Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: Differential Effects of vIL6 and hIL6 on the frequency and distribution of KSHV infection in B cells. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated and analyzed at 3 dpi for the distribution of KSHV infection within B cell lineages (Table ) using the GFP reporter by flow cytometry. (A) percent of viable CD19 + B lymphocytes that were GFP+ in each condition. Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.04 comparing no treatment to IL6 neutralization in KSHV‐WT infection. (B) Percent of GFP+ plasmablasts; paired T ‐test p = 0.05 comparing WT to ∆K2 in untreated cultures (C) percent of GFP+ classical memory cells; Two‐way repeated measures ANOVA p = 0.03, F = 5.7 for Nab treatment, p = 0.03, F = 5.9 for virus strain; paired T ‐test p = 0.04 comparing WT to ∆K2 in untreated cultures. (D) Percent of GFP + IL6 + CD19+ cells; paired T ‐test p = 0.03 comparing WT and ∆K2 infection in the untreated cultures. (E) Correlation between total infection (GFP + CD19 + ) and GFP within plasmablast using pearson's method. (F) Correlation between total infection (GFP + CD19 + ) and GFP within classical memory using pearson's method. (G) lack of correlation between total infection (GFP + CD19 + ) and GFP within IL6 + B cells. (H) Percent of GFP + MZ‐like cells; Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.03 comparing no treatment to IL6 neutralization in KSHV‐∆K2 infection. (I) Correlation between total infection (GFP + CD19 + ) and GFP within MZ‐like using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Isolation, Derivative Assay, Flow Cytometry, Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 impacts hIL6 expression in B cell subsets. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated. At 3 dpi supernatants were analyzed for secreted hIL6 and cells were analyzed for hIL6 expression within B cell subsets by ICCS. (A) Frequency of IL6 + B cells. Two‐way repeated measures ANOVA p = 0.002, F = 7.6 for virus strain, post hoc paired T ‐test p = 0.006 comparing KSHV‐WT to ∆K2 in the no treatment condition. (B) Secreted hIL6 showed the same statistical effect as IL6+ within B cells ( p = 0.01). (C) Frequency of IL6+ within B cell subsets with the following significant effects. Plasma cell: Two‐way repeated measures ANOVA p = 0.02, F = 4.4 for virus strain, post hoc paired T ‐test p = 0.05 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition. Double negative memory: paired T ‐test p = 0.0 comparing KSHV‐WT to ∆K2 in the no treatment condition. Naive: Two‐way repeated measures ANOVA p = 0.03, F = 3.9 for virus strain, post hoc paired T ‐test p = 0.02 comparing KSHV‐WT to ∆K2 in the no treatment condition. Transitional: Two‐way repeated measures ANOVA p = 0.003, F = 7.3 for virus strain, post hoc paired T ‐test p = 0.04 comparing KSHV‐WT to ∆K2 in the no treatment condition. Germinal Center: Two‐way repeated measures ANOVA p = 0.002, F = 7.7 for virus strain, post hoc paired T ‐tests p = 0.02 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition, p = 0.01 comparing KSHV‐WT to ∆K2 in the no treatment condition.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Expressing, Isolation, Infection, Derivative Assay, Virus, Clinical Proteomics

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 differentially alter secretion of TNF‐α, BAFF and IL‐10 during KSHV infection. Supernatants from 11 unique tonsils infected and treated as in experiments described in Figure were harvested at 3 dpi and clarified of cellular debris by centrifugation. Concentrations of 13 cytokines related to B cell activation and differentiation were determined using the Biolegend Legendplex Human B cell panel for each supernatant. Cytokines significantly altered by experimental parameters were (A) BAFF with 2‐way repeated measures ANOVA showing significant interaction of virus infection and hIL‐6 neutralization ( p = 0.03, F = 4.4) and significantly different pairwise comparison via T‐test comparing Mock+IL6 NAb and KSHV‐∆K2 + IL6 Nab ( p = 0.039). (B) TNF‐α with 2‐way repeated measures ANOVA showing significant effect of virus condition ( p = 0.05, F = 3.6) and significant pairwise comparisons via T ‐test comparing Mock and KSHV‐WT ( p = 0.008) and KSHV‐WT with KSHV‐∆K2 ( p = 0.01) with no hIL6 neutralization; (C) IL‐10 with significant pairwise comparisons via T‐test comparing Mock and KSHV‐WT ( p = 0.02) with no hIL6 neutralization.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Centrifugation, Activation Assay, Virus, Neutralization, Comparison

