Journal: iScience

Article Title: Defects in NK cell immunity of pediatric cancer patients revealed by deep immune profiling

doi: 10.1016/j.isci.2024.110837

Figure Lengend Snippet:

Article Snippet: Anti-ILT4 (clone 42D1, conjugated to 161Dy) , Standard BioTools , Cat#3161019B.

Techniques: Purification, Clinical Proteomics, Recombinant, Blocking Assay, Staining, Saline, Mass Cytometry, Software, Cytometry

Journal: PLoS ONE

Article Title: Downregulation of Immunoglobulin-Like Transcript-4 (ILT4) in Patients with Psoriatic Arthritis

doi: 10.1371/journal.pone.0092018

Figure Lengend Snippet: ( A ) Cells were gated for monocytes based on their forward-scatter/side-scatter profile. Controls with isotype matched irrelevant mAbs consistently showed <1% of positive cells. The data represent mean±SD (error bars). MFI values of ILT4 expression in CD40L-stimulated cells were as follows. Freshly isolated monocytes: controls, 44.6±9.8, PsA, 40±12.3, P >0.05. CD40L-monocytes: controls, 97.5±29.8, PsA, 57.1±15, P <0.005. Comparison between means was performed by Student's unpaired t test. All P values are 2-tailed. P values <0.05 were considered significant. ( B ) Correlation between monocyte ILT4 expression and DAS44. ILT4 expression negatively correlated to DAS44 ( P = 0.02). Correlations were evaluated by the Pearson parametric for the univariate analysis.

Article Snippet: 2×10 5 cells were incubated with the following antibodies at appropriate dilutions for 30 min at 4°C in the dark: R-phycoerythrin-cyanine (PC5)-conjugated anti-CD14 (Immunotech), phycoerythrin (PE)-conjugated anti- ILT4 (Immunotech, Marseille, France, clone: PN A22334), fluorescein isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti CD86 (BD Biosciences San Josè CA), and anti-TNF-α (R&D system, San Diego CA).

Techniques: Expressing, Isolation, Comparison

Journal: PLoS ONE

Article Title: Downregulation of Immunoglobulin-Like Transcript-4 (ILT4) in Patients with Psoriatic Arthritis

doi: 10.1371/journal.pone.0092018

Figure Lengend Snippet: Dot plots showing CD14/ILT4 co-expression in CD40L monocytes from representative control and PsA subjects.

Article Snippet: 2×10 5 cells were incubated with the following antibodies at appropriate dilutions for 30 min at 4°C in the dark: R-phycoerythrin-cyanine (PC5)-conjugated anti-CD14 (Immunotech), phycoerythrin (PE)-conjugated anti- ILT4 (Immunotech, Marseille, France, clone: PN A22334), fluorescein isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti CD86 (BD Biosciences San Josè CA), and anti-TNF-α (R&D system, San Diego CA).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Downregulation of Immunoglobulin-Like Transcript-4 (ILT4) in Patients with Psoriatic Arthritis

doi: 10.1371/journal.pone.0092018

Figure Lengend Snippet: Sera from each of the 16 patients with PsA and of the 15 controls were incubated with elutriated monocytes from a single healthy donor. Sera from patients and controls were obtained at baseline. Medications of patients at baseline are reported in . FCS was used as negative control. The experiments were performed twice, using cells from two different healthy donors; each experiment was carried out in triplicate wells. The data represent mean±SD (error bars). Comparison between means was performed by Student's unpaired t test. MFI values of ILT4 expression in CD40L-stimulated and, in parenthesis, unstimulated cells were as follows: FCS-exposed, 145±13.9 (34.5±12.4); normal serum-exposed, 120.3±11.2 (30.9±13.7); patient's serum-exposed 33.6±8.4 (36.1±14.6). CD40L: FCS-exposed vs. normal serum-exposed, P >0.05; normal serum-exposed vs. patient's serum-exposed, P <0.001. Unstimulated: FCS-exposed vs. normal serum-exposed, and normal serum-exposed vs. patient's serum-exposed, P >0.05.

Article Snippet: 2×10 5 cells were incubated with the following antibodies at appropriate dilutions for 30 min at 4°C in the dark: R-phycoerythrin-cyanine (PC5)-conjugated anti-CD14 (Immunotech), phycoerythrin (PE)-conjugated anti- ILT4 (Immunotech, Marseille, France, clone: PN A22334), fluorescein isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti CD86 (BD Biosciences San Josè CA), and anti-TNF-α (R&D system, San Diego CA).

