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) Coomassie-stained SDS-PAGE of 0.5 μg of purified Shp2 variants (B) Coomassie-stained SDS-PAGE of 0.5 μg of purified PD1 variants.

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: Staining, SDS Page, Purification

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, confocal images of h Shp2 WT (green) mixed with the indicated h PD1 ICD (magenta) in LLPS buffer. Right, the OD 600 values of the indicated h Shp2: h PD1 ICD mixture. [Shp2] = [PD1 ICD ] = 8 μM. n = 3 independent experiments. (B) Time-lapse bright field and confocal images showing a condensate fusion event in a mixture of 8 μM h Shp2 WT (green) and 8 μM p- h PD1 ICD (magenta). Time zero marks the start of image acquisition. (C) Left, time-lapse confocal images showing FRAP of h Shp2 WT (green) and h PD1 ICD (magenta) within the indicated condensate, which was photo-bleached at time zero. Right, FI time courses in the bleached area. FI values were normalized to those of a non-bleached h Shp2:p- h PD1 ICD condensate. [Shp2] = [pPD1 ICD ] = 8 μM. n = 3 condensates. Scale bars: 5 μm (A, C) or 1 μm (B, C inset). 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:

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) Representative confocal images of h Shp2 C459E (green) mixed with either non-phosphorylated h PD1ICD or p- h PD1ICD (magenta). [Shp2] = [PD1 ICD ] = 8 μM. (B) Representative time-lapse confocal images of h Shp2:p- h PD1condensates containing the indicated Shp2 variant. Condensates were photo-bleached at time zero. [Shp2] = [PD1 ICD ] = 8 μM. (C) Time courses of Shp2 (left) or PD1 ICD (right) FI of the photo-bleached area in B. FIs were normalized to a non-bleached PD1:Shp2 condensates nearby. n = 3 condensates. Scale bars: 5 μm (A) or 1 μm (B). 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: Variant Assay

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) Cartoon depicting a model in which pPD1 induces a close-to-open conformational transition in Shp2. The basic amino acids that mediate Shp2 self-association (R362/K364) are highlighted as “+”. (B) Spot matrix plots showing the OD 600 for h Shp2 WT mixed with either h PD1 ICD or p- h PD1 ICD across varying concentrations of Shp2 and NaCl. [PD1 ICD ] = 8 μM. From left to right, [Shp2] were 0, 2, 4, 8, 12 μM, from top to bottom [NaCl] were 75, 125, 200, 400, and 600 mM. (C) Confocal images of condensates formed by Shp2 WT and p- h PD1 ICD in LLPS buffer containing the indicated [NaCl]. (D) Time courses for the OD 600 of Shp2 WT :p- h PD1 ICD mixture before and after changes in [NaCl] upon injection of NaCl-containing LLPS buffer. [Shp2] = [PD1 ICD ] = 8 μM. (E) Effect of Shp2 REKE mutations on h PD1: h Shp2 LLPS. Left, OD 600 of h Shp2 WT or h Shp2 REKE mixed with indicated [p- h PD1 ICD ]. Right, representative confocal images of the condensates in h PD1: h Shp2 WT mixture and h PD1: h Shp2 REKE mixture containing 8 μM of each. n = 3 independent experiments. (F) Same as E except that m Shp2 variants were used. n = 3 independent experiments. 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: 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 Shp2 catalytic activity assay, where recombinant h Shp2 was incubated with p- h PD1 ICD to induce the open conformation. Shp2 mediated dephosphorylation of the substrate DiFMUP produces the fluorescent DiFMU. (B) Representative time course of [DiFMU] upon mixing with the indicated Shp2 variants. To prevent PD1:Shp2 LLPS, [ h Shp2] and [p- h PD1 ICD ] were kept at 20 nM and 30 nM, respectively. (C) Initial velocity (V0) of [DiFMU] increase plotted against [DiFMUP] for the indicated Shp2 variants, fitted by the Michaelis-Menten model. n = 3 independent experiments. (D) Dot plots summarizing K M and k cat values calculated from C. n = 3 independent experiments. (E) Same as A, except that pre-phosphorylated h CD3ζ (p- h CD3ζ) was used as a substrate. (F) Representative IB showing the time-course of phosphorylation status of h CD3ζ upon mixing with 4 μM p- h PD1 ICD and 0.4 μM h Shp2 WT or h Shp2 REKE . (G) Same as F, except that h CD3ζ was mixed with 4 μM p- h PD1 ICD and 4 μM h Shp2 WT or h Shp2 REKE . (H) Time course of % p- h CD3ζ-GFP calculated from F (left) or G (right). n = 3 independent experiments. (I) Left, representative confocal images of h Shp2 WT (orange) and GFP(0)-tagged proteins (green) mixed with p- h PD1 (magenta) under the LLPS condition. Right, violin plots summarizing the GFP enrichment in the Shp2 condensates shown on the left. n = 5 independent experiments. (J) Left, representative confocal images of synaptic PLA signals (magenta) and bright field signals (grey) of Jurkat cells co-expressing V5-tagged h PD1 and the indicated ALFA-tagged receptors and h Shp2 variants. Right, violin plots summarizing the number of PLA puncta in individual cells shown on the left. n = 118-171 cells. (K) Model depicting how PD1:Shp2 LLPS organizes the inhibitory signalosome. PD1 phosphorylation promotes Shp2 condensation into dynamic LLPS compartments that enhance PD1 clustering, maintain liquidity through catalysis, and achieve selective substrate inclusion (CD3ζ/CD28) and exclusion (TIGIT). Scale bars: 2 μm (I) or 10 μm (J). Data are mean ± SD. *P < 0.05; ***P < 0.001; ns, not significant; 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: Activity Assay, Recombinant, Incubation, De-Phosphorylation Assay, Phospho-proteomics, 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 Shp2 catalytic activity assay, where recombinant h Shp2 was incubated with p- h PD1 ICD to induce the open conformation. Shp2 mediated dephosphorylation of the substrate DiFMUP produces the fluorescent DiFMU. (B) Representative time course of DiFMU FI upon mixing with 20 nM h Shp2 WT and the varying concentrations of [p- h PD1 ICD ]. (C) Initial velocity (V0) of DiFMU FI increase plotted against [p- h PD1 ICD ] calculated from B.