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 cooperatively support B cell viability during KSHV infection but have differential effects on subset frequencies. Naïve B cells were extracted from a distinct set of 15 tonsils and subjected to infection with either Mock, KSHV‐WT, or KSHV‐ΔK2. The cultures were either supplemented with IL6‐neutralizing antibodies or remained untreated. At 3dpi, the cultures were examined by flow cytometry to assess B cell viabilities and the frequencies of B cell subsets. (A) Percent of viable CD19 + . Two‐way repeated measures ANOVA p = 0.01, F = 5.3 for virus strain, p = 0.04, F = 5.5 for hIL6 neutralization and p = 0.03, F = 3.9 for the interaction of the two variables. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.01 comparing no treatment to hIL6 neutralization in Mock cultures, p = 0.01 comparing Mock with KSHV‐WT in untreated cultures and p = 0.004 comparin Mock with ∆K2 in untreated cultures. (B) Frequency of naïve B cells Two‐way repeated measures ANOVA p = 0.05, F = 4.7 for hIL6 neutralization and p = 0.04, F = 3.6 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.018 comparing Mock to ∆K2 in the no treatment condition and p = 0.01 comparing untreated to hIL6 neutralization in Mock infected cultures. (C) Frequency of germinal center B cells. Two‐way repeated measures ANOVA p = 0.03, F = 4.1 for virus strain. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.019 comparing Mock to ∆K2 in the no treatment condition. (D) Frequency of plasmablasts. Two‐way repeated measures ANOVA p = 0.00006, F = 14.1 for virus strain and p = 0.001, F = 9.0 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.03 comparing Mock and WT, p = 0.03 comparing Mock and ∆K2 in the no treatment conditions and p = 0.002 comparing Mock and WT, p = 0.04 comparing Mock and ∆K2 and p = 0.004 comparing WT and ∆K2 in the hIL6 neutralized conditions.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Flow Cytometry, Virus, Neutralization

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: hIL6 and vIL6 have separable effects on the differentiation of plasmablast and germinal center B cells from naïve B cells during infection. I B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) proportional distribution of dye+ B cells amongst B cell subsets (B) frequency of undifferentiated naïve B cells. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.031 comparing Mock to ∆K2 in the untreated condition. (C) Frequency of GC B cells differentiated from naïve. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for virus strain, p = 0.03, F = 3.8 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.039 comparing Mock to ∆K2 and p = 0.035 comparing WT to ∆K2 in the untreated conditions. (D) Frequency of plasmablast differentiated from naïve. Two‐way repeated measures ANOVA p = 0.00004, F = 10.2 for virus strain, p = 0.05, F = 3.9 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.012 comparing Mock to ∆K2 and p = 0.014 comparing Mock to WT in the untreated conditions, p = 0.007 comparing Mock to WT and p = 0.011 comparing WT to ∆K2 in the hIL6 neutralized conditions. (E) Correlation between differentiation of GCB and total frequency of GCB using pearson's method. (F) Correlation between differentiation of plasmablast and total frequency of plasmablast via pearson's method. (G) Correlation between differentiation of GCB and IL6 + GCB within each virus condition using pearson's method. (H) Correlation between GCB differentiation and total KSHV infection using pearson's method. (I) Correlation between differentiation of GCB and KSHV infection of plasmablast using pearson's method. (J) Correlation between differentiation of GCB and KSHV infection of classical memory using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: KSHV infection drives functional maturation of B cells during ex vivo infection, but vIL6 suppresses differentiation of IgG+ plasma cells during KSHV infection. Naive B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers including Ig isotype, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) Frequency of differentiated IgG+ B cells normalized to each sample's mock value. Two‐way repeated measures ANOVA conducted before data normalization: p = 0.00008, F = 15.4 for virus strain, p = 0.04, F = 5.1 for hIL6 neutralization and p = 0.002, F = 7.4 for interaction of virus strain and hIL6 neutralization. (B) frequency of differentiated IgG+ plasma cells. Paired T ‐test p = 0.03 comparing WT and ∆K2 in the untreated condition. (C) Concentrations of IgG isotypes at 3 dpi were determined in clarified supernatants from eight samples via Legendplex immunoassay. Two‐way repeated measures ANOVA showed significant effect of virus strain on supernatant concentrations of IgG1 ( p = 0.036, F = 6.4) and significant effect of virus strain ( p = 0.04, F = 5.99) and significant interaction of virus strain and IL6 neutralization ( p = 0.04, F = 6) on supernatant concentrations of IgG3.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Functional Assay, Ex Vivo, Clinical Proteomics, Neutralization, Virus