Techniques: Incubation, Medications, Negative Control, Comparison, Expressing

Journal: PLoS ONE

Article Title: Downregulation of Immunoglobulin-Like Transcript-4 (ILT4) in Patients with Psoriatic Arthritis

doi: 10.1371/journal.pone.0092018

Figure Lengend Snippet: The box plots represent the expression of ILT4 analyzed by FACS on CD14 + gated cells as percentage of positive cells. Controls with isotype matched irrelevant mAbs consistently showed <1% of positive cells. The data represent means±SD (error bars) Comparison between means was performed by Student's unpaired t test.

Article Snippet: 2×10 5 cells were incubated with the following antibodies at appropriate dilutions for 30 min at 4°C in the dark: R-phycoerythrin-cyanine (PC5)-conjugated anti-CD14 (Immunotech), phycoerythrin (PE)-conjugated anti- ILT4 (Immunotech, Marseille, France, clone: PN A22334), fluorescein isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti CD86 (BD Biosciences San Josè CA), and anti-TNF-α (R&D system, San Diego CA).

Techniques: Expressing, Comparison

Journal: PLoS ONE

Article Title: Downregulation of Immunoglobulin-Like Transcript-4 (ILT4) in Patients with Psoriatic Arthritis

doi: 10.1371/journal.pone.0092018

Figure Lengend Snippet: The box plots represent the spontaneous (A) and LPS-induced (B) intracellular production of TNF-α analyzed by FACS as percentage of positive cells. Appropriate controls with isotype matched irrelevant mAbs were carried out and consistently showed <1% of positive cells. The data represent means±SD (error bars). Comparison between means was performed by Student's unpaired t test. (C) LPS-induced TNF-α production by total monocyte population from a representative control subject and a patient with PsA. CD40L-activated monocytes were stimulated with LPS and stained with PE-anti-ILT4 for the determination of surface phenotype. Intracellular TNF-α was detected by staining with FITC-anti-TNF-α. The data are displayed as dot plots.

Article Snippet: 2×10 5 cells were incubated with the following antibodies at appropriate dilutions for 30 min at 4°C in the dark: R-phycoerythrin-cyanine (PC5)-conjugated anti-CD14 (Immunotech), phycoerythrin (PE)-conjugated anti- ILT4 (Immunotech, Marseille, France, clone: PN A22334), fluorescein isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti CD86 (BD Biosciences San Josè CA), and anti-TNF-α (R&D system, San Diego CA).

Techniques: Comparison, Control, Staining

Journal: PLoS ONE

Article Title: Downregulation of Immunoglobulin-Like Transcript-4 (ILT4) in Patients with Psoriatic Arthritis

doi: 10.1371/journal.pone.0092018

Figure Lengend Snippet: ( A ) Median and interquartile range of percentage of monocyte expressing ILT4, CD40, CD80 and CD86 from sixteen patients at baseline vs. week 12 were as follows: ILT4, 45.5 (34–57) vs. 57 (47.5–65.5), P = 0.016; CD40, 15 (10–18.5) vs. 11 (7–12.5), P = 0.025; CD80, 16.6 (12.5–19.5) vs. 12 (8–15), P = 0.015; CD86, 65.5 (49.5–75) vs. 49.5 (41.5–61.5), P = 0.036. Comparison between means was performed by Student's unpaired t test. ( B ) Correlation between monocyte ILT4 expression and ΔDAS44 at week 12 after adalimumab. ILT4 expression directly correlates to ΔDAS44 ( P = 0.02) Correlations were evaluated by the Pearson parametric for the univariate analysis.

Article Snippet: 2×10 5 cells were incubated with the following antibodies at appropriate dilutions for 30 min at 4°C in the dark: R-phycoerythrin-cyanine (PC5)-conjugated anti-CD14 (Immunotech), phycoerythrin (PE)-conjugated anti- ILT4 (Immunotech, Marseille, France, clone: PN A22334), fluorescein isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti CD86 (BD Biosciences San Josè CA), and anti-TNF-α (R&D system, San Diego CA).

