Journal: Nature

Article Title: Expanding the cytokine receptor alphabet reprograms T cells into diverse states

doi: 10.1038/s41586-025-09393-1

Figure Lengend Snippet: a , Orthogonal chimeric receptor constructs were introduced into WT mouse T cells via YFP-encoding retroviral vectors. Expression levels of each receptor were assessed by YFP fluorescence using flow cytometry. b–e , Orthogonal receptor expressing (YFP + ) C57BL/6 WT CD3 + T cells were stimulated with MSA fusions of MSA-oIL2 for 20 min. Cells were analysed for pSTAT using flow cytometry. Data are presented as the MFI of pSTAT from YFP + T cells plotted against the log 10 concentration of MSA-oIL2. b , pSTAT-1, -3, -4, -5, and -6 signalling dose-response curves of orthogonal IL-2Rβ ECD–ICD from o4R, o7R, o9R, and o21R transduced T cells (n = 2 biologically independent samples). c , pSTAT-1, -3, and -5 signalling dose-response curves of orthogonal IL-2Rβ ECD–ICD from oIFNAR2, oIFNGR1, and oIFNLR1 transduced T cells (n = 2 biologically independent samples). d , pSTAT-1, -4, and -5 signalling dose-response curves of o10R, o20R, and o22R transduced T cells (n = 2 biologically independent samples). e , pSTAT-1, -3, and -5 signalling dose-response curves of orthogonal IL-2Rβ ECD–ICD from oGCSFR and oEPOR transduced T cells (n = 3 biologically independent samples). All data represent mean ± s.e.m.

Article Snippet: Platinum-E (Plat-E) and Platinum-GP (Plat-GP) retroviral Packaging Cell Line were purchased from Cell Biolabs.

Techniques: Construct, Retroviral, Expressing, Fluorescence, Flow Cytometry, Concentration Assay

Journal: Nature

Article Title: Expanding the cytokine receptor alphabet reprograms T cells into diverse states

doi: 10.1038/s41586-025-09393-1

Figure Lengend Snippet: a – f , Orthogonal chimeric receptors were introduced into YT-1 cells using YFP-encoding retroviral vectors. Cells were stimulated with MSA-human orthogonal IL2 (MSA-hoIL2) for 20 min and analysed for pSTAT via flow cytometry (n = 2 biological independent samples). Data are presented as the MFI of pSTAT from YFP + T cells, plotted against the log 10 concentration of MSA-hoIL2. a – c , pSTAT1 ( a ), pSTAT3 ( b ), and pSTAT5 ( c ) signalling dose-response curves for human o2R (ho2R), ho10R, ho22R, and hoGCSFR-transduced YT-1 (CD25 + ) cells. d – f , pSTAT1 ( d ), pSTAT3 ( e ), and pSTAT5 ( f ) signalling dose-response curves for ho2R, ho10R, ho22R, and hoGCSFR-transduced γ c knockout (KO) YT-1 cells. g , ho2R were transduced into CD25 + YT-1 cells. Cells were stimulated with orthogonal IL-2 (oIL-2) or oIL-2-TR (carrying the Q126T and S130R γ c interface mutations) for 20 min and analysed for pSTAT via flow cytometry. Data are presented as the MFI of pSTAT from YFP + T cells. h , CD19 CAR T cells transduced with the indicated CAR-encoding construct (n = 2 biological independent samples). CD19_28z: a murine CD19 CAR (1D3 clone); CD19_28z_YRHQ: CD19 CAR with a STAT3-binding motif (YRHQ) at the C-terminus; CD19_28z_22 R CTD: CD19 CAR with the C-terminal domain (CTD) of IL-22R fused at the C-terminus. CAR T cells were stimulated with CD19 + B cells or treated with IL-21 (50 ng/mL) served as a control. Shown are MFI of pSTAT3 over time. i , Normalized mRNA expression levels of indicated genes in mouse T cells transduced with o2R lacking ICD (NoICD) or oIFNGR1, following 6- or 48-hour stimulation (unstimulated [NS] or stimulated with MSA-oIL-2) (n = 1 biological independent sample). All data represent mean ± s.e.m. and are analysed by one-way ANOVA with Tukey’s post-test ( h ).

Article Snippet: Platinum-E (Plat-E) and Platinum-GP (Plat-GP) retroviral Packaging Cell Line were purchased from Cell Biolabs.

Techniques: Retroviral, Flow Cytometry, Concentration Assay, Knock-Out, Transduction, Construct, Binding Assay, Control, Expressing

Journal: Nature

Article Title: Expanding the cytokine receptor alphabet reprograms T cells into diverse states

doi: 10.1038/s41586-025-09393-1

Figure Lengend Snippet: a , NY-ESO-1 TCR and ho4R constructs were introduced via retroviral vectors. Expression levels of NY-ESO-1 TCR and ho4R were assessed by EGFR and YFP expression using flow cytometry, respectively. Shown are representative flow cytometry plots of transduction efficiency. b , Frequencies of IFN-γ + subpopulation among CD4 + (left) and CD8 + (right) NY-ESO-1 TCR-T cells (n = 4 biologically independent samples). c , MFI of CXCR3 (left) and CCR4 (right) among CD3 + NY-ESO-1 TCR-T cells (n = 4 biologically independent samples). d , The experimental timeline for Fig. . e , Experimental setting is described in Fig. . Relative body weight of mice post indicated treatment. f , Representative flow cytometry histograms showing GATA-3 expression level. g , NT or ho4R NY-ESO-1 TCR-T cells (unmodified, GATA3 knockout, or treated with the indicated antibodies) were co-cultured with A375 cells at an E:T ratio of 1:2 for 48 h (n = 3 biologically independent samples). Shown are the counts of viable A375 tumour cells. All data represent mean ± s.e.m. and are analysed by two-tailed Student’s t-test ( b , c ) or one-way ANOVA with Tukey’s post-test ( g ). The diagram in d was created using BioRender.

Article Snippet: Platinum-E (Plat-E) and Platinum-GP (Plat-GP) retroviral Packaging Cell Line were purchased from Cell Biolabs.

Techniques: Construct, Retroviral, Expressing, Flow Cytometry, Transduction, Knock-Out, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Expanding the cytokine receptor alphabet reprograms T cells into diverse states

doi: 10.1038/s41586-025-09393-1

Figure Lengend Snippet: a , CD19 CAR, ho22R, hoGCSFR constructs were introduced via retroviral vectors. Expression levels of CD19 CAR were measured by Myc-tag fluorescence using flow cytometry. Expression levels of ho22R and hoGCSFR were measured by YFP fluorescence using flow cytometry. Shown are representative flow cytometry plots of transduction efficiency. b , pSTAT-1, -3, -4, and -5 signalling dose-response curves (n = 2 biologically independent samples). Data are presented as the MFI of pSTAT against the log 10 concentration of MSA-hoIL2. c , d , CD19 CAR T, ho22R CD19 CAR T, and hoGCSFR CD19 CAR T cells were cocultured with NALM6-Luciferase (NALM6-Luc) cells or Raji cells at different E:T ratios in the presence of MSA-hoIL2 (100 nM) for 24 h (n = 3 biologically independent samples). Shown are the percentages of killing of NALM6-Luc cells ( c ) and Raji cells ( d ). e–g , CD19 CAR T, ho22R CD19 CAR T, and hoGCSFR CD19 CAR T cells were cultured in MSA-hoIL2 (100 nM) supplemented T cell medium for 3 days (n = 3 biologically independent samples). e , Shown are MFI of CD62L and CD45RA among CD19 CAR T cells. f , Representative flow cytometry plots showing the gating strategy for CD45RA + CD62L + CCR7 + CD95 + subpopulations in NT, hoGCSFR, and ho22R CD3 + NY-ESO-1 TCR-T cells. g , Frequencies of CD45RA + CD62L + CCR7 + CD95 + cells. h–j , NT and oGCSFR CD8 + human T cells were stimulated with MSA-hoIL2 (500 nM) for 72 h, followed by flow cytometry analysis (n = 6 biologically independent samples). h , Representative flow cytometry plots of Mac-1 + and CD66b + cells among CD8 + T cells. i , j , Shown are the frequencies of Mac-1 + ( i ) and CD66b + ( j ) cells among CD8 + T cells. All data represent mean ± s.e.m. and are analysed by two-tailed Student’s t-test ( i , j ), one-way ANOVA ( g ), or two-way ( c–e ) ANOVA with Tukey’s post-test.

Article Snippet: Platinum-E (Plat-E) and Platinum-GP (Plat-GP) retroviral Packaging Cell Line were purchased from Cell Biolabs.

Techniques: Construct, Retroviral, Expressing, Fluorescence, Flow Cytometry, Transduction, Concentration Assay, Luciferase, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Expanding the cytokine receptor alphabet reprograms T cells into diverse states

doi: 10.1038/s41586-025-09393-1

Figure Lengend Snippet: a , NY-ESO-1 TCR, ho2R, ho22R, and hoGCSFR constructs were introduced via retroviral vectors. Expression levels were measured by EGFR (NY-ESO-1 TCR) or YFP fluorescence (ho2R, ho22R, and hoGCSFR) using flow cytometry. Shown are representative flow cytometry plots of transduction efficiency. b , pSTAT-1, -3, -4, and -5 signalling dose-response curves (n = 2 biologically independent samples). Data are presented as the MFI of pSTAT against the log 10 concentration of MSA-hoIL2. c , Fold growth of CD3 + NY-ESO-1 TCR-T cells post 3-day culture in the presence of MSA-hoIL2 (100 nM) (n = 4 biologically independent samples). d–f , NT T cells, NY-ESO-1 TCR-T cells, or NY-ESO-1 TCR-T cells transduced with ho2R, ho22R, or hoGCSFR were cocultured at a 1:1 E:T ratio with the HLA*0201 + NY-ESO-1 + melanoma cell line (nRFP-M407) in the presence of MSA-hoIL2 (100 nM). nRFP-M407 cells were reintroduced after 48 h of coculture (blue arrows) in the presence of MSA-hoIL2 (100 nM). After three rounds of tumour challenge, cells were collected for flow cytometry analyses. d , Shown is the normalized tumour cell confluence (n = 4 biologically independent samples). e , Frequencies of Granzyme B + (left) and IFN-γ + (right) among CD3 + NY-ESO-1 TCR-T cells (n = 3 biologically independent samples). f , Frequencies of CD45RA + CD62L + CCR7 + CD95 + among CD3 + NY-ESO-1 TCR-T cells (n = 3 biologically independent samples). g , Experimental setting is described in Fig. . Shown are the individual tumour growth curves. Indicated are the numbers of tumour-free mice per total number of mice in each group. All data represent mean ± s.e.m. and are analysed by one-way ANOVA ( e , f ) or two-way ( c , d ) ANOVA with Tukey’s post-test.

Article Snippet: Platinum-E (Plat-E) and Platinum-GP (Plat-GP) retroviral Packaging Cell Line were purchased from Cell Biolabs.

Techniques: Construct, Retroviral, Expressing, Fluorescence, Flow Cytometry, Transduction, Concentration Assay