Extended Data Fig. 10: Human chimeric orthogonal IL-2Rβ-ECD–IL-9R-ICD drives a stem-like phenotype and polyfunctionality in T cells even after repetitive antigen-specific tumour challenge.
From: Potentiating adoptive cell therapy using synthetic IL-9 receptors
a, Flow plots of normal healthy donor T cells co-transduced with either ho2R and NYESO1-TCR (left) or ho9R and NYESO1-TCR (right), as detected by anti-human Vβ13.1 antibody that recognizes the β chain of the NYESO1-TCR clone 1G4 and YFP (internal marker of the o2R or o9R vectors). Co-transduction efficiencies are shown. b, pSTAT signalling dose response of untransduced or ho2R (red) or ho9R (purple) or untransduced (UTD; grey) expressing activated human T cells (donor 651) stimulated with MSA-hoIL2 (top panel) or MSA-hIL2 (bottom panel) for 20’. Data are shown as mean fluorescence ± SEM, n = 2. c, Gating strategy for immunophenotyping of YFP+ population of ho2R–NYESO1-TCR and ho9R–NYESO1-TCR T cells shown in Fig. 4c. d, Immunophenotype of ho2R–NYESO1-TCR and ho9R–NYESO1-TCR engineered human T cells after six days in culture with MSA-hoIL2 (1 μM). Shown are representative plots of CD45RA and CD27 expression gated on YFP+ T cells (top row), and CD95 and CCR7 expression gated on the CD45RA+CD27+ population (indicated by arrow and dotted line) corresponding to bar plot in Fig. 4c. e, Immunophenotype of ho2R–NYESO1 TCR and ho9R–NYESO1 TCR engineered human T cells after two days in culture with MSA-hoIL2 (1 μM). Shown is bar plot quantification of TSCM cells as a percentage of the YFP+ population (mean ±SD, n = 3 biological replicates/group). **, p < 0.01 (unpaired t-test, two-sided). f, T cells from Fig. 4e were collected and restimulated with either αCD3/αCD28 dynabeads, melanoma cell line M407 (HLA*0201+NY-ESO-1+) or M263 (HLA*0201+NY-ESO-1−). IFNγ, TNF and IL-2 were quantified among YFP+ CD4 T cells (CD8-) by ICS (mean ±SD, n = 3 biological replicates/group). g, Gating strategy for flow sorting of healthy donor T cells co-transduced with lentiviruses expressing anti-mesothelin CAR (M5) and ho2R-GFP/ho9R-GFP (left plots). Right plots indicate MFI of orthogonal IL-2Rβ in double-positive cells.
