Figure 5.

PD-1 mAb further enhances CTL lysis of engineered antigen-positive MOC1 cells following treatment with scL-53. A, to evaluate if scL-53 treatment enhances antigen presentation and antigen-specific CTL lysis, we performed experiments with MOC1 cells engineered to express full-length ovalbumin as a defined model antigen. SIINFEKL presentation via H2-K^b on the surface of MOC1ova cells was assessed via flow cytometry after treatment with scL-empty or scL-53 (10 ng per 1 × 10⁴ cells for 4 hours then incubated for 24 hours; representative histograms on left, quantification on right). B, CTL-mediated lysis of MOC1ova cells following scL-53 treatment as in A was measured using a standard CR⁵¹-release assay, (SIINFEKL pulsed untreated EL-4 cells used as a positive control). C, PD-1 expression was assessed via flow cytometry on the surface of SIINFEKL-specific CTLs generated from OT-1 splenocytes. D, CTL-mediated lysis of MOC1ova cells following treatment with scL-53, scL-vec or scL-empty as in A with or without PD-1 mAb (clone RMP1-14, 1 μg/mL) or isotype control (rat IgG2 Ab) was measured using a real-time impedance-based cytotoxicity assay. OT-1 CTLs were added at the indicated vertical black line. scL treatment times indicated by grey shaded box. Conditional growth curves shown on left, quantification 24 hours after the addition of the CTLs on the right (vertical dashed line). Quantification of loss of cell index quantified on right. All data are representative results from one of at least three independent experiments. **, p < 0.01; ***, P,0.001.