Journal: Brain, behavior, and immunity

Article Title: Bi-allelic NRXN1α deletion in microglia derived from iPSC of an autistic patient increases interleukin-6 production and impairs supporting function on neuronal networking.

doi: 10.1016/j.bbi.2024.09.001

Figure Lengend Snippet: Fig. 3. NRXN1α deletion promotes upregulation IL6 gene expression and IL6 secretion in iPSC-derived microglia. (A) RT-qPCR analysis reveals an upregulation of mRNA expression for IL6, TNF, and IL1B, in NRXN1α-del iPSC-microglia, as compared to control iPSC-microglia. (B) Analysis of IL6 secretion by ELISA shows increased concentrations in the medium collected from in NRXN1α-del iPSC-microglia, as compared to control iPSC-microglia. (n = 5 biological replicates; data are shown as mean ± sem). A, * p < 0.05, one sample t-test vs. 1 (“no difference” as null hypothesis). B, * p < 0.05, 2-sample t-test.

Article Snippet: Similarly, 200 ng/ml of IL6 neutralizing antibody (Human IL6 Mab-2061, Clone 1936, R&D Systems, Inc) was added to NRXN1α-del iPSC-NES/NRXN1α-del − iPSC microglia co-culture for 15 days.

Techniques: Gene Expression, Derivative Assay, Quantitative RT-PCR, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain, behavior, and immunity

Article Title: Bi-allelic NRXN1α deletion in microglia derived from iPSC of an autistic patient increases interleukin-6 production and impairs supporting function on neuronal networking.

doi: 10.1016/j.bbi.2024.09.001

Figure Lengend Snippet: Fig. 7. Increased IL6 secretion by NRXN1α-deficient microglia contributes to the negative effects on neuronal maturation and network development. (A) The addition of recombinant IL6 (rIL6) in the culture medium of control iPSC-NES+control iPSC-microglia co-cultures impairs single cell burst activity (decreased burst percentage and shorter burst duration), as well as neuronal network activity (decreased network burst percentage). (B) The addition of a neutralizing antibody against IL6 (naIL6) in the culture medium of NRXN1α-del iPSC-NES+NRXN1α-del iPSC-microglia co-cultures promotes single cell burst activity (increased burst percentage). (C) RT-qPCR analysis detects consistent mRNA expression for IL6R and IL6ST in differentiating iPSC-NES in monoculture. mRNA expression for both IL6R and IL6ST was downregulated in NRXN1α-del iPSC-NES as compared to control iPSC-NES. (D) Quantitative measurement of soluble IL6 receptor (sIL6R) by ELISA in the culture medium of iPSC-microglia. The concentration of sIL6R was significantly lower in NRXN1α-del iPSC-microglia as compared to control iPSC-microglia. A- C: n = 3 replicates, 8 wells/replicate; D: n = 4 replicates. * p < 0.05, 2-sample t-test.

Article Snippet: Similarly, 200 ng/ml of IL6 neutralizing antibody (Human IL6 Mab-2061, Clone 1936, R&D Systems, Inc) was added to NRXN1α-del iPSC-NES/NRXN1α-del − iPSC microglia co-culture for 15 days.

Techniques: Recombinant, Control, Activity Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay