B-cell fate decisions following influenza virus infection

Abstract

Rapidly induced, specific Ab generated in extrafollicular foci are important components of early immune protection to influenza virus. The signal(s) that prompt B cells to participate in extrafollicular rather than germinal center responses are incompletely understood. To study the regulation of early B-cell differentiation events following influenza infection, we exploited earlier findings of a strong contribution of C12 idiotype-expressing B cells to the primary HA-specific response against influenza A/PR/8/34. Using an idiotype-specific mAb to C12 and labeled HA, in conjunction with multicolor flow cytometry, we followed the fate of C12Id-expressing influenza HA-specific B cells in WT BALB/c mice, requiring neither genetic manipulation nor adoptive cell transfer. Our studies demonstrate that HA-specific C12Id(+) B cells are phenotypically indistinguishable from follicular B cells. While they induced both extrafollicular and germinal center responses, extrafollicular responses were strongly predominant. Provision of increased HA-specific T-cell help increased the magnitude of the extrafollicular response, but did not shift the C12Id(+) response toward germinal center formation. Collectively the data are consistent with the hypothesis that B-cell fate determination following activation is a stochastic process in which infection-induced innate signals might drive the preferential expansion of the early extrafollicular response.

Figure 1. T-dependent rapid antibody responses encoded by C12Id are generated predominately in the regional lymph nodes following influenza infection

(A) Shown are mean ± SD relative units Ig/ml serum of virus-specific C12Id⁺ (left panel, closed squares) and total antiviral (right panel, open circles) antibodies as determined by ELISA of four individual mice bled at indicated time-points before and after infection with influenza virus A/PR8. Relative units were calculated by comparison to binding of a virus-specific C12Id-expressing and non-C12Id-expressing mAb, respectively. (B) Shown are mean frequencies of virus-specific C12Id⁺ (upper panel) and virus-specific total Ig-secreting cells as determined by ELISPOT assays set up in duplicate of mediastinal lymph nodes (MedLN), lung tissue, and spleen from individual mice (n = 4) at indicated time points before and after infection. (C) Sera from groups (n = 4) of BALB/c (striped bars) and T cell-deficient (nu/nu) (filled bars) mice were taken before and at the indicated times after intranasal infection with influenza A/PR8. Virus-specific C12Id⁺ antibody levels were determined by ELISA and expressed as relative units (mean ± SD) calculated by comparison to a standard hyperimmune serum.

Figure 2. C12Id⁺ lymph node B cells display a follicular B cell phenotype

(A) Nine-color FACS analysis was conducted on pooled peripheral (PLN) and MedLN of non-infected BALB/c mice to determine the phenotype of C12Id⁺ lymph node B cells. Shown are 5% contour plots and histograms of CD19⁺ B cells following exclusion of CD3⁺ auto-fluorescent and dead cells. Numbers indicate frequencies of cells in the depicted gates. MedLN (grey filled) and PLN (solid line, no fill) B cells were further separated into C12Id⁺ (upper panel) and C12Id^- (lower panel), and expression of CD21, CD23, CD1 and CD9 was determined. Splenic CD19⁺ CD23^low/-, CD21^hi marginal zone (MZ) B cells (dashed line, no fill) served as controls. (B) Activation marker expression on C12Id expressing and non-expressing B cells was determined on MedLN B cells at day 4 of influenza virus A/PR8 infection. Shown are 5% contour plots to depict staining of C12Id (top) and overlay histogram profiles (bottom) of C12Id⁺ (upper) and C12Id^- (lower panels) B cells (solid line, filled histograms) identified as shown in (A). Expression levels of indicated markers were compared to that of splenic CD19⁺ CD21^hi CD23^low/- marginal zone B cells (dashed line, no fill) and PLN (solid line, no fill) B cells from uninfected mice.

