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

Journal: Frontiers in Immunology

Article Title: Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex

doi: 10.3389/fimmu.2021.745132

Figure Lengend Snippet: List containing all antibodies utilized for surface staining of mass cytometry samples.

Article Snippet: The master mix for blood and spleen samples analyzing immune subsets during the trauma response contained fluorescently labeled antibodies specific to CD11c (VioBlue, Miltenyi Biotec, 130-110-843), CD8a (VioGreen, Miltenyi Biotec, 130-109-330), CD3 (FITC, Miltenyi Biotec, 130-119-798), CD11b (PE, Miltenyi Biotec, 130-113-806), CD45R/B220 (PerCP-Vio700, Miltenyi Biotec, 130-102-218), CD4 (PE-Vio770, Miltenyi Biotec, 130-123-894), Ly-6C (APC-Vio770, Miltenyi Biotec, 130-111-919) and CD192/CCR2 (APC, Miltenyi Biotec, 130-119-658).

Techniques: Staining, Mass Cytometry, Marker

Journal: Frontiers in Immunology

Article Title: Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex

doi: 10.3389/fimmu.2021.745132

Figure Lengend Snippet: General gating strategy for identified immune cell subsets and the used marker combination for definition.

Article Snippet: The master mix for blood and spleen samples analyzing immune subsets during the trauma response contained fluorescently labeled antibodies specific to CD11c (VioBlue, Miltenyi Biotec, 130-110-843), CD8a (VioGreen, Miltenyi Biotec, 130-109-330), CD3 (FITC, Miltenyi Biotec, 130-119-798), CD11b (PE, Miltenyi Biotec, 130-113-806), CD45R/B220 (PerCP-Vio700, Miltenyi Biotec, 130-102-218), CD4 (PE-Vio770, Miltenyi Biotec, 130-123-894), Ly-6C (APC-Vio770, Miltenyi Biotec, 130-111-919) and CD192/CCR2 (APC, Miltenyi Biotec, 130-119-658).

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex

doi: 10.3389/fimmu.2021.745132

Figure Lengend Snippet: Influence of diet-induced obesity on circulating murine immune cells. Uniform manifold approximation and projection (UMAP) with clusters from FlowSOM analysis (Ly6G, CD115, CD4, CD11b, CD19, CD3e, TCRgd, Ly6C, NKp46, CD8a, NK1.1, B220, and CD11c) in mice receiving either low-fat diet (LFD) or high-fat diet (HFD) (A) . Z -score normalized heatmap indicating percentages of immune cell populations in lean and obese mice (B) as well as their actual percentages (C) . Statistics: unpaired two-tailed Student’s t -test to compare the populations between lean and obese mice. ▪ indicates p ≤ 0.1, * indicates p ≤ 0.05, ** indicates p ≤ 0.01. Sample sizes: LFD CTRL = 5, HFD CTRL = 5. Data are displayed as mean ± SEM. ns, not significant.

Article Snippet: The master mix for blood and spleen samples analyzing immune subsets during the trauma response contained fluorescently labeled antibodies specific to CD11c (VioBlue, Miltenyi Biotec, 130-110-843), CD8a (VioGreen, Miltenyi Biotec, 130-109-330), CD3 (FITC, Miltenyi Biotec, 130-119-798), CD11b (PE, Miltenyi Biotec, 130-113-806), CD45R/B220 (PerCP-Vio700, Miltenyi Biotec, 130-102-218), CD4 (PE-Vio770, Miltenyi Biotec, 130-123-894), Ly-6C (APC-Vio770, Miltenyi Biotec, 130-111-919) and CD192/CCR2 (APC, Miltenyi Biotec, 130-119-658).

Techniques: Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex

doi: 10.3389/fimmu.2021.745132

Figure Lengend Snippet: Analysis of essential components of signal transduction of the inflammatory reflex in the spleen. Occurrence of ChAT + CD4 + T cells in the spleen of lean and obese mice (A) . Statistics: Comparison of lean and obese mice was achieved by an unpaired two-tailed Student’s t -test. Comparison of TNF-α + splenic macrophages after LPS stimulation with or without different concentrations of nicotine treatment to the control (B) were analyzed using a two-way ANOVA with repeated measurements followed by an uncorrected Fisher’s LSD test. * indicates p ≤ 0.05. Sample sizes: Each group at for each analysis and condition: n = 5. Data are displayed as mean ± SEM.

