Fig. 7: In vitro PGRN blockade promotes CD8 antitumor cytotoxicity via MHCI regulation.

a Genetic strategy to induce spatially and temporally controlled GP33 expression by tamoxifen-mediated activation of CreERT2 in pancreatic cells harboring mutant Kras^G12D and loss of Tp53. Ptf1a^wt/flp; Kras^wt/FSF-G12D; p53^frt/frt (FKP) mice crossed to Gt(ROSA)26Sor^{tm3(CAG-Cre/ERT2)Das} (R26^{FSF-CAG−CreERT2}) and Gt(ROSA)26Sor^{tmloxP-STOP-loxP-GP-IRES-YFP} (R26^LSL-GP) strains to generate FKPC2GP mice. b Experimental setup for co-culture of GP82, LCMV-gp33-expressing cell line derived from FKPC2GP tumor, and the gp33-reactive T cells isolated from the spleen of P14-TCR-Tg mice. c LCMV-gp33 (GP) expression in GP82 cells treated with tamoxifen (25 μM) or vehicle control DMSO for 2 days was assessed by flow cytometry (n = 4 independent experiments). Two-tailed Mann–Whitney test. Tam: Tamoxifen. d Cellular PGRN level in GP82 cells treated with or without PGRN Ab or mIg (100 μg/ml) was assessed by flow cytometry (n = 4 independent experiments). One-way ANOVA, Kruskal–Wallis test. e Surface expression of MHCI marker H-2Db on GP82 cells treated with or without PGRN Ab or mIg (100 μg/ml) was assessed by flow cytometry (n = 6 independent experiments). One-way ANOVA, Kruskal–Wallis test. f IF staining of MHCI marker H-2Db of GP82 upon treatment with or without PGRN Ab or mIg (100 μg/ml). White arrowheads indicate the membraneous staining of MHCI. (n = 3 independent experiments). Representative images are shown. g Microscopic images of GP82 cells and LCMV-gp33-reactive T cells (CFSE-labeled, green) after 2 days of co-culture. When anti-MHCI (H-2Db) neutralizing antibody (MHCI Ab, MAb) was included in the treatment, MHCI Ab was added 1 h after PGRN Ab (PAb) treatment. T cells were then added 1 h after MHCI Ab treatment. White arrowheads indicate T-cell clusters accumulated at GP82 cells. (n = 6 independent experiments). Representative images are shown. h Cytotoxicity level (PI⁺ %) of GP82 cells upon co-culture with LCMV-gp33-reactive T cells (n = 6 independent experiments performed with LCMV-gp33-reactive T cells isolated from six different mice). T: T cells; Tu: GP82 tumor cells; Tu*GP: LCMV-gp33-induced GP82 tumor cells; PAb: PGRN Ab (100 μg/ml); MAb: MHCI Ab (100 μg/ml). One-way ANOVA, Kruskal–Wallis test. For PAb vs Mab: Two-tailed Mann–Whitney test. i Percentage of CD8⁺ cells that are positive for cytotoxic markers granzyme B (GzmB), TNF, and IFNg (% in total T cells) assessed by flow cytometer (n = 6 independent experiments performed with LCMV-gp33-reactive T cells isolated from six different mice). One-way ANOVA, Kruskal–Wallis test. For PAb vs Mab: Two-tailed Mann–Whitney test. Mean + SD are shown. MFI mean fluorescence intensity. Scale bar unit: μm.