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. 2019 May 16;10:1105. doi: 10.3389/fimmu.2019.01105

Figure 2.

Figure 2

Isolation and characterization of Borrelia VlsE-C6-reactive memory B cells. (A) Sera (dilution 1:100) from 255 healthy donors with a high risk for tick bites and hence for Borrelia seroconversion were screened with both a commercial immunoblot (Sekizui Virotech) and our in-house VlsE-C6 peptide ELISA. Peptides from three Borrelia strains (IP90, PT7, B31) were tested. Samples are grouped according to their reactivity in the commercial immunoblot (negative to highly positive: VlsE−, VlsE+, VlsE++, VlsE+++). Values from the in-house ELISA are plotted on the y-axis. ELISA values were normalized to two negative control samples (Ctrl) included on each plate. (B) Labeling of memory (CD27+IgD−) and naïve (CD27−IgD+) B cells from seropositive and seronegative donors with VlsE-C6-Neutravidin tetramers (52). Background staining with biotin tetramers only was subtracted to give the values shown. Reactivities of samples of the different groups were compared by One-way Analysis of Variance test followed by Tukey's Multiple Comparison test. Results are from five independent experiments including data from nine seropositive and five seronegative donors. Each datapoint represents the percent reactive cells with individual peptides. For an initial assessment, three seropositive and three seronegative donors were only screened with the IP90 peptide (Supplementary Table 1). Therefore, there are 3x1(IP90) + 6x3(IP90, PT7, B31) = 21 datapoints for seropositive donors and 3x1(IP90) + 2x3(IP90, PT7, B31) = 9 datapoints for seronegative donors. (C) Tetramers containing the individual peptides were pooled and acute Lyme disease patients' samples stained with otherwise the same protocol. Only part of the healthy controls (n = 6, average age: 52, 33% female) and all of the acute patients (n = 11, average age: 55, 82% female) were sampled at different timepoints over 1 month period shown as T0, T1, and T2. Samples from single timepoints (T0 for both acute and healthy controls) were compared using a One-tailed Mann Whitney test. Two-tailed unpaired t-test showed no difference in age between both groups. (D) The percentage of CD27+ and CD27+IgD- memory B cells reacting with VlsE-C6 peptide tetramers of the acute patients shown in panel 2C, presented by time since onset of symptoms. Spearman's Rank Correlation Hypothesis Testing was done manually in Excel. *p = 0.01–0.05, ***p = 0.0001–0.001.