Journal: Scientific Reports

Article Title: Combining DNMT and HDAC6 inhibitors increases anti-tumor immune signaling and decreases tumor burden in ovarian cancer

doi: 10.1038/s41598-020-60409-4

Figure Lengend Snippet: DNMT1 protein levels are decreased by combination treatment of DNMTi and HDAC6i. ( A ) Ovarian cancer cell lines were treated as in Fig. and protein was extracted at Day 7 after treatment with IFN-gamma (IFN-γ+) (to assess MHC I and PD-L1 expression, in later figures) or control (IFN-γ -). Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( B ) The TykNu cell line was treated as in ( A ) and the protein synthesis cycloheximide added to cells on Day 7 for 0, 4, and 8 hours at 10 μM as indicated on the blot. Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( C ) Stable knockdowns of the HDAC6 protein were generated in the ID8 Trp53+/+ and Trp53−/− cell lines . Protein was extracted and immunoblots were run for the DNMT1 protein with B-actin as a loading control. Immunoblot membranes were probed for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( D ) Immunoblot showing knockdown of HDAC6 protein with a-Tubulin as a loading control. Protein was extracted and immunoblots were run for the HDAC6 protein with B-actin as a loading control. Immunoblots were probed for HDAC6 (131 kDa) and tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( E ) Ovarian cancer cell lines were treated as in Fig. and RNA was extracted at Day 7. qRT-PCR was run for DNMT1, DNMT3a, and DNMT3b and TBP was used as a reference gene. *p < 0.05 compared to Mock.

Article Snippet: The antibodies used for immunoblotting included: DNMT1 (Sigma, D4692), PD-L1 (ProSci, 4059), HDAC1 (Cell Signaling, 2062), HDAC2 (Cell Signaling, 2540), HDAC6 (Assay Biotech, C0026), acetyl-alpha Tubulin (Cell Signaling, 3971), alpha-Tubulin (Cell Signaling, 3873).

Techniques: Expressing, Control, Isolation, Western Blot, Generated, Knockdown, Quantitative RT-PCR

Journal: Cellular and Molecular Immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Computer-aided homology modeling. (a) The ribbon structure of the human PD-1 and PD-L1 complex based on the computer-guided modeling and docking methods. (b) The fine structure of the binding mode between human PD-1 and PD-L1. The purple sticks denote the key amino-acid residues in human PD-L1, whereas the green balls and sticks denote the key amino-acid residues in human PD-1. (c) The 3-D theoretical structure of IgHV3-33*01, where the balls and sticks denote H28, H31 and H32. (d) The 3-D theoretical structure of IgHK1-11*01, where the balls and sticks denote L27, L50 and L91.

Article Snippet: The CD274 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014143","term_id":"1519243726","term_text":"NM_014143"}} NM_014143 ) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Binding Assay

Journal: Cellular and Molecular Immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Preliminary functional assessment of FV78. (a) Binding activity of FV78 to PD-1/Fc. (b) Epitope comparability of FV78 and MIL75 binding to PD-1/Fc by ELISA assay. (c) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L1 to PD-1 by ELISA assay. (d) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L1 to PD-1 by FACS assay. (e) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L2 to PD-1 by ELISA assay. (f) Evaluation of the cross-reactive binding activity of FV78 to other CD28 family members.

Article Snippet: The CD274 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014143","term_id":"1519243726","term_text":"NM_014143"}} NM_014143 ) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Functional Assay, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Two siblings homozygous for a splice-site variant of CD274 . (A) The pedigree. Black symbols indicate affected individuals. Genotypes for the CD274 allele are also shown. WT: wild-type. M: mutant. E?: unknown. (B) Validation of the variant by Sanger sequencing. (C) Gene-level negative selection. PDCD1 , CD274 , and PDCD1LG2 (encoding PD-1, PD-L1, and PD-L2, respectively) are not under negative selection, as shown by CoNeS score , as also reported for other genes for which mutations underlie AR IEI. CTLA4 is also shown, as an example of another gene under negative selection. (D) Population genetics of PDCD1 , CD274 , and PDCD1LG2 . The MAF and CADD scores for all non-synonymous variants found in the gnomAD database are depicted. All biallelic variants are labeled with their predicted protein-level consequences. The horizontal dotted line indicates the MSC ( ; ).