Figure 3. C12Id⁺ B cells generate strong extrafollicular foci responses

(A) Immunohistochemistry (left) and double immunofluorescence (right) was done to identify C12Id antibody-expressing cells in MedLN collected from BALB/c mice on day 10 after infection with influenza A/PR8. Immunohistochemistry identifies the accumulation of C12Id⁺ cells (red, arrows) in the medullary cords (M) but not in germinal centers (GC) (Bar = 50 μm). Double immunofluorescence shows double-positive C12Id⁺ (green cytoplasmic staining) and CD138+ positive (red staining and arrows) cells in the medulla (M) of the MedLN (Bar = 20 μm). (B) Shown are pseudocolor FACS plots depicting CD45R (B220) and C12Id-expression on MedLN B cells collected at indicated times after infection, after gating on live (propidium iodide^neg), non-dump (CD3/4/8/F4/80^neg) CD19⁺ CD45R^+/low cells (not shown). Scatter plot shows frequencies of C12Id⁺ (triangle) and C12Id^- (square) cells among CD45R^low lymph node (LN) B cells from BALB/c mice before and after infection (n = 3 - 4). Distal lymph nodes are PLN (inguinal and axillary) from day 7 and 14-infected mice. Each symbol represents data from an individual mouse. Horizontal line indicates the data mean. (C) Analysis of CD138 expression of HA-(A/PR8) specific C12Id⁺ plasmablasts in MedLN of mice infected for 7 days with influenza A/PR8 and treated in vivo with 0.25mg Brefeldin A for 6h prior to tissue collection. Shown are pseudo-color plots from a representative 5-color flow cytometric analysis of CD19⁺ C12Id⁺ B cells, identified by lack of expression for CD3, 4, 8, and F4/80 and expression of CD19 and C12Id (not shown). Virus-specific cells identified by binding to HA showed reduced expression of CD45R (B220) (upper panel, box). A subset of these cells was CD138⁺ (syndecan; lower panel). Number Indicates mean frequencies ± SD CD138⁺ cells among C12Id⁺ HA⁺ B cells from 4 individual mice. (D) Shown are pseudo-color FACS plots for gating of CD19⁺ (left panel) surface and intracytoplasmic HA-A/PR8 binding (s+ icp) (middle panel) cells that express the C12Id (right panel) in MedLN of 7 day influenza virus A/PR8-infected mice. Number in right panel indicates the mean frequency ± SD of C12Id⁺ HA-specific cells analyzed in MedLN of four individual mice.

Figure 4. C12Id⁺ B cells can form germinal centers

(A) Analysis of germinal center formation among C12Id⁺ and C12Id- MedLNs of mice infected for 140 days with influenza A/PR8. Shown are 5% contour plots with outliers including an unstained control (left panels) from a representative 7-color flow cytometric analysis of C12Id⁺ and C12Id^- live B cells identified by lack of expression for CD3, 4, 8 and F4/80, expression of CD19 and high expression of CD45R (not shown). Germinal center B cells among C12Id⁺ and C12Id^- cells were identified as CD38^low CD24^high (boxed, right panels). Numbers indicate frequencies of cells in the depicted gates. Each plot on the right represents results from one of 4 mice per time point. Results are summarized in (B). Scatter plots show frequencies of germinal center cells among all (left panel), C12Id^- (middle) and C12Id⁺ (right) B cells. Frequencies of germinal center B cells in peripheral (distal) lymph nodes taken from day 7 and 14 infected mice are shown for comparison. Each symbol represents data from an individual mouse. Mean values for each time point are indicated by a horizontal line.

Figure 5. CD4 T cells enhance the magnitude but not the quality of the C12Id⁺ response

(A) Shown is a scatter plot indicating frequencies of virus-specific C12Id⁺ antibody-secreting cells in MedLN of day 7 influenza virus A/PR8-infected BALB/c mice that received 2.3 × 10⁶ CD4 T cells from either wild type (closed squares) or TCR-transgenic TS-1 mice (open circles), or PBS alone (control, stars) 12h prior to infection as assed by ELISPOT. (B) Shown is the FACS-gating strategy to determine the frequencies of C12Id⁺ plasma blasts in MedLN of mice as in (A). Live MedLN B cells were identified by lymphocyte FSC/SSC (not shown) lack of expression of CD3, 4, 8, F4/80 (dump), and expression of CD19. Cells were further gated on staining for CD45R (B220) and binding to HA-A/PR8 (surface and intracytoplasmic (s + icp)). Virus-specific C12Id⁺ plasmablasts were identified as B220^lo HA⁺ surface and/or intracytoplasmic (s+icp) C12Id positive. Numbers indicate frequencies of cells in the depicted gates. Results are summarized in the scatter plot below. Each symbol represents results from one animal. Horizontal lines indicate mean frequencies. (C) Shown is the FACS-gating strategy to determine frequencies of C12Id⁺ germinal center B cells in MedLN from individual mice of groups similar to those shown for A and B, expect that controls did not receive PBS prior to infection. HA-specific C12Id⁺ germinal center B cells were identified as live, non-dump, CD19⁺ (left panel), HA⁺ CD45R (B220)^high (second from left), CD24⁺ CD38^lo (third) and C12Id⁺ (s+icp, right panel). FACS analysis gates were set based on control stains that lacked only the one marker of interest (shown for HA-stain and data not shown). Numbers indicate frequencies of cells in the depicted gates. Scatter plots summarize the results. Each symbol represents data from individual animals and horizontal line shows mean of the group. Results are from one of two independent experiments that yielded similar results. Statistical significance was calculated using the Student's t test.