Memory B cells in the lung participate in protective humoral immune responses to pulmonary influenza virus reinfection

Abstract

After pulmonary virus infection, virus-binding B cells ectopically accumulate in the lung. However, their contribution to protective immunity against reinfecting viruses remains unknown. Here, we show the phenotypes and protective functions of virus-binding memory B cells that persist in the lung following pulmonary infection with influenza virus. A fraction of virus-binding B-cell population in the lung expressed surface markers for splenic mature memory B cells (CD73, CD80, and CD273) along with CD69 and CXCR3 that are up-regulated on lung effector/memory T cells. The lung B-cell population with memory phenotype persisted for more than 5 mo after infection, and on reinfection promptly differentiated into plasma cells that produced virus-neutralizing antibodies locally. This production of local IgG and IgA neutralizing antibody was correlated with reduced virus spread in adapted hosts. Our data demonstrates that infected lungs harbor a memory B-cell subset with distinctive phenotype and ability to provide protection against pulmonary virus reinfection.

Fig. 1.

Kinetics and surface phenotypes of HA-binding IgM/D⁻ B-cell populations. (A) Cells recovered at the indicated time points after infection were subjected to flow-cytometric analysis (n = 4–5). Representative flow data for HA-binding/CD38 expression by IgM/D⁻dump⁻ B cells (Fig. S1) are shown. (B) Absolute cell number of CD38⁺ and CD38⁻ cells within the lymphocyte gate was plotted. (C) The frequencies of cells expressing CD69, CXCR3, or IgA are plotted. In B and C, each circle represents the result for an individual mouse (lung and spleen) and two to four pooled mice (MLN). **P < 0.01.

Fig. 2.

Reactivation and terminal differentiation of the memory-like B-cell population in lung following secondary infection. (A) BALB/c mice were infected twice, with an 80-d interval. Mice were treated intraperitoneally with 1 mg BrdU for 48 h before analysis, and cells from the indicated organs were subjected to intracellular analysis for BrdU. Representative flow data for BrdU staining of HA-binding IgM/D⁻CD38⁺ B cells are presented (n = 3). (B) HA-binding/CD138 expression among B220^dulldump⁻ cells and IgA expression among HA-binding CD138⁺ cells are presented (n = 3–5).

Fig. 3.

Protective function of the memory-like B-cell population against secondary infection. (A) CB17-scid mice were injected intranasally with CpG and i.v. with BALB/c splenocytes. Ten days after treatment, lung sections were stained with HE (A) or B220 (green), CD90 (red), and DAPI (blue) (B). A representative section (n = 6) is presented. Arrowheads indicate iBALT-like structures. (Scale bar: 500 μm in A and 100 μm in B). (C) HA-binding IgM/D⁻CD38⁺ B cells were sorted from pooled lungs and spleens (n = 10). (D and E) CpG-treated (D) or untreated (E) CB17-scid mice were reconstituted with either lung or splenic memory-like B-cell population (3,000 cells per mouse) together with splenic B cells from naïve mice and CD4⁺ T cells from infected mice. Splenic naïve B cells were added to prevent the loss of small numbers of memory-like B-cell population after sorting. On day 6 postinfection, virus titers in BALFs were determined. (F and G) HA-binding IgG and IgA-secreting plasma cells in the lungs of CpG-treated (F) or untreated (G) mice were enumerated by ELISPOT. (H) HA-binding IgG and IgA Ab titers in BALFs of CpG-treated mice were estimated by ELISA. In D–H, each circle represents the result for an individual mouse. *P < 0.05; **P < 0.01.

Fig. 4.

Relative contribution of IgG and IgA Abs in BALFs to virus-neutralization. (A) Seven days after secondary challenge of BALB/c mice, anti-HA IgG and IgA Ab titers in BALFs were detected by ELISA. Each line represents the result for an individual mouse. (B) Virus-neutralization Ab titers in BALFs were determined by in vitro assay using MDCK cells. In B, each circle represents the result for an individual mouse. *P < 0.05; **P < 0.01.

Fig. 5.

Virus-dependent recruitment of IgA-secreting plasma cells from IgA⁻ memory B cells. (A) CB17-scid mice were reconstituted similarly to Fig. 3D and boosted with inactivated viruses. At day 7 after boosting, the numbers of HA-binding IgG⁺ and IgA⁺ plasma cells were enumerated and plotted. (B) The frequencies of IgA⁺ plasma cells among IgG⁺/IgA⁺ plasma cells are shown. (C) IgA⁺ and IgA⁻ memory B cells were sorted from pooled lung cells and splenocytes (n = 10). (D) The recipient mice were reconstituted with 200 IgA⁺ or IgA⁻ memory B cells in the lungs together with B cells and CD4⁺ T cells, and the numbers of IgG⁺ and IgA⁺ plasma cells were determined after boosting. (E) Mice were reconstituted with IgA⁻ memory B cells in the lungs, and the numbers of IgG⁺ and IgA⁺ plasma cells were determined after boosting with inactivated viruses or rHA. In A, B, D, and E, each circle represents the result for an individual recipient mouse. *P < 0.05; **P < 0.01.