Journal: medRxiv

Article Title: Human LUBAC deficiency leads to autoinflammation and immunodeficiency by dysregulation in TNF-mediated cell death

doi: 10.1101/2022.11.09.22281431

Figure Lengend Snippet: (a) NF-κB induction assay. EBV-immortalized lymphoblast (EBV-B) cells from SHARPIN κ deficient patient showed attenuated phosphorylation of IKKα/βI Bα and JNK after CD40L, stimulation. (b) mRNA expression of NF-κ stimulation. The experiment was performed in triplicates, and the expression levels were normalized to GAPDH . (c) mRNA expression of AICDA gene (encoding AID), normalized to GAPDH . CD19 + primary B cells were enriched by anti-CD19 magnetic beads and stimulated with CD40L for 24h. The experiment was performed in quadruplicates. (d) Proliferation assay using primary B cells from LUBAC deficient patients. PBMCs were stained with CellTrace Violet, cultured with CD40L and IL21 for 96 h and analyzed by flow cytometer. (e) Cell death assay by SYTOX staining of primary B cells from LUBAC deficient patients. PBMCs were cultured with CD40L and IL-21 for 96 h. (f-g) Cleaved caspase-3 and BCL6 immunohistochemistry staining of (f) adenoid from SHARPIN deficient patient and (g) axillary lymph node from a HOIP deficient patient, compared with control tissues. The number of cleaved caspase-3 positive cells per follicle was normalized by CD79a + follicle area. (h-j) BCR sequencing from naïve and memory B cells, FACS-sorted from peripheral blood. (h) Relative frequencies of the 100 most abundant IGH clonotypes in LUBAC deficient patients and five age- matched healthy controls. (i-j) Somatic hypermutation (SHM) quantification in sorted B cells. SHM in the entire V region (i) and the CDR3 region (j) of the IGH gene were normalized by the nucleotide length of each clone. Normalized SHM of top 100 IGH clonotypes per each sample are demonstrated. * p <0.05, ** p <0.01, *** p < 0.001 (Student’s t-test). The data are representative of two (a-c) or three (d-e) independent experiments.

Article Snippet: For proliferation assays, PBMCs were incubated with CellTrace Violet (Cell Proliferation Kit, Thermo) and stimulated with anti-IgM (Jackson Lab; 109-006-129), CD40L (Enzo; ALX522-110-C010), IL-21 (Peprotech; 200-21), CpG (Enzo; ALX522-025-C010), anti-CD3 (eBioscience; 16-0037-85), anti-CD28 (eBioscience; 16-0289-85) and PHA (Sigma; L9017).

Techniques: Expressing, Magnetic Beads, Proliferation Assay, Staining, Cell Culture, Flow Cytometry, Immunohistochemistry, Sequencing

Journal: Frontiers in Immunology

Article Title: Role of Programmed Cell Death Protein-1 and Lymphocyte Specific Protein Tyrosine Kinase in the Aryl Hydrocarbon Receptor- Mediated Impairment of the IgM Response in Human CD5 + Innate-Like B Cells

doi: 10.3389/fimmu.2022.884203

Figure Lengend Snippet: TCDD-mediated increase in the percentage of LCK + human CD5 + ILBs and the suppression of the IgM response. Human CD5 +/- B cells were activated with CD40L, IL-21, and IL-2 and treated with Veh (0.02% DMSO), or TCDD (10 nM) on day 0 and cultured for 7 days. Cells and culture supernatants were collected and assessed for LCK and LCK Y505 phosphorylation by flow cytometry and IgM secretion via ELISA and ELIspot. (A) Correlation of percent CD5 + B cells and percent LCK + B cells; (B) Representative flow cytometry plots of CD5 + and LCK + cells; (C) Percentage of LCK + B cells within the CD5 +/- populations on day 0; (D) Representative flow cytometry plots of LCK + cells within CD5 +/- populations; (E) Percentage of LCK + B cells within CD5 +/- populations on day 1, 3, 4 and 7 with Veh or TCDD treatment; (F) Percentage of pLCK (Y505) + B cells within CD5 +/- populations on day 1, 3, 5 and 7 with Veh or TCDD treatment; (G) Representative ELIspot wells showing IgM secreting cells measured within CD5 +/- populations with Veh or TCDD (10 nM) treatment on day 7; (H) Number of IgM secreting cells; and (I) IgM concentration from culture supernatants from CD5 +/- B cells treated with Veh or TCDD. Determinations were made using B cells from 6 human donors (N = 6). For (E, F) , data were normalized to the CD5 - Veh on day 1. For (H, I) , data were normalized to CD5 - Veh. Significant differences are indicated by * p < 0.05 and ** p < 0.01 (Student’s T test or two-way ANOVA following with Fisher’s LSD post hoc test).

