Toll-like receptor 7 and 9 defects in common variable immunodeficiency

Abstract

Background: Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia, reduced numbers of peripheral blood isotype-switched memory B cells, and loss of plasma cells.

Objective: Because Toll-like receptor (TLR) activation of B cells can initiate and potentially sustain normal B cell functions, we examined functional outcomes of TLR7 and TLR9 signaling in CVID B cells.

Methods: TLR7-mediated, TLR7/8-mediated, and TLR9-mediated cell proliferation, isotype switch, and immunoglobulin production by control and CVID B cells or isolated naive and memory B cell subsets were examined. We quantitated TNF-alpha, IL-6, and IL-12 production in response to TLR1-9 ligands and measured IFN-alpha production by TLR7-stimulated PBMCs and isolated plasmacytoid dendritic cells (pDCs). IFN-beta mRNA expression by TLR3-stimulated fibroblasts was assessed.

Results: Unlike CD27(+) B cells of controls, TLR7-activated, TLR7/8-activated, or TLR9-activated CVID B cells or isolated CD27(+) B cells did not proliferate, upregulate CD27, or shed surface IgD. TLR-stimulated CVID B cells failed to upregulate activation-induced cytosine deaminase mRNA or produce IgG and IgA. TLR7-stimulated PBMCs and pDCs produced little or no IFN-alpha. Reconstituting IFN-alpha in TLR7-stimulated CVID B-cell cultures facilitated proliferation, CD27 upregulation, and isotype switch. These TLR defects are restricted because CVID PBMCs stimulated with TLR ligands produced normal amounts of TNF-alpha, IL-6, and IL-12; TLR3-mediated expression of IFN-beta by CVID fibroblasts was normal.

Conclusion: Defective TLR7 and TLR9 signaling in CVID B cells and pDCs, coupled with deficient IFN-alpha, impairs CVID B cell functions and prevents TLR-mediated augmentation of humoral immunity in vivo.

FIG 1

Impaired CVID CD27⁺ B cell proliferation and differentiation. Isolated CD27⁺ and CD27⁻ CVID or control B cells were cultured with (Loxoribine) or without (No Stim) 500 mmol/L loxoribine for 6 days. CSFE dye dilution and cell surface expression of CD19 (top panel), CD27 (middle panel), and IgD (bottom panel) were tracked with each cell division. These data are representative of 12 subjects with CVID and 7 control subjects.

FIG 2

A, Deficient AID expression in TLR-stimulated CVID B cells. AID expression by real-time PCR in isolated total B cells from subjects with CVID (n = 10–13) and control subjects (n = 11) after 72 hours of stimulation with ODN2006, loxoribine, or CL097. AID mRNA copy number was expressed as copy number per microgram of RNA normalized to β-actin. B, AID expression in TLR9-stimulated CD27⁻ and CD27⁺ B cells. AID expression by RT-PCR in isolated CD27⁺ and CD27⁻ B cells from subjects with CVID (n = 6) and control subjects (n = 5) after 72 hours of stimulation with ODN2006. AID mRNA was analyzed by agarose gel electrophoresis in comparison with β-actin. One representative subject with CVID and a control subject are depicted.

FIG 3

Impaired TLR-induced IgG and IgA production by CVID B cells. CVID or control PBMCs were stimulated with ODN2006 (11 controls, 24 CVID), control ODN2137 (data not shown), loxoribine (11 controls, 24 CVID), or CL097 (6 controls, 16 CVID). IgG and IgA production was assessed by ELISA after 13 days.

FIG 4

A, Impaired production of IFN-α by TLR7-stimulated CVID PBMCs. CVID (n = 14–24) and control (n = 9–10) PBMCs were cultured with 10 μmol/L, 300 μmol/L, or 500 μmol/L loxoribine for 24 hours. B, Impaired production of IFN-α by TLR7-stimulated CVID pDCs. Isolated CVID (n = 27–34) and control (n = 20) pDCs were stimulated with 10 μmol/L, 300 μmol/L, or 500 μmol/L loxoribine for 48 hours.

FIG 5

TLR3-mediated IFN-βmRNA production by CVID fibroblasts is spared. Fibroblasts from subjects with CVID (n = 4) and control subjects (n = 2) were either untreated (−) or stimulated (+) with poly(I:C) for 24 hours. IFN-β mRNA was analyzed by agarose gel electrophoresis in comparison with β-actin. Data from 2 subjects with CVID and a representative control are shown.

FIG 6

A, Addition of IFN-α amplifies TLR7-stimulated proliferation and differentiation of CD27⁺ and CD27⁻ B cells. Isolated CD27⁺ and CD27⁻ CVID or control B cells were stimulated with 500 μmol/L loxoribine in the presence or absence of 1000 U/mL IFN-α for 6 days. CSFE dye dilution and cell surface expression of CD19, CD27, and IgD were tracked with each cell division. These data are representative of 12 subjects with CVID and 7 control subjects. B, TLR7-mediated isotype switching can be augmented in the presence of IFN-α. Total CD19⁺ B cells were stimulated with 500 μmol/L loxoribine in the presence or absence of IFN-α for 6 days. CD27 and IgD surface expression was examined by flow cytometry to determine the percentage of isotype-switched CD27⁺IgD⁻ B cells (lower right quadrant of the inset contour plot).