Journal: STAR Protocols

Article Title: Protocol for phenotyping mouse myeloid and lymphoid cells by mass cytometry

doi: 10.1016/j.xpro.2025.103684

Figure Lengend Snippet: Surface and intracellular antibody cocktails for antibody titrations

Article Snippet: Anti-mouse CD4 (RM4-5)-145Nd-100 tests , Standard BioTools , Cat#3145002B; RRID: AB_2687832.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for phenotyping mouse myeloid and lymphoid cells by mass cytometry

doi: 10.1016/j.xpro.2025.103684

Figure Lengend Snippet: Summary table of markers expressed for each cell type in mouse lungs, spleen and peritoneal lavage

Article Snippet: Anti-mouse CD4 (RM4-5)-145Nd-100 tests , Standard BioTools , Cat#3145002B; RRID: AB_2687832.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for phenotyping mouse myeloid and lymphoid cells by mass cytometry

doi: 10.1016/j.xpro.2025.103684

Figure Lengend Snippet: UMAP plots of mouse cells analyzed by mass cytometry UMAP is a dimensionality reduction technique, which allows summarizing n dimensions data into two dimensions. These graphs show the UMAP plots for cells isolated from thawed (A) and fresh (B) spleen of mice infected with Echinococcus multilocularis , cells isolated from fresh spleen of uninfected mouse (C), thawed (D) and fresh (E) peritoneal cells of mice infected with E. multilocularis , and cells isolated from fresh lungs of uninfected mouse (F). Gates show B cells (green), T CD4 + cells (orange), T CD8 + cells (blue), monocytes/macrophages/dendritic cells (red), eosinophils (pink), neutrophils and precursors (black) and undetermined populations (gray). Thawed cells (especially myeloid cells) had modified expression of some markers, compared to fresh cells (A vs. B, and D vs. E, respectively). This modified slightly their UMAP distribution and can interfere with analysis and interpretation process.

Article Snippet: Anti-mouse CD4 (RM4-5)-145Nd-100 tests , Standard BioTools , Cat#3145002B; RRID: AB_2687832.

Techniques: Mass Cytometry, Isolation, Infection, Modification, Expressing

Journal: STAR Protocols

Article Title: Protocol for phenotyping mouse myeloid and lymphoid cells by mass cytometry

doi: 10.1016/j.xpro.2025.103684

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD4 (RM4-5)-145Nd-100 tests , Standard BioTools , Cat#3145002B; RRID: AB_2687832.

