Early B-cell activation after West Nile virus infection requires alpha/beta interferon but not antigen receptor signaling

Abstract

The B-cell response against West Nile virus (WNV), an encephalitic Flavivirus of global concern, is critical to controlling central nervous system dissemination and neurological sequelae, including death. Here, using a well-characterized mouse model of WNV infection, we examine the factors that govern early B-cell activation. Subcutaneous inoculation with a low dose of replicating WNV results in extensive B-cell activation in the draining lymph node (LN) within days of infection as judged by upregulation of the surface markers CD69, class II major histocompatibility complex, and CD86 on CD19(+) cells. B-cell activation in the LN but not the spleen was dependent on signals through the type I alpha/beta interferon (IFN-alpha/beta) receptor. Despite significant activation in the draining LN at day 3 after infection, WNV-specific B cells were not detected by immunoglobulin M enzyme-linked immunospot analysis until day 7. Liposome depletion experiments demonstrate that B-cell activation after WNV infection was not affected by the loss of F4/80(+) or CD169(+) subcapsular macrophages. Nonetheless, LN myeloid cells were essential for control of viral replication and survival from infection. Overall, our data suggest that the massive, early polyclonal B-cell activation occurring in the draining LN after WNV infection is immunoglobulin receptor and macrophage independent but requires sustained signals through the type I IFN-alpha/beta receptor.

FIG. 1.

B-cell activation in the draining LN. A. Expression of CD69 on CD19⁺ B cells after WNV-NY infection in wild-type (WT) and IFN-α/βR^−/− mice. The data reflect averages from 5 to 10 mice per time point. B. Total number of CD69⁺ CD19⁺ B cells in the ILN after WNV-NY infection. C and D. Expression of CD69 on CD4⁺ (C) and CD8⁺ (D) T cells after WNV-NY infection. E. Measurement of B-cell proliferation in ILN at the indicated days after WNV infection or mitogenic stimulus (anti-CD40 plus IL-4) as judged by Ki-67 expression. F. Expression of CD69 on CD19⁺ B cells in wild-type and IFN-α/βR^−/− mice 3 days after infection with WNV-MAD or WNV-NY. G. Effect of neutralizing anti-IFN-α/βR MAb (MAR1-5A3) on CD69 expression on CD19⁺ B cells after WNV infection. Mice were treated with MAb (or isotype control) at the indicated times relative to infection, and B cells were analyzed at 3 days after infection. H. Expression of CD69 on CD19⁺ B cells in HELMET mice 3 days after WNV infection. The dotted line represents the basal level of expression in naïve wild-type mice. Asterisks indicate time points at which differences are statistically significant (**, P < 0.01; ***, P < 0.001). I. Expression of CD69 on CD19⁺ B cells from the ipsilateral popliteal lymph node (PLN), ipsilateral ILN, and contralateral ILN node 2 days after WNV infection.

FIG. 2.

B-cell activation in the spleen. A. Expression of CD69 on CD19⁺ splenic B cells after WNV-NY infection in wild-type and IFN-α/βR^−/− mice. The data reflect averages from 5 to 10 mice per time point. B. Total number of CD69⁺ CD19⁺ B cells in the spleen after WNV-NY infection. C and D. Expression of CD69 on CD4⁺ (C) and CD8⁺ (D) T cells after WNV-NY infection. E. Measurement of B-cell proliferation in the spleen at the indicated days before or after WNV infection as judged by Ki-67 expression. F. Expression of CD69 on CD19⁺ B cells in HELMET mice 3 days after WNV infection. G. Effect of neutralizing anti-IFN-α/βR MAb (MAR1-5A3) on CD69 expression on splenic B cells after WNV-NY infection. Mice were treated with MAb (or isotype control) at the indicated times relative to infection, and B cells were analyzed at 3 days after infection. The dotted line represents the basal level of expression in naïve wild-type mice. Asterisks indicate time points at which differences are statistically significant (*, P < 0.05; **, P < 0.01).

FIG. 3.

