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: