Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a Rab11 vesicles perform kiss-and-run dependent on FERARI. Movie stills showing kiss-and-run of endogenously GFP-tagged Rab11 vesicles (arrow) in ctr KO ( n = 41) and vipas39-KO ( n = 25) HeLa cells (see also Supplementary Movie ). Scale bar: 2 µm. b Rab11 vesicles dock on SNX1 networks with residence times in distinct intervals. Groups of vesicles with similar residence times appear as peaks (see also Supplementary Fig. for single-vesicle graph, mock : n = 41, vipas39-KO : n = 25). This pattern is abolished in vipas39 -KO cells (see Supplementary Fig. for ehd1-KO and rab11fip5-KO additional data). One-way multiple comparison ANOVA test P -values: mock-vipas39-KO P = 1.98E-09, mock-ehd1-KO P = 1.6E-10, mock-rab11fip5-KO P = 1.58E-09. c List of FERARI genes in C. elegans and H. sapiens . The human nomenclature is used throughout the manuscript. The model organism is indicated by a human or worm icon next to the data. d Movie stills showing kiss-and-run of Rab5 vesicles (arrow) in wild-type worms ( n = 53). Scale bar: 2 µm. e Movie stills showing kiss-and-run of Rab5 vesicles (arrow) in ehd1(RNAi) worms ( n = 33) (see also Supplementary Movie ). Scale bar: 2 µm. f Rab5 vesicles dock on SNX1 networks with residence times in distinct intervals (see also Supplementary Fig. for single-vesicle graph, n = 53). This pattern is abolished in ehd1 ( n = 33) and fip5 ( n = 35) knock-downs (see Supplementary Fig. for single-vesicle plots). One-way multiple comparison ANOVA test P -values: mock-ehd1 P = 4.63E-05, mock-fip5 P = 3.83E-05. g Residence times intervals in Rab5 vesicles docking to mCherry-EHD1 compartments ( wild-type from f as comparison, n = 63) (see Supplementary Fig. ). Unpaired two-tailed t -test: P = 0.8467. h Rab5 exhibits kiss-and-run behavior in HeLa cells stably expressing mApple-Rab5 and transiently expressing GFP-SNX1. Movie stills show representative vesicles for ctr KO ( n = 41) and vipas39- KO ( n = 23) cells (arrow) (see also Supplementary Movie ). Scale bars: 2 µm. i Binning of vesicles from (h) reveals distinct intervals of residence times for Rab5 vesicles ( mock : n = 41, vipas39-KO : n = 23). This pattern is abolished in vipas39-KO cells (see Supplementary Fig. for single-vesicle plots). Unpaired two-tailed t -test: P = 0.000118.

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Two Tailed Test, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a GFP-Rab10 compartments docking onto mCherry-SNX1 networks in worm intestine (see also 3D projection, Supplementary Movie ). Regions of co-localization are indicated by arrows. Quantification of co-localization by Mander’s coefficients ( n = 10). Data are presented as mean values +/– SD. M1 denotes the overlap between SNX1 and Rab10, and M2 between Rab10 and SNX1. One-way multiple comparison ANOVA test P -values: M1 SNX1-Rab10 vs. SNX1-DHS-3 P = 1.94E-08, M1 SNX1-Rab10 vs. SNX1-MANS P = 9.62E-09, M2 SNX1-Rab10 vs. SNX1-DHS-3 P = 4.37E-13, M2 SNX1-Rab10 vs. SNX1-MANS P = 4.43E-13. Scale bar: 2 µm. b Genetic interaction between FERARI subunits and Rab10. rab10(ok1494) causes accumulation of SNX1 compartments. RNAi of vps45 and vipas39 but not vps33B (CHEVI) show additional enlargement of SNX1 (arrowheads). n = 20 worms from 3 experiments (for quantification see Supplementary Fig. ). Scale bar: 10 µm. c Western blot of CRISPR–Cas9-mediated KO of Rab10 in HeLa cells ( n = 3 independent experiments). d Size of the endogenous SNX1 structure is enlarged in rab10 KO cells. SNX1 was detected by immunofluorescence. Violin plot showing the enlarged volume of the SNX1 structure in ctr and rab10 KO cells (volume of >14,700 particles was measured from each group from three independent experiments, median indicated by the red line). Unpaired two-tailed t -test: P = 1.46E-17. Scale bars 10 µm. e Movie stills for Rab10 vesicles (arrow) showing kiss-and-run in ehd1(RNAi) ( n = 40) and wild-type worms ( n = 51) (Supplementary Movie ). “N”: nucleus with high mCh-SNX1 signal. Scale bar: 2 µm. f Rab10 vesicles show residence times with quantal increases ( n = 51). This behavior is abolished in ehd1 ( n = 40) and fip5 ( n = 36) knock-downs (Supplementary Fig. for single-vesicle plots). One-way multiple comparison ANOVA test P -values: mock-ehd1 P = 7.23E-08, mock-fip5 P = 4.29E-10. g Kiss-and-run of Rab10 on sorting compartments is conserved in metazoans. Residence times of Rab10 vesicles exhibit characteristic intervals abolished in vipas39- KO cells (see Supplementary Fig. for single-vesicle plots). Unpaired two-tailed t -test: P = 0.000128. h Movie stills of mCherry-tagged Rab10 vesicles (arrow) showing kiss-and-run on SNX1 (GFP-tagged) compartment in ctr KO and vipas39- KO HeLa cells ( n = 3 independent experiments, mock : n = 53, vipas39-KO : n = 22 vesicles) (see Supplementary Movie ). Scale bar: 2 µm.

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Western Blot, CRISPR, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a Co-localization between GFP-Rab11 and endogenous SNX1 is reduced in snx5 + 6 double knock out cells. Antibodies against GFP and SNX1 were used to detect Rab11 and SNX1 by immunofluorescence, respectively. Scale bars 10 µm; 5x magnified. Pearson’s coefficient from n = 65 and n = 71 cells from ctr KO and snx5 + 6 KO cells, respectively. Data are presented as single data points with median and min to max whiskers. Unpaired two-tailed t -test P = 2.5316E-07. b Size of the GFP-Rab11-positive structures is increased in snx5 + 6 KO cells in comparison with the ctr KO cells ( n = 3 independent experiments) Scale bars 10 µm; magnification 5x. c Volume of the GFP-Rab11-positive particles in both ctr and KO cells were determined (volume of >15,000 particles were measured from each group from three independent experiments, median is shown by the red line). Unpaired two-tailed t -test P = 3.05E-73. d CI-MPR trafficking is impaired in snx 5 + 6 KO cells. CI-MPR (red) is mostly localized in TGN (green) area in ctr KO cells and is dispersed in snx 5 + 6 KO cells ( n = 3 independent experiments, n = 150 cells per condition). Pearson’s coefficient measurements are shown on the right. Unpaired two-tailed t -test P = 1.97E-51. Scale bars 10 µm. e CI-MPR localization remain unchanged in vipas39 KO cells ( n = 3 independent experiments, n = 150 cells per condition). Scale bars 10 µm; magnification 5x. Co-localization was quantified by Pearsons’s coefficients. Unpaired two-tailed t -test P = 0.0536. f Immunoprecipitation data showing that endogenous SNX6 (left panel), but not SNX5 (right panel), binds to FERARI members Rabenosyn-5 and VIPAS39 in HEK-293 cells ( n = 3 independent experiments). g C. elegans SNX6 interacts with RABS-5 (but not with other FERARI subunits) in Y2H assays. Quantification is shown in Table ( n = 3 growth experiments with n = 6 independent yeast colonies each).

