A fundamental defect in CVID is a block in B cell maturation, inhibiting immunoglobulin production and the development of plasma cells. Recent efforts have focused on elucidating the molecular defects that prevent normal heavy-chain isotype class-switching and somatic hypermutation in CVID. With the exceptions of autosomal recessive defects in a minority of subjects1,3
and the association with selected mutations in transmembrane activator and calcium ligand interactor,4–6
the pathogenesis of the CVID syndrome in most cases remains unknown. TLR activation leads to terminal B cell differentiation and immunoglobulin production41–43
even in the absence of antigen exposure,44,45
but we had found that CpG-ODN failed to activate TLR9 signaling in CVID B cells or promote IL-10 and IL-6 secretion. In the current studies, we find that functions of the related endosomal receptor, TLR7, on B cells is even more impaired, leading to poor B cell proliferation, lack of maturation, isotype switch, and IgG and IgA production. Naive B cells express lower levels of TLRs, whereas memory B cells constitutively express TLR7 and 9 at higher levels.11,20,38,43
The somewhat smaller proportion of circulating memory CD27+
B cells in CVID could account for the reduced TLR responses in CVID; however, we show that even isolated CD27+
CVID B cells have severely defective responses compared with CD27+
B cells of control subjects. These data suggest that intrinsic defects of TLR activation either accompany or lead to ineffective generation of antibody-producing isotype-switched B cells in CVID, whereas other TLR-mediated responses remain intact. Furthermore, these defects are not restricted to B cells because CVID pDCs produce little IFN-α on TLR7 and TLR929
Because TLR7 and TLR9 signaling pathways are markedly defective in CVID, we suggest that 1 or more effector molecules common to these related activation pathways might be abnormal. TLR signaling requires an array of adapter molecules including myeloid differentiation factor (MyD88), myeloid differentiation factor adapter-like protein, Toll and IL-1 receptor domain–continuing adapter-inducing IFN-β (TRIF), and TRIF adaptor molecule. The potential importance of TLR function in B cell responses has been suggested by the reduction or elimination of T-dependent antibody responses in MyD88 knockout mice.46
Functional differences and variable expression patterns of TLRs on different cell types have been important in dissecting mutations of the genes encoding components of the human TLR pathways, including nuclear factor-κB (NF-κB) essential modulator (NEMO/IKKγ), IL-1 receptor-associated kinase 4 (IRAK-4), inhibitor of NF-κB, alpha (IκBα), MyD88, UNC93B, and TLR3.47–52
Mutations in these genes result in discrete immune defects that predispose the patient to selected bacterial, viral, and/or myco-bacterial infections. Because CVID PBMCs had normal TNF-α, IL-12, and IL-6 production, the intracellular signaling proteins such as MyD88 or NEMO/IKKγ that mediate NF-κB activation by multiple TLRs were more likely to be preserved. However, even here, exceptions occur: although NEMO mutation classically manifests with defects in production of TNF-α and IL-6,48
Salt et al52
identified 1 case with spared inflammatory cytokine production. The defects in CVID are somewhat similar to those found in subjects with mutations in the endoplasmic membrane protein UNC93B; the PBMCs of these patients also have impaired production of IFN-α on TLR7, TLR8, and TLR9 signaling but normal production of both TNF-α and IL-6.49
However, in contrast with subjects with mutations in UNC93B, severe herpes simplex or other systemic viral infections are not common in CVID, possibly because the TLR3 pathway in CVID appears to be spared. We show that TLR3 activation of CVID PBMCs and fibroblasts leads to normal production of IFN-α and IFN-β, potentially permitting adequate viral protection. Although TLR7, TLR8, and TLR9 signal via the common adaptor protein MyD88, TLR3 signals via an MyD88-independent pathway involving TRIF and interferon response factor (IRF3).14
These differences may aid in further dissecting the TLR defects in CVID. Considering the endocytic location of these receptors,14
it is also possible that the intracellular trafficking, delivery, and/or processing of TLR ligands may be impaired in CVID.
A critical link between pDC and normal B cell function is the production of IFN-α. Similar to B cells, pDCs express high levels of TLR7 and TLR9; viral or ligand stimulation of these receptors normally results in the secretion of large amounts of type 1 IFNs. Although IFN-α is generally viewed as an antiviral or antiproliferative cytokine,53
IFN-α enhances B cell sensitivity to TLR7/8-induced activation, primes B cells by enhancing responsiveness to BCR cross-linking, and further augments TLR-mediated isotype switch and immunoglobulin secretion.32,54–56
Because we found that CVID PBMCs and pDCs produce little or no IFN-α when exposed to these TLR ligands, we tested whether adding IFN-α to TLR7-stimulated cell cultures might restore some facets of B cell function. In fact, IFN-α enhanced TLR7-induced CVID naive and, in particular, memory B cell proliferation in most subjects and increased the percentage of isotype-switched memory B cells in these cultures. On the other hand, restoration of IFN-α alone is not likely to improve IgG and IgA production in vitro
because normal B cells triggered by TLR7 require additional cytokines, such as IL-2 and IL-10, to produce immunoglobulin.26
However, these data suggest that the loss of IFN-α is an important component of this immune deficiency phenotype.
We demonstrate here that the CVID phenotype includes a selected impairment of responses of TLR7 and TLR9, the predominant TLRs expressed in pDCs and B cells. These defects include loss of cell activation, proliferation, and cytokine production by B cells and pDCs; as a result, CVID B cells forfeit the reinforcement that environmental TLR ligands provide normal human B cells. Our data suggests that the defects in TLR signaling in CVID could play a substantial role in the pathogenesis of this immunodeficiency.
- Functional defects in TLR7 and 9 signaling in CVID lead to impaired B cell proliferation and differentiation and subsequent loss of switched memory B cells.
- Common variable immunodeficiency plasmacytoid dendritic cells produce little IFN-α, but reconstitution of this cytokine in TLR7-stimulated B cell cultures can induce proliferation, CD27 expression, and, in some cases, isotype switch.