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Variant Assay, Mutagenesis, Sequencing, Selection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of the effect of the CD274 splice-site variant on mRNA splicing in an overexpression system. (A) Schematic diagram of an exon-trapping assay. A region of genomic DNA flanking the fourth exon of the canonical CD274 isoform with or without the c.682+1G>A splice-site in the homozygous state was inserted into the pSPL3 vector. The plasmids were used to transfect HEK293T cells and, 24 h later, the spliced mRNA product was recovered by RT-PCR and TOPO cloning, and subjected to Sanger sequencing. (B) Exon trapping. The schematic diagram shows the four types of cDNA identified, with the number of nucleotides in each region indicated. Representative data from two experiments are shown. (C) A schematic diagram of the CD274 mRNA and PD-L1 protein. Exon 1 is omitted because it contains no coding sequence. The red rectangle depicts the 51-amino acid in-frame deletion caused by the c.682+1G>A variant. SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain. Source data are available for this figure: .

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Variant Assay, Over Expression, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of the PD-L1 protein with in-frame deletion in an overexpression system. (A and B) PD-L1 protein levels. Raji B-lymphoma cells were lentivirally transduced with cDNA encoding the WT or a mutant PD-L1 isoform, or with EV, and were then subjected to selection on puromycin. PD-L1 protein levels were determined by (A) immunoblotting and (B) flow cytometry with monoclonal antibodies (mAb) against PD-L1. In B, a vertical dotted line within a histogram indicates the median. Representative results from two experiments are shown. (C and D) PD-1:PD-L1-mediated suppression assay. (C) Schematic diagram. HuT78 T-lymphoma cells lentivirally transduced with EV or with WT PD-1 were cocultured with Raji cells transduced with EV or a WT or mutant PD-L1 isoform for 24 h without stimulation or with blinatumomab (CD3-CD19 bispecific antibody, BiTE). Secretion inhibitors were added for the last 6 h. IFN-γ production was quantified by intracellular flow cytometry. The effect of anti-PD-L1 neutralizing mAb (equivalent to atezolizumab) or its isotype control was also assessed in this system. (D) Summary plot. The readout (percentage of IFN-γ + HuT78 cells) was normalized against the mean in the “BiTE plus anti-PD-L1 antibody” group. Results from two independent experiments with 12 technical replicates in total were compiled. Statistical significance was determined for differences between EV and each PD-L1 construct in BiTE-stimulated conditions by two-tailed Wilcoxon’s rank sum tests with FDR adjustment. n.s., not significant. ****, P < 0.0001. Source data are available for this figure: .