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Knock-Out, Immunofluorescence, Two Tailed Test, Immunoprecipitation

Journal: Gene Therapy

Article Title: First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model

doi: 10.1038/s41434-022-00313-w

Figure Lengend Snippet: a RT-PCR gel of mGHR mRNA expression. 18S rRNA housekeeping gene was used as loading control. b Western blot of mGHR protein expression. Actin housekeeping gene was used as loading control. c Immunofluorescence staining of mGHR expression, scale bars are 50 µm and nuclei were stained with DAPI. HepG2 cells were transfected with pAAV-HLP-mGHR consisted of hybrid liver-specific promoter (HLP) driving the expression of mGHR. Untransfected control and cells transfected with pAAV-HLP-Luc expressing luciferase gene were used as negative control. Analyses were performed 48 h post-transfection.

Article Snippet: The mGHR PCR fragment was amplified from cloning vector MG50043-M (Sino Biological, Beijing, China) containing mouse growth hormone receptor/GHR/GHBP transcript variant 1 gene ORF cDNA clone using forward primer with introducing NotI site: 5’-ATAAGAATGCGGCCGCACCATGGATCTTTGTCAGGTCTTC and reverse primer with introducing XhoI site: 5’-CCGCGCTCGAGCTACTGCATGATTTTGTTCAGTTG.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Negative Control

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Left, cartoon depicting a human-derived coculture assay containing Jurkat expressing TCR, CD28 and mGFP-tagged hu PD1 or mo PD1, and SEE-pulsed Raji APCs expressing MHCII, CD86, and mCherry-tagged hu PDL1 or mo PDL1. Right, FACS histograms showing the expression of indicated PD1-mGFP or PDL1-mCherry variant on the Jurkat or Raji cells. (B) Atezolizumab dose-responses of secreted IL-2 in the indicated Jurkat:Raji coculture in the presence of 0.1 ng/mL SEE. Data were normalized to the IL-2 amounts at the highest [atezolizumab]. (C) Superantigen dose-responses of % IL-2 inhibition mediated by either hu PD1: hu PDL1 or mo PD1: mo PDL1 pair in the Jurkat:Raji coculture assay depicted in A . Raji cells were pre-treated with 0 or 120 µg/mL Atezolizumab. (D) Left, cartoon depicting a mouse-derived coculture assay containing DO11.10 cells expressing TCR, CD28 and mGFP-tagged hu PD1 or mo PD1, and OVA 323-339 -pulsed A20 APCs expressing MHCII, CD86, and mCherry-tagged hu PDL1 or mo PDL1. Right, FACS histograms showing the expression of indicated PD1-mGFP or PDL1-mCherry variant on the DO11.10 or A20 cell. (E) OVA 323-339 dose-responses of % IL-2 inhibition mediated by either hu PD1: hu PDL1 or mo PD1: mo PDL1 pair in the DO11.10:A20 coculture assay depicted in D . Data are mean ± SD from three independent experiments. ***P < 0.001; two-way ANOVA.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Derivative Assay, Co-culture Assay, Expressing, Variant Assay, Inhibition

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: Left, FACS histograms showing hu PD1 expressions on anti-CD3ε/anti-CD28 stimulated human primary CD4 + T cells and on stable Jurkat cells transduced with hu PD1 driven by either an dSV40 (weak) or an SFFV (strong) promoter. Right, PD1 densities on human primary CD4 + T cells plotted against duration of stimulation by anti-CD3/anti-CD28 beads. Dashed lines indicate the densities of SFFV- and dSV40-driven PD1 expressions on Jurkat cells. PD1 densities were calculated using molecular numbers quantified with a Quantum PE MESF kit and diameters of cells (human primary CD4 + T cell: 4.8 µm, Jurkat cells: 15 µm).

