green line).

The tetramers were added at 1 min (arrow) and the sample re-analyzed for a further 4 min. The H-2Kb/OVA tetramer trace is taken from a 5 min time course and overlaid here as a representation of a responding population. Response to the H-2Kb/OVA tetramers in the absence of CD8 antibodies is shown (blue line), as well as response to H-2Kb/SIYR tetramers (black line) as a negative control. All traces indicate the median of the responding population.

Shore, D. A., Issafras, H., Landais, E., Teyton, L., & Wilson, I. A. (2008). The crystal structure of CD8 in complex with YTS156. 7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 αβ to MHC class I. Journal of molecular biology, 384(5), 1190-1202.

Figure 2 Equivalent plot to show relative Ca²⁺ mobilization in OT-I RAG-splenocytes treated with H-2Kb/OVA tetramers after pre-staining now with only the Fabs of mAbs 53.6.7 (red line), YTS105.18 (purple line) or YTS156.7.7 (green line).

The baselines for all traces are normalized to the response to H-2Kb/OVA tetramers in the absence of any Fabs (blue line).

Shore, D. A., Issafras, H., Landais, E., Teyton, L., & Wilson, I. A. (2008). The crystal structure of CD8 in complex with YTS156. 7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 αβ to MHC class I. Journal of molecular biology, 384(5), 1190-1202.

Figure 3 sCD8αβ was determined as having correctly folded native structure by binding to a panel of monoclonal antibodies specific for the CD8α (YTS105.18 and YTS169) and CD8β subunit (YTS156.7)

sCD8αβ was determined as having correctly folded native structure by binding to a panel of monoclonal antibodies specific for the CD8α (YTS105.18 and YTS169) and CD8β subunit (YTS156.7), as determined by surface plasmon resonance (SPR) using a BIAcore 2,000 instrument (BIAcore). mAbs YTS105.18 and YTS169 came from our laboratory (L.T.). Approx. 10,000 response units (RU) of anti-rat IgG Fc (Pierce Biotechnology) was bound to a CM-5 sensor chip by standard amine conjugation. mAbs were passed over the surface of individual flow cells in PBS until an equivalent amount (approx. 250-350 RU) had been captured in each flow cell. Soluble CD8αβ IgSF was subsequently passed over each flow cell in the buffer at concentrations of 0, 3.25, 7.5, 15 and 30 nM. Both CD8 and mAbs were stripped from the surface by injection of 20 mM HCl following each injection.

Shore, D. A., Issafras, H., Landais, E., Teyton, L., & Wilson, I. A. (2008). The crystal structure of CD8 in complex with YTS156. 7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 αβ to MHC class I. Journal of molecular biology, 384(5), 1190-1202.

Figure 4 (a) OT-I splenocytes were incubated with a panel of antibodies specific for mouse CD8α (KT12, 53.6.7, YTS105.18) or CD8β (YTS156.7, 53.5.8)

(a) OT-I splenocytes were incubated with a panel of antibodies specific for mouse CD8α (KT12, 53.6.7, YTS105.18) or CD8β (YTS156.7, 53.5.8), before being stained with 50 nM H-2Kb/OVA PE-tetramers (dark line, right panel), or with a PE-conjugated secondary antibody (dark line, left panel). The staining with tetramers of H-2Kb/OVA (right, dark grey) and tetramers of H-2Kb/SIYR (right, light grey) in absence of anti-CD8 antibody, as well as the background of the secondary antibody (left, light grey), are shown as controls. (b) Mean fluorescence of H-2Kb/OVA PE-tetramer staining on OT-I splenocytes in the presence of increasing concentrations of anti-CD8. The experiment was performed as in (a).

Shore, D. A., Issafras, H., Landais, E., Teyton, L., & Wilson, I. A. (2008). The crystal structure of CD8 in complex with YTS156. 7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 αβ to MHC class I. Journal of molecular biology, 384(5), 1190-1202.