Efficient Acquisition of Fully Human Antibody Genes against Self-Proteins by Sorting Single B Cells Stimulated with Vaccines Based on Nitrated T Helper Cell Epitopes

Figure 6

B cells transferred into HIS mice displayed somatic hypermutation and underwent antibody class switching in the stimulation of NitraTh-based vaccines. NCG mice were engrafted with PBMCs on day 0 and DCs on day 7 to build DC-HIS mice. Then, DC-HIS mice were immunized with HER2-NitraTh or HER2-Th on days 7 and 21. Splenocytes were harvested from vaccinated mice on day 28 for sorting CD3⁻CD19⁺CD27⁺HER2⁺ B cells. CD3⁻CD19⁺ naïve B cells were sorted from peripheral blood in the same donor. The VH and VL genes of these cells were amplified by RT-PCR with specific primers. (a) Bar graphs depicted the frequencies of the VH-CDR3 length with ≤9, 10 to 14, 15 to 19, and ≥20 amino acids (aas). (b) Pie charts showed the proportion of VH and VL with 0 to 1, 3 to 5, 6 to 9, and ≥10 somatic mutations. (c) The ratio of the replacement (blue bar) and silent mutations (orange bar) in IGHV-FWRs and CDRs was calculated in vaccinated mice as mutated nucleotides per total base pairs analyzed. The R/S ratio for each region was indicated. (d) Pie charts depicted the frequencies of the antibody gene types of naïve B cells and HER2⁺ B cells. (e) Eight IgG sequences from HER2⁺ B cells were cloned into pCDNA3.1 vector and transformed into the HEK293 cell line. Transfected HEK293 cells were tested by ELISpot assay to confirm that the antibodies encoded by the obtained sequences can bind with HER2. (f) FACS determined the specific binding capacity of the antibody produced by clone B to SK-BR-3.

(a)

(b)

(c)

(d)

(e)

(f)