Article Snippet: The treated ILBs were then activated by soluble human CD40 ligand (100 ng/mL) (Enzo, Farmingdale, New York) and supplied with recombinant human cytokines IL-2 (1 ng/ml) (Roche Applied Science, Indianapolis, Indiana) and IL-21 (100 ng/mL) (R&D system, Minnesota) for a total of 7 days.

Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Effects of Vitamin D and Dexamethasone on Lymphocyte Proportions and Their Associations With Serum Concentrations of 25-Hydroxyvitamin D 3 In Vitro in Patients With Multiple Sclerosis or Neuromyelitis Optica Spectrum Disorder

doi: 10.3389/fimmu.2021.677041

Figure Lengend Snippet: Changes in the proportion of lymphocytes in PBMC samples in response to nonspecific stimulation with LPS+CD40L+CpG-ODN with or without 1,25(OH) 2 D 3 , and/or steroid (dexamethasone) in HCs and in patients with MS or NMOSD. Stimulation with 1,25(OH) 2 D 3 significantly reduced the percentages of (A) total lymphocytes and (B) T cells in HCs. However, stimulation with 1,25(OH) 2 D 3 followed by steroid caused significant decreases in the percentages of (A) total lymphocytes and (B) T cells in the MS and NMOSD groups compared with those achieved by stimulation with steroid treatment alone. 1,25(OH) 2 D 3 stimulation did not alter the proportions of (C) B cells or (D) Breg, or (F) the Bmem/Breg ratio in any of the three study groups. Stimulation with 1,25(OH) 2 D 3 followed by steroid significantly increased the proportion of (E) Bmem in PBMC samples compared with 1,25(OH) 2 D 3 stimulation alone in the NMOSD group. Data are mean and standard-error values. Statistically significant differences are indicated by horizontal bars: * p <0.05, ** p <0.01, *** p <0.001. 1,25(OH) 2 D 3 , 1,25-dihydroxyvitamin D 3 ; Bmem, CD19 + CD27 + memory B cell; Breg, CD19 + CD24 + CD38 + regulatory B cell; CD40L, CD40 ligand; CpG-ODN, cytosine phosphate guanosine oligodeoxynucleotides; HC, healthy control; LPS, lipopolysaccharide; MS, multiple sclerosis; NMOSD, neuromyelitis optica spectrum disorder; PBMC, peripheral blood mononuclear cell.

Article Snippet: They were then incubated with a nonspecific stimulation mixture of 10 ng/ml lipopolysaccharide (LPS; Sigma ldrich), 100 ng/ml CD40 ligand (CD40L; Enzo Biochem, New York, NY, USA), and 5 nM cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODN 2006; Invivogen, USA), either alone (LPS+CD40L+CpG-ODN) or in combination with steroid (dexamethasone, 10 nM, Sigma Aldrich), 1,25(OH) 2 D 3 (1 μM, Sigma Aldrich), or both steroid and 1,25(OH) 2 D 3 .

