Journal: Frontiers in Immunology

Article Title: Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation

doi: 10.3389/fimmu.2022.803229

Figure Lengend Snippet: MS severity correlates with the elevated level of transitional CD19 + CD24 high CD38 high Bregs in peripheral blood. (A) Total CD19 + B cell pool and transitional regulatory CD19 + CD24 high CD38 high subpopulation (tBregs) from peripheral blood were sorted individually for subsequent RNA isolation and RT-PCR amplification of IGVH, IGVK and IGVL followed by high-throughput sequencing and subsequent bioinformatic clustering and analysis. (B) The percentage and (C) absolute cell count of transitional Bregs in MS patients (MS) and healthy donors (HD). BMS denotes benign multiple sclerosis, HAMS is highly active multiple sclerosis. Data are shown as mean values, interquartile range, and p -values. The statistical significance was evaluated with the Mann Whitney test (only significant p -values are shown).

Article Snippet: B cells were washed twice with a cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer with the addition of 10 μl IL-10 detection antibody (APC) (IL-10 secretion assay, Miltenyi Biotec) and 10 μl of the antibody mix (α-CD19-PE-Cy7, α-CD24-PE, and α-CD38-AlexaFluor700).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Next-Generation Sequencing, Cell Counting, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation

doi: 10.3389/fimmu.2022.803229

Figure Lengend Snippet: Frequencies of the CD27-positive B cells in peripheral blood of MS patients and healthy individuals. (A) The percentage of CD27-positive activated memory-like transitional cells in CD19 + CD24 high CD38 high tBreg subpopulation and (B) the frequency of memory (CD19 + CD24 +/high CD38 +/low CD27 + ), naïve (CD19 + CD24 + CD38 +/low CD27 - ) or memory Breg (CD24 high CD27 + ) among peripheral B cells in multiple sclerosis patients (MS) and healthy donors (HD). The bottom panel shows the gating strategy of flow cytometric analysis. (C) Age and gender comparison of MS and HD analyzed in the same experiment. The difference in cell frequency was analyzed by Mann Whitney test. Statistically significant p -values are shown.

Article Snippet: B cells were washed twice with a cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer with the addition of 10 μl IL-10 detection antibody (APC) (IL-10 secretion assay, Miltenyi Biotec) and 10 μl of the antibody mix (α-CD19-PE-Cy7, α-CD24-PE, and α-CD38-AlexaFluor700).

Techniques: Comparison, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation

doi: 10.3389/fimmu.2022.803229

Figure Lengend Snippet: Delayed maturation of CD24 high CD38 high transitional B lymphocytes in the MS patients. (A) Mutation frequency for V H , Vκ, and Vλ genes; (B) the number of unique clonotypes per sorted cells; and (C) the tBreg/total B cell clonotype ratio of the total blood B cells and CD24 high CD38 high transitional Bregs from the multiple sclerosis patients (MS) and healthy donors (HD). Mutation frequency refers to the percentage of clonotype sequence different from the corresponding germline V- and J-segment sequences excluding CDR3 region. BMS denotes benign multiple sclerosis, HAMS is highly active multiple sclerosis. Bar and line plots represent a median and interquartile range. Statistical significance of the differences between donor groups was assessed using the Mann-Whitney test (A, B) and ratio paired T -test (C) . p -values <.05 after correction for multiple comparisons were considered statistically significant and designated with asterisks.

Article Snippet: B cells were washed twice with a cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer with the addition of 10 μl IL-10 detection antibody (APC) (IL-10 secretion assay, Miltenyi Biotec) and 10 μl of the antibody mix (α-CD19-PE-Cy7, α-CD24-PE, and α-CD38-AlexaFluor700).

Techniques: Mutagenesis, Sequencing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation

doi: 10.3389/fimmu.2022.803229

Figure Lengend Snippet: Differences in CDR3 length. The distribution of B cell subset CDR3 amino acid length for heavy (A) , kappa (C) , and lambda (D) light chains. Bar and line plots show mean ± SD. (B) The CDR3 amino acid length distribution for IGVH clonotypes in different B cell subsets. To balance the sample size, an equal number of clonotypes ( n = 1000) were randomly sampled from each donor repertoire. Rare clonotypes with CDR3 length <6 a.a. or >35 a.a. were excluded. Mean values are displayed by numbers. The difference in CDR3 length between tBreg and total B cell fraction was analyzed by the ratio paired T -test. The difference in CDR3 length between donor groups was assessed using the Mann Whitney test. Only statistically significant p -values are indicated. The total CD19 + B cell pool and transitional regulatory CD19 + CD24 high CD38 high subpopulation from peripheral blood are designated as B cells and tBregs, respectively. ns , not significant.

Article Snippet: B cells were washed twice with a cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer with the addition of 10 μl IL-10 detection antibody (APC) (IL-10 secretion assay, Miltenyi Biotec) and 10 μl of the antibody mix (α-CD19-PE-Cy7, α-CD24-PE, and α-CD38-AlexaFluor700).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation

doi: 10.3389/fimmu.2022.803229

Figure Lengend Snippet: Frequencies of the IL-10–positive B cells in peripheral blood of MS patients and healthy individuals after rapid CpG stimulation. (A) The percentage of IL-10–positive cells in CD19 + CD24 high CD38 high tBreg subpopulation and total B cells. (B) The percentage of CD19 + CD24 high CD38 high tBreg in IL-10–positive and IL-10–negative B cells. Results are expressed as a median, interquartile range, and p -values analyzed by ratio paired T -test. Statistical significance of the differences between donor groups was assessed by the Mann Whitney test, and significance of differences between B cell subpopulations was assessed by the ratio paired T -test. Statistically significant p -values ( p <.05) are shown.

Article Snippet: B cells were washed twice with a cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer with the addition of 10 μl IL-10 detection antibody (APC) (IL-10 secretion assay, Miltenyi Biotec) and 10 μl of the antibody mix (α-CD19-PE-Cy7, α-CD24-PE, and α-CD38-AlexaFluor700).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation

doi: 10.3389/fimmu.2022.803229

Figure Lengend Snippet: Abnormalities in transitional B cell maturation in MS. An elevated frequency of CD19 + CD24 high CD38 high transitional B cells (TrB) observed in MS patients is characterized by greater germline identity compared with healthy donors. There are least three possible coexisting or independent scenarios explaining these findings: ( i ) deficient maturation of TrB, ( ii ) delayed TrB maturation, and ( iii ) an increased number of TrB compensating deficient maturation.

Article Snippet: B cells were washed twice with a cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer with the addition of 10 μl IL-10 detection antibody (APC) (IL-10 secretion assay, Miltenyi Biotec) and 10 μl of the antibody mix (α-CD19-PE-Cy7, α-CD24-PE, and α-CD38-AlexaFluor700).

Techniques:

Journal: PLOS Pathogens

Article Title: Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages

doi: 10.1371/journal.ppat.1011691

Figure Lengend Snippet: (A) Timeline of infections with HP by oral gavage and MHV68 by intraperitoneal injection. The timepoints shown in (B-G) are outlined by a red box. Partially created in BioRender. (B) Quantification of flow cytometric analysis of the number of LPMs and SPMs at day 28 of MHV68 infection. Data are pooled from 3 independent experiments (n = 11-13/group, mean ± standard deviation). Each dot represents an individual mouse. (C-F) Quantification of flow cytometric analysis of MHV68-infected CD11b+ PECs at day 28 of MHV68 infection. CD11b+ cells were isolated with CD11b+ beads and MACs columns before staining. LPMs were gated as CD19- CD11b hi ICAM-2 hi . SPMs were gated as CD19- CD11b+ ICAM-2 lo . B cells were gated as CD19+. Data are pooled from 3 independent experiments (n = 11-13/group, mean ± standard deviation). Each dot represents an individual mouse. (C) Total number of MHV68-infected CD11b+ PECs. (D) Representative flow plots of MHV68+ LPMs. (E) Quantification of number of MHV68-infected macrophages and B1 B cells. (F) Proportion of MHV68-infected cells out of the parent populations. (G) Total PECs from days 28–31 of MHV68 infection were subjected to limiting dilution PCR analysis to detect frequency of viral genomes. Data are pooled from 4 independent experiments (3 mice pooled/ group, mean± standard deviation). (H) LPMs were sorted from mice at days 28 and 29 of MHV68 infection as CD19- SiglecF- Ly6G- F4/80+ CD11b hi ICAM-2 hi . Sorted cells were subjected to limiting dilution PCR analysis to detect frequency of viral genomes. Data are pooled from 2 independent experiments (4–8 mice were pooled for each group before sorting, mean± standard deviation). (B, D, E) P-values, 2-way ANOVA, Tukey’s multiple comparisons. (C) P-values, unpaired t-test. (G, H) Dotted line represents Poisson distribution. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend).

