Fig. 6

Tumors upregulate NMD to evade ICB therapy induced immune response. (A) Top: treatment schedule. C57/BL6 mice were implanted with Panc02.gCtrl on the right flank and SMG1^KD on the left flank. Both cell lines expressed the NMD reporter plasmid that contains SIINFEKL under the control of a PTC, mimicking a tumor antigen under NMD pressure. Anti CTLA-4 + anti PD-1 combination treatment was injected intraperitoneally as indicated. Bottom: On day 14 mice were sacrificed and cells from tumor-draining lymph nodes were isolated and IFN-γ ELISPOT assay against SIINFEKL peptide was carried out. Human gp100-derived peptide KVPRNQDWL was used as negative control. n = 3-6/group. (B) Individual ELISPOT wells from (A) classified in strong, medium or non-responders. (C) Treatment schedule of (D). (D) C57/BL6 mice were implanted with Panc02.gCtrl (right flank) or SMG1^KD (left flank) cells expressing luciferase-SIINFEKL reporter plasmid and radiance was measured over time. Mice were treated with 100 μg of each: anti CTLA-4 and anti PD-1 antibodies (A). On day 15 we depleted CD8 and CD4 T cells by injecting CD4 and CD8 antibodies (200 μg of each, clones GK1.5 and 53-6.7 respectively). n = 7. (E) Images showing luciferase intensity on days 7, 15 and 18. (F) luciferase levels on day 18 comparison between control tumor (left) and SMG1^KD (right) from (F). p-values are shown for 2-way ANOVA with Bonferroni’s post-hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Mechanism proposed for the luciferase changes observed in anti-CTLA-4 + anti-PD-1-treated Panc02.gCtrl tumors. ICB therapy elicits a strong immune response and is sensed by the tumor cell. Cell with unaltered NMD trigger upregulation of NMD (reflected in a decrease of luciferase signal) in tumors to repress the presentation of PTC-controlled antigens (SIINFEKL in our scenario) by degrading their mRNAs via this surveillance mechanism. When the immune response is evaded by tumor cells, they recover normal NMD activity which we detected as a luciferase signal increase. (H) Mechanism proposed for anti-CTLA-4 + anti-PD-1-treated mice bearing NMD-reporter-expressing Panc02.SMG1^KD tumor cell line. In contrast with control cells (C), SMG1^KD cells are unable to modulate NMD activity. In this case, the immune response is capable of eliminating SIINFEKL⁺ tumor cells