Figure 6. NLGP protects CD8⁺ T cells from anergy within TME.

MNCs were isolated from normal mice and exposed to PBS-TME/NLGP-TME for 120 hrs and ionomycin for 48 hrs, as a positive control in vitro. A. Then CD8⁺ T cells were purified by MACS to isolate total RNA. RT-PCR analysis was performed for different anergy related genes, B.1. CD8⁺ T cells were directly purified from tumors of PBS and NLGP treated mice in in vivo condition and total RNA was isolated. Different anergy related genes were analyzed at transcriptional level by RT-PCR, B.2. Densitometric analysis was performed in each case. C. pNFAT and NFAT were analyzed at protein level, purified from CD8⁺ T cells as mentioned in A, by Western blotting D.1. MNCs were isolated from normal mice and exposed to PBS-TME and NLGP-TME for 120 hrs in vitro. Then CD8⁺ T cells were purified by MACS to isolate total RNA and protein. RT-PCR analysis was performed for Fas-R, cFlip, D.2. Densitometric analysis was performed in each case. E. and Western blot for Fas-R, cFlip, FasL, F. and activated Caspase3 and Caspase8. G. MNCs were purified from tumors of PBS and NLGP treated mice and assessed for FasR⁺CD8⁺ T cells by flow cytometry. *p<0.001.