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Over Expression, Transduction, Mutagenesis, Selection, Western Blot, Flow Cytometry, Suppression Assay, Control, Construct, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of endogenously expressed CD274 mRNA and PD-L1 protein in the patients’ leukocytes. (A, B, and D) Bulk RNASeq analysis. PBMCs from the two PD-L1-deficient siblings (ages 11 and 10 years), and adult and age-matched controls were either left non-stimulated or were stimulated with lipopolysaccharide (LPS), anti-CD2/CD3/CD28 mAb cocktail, or phorbol 12-myristate 13-acetate and ionomycin (P/I) for 24 h. (A) A schematic diagram of the CD274 mRNA exon 3-4-5 splice junctions in the cells of healthy donors (canonical) and the patients (alternative). (B) Ratio of read counts supporting the canonical and alternative exon 4–5 splice junction to read counts for the exon 3–4 splice junction. (C) RT-PCR products with a primer pair amplifying the whole CD274 coding sequence derived from the total RNA of PBMCs stimulated with LPS for 24 h. (D) Expression levels (transcripts per million reads; TPM) for each CD274 exon. (E) Western blot analysis for PD-L1 in PBMCs. PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years), their mother, and two healthy controls were either left non-stimulated or were stimulated with PHA overnight. Cell lysates were either left untreated or were treated with PNGase F, as indicated. (F) Densitometry results for the western blot shown in E. Values are normalized against the density of the loading control (HSP90). (G) Surface PD-L1 expression. PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years) and adult and age-matched controls were either left non-stimulated or were stimulated with IFN-α2, LPS, or anti-CD3/CD28 mAb-conjugated beads for 24 h. The level of PD-L1 expression on the surface of the cells of the different leukocyte subsets was determined by flow cytometry with two different mAbs against human PD-L1. (H) IFN-γ neutralization assay. PBMCs from healthy controls were either left non-stimulated or were stimulated with anti-CD3/CD28 mAb-conjugated beads for 24 h in the presence of anti-IFN-γ neutralizing mAb or its isotype control. PD-L1 levels were determined by flow cytometry with the 29E.2A3 clone. The horizontal dotted line indicates the level of background fluorescence determined with an isotype control for 29E.2A3. In B and G, bars represent the mean and SEM. In A–D and G, the experiments were performed once. In E and F, representative data from two experiments are shown. In H, results from three experiments (six donors in total) with technical duplicates are compiled. Statistical significance was determined for differences between IFN-γ neutralization and isotype control in anti-CD3/CD28-stimulated conditions by two-tailed paired Wilcoxon signed rank tests with FDR adjustment. **, P <0.01. Source data are available for this figure: .

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Derivative Assay, Expressing, Western Blot, Control, Flow Cytometry, Neutralization, Fluorescence, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of CD274 mRNA in the patients’ leukocytes. (A) Schematic diagram of the design of RT-PCR primers and TaqMan probes. (B) RT-PCR on the CD274 CDS from total RNA extracted from PBMCs stimulated with LPS for 24 h. The 153nt deletion observed in bulk RNASeq data was confirmed by Sanger sequencing. (C) Bulk RNASeq for stimulated PBMCs. PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years), an age-matched control, and several healthy adult controls were either left unstimulated or were stimulated with LPS, anti-CD2/3/28 mAb cocktail, or PMA/ionomycin (P/I) for 24 h. Total RNA was