Essential role of the initial activation signal in isotype selection upon deletion of a transcriptionally committed promoter

Abstract

Class switch recombination (CSR), which targets exclusively the constant region of the immunoglobulin heavy chain (IgH) locus, plays an important role in humoral immunity by generating different antibody effector functions. The IgH constant locus contains multiple genes controlled by isotype (I) promoters induced by extracellular signals that activate specific I promoters, leading to B cell commitment. However, it is unknown whether after initial commitment to one promoter, non-responsive I promoters are irreversibly silent or if they can be activated after exposure to their specific inducers. Here, we studied the murine cell line CH12, which is committed to produce IgA in response to TGF-β. We show that, although other promoters than Iα are transcriptionally inactive, