Recombinase-mediated cassette exchange as a novel method to study somatic hypermutation in Ramos cells

Abstract

Activation-induced cytidine deaminase (AID) mediates the somatic hypermutation (SHM) of immunoglobulin (Ig) variable (V) regions that is required for the generation of antibody diversity and for the affinity maturation of the antibody response against infectious agents and toxic substances. AID preferentially targets WRC (W = A/T, R = A/G) hot spot motifs, particularly WGCW motifs that create overlapping hot spots on both strands. In order to gain a better understanding of the generation of antibody diversity and to create a platform for the in vitro generation of affinity-matured antibodies, we have established a system involving recombinase-mediated cassette exchange (RMCE) to replace the V region and its flanking sequences. This makes it possible to easily manipulate the sequence of the Ig gene within the endogenous heavy chain of the Ramos human Burkitt's lymphoma cell line. Here we show that the newly integrated wild-type (WT) VH regions introduced by RMCE undergo SHM similarly to non-RMCE-modified Ramos cells. Most importantly, we have shown that introducing a cluster of WGCW motifs into the complementary determining region 2 (CDR2) of the human heavy chain V region significantly raised the mutation frequency and number of mutations per sequence compared to WT controls. Thus, we have demonstrated a novel platform in Ramos cells whereby we can easily and quickly manipulate the endogenous human VH region to further explore the regulation and targeting of SHM. This platform will be useful for generating human antibodies with changes in affinity and specificity in vitro.

Importance: An effective immune response requires a highly diverse repertoire of affinity-matured antibodies. Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Although a great deal has been learned about the regulation of AID, it remains unclear how it is preferentially targeted to particular motifs, to certain locations within the Ig gene and not to other highly expressed genes in the germinal center B cell. This is an important question because AID is highly mutagenic and is sometimes mistargeted to other highly expressed genes, including proto-oncogenes, leading to B cell lymphomas. Here we describe how we utilize recombinase-mediated cassette exchange (RMCE) to modify the sequence of the endogenous heavy chain locus in the Ramos Burkitt's lymphoma cell line. This platform can be used to explore the regulation and targeting of SHM and to generate human antibodies with changes in affinity and specificity in vitro.

FIG 1

Schematic of the recombinase-mediated cassette exchange strategy. (A) Homologous recombination to create the Hyg-TK Ramos cell line. A schematic of the productive endogenous IgH allele includes the leader exon (L), the variable region (VDJ), Eμ, Sμ, and Cμ. The 5′ and 3′ homologous arms, the lox sites, and the location of the Hyg-TK gene are depicted in the targeting construct. (B) Southern blot of 5 independently isolated Hyg-TK RMCE clones and a control (lane C) IgM⁺ wild-type non-RMCE Ramos clone. DNA was digested with NotI and NsiI, shown in panel A, and hybridized with a Cμ probe depicted by the black diagonal line in panel A. (C) PCR analysis using genomic DNA of the same clones described in panel B using primers P1 and P2 shown in panel A.

FIG 2

Schematic of the Cre-mediated recombination to create the replaced IgH at the endogenous locus. The pentagon represents the addition of Cre, and the arrows demonstrate the transfection of WT A to create the primary replacement and the transfection of WT B to create the secondary replacement. The asterisk indicates the C-to-G mutation within VDJ of WT B.

FIG 3

Insertion of a cluster of hot spots into the endogenous heavy chain gene of Ramos cells increases V region mutation. (A) Schematic of the replaced IgH locus with primer pairs used for amplification and sequencing indicated by arrows (P3 to P8). (B) Relative AID transcript levels in the various subclones shown in the bar graph with standard deviations. (C) A bar graph showing the frequencies of mutations depicted in Table 1 from the promoter, V, and constant regions. Error bars represent the standard deviations between 2 clones. (D) Pie charts representing the number of mutations per sequence; the number of sequences analyzed is depicted in the center of each circle.

FIG 4

Distribution of mutations in WT and HS clones. Distribution of total mutations in the 458-bp VDJ region of HS A and B clones compared to WT A. The red line indicates the boundary of the hot spot cluster. Some sequences were dominated by a single highly mutated site which was removed from the analysis and replaced by the blue line in order to display the results on the same y axis scale. The black boxes represent the WGCW motifs. In order to increase the number of mutations examined, additional sequences from WT A and HS A and B subclones not shown in the previous figures and Table 1 were added to this figure.