Journal: Journal for Immunotherapy of Cancer
Article Title: Epitope spreading driven by the joint action of CART cells and pharmacological STING stimulation counteracts tumor escape via antigen-loss variants
doi: 10.1136/jitc-2021-003351
Figure Lengend Snippet: Perf-deficiency in CART cells impaired the Ag spreading and the local and abscopal effects of CART/STING-L combination. (A) WT or Perf-KO CD8 or CD4 T cells expressing CART-PSMA or CART-CD19 (mock) were cultured in vitro with B16-PSMA tumor cells at a ratio of 1:1 (CART cell:tumor cell) (test wells). As a control, tumor cells were cultured alone (medium, ctrl wells). The percentage of dead tumor cells (7AAD + CD45 − ) in total tumor cells (CD45 − ) was analyzed 12 hours later by flow cytometry. The graph shows the killing activity of T cells as percentage of specific lysis calculated as described in the Methods section. Representative flow cytometry dot plots showing percentage of dead tumor cells in the culture conditions with WT or Perf-KO CART-PSMA CD8 T cells are also included. (B, C) WT mice (n=10 mice/group) bearing bilateral tumors were left NT or received an intravenous dose of WT or Perf-KO CART-gp75 cells along with STING-L as in . (B) Size of right tumor on day 4 of ACT (day 8 of tumor implantation) before STING-L injection. (C) Tumor growth kinetics of STING-L-treated and STING-L-non-treated tumors (left and middle panels) and survival rate curves (right). (D) Percentage of M8 (left) and OVA (right) tetramer + cells within endogenous peripheral blood CD8 T cells on day 14 of tumor implantation as in . (A) One experiment was representative of two experiments. (B–D) Two compiled experiments. Data are represented as mean±SD (A) and mean±SEM (B, C, left and middle panels; D). Non-parametric Mann-Whitney test, two-tailed (A, B, D). Non-linear regression (curve fit) (C, left and middle panels). Log-rank (Mantel-Cox) test (C, right panel). ACT, adoptive T-cell transfer; Ag, antigen; CART, chimeric antigen receptor T; KO, knockout; ns, not significant; NT, untreated; PSMA, Perf, perforin; PSMA, prostate-specific membrane antigen; STING-L, stimulator of interferon gene ligand; WT, wild type.
Article Snippet: Untouched CD8 and CD4 T cells were sorted using the negative selection EasySep Mouse CD8 or CD4 T-cell isolation kits (StemCell), respectively.
Techniques: Expressing, Cell Culture, In Vitro, Control, Flow Cytometry, Activity Assay, Lysis, Tumor Implantation, Injection, MANN-WHITNEY, Two Tailed Test, Knock-Out, Membrane
