Journal: Journal for Immunotherapy of Cancer
Article Title: Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in ovarian cancer enhances therapeutic efficacy
doi: 10.1136/jitc-2021-003959
Figure Lengend Snippet: Expression of a Fas-IFP enhances accumulation of CD8 T cells in vivo (A) Schematic of monomeric endogenous Fas receptor, a Fas-4-1BB tm immunomodulatory fusion protein, and vectors encoding TCR 1045 or TCR 1045 and a Fas-4-1BB tm immunomodulatory fusion protein (IFP). Genes are separated by P2A or codon-optimized (co) P2A elements to ensure equimolar expression. (B) CD95 (Fas) and Vβ9 expression on transduced P14 Thy1.1 + CD8 T cells. T cells were transduced with retroviral vectors containing TCR 1045 or TCR 1045 and Fas-4-1BB tm IFP and re-stimulated with irradiated splenocytes pulsed with Msln 406-414 in the presence of IL-2. Cells were screened by flow cytometry 7 days after restimulation. Data representative of >5 independent experiments. Quadrant values indicate mean and SD from five independent experiments. (C) Intracellular cytokine staining for IL-2 production by P14 Thy1.1 + CD8 T cells expressing either TCR 1045 (gray) or TCR 1045 and Fas-4-1BB tm IFP (red) after 5-hour stimulation with Msln 406-414 peptide presented as a percent of engineered T cells (left panel) and as geometric mean fluorescence intensity (gMFI, right panel). Cumulative data from three independent experiments (1–3 technical replicates per experiment). Unpaired t-test, **p=0.0040. n.s.=not significant. (D, E) In vitro intracellular cytokine staining for IL-2 production by P14 Thy1.1 + TCR 1045 (gray) or TCR 1045 /Fas-4-1BB tm IFP (red) CD8 T cells, with or without FasL blockade (clone: Kay10), after 5 hour stimulation with Msln 406-414 peptide. (D) Representative flow cytometry plots from one experiment. The average percentage of IL-2 + cells is displayed for each condition (samples run in triplicate). (E) Data are displayed as a percent difference between cells treated with FasL blocking Ab (clone: Kay10) compared with cells treated with isotype control Ab. Cumulative data from four independent experiments. Paired t-test. *P<0.05. (F) Immunohistochemistry (IHC) staining for CD3 + T cells in ID8 VEGF tumors 21 days after T cell transfer. P14 Thy1.1 + CD8 T cells expressing TCR 1045 or TCR 1045 and Fas-4-1BB tm IFP were injected i.p. into ID8 VEGF tumor-bearing mice. Mice were pre-treated with 180 mg/kg cyclophosphamide >6 hours prior to cell transfer. All mice received 1×10 7 engineered T cells, 5×10 7 irradiated splenocytes pulsed with Msln 406-414 peptide, and daily IL-2 s.c. for 10 days (1×10 4 IU). CD3 staining in untreated mice (white bar) included as a reference for endogenous T cell infiltration. Cumulative data from three independent experiments, n=7 per group. One-way ANOVA Tukey’s multiple comparisons test, *p=0.0442. n.s.=not significant G) Intracellular flow cytometry staining for Ki67 expression in Thy1.1 + CD8 + Vβ9 + T cells 7 days after in vitro restimulation of engineered T cells. Data from four independent experiments. Unpaired t-test. H–N) Thy1.1 + or Thy1.1/1.2 + P14 T cells expressing TCR 1045 (gray) or TCR 1045 and Fas-4-1BB tm IFP (red) were co-transferred at a 1:1 ratio into ID8 VEGF tumor-bearing mice with 5×10 7 irradiated splenocytes pulsed with Msln 406-414 peptide, and daily s.c. IL-2 (1×10 4 IU). at 3 or 7 days after co-transfer, T cells were isolated from tumors, enumerated and evaluated by flow cytometry for indicated markers. (H) Proportion of donor T cells within ID8 VEGF tumors after co-transfer. Data pooled from three independent experiments, n=8 (day 3) or n=7 (day 7) mice per group. Two-way ANOVA for multiple comparisons. n.s. (TCR 1045 from pre-transfer to D3 and TCR 1045 /Fas-4-1BB tm IFP pre-transfer to D3). ****p<0.0001 (TCR 1045 from D3 to D7 and TCR 1045 /Fas-4-1BB tm IFP from D3 to D7). (I) Quantification of intratumoral donor T cells. Counts of the cotransferred T cell populations from each individual mouse are connected with a line. Paired t-test. *P<0.05. (J) Flow cytometry staining of intratumoral endogenous and donor T cells three or 7 days after transfer for Ki67 expression and EdU incorporation. EdU was injected i.p. into mice 24 hours prior to T cell isolation. 1-way ANOVA for multiple comparisons. ****p<0.0001. n.s.=not significant. (K) Quantification of EdU + Ki67 + intratumoral donor T cells. Counts of the cotransferred cell populations from each individual mouse are connected with a line. Paired t-test. *P<0.05. (L) Flow cytometry staining of intratumoral donor T cells three or 7 days after transfer for Annexin V and Propidium Iodide (PI). Cell populations from the same mouse are connected with a line. Data from three independent experiments, n=8 (day 3) or n=9 (day 7). Paired t-test. **P<0.01. (M) qPCR for Bcl2 expression in TCR 1045 (gray) or TCR 1045 /Fas-4-1BB tm IFP (red) T cells 8 days after restimulation in vitro. Data pooled from four independent experiments. Unpaired t-test **p=0.0016. (M) Flow cytometry staining of intratumoral engineered T cells 3 days after transfer for intracellular Bcl2. Data are displayed relative to expression in endogenous CD8 T cells from the spleen of the same mouse. Cell populations from the same mouse are connected with a line. Data from three independent experiments, n=5. Paired t-test. *P=0.0376. All error bars represent SD. ANOVA, analysis of variance.
Article Snippet: The codon-optimized murine Vβ9 and Vα4 TCR chains, connected by a porcine teschovirus-1 2A element (“P2A”; Life technologies), recognize the Msln 406-414 epitope presented in H2-D b , and were cloned from the Mig-R1 retroviral vector into the pENTR vector and subsequently Gateway cloned into the pMP71 retroviral vector for all TCR 1045 studies.
Techniques: Expressing, In Vivo, Transduction, Irradiation, Flow Cytometry, Staining, Fluorescence, In Vitro, Blocking Assay, Immunohistochemistry, Injection, Isolation, Cell Isolation
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