Techniques:

Journal: Frontiers in Immunology

Article Title: Effects of Vitamin D and Dexamethasone on Lymphocyte Proportions and Their Associations With Serum Concentrations of 25-Hydroxyvitamin D 3 In Vitro in Patients With Multiple Sclerosis or Neuromyelitis Optica Spectrum Disorder

doi: 10.3389/fimmu.2021.677041

Figure Lengend Snippet: Changes in the mRNA expression of VDR , CYP27B1 , CYP24A1 , and IL-10 in PBMCs in response to stimulation with LPS+CD40L+CpG-ODN (nonspecific) with or without 1,25(OH) 2 D 3 . In HCs and patients with MS or NMOSD, stimulation with 1,25(OH) 2 D 3 increased the mRNA expression of CYP24A1 and decreased that of CYP27B1 over those observed in samples stimulated with LPS+CD40L+CpG-ODN alone. * p <0.05, ** p <0.01, *** p <0.001. 1,25(OH) 2 D 3 , 1,25-dihydroxyvitamin D 3 ; CD40L, CD40 ligand; CpG-ODN, cytosine phosphate guanosine oligodeoxynucleotides; HC, healthy control; LPS, lipopolysaccharide; MS, multiple sclerosis; NMOSD, neuromyelitis optica spectrum disorder; PBMC, peripheral blood mononuclear cell.

Article Snippet: They were then incubated with a nonspecific stimulation mixture of 10 ng/ml lipopolysaccharide (LPS; Sigma ldrich), 100 ng/ml CD40 ligand (CD40L; Enzo Biochem, New York, NY, USA), and 5 nM cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODN 2006; Invivogen, USA), either alone (LPS+CD40L+CpG-ODN) or in combination with steroid (dexamethasone, 10 nM, Sigma Aldrich), 1,25(OH) 2 D 3 (1 μM, Sigma Aldrich), or both steroid and 1,25(OH) 2 D 3 .

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of a Sensitive Human Immunological Target of Aryl Hydrocarbon Receptor Activation: CD5 + Innate-Like B Cells

doi: 10.3389/fimmu.2021.635748

Figure Lengend Snippet: Frequency of CD5 expressing B cells in circulation predicts donor sensitivity to TCDD-mediated IgM suppression. CD19 + naïve human B cells isolated from PBMC by magnetic separation were activated with sCD40L, IL-21, and IL-2 and treated with TCDD (10nM) or Veh (0.04% DMSO) as comparator for 7 days. On day 7 cells were collected and either surface stained for CD5 protein expression or assayed for IgM secretion by ELISPOT. The %IgM suppression for a given donor was calculated by normalizing each by the number of IgM + spots in the TCDD-treated samples to the number of spots in each donors Veh control. A representative flow plot of CD19 expressing CD5 + B cells is shown in panel (A) CD5 + cells were identified in the lymphocyte, singlet gate by gating on live CD19 + cells. The %IgM suppression was then graphed against the frequency of CD5 expression and is shown in panel (B) A linear regression analysis was performed to calculate slope, best fit (R), and significance. Data are from 7 independent experiments assessing a total of 18 human donors.

Article Snippet: Purified B cells were activated with 100 ng/mL soluble CD40 Ligand (Enzo, Farmingdale, NY), 100ng/mL rIL-21 (R&D Systems, Minneapolis, MN) and 1 ng/mL rIL-2 (Roche Applied Science, Indianapolis, IN) and seeded at a density of 1x10 6 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% human AB serum (Valley Biomedical, Winchester, VA), 50 μM of 2-mercaptoethanol (ThermoFisher, Lafayette, CO) and 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA).

