Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting

Abstract

Germinal centers (GCs) represent the main sites for the generation of high-affinity, class-switched antibodies during T cell-dependent antibody responses. To study gene function specifically in GC B cells, we generated Cgamma1-cre mice in which the expression of Cre recombinase is induced by transcription of the Ig gamma1 constant region gene segment (Cgamma1). In these mice, Cre-mediated recombination at the fas, Igbeta, IgH, and Rosa26 loci occurred in GC B cells as early as 4 days after immunization with T cell-dependent antigens and involved >85% of GC B cells at the peak of the GC reaction. Less than 2% of IgM(+) B cells showed Cre-mediated recombination. These cells carried few Ig somatic mutations, expressed germ-line Cgamma1- and activation-induced cytidine deaminase-specific transcripts and likely include GC B cell founders and/or plasma cell precursors. Cre-mediated recombination involved most IgG1, but also a fraction of IgG3-, IgG2a-, IgG2b-, and IgA-expressing GC and post-GC B cells. This result indicates that a GC B cell can transcribe more than one downstream C(H) gene before undergoing class switch recombination. The efficient induction of Cre expression in GC B cells makes the Cgamma1-cre allele a powerful tool for the genetic analysis of these cells, as well as, in combination with a suitable marker for Cre-mediated recombination, the tracking of class-switched memory B and plasma cells in vivo. To expedite the genetic analysis of GC B cells, we have established Cgamma1-cre F(1) embryonic stem cells, allowing further rounds of gene targeting and the cloning of compound mutants by tetraploid embryo complementation.

Fig. 1.

Knockin of the Cre transgene into the Cγ1 locus. (a) Strategy to insert the IRES-Cre cassette into the mouse Cγ1 locus. The targeted locus is depicted before and after removal by FLP recombinase of the frt-flanked selection marker. BamHI (B) sites within the targeted genomic region are indicated. (b) Southern blot analysis, using probe a, of BamHI-digested tail DNA from WT (lane 1) and Cγ1-cre heterozygous mice, before (lane 2) and after (lane 3) removal of the selection marker.

Fig. 2.

Cre-mediated recombination in B cell subsets of Cγ1-cre mice. (a) Status of the rag-2 allele. wt, wild type; fl, floxed; Δ, deleted) in IgM⁺ and IgG1⁺ B cells sorted from cultures of Cγ1-cre; Rag-2^fl/+ B cells. (b) Percentage of EYFP⁺ cells within gated splenic B cell subsets of Cγ1-cre/+; R26-EYFP mice. (c) Percentage of gated IgG1⁺EYFP⁺ within the GC B (IgG1^loCD38^lo; Upper) and memory B (IgG1⁺CD38⁺; Lower) cell subsets of Cγ1-cre/+; R26-EYFP and Cγ1-cre/+ control mice. EYFP gates were set based on controls. (d) Immunofluorescence analysis of SPL sections from day-10 SRBC-immunized Cγ1-cre/+; R26-EYFP (Upper) and R26-EYFP control animals (Lower) stained as indicated. Results shown in b–d are representative of at least four independent experiments. Experiments shown in a were repeated twice.

Fig. 3.

Early induction of Cre-mediated recombination in GC responses of Cγ1-cre mice. (a) Percentage of EGFP⁺ cells among gated splenic PNA^hiFas^hi GC B cells at different time points after NP-CG immunization. Two representative Cγ1-cre; Igβ^fl-EGFP mice are shown. (b) NP- and allotype–specific serum IgG1 titers measured >28 days after NP-CG immunization of Cγ1-cre heterozygous (○) and CB6F1 (•) control mice; bars indicate geometric mean of IgG1 levels. (c) Percentage of boxed IgG1⁺ B cells in Cγ1-cre mice carrying either the B1-8i or B1-8f alleles. (d) Percentage of β-gal⁺ cells among gated B220⁺IgG1⁺ B cells of Cγ1-cre mice carrying the indicated B1-8 alleles. Two mice per group are shown. β-gal gates were defined based on control mice. Results shown are representative of at least three independent experiments

Fig. 4.

Germ-line Cγ1 transcription induces Cre-mediated recombination in resting Cγ1-cre B cells. (a) EYFP⁺IgM⁺ B cells in Cγ1-cre; R26-EYFP mice are divided into two subsets according to Fas expression. (b) RT-PCR analysis to detect IgH germ-line (GLT), circle (CT)-switched and AID-specific transcripts in IgM⁺EYFP⁻ (lane 1), IgM⁺EYFP⁺Fas^lo (lane 2), and IgM^loEYFP⁺Fas^hi (lane 3) sorted B cells. cDNA from IgG1⁺ A20 B lymphoma cells (lane 4) and IgM⁺ c-MYC transformed pre-GC mouse B lymphoma cells were included as controls; arrows indicate specific PCR fragments. (c) Percentage of EGFP⁺ Cγ1-cre; Igβ^fl-EGFP/+ and Igβ^fl-EGFP/+ control B cells throughout a 3-day culture period in the presence of the indicated amounts of IL-4. Results shown are representative of at least three independent experiments

Fig. 5.

Cγ1-cre B cells undergoing Cre-mediated recombination in vivo express multiple Ig isotypes. (a) Ig switch transcripts in sorted MLN GC B cells from two Cγ1-cre; R26-EYFP mice detected by RT-PCR. 2470.1, c-MYC-transformed, IgM⁺ primary B lymphoma cells and IgG1⁺ A20 mouse B lymphoma cells were included as controls. (b) EYFP expression in Peyer’s patches Fas^hiIgA⁺ GC B cells of Cγ1-cre; R26-EYFP mice. (c) Immunofluorescence analysis of SPL sections from Cγ1-cre; R26-EYFP mice 10 days after SRBC immunization, stained for EYFP (red) and each one of the indicated IgH isotypes (green). Arrows indicate coexpressing cells. (d) Percentage of EGFP⁺ cells 4 days after treating Cγ1-cre; Igβ^fl-EGFP/+ B cells with different stimuli promoting CSR. (e) Percentage of EGFP⁺ cells among isotype-switched B cells in Cγ1-cre; Igβ^fl-EGFP/+ mice (continuous black line). Stimulated Igβ^fl-EGFP/+ B cells were used as negative controls for EYFP expression (dotted line). Results shown in a–d are representative of at least two independent experiments.