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Transduction

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) FACS histograms showing expressions of endogenous mo PD1, mo PDL1, mo CD28, mo CD80, and mo CD86 on DO11.10 T cell hybridoma and A20 cells before and after the KO of indicated genes. Isotype antibodies were used as controls. (B) FACS histograms showing mo CD80 and mo CD86 levels on A20 cells transduced with indicated mCherry-tagged PDL1 variants. Controls for mo CD80 and mo CD86 staining were generated with isotype antibodies. Control for PDL1-mCherry corresponded to untransduced PDL1 −/− A20 cells.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Transduction, Staining, Generated, Control

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) FACS histograms showing the expressions of indicated mGFP-tagged PD1 or mCherry-tagged PDL1 variants on Jurkat or Raji cells used for cocultures. (B) Left, a cartoon depicting the PD1:PDL1 pairs tested in five parallel Jurkat:Raji coculture assays. Right, bar graphs showing the % inhibition of IL-2 secretion and of CD69 expression by the PD1:PDL1 pairs indicated on the left (n = 6 independent experiments). (C) FACS histograms showing the expressions of indicated mGFP-tagged PD1 or mCherry-tagged PDL1 variants on DO11.10 or A20 cells used for cocultures. (D) Left, a cartoon depicting the PD1:PDL1 pairs tested in five parallel A20:DO11.10 coculture assays. Right, a bar graph showing the % inhibition of IL-2 secretion by the PD1:PDL1 pairs indicated on the left (n = 3 independent experiments). (E) Left, diagram showing the purification method for human primary CD4 + T cells. Right, FACS histograms of CD3 and CD4 expressions on hu PBMC and CD4 + T cells. (F) Top, cartoon depicting a coculture assay containing CD4 + T cells expressing TCR, CD28, and mGFP ECD -TMD-PD1 ICD , and SEB-pulsed Raji APCs expressing MHCII, CD86, and GFPNb-TM-TagBFP. Bottom, FACS histograms showing the expression of indicated PD1 chimera or GFPNb-TM-TagBFP on CD4 + T or Raji cells, respectively. (G) A dot plot showing % inhibition of IL-2 secretion by the PD1 chimera:GFPNb association in CD4 + T cell:Raji coculture depicted in F (n = 3 independent experiments). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Inhibition, Expressing, Purification, Co-culture Assay

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Representative BLI data obtained for the indicated PD1:ligand pairs with the PDL1 or PDL2 immobilized on the sensor chip and increasing concentrations of PD1-ECD presented in the solution. (B) K d values of the indicated PD1:ligand pairs based on the three independent BLI experiments. (C) Left, representative confocal images of a conjugate between Jurkat expressing the indicated PD1 variants (green) and Raji expressing the indicated PDL1 variants (red) with or without anti-PDL1 (atezolizumab). Right, a dot plot showing the synaptic enrichment score of each PD1 variant in the presence or absence of atezolizumab (n = 40 cells). (D) A cartoon depicting a cell-SLB assay, in which Jurkat cells expressing mGFP-tagged PD1 variant interacted with a SLB functionalized with anti- hu CD3ε and hu / mo PDL1-His. PD1 microclusters were visualized via TIRF-M. (E) Top, representative TIRF images showing microclusters of indicated mGFP-tagged PD1 variant in a Jurkat cell that contacted the SLB functionalized with 3 nM hu PDL1 or mo PDL1 that matched the species of the ECD of the corresponding PD1 variant. Bottom, dot plots showing the clustering indices of all five PD1 variants (See methods) (n = 40 cells). (F) Dose-response curves showing PD1 clustering indices of the indicated PD1 variant plotted against [PDL1] (n = 3 independent experiments). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing, Variant Assay

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: Left, cartoon depicting a TIRF-M assay visualizing PD1 ECD -attached, bodipy-labeled LUVs captured by PDL1 ECD -attached SLB. Middle, representative TIRF images of LUVs (bodipy) and SLBs (DiD) in the presence or absence of anti-PDL1 blockade antibody (atezolizumab). Right, normalized F.I. of LUVs in the TIRF field under indicated conditions. Scale bars: 5 µm. Data are mean ± SD from three independent experiments. **P < 0.01; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Labeling