Article Snippet: The master mix for blood and spleen samples analyzing immune subsets during the trauma response contained fluorescently labeled antibodies specific to CD11c (VioBlue, Miltenyi Biotec, 130-110-843), CD8a (VioGreen, Miltenyi Biotec, 130-109-330), CD3 (FITC, Miltenyi Biotec, 130-119-798), CD11b (PE, Miltenyi Biotec, 130-113-806), CD45R/B220 (PerCP-Vio700, Miltenyi Biotec, 130-102-218), CD4 (PE-Vio770, Miltenyi Biotec, 130-123-894), Ly-6C (APC-Vio770, Miltenyi Biotec, 130-111-919) and CD192/CCR2 (APC, Miltenyi Biotec, 130-119-658).

Techniques: Transduction, Comparison, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Increased expression of the Th17-IL-6R/pSTAT3/BATF/RorγT-axis in the tumoural region of adenocarcinoma as compared to squamous cell carcinoma of the lung

doi: 10.1038/srep07396

Figure Lengend Snippet: (a),(b)- Flow cytometry analysis of IL-6R in the lungs of naïve as compared to LL/2-luc-M38 injected wild-type mice (N naive = 10, N LL/2 = 11). a-Percentage of IL6R+CD4+ T-cells gated on lymphocytes. b- Percentage of IL-6R+cells gated on CD4+CD25+Foxp-3+ T-cells. Dot plots depict the gating strategy. Bar charts show the mean percentage of gated cells. Data are shown as mean values +/− s.e.m. using student's t -test *** P,0.001 .

Article Snippet: Isolation of CD4 + T-cells from total lung cells was performed using anti-mouse CD4 Microbreads (Miltenyi Biotec, Bergisch-Gladbach, Germany) by positive selection according to manufacturer's protocol.

Techniques: Flow Cytometry, Injection

Journal: Scientific Reports

Article Title: Increased expression of the Th17-IL-6R/pSTAT3/BATF/RorγT-axis in the tumoural region of adenocarcinoma as compared to squamous cell carcinoma of the lung

doi: 10.1038/srep07396

Figure Lengend Snippet: (a)- Quantitative Real-time PCR analysis of BATF (Basic leucine zipper transcription factor, ATF-like) mRNA expression in the tumoural and control lung area of patients with ADC (adenocarcinoma) and SCC (squamous cell carcinoma) (N (ADC control ) = 21; N (ADC tumoural ) = 20; N (SCC control ) = 17; N (SCC tumoural ) = 17). Data are shown as mean values ± s.e.m. using student's t -test * P ,0.05; ** P ,0.01. (b),(c)- Analysis of BATF protein expression by Immunohistochemistry (IHC) in the tumoural region of ADC (b) as compared to the tumoural region of SCC (c). d- Luciferase measurement of 2 representative mice injected with LL/2-luc-M38 cells. e,f- Quantitative Real-time PCR analysis of Batf and Stat3 mRNA expression in CD4+ T-cells, isolated from the lungs of naïve as compared to tumour bearing wild-type mice and stimulated with anti-CD3 and anti-CD28 antibody. Data are shown as mean values ± s.e.m. using student's t -test * P ,0.05 (N = per group).

Article Snippet: Isolation of CD4 + T-cells from total lung cells was performed using anti-mouse CD4 Microbreads (Miltenyi Biotec, Bergisch-Gladbach, Germany) by positive selection according to manufacturer's protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Immunohistochemistry, Luciferase, Injection, Isolation

Journal: Scientific Reports

Article Title: Increased expression of the Th17-IL-6R/pSTAT3/BATF/RorγT-axis in the tumoural region of adenocarcinoma as compared to squamous cell carcinoma of the lung

doi: 10.1038/srep07396

Figure Lengend Snippet: (a)- Experimental design is shown. (b)- Flow cytometry analysis of Foxp-3+CD25+ cells gated on CD4+ lung T-cells from naïve or tumour bearing wild-type mice injected with LL/2-luc-M38 cells and treated with anti-IL6R antibody or IgG1 isotype control as compared to untreated mice. Data are shown as mean percentage ± s.e.m. using student's t -test * P ,0.05; ** P ,0.01 (N = 10–11 per group). (c),(d)-Gene array analysis of RNA, isolated from CD4 + lung T-cells of tumour bearing wild-type mice treated with anti-IL6R antibody or untreated. The fold-change of the mRNA expression of significantly altered genes is shown as a heatmap (c) and as a bar chart (d) (N = 3 per group). (e),(f),(g)- Quantitative Real-time PCR analysis of Il17 (e), Rorc (f) and Batf (g) mRNA expression in CD4+ T-cells, cultured with anti-CD3 and anti-CD28 antibody. CD4 + T-cells were isolated from the lungs of naïve or tumour bearing wild-type mice injected with LL/2-luc-M38 cells and treated with anti-IL6R antibody or IgG1 isotype control compared to untreated mice. Data are shown as mean values ± s.e.m. Sudent's t-test was used. * P,0.05 (N = 10–11 per group).

