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: Nature Communications

Article Title: Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis

doi: 10.1038/s41467-022-33006-4

Figure Lengend Snippet: a CD4 + T cell differentiation ratio in gate of CFSE − cells in T cell sample of healthy donors 1. T cells of healthy donors 1 were induced by anti-CD3 antibody (5 μg/mL) and anti-CD28 antibody (5 μg/mL) for 3 days or 12 days with/without treatment. b CD4 + T cell differentiation ratio in gate of total cells and gate of CFSE − cells in T cell sample of healthy donors 2. T cells of healthy donors 2 were induced by anti-CD3 antibody (5 μg/mL) and anti-CD28 antibody (5 μg/mL) for 9 days with or without treatment. c CD4 + T cell differentiation ratio in gate of total cells and gate of CFSE − cells in T cell sample of cancer patient donors 1. T cells of cancer patient donors 1 were induced by anti-CD3 antibody (5 μg/mL) and anti-CD28 antibody (5 μg/mL) for 3 days with or without treatment. In experiments of a – c , CFSE − cells, CD4 + cells, CD8 + cells were assayed by flow cytometry and analyzed their percentage in the corresponding gate after treatment. Data are presented as the mean ± s.d. from three or four biological replicate per group. Statistical difference was determined by unpaired two-tailed Student’s t-test. Significant difference p -value is < 0.05.

Article Snippet: Subsequently, the proportion of CD4 + and CD8 + cells was determined by flow cytometry (Beckman Coulter, FC500) according to manufacturer’s instruction of PE-Cyanine7 anti-Human CD8a and PE anti-Human CD4 antibodies (Tonbo Biosciences).

Techniques: Cell Differentiation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis

doi: 10.1038/s41467-022-33006-4

Figure Lengend Snippet: a micro-CT images of the trabecular part of distal femur of rat treated with S3-15 shows higher bone density than rat not treated (OVX). Histological analysis of trabecular bone parameters including BV/TV, Tb.Th, Tb.N and Tb.Sp demonstrate that S3-15 improve significantly the morphometric characteristics of trabecular bone. b Serum PINP, CTX-I, OCN, and Glu-Ocn osteoporosis related marker levels were decreased in rats treated with S3-15 . c micro-CT images of the trabecular part of distal femur of mouse treated with S3-15 shows higher bone density than mouse not treated (OVX). Histological analysis of trabecular bone parameters including BV/TV, Tb.Th, Tb.N, and Tb.Sp demonstrate that S3-15 improve significantly the morphometric characteristics of trabecular bone. Biomechanical testing shows S3-15 treated mice improving ultimate load. d , e S3-15 can improve the imbalance of immune cell ratio caused by osteoporosis. In peripheral blood total CD3 + , CD8 + and CD3 + CD8 + T cells are decreased in osteoporosis mice, (left). In CD3 + T cells subset, osteoporosis mice show an increased percentage of CD3 + CD4 + T cells and a decreased percentage of CD3 + CD8 + T cells, that results in the increased the value of CD3 + CD4 + / CD3 + CD8 + (right). S3-15 treatment can reverse those abnormal indicators. f S3-15 alleviating spleens swelling in mice with osteoporosis. g Spleens of sham, OVX and S3-15 treated mice are got and applied splenic lymphocyte transformation experiment. Spleen lymphocyte transforming index is lower in osteoporosis mice than sham mice and can improve to normal level after S3-15 treatment for 4 weeks. Rat number of sham and OVX + S3-15 group, n = 9. Rat number of OVX group, n = 10. In mouse study, n = 8 mice per group. Data are expressed as means ± s.d. Statistical difference for ultimate load was determined by one-way ANOVA followed by Uncorrected Fisher’s LSD, and the others were followed by Tukey’s multiple comparisons test. Significant difference p -value is < 0.05. Scalar bar, 500 μm.

Article Snippet: Subsequently, the proportion of CD4 + and CD8 + cells was determined by flow cytometry (Beckman Coulter, FC500) according to manufacturer’s instruction of PE-Cyanine7 anti-Human CD8a and PE anti-Human CD4 antibodies (Tonbo Biosciences).

Techniques: Micro-CT, Marker, Transformation Assay