Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: Inflammation and the NKG2D system in H. pylori infection and gastric cancer. Stomach biopsies from healthy controls (Healthy), H. pylori gastritis cases (HpG) and stomach adenocarcinoma cases (Cancer) were immune-phenotyped. (A, B) H&E and IHC staining of leucocytes (CD45), CTLs (CD8) and NK cells (CD56, NKp46). (A) Representative images, scale bars: 100 µm. (B) Quantification of positive cells per mm 2 of tissue n=10-30 per group, the data do not follow a normal distribution, median ± interquartile range, Kruskal-Wallis test and Dunn’s test. (C) qPCR analysis of NKG2D , MICA and MICB , n=8-16 per group. The data passed Shapiro-Wilk normality test, mean ± SD, one-way ANOVA and Tukey’s test. *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001. ns, not significant.

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Infection, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: H. pylori -dependent modulation of NKG2D-L expression and soluble release via proteolytic shedding. MKN28 cells were challenged with C. acnes , butyrate, and H. pylori WT for 24 and 48 h (A) MICA and MICB mRNA levels were determined by qPCR. (B) Soluble MICA and MICB levels in cell culture supernatants were determined by ELISA. Experiments were performed three times. Mean ± SD, one-way ANOVA and Tukey’s test (ns, not significant, *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001). (C) MKN28 and AGS cells were challenged with H. pylori WT or 2 mM butyrate for 48 h Simultaneously, cells were treated with either 10 µmol/L batimastat dissolved in DMSO (= batimastat +) or with DMSO alone as solvent control (= batimastat –). Soluble MICA and MICB proteins in cell culture supernatants were determined by ELISA. Experiments were performed three times. Mean ± SD, t-test of batimastat – vs. batimastat +, for each treatment group (ns, not significant, *P <0.05; ***P <0.001; ****P <0.0001).

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Solvent, Control

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: H. pylori virulence factor-dependent modulation of NKG2D-L expression and soluble release. MKN28 cells were challenged with H. pylori WT and isogenic mutants ΔcagA , ΔcagL and ΔvacA for 24 and 48 h (A) MICA and MICB mRNA levels were determined by qPCR. (B) Soluble MICA and MICB levels in cell culture supernatants were determined by ELISA. (C–E) AGS-MICA cells were challenged with H. pylori WT and isogenic mutants ΔcagA , ΔcagL and ΔvacA for 24 h (C) MICA mRNA levels were determined by qPCR. (D) MICA protein in cell lysates was determined by Western Blot. (E) Soluble MICA protein in cell culture supernatants was determined by ELISA. Experiments were performed three times. Mean ± SD, one-way ANOVA and Tukey’s test (ns, not significant, *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001).

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: Scheme highlighting the NKG2D system modulation by H. pylori . Healthy epithelia store MICA and MICB proteins intracellularly. Upon stress, MICA/B are expressed at the cell surface and bound by the immunoreceptor NKG2D, which activates immune attack by NKG2D-harboring effector cells. During H. pylori infection, virulence factors CagA and VacA modify the expression and proteolytic shedding of MICA/B. Soluble MICA/B proteins lead to immune evasion by binding NKG2D, which results in internalization and downregulation of NKG2D and a suppression of lymphocyte cytotoxicity. Hypofunctional NK and cytotoxic T cells in the stomach lamina propria could allow transformed cells to escape immune surveillance and facilitate tumor development. Scheme drawn with BioRender ( https://biorender.com ).

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Infection, Expressing, Binding Assay, Transformation Assay

Journal: Journal for immunotherapy of cancer

Article Title: Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma.

doi: 10.1136/jitc-2023-007886

Figure Lengend Snippet: Figure 2 sMICA is increased in patients with MM and to a higher concentration in BM than in PB. (A) Peripheral blood (PB) and bone marrow (BM) plasma of healthy (n=9), patients with monoclonal gammopathy of undetermined significance (MGUS) (n=30), smoldering multiple myeloma (SMM) (n=16), and multiple myeloma (MM) (n=74) were examined for soluble MICA (sMICA) with an ELISA. (B) BM plasma of healthy (n=6) and patients with MM (n=74) were assessed for soluble MICB (sMICB), soluble ULBP1 (sULBP1), soluble ULBP2 (sULBP2), and soluble ULBP3 (sULBP3) with an ELISA. Each dot indicates a value obtained from one patient. P values were determined versus PB, healthy, MGUS, or SMM using the Mann-Whitney U test. n.s., not significant. sMICA/B, soluble major histocompatibility complex class I chain-related molecule A/B; sULBP1-3, soluble UL16- binding proteins 1-3.