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) A cartoon depicting a cell-SLB assay using Jurkat expressing PD1-mGFP. (B) Time lapse TIRF images visualizing microclusters of indicated PD1 variants. Scale bars: 5 µm.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Left, IL-2 secretion from human CD8 + T cells stimulated by SEB-loaded hu PDL1-expressing Raji cells, with or without the presence of anti-PD1 blockade antibody (pembrolizumab), with or without 5 µM SHP099. Right, degree of PD1-mediated inhibition of IL-2 secretion with or without 5 µM SHP099, calculated based on data on the left. (B) Same as A except using mouse CD8 + T cells isolated from the splenocytes of P14 mice, and using anti- mo PD1 RMP1-14 to block PD1 signaling. (C) Degrees of SHP099 mediated inhibition of endogenous PD1 function in human and mouse CD8 + T cells calculated based on data from A and B . (D) Same as B except using Pdcd1 −/− P14 mouse CD8 + T cells engineered to express ICD-humanized mo PD1. (E) Same as B except using Pdcd1 −/− P14 mouse CD8 + T cells reconstituted with WT mo PD1. (F) Degrees of SHP099 mediated inhibition of exogenous PD1 function in Pdcd1 −/− P14 mouse CD8 + T cells calculated based on data from D and E . Data are mean ± SD from three independent experiments. **P<0.01, ***P < 0.001, ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing, Inhibition, Isolation, Blocking Assay

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) A cartoon showing a cell-SLB assay for TIRF imaging of PD1:Shp2 association in Jurkat or human CD4 + T cells. ( B ) Left, representative TIRF images of the indicated mGFP-tagged PD1 variants (green) and anti-Shp2 (magenta) at the interface of a Jurkat cell and the SLB as depicted in A . Right, dot plots showing the anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). ( C ) Same as B , except that human CD4 + T cells expressing the indicated PD1 variants were imaged (n = 40 cells). ( D, E ) Same as A, B , except that DO11.10 cells were observed. ( F ) AA sequence alignment of hu PD1 ICD and mo PD1 ICD , with ITIM, PRS, PEQ/H, and ITSM highlighted. ( G ) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp2 tSH2 interaction. ( H ) Left, representative time courses of SC505 (Shp2 tSH2 ) F.I. at increasing concentrations of hu PD1 ICD . Right, % SC505 quenching 30 min after ATP addition plotted against the [ hu PD1 ICD ] and [ mo PD1 ICD ] (n = 3 independent experiments). ( I ) Bar graphs summarizing apparent K d of Shp2 tSH2 interaction with indicated PD1 ICD variants determined via assays shown in G and H (n = 3 independent experiments). ( J ) Left, representative TIRF images showing Shp2 recruitment to microclusters of indicated PD1 variants in a Jurkat-SLB assays. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Imaging, Expressing, Sequencing, Reconstitution Assay

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) FACS histograms showing the expressions of indicated mGFP-tagged PD1 variant and mCherry-tagged huShp2 in human primary CD4 + T cells. T cells expressing both mCherry-huShp2 and the indicated PD1 variant were sorted and used in cell-SLB assays. (B) Left, representative TIRF images showing microclusters of indicated PD1-mGFP variants and mCherry-Shp2. Right, dot plots showing mCherry (Shp2) F.I. normalized to (mGFP) PD1 F.I. in TIRF images. Scale bars: 5 µm. Data are mean ± SD from n = 30 cells. ***P < 0.001; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Variant Assay, Expressing

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp1 tSH2 interaction. Same as except using hu Shp1 tSH2 rather than hu Shp2 tSH2 . (B) % ATP-triggered quenching of Shp1 tSH2 fluorescence plotted against either [ hu PD1-ICD] or [ mo PD1-ICD]. (C) K d values of PD1:Shp1 tSH2 interaction determined via fitting the data in B . Data are mean ± SD from three independent experiments. *P < 0.05; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Reconstitution Assay, Fluorescence