Techniques: Expressing, Comparison

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: Correlation between pEGFR/ILT4 co-expression and clinicopathological characteristics in human NSCLC (n = 80)

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Mutagenesis, Expressing

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 expression in NSCLC cells was induced by EGFR activation. (A-B) ILT4 expression in tumor cells of NSCLC tissues was positively correlated with pEGFR levels by IHC analysis. Tumor samples were sequentially sectioned and stained with ILT4 and pEGFR primary antibodies (A) Representative images of ILT4 and pEGFR co-localization, brown granules define positive staining. (B) Statistical results from 80 patients. Scale bar: 20 µm. (C-D) Inhibition of EGFR activation using gefitinib significantly decreased mRNA (C) and protein (D) expression of ILT4 in a concentration-dependent manner in PC9 and HCC827 cells harboring activating EGFR mutation. PC9 and HCC827 cells were treated with a gradient concentration of gefitinib for 24 h before real-time PCR and Western blot analyses. (E-F) Inhibition of EGFR activation using osimertinib but not gefitinib markedly reduced mRNA (E) and protein (F) expression of ILT4 in EGFR T790M-mutant H1975 cells. The cells were treated with osimertinib (0.1 µM) or gefitinib (0.1 µM) for 24 h, and mRNA and protein expression of ILT4 were analyzed by real-time PCR and Western blotting. (G-H) Knockdown of EGFR in PC9 and H1975 cells decreased ILT4 mRNA (G) and protein (H) levels. PC9 and H1975 cells were transfected with specific EGFR siRNA, and the mRNA or protein expression of ILT4 and EGFR was analyzed 72 h after transfection using real-time PCR and Western blotting. (I-J) EGF stimulation of EGFR wild-type H1299 cells elevated ILT4 expression in both concentration- (I) and time-dependent (J) manners. (I-J) Average results from 3 independent experiments. H1299 cells were treated with different concentrations of EGF for 24 h or with 100 ng/mL EGF for different durations and Western blotting was performed to determine ILT4, EGFR, and pEGFR levels. (K-L) Overexpression of EGFR in H1299 cells increased pEGFR, ILT4 (L) and EGF (K) levels determined by Western blotting or real-time PCR. ILT4 and pEGFR expression were detected 48 h after transfection of EGFR overexpression plasmid using Western blotting, while EGF level was detected by real-time PCR. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Gef: gefitinib; NC: normal control; OE: overexpression plasmid; Osi: osimertinib; si: small interfering RNA.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Expressing, Activation Assay, Staining, Inhibition, Concentration Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Transfection, Over Expression, Plasmid Preparation, Control, Small Interfering RNA

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: Activated ERK and AKT signaling mediated EGFR-induced ILT4 expression. (A) MAPK was among the most significantly altered signaling pathways upon treatment with osimertinib in H1975 cells with osimertinib (0.2 µM) for 24 h. mRNA was extracted and microarray was performed for cluster analysis of altered signaling pathways. (B) EGFR expression in NSCLC tissues was positively correlated with EKR (r = 0.44), NF-κB1 (r = 0.38), P65 (r = 0.37), and AKT1(r = 0.32) in the TCGA database. The online tool GEPIA was used to analyze the correlation of EGFR with key modulators of MAPK, NF-κB, and AKT signaling pathways. A correlation coefficient (r) of > 0.3 was considered significant. ( C ) pEGFR expression in lung adenocarcinoma and squamous cell carcinoma was positively correlated with activation of MAPK1/3, AKT1/2/3, and NF-κB1 in the RPPA database. The online tool cBioportal and Spearman correlation coefficient were used to evaluate the relevance. (D-E) Treatment with a gradient concentration of gefitinib or osimertinib inhibited ERK1/2, P65, and AKT phosphorylation in PC9 (D) or H1975 (E) cells. PC9 or H1975 cells were treated with different concentrations of gefitinib or osimertinib for 24 h and Western blotting was performed to determine the phosphorylation of signaling molecules. (F) EGF stimulation activated ERK1/2, P65, and AKT phosphorylation in H1299 cells treated with 100 ng/mL EGF for 24 h before Western blotting. (G-I) Treatment with ERK (U0126) or AKT (MK2206) inhibitor rather than NF-κB (PDTC) inhibitor decreased ILT4 expression in both PC9 and H1975 cells treated with different concentrations of specific inhibitors for 72 h and Western blotting was performed. The average results from 3 independent experiments are shown. Gef: gefitinib; Osi: osimertinib.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Expressing, Microarray, Activation Assay, Concentration Assay, Western Blot