Article Snippet: The concentration of soluble NKG2D ligands in plasma or supernatant was measured by DuoSet ELISA (R&D Systems, Minnesota, USA) for human MICA (DY1300), MICB (DY1599), ULBP1 (DY1380), ULPB2 (DY1298) and ULBP3 (DY151705), using the procedures recommended by the manufacturers.

Techniques: Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Immunopeptidomics, Binding Assay

Journal: Journal for immunotherapy of cancer

Article Title: Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma.

doi: 10.1136/jitc-2023-007886

Figure Lengend Snippet: Figure 3 sMICA concentration is positively correlated with plasma cell burden but negatively correlated with NKG2D+ NK cells in MM patients. (A) Correlation of the NKG2D ligand positive cells among the gated plasma cells and the level of soluble NKG2D ligands in BMMCs and paired plasma of pooled patients with MGUS, SMM, and MM at diagnosis (n=22). (B–E) Correlation of BM plasma levels of soluble NKG2D ligands in diagnostic BM aspirates of patients with MM (n=74) examined with an ELISA and percentage of BM plasma cells in diagnostic BM aspirates (B) CD56dimCD16+ NK cells among BMMCs (C) NKG2D+ NK cells among CD56dimCD16+ NK cells (D) and DNAM-1+ NK cells among CD56dimCD16+ NK cells (E) in paired samples assessed by flow cytometry analysis. Each dot indicates a value obtained from one patient. Correlation coefficients (R) and P values were determined by Spearman’s rank-order correlation test. BM, bone marrow; BMMC, BM mononuclear cell; DNAM-1, DNAX accessory molecule-1; MM, multiple myeloma; NK, natural killer; NKG2D, natural killer group 2D; sMICA/B, soluble major histocompatibility complex class I chain-related molecule A/B; sULBP, soluble UL16-binding protein.

Article Snippet: The concentration of soluble NKG2D ligands in plasma or supernatant was measured by DuoSet ELISA (R&D Systems, Minnesota, USA) for human MICA (DY1300), MICB (DY1599), ULBP1 (DY1380), ULPB2 (DY1298) and ULBP3 (DY151705), using the procedures recommended by the manufacturers.

Techniques: Concentration Assay, Clinical Proteomics, Biomarker Discovery, Diagnostic Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunopeptidomics, Binding Assay

Journal: Journal for immunotherapy of cancer

Article Title: Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma.

doi: 10.1136/jitc-2023-007886

Figure Lengend Snippet: Figure 5 huB10G5 cleared bortezomib-induced increase in sMICA in culture supernatant of SKO-007-MICA cells. (A) Representative flow cytometry plots of surface MICA expression in SKO-007-MICA cells after treatment with DMSO (0.2%), bortezomib (V, 2.5 nM), lenalidomide (R, 20 µM), dexamethasone (d, 10 µM), daratumumab (D, 10 µg/mL), VRd, or D-VRd for the indicated time period. (B) Per cent MICA positive cells among gated live cells measured by flow cytometry after treatment with indicated drugs. (C) Concentration of sMICA examined with ELISA in culture supernatant of SKO-007-MICA cells after treatment with IgG (5 µg/mL) or huB10G5 (5 µg/mL) for the indicated time period. P values for sMICA were determined versus DMSO using the Mann-Whitney U test. *P<0.05. DMSO, Dimethyl Sulfoxide; MICA, major histocompatibility complex class I chain-related molecule A; sMICA, soluble MICA.

Article Snippet: The concentration of soluble NKG2D ligands in plasma or supernatant was measured by DuoSet ELISA (R&D Systems, Minnesota, USA) for human MICA (DY1300), MICB (DY1599), ULBP1 (DY1380), ULPB2 (DY1298) and ULBP3 (DY151705), using the procedures recommended by the manufacturers.

Techniques: Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Immunopeptidomics