used to prepare libraries for bulk RNASeq. TPM is shown for each CD274 exon. (D) Quantitative PCR on the cDNA derived from the total RNA extracted from the LPS-stimulated PBMCs analyzed in C. GUSB was used as an endogenous control. Ratios of results for two TaqMab probes, targeting the exon 1–2 or 6–7 junction, are shown. The non-stimulated conditions were analyzed twice (technical replicates). (E) Bulk RNASeq on whole-blood leukocytes. Freshly drawn venous blood samples from the two PD-L1-deficient siblings (aged 11 and 10 years) and age-matched controls were used for total RNA extraction. Globin-depleted total RNA was used for sequencing. TPMs per region ( CD274 exons 1–4 and 5–7) are shown. (F) IFN-γ neutralization assay. PBMCs from healthy controls were either left non-stimulated or were stimulated with anti-CD3/CD28 mAb-conjugated beads for 24 h in the presence of anti-IFN-γ neutralizing mAb or its isotype control. PD-L1 levels were determined by flow cytometry with the 29E.2A3 clone. The fold-change decrease in PD-L1 MFI was calculated. In A–E, the experiments were performed once. In F, results from three experiments (six donors in total) with technical duplicates are compiled. The statistical significance of the difference between anti-IFN-γ and isotype control treatments was determined for each set of conditions in two-tailed Wilcoxon’s signed-rank tests with FDR adjustment. n.s., not significant. **, P < 0.01.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Control, Real-time Polymerase Chain Reaction, Derivative Assay, RNA Extraction, Neutralization, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Immunophenotyping analysis of PD-L1-deficient leukocytes. Freshly drawn whole-blood leukocytes were analyzed by flow cytometry. (A) Absolute cell numbers were determined with Trucount Absolute Counting Tubes. (B–D) Frequencies of the given leukocyte subsets within each parental subset (indicated at the top of the plots). (E) Gating strategy for circulating T FH (cT FH ) cells. (F) Representative plots for cT FH cells. (G) Percentage of cT FH cells. In A–D and G, bars represent the mean and SEM.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Immunophenotyping analysis of PD-1- and PD-L1-deficient leukocytes. Freshly thawed PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years), their mother, and adult and age-matched controls were immunophenotyped by flow cytometry. PBMCs from the previously described PD-1-deficient child (aged 11 years) were also analyzed with the same panel. (A) Proportions of leukocyte subsets in PBMCs. (B) CD4 + αβ T lymphocyte subsets. (C) CD8 + αβ T lymphocyte subsets. (D and E) Proportions of activated T lymphocyte subsets in (D) PBMCs and (E) each parental subset. Bars represent the mean and SEM.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of blood αβTCR repertoire in the PD-1- and PD-L1-deficient patients. The complementarity-determining region 3 (CDR3) sequences in the TRAV and TRBV regions were reconstructed with MiXCR from bulk RNASeq datasets for whole-blood leukocytes from the two PD-L1-deficient siblings (aged 11 and 10 years), their mother, and adult and age-matched controls. For TRBV , the previously published Adaptive ImmunoSeq data for genomic DNA from the whole-blood leukocytes of the PD-1-deficient child (aged 10 years), his healthy brother (aged 6 years), and three healthy controls are also shown for comparison. (A) CDR3 length and physicochemical properties. The median values for each individual are shown. (B and C) CDR3 clonotype diversity. (D and E) Properties of the distribution of CDR3 clonotype sizes. Bars represent the mean and SEM.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Comparison