Techniques: Purification, Blocking Assay, Recombinant, Sterility, Staining, Red Blood Cell Lysis, Antibody Labeling, Software, Membrane, Transferring, Spectrophotometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 2. Characteristics of the HHR thymus and differentiation status of thymocytes in the thymus. (A) Graphs show the weights and cellularities of SDR and HHR thymi at 4–5 wk of age. Three SDRs and HHRs were used for each analysis. Data are shown as mean 6 SD. *p , 0.05. (B) Histological sections of thymi from 4-wk-old SDRs and HHRs were stained with H&E. Scale bars represent 1 mm. Representative results of two SDRs and HHRs are shown. (C) The differentiation status of thymocytes was analyzed with a flow cytometer. Representative results of three SDRs and HHRs are shown. An arrow indicates a population of DP thymocytes with decreased CD4 levels. Averaged values from three independent flow cytometric analyses for the proportions of DP, CD4-SP, CD8-SP, and DN thymocytes are shown in the lower graph. Data are shown as mean 6 SD. (D) Expression levels of Cd4 and Cd8 genes in the thymus were analyzed by real-time RT-PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Staining, Cytometry, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 3. nTreg numbers in the HHR thymus. (A) Expression levels of Cd25 and Foxp3 genes in CD4-SP thymocytes were analyzed by real-time RT- PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) Upper, The proportion of CD4+CD25+ cells in the thymus was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of CD4+CD25+ cells are shown in the graph. Data are shown as mean 6 SD. Lower, The proportion of Foxp3+ cells in the CD4+CD25+ cell fraction was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of Foxp3+ cells in the CD4+CD25+ cell fraction are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. (C) Upper, Estimated values for the proportion of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of Foxp3+ cells in the CD4+CD25+ cell fraction obtained from flow cytometric analysis and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. Lower, Estimated values for the absolute number of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of total thymus cell number (Fig. 2A) and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 5. Loss of Ly49s3 gene expression in HHR thymic cDCs. (A) Left, Genome-wide microarray CGH analysis, performed with genomic DNA from SDR and HHR livers, shows the deletion of four Ly49 family genes—Ly49s4, Ly49i4, Ly49s3, and Ly49i3—in chromosome 4 at the q42 region (shaded area). Data shown are representative of two independent analyses. Right, Genomic PCR with DNA from SDR and HHR livers was performed to confirm the deletion of DNA in this region. As an internal standard, the Ccr4 gene, located at chromosome 8q32, was used. (B) Left, RT-PCR analysis of the expression of the Ly49s3 gene was performed with total RNA from SDR and HHR thymi. As an internal standard, the Gapdh gene was used. Middle, RT- PCR analysis of Ly49s3 gene expression in DP, CD4-SP, and CD8-SP thymocytes of the SDR thymus was performed with total RNA from the cells. Note that it was not possible to isolate pure DN thymocytes by positive and/or negative selection using CD4 and CD8a microbeads because the remaining cells after the selection of DP, CD4-SP, and CD8-SP cells are a mixture of DN thymocytes and all of the other types of cells. Right, RT-PCR analysis of Ly49s3 gene expression in cDCs of SDR and HHR thymi was performed with total RNA from the cells.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Gene Expression, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 6. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture. (A) A total of 1 3 106 CD4-SP thymocytes isolated from the SDR thymus were cultured with 2.5 3 105 cDCs from the SDR thymus, and 1 3 106 CD4-SP thymocytes from the HHR thymus were cultured with 2.5 3 105 cDCs from the HHR thymus. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) To confirm the expression levels of Foxp3 and CD25, an additional mixed-cell culture experiment was performed, and the proportion of Foxp3+ or CD25+ cells was determined by flow cytometric analysis. (C) As control experiments, 1 3 106 CD4-SP thymocytes were cultured alone for 3 d and the same real-time RT-PCR analyses as above were performed. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for SDR cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (D) Effects of cDCs on nTreg marker gene expression. The expression levels of nTreg marker genes in mixed-cell (A) and control cultures (C) are shown in the same graph relative to the value for the SDR control culture, which is set at 1. Statistical analysis was performed between the induction ratios of gene expression. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 1 3 106 CD4+CD82CD252 thymocytes isolated from SDR and HHR thymi were cultured with 2.5 3 105 cDCs isolated from SDR and HHR thymi, respectively. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 7. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture using Ly49s3-expressing HHR thymic cDCs. (A) The FLAG- tagged Ly49s3 structure is schematically represented. R: arginine residue. (B) Left, 293T cells were transfected with recombinant vectors before being packaged into the virus, and cell lysates were subjected to Western blot analysis with the anti-FLAG M2 Ab. Right, The proteins on the membrane were stained with fast green. (C) Left, cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3, and the expression of the fusion protein on the surface of cDCs was confirmed by fluorescence microscopic observations with the anti-FLAG M2 Ab. Right, Phase contrast appearance of the identical cells shown in the left photographs. Note dendrites on the surface of the cells. The cells were suspended in buffer and all the photos were taken. Original magnification 3800. (D) A total of 2.5 3 105 cDCs isolated from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 (Ly) or the mock vector (Mo) and then mixed with 1 3 106 CD4-SP thymocytes isolated from the HHR thymus. At 3 d later, total RNAwas extracted and the expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture, using cDCs transduced with the mock vector, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 2.5 3 105 cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 and then mixed with 1 3 106 CD4-SP thymocytes from the HHR thymus. Next 5 mg of anti-rat MHC class I Ab or normal IgG was added to the culture. At 3 d later, total RNA was extracted, and the expression levels of nTreg marker genes and MHC class II genes were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture in the presence of normal IgG, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (F) Summary of the experiments performed using the lentiviral vector of the Ly49s3 gene and anti-MHC class I Ab. The level of each gene is shown relative to the value for mock-transduced cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Residue, Transfection, Recombinant, Virus, Western Blot, Membrane, Staining, Transduction, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: GRMD muscle (cranial sartorius) was triple stained for Neu5Gc (green), Pax7 (A), CD11b (B), CD4 (C), CD8 (D) (all red), and DAPI (blue). Arrows indicated co-staining of Neu5Gc and Pax7 (in A), CD11b (in B) or CD4 (in C). Bar is 100 µm (A) and 50 µm (B–D) for all panels.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: (A) Cranial sartorius muscle sections from GR and GRMD dogs were stained with markers for satellite cells (Pax7), T lymphocytes (CD4 or CD8) or macrophages (CD11b) and quantified for numbers of cells stained per 40X visual field. (B) The percentage of cells co-stained for Neu5Gc and CD4, CD8, CD11b or Pax7 in GRMD muscles was quantified. Errors are SEM. ***P<0.001, for each GR vs. GRMD comparison in A.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining, Muscles, Comparison

