Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD57 + GC-Th cells are more efficient than other tonsil CD4 + T cell subsets in helping B cells. (A) CD4 + T cell subsets were cultured with total tonsil CD19 + B cells for 7 days in the presence of SEB. Naïve B cells (C) or GC-B cells (D) were cultured with equal numbers of CD57 + GC-Th cells or other T cell subsets (CD57 - , CD57 - CD69 + and CD57 - CD69 - T cells) for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 5 independent experiments were combined and the averages are shown with standard errors. Relative production levels to CD57 + GC-Th cells are shown. *Significant differences from CD57 + GC-Th cells. The absolute Ig production levels (ng/ml) in panel A (GC-Th + Total B cells) were 5737 ± 1764 (IgM), 2111 ± 1185 (IgG), 577 ± 186 (IgA), and 4.8 ± 2.1 (IgE). The absolute Ig production levels (ng/ml) in panel B (GC-Th cells + naïve B cells) were 2045 ± 697 (IgM), 63 ± 21 (IgG), 40 ± 23 (IgA), and 2.9 ± 1.2 (IgE). The average levels (ng/ml) of Ig produced in the cultures of GC-Th cells and GC B cells were 750 ± 279 (IgM), 175 ± 52 (IgG), 51 ± 13 (IgA), and 1.0 ± 0.5 (IgE). (D) Isotype composition of the Ig induced by CD57 + GC-Th cells. Naïve B cells or GC-B cells were cultured with equal numbers of CD57 + GC-Th cells or CD57 - CD69 + T cells for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 4 independent experiments were combined and the averages are shown with standard errors. *Significant differences between naïve and GC-B cells, but not between the two T cell subsets, were observed.

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD57 + GC-Th cells have the capacities to induce AID expression and to support CSR in B cells. IgD + CD38 - naïve B cells were cultured with CD57 + GC-Th cells for indicated time periods followed by RT-PCR analysis for (A) AID expression and (B) CSR. The sizes of specific PCR products are 152 bp (IgM); 416 bp (IgG1, G2, G3), 904 bp (IgG4); 904 bp (IgA1); 891 bp (IgA2); and 179 bp (IgE). Shown are productive recombination products. (C) The expression kinetics of AID and productive IgG3 transcripts over an 8 day period are shown together in a graph. In this panel, normalized expression levels calculated after dividing the levels of AID amplification by β-actin levels are shown. The time gap to reach the peak levels of the expression between AID and productive IgG3 transcripts is shown by an arrow. Representative data from at least three independent experiments are shown (panels A and B). (D) Identification of extrachromosomal reciprocal DNA recombination products. Naïve B cells were cultured with CD57 + GC-Th cells for indicated time periods and were processed to isolate genomic DNA. Fresh GC-B cells were examined for positive controls. The switch circles were detected by a nested PCR method. Representative data out of three independent experiments are shown. (E) Detection of switch circles by a DC-PCR technique. Naive B cells, CD38 + GC-B cells and naïve B cells cultured with GC-Th cells for 5 days were examined for the presence of γ3 and α1/2 switch circles.

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Nested PCR

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD40L and cytokines in regulation of the helper activity of CD57 + GC-Th cells. (A and B) Effects of endogenous CD40L and cytokines on the helper activity of CD57 + GC-Th cells were determined. In cultures of CD57 + GC-Th cells with naïve or GC-B cells, neutralizing antibodies to IL-4, IL-10, IFN-γ or CD40L or control antibodies (mouse IgG1) were added. *Significant differences from the control group (control antibody). (C and D) Effects of exogenously added cytokines on the helper activity of CD57 + GC-Th cells were determined. To cultures of CD57 + GC-Th cells with naïve or GC-B cells, IL-4, IL-10, IFN-γ and TGF-β1 were added separately. Cells were cultured for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Relative Ig secretion levels (the medium control = 1) obtained from 9 independent experiments were combined, and averages and standard errors are shown. *Significant differences from the control group (medium).

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Activity Assay, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

doi: 10.1016/j.celrep.2023.112766

Figure Lengend Snippet: Figure 3. Titration of rtN1- and rtN2-spe- cific serum IgG in ELISA The sera samples collected 2 weeks after (A and D) prime immunization, (B and E) first booster, and (C and F) second booster were titrated for (A–C) rtN1 and (D–F) rtN2. Each dot represents the serum antibody titer from one mouse (n = 5 per group). Bars represent the mean values of serum antibody titers. The dash line represents the limit of detection, corresponding to the initial serum dilution. The positive wells were determined as OD values above mean +2 3 SD of the OD value of the initial pre-immune sera dilution. Statistical signifi- cance was analyzed by t test for (B), (C), (E), and (F). *p < 0.05, **p < 0.01, and ****p < 0.001. All experiments were repeated twice using the same serum samples and showed similar results.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-63His tag Sino Biological Cat#105327-MM02T-H Rabbit polyclonal anti-N1 Sino Biological Cat#11058-R001 Rabbit polyclonal anti-N2 Sino Biological Cat#40017-RP01 HRP-conjugated goat anti-human IgG Proteintech Group Cat#SA00001-17 HRP-conjugated goat anti-mouse IgG Proteintech Group Cat#SA00001-1 HRP-conjugated goat anti-mouse IgG1 Proteintech Group Cat#SA00012-1 HRP-conjugated goat anti-mouse IgG2a Proteintech Group Cat#SA00012-2 HRP-conjugated goat anti-mouse IgG3 Proteintech Group Cat#SA00012-5 HRP-conjugated goat anti-mouse IgM Proteintech Group Cat#SA00012-6 HRP-conjugated peanut agglutinin Sigma-Aldrich Cat#L7759 Bacterial and virus strains E.coli DH5a competent cells Lab stock N/A E.coli DH10Bac competent cells Lab stock N/A A/Hong Kong/8/1968 (H3N2) ATCC Cat#VR544TM A/New Jersey/8/1976 (H1N1) ATCC Cat#VR897TM A/Puerto Rico/8/1934 (H1N1) Haiyan Chang, Hunan Normal University N/A A/reassortant/NYMC X179A (H1N1) Haiyan Chang, Hunan Normal University N/A A/Guizhou/54/1989 (GZ89, H3N2) Haiyan Chang, Hunan Normal University N/A A/Puerto Rico/8/1934 (H1N1) Haiyan Chang, Hunan Normal University N/A Biological samples Human convalescent sera samples Yao-Qing Chen’s laboratory stock N/A Chemicals, peptides, and recombinant proteins N1P1 Top-peptide N/A N1P2 Top-peptide N/A N1P3 Top-peptide N/A N1P4 Top-peptide N/A N2P1 Top-peptide N/A N2P2 Top-peptide N/A N2P3 Top-peptide N/A N2P4 Top-peptide N/A rtN1 This paper N/A rtN2 This paper N/A KLH Solarbio Cat#K8160 Receptor destroying enzyme Denka Seiken Cat#340122 Fetuin from fetal bovine serum Sigma-Aldrich Cat#F2379 Incomplete Freund’s adjuvant Sigma-Aldrich Cat#F5506 Critical commercial assays ReadiLinkTM KLH Conjugation Kit AAT Bioquest Cat#5502 Mouse IFNg ELISPOT Kit BD Biosciences Cat#551083 Mouse IL-4 ELISPOT Kit Dakewe Cat#DKW22-2040-500 (Continued on next page) Cell Reports 42, 112766, July 25, 2023 11

Techniques: Titration, Enzyme-linked Immunosorbent Assay