Loss of pre-existing humoral immunity to Influenza A virus is dependent on FcγRIIB.

<p>Female C56BL/6 (•) and FcγRI,II,III^−/− (Δ) mice aged 8–10 weeks were infected by intranasal instillation of 250 HAU of PR8. 150 days later, mice were infected with <i>P. chabaudi</i>. <b>A.</b> Representative FACS plot showing the relative MFI of FcγRIIB (CD32) expression on B220⁻ CD138⁺ bone marrow LLPCs compared to an isotype control. <b>B</b> and <b>C</b> 8 days after <i>P. chabaudi</i> infection, bone marrow from femur pairs was obtained. <b>B.</b> Total bone marrow cellularity was determined and <b>C,</b> HA-specific ASCs were quantified using ELISpot [FcγRI,II,III^−/− (Δ): n = 6 (d150/0) and 8 (d158/8); C56BL/6 (•): n = 3 (d150/0) and 8 (d158/8)]. Data were pooled from two independent experiments. <b>D.</b> Venous blood was obtained from C56BL/6 and FcγRI,II,III^−/− mice on days 0, 35 and 56 of <i>P. chabaudi</i> infection and processed for serum. HA-specific IgG antibodies were quantified by ELISA. Data was obtained from one experiment [FcγRI,II,III^−/− (Δ): n = 5 (d150/0), 10 (d185/35) and 10 (d196/56); C56BL/6 (•): n = 5 (d150/0), 7 (d185/35) and 7 (d196/56)]. <b>E–H.</b> 8–10 week old female C56BL/6 (•) and FcγRIIB^−/− (Δ) mice were infected by intranasal instillation of 250 HAU of PR8. 56 days later, mice were infected with <i>P. chabaudi</i> as previously described. <b>E.</b> 5 and 8 days after <i>P. chabaudi</i> infection, bone marrow from femur pairs were obtained and total bone marrow cellularity was determined. <b>F.</b> 8 days after <i>P. chabaudi</i> infection, HA-specific ASCs were quantified using ELISpot. 5 days after <i>P. chabaudi</i> infection, % Annexin expression was determined on <b>G,</b> total bone marrow cells and <b>H,</b> bone marrow LLPCs. Data was obtained from one experiment [FcγRIIB^−/− (Δ): n = 4(d56/0), 5(d61/5) and 5(d64/8); C56BL/6 (•): n = 3(d56/0), 5(d61/5) and 5(d64/8)]. Error bars indicate the median ± S.E.M. and statistical values were calculated using a two-tailed Mann-Whitney test. Non-significant values (P>0.05) are not indicated on the graphs.