Techniques: Expressing, Isolation, Staining, Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Identification of a Sensitive Human Immunological Target of Aryl Hydrocarbon Receptor Activation: CD5 + Innate-Like B Cells

doi: 10.3389/fimmu.2021.635748

Figure Lengend Snippet: Both CD5 + and CD5 - are negative for markers of activation, CD80 and CD86, directly ex vivo , which is increased following activation with CD40L and IL-21. CD5 + and CD5 - B cells were isolated and activated as previously described, taken at day 0 for purity stain and quantification of CD80 and CD86, or cultured in complete RPMI supplemented with IL-2 alone. At each indicated time point, cells were collected and surface stained for CD19, CD5, CD80, and CD86. For comparison of cell types, data is on gated CD19 + cells within the live lymphocyte gate. Representative flow plots for CD80 and CD86 expression at select times is shown in (A) . Cell surface CD80 + , CD86 + , CD80 + CD86 + or CD80 - CD86 - protein expression over time is shown for CD5 + B cells in panel (B) and CD5 - B cells in panel (C) . Secreted IgM from CD5 +/- B cells +/- IL-21 and CD40L are shown in panel (D) . Data shown are from 3 independent experiments assessing a total of 5 human donors. Significant differences compared to day 0 were determined within each cell type by a one-way ANOVA with a Tukey’s posttest where *p < 0.05, **p < 0.01, and ***p < 0.001. Significance between cell types at a given timepoint was determined with a two-way ANOVA with a Tukey’s posttest where a p < 0.05. Significance for IgM accumulation in culture supernatants between activated and non-activated cells was determined with a one-way ANOVA with a Tukey’s posttest where *p < 0.05 and ***p < 0.001.

Article Snippet: Purified B cells were activated with 100 ng/mL soluble CD40 Ligand (Enzo, Farmingdale, NY), 100ng/mL rIL-21 (R&D Systems, Minneapolis, MN) and 1 ng/mL rIL-2 (Roche Applied Science, Indianapolis, IN) and seeded at a density of 1x10 6 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% human AB serum (Valley Biomedical, Winchester, VA), 50 μM of 2-mercaptoethanol (ThermoFisher, Lafayette, CO) and 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA).

Techniques: Activation Assay, Ex Vivo, Isolation, Staining, Cell Culture, Expressing

Journal: Frontiers in Immunology

Article Title: Identification of a Sensitive Human Immunological Target of Aryl Hydrocarbon Receptor Activation: CD5 + Innate-Like B Cells

doi: 10.3389/fimmu.2021.635748

Figure Lengend Snippet: Human CD5 + B cells respond to T-dependent and T-independent activators. Human CD19 + naïve B cells were isolated from PBMC and separated into CD5 + and CD5 - populations as previously described. Cells were then activated with IL-21, IL-2, and either CD40L or indicated TLR agonist. After the 7-day culture period, cells were collected and IgM secretion assessed via IgM ELISPOT. For quantification of CD40 and TLR9, freshly isolated CD19 + B cells were stained for CD19, CD5, and CD40 surface expression. Cells were then fixed and permeabilized and stained intracellularly for TLR9. Representative ELISPOT wells from a given donor are shown in panel (A) Averaged results from 3 independent experiments assessing a total of 8 human donors are shown in panel (B) Representative flow plots showing CD5, CD40, and TLR9 are shown in panel (C) Averaged results from 2 independent experiments assessing a total of 9 human donors are shown in panels (D–G) . A repeated measures ANOVA with a Tukey’s posttest was used to determine significance in panel B where * indicates significant differences compared to CD40L activation within each cell type. “a” indicates significant differences compared to TLR9 activation within each cell type. *p < 0.05, **p < 0.01, a p < 0.05, and aa p < 0.01. A paired t-test was used to determine significance in panels (D–G) where **p < 0.01 and ***p < 0.001.

Article Snippet: Purified B cells were activated with 100 ng/mL soluble CD40 Ligand (Enzo, Farmingdale, NY), 100ng/mL rIL-21 (R&D Systems, Minneapolis, MN) and 1 ng/mL rIL-2 (Roche Applied Science, Indianapolis, IN) and seeded at a density of 1x10 6 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% human AB serum (Valley Biomedical, Winchester, VA), 50 μM of 2-mercaptoethanol (ThermoFisher, Lafayette, CO) and 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA).

Techniques: Isolation, Enzyme-linked Immunospot, Staining, Expressing, Activation Assay