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: Activity Assay, Recombinant, Incubation, De-Phosphorylation Assay

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: Left, representative confocal images of the mixture of p- h PD1 (magenta), h Shp2 WT (orange), and the indicated charge-modified GFP (green). Right, dot plots summarizing the GFP enrichment in the PD1:Shp2 condensates shown on the left. n = 5 independent experiments. Scale bars: 2 μm. Data are mean ± SD. ***P < 0.001; ns, not significant; 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: Modification

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: Flow cytometry showing the expression of V5-tagged h PD1 (left), ALFA-tagged receptors (middle), and mCherry-tagged h Shp2 (right) in the indicated Jurkat cells.

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: Flow Cytometry, 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) 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

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: OD 600 of 8 μM h Shp2 WT or m Shp2 WT mixed with either pre-phosphorylated h PD1 ICD or m PD1 ICD at the indicated concentration. n = 3 independent experiments. Data are mean ± SD. **P < 0.01; 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: Concentration Assay

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) Flow cytometry gating strategies to measure %IFNγ + TNFα + P14 cells in P14: DC2.4 cocultures. (B) Representative flow cytometry plots showing the IFNγ and TNFα intracellular staining in the indicated P14 cells in P14: DC2.4 cocultures. (C) Dot plots showing %IFNγ + TNFα + P14 cells under the indicated conditions calculated from B. (D) PD1-mediated % inhibition on % IFNγ + TNFα + P14 cells under the indicated conditions calculated from C. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; 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: Flow Cytometry, Staining, Inhibition

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: Flow cytometry histograms showing the expression of ex PD1 (left) and ex Shp2 (right) in the indicated P14 cells.

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: Flow Cytometry, Expressing

Journal: Oncoimmunology

Article Title: Prednisolone modulates CD8⁺ and regulatory T-cell activity to dampen response to immune checkpoint inhibitor therapy in melanoma

doi: 10.1080/2162402X.2026.2643494

Figure Lengend Snippet: Systemic immune activation is impaired early during treatment with PRED. (a) Left: Plasma levels of indicated cytokines early during PRED treatment (EDT; 0.9 mg/kg PRED or vehicle control). Each column represents a single mouse. Fluorescence intensity (FI) values were log 2 transformed and a heatmap was generated in Morpheus. The color scale indicates relative marker expression. Right: Volcano plot displaying differential cytokine expression between Combi-ICI + PRED vs Combi-ICI + DMSO-treated mice. Cytokine expression values were compared using limma’s moderated t-tests with Benjamini-Hochberg false discovery rate (BH-FDR) adjustment; p -values and q-values (BH-adjusted p -values) are shown in Supplementary Table S2. Red dots indicate downregulation and black dots, upregulation. Dotted lines indicate cut off values for y-axis log 10 (q values; <0.05) and x-axis; log 2 (Fold change; >1.5). Significant cytokine changes highlighted in a circle. (b−h) PBMCs collected early during treatment (EDT; 0.9 mg/kg PRED or vehicle control) or late during treatment (LDT; 0.5 mg/kg PRED or vehicle control) were profiled by flow cytometry for T-cell activation. (b) Representative flow profiles (left) and a summary (right) of proliferation (Ki67 + ) or activation (Ki67 + PD1 + ) of CD8 + T-cells EDT and LDT. (c) CD8 + Tem gating (left panel), and a summary of proliferation (Ki67 + ) or activation (Ki67 + PD1 + ) of CD8 + Tem cells EDT and LDT (right panels). (d−h) Proliferation (Ki67 + ) or activation (Ki67 + PD1 + ) of CD8 + Tcm (d), naïve CD8 + T-cells (e), CD4 + Foxp3 – Tconv cells (f), naïve CD4 + Foxp3 – Tconv (g) and NK cells (h). Orange dots indicate delayed progressors (DP). Data pooled from two independent experiments, mean ± SD is shown. Statistical comparisons were determined using ordinary ANOVA with post-hoc Bonferroni multiple comparison test; significant p -values are shown.

Article Snippet: Mice received three cycles of antibodies against PD1 (Cat# BE0146, clone RMP1-14, 300 μg/mouse; BioXCell, Lebanon, NH) and CTLA4 (Cat# BE0131, clone 9H10, 200 μg/mouse, BioXCell) or matched isotype control antibodies (Cat# BE0089, clone 2A3 and Cat# BE0087, Syrian hamster IgG; both from BioXCell).

Techniques: Activation Assay, Clinical Proteomics, Control, Fluorescence, Transformation Assay, Generated, Marker, Expressing, Flow Cytometry, Comparison