Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system

Abstract

"Natural" Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposure per se is not sufficient to increase natural antibody production.

Figure 1

The peritoneal cavity of allotype chimeras contains B-1 and B-2 cells of differing allotypes. Shown are 5% contour plots of PerC cells from the indicated mouse strains after gating for live, B220⁺, CD4⁻, CD8⁻, F4/80⁻ cells. Cells were stained either for total or allotype-specific IgM and IgD. Frequencies of B-1 cells (IgM^hi, IgD^lo) and B-2 cells (IgM^lo, IgD^hi) were calculated from the indicated gates. These gates were chosen to exclude CD23⁺ CD43⁻ CD5⁻ cells in the B-1 cell gate, and CD11b⁺, CD5⁺ cells in the B-2 cells gate (data not shown). In chimeras all Igh^a-expressing cells are B-1 cells, and the majority of Igh^b-expressing cells are B-2 cells.

Figure 2

Spleens of allotype chimeras contain normal frequencies of B cell subpopulations. Splenic single cell suspensions from the indicated mouse strains were stained and gated for live, CD4⁻, CD8⁻, F4/80⁻ B220⁺ cells. Further gating was performed as indicated. Frequencies of the gated cells are calculated as percent of total cells within each gate. (Upper) Similar frequencies of immature/transitional (Imm./Tr.). B, marginal zone B (MZ) and B-1 cells are found in spleens of C.B-17 mice and allotype chimeras. (Lower) B220⁺, CD43⁺, CD5⁺ (B-1) spleen cells from chimeric mice express Igh^a, and B220⁺, CD43⁻, CD5⁻ (B-2) cells express Igh^b.

Figure 3

Serum from allotype chimeras contains B-1 and B-2 cell-derived Ig. Concentrations of a- and b-allotype IgM, IgG2a, and IgA in sera of allotype chimeras (n = 13) and controls (for Igh^a: BALB/c mice, n = 12; for Igh^b: C.B-17 mice, n = 11) were determined by ELISA. In chimeras, Igh^a is PerC donor-derived and therefore produced by B-1 cells (see Figs. 1 and 2), and Igh^b is host-derived. Data are shown as interquartile boxes; disconnected bars above and below the boxes indicate the 90th and 10th percentiles, respectively. ■, Data from individual mice. ELISA were highly specific, as measurement of sera from the nonrelevant allotype were below the threshold of detection for the assays (<0.01 μg/ml for IgM and IgG2a, <0.001 μg/ml for IgA). Statistical significance was tested with the Wilcoxon Rank test (P < 0.05); n.s., not significant.

Figure 4

Natural antiviral Ig is derived from B-1 cells, acquired antiviral Ig from B-2 cells. Sera of allotype chimeras (n = 13) and control mice (for Igh^a, BALB/c mice, n = 12; for Igh^b, C.B-17 mice, n = 11) were tested before and seven of the chimeras and all controls were also tested at day 7 after influenza virus infection for levels of influenza virus-specific Igh^a and Igh^b, IgM, and IgG2a. Units anti-influenza Ig were calculated in relation to standard hyperimmune sera. Data are shown as interquartile boxes; disconnected bars above and below the boxes indicate the 90th and 10th percentiles. ■, Data from individual mice. n.s., not significant. In chimeras, Igh^a is PerC donor-derived and therefore produced by B-1 cells.

Figure 5

Influenza virus infection induces antiviral IgM without affecting serum levels of natural antiviral IgM. Sixteen allotype chimeras were infected with influenza virus and groups of eight mice were bled on alternating days as indicated. Serum levels of anti-influenza virus-specific IgM^a (PerC donor-derived) and IgM^b (host-derived) were determined by ELISA. ●, Mean levels; ■, data from individual mice. Bars = SD.