Journal: bioRxiv

Article Title: Dual-inactivation of Regnase-1 and SOCS1 rewires exhausted CD8 + T cell fate to enhance anti-tumor functionality

doi: 10.64898/2026.01.21.700812

Figure Lengend Snippet: CRISPR/Cas9 engineered TIL (eTIL ® ) were manufactured to inactivate SOCS1 (KSQ-001EX), Regnase-1 (sgRegnase-1), or both SOCS1 and Regnase-1 (KSQ-004EX) with non-electroporated controls used as a benchmark (No EP). (A) Percent killing of A375-mOKT3 tumor spheroids by indicated eTIL. (B) Production of IFNγ from during eTIL co-culture with A375-mOKT3 tumor spheroids. (C) Repeat stimulation assay assessing the ability of eTIL manufactured from a treatment-refractory melanoma donor to kill A375-mOKT3 cells following each stimulation over time. (D) eTIL bulk RNA-Seq, with Principle Components (PC) PC1, PC2 and PC3 depicted. No EP contains 30 independent donors; KSQ-001EX contains 26 independent donors; sgRegnase-1 contains 13 independent donors, and KSQ-004EX contains 25 independent donors. (E) Select DEGs between pairwise eTIL comparisons are depicted, with log fold change depicted by heatmap. (F) The number of DEGs between the eTIL comparisons are depicted by Venn diagram. (G) Transcription factor activity analysis from eTIL bulk RNA-Seq (H) Heatmap of log fold changes of top DEGs between edited TILs versus their respective controls in both human and mouse. Statistical significance between treatment groups was determined using a two-way ANOVA in with ns = no significance, * = p value < 0.05, ** = p value < 0.01, and *** = p value < 0.001. For and , the * in the heatmap represent the adj-p value (Benjamini Hochberg) for pairwise differential expression analysis with DESeq2 where *** = adj-p value < 0.001; ** = 0.001- 0.01; * = 0.01-0.05 and ‘.’ = 0.05-0.1. For , the *s represent adj-p values (Benjamini Hochberg) for transcription factor activity analysis. is representative of n=16 independent donors, is representative of n=7 independent donors. See also Figure S10.

Article Snippet: The A375 human melanoma cell line was obtained from ATCC and engineered to express either a low-affinity or high-affinity membrane associated anti-CD3 binding domain from clone OKT3 (mOKT3) together with RFP as previously described (Schlabach et al. 2023), with the low-affinity A375-mOKT line used for in vitro spheroid cytotoxicity and IFN- release assays, and the high-affinity A375-mOKT3 line used for the chronic stimulation assay[ ].

Techniques: CRISPR, Co-Culture Assay, RNA Sequencing, Activity Assay, Quantitative Proteomics