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CD19 controls Toll-like receptor 9 responses in human B cells
Henner Morbach1, Jean-Nicolas Schickel1, Charlotte Cunningham-Rundles2
1Department of Immunobiology, Yale University School of Medicine, New Haven, Conn.
Insights
CD19 is crucial for Toll-like receptor 9 (TLR9) function in human B cells. This molecule integrates B-cell receptor (BCR) and TLR9 signaling, impacting immune responses and potentially common variable immunodeficiency.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- CD19 is a B cell-specific costimulatory molecule vital for B-cell receptor (BCR) responses.
- Mutations in the CD19 gene are linked to common variable immunodeficiency (CVID) in humans.
- B-cell responses mediated by BCR and Toll-like receptor 9 (TLR9) are often impaired in CVID patients.
Purpose of the Study:
- To investigate the necessity of CD19 for TLR9 function in human B cells.
- To elucidate the role of CD19 in integrating BCR and TLR9 signaling pathways.
Main Methods:
- Flow cytometry was used to assess B cell activation markers (CD86, TACI, CD23) after anti-IgM or CpG stimulation.
- Phospho flow cytometry, immunoblotting, and co-immunoprecipitation analyzed signaling molecule phosphorylation and interactions.
- Experiments utilized B cells from patients with CD19 gene defects and a B-cell line with CD19 knockdown.
Main Results:
- B cells with defective CD19 alleles exhibited impaired upregulation of activation markers following TLR9 stimulation.
- TLR9 stimulation normally induces CD19 phosphorylation via MYD88/PYK2/LYN complexes, facilitating PI3K recruitment and BTK/AKT phosphorylation.
- Inhibition or deficiency of PI3K, AKT, or BTK mimicked TLR9 activation defects seen in CD19-deficient cells.
Conclusions:
- CD19 is essential for TLR9-induced B-cell activation.
- The CD19/PI3K/AKT/BTK axis is a critical pathway for integrating BCR and TLR9 signaling in human B cells.
Background:
CD19 is a B cell-specific molecule that serves as a major costimulatory molecule for amplifying B-cell receptor (BCR) responses. Biallelic CD19 gene mutations cause common variable immunodeficiency in human subjects. BCR- and Toll-like receptor (TLR) 9-induced B-cell responses are impaired in most patients with common variable immunodeficiency.
Objective:
We sought to analyze whether CD19 is required for TLR9 function in human B cells.
Methods:
Expression of surface activation markers was assessed after anti-IgM or CpG stimulation by using flow cytometry on B cells from patients with 1 or 2 defective CD19 alleles, which decrease or abrogate CD19 expression, respectively. The phosphorylation or interaction of signaling molecules was analyzed by using phospho flow cytometry, immunoblotting, or co-immunoprecipitation in CD19-deficient or control B cells and in a B-cell line in which CD19 has been knocked down with lentivirus-transduced short hairpin RNA.
Results:
B cells from subjects with 1 or 2 defective CD19 alleles showed defective upregulation in vitro of CD86, transmembrane activator and CAML interactor (TACI), and CD23 activation markers after TLR9 stimulation. TLR9 ligands normally induce phosphorylation of CD19 through myeloid differentiation primary response gene-88 (MYD88)/proline-rich tyrosine kinase 2 (PYK2)/LYN complexes, which allows recruitment of phosphoinositide 3-kinase (PI3K) and phosphorylation of Bruton tyrosine kinase (BTK) and AKT in human B cells with a different kinetic than that of BCRs. In addition, inhibition of PI3K, AKT, or BTK, as well as BTK deficiency, also resulted in TLR9 activation defects in B cells similar to those in patients with CD19 deficiency.
Conclusion:
CD19 is required for TLR9-induced B-cell activation. Hence CD19/PI3K/AKT/BTK is an essential axis integrating BCRs and TLR9 signaling in human B cells.
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