Journal: Cell reports
Article Title: Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism.
doi: 10.1016/j.celrep.2024.114245
Figure Lengend Snippet: Figure 1. Inhibited IgG recall responses following TcdB2 treatment (A) Female B6 mice (n = 5 per group) were given PBS vehicle or 1 ng TcdB2 (i.p.) and then immunized with 10 mg of B2D/Alum (subcutaneously [s.c.]) after 5 h. Mice were bled on days 14, 28, 42, and 67 (primary). On day 67, mice were boosted with 10 mg of B2D in PBS (s.c.) and then bled on day 81 (recall). (B) Serum B2D-specific IgM, IgG1, IgG2b, and IgG2c endpoint recall titers were determined by ELISA. All control mice had an IgG2c booster response, while 1 of 5 TcdB2-treated mice responded. Graphs show comparison of mean ± SD endpoint titers pre and post booster vaccine administration, and significant differ- ences were determined by a matched-pairs t test. Further, Mann-Whitney U tests were used to compare post-booster vaccine titers between control and TcdB2- treated groups (for IgG1, p = 0.036). (C) CHO cells were incubated with TcdB2, sera, or sera and TcdB2 for 24 h. Cell viability was determined using the CCK-8 assay as a measure of TcdB2 neutralization. The graph shows mean ± SD cell viability using recall sera from control (n = 5) and TcdB2-treated (n = 6) groups, and significance was deter- mined by unpaired t test. Other data related to this figure are depicted in Figure S1.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP anti-mouse IgM Southern Biotech Cat# 1020-05 RRID: AB_2794201 HRP anti-mouse IgG1 Southern Biotech Cat# 1070-05 RRID_2650509 HRP anti-mouse IgG2b Southern Biotech Cat# 1090-05 RRID: AB_2794521 HRP anti-mouse IgG2c Southern Biotech Cat# 1079-05 RRID: AB_2794466 HRP anti-mouse IgA Southern Biotech Cat # 1040-05 RRID# AB_2714213 Anti-mouse/rat/human GAPDH, clone 6C5 Abcam Cat # Ab8245 RRID: AB_2107448 Biotin anti-mouse CXCR5, clone 2G8 BD Biosciences Cat # 551960 RRID: AB_394301 FITC anti-mouse B220, clone RA3-6B2 BD Biosciences Cat # 553088, RRID: AB_394618 FITC anti-mouse CD21/23, clone 7G6 BD Biosciences Cat# 553818 RRID: AB_395070 APC anti-mouse CD5, clone 53–7.3 BD Biosciences Cat# 550035 RRID: AB_398457 Anti-Rac1 (multi species), clone 102/Rac1 BD Biosciences Cat# 610651 RRID: AB_397978 APC-Cy7 anti-mouse CD19, clone 6D5 Biolegend Cat# 152411 RRID: AB_2922473 Alexa 488 anti-mouse/human-Ki67, clone 17F6 Bioegend Cat# 151204 RRID# AB_2566800 PE anti-mouse CD19, clone 1D3 eBiosciencess Cat# 12-0193-82 RRID: AB_657659 APC anti-mouse CXCR4, clone 2B11 eBioscience Cat# 17-9991-82 RRID AB_10670878 V450 anti-mouse CD4, clone GK1.5 Tonbo Cat # 75-0041 RRID: AB_2621927 Alexa 750 anti-mouse CD93, clone 223437 R&D Systems Cat# FAB1 696S RRID: AB_2935869 Anti-mouse FcR, clone 2.4G2 BioXCell Cat# BE0307 RRID: AB_2736987 InVivoPlusTM isotype control mAb, clone 2A3 BioXCell Cat# BP0089 RRID: AB_1107769 InVivoPlusTM anti-mouse CD40 mAb, clone FGK4.5 BioXCell Cat# BP0016-2 RRID: AB_1107647 Bacterial and virus strains C. difficile R20291 strain N/A Chemicals, peptides, and recombinant proteins TcdB2: wild type recombinant C. difficile toxin Bacillus megaterium expression system, in house N/A TcdB2 D270N: glucosyltransferase-null recombinant C. difficile toxin Bacillus megaterium expression system, in house N/A (Continued on next page) 18 Cell Reports 43, 114245, May 28, 2024
Techniques: Enzyme-linked Immunosorbent Assay, Control, Comparison, MANN-WHITNEY, Incubation, CCK-8 Assay, Neutralization
SouthernBiotech is a verified supplier 
![Figure 1. Inhibited IgG recall responses following TcdB2 treatment (A) Female B6 mice (n = 5 per group) were given PBS vehicle or 1 ng TcdB2 (i.p.) and then immunized with 10 mg of B2D/Alum (subcutaneously [s.c.]) after 5 h. Mice were bled on days 14, 28, 42, and 67 (primary). On day 67, mice were boosted with 10 mg of B2D in PBS (s.c.) and then bled on day 81 (recall). (B) Serum B2D-specific IgM, IgG1, <t>IgG2b,</t> and IgG2c endpoint recall titers were determined by ELISA. All control mice had an IgG2c booster response, while 1 of 5 TcdB2-treated mice responded. Graphs show comparison of mean ± SD endpoint titers pre and post booster vaccine administration, and significant differ- ences were determined by a matched-pairs t test. Further, Mann-Whitney U tests were used to compare post-booster vaccine titers between control and TcdB2- treated groups (for IgG1, p = 0.036). (C) CHO cells were incubated with TcdB2, sera, or sera and TcdB2 for 24 h. Cell viability was determined using the CCK-8 assay as a measure of TcdB2 neutralization. The graph shows mean ± SD cell viability using recall sera from control (n = 5) and TcdB2-treated (n = 6) groups, and significance was deter- mined by unpaired t test. Other data related to this figure are depicted in Figure S1.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1377/pm38761377/pm38761377__page3_image1.jpg)