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: Anti-IL-9 antibody treatment decreased mast cell infiltration of the CNS. Purified cells (mainly composed of mast cells) were harvested from the CNS on days 0, and 5, 15, and 20 after EAE immunization. CD45 + CD117 + mast cells were detected by flow cytometry. CNS mast cells maintained a high level throughout the disease course in EAE group. However, mast cells were significantly reduced from day 5, and remained low in anti-IL-9 Abs group, compared with IgG group. Data are shown as mean ± SE. * P < 0.01, anti-IL-9 Abs group versus IgG group. CNS: Central nervous system; EAE: Experimental autoimmune encephalomyelitis; SE: Standard error; IL: Interleukin.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: Purification, Flow Cytometry

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: IL-9 blockade reduced production of chemokine recruiting mast cells in the CNS. Five days after immunization, mRNA expressions of Pecam1, SCF, Vcam-1, CCL2, and CCL5 in CNS tissue of EAE mice were detected by RT-PCR. After IL-9 neutralization, mRNA expressions of CCL5 and Vcam-1 were significantly decreased in anti-IL-9 Abs group, compared with IgG group. * P < 0.01. Pecam1: Platelet and endothelial cell adhesion molecule 1; SCF: Supercoiling factor; Vcam-1: Vascular cell adhesion molecule 1; CCL2: C-C motif chemokine ligand 2; CCL5: C-C motif chemokine ligand 5; CNS: Central nervous system; EAE: Experimental autoimmune encephalomyelitis; IL: Interleukin; RT-PCR: Reverse transcription-polymerase chain reaction; mRNA: Messenger RNA.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Neutralization

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: In vitro , the effect of anti-IL-9 antibody on splenic mast cells. Splenocytes were harvested from experimental autoimmune encephalomyelitis mice 5 days after MOG immunization. After co-culture with anti-IL-9 antibody or anti-mouse IgG for 7 h, mast cell number was counted by flow cytometry. Splenic mast cells cultured with anti-IL-9 antibody showed significantly lower levels in a dose-dependent manner. This trend was particularly evident with anti-IL-9 antibody concentrations up to 20 μg/ml. * P < 0.05; † P < 0.01. IL: Interleukin; MOG: Myelin oligodendrocyte glycoprotein.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Cell Culture

