Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• High-affinity antibodies are crucial for adaptive immunity.
• Somatic hypermutation (SHM) introduces mutations in immunoglobulin (Ig) variable (V) regions to enhance antibody affinity.
• The precise mechanisms and DNA polymerases involved in SHM, particularly at V-region hotspots, remain incompletely understood.

Purpose of the Study:

• To investigate the role of DNA polymerases iota, eta, and zeta in somatic hypermutation (SHM).
• To determine the expression patterns of these polymerases in response to stimuli mimicking in vivo SHM conditions.
• To assess the mutational fidelity of DNA polymerase iota at specific DNA sequences and sequence contexts.

Main Methods:

• Cultured Burkitt's lymphoma (BL)2 cells were used to measure mRNA expression of DNA polymerases iota, eta, and zeta.
• Cells were co-stimulated with T cells and IgM crosslinking to mimic in vivo SHM conditions.
• DNA polymerase iota fidelity was assessed at RGYW hotspots and non-hotspots under various DNA damage conditions (nicks, gaps, double-strand breaks).

Main Results:

• Co-stimulation with T cells and IgM crosslinking induced a 4-fold increase in pol iota mRNA within 12 hours.
• Pol iota exhibited a 3-fold higher error rate at hotspots compared to non-hotspots, forming T x G mispairs consistent with C to T transitions.
• While pols iota, eta, and zeta were present under all conditions, SHM induction required both T cell and IgM stimuli, indicating polymerase presence alone is insufficient.

Conclusions:

• T cell and IgM crosslinking-dependent induction of pol iota suggests a potential SHM triggering event.
• DNA polymerase iota plays a role in generating mutations at SHM hotspots, contributing to C to T transitions.
• The coordinated action of T cell and IgM signals is essential for initiating SHM, highlighting the complexity of this immune process.