Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.

Affinity maturation of the humoral immune response is caused by single base changes that are introduced into the V regions of the Ig genes during a brief period of B cell differentiation. It has recently become possible to study V region mutation in some human Burkitt's lymphoma cell lines that mutate their V regions and express surface markers that suggest they arose from the malignant transformation of germinal center B cells. Ramos Burkitt's cells constitutively mutate their V regions at a rate of approximately 2 x 10(-5) mutations/bp/generation. However, the sequencing of unselected V regions suggested that our Ramos cell line was progressively losing its ability to undergo V region hypermutation. To accurately quantify this process, subclones with different nonsense mutations in the mu heavy chain V region were identified. Reversion analysis and sequencing of unselected V regions were used to examine the clonal stability of V region hypermutation. Even after only 1 month in culture, stable and unstable subclones could be identified. The identification of mutating and non-mutating subclones of Ramos provided a unique opportunity to identify factors involved in the mutational process. Differential gene expression between mutating and non-mutating Ramos clones was examined by RT-PCR and cDNA microarray analyses. We found that the expression of activation-induced cytidine deaminase (AID), a putative cytidine deaminase, correlated with mutation rates in Ramos subclones. These results suggest that the hypermutation phenotype is inherently unstable in Ramos and that long culture periods favor outgrowth of non-mutating cells that express lower levels of AID.

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

High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10(-5)--10(-3) per base pair per generation, about 10(6)-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases iota, eta, and zeta by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5-10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumed in vivo requirements for SHM. An approximately 4-fold increase pol iota mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols eta and zeta occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol iota was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol iota formed T x G mispairs at a frequency of 10(-2), consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol iota at 12 h may indicate an SHM "triggering" event has occurred. However, pols iota, eta, and zeta are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.