Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses

Abstract

B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.

Figure 1. pDCs mediate human B cell activation and plasmablast induction by RV in vitro.

(A) The necessity of pDCs for the induction of CD69 expression on human B cells within indicated PBMC preparations was assessed by flow cytometry 12 hours following mock (black) or RV stimulus. Blue, total PBMCs; green, pDC-depleted PBMCs; red, reconstituted pDC-depleted PBMCs. One experiment representative of 3 donors is shown. (B) The sufficiency of pDCs to induce B cell activation by RV was assessed by flow cytometry for expression of CD69 by purified human B cells 12 hours following mock (white) or RV (black) stimulus in the presence or absence of pDCs, as indicated. **P = 0.008, Wilcoxon signed rank test; n = 9. (C) The necessity of primary human pDCs to induce plasmablasts (CD3^-CD19⁺CD20^loCD38⁺ cells) was assessed by flow cytometry 4 and 5 days after the indicated stimulus. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, repeated measures ANOVA with Neuman-Keuls multiple comparison test; n = 3. (D) The percentage of B cells expressing extracellular IgA at the indicated times after mock (white) or RV (black) stimulus. *P < 0.05, paired t test; n = 3.

Figure 2. pDCs mediate human B cell activation by soluble type I IFN.

(A) B cell activation was assessed by flow cytometry analysis of CD69 expression by B cells cultured for 12 hours in the presence (+) or absence (–) of pDCs or RV as indicated. Cells were cocultured, allowing pDC/B cell contact (white), or with pDCs plated within Transwell inserts (black). *P < 0.05, Mann-Whitney; n = 3–4. (B) Representative histograms of CD69 expression by purified human B cells 12 hours after stimulus with 1:20 dilutions of supernatants from overnight cultures of pDCs exposed to mock (red) or RV (blue) stimulus. One experiment representative of 10 is presented. (C) CD69 expression was assessed by flow cytometry following overnight stimulus of purified B cells with supernatants from mock or RV-stimulated pDCs in the presence of the indicated neutralizing antibodies (αIFN-α, αIFN-β or αIFN-α, αIFN-β, and αIFN-receptor, indicated as “type I IFN”). The resulting increase in B cell activation is expressed as a percentage of that observed with the appropriate isotype control. **P = 0.002 vs. isotype, αIFN-α and αIFN-β, repeated measures ANOVA with Tukey’s multiple comparison test; n = 3. (D) CD69 expression by purified B cells following overnight stimulus with mock-stimulated pDC supernatant (black), RV-stimulated pDC supernatant (green), RV-stimulated pDCs (blue), or IFN-α (red). Data depict 1 donor and are representative of 6 experiments.

Figure 3. Identification of IFN-α⁺ cells following RV infection of total PBMCs.

(A) Intracellular staining for IFN-α was performed on total PBMCs 6 or 12 hours following RV infection. IFN-α was only observed in viable lineage HLA-DR⁺ cells, as indicated. Within this population, IFN-α staining was uniquely detectable within the pDC (CD123⁺CD11c^–), but not myeloid DC (CD123^–CD11c⁺), population. Similar results are observed with IFN-β; n = 3–4. (B and C) Percentage IFN-α–positive (B) or IFN-β–positive (C) cells in the indicated populations; n = 3.

Figure 4. pDCs and type I IFN signaling contribute to in vivo B cell activation following murine RV infection.

(A and B) Percentage of CD69⁺ B cells in the Peyer’s patches (A) or MLN (B) of C57BL/6 mice depleted of pDCs or treated with isotype control 3 days after mock (white) or virulent murine RV (black) infection. (C and D) B cell activation in the Peyer’s patches (C) and MLN (D) of wild-type SV129 or IFN-R knockout mice, 4 dpi. *P = 0.03; **P = 0.004; ***P = 0.0001, t test; n = 5 per group.

Figure 5. pDC-deficiency impairs in vivo serum antibody responses to RV.

Serum RV–specific IgG (A), IgA (B), and IgM (C) were assessed by ELISA in isotype control (white) and PDCA-1–depleted (black) mice at the indicated dpi. *P ≤ 0.05; ***P ≤ 0.001, t test; n = 5 per group.

Figure 6. pDC deficiency impairs in vivo mucosal IgA responses to RV.

Fecal IgA was assessed by ELISA in (A) isotype control (black), anti-PDCA-1–treated (white), or anti-SiglecH–treated (gray) CD8 knockout or (B) Wild-type (black) or BDCA2-DTR (white) mice at the indicated dpi. *P ≤ 0.05; **P ≤ 0.01 ***P ≤ 0.001, Mann-Whitney; n = 3–5 per group.

Figure 7. The defective pDC response enhances rotaviral shedding.

(A) Fecal shedding of murine RV was assessed by ELISA in isotype control (black), anti-PDCA-1–treated (green) or anti-SiglecH–treated (orange) CD8 knockout mice at the indicated dpi. *P < 0.05; ***P < 0.001, Mann-Whitney; n = 5 per group. (B) Fecal shedding of murine RV 3 dpi in wild-type (black) or BDCA2-DTR (white) mice; Mann-Whitney.

Figure 8. Model of B cell activation in response to RV.

Immature pDCs sense RV in vivo and in vitro, leading to type I IFN production by pDCs. pDC-derived type I IFN activates naive B cells, ultimately leading to the production of RV-specific IgA in the intestines.