Phenotyping of activated B cells in LN and spleen after WNV infection. A and B. B-cell activation in the draining LN at day 3 after WNV infection was assessed by immunostaining for increased surface expression of MHC class II (A) and CD86 (B). Three-color staining was performed with CD19⁺ CD69⁺ and CD19⁺ CD69⁻ cells. Asterisks indicate differences that are statistically significant (**, P < 0.01; ***, P < 0.001). D and E. WNV infection induces CD69 expression on CD19⁺ B cells. A representative flow cytometry panel of LN cells from uninfected (D) and WNV-infected (E) mice is shown. F. All of the CD69⁺ B cells in the LN showed a mature (surface IgD⁺ IgM⁺) phenotype. G to I. CD69 staining on different B-cell subsets in the spleen at day 4 after WNV infection. (H and I) Follicular (CD21^high CD23^high) (H) and marginal zone (CD21^high CD23^low) (I) B cells were determined by flow cytometry after gating (G). The average values for activation of these B-cell subsets are indicated in panel C and reflect results for at least five mice per condition.

FIG. 4.

B-cell activation after MNV infection. CD69 expression on CD19⁺ B cells from the draining mesenteric lymph node (A) or spleen (B) after peroral infection with MNV. Each time point represents the average of at least five mice.

FIG. 5.

Antigen-specific IgM ELISPOT in the ILN after WNV infection. A. Three or 7 days after infection with WNV, cells from the ILN were harvested and analyzed using an IgM ELISPOT against specific (WNV E protein) or nonspecific (ovalbumin) protein. The data are expressed as the number of antigen-specific B cells producing IgM per 10⁶ cells and are averages of at least six mice per time point. B. A digital photographic example of an ELISPOT from B cells harvested at day 7 after WNV infection. The left well represents B cells that recognize WNV-E, and the right well represents nonspecific B cells that bind ovalbumin.

FIG. 6.

Effects of LN macrophage depletion on B-cell activation and humoral response after WNV infection. Mice were treated with clodronate- or PBS-containing liposomes (CLL or PBSL, respectively) 1 week prior to infection with WNV-NY. A. Evidence of depletion of F4/80⁺ and CD169⁺ macrophages in the ILN by clodronate liposomes immediately prior to infection. Samples were processed by multicolor flow cytometry. Depletion with clodronate-containing liposomes reduced the number of F4/80⁺ and CD169⁺ macrophages by 78 and 76%, respectively. B and C. Effect of clodronate or PBS liposomes on B-cell activation as judged by CD69 expression after WNV infection in the ILN (B) and spleen (C). Cells were processed 3 (ILN) or 4 (spleen) days after infection. D. Time course of appearance of WNV RNA in the draining LN as judged by fluorogenic quantitative reverse transcription-PCR. The data are expressed as Ct values, where a value of 40 is considered negative. E and F. Effect of clodronate on WNV-specific IgM (E) and IgG (F) titers at days 4 and 8 after infection. Data are expressed as reciprocal log endpoint titers after regression analysis and reflect at least eight mice per group. G. IgG subclass analysis after clodronate- or PBS-liposome treatment. Samples were from day 8 after infection. No statistically significant differences were observed. H. Relative neutralizing activity of serum from mice treated with clodronate or PBS liposomes at day 8 after WNV infection. Samples were analyzed using a previously described reporter virus particle assay on Raji-DC-SIGN-R cells (40). Data are expressed as the EC₅₀ titer (concentration at which 50% neutralization occurs) and reflect results for five mice per group. Asterisks represent statistically significant differences (**, P < 0.01; ***, P < 0.001).

FIG. 7.

Effects of LN macrophage depletion on WNV pathogenesis. Mice were treated with clodronate- or PBS-containing liposomes (CLL or PBSL, respectively) as described above and then infected with 10² PFU of WNV-NY. Animals were monitored for survival (n = 11 per group; P < 0.0001) (A) or viral burden as measured by plaque assay from serum at day 3 (B) or brain at day 8 (C) after infection. The dotted lines indicate the limit of sensitivity of the assay.