Journal: bioRxiv

Article Title: PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

doi: 10.64898/2026.03.09.710629

Figure Lengend Snippet: (A) Schematic depicting the experimental set up for the cell–SLB assay, where a PD1/Shp2 DKO Jurkat cell expressing h PD1-GFP along with mCherry- h Shp2 contacts an SLB presenting α- h CD3ε (Okt3) and h PDL1. (B) Fusion events of h PD1 microclusters. Left, a representative confocal image of h PD1 microclusters in an SLB-bound Jurkat ( h PD1-GFP) cell. Right, zoomed in time-lapse images of the boxed area shown on the left. Time zero indicates the beginning of image acquisition. (C) Left, a confocal image of mCherry- h Shp2 in the same cell as in B. Right, time-lapse images of the boxed area shown on the left. (D) FRAP of h PD1 microclusters. Left, a confocal image of h PD1 microclusters in an SLB-bound Jurkat ( h PD1-GFP) cell. Middle, zoomed in time-lapse images of the boxed area of the left image, with the circled cluster photobleached at time zero. Right, time course of the fluorescent intensity (FI) of the circled cluster normalized to the FI of the adjacent unbleached cluster. n = 3 cells. (E) FRAP of mCherry- h Shp2 in the same cell as in D. n = 3 cells. Scale bars: 5 µm. Data are mean ± SD.

Article Snippet: Biotin α-human CD3ε (clone Okt3, #317320), Alexa Fluor 647 α-human PD1 (clone NAT105, #367419), Alexa Fluor 488 α-mouse CD45 (clone 30-F11, #103121), BV421 α-mouse IFNγ (clone XMG1.2, #505829), and BV711 α-mouse TNFα (clone MP6-XT22, #506349) were purchased from BioLegend. α-mouse CD3ε (clone 145-2C11, #BE00011), α-mouse CD28 (clone 37.51, #BE0015-1), α-human PDL1 (Atezolizumab, #SIM0009), α-mouse PD1 (clone RMP1-14, #BE0146), and α-Trinitrophenol isotype control (#BE0089) were purchased from Bio X Cell.

Techniques: Expressing

Journal: bioRxiv

Article Title: PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

doi: 10.64898/2026.03.09.710629

Figure Lengend Snippet: (A) Schematic depicting a PD1 KO Jurkat cell expressing GFP-tagged h PD1 stimulated on an SLB containing α- h CD3ε (Okt3) and h PDL1. (B) Flow cytometry showing the α-PD1 or GFP fluorescence of Jurkat cells expressing the indicated h PD1 variants. (C) Left, representative confocal images of GFP-tagged h PD1 variants in SLB-bound Jurkat cells as depicted in A. Right, h PD1 cluster indices under the indicated conditions. n = 58-79 cells. Scale bars: 5 µm. Data are mean ± SD. ***P < 0.001; student’s t-test.

Article Snippet: Biotin α-human CD3ε (clone Okt3, #317320), Alexa Fluor 647 α-human PD1 (clone NAT105, #367419), Alexa Fluor 488 α-mouse CD45 (clone 30-F11, #103121), BV421 α-mouse IFNγ (clone XMG1.2, #505829), and BV711 α-mouse TNFα (clone MP6-XT22, #506349) were purchased from BioLegend. α-mouse CD3ε (clone 145-2C11, #BE00011), α-mouse CD28 (clone 37.51, #BE0015-1), α-human PDL1 (Atezolizumab, #SIM0009), α-mouse PD1 (clone RMP1-14, #BE0146), and α-Trinitrophenol isotype control (#BE0089) were purchased from Bio X Cell.

