Fig. 3. Selection of broadly reactive IgG is regulated differently from strain-specific IgG.

a B220⁺IgM⁻IgD⁻Dump⁻ B cells were obtained from spleen of the mice immunized with inactivated A/Narita/1/2009 (Vaccine, white bars; n = 3) and from MLN in the A/Narita/1/2009 infected mice (Infection, black bars; n = 3). Binding to A/Narita/1/2009-HA and/or A/PR/8/1934-HA was determined by flow cytometry analysis. b The data indicate binding to HA in flow cytometry analysis (left) and the number of single or dual-binding B cells (right) in spleen (white bars) or MLN (black bars) from the mice infected with A/Narita/1/2009 (n = 5). c Narita-HA binding B cells (2000 cells each) were isolated from vaccinated mice (spleen, white bar; n = 3) and infected mice (MLN, black bar; N = 3). The V-D-J combinations are shown by connected color bands between V and D (left), V and J (middle), and D and J (right). d V_h gene usage of A/Narita/1/2009-HA binding B cells in spleen from three vaccinated mice (Vaccine n = 3) or MLN (Infection n = 2) from two infected mice with A/Narita/1/2009. Total RNA isolated from sorted A/Narita/1/2009-HA binding B cells served as the template for cDNA synthesis followed by PCR to amplify IgG variable region genes. Amplified IgG gene libraries were sequenced by MiSeq. The IgG sequences were analyzed with the ImMunoGeneTics (IMGT) HighV-QUEST. e Seven hundred twenty HA probe⁺ B cells were single sorted and cultured on NB21.2D9 feeder cells with IL-4 in individual wells. After 20 days of culture, 303 IgG-producing cells were obtained. The binding of antibodies produced by these cells to A/Narita/1/2009-HA, A/PR/8/1934-HA, A/H1N1pdm09 derived LAH (blue), or rHA2 (red) was measured by ELISA. Finally, reliable binding and V_H genetic information were obtained for 101 clones. Black circles indicate clones showing no binding to LAH or rHA2. Y-axis represents the ratio of the OD₄₅₀ value in the sample vs the OD₄₅₀ value obtained with IgG [anti-Narita-HA (1 µg/ml), anti-PR8-HA (1 µg/ml), anti-LAH (1 µg/ml), and rHA-2 (3 µg/ml)]. f Amino acid sequences encoded by V_h genes in single or dual-HA binding B cell clones in the region from CDR1 to CDR3 are shown with germline amino acid sequence (unhighlighted). Red boxes indicated CDR regions. The positions where amino acid substitutions occur are indicated by the red font. g Number of amino-acid mutations in the region extending from CDR1 to CDR3 of single or dual-binding B cell clones (single, blue dots; n = 18, dual, red dots; n = 31) as determined by referring to the germline sequences of the corresponding V_h in the IMGT database. *p < 0.05, **p < 0.01 by unpaired, two-tailed t-test (Vaccine group with Infection group in a and c, Spleen group with MLN group in b, Single group with Dual group in g). All error bars represent SEM.