Journal: Molecular Neurodegeneration

Article Title: Gut-first Parkinson’s disease is encoded by gut dysbiome

doi: 10.1186/s13024-024-00766-0

Figure Lengend Snippet: Gut immunity remodeling in PD. A Representative immunofluorescence images of transverse mouse ileum sections stained with anti-CD11b. B Quantification of CD11b + cells per mm 2 in the ileum ( n = 5–9 mice per group). C , D Measurement of specific inflammatory cytokines by ELISA. C TNF ( n = 4–13 mice per group), ( D ) IL-6 ( n = 4–5 mice per group). E Representative immunofluorescence images of human terminal ileum sections stained with anti-CD11b. F Quantification of CD11b + cells per mm 2 in the ileum ( n = 4–5). G Representative photomicrographs images of transverse ileum sections stained with anti-CD4. H Quantification of CD4 + cells per mm 2 in the ileum ( n = 5–8 mice per group). I Representative immunofluorescence images of Th17 cells (CD4 + /IL17 + ) in transverse sections of the ileum. J Quantification of Th17 cells (CD4 + /IL17 + ) cells per mm 2 in the ileum ( n = 4–5 mice per group). K IL-17 levels (pg/mL) in the ileum ( n = 6–15 mice per group) measured by ELISA. L Representative images of human terminal ileum sections stained with anti-IL-17 and CD4. M Quantification of CD4 + /IL - 17 + cells per mm. 2 in human ileum ( n = 4–5). Data for histological analysis and IL - 17 determination were obtained from different animal cohorts. * p < 0.05, ** p < 0.01, *** p < 0.001, using one-way ANOVA with Dunnet´s test ( B , D , H and J , K ) or Kruskal–Wallis with Dunn´s test ( C ) and unpaired Student´s t - test ( F and M ). Data are expressed as mean ± SEM.. Scale bars are 50 µm. See also Tables S3-S4 and Figures S3

Article Snippet: PBMC pellet was incubated with anti-mouse CD45 PerCP (clone 30F11), anti-mouse CD3 FITC (clone REA641), anti-mouse CD4 APC (clone REA604) and anti-mouse CD8 PE (clone REA601) (1/50) (Miltenyi biotec) for 10 min at 4 oC.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Gut-first Parkinson’s disease is encoded by gut dysbiome

doi: 10.1186/s13024-024-00766-0

Figure Lengend Snippet: Systemic inflammation and permeabilization of the blood–brain barrier. A Representative dot plots of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + populations in serum samples by flow cytometry. B Quantification of the CD4/CD8 ratio ( n = 7–9 mice per group). C - E Measurement of specific inflammatory cytokines in mouse plasma by ELISA. (C) IFNγ ( n = 4–6 mice per group), ( D ) IL-6 levels ( n = 4–6 mice per group) and ( E ) IL - 17 levels ( n = 3–8 mice per group). F Representative immunohistological images of SN coronal sections stained with IgG. (G) Quantification of IgG-positive microvascular leakage per mm 2 in the SN ( n = 5 mice per group). * p < 0.05, ** p < 0.01, using one-way ANOVA with Dunnet´s test ( C - E and G ) or Kruskal–Wallis with Dunn´s test ( B ). Data are mean ± SEM. Scale bars are 50 µm and 500 µm (upper panel). See also Figure S6

Article Snippet: PBMC pellet was incubated with anti-mouse CD45 PerCP (clone 30F11), anti-mouse CD3 FITC (clone REA641), anti-mouse CD4 APC (clone REA604) and anti-mouse CD8 PE (clone REA601) (1/50) (Miltenyi biotec) for 10 min at 4 oC.

Techniques: Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

doi: 10.1038/s44321-024-00098-y

Figure Lengend Snippet: ( A ) Single-cell sequencing analysis of biopsies from non-small cell lung cancer (NSCLC) patients. Panels indicate the expression of PDCD1 , LAG3 and CBLB and CBLC analyzed from the single-cell lung cancer extended atlas (LuCA) (Salcher et al, ) repository as indicated. ( B ) Dot plot with the percentage of CD4 and CD8 T-cells that co-express PD-1 and LAG-3 after ex vivo activation, from healthy donors ( n = 8) and NSCLC patients ( n = 10). Statistical comparisons were performed by the Mann–Whitney test. Error bars correspond to ±SD ( C ) CBL-B expression by mean fluorescent intensities in CD4 and CD8 T-cells from a sample of non-responder NSCLC patients ( n = 4), activated ex vivo in the presence of the indicated treatments. Shown data from total CD4 and CD8 gated populations. Statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests. Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( D ) Same as ( C ) but for C-CBL expression. Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( E ) Percentage of proliferating CD4 T cells (left) and CD8 T cells (right) from a sample of high PD-1/LAG-3 co-expression patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. Statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests ( n = 5). Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( F ) Flow cytometry histograms of SATB1, Phospho SMAD 2/3, LCK and ZAP70 expression. Gates were established according to unstained controls in T-cells from a sample of non-responder NSCLC patients. Percentage of expression and Mean Fluorescence Intensity values are indicated. Data information: Statistical comparisons are shown in the graph as indicated in Methods. Briefly, for ( B ) statistical comparisons were performed by the Mann–Whitney test. For ( C – E ), statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests. Error bars correspond to ±SD. **, ***, ****, indicate P < 0.01, P < 0.001 and P < 0.0001 differences. .

Article Snippet: The following antibodies were used at 1:50 dilution unless otherwise stated: CD4-APC-Vio770 (clone M-T466, Miltenyi), CD3-APC (clone REA613, Milenyi Biotec), CD28-PECy7 (clone CD28.2, Biolegend), PD-1-PE (clone EH12.2H7, Biolegend), CD8-FITC (clone SDK1, Biolegend), LAG3-PE (clone 11C3C65, Biolegend), LAG3-PerCP-Cy5.5 (clone 11C3C65, Biolegend), CD4-FITC (clone REA623, Milteny), CD3-PerCP-Cy5.5 (clone T100, TONBO), CD4-PE-Vio770 (clone REA261, Milteny), CD223-PerCP-Cy5 (clone 11C3C65, Biolegend), PD-1-Pacific Blue (clone EH12.2H7, Biolegend).

Techniques: Sequencing, Expressing, Ex Vivo, Activation Assay, MANN-WHITNEY, Flow Cytometry, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

doi: 10.1038/s44321-024-00098-y

Figure Lengend Snippet: ( A ) Schematic design of the experiment. BALB/c female mice were randomly allocated and subcutaneously injected with 2 × 10 6 Lung adenocarcinoma (Lacun3) cells per animal. 100 µg of anti-PD-1, 100 µg of anti-LAG3, 30 mg/kg of CBL-Bi and the corresponding depletion antibodies were administered intraperitoneally at days 0, 2, 6, 9, 13 and 15 as indicated in the figure. NK, CD4, and CD8 T‐cell depletions were carried out by intraperitoneal administration of 100 μg of anti‐mouse CD8a, CD4 or NK1.1 antibody. Mice were humanely sacrificed when tumor size reached ~150–200 mm 2 , or when tumor ulceration or discomfort were observed. ( B ) Kaplan–Meier survival plot of mice under the indicated treatments or depletion (percent). Statistical significance was tested with the Log-rank test. ( C ) Evolution of mean tumor size following the indicated treatments (left). Tumor volumes 9 days after treatment initiation (right). Error bars correspond to ±SEM (left) and box and whiskers with min to max values (right), computing the minimum, maximum, median and quartiles for 25th and 75th percentiles. The whiskers go down to the smallest value and up to the largest ( n = 6 mice per group). Data information: Statistical comparisons were carried out by a two-way ANOVA followed by pair-wise Tukey tests. ( D ) Tumor growth of individual mice in the indicated treatment groups ( n = 6 mice per group). Statistical comparisons are shown in the graph as indicated in Methods. Data information: Briefly, for ( B ), survival was represented by Kaplan–Meier plots and analyzed by log-rank test. For ( C ), statistical comparisons were carried out by a two-way ANOVA followed by pair-wise Tukey tests. *, **, ****, indicate P < 0.05, P < 0.01, and P < 0.0001 differences. .

Article Snippet: The following antibodies were used at 1:50 dilution unless otherwise stated: CD4-APC-Vio770 (clone M-T466, Miltenyi), CD3-APC (clone REA613, Milenyi Biotec), CD28-PECy7 (clone CD28.2, Biolegend), PD-1-PE (clone EH12.2H7, Biolegend), CD8-FITC (clone SDK1, Biolegend), LAG3-PE (clone 11C3C65, Biolegend), LAG3-PerCP-Cy5.5 (clone 11C3C65, Biolegend), CD4-FITC (clone REA623, Milteny), CD3-PerCP-Cy5.5 (clone T100, TONBO), CD4-PE-Vio770 (clone REA261, Milteny), CD223-PerCP-Cy5 (clone 11C3C65, Biolegend), PD-1-Pacific Blue (clone EH12.2H7, Biolegend).

Techniques: Injection