Toll-like receptor 7–induced naive human B-cell differentiation and immunoglobulin production

doi:10.1016/j.jaci.2008.09.018

Journal of Allergy and Clinical Immunology

Volume 123, Issue 1, January 2009, Pages 224-230.e4

https://doi.org/10.1016/j.jaci.2008.09.018 Get rights and content

Background

Toll-like receptors contribute to the establishment of adaptive immune responses.

Objective

The reported studies were conducted to examine the effects of the Toll-like receptor (TLR)–7 ligand, resiquimod, on human naive B-cell differentiation.

Methods

Naive human B cells were cultured with resiquimod in the presence or absence of IL-2 and IL-10. Secreted IgM and IgG were measured by ELISA, and IL-6, IL-10, and IFN-α were measured by a multiplex protein array. Cell proliferation was assessed by measuring [³H]thymidine uptake. mRNA for activation-induced cytidine deaminase and I_γ1-C_μ circle transcripts was measured by means of RT-PCR.

Results

Resiquimod induced the production of IgM and, to a lesser extent, IgG by naive human B cells in association with the secretion of IL-6 and IL-10, and a weak proliferative response. IL-2 and IL-10 synergized with resiquimod in markedly augmenting resiquimod-induced IgM and IgG production and proliferation. Resiquimod also stimulated production of IgG by B cells isolated from the blood of a patient with the X-linked hyper-IgM syndrome, with a greater response when these cells were costimulated with IL-2 and IL-10. The stimulated naive B cells from healthy volunteers displayed molecular evidence of immunoglobulin class-switch recombination—namely the appearance of activation-induced cytidine deaminase and I_γ1-C_μ circle transcripts.

Conclusion

Perturbation of TLR-7 on naive human B cells can lead to the induction of immunoglobulin class switch and IgG production in the absence of B-cell receptor cross-linking and CD40-CD40L interaction. The results are relevant to vaccine development and mechanisms by which microbial infection may lead to autoimmunity.

Section snippets

Isolation of human B-cell populations

PBMCs were separated on Ficoll-Hypaque density gradients from 60 mL blood obtained from healthy volunteers age 25 to 50 years and 1 patient with the X-linked hyper-IgM syndrome (kindly provided by Dr Kathleen Sullivan, Children's Hospital, Philadelphia, Pa). The PBMCs were suspended in cold wash buffer (1% FCS/1% NaN₃/PBS). After centrifugation at 4°C, the cell pellets were resuspended at 0.2 × 10⁶ cells/160 μL. After blocking nonspecific binding with mouse IgG, the cells were stained with the

Immunoglobulin production by naive CD19⁺CD27⁻ B cells

Addition of resiquimod to the naive CD19⁺CD27⁻ B cells (>98% pure by flow cytometry, data not shown) induced secretion of IgM (range, 82-1050 ng/mL; n = 5) and IgG (range, 14-158 ng/mL), whereas both isotypes were undetectable in the supernatants of unstimulated CD19⁺CD27⁻ B cells (Fig 1). Addition of IL-2 and IL-10 to naive B-cell cultures failed to stimulate IgM and IgG production. However, when added along with resiquimod, the cytokines demonstrated a synergistic effect on both IgM

Resiquimod-induced immunoglobulin production by human B cells

The results of this study are highlighted by the finding that resiquimod stimulated the differentiation of naive human B cells to immunoglobulin-secreting cells. This imidazoquinolone and oxidized guanosines are synthetic human TLR-7 ligands,19, 20 whereas the natural ligand for TLR-7 is single-stranded RNA.21, 22, 23 Interestingly, resiquimod induced the production of small amounts of IgG, in addition to IgM, by naive human B cells cultured in the absence of BCR cross-linking and addition of

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: Supported by the Immunology Research Fund, University of Pennsylvania School of Medicine.

: Disclosure of potential conflict of interest: A. I. Levinson has consulting arrangements with Talecris and Baxter, has received research support from Novartis and the National Institutes of Health, and has served as an expert witness in vaccine injury litigation. C. H. Pletcher has served as a member of the International Society for the Advancement of Cytometry and the Great Lakes Imaging and Flow Cytometry Association. The rest of the authors have declared that they have no conflict of interest.

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Mechanisms of allergy and clinical immunologyToll-like receptor 7–induced naive human B-cell differentiation and immunoglobulin production

Background

Objective

Methods

Results

Conclusion

Section snippets

Isolation of human B-cell populations

Immunoglobulin production by naive CD19+CD27− B cells

Resiquimod-induced immunoglobulin production by human B cells

Sem Immunol

Blood

Blood

Trends Immunol

Adv Immunol

J Allergy Clin Immunol

Toll-like receptors

Annu Rev Immunol

TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses

J Immunol

Control of B-cell responses by Toll-like receptors

Nature

Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors

Nature

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement

J Exp Med

Distinct response of human B cell subpopulations in recognition of an innate immune signal, CpG DNA

J Immunol

Maintenance of serological memory by polyclonal activation of human memory B cells

Science

CpG DNA induces IgG class switch DNA recombination by activating human B cells through an innate pathway that requires TLR9 and cooperates with IL-10

J Immunol

Plasmacytoid dendritic cells control TLR7 sensitivity of naïve B cells via type I IFN

J Immunol

Staphylococcus aureus Cowan I: potent stimulus of immunoglobulin M rheumatoid factor production

J Clin Invest

Dendritic cell ontogeny: a human dendritic cell lineage of myeloid origin

Proc Natl Acad Sci U S A

Mechanisms of allergy and clinical immunology
Toll-like receptor 7–induced naive human B-cell differentiation and immunoglobulin production

Immunoglobulin production by naive CD19⁺CD27⁻ B cells