Article Snippet: Isolation of CD4 + T-cells from total lung cells was performed using anti-mouse CD4 Microbreads (Miltenyi Biotec, Bergisch-Gladbach, Germany) by positive selection according to manufacturer's protocol.

Techniques: Flow Cytometry, Injection, Control, Isolation, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Experimental and Therapeutic Medicine

Article Title: Application of red clover isoflavone extract as an adjuvant in mice

doi: 10.3892/etm.2019.8315

Figure Lengend Snippet: Effect of RCIE or alum adjuvant on CD4 + and CD8 + T cell populations in the peripheral blood of OVA-immunized mice. Representative flow cytometry dot plots showing the population of CD4 + and CD8 + T cells in the peripheral blood from mice in the (A) Saline, (B) OVA, (C) OVA + alum, (D) OVA + 50 µg RCIE, (E) OVA + 100 µg RCIE and (F) OVA + 200 µg RCIE groups. (G) Quantified flow cytometry data. Values are presented as mean ± SEM (n=10). *P<0.05 vs. OVA. OVA, ovalbumin; alum, aluminum hydroxide gel; RCIE, red clover isoflavone extract; PE, phycoerythrin.

Article Snippet: The ELISA kits (IFN-γ, cat. no. F10660; TNF-α cat. no. F11630; IL-2 cat. no. F10780; IL-4 cat. no. F10810; and IL-5 cat. no. F10820) were from Shanghai Westang Biotechnology Co., Ltd. FITC/phycoerythrin (PE) rat anti-mouse CD4/CD8 antibodies (cat. no. FMD001-050) were purchased from Beijing 4A Biotech Co., Ltd. RNAiso plus ® (cat. no. 9108), First Strand cDNA Synthesis kit (cat. no. RR036A) and SYBR ® Premix Ex Taq™ were purchased from Takara Biotechnology Co., Ltd. Pathogenic porcine Escherichia coli ( E. coli ) ( ) was kindly provided by Dr Jingxuan Ni (Longyan University).

Techniques: Flow Cytometry

Journal: PLoS ONE

Article Title: A Single Intramuscular Vaccination of Mice with the HSV-1 VC2 Virus with Mutations in the Glycoprotein K and the Membrane Protein UL20 Confers Full Protection against Lethal Intravaginal Challenge with Virulent HSV-1 and HSV-2 Strains

doi: 10.1371/journal.pone.0109890

Figure Lengend Snippet: (A) Colorimetric ELISA based analysis of HSV-1 reactive polyclonal IgG produced 21 days post vaccination n = 20. Statistical comparison conducted by SAS using the T test Procedure. Bars represent the 95% confidence interval about the mean (B) Titration of serum neutralizing fixed PFU of HSV-1 (McKrae) normalized to a no serum control n = 5. Significant reduction in PFU observed 1∶160, 1∶80, 1∶40, and 1∶20 dilutions of the sera. Statistical comparison conducted by SAS using The Mixed Procedure and Differences in Least Squares Means. Bars represent the 95% confidence interval about the mean. (C) Cross reactive neutralization of HSV-1 (McKrae) and HSV-2 (G) at a 1∶20 dilution of sera from vaccinated and mock inoculated mice. Percent neutralization normalized to no serum controls. Statistical comparison conducted by SAS using The Mixed Procedure and Differences in Least Squares Means. Bars represent the 95% confidence interval about the mean. Significant differences noted as * p≤0.05, ** p≤0.01, or *** p≤0.0001.

Article Snippet: Labeled cells were then cultured at a concentration of 10 5 cells/well in a 96 well U-bottom plate and incubated at 37°C and 5% CO 2 for 7 days in the presence of pooled peptides specific to either HSV-1 or HSV-2 at a concentration of 10 µg/mL Cells were then stained with polyclonal anti-mouse CD4 antibody conjugated to PE (BD Biosciences) and polyclonal anti-mouse CD8a antibody conjugated to APC (BD Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay, Produced, Titration, Neutralization