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Single-cell transcriptomic analysis of PD-1- and PD-L1-deficient leukocyte subsets. scRNASeq was performed on cryopreserved PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years), their mother, and adult and age-matched controls. Previously generated datasets for healthy and diseased controls, including the PD-1-deficient child and his healthy brother, were also integrated into the analysis ( ; ). (A) Clustering. Graph-based clustering was conducted after the removal of batch effects with Harmony . Clusters were identified with SingleR guided by the Monaco datasets , followed by manual inspection. (B) Representative gene expression profiles. (C) Pseudobulk DE analysis. Individuals with PD-1 or PD-L1 deficiency were compared with age-matched controls, including the PD-1-deficient patient’s brother and an age-matched control for the PD-L1-deficient siblings. DE genes (DEGs) were defined as genes with FDR-adjusted P values <0.1. cDC1 was omitted because too few cells were captured for the PD-1-deficient patient. The numbers of DEGs per cell type are shown on a bar chart. (D) DEGs common to other monogenic etiologies of autoimmune or autoinflammatory disorders. For each condition, patients with monogenic disease were compared with age-matched controls. The numbers of DEGs (FDR-adjusted P value <0.1) common to (1) PD-1 or PD-L1 deficiency and (2) one of the four known monogenic forms of autoimmunity or autoinflammatory diseases are shown for each cell type. (E) DEGs common to the PD-1- and PD-L1-deficient leukocyte subsets. Here, DEGs are defined as genes with |log 2 FC| > 2 relative to age-matched controls. (F) Geneset overrepresentation analysis. DEGs upregulated in the classical or non-classical monocytes of PD-1- and PD-L1-deficient patients relative to age-matched controls were projected onto the Gene Ontology Molecular Function (GO MF) gene sets. GO MF gene sets for which significant enrichment was detected are shown. (G and H) SCENIC regulon activity analysis on (G) Vδ2 + γδ T cells and (H) monocytes (classical and non-classical combined). Single-cell regulon activities were aggregated to obtain a mean level of activity per cell type and per individual. The regulons most strongly differentially regulated in individuals with PD-1 and PD-L1 deficiencies relative to age-matched controls, as determined by two-tailed Wilcoxon’s rank sum test, are shown. Bars represent the mean and SEM.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Generated, Expressing, Control, Activity Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Single-cell transcriptomic analysis. scRNASeq was performed on cryopreserved PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years), their mother, and adult and age-matched controls. Previously generated datasets for healthy and diseased controls, including the PD-1-deficient child and his healthy brother, were also integrated into the analysis ( ; ). Cell subsets were identified by unsupervised clustering followed by automated (i.e., SingleR) and manual annotation. (A) Frequencies of transcriptionally determined leukocyte subsets. (B–D) Pseudobulk DE analysis was performed to compare individuals with PD-1 or PD-L1 deficiency and age-matched controls, including the PD-1-deficient patient’s brother and an age-matched control for the PD-L1-deficient siblings. DE genes (DEGs) were defined as genes with |log 2 FC| > 2 relative to age-matched controls. (B) Geneset overrepresentation analysis. DEGs upregulated in Vδ2 + γδ T cells from PD-1- and PD-L1-deficient patients relative to age-matched controls were projected onto the gene ontology (GO) gene sets (BP for biological process, MF for molecular function, and CC for cellular component). GO gene sets for which significant enrichment was detected are shown. (C and D) Gene network plots for (C) Vδ2 + γδ T cells and (D) monocytes (classical and non-classical combined). DEGs contributing to a given GO term are connected by edges.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of the cellular responses of PD-1- and PD-L1-deficient leukocytes in vitro. (A–E) PBMC stimulation assay. PBMCs from the two PD-L1-deficient siblings (aged 11 and 10 years), their mother, and adult and age-matched controls were either left non-stimulated or were stimulated for 24 h. Bulk RNASeq was performed. (A) PCA. (B) GSEA. Genes were ranked based on their fold-change induction (stimulated versus non-stimulated) in PD-L1-deficient cells relative to either healthy controls or the heterozygous mother. Only significant results (FDR-adjusted P value <0.05) from 50 Hallmark gene sets are shown. Gene sets were reordered by hierarchical clustering for visualization purposes. (C) Differential gene induction. Genes related to cytokines or their receptors downregulated in PD-L1-deficient cells relative to control cells are labeled. (D and E) Transcription factor (TF) activity inference analysis based on the CollecTRI gene regulatory network database. (D) PCA. (E) Scaled activity for the TFs known to regulate IFNG mRNA levels lying in the top 30 for the loading of PC1 in D. (F and G) T-blast stimulation assay. (F) T-blasts were stimulated for 4 h in the presence of secretion inhibitors. Cytokine production was quantified by intracellular flow cytometry. Technical duplicates were prepared. (G) T-blasts were stimulated for 4 h, and cytokine secretion was quantified by multiplex ELISA. The age-matched controls include the healthy brother of the PD-1-deficient child. In A–E, the experiments were performed once. In F, representative data from two experiments are shown. In G, data from three experiments with technical replicates for PD-1-deficient cells (14 replicates for N = 1) and PD-L1-deficient cells (duplicates for N = 2) are compiled. In A, D, F, and G, the bars represent the mean and SEM. In F, statistical significance was determined for differences between all healthy controls combined and the PD-1/PD-L1-deficient patients combined by two-tailed Wilcoxon’s rank sum tests with FDR adjustment. *, P < 0.05.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: In Vitro, Control, Labeling, Activity Assay, Flow Cytometry, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency

doi: 10.1084/jem.20231704

Figure Lengend Snippet: Analysis of the cellular responses of PD-1- and PD-L1-deficient T lymphocytes in vitro. T-blasts from healthy donors, a PD-1-deficient patient, and the PD-L1-deficient siblings and their heterozygous mother were either left non-stimulated or were stimulated with anti-CD2/CD3/CD28 mAb cocktail or PMA/ionomycin (P/I) for 4 h. (A–C) Intracellular cytokine levels were measured by flow cytometry. (D) Secreted cytokine levels were measured by multiplex ELISA. In B–D, bars represent the mean and SEM. In B, representative data from two experiments are shown. In C, the experiment was performed once. In D, data from three experiments with technical replicates for PD-1-deficient cells (14 replicates for N = 1) and PD-L1-deficient cells (duplicates for N = 2) are compiled.

Article Snippet: HuT78 cells (1 × 10 5 cells per well) and Raji cells (1 × 10 5 cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen).

Techniques: In Vitro, Flow Cytometry, Multiplex Assay, Enzyme-linked Immunosorbent Assay