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 in EGFR-activated tumor cells promoted TAM recruitment and M2-like polarization. (A-B) Patients with high ILT4 expression in tumor cells of NSCLC tissues showed markedly elevated infiltration of CD68 + TAMs by IHC analysis. (A) Representative images of ILT4 expression and TAM infiltration, brown granules represent positive staining. (B) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 (median) and ≥ 6. Scale bar: 20 µm. (C-D) TAMs induced by PC9 and H1975 cells displayed increased migration ability compared with parental macrophages, whereas ILT4 knockdown in PC9 and H1975 cells restricted tumor-induced TAM migration. Tumor cells were first transfected with lentivirus carrying ILT4 shRNA for 48 h, and CM was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (C) Images of migrated cells and (D) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. ### , p < 0.001 compared with the LV-shNC group. (E-F) ILT4 knockdown in PC9 and H1975 cells decreased the secretion of CCL2 and CCL5. (E) showed ELISA results of CCL2, (F) showed CCL5. (G-H) Recombinant human CCL2 or CCL5 reversed ILT4 knockdown and decreased macrophage migration. 100 ng/mL recombinant human CCL2 or 200 ng/mL CCL5 were added to the CM of ILT4 knockdown PC9 and H1975 cells to induce TAM migration. The migration ability of TAMs was evaluated by the Transwell migration assay. (G) Images of migrated cells. (H) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. compared with LV-shNC group. (I-J) Patients with high ILT4 expression in tumor cells displayed more frequent CD206 + but less CD86 + TAM accumulation in tumor tissues by IHC analysis. (I) Representative images of CD68 + TAMs, CD206 + M2-like TAMs and CD86+ M1-like TAMs frequencies; brown granules represent positive staining. (J) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 and ≥ 6 respectively. Scale bar: 20 µm. (K-L) ILT4 knockdown in PC9 and H1975 cells decreased M2-like markers (CD163, CD206, IL-10 and Arg1) and increased M1-like markers (CD80, CD86, IL-12 and TNFα) in TAMs by real-time PCR (K) and flow cytometry (L). TAMs were induced by CM as in (D) for 24 h. (M-N) TAMs induced by ILT4-downregulated PC9 and H1975 cells promoted the proliferation (M) and IFN-γ expression (N) of T cells. For T cell proliferation assay, CD3 + T cells separated from fresh PBMCs were first pre-activated by anti-CD3 for 24 h and stained with CFSE (1:1000), and then co-cultured for 4 days with TAMs induced by CM of ILT4-downregulated PC9 and H1975 cells. Flow cytometry was performed to determine CFSE strength. For IFN-γ expression, pre-activated CD3 + T cells were co-cultured with TAMs as described above for 48 h and IFN-γ levels were analyzed by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Arg1: arginase 1; CM, conditioned medium; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Expressing, Staining, Migration, Knockdown, Transfection, shRNA, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Flow Cytometry, Proliferation Assay, Cell Culture, Control

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 in EGFR-activated tumor cells impaired the proliferation and cytotoxicity of T cells. (A-B) High ILT4 expression in tumor cells of NSCLC tissues was correlated with decreased infiltration and IFN-γ generation in CD3 + T cells detected by IHC analysis. (A) Images of ILT4 expression, T cell infiltrates, and IFN-γ levels, brown granules represent positive staining. (B) Average numbers of T cells in 80 patients. The cutoff scores for ILT4-high and -low were the same as in Figure I. Scale bar: 20 µm. (C) Patients with high ILT4 expression displayed significantly decreased IFN-γ levels in tumor-infiltrating T cells compared with ILT4-low patients by IHC analysis. The histogram shows the average proportion of IFN-γ + T cells from 80 patients. (D-E) T cells co-cultured with ILT4-downregulated PC9 and H1975 cells showed increased proliferation ability compared with the counterpart group. PC9 and H1975 cells were first transfected with ILT4-knockdown lentivirus for 48 h, and then co-cultured with CD3 + T cells for 4 days at 1:2 ratio. The proliferation ability of T cells was assessed by the CFSE proliferation assay. (D) Flow cytometry results and (E) Average results from 3 independent experiments. (F-G) Transfection of ILT4-knockdown lentivirus in PC9 and H1975 cells inhibited T cell apoptosis compared with the control lentivirus group. Flow cytometry was performed to assess apoptotic T cells after co-culturing with different tumor cells. (F) Representative results for T cell apoptosis and (G) Results from 3 independent experiments. (H-I) ILT4 knockdown in PC9 and H1975 cells increased IFN-γ production in T cells. T cells co-cultured with tumor cells were collected and evaluated by flow cytometry to determine IFN-γ expression levels. (H) Flow cytometry results and (I) Average results from 3 independent experiments. (J) T cells co-cultured with ILT4-downregulated PC9 and H1975 cells released more IFN-γ into the supernatant than those co-cultured with control tumor cells. IFN-γ secretion in the same supernatant as in (F-G) by ELISA. (K) T cells co-cultured with ILT4-downregulated PC9 and H1975 cells displayed increased cytolytic ability compared with those co-cultured with control tumor cells. T cells were co-cultured with ILT4-downregulated or control PC9 and H1975 cells at different E: T ratios for 4 days, and the cytolysis assay was used to assess the killing ability of T cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E:T ratio: Effector: target cell ratio; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Expressing, Staining, Cell Culture, Transfection, Knockdown, Proliferation Assay, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, shRNA