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) Phylogeny of 236 vertebrate species, color-coded based on the pre-ITSM sequence of PD1. ( B ) Left, a phylogenetic tree of pre-ITSM sequence found in PD1 orthologs. Middle, representative TIRF images of microclusters of GFP-tagged hu PD1 variant bearing the indicated pre-ITSM sequence (green) and endogenous Shp2 (magenta) in an anti- hu CD3ε/ hu PDL1 stimulated Jurkat cell. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. in Jurkat cells expressing the indicated hu PD1 variant (n = 40 cells). ( C ) The subtree of the 107-species mammal phylogeny spanning the rodent clade and the primate clade. The branch lengths were inferred based on PD1 codon sequence evolution. The marked nodes are mouse, human or the ones with reconstructed ancestral sequences. The color codes show the inhibition ability of the sequences at respective nodes, based on data in D. ( D ) Left, FACS histograms showing the expressions of hu PD1 chimera harboring an ICD corresponding to the indicated node, on Jurkat cells. Right, % inhibition of IL-2 secretion by the indicated hu PD1 chimera upon stimulating Jurkat ( hu PD1 chimera) with Raji ( hu PDL1). The ITIM and pre-ITSM type is labeled: hu , human-like, mo , mouse like (n = 5 independent experiments). Data are mean ± SD. Scale bars: 5 µm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Sequencing, Variant Assay, Expressing, Inhibition, Labeling

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) AA sequence alignment of ITIM-ITSM region in PD1 of five selected species, with the pre-ITSM region annotated with a box. (B) Left, FACS histograms showing the expressions of mGFP-tagged hu PD1 variants that harbored the indicated PD1 ICD in five engineered Jurkat lines. Right, bar graphs showing % inhibition of IL-2 secretion mediated by the indicated hu PD1 variants. The five Jurkat lines were stimulated in parallel using SEE-pulsed, hu PDL1-expressing Raji cells, with or without Atezo (n = 3 independent experiments). Data are mean ± SD. *P < 0.05; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Sequencing, Inhibition, Expressing

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) Schematic of an adoptive transfer experiment using Pdcd1 −/− P14 CD8 + T cells and B16.gp33 cells. Pdcd1 −/− P14 cells were retrovirally transduced with exoPD1 and Thy1.1, and adoptively transferred to mice bearing B16.gp33 melanoma. ( B ) Surface staining of indicated mo PD1 variants and Thy1.1 on P14 cells transferred to mice in A . ( C ) Tumor growth curves in mice received 1 million Pdcd1 −/− P14 cells expressing either mo PD1 or mo PD1 hu ICD (n = 2-4 mice). ( D ) Number of Thy1.1+ intratumoral CD8 + T cells (n = 5 tumors). ( E ) % TCF-1+, % TIM-3+, % IFNγ+, and % GzmB+ population within TOX+ intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD , based on flow cytometry; n = 4-5 tumors. ( F ) Expressions of the indicated genes in Tox + intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD , based on scRNAseq. ( G ) UMAP showing the Tox + intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD and their cell clusters. ( H ) Dot plot of indicated gene expressions in the cell subsets identified in G . ( I ) % population of the indicated subsets of intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD ; n = 4-6 tumors. ( J ) B16.gp33 tumor growth curves in mice that received 1.2 million P14 cells containing mo PD1 WT (left) or mo PD1 hu ICD (right), in response to treatment of either anti-PD1 (blue) or isotype control (magenta); n = 7 mice per group. ( K ) Model showing how PD1 humanization affects the precursor-to-terminal differentiation of Tox + intratumoral CD8+ T cells. Data are mean ± SEM ( C , D , E , I , and J ). For violin-box plots ( F ), black bars are median, the boxes are 25% to 75% interquartile range, and the whiskers stand for minimum to maximum values excluding outliers. *P < 0.5; **P < 0.01; ***P < 0.001; ns, not significant; two-way ANOVA ( C and J ), student’s t-test ( D , E , and I ), or Wilcoxon signed-rank test ( F ).