Techniques: Injection, Infection, Standard Deviation, Isolation, Staining

Journal: PLOS Pathogens

Article Title: Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages

doi: 10.1371/journal.ppat.1011691

Figure Lengend Snippet: (A) C57BL/6J mice were infected with 100 L3 larvae of HP by oral gavage. Quantification of flow cytometric analysis of LPMs in C57BL/6 mice at the indicated days post HP infection. LPMs were gated as CD3- CD19- Siglec F- CD11b hi ICAM-2 hi . Data are pooled from 3 independent experiments (n = 9-10/group, mean ± standard deviation). (B) C57BL/6J mice were left untreated and uninfected (Uninfected), intraperitoneally injected with sterile PBS (Vehicle) or intraperitoneally infected with 10 6 PFU of MHV68 (MHV68+). PECs were collected on day 4 of MHV68 infection and the number of LPMs was quantified by flow cytometry. LPMs were gated as CD3- CD19- Siglec F- CD11b hi ICAM-2 hi . Data are pooled from 2 independent experiments (n = 10/group, mean ± standard deviation) (C) Timeline of infections with HP by oral gavage and MHV68 by intraperitoneal injection (10 6 plaque forming units (PFU)). The time points shown in (D-K) are outlined by a red box. Partially created in BioRender. (D) Quantification of flow cytometric analysis of LPMs in C57BL/6 mice at days 2, 4, and 7 post MHV68 infection. LPMs were gated as CD19- CD11b hi ICAM-2 hi . Data are pooled from 3 independent experiments (n = 10–11 MHV68+ groups, n = 4 HP+ only group, n = 2 uninfected group, mean ± standard deviation). (E-J) Mice were infected as in (C) with 10 6 PFU of MHV68.ORF73β-lactamase virus. Quantification of flow cytometric analysis of MHV68-infected PECs at days 2, 4, and 7 post MHV68 infection. Data are pooled from 3 independent experiments (n = 10-11/group, mean ± standard deviation). (E) Representative flow plots of the total β-lactamase-positive (MHV68+) PECs. (F) Quantification of the total number of MHV68-infected PECs. (G) Number of MHV68-infected LPMs. (H) Proportion of LPMs that are MHV68-infected. (I) Number of MHV68-infected SPMs. SPMs were gated as CD19- CD11b+ ICAM-2 lo . (J) Number of MHV68-infected B cells. B cells were gated as CD19+. Each dot represents an individual mouse. (K) MHV68 titers determined by plaque assay of PECs at day 1–7 of infection. Data are pooled from 5 independent experiments (n = 6–7 day 1, n = 11–14 days 2 and 4, n = 3–4 day 7, mean ± standard deviation). P-values, 2-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend).

Techniques: Infection, Standard Deviation, Injection, Sterility, Flow Cytometry, Virus, Plaque Assay

Journal: PLOS Pathogens

Article Title: Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages

doi: 10.1371/journal.ppat.1011691

Figure Lengend Snippet: (A) Timeline of intraperitoneal IL-4 complex (IL-4c), thioglycolate (Thio) treatments and MHV68.ORF73β-lactamase intraperitoneal infection (10 6 plaque forming units (PFU)). 5μg of IL-4 and 25 μg α-IL-4 were complexed and injected for LPM expansion. 3.8% sterile thioglycolate broth was used to expand SPMs. The time point shown in (B-H) is outlined by a red box. (B) Quantification of flow cytometric analysis of LPMs, SPMs, and B cells at 2 days post MHV68 infection. LPMs were gated as CD19- CD11b hi ICAM-2 hi . SPMs were gated as CD19- CD11b+ ICAM-2 lo . B cells were gated as CD19+. Data are pooled from 3 independent experiments (n = 10-12/group, mean ± standard deviation). (C) Representative flow plots of the macrophage populations with the different treatments. All mice were infected with MHV68.ORF73β-lactamase. (D-F) Mice were infected as in A with 10 6 PFU of MHV68.ORF73β-lactamase virus. Quantification of flow cytometric analysis of MHV68-infected PECs at day 2 post MHV68 infection. Data are pooled from 3 independent experiments (n = 10-12/group, mean ± standard deviation). (D) Quantification of the total number of MHV68-infected PECs. (E) Proportion of total MHV68-infected PECs for each treatment. (F) Number of MHV68-infected LPMs, SPMs, and B cells. (G) Proportion of MHV68-infected LPMs and SPMs out of the LPM and SPM populations, respectively. (H) Proportion of MHV68-infected LPMs and SPMs out of the LPM and SPM populations, respectively. 2-way ANOVA analysis compared proportion of LPMs to SPMs for each treatment. Each dot represents an individual mouse. (B, F, G, H) P-values, 2-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (D, E) P-values, Ordinary 1-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend).