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: Combined ILT4 and PD-L1 blockade in EGFR-activated NSCLC cells synergistically improved TAM- and dysfunctional T cell-mediated immunosuppression. (A-B) ILT4 or PD-L1 blockade in PC9-GR and H1299 cells inhibited the migration ability of TAMs, while combined ILT4 or PD-L1 blockade showed a stronger suppressive effect compared with either antibody alone. PC9-GR and EGF-preactivated H1299 cells were first pretreated with anti-ILT4 (500 ng/mL)/anti-PD-L1 (500 ng/mL)/combined anti-ILT4 (250 ng/mL) and anti-PD-L1 (250 ng/mL) for 8 h. CM from these cells was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (A) Images of TAM migration, and (B) Average results from 3 independent repetitions. Scale bar: 50 µm. (C-F) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells decreased the expression and secretion of CCL2 and CCL5, while combined blockade showed a synergistic effect. (C-D) Decreased CCL2 and CCL5 mRNA expression detected by real-time PCR, (E-F) Decreased CCL2 and CCL5 secretion detected by ELISA. Tumor cells were treated and conditioned media were collected as described in (A). (G-I) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells reversed tumor-induced M2-like markers in TAMs, while combined ILT4 and PD-L1 blockade displayed more significant reversion compared with either blockade alone. TAMs were induced as described in (A), and M2-like markers in TAMs were determined by flow cytometry. (G) Flow cytometry results and (H-I) Average results of CD163 and CD206 from 3 independent experiments. (J-K) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells increased T cell proliferation, while combined ILT4 or PD-L1 blockade showed a synergistic effect on T cell proliferation. PC9-GR and H1299 cells were treated as mentioned in (A), and co-cultured with anti-CD3-preactivated CD3 + T cells for 4 days at the ratio of 1:2. CFSE proliferation assay was performed to determine the proliferation of T cells. (J) Images of flow cytometry and (K Average results from 3 independent experiments. (L-M) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells suppressed T cell apoptosis, while combined ILT4 and PD-L1 blockade showed a more significant suppression than either antibody alone. T cells co-cultured with tumor cells for 2 days were evaluated for apoptotic T cells by flow cytometry. (L) Flow cytometry results and (M) Average results from 3 independent experiments. (N) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells promoted IFN-γ secretion in co-cultured T cells, while combined blockade released more abundant IFN-γ level than either antibody alone. T cells as in (J) were evaluated for IFN-γ level by ELISA. (O) T cells co-cultured with PC9-GR and H1299 cells pretreated with anti-ILT4 or anti-PD-L1 had stronger tumoricidal ability compared with those co-cultured with IgG-pretreated tumor cells, and T cells co-cultured with combination antibody-pretreated tumor cells showed the strongest tumor cytolytic activity. Tumor cells were pretreated with different antibodies as mentioned in (A) and then co-cultured with tumor cells at an E:T ratio of 5:1. Cytolysis assays were employed to determine the killing ability of T cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Anti: neutralization antibody; CM: conditioned medium.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Migration, Transwell Migration Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Culture, Proliferation Assay, Activity Assay, Neutralization