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

doi: 10.64898/2026.03.09.710629

Figure Lengend Snippet: (A) Left, schematic depicting a Jurkat cell expressing h PD1-GFP along with either mCherry-tagged h Shp2 WT or h Shp2 REKE , contacting an SLB presenting Okt3 and h PDL1. Right, flow cytometry showing GFP and mCherry fluorescent intensities of the indicated Jurkat cells. (B) Left, representative confocal images showing PD1 (green) and Shp2 (magenta) at the interface of Jurkat cells and SLBs shown in A. Right, violin plots showing the synaptic fluorescent intensities or clustering indices of PD1 or Shp2. (C) Schematic depicting a P14:DC2.4 coculture assay. Cas9 + P14 cells were retrovirally transduced with Shp2-targeting gRNA, along with exogenous Shp2 ( ex Shp2: mScarlet- m Shp2 WT or m Shp2 REKE ) and exogenous PD1 ( ex PD1: m PD1 WT or m PD1 h ICD), and cocultured with gp 33-41 peptide-pulsed PDL1 + DC2.4 cells. (D) Flow cytometry showing the PD1 expression on the indicated P14 cells prepared in C. (E) IBs showing the endo Shp2 or ex Shp2 expression in the indicated P14 cells prepared in C. (F) % inhibition on IFNγ secretion under the indicated conditions calculated from . (G) Schematic depicting an ACT murine melanoma model. PD1 KO Cas9 + P14 cells were retrovirally transduced with Ptpn11 -targeting gRNA, alongside ex PD1 ( m PD1 h ICD or m PD1 h ICD FF ) and ex Shp2 (mScarlet- m Shp2 WT or m Shp2 REKE ), and intravenously injected (i.v.) into host mice bearing B16-gp33 melanoma. n = 6–8 mice. (H) Tumor growth curves in mice that received P14 cells expressing the indicated ex PD1 and ex Shp2. (I) Endpoint tumor sizes in H. Scale bars: 5 μm. Data are mean ± SD. *P < 0.05; **P < 0.01; ns, not significant; student’s t-test (B, F, and I) or two-way ANOVA (H).

Article Snippet: Biotin α-human CD3ε (clone Okt3, #317320), Alexa Fluor 647 α-human PD1 (clone NAT105, #367419), Alexa Fluor 488 α-mouse CD45 (clone 30-F11, #103121), BV421 α-mouse IFNγ (clone XMG1.2, #505829), and BV711 α-mouse TNFα (clone MP6-XT22, #506349) were purchased from BioLegend. α-mouse CD3ε (clone 145-2C11, #BE00011), α-mouse CD28 (clone 37.51, #BE0015-1), α-human PDL1 (Atezolizumab, #SIM0009), α-mouse PD1 (clone RMP1-14, #BE0146), and α-Trinitrophenol isotype control (#BE0089) were purchased from Bio X Cell.

Techniques: Expressing, Flow Cytometry, Co-culture Assay, Transduction, Inhibition, Injection

Journal: bioRxiv

Article Title: PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

doi: 10.64898/2026.03.09.710629

Figure Lengend Snippet: (A) Schematic depicting a Jurkat cell expressing h PD1-GFP with either mCherry- h Shp2 WT or h Shp2 REKE , cocultured with SEE-loaded, h PDL1-expressing, CD80 KO Raji cells. (B) Left, IL-2 secreted in the Jurkat:Raji coculture shown in a in the presence or absence of α- h PDL1. Right, % inhibition of IL-2 secretion mediated by the indicated h PD1 variant calculated from the left. [SEE] = 0.06 ng/mL. n = 3 independent experiments. Data are mean ± SD. ***P < 0.001; student’s t-test.

Article Snippet: Biotin α-human CD3ε (clone Okt3, #317320), Alexa Fluor 647 α-human PD1 (clone NAT105, #367419), Alexa Fluor 488 α-mouse CD45 (clone 30-F11, #103121), BV421 α-mouse IFNγ (clone XMG1.2, #505829), and BV711 α-mouse TNFα (clone MP6-XT22, #506349) were purchased from BioLegend. α-mouse CD3ε (clone 145-2C11, #BE00011), α-mouse CD28 (clone 37.51, #BE0015-1), α-human PDL1 (Atezolizumab, #SIM0009), α-mouse PD1 (clone RMP1-14, #BE0146), and α-Trinitrophenol isotype control (#BE0089) were purchased from Bio X Cell.

Techniques: Expressing, Inhibition, Variant Assay