Techniques: Infection, Injection, Sterility, Standard Deviation, Virus

Journal: PLOS Pathogens

Article Title: Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages

doi: 10.1371/journal.ppat.1011691

Figure Lengend Snippet: Mice were raised on a vitamin A deficient diet or a control diet and infected with HP followed by the MHV68.ORF73β-lactamase reporter virus. PECs were collected on day 2 and day 4 of MHV68 infection. (A) Timeline of infections with HP by oral gavage and MHV68 by intraperitoneal injection (10 6 plaque forming units (PFU)). The time points shown in (B-H) are outlined by a red box. Partially created in BioRender. (B) Representative flow plots of LPMs (ICAM-2 hi CD11b hi CD19-) and SPMs (CD11b+ ICAM-2 lo CD19-) in each of the infection states and diets at day 2 of MHV68 infection. (C) Quantification of flow cytometric analysis of LPMs at days 2 and 4 of MHV68 infection. LPMs were gated as CD19- CD11b hi ICAM-2 hi . (D) Quantification of flow cytometric analysis of SPMs at days 2 and 4 of MHV68 infection. SPMs were gated as CD19- CD11b+ ICAM-2 lo . Data are pooled from 2 independent experiments (n = 7-12/group, mean ± standard deviation). Each dot represents an individual mouse. P-values, 2-way ANOVA, Tukey’s multiple comparisons. (E-H) Quantification of flow cytometric analysis of MHV68-infected PECs at day 2 and 4 of MHV68 infection. Data are pooled from 2 independent experiments (n = 7-12/group, mean ± standard deviation). Each dot represents an individual mouse. (E) Total number of MHV68-infected PECs. (F) Number of MHV68-infected LPMs. (G) Proportion of MHV68-infected LPMs out of total LPMs. (H) Number of MHV68-infected SPMs. (C-F) P-values, 2-way ANOVA, Tukey’s multiple comparisons. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend).

Techniques: Infection, Virus, Injection, Standard Deviation

Journal: PLOS Pathogens

Article Title: Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages

doi: 10.1371/journal.ppat.1011691

Figure Lengend Snippet: (A) Timeline of intraperitoneal IL-4 complex (IL-4c), thioglycolate (Thio) treatments and MHV68.ORF73β-lactamase intraperitoneal infection (10 6 plaque forming units (PFU)). 5μg of IL-4 and 25 μg α-IL-4 were complexed and i.p. injected for LPM expansion. 3.8% sterile thioglycolate broth was i.p. injected to expand SPMs. The time point shown in (B-E) is outlined by a red box. (B) Quantification of flow cytometric analysis of LPMs and SPMs 28 days post MHV68 infection. LPMs were gated as CD19- CD11b hi ICAM-2 hi . SPMs were gated as CD19- CD11b+ ICAM-2 lo . Data are pooled from 2 independent experiments (n = 9/group, mean ± standard deviation). (C-E) Mice were infected as in A with 10 6 PFU of MHV68.ORF73β-lactamase virus. CD11b+ PECs were isolated to enrich for MHV68+ cells. Quantification of flow cytometric analysis of MHV68-infected PECs at day 28 post MHV68 infection. Data are pooled from 2 independent experiments (n = 9/group, mean ± standard deviation). (C) Quantification of the total number of MHV68-infected CD11b+ PECs. P-values, 1-way ANOVA, Tukey’s multiple comparisons. (D) Number of MHV68-infected LPMs and SPMs. (E) Proportion of MHV68-infected LPMs and SPMs of their respective parent populations. Each dot represents an individual mouse. (B, D, E) P-values, 2-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend).

Techniques: Infection, Injection, Sterility, Standard Deviation, Virus, Isolation

Journal: bioRxiv

Article Title: Th2 Cytokine Modulates Herpesvirus Reactivation in a Cell Type Specific Manner

doi: 10.1101/2020.09.30.321778

Figure Lengend Snippet: A. Il4rα f/f x LysMcre+ and Il4rα f/f x LysMcre- mice were intraperitoneally infected with 10 6 PFU wild-type MHV68. Peritoneal lavage was collected 37 days post infection, and flow cytometry was performed to detect IL-4R on peritoneal cells. Cells that have high expression of CD11b are macrophages and CD11b low or negative cells include B cells. Contour plots are representative of one experiment. B. Mice received both IL-4c and anti-IFNγ 28 days post-infection. Another dose of IL-4c was administered two days after the first treatment and then mice were euthanized at 5 days post-treatment. Peritoneal exudate cells were processed into single-cell suspensions and were subjected to nested-PCR analysis to assess the frequency of cells harboring viral genomes. Groups of 3-5 mice were pooled for each infection and analysis. The data are pooled from 3 independent experiments.

Article Snippet: Cells were stained with the following: BV605-α-CD19 (1D3, BD Biosciences), APC-Cy7-α-CD19 (1D3, BD Biosciences), Per-CpCy5.5-α-F4/80 (BM8.1 Tonbo), APC-Cy7-α-F4/80 (BM8, Biolegend), Fitc-α-CD11b (M1/70, Biolegend), PE-Cy7-α-CD11b (M1/70, Tonbo), PE-α-IL4Rα/CD124 (I015F8, Biolegend), Violetfluor 450-α-CD3 (17A2, Tonbo).

Techniques: Infection, Flow Cytometry, Expressing, Nested PCR