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Adoptive Transfer Assay, Transduction, Staining, Expressing, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) A diagram depicting an adoptive transfer experiment using Cas9 +/- P14 CD8 + T cells and B16.gp33 cells. Cas9 +/- P14 CD8 + T cells were retrovirally transduced with sgRNA targeting Pdcd1 , exoPD1, and Thy1.1 to KO the Pdcd1 and reconstitute with exoPD1. Thy1.1 + CD8 + T cells were sorted and adoptively transferred to mice bearing B16.gp33 melanoma. (B) Pdcd1 KO scores of Cas9 +/- P14 CD8 + T cells calculated as described in Methods (n = 3 independent experiments). (C) Surface staining of indicated mo PD1 variants and Thy1.1 on T cells transferred to mice in A . (D) Tumor growth curves in individual mice received 0.5 million Cas9 +/- Pdcd1 −/− P14 CD8 + T cells expressing the indicated mo PD1 variant. Averaged tumor growth curves are shown in the bottom-right graph (n = 2-4 tumors). Data are mean ± SD ( B ) or SEM ( D ). *P < 0.05; **P < 0.01; ns, not significant; two-way ANOVA

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Adoptive Transfer Assay, Transduction, Staining, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) % IFNγ+, % GzmB+, and %TOX+ of the intratumoral P14 T cells expressing either mo PD1 WT or mo PD1 hu ICD measured by flow cytometry (n = 4-6 tumors). (B) Schematic showing scRNA-seq comparing intratumoral P14 Thy1.1+ CD8+ T cells expressing either mo PD1 WT or mo PD1 hu ICD . Each mouse was adoptively transferred with 2 million P14 cells at 7 days after B16.gp33 inoculation. Another 7 days later, intratumoral P14 cells were purified, hash-tagged, and subjected to scRNA-seq. (C) UMAP showing the intratumoral P14 Thy1.1+ cells expressing mo PD1 WT (orange) or hu PD1 hu ICD (blue). (D) Expressions of the indicated transcripts in the intratumoral P14 Thy1.1+ cells expressing either mo PD1 WT or mo PD1 hu ICD . (E) UMAP showing cell clusters (left) and the indicated gene expressions (right) of intratumoral P14 cells. Stem-like exhausted cells (cluster 2; Tox high , Tcf7 high ), Tex1 (cluster 0; Tcf7 low , Mki67 high ), Tex2 (cluster 1; Tcf7 low , Mki67 low ), Prf1 -high effector cells (cluster 3; Tox low , Pfr1 high , Gzmb high ), and Gzma/k -high effector cells (cluster 4; Tox low , Gzmb high , Gzma high , Gzmk high ) are annotated. (F) Left, % population of the indicated cell clusters in E for intratumoral P14 cells expressing either mo PD1 or mo PD1 hu ICD . Right, summarized % population of Tox high cell clusters shown in the left (n = 4-6 tumors). Data are mean ± SEM ( A and F ), or violin-box plot ( D ), in which the black bars are median, boxes are 25% to 75% interquartile range, whiskers are minimum to maximum values excluding outliers. *P < 0.05; ns, not significant; FDR, false discovery rate; student’s t-test ( A and F ), or Wilcoxon signed-rank test ( D ).

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing, Flow Cytometry, Purification

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) UMAP showing the cell cycle phases of the five subclusters of Tox + P14 cells expressing either mo PD1 WT or mo PD1 huICD . (B) Violin plots showing the cell cycle phase of the five subclusters of Tox + P14 cells.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Schematic of the adoptive transfer experiment. Pdcd1 −/− P14 cells were retrovirally transduced with exoPD1 and Thy1.1, and adoptively transferred to mice bearing B16.gp33 melanoma. The host mice then received anti-PD1 or isotype control on day 10, 14, and 17. (B) Tumor growth curves of individual mice that received 1.2 million Pdcd1 −/− P14 T cells expressing either mo PD1 or mo PD1 hu ICD , and treated with either anti-PD1 or isotype control antibody.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Adoptive Transfer Assay, Transduction, Control, Expressing