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 blockade suppressed tumor progression and immune escape in TKI-resistant EGFR mutant NSCLC in vivo . (A-B) ILT4 knockdown markedly inhibited PC9-GR growth in humanized NSG mice. However, PD-L1 blockade accelerated PC9-GR growth and combined blockade of both molecules showed no tumor inhibition compared to the control group. PC9-GR cells (3×10 6 cells/mouse) transfected with lentiviruses carrying ILT4-knockdown or control shRNA were subcutaneously injected into 6-8-week female NSG mice. Day 7 after tumor inoculation, 2×10 7 PBMCs from healthy volunteers were transplanted into each mouse via the tail vein. Anti-PD-L1 or control IgG (200 µg/mouse) were intraperitoneally injected into the tumor-bearing mice every 4 days from the day of PBMC transplant. Tumor sizes were measured every 4 days and presented as mean ± SD (n = 8 mice/group). (A) Tumor growth rate in each group and (B) Tumor images from each mouse. Scale bar: 1cm. (C) Tumors in ILT4 knockdown group showed significantly smaller tumor weight than in the control group. However, PD-L1 blockade led to higher while combined blockade generated similar tumor weight compared with the control group. The histogram shows the mean ± SD of tumor weights from each group at the experiment endpoint (n = 8 mice/group). (D-E) ILT4 knockdown increased CD3 + T cells in leukocytes in both spleens (D) and blood (E) of tumor-bearing mice. However, PD-L1 blockade alone or in combination with ILT4 inhibition did not affect CD3 + T cells in these organs. The frequency of CD3 + T cells in tumor spleens and blood were determined by flow cytometry. (F-G) ILT4 knockdown promoted the infiltration of CD3 + T cells in tumors by IHC staining. However, PD-L1 blockade alone or in combination with ILT4 inhibition did not alter the number of CD3 + T cells in tumor tissues. (F) Images of T cell infiltrates and (G) Average results from 8 mice in each group. Scale bar: 20 µm. (H-I) The IFN-γ levels in CD3 + T cells from blood, spleens, and tumors were remarkably increased by ILT4 knockdown compared with PD-L1 blockade or combined ILT4 and PD-L1 blockade. (H) The IFN-γ expression in blood and spleens were analyzed by flow cytometry, and (I) in tumors by IHC. (J-K) The distribution of CD4 + and CD8 + T cell subsets in mouse blood (J) and spleens (K) was not altered by inhibition of ILT4 or PD-L1 or both molecules as determined by flow cytometry analysis. (L-M) The IFN-γ expression in both CD4 + and CD8 + T cells was augmented by ILT4 knockdown but not by anti-PD-L1 or combination therapy. Results are shown in spleens (L) and blood (M). The level of IFN-γ expression in both organs was evaluated by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA; ns: no significance.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: Mutagenesis, In Vivo, Knockdown, Inhibition, Control, Transfection, shRNA, Injection, Generated, Flow Cytometry, Immunohistochemistry, Expressing

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 blockade enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type NSCLC in vivo . (A-B) ILT4 or PD-L1 blockade markedly slowed H1299 growth in humanized NSG mice compared with the control group, and combination treatment generated the lowest tumor growth rate. EGFR-overexpressing H1299 cells (3×10 6 cells/mouse) were first transfected with ILT4 knockdown or control lentiviruses, and then inoculated into the right flanks of 6-8-week female NSG mice. On day 7 post tumor inoculation, 2×10 7 PBMCs from healthy volunteers were intravenously injected into each mouse. Anti-PD-L1 or control IgG (200 µg/mouse) were intraperitoneally injected into the tumor-bearing mice every 4 days from the day of PBMC transplant. Tumor sizes were measured every 4 days and presented as mean ± SD (n = 8 mice/group). (A) Tumor growth rate in each group and (B) Tumor images for each mouse. Scale bar: 2 cm. (C) Tumors in ILT4 or PD-L1 blockade group showed significantly smaller tumor weight than the control group, while combination therapy showed synergy in tumor inhibition. The histogram shows the mean ± SD of tumor weights from each group at the experiment endpoint (n = 8 mice/group). (D-E) Spleen, blood and tumor tissues in ILT4 or PD-L1 blockade group showed significantly higher CD3 + T cell frequency than the control group, while the combination therapy group showed the highest CD3 + T cell infiltrates. (D) The CD3 + T cell frequency in mouse spleen and blood was detected by flow cytometry and (E) tumors were detected by IHC respectively. (F-G) ILT4 or PD-L1 blockade increased IFN-γ expression in T cells of spleen, blood and tumors relative to the control group, and combination therapy yielded the highest IFN-γ in T cells. The IFN-γ expression in mouse spleen, blood and tumors was detected by flow cytometry and IHC, respectively. (H-I) The distribution of CD4 + and CD8 + T cell subsets in spleen (H) and blood (I) was not altered by single or combination blockade of ILT4 and PD-L1 as determined by flow cytometry (J-K) ILT4 or PD-L1 blockade increased IFN-γ expression in CD4 + and CD8 + T cells of spleen (J) and blood (K) with the highest IFN-γ level in combination therapy group by flow cytometry analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001. LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA; ns: no significance.

Article Snippet: The used primary antibodies were as follows: anti-ILT4 (1:1000; Abnova; Cat No. H00010288-B01), anti-EGFR (1:1000; Abcam; Cat No.ab52894), anti-phospho-EGFR (Tyr1068) (1:1000; Abcam; Cat No. ab40815), anti-PD-L1 (1:1000; Proteintech; Cat No.66248-1-Ig), anti-PIR-B antibody (1:1000; Immunoway; Cat No. YN1914), anti-Ki67 antibody (1:1000; Abcam; Cat No. ab16667), anti-ERK1/2 (1:1000; CST; Cat No. 4695), anti-phospho-ERK1⁄2 (Thr202⁄Tyr204) (1:1000; CST; Cat No. 4370), anti-AKT (1:1000; Abcam; Cat No. ab8805), anti-phospho-AKT (Ser473) (1:1000; Abcam; Cat No. ab81283), anti-phospho-P65 (S536) (1:1000; Abcam; Cat No. ab76302) and anti-P65 (1:1000; Abcam; Cat No. ab16502).

Techniques: In Vivo, Control, Generated, Transfection, Knockdown, Injection, Inhibition, Flow Cytometry, Expressing, shRNA

Journal: Frontiers in Immunology

Article Title: Neuropilin-1 Acts as a Receptor for Complement Split Products

doi: 10.3389/fimmu.2019.02209

Figure Lengend Snippet: Interaction between NRP1 and C4d deposited to surfaces via thioester binding following classical complement activation. (A) C4d biotinylated at its intrinsic thioester carbonyl via amine-PEG2-biotin reagent (ih-C4d-bio) was immobilized to a solid phase either via streptavidin (+SA) or in random orientation without SA (–SA) and detected by ELISA using a monoclonal C4d antibody. (B) Interaction between C4d, immobilized to a solid phase as shown in (A) , and rh-NRP1-Ig and rh-ILT4-Ig. Rh-CTLA-4-Ig fusion proteins and goat anti-human-IgG (ctrl) served as negative controls. (C) C4d biotinylated via its intrinsic thioester was tetramerized using streptavidin-APC and probed with BW cells expressing ILT4 or NRP1 and analyzed via flow cytometry. (D) Antibody-coated sheep erythrocytes (EA) bearing C4d were prepared using purified C1, C4, C4 binding protein (C4BP), and factor I (FI) as described in Material and Methods. Deposition of C4d was detected with a monoclonal C4d antibody (upper panel). Interaction between C4d-loaded EA and control EA with immunoglobulin fusion proteins (rh-ILT4-Ig; rh-NRP1-Ig) is shown in the lower panel. (E) Binding of goat anti-human-IgG (ctrl) or rh-NRP1-Ig (20 or 60 μg/ml) to C4d-EA in the presence or absence of an anti-C4d and anti-C4c antibodies. Data shown are representative for two independently performed experiments. gMFI, geometric mean of fluorescence intensity.

Article Snippet: PE-conjugated ILT4 antibody was purchased from Becton Dickinson (Franklin Lakes, NJ).

Techniques: Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Purification, Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: Neuropilin-1 Acts as a Receptor for Complement Split Products

doi: 10.3389/fimmu.2019.02209

Figure Lengend Snippet: NRP1 interacts with MHC class I molecules . (A) Binding of tetramers representing MHC class I molecule HLA-A*02:01 (CMV CLGGLLTMV) with BW control cells and BW cells expressing NRP1 or ILT4 in the presence or absence of a polyclonal NRP1 antibody via flow cytometry. (B) Data of two independently performed binding experiments is shown. gMFI, geometric mean of fluorescence intensity.

Article Snippet: PE-conjugated ILT4 antibody was purchased from Becton Dickinson (Franklin Lakes, NJ).

Techniques: Binding Assay, Control, Expressing, Flow Cytometry, Fluorescence