Protective antiviral antibody responses in a mouse model of influenza virus infection require TACI

Abstract

Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.

Figure 1. Kinetics of ASCs during influenza virus infection.

(A) Naive and influenza virus PR8–infected BLIMP-1–YFP mice (day 21 after infection) were analyzed for YFP⁺CD138⁺ ASCs in lung-draining medLN, lungs, and BM. Cells expressing CD4, CD8, and CD11b were excluded. Dot plots with percentages shown are representative of n = 3–5 mice from at least 2 independent experiments. (B) Level of YFP expression in ASCs from medLN, lungs and BM isolated from naive and influenza-infected BLIMP-1–YFP mice. Histograms are based on CD4^–CD8^–CD11b^– cells and representative of n = 5–7 mice. (C) Kinetics of YFP⁺ ASCs during influenza virus infection. Data with mean ± SEM are representative of n = 3–5 mice per time point. (D) Immunofluorescence of lungs from naive and infected BLIMP-1–YFP mice stained for B220 (red). Original magnification, ×20, Scale bar: 100 μm.

Figure 2. ASCs in lungs of influenza virus–infected BLIMP-1–YFP mice are sensitive to irradiation.

(A) Influenza virus PR8–infected BLIMP-1–YFP mice (n = 3–9 mice/group) were subjected to whole-body irradiation (7.5 Gy) at 6 days, 3–4 weeks, or 4 months p.i. and analyzed 10 days later together with non-irradiated controls. (B) Total YFP⁺ ASCs in lungs, spleen, and BM were determined by flow cytometry. (C) PR8-specific ASCs were determined by ELISPOT. (D) PR8-specific IgG Ab titers in sera from control and irradiated mice were measured by ELISA. All data with mean ± SEM represent at least 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. TACI-Fc treatment of BLIMP-1–YFP mice 1 month after influenza virus infection dramatically reduces virus-specific ASCs.

(A) Thirty-five days after infection with influenza virus PR8, BLIMP-1–YFP mice were treated with 100 μg TACI-Fc (n = 7 mice) or human control IgG Ab (co-Ig) (n = 4 mice) every 2–3 days until analysis at day 48 p.i. (B) YFP⁺ ASCs were enumerated in spleen, medLN, lungs, and BM. (C) PR8-specific ASCs were determined by ELISPOT. (D) PR8-specific IgG in serum and (E) PR8-specific IgG and IgA Ab titers in BAL from mice treated with TACI-Fc and co-Ig were determined by ELISA. All data with mean ± SEM represent 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Maintenance of virus-specific Abs to influenza virus requires TACI.

(A) Sera from WT and TACI^–/– mice were assayed for PR8-specific IgM and IgG by ELISA. Blood samples were collected from naive mice (n = 3 mice) and on days 8, 18, 33, and 60 p.i. (n = 3–12 mice per time point). (B) PR8-specific IgM, IgG, and IgA Ab titers in BAL were measured at days 8 and 32–34 p.i. (C) Total IgG and IgA in BAL from WT and TACI^–/– mice (n = 8–11 mice/group) were determined (days 32–34 p.i.). Data with mean ± SEM are representative of at least 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Maintenance but not early generation of virus-specific ASCs is impaired in TACI^–/– mice.

(A) MedLNs from WT and TACI^–/– mice (n = 10–14 mice/group) were harvested 6–8 days p.i. and assayed for PR8-specific IgM, IgG, and IgA ASCs by ELISPOT. (B) PR8-specific IgG and IgA ASCs from indicated organs of WT and TACI^–/– mice were determined by ELISPOT at days 32–34 p.i. (n = 5–8 mice/group). (C) WT and TACI^–/– mice (n = 5 mice/group) received 1,000 HAU/100 μl purified PR8 virus intravenously, and PR8-specific IgG ASCs were enumerated 30 days later in BM and spleen. Data with mean ± SEM are representative of at least 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Induction but not maintenance of the antibody response to influenza is dependent on T cell help.

(A) WT and TACI^–/– mice (n = 7–8 mice/group) were infected with influenza virus PR8. Starting on day 3 p.i., WT and TACI^–/– mice received 5 i.p. injections of MR1 or hamster Ig control Ab (150 μg/mouse) in 3-day intervals. Blood samples were collected prior to (day 0) and days 8, 14, 21, 30, and 45 after infection and PR8-specific IgG Ab titers were determined by ELISA. (B–D) BLIMP-1–YFP mice were infected with influenza virus PR8 and treated 3 times in 3-day intervals starting on day 38 p.i. with MR1 or hamster Ig Ab (150 μg/mouse). At day 46 p.i., spleen, medLN, lungs, and BM were harvested. Diagram in panel B applies to panels B–E. (B) YFP⁺ ASCs were enumerated by flow cytometry and (C) PR8-specific ASCs determined by ELISPOT. (D) PR8-specific IgG Ab titers in serum and (E) IgG and IgA in BAL were measured by ELISA. All data with mean ± SEM combine the results of 2 independent experiments. *P < 0.05, ***P < 0.001.

Figure 7. Reduction of BLyS- and APRIL-expressing CD11b⁺CD11c^– cells in lungs of TACI^–/– mice after influenza virus infection.

(A) Flow cytometry plots with percentages of CD11b⁺CD11c⁺ and CD11b⁺CD11c^– cells in lungs, BAL, and spleen isolated from WT or TACI^–/– mice at day 34 p.i. (B) Frequency of CD11b⁺CD11c^– and CD11b⁺CD11c⁺ cells in various organs of WT and TACI^–/– mice at day 34 p.i. Data with mean ± SEM combine the results of 3 independent experiments (n = 8 mice/group). (C) Identification of cell subsets in lungs of mice based on expression of CD11b, CD11c, and Gr-1 used for purification by FACS. DN, double negative (CD11b^–CD11c^–). (D) Real-time PCR analysis of BLyS and April mRNA expression in cell subsets sorted from lungs as shown in C of naive mice (solid bars) and mice 3–4 weeks p.i. Data with mean ± SEM are from 2 independent experiments. *P < 0.05.

Figure 8. TACI^–/– mice have reduced Ab-mediated protection.

(A) HI assay with sera from WT and TACI^–/– mice collected 3 months after infection with influenza virus PR8. (B) Sera from WT and TACI^–/– mice (n = 8–9 mice/group) 2–3 months after infection were tested for binding to PR8 (dashed line) and SW (solid line) virus by ELISA. As a control, sera from naive mice are shown at a dilution of 1:200. (C) Two to 3 months after primary infection with influenza virus PR8, WT and TACI^–/– mice were challenged with 2,000 TCID₅₀/30 μl of SW virus, and lungs were assayed for viral titers 5 days later. (D) Viral titers in lungs from PR8-infected WT and TACI^–/– mice 5 days after challenge with SW virus. Dotted line represents limit of detection for this assay. Data with mean ± SEM summarize 2 independent experiments. ***P < 0.001.