Extended Data Fig. 2: Overexpression of NFAT5 in tumor specific P1A cell led to reduced tumor control.
a) The four murine isoforms of NFAT5 were aligned on the software Geneious using the entries from GenBank and Ensemble. Domains described by Cheung et al. were aligned against mouse isoforms 203, whose length corresponds to human isoform C. NES: Nuclear export signal, AD1: Activation domain 1, AES: Auxiliary export signal, DRL: DNA recognition loop, DD: Dimerization domain, RHD: Rel homology region, AD2: Activation domain 2, AD3: Activation domain 3. b) Retroviral vector where the various isoforms were cloned. c) Nfat5 mRNA levels in CD8+ T cells transduced with a vector encoding for NFAT5 isoform A (NFAT5 A) (left) or control eGFP (right) and sorted by flow cytometry according to GFP expression. d) Timeline of the experiment. Activated TCRP1A CD8+ T cells were transduced, sorted on the base of GFP expression and transferred into Rag1−/−B10D2 mice seven days post P511 tumor engraftment. CD8+ T cells were sorted for RNA-sequencing on day 14. e) Tumor growth in mice transferred with TCRP1A CD8+ T cells transduced and sorted according to GFP expression with the indicated constructs (control eGFP, NFAT5 A, NFAT5 A lacking the DNA binding domain (DBD), NFAT5 D or NFAT1 CA-RIT). n = 5 mice per group (left) n = 5 mice eGFP, n = 6 mice NFAT5 IsoA, n = 7 mice NFAT5 noDBD, and n = 5 NFAT5 IsoD (middle, right). Two-ways ANOVA was performed. Error bars represent SEM. f) Bioluminescence of the luciferase-expressing TCRP1A CD8+ T cells after injection of the mice with luciferin. Each dot is representing one mouse. g) PC analysis of sorted GFP+ CD8+ TILs transduced with control eGFP, NFAT5 A, or NFAT1 CA-RIT. Each dot is representing one mouse. h, Venn diagram showing the number of genes upregulated in NFAT5 A transduced CD8+ TILs, NFAT1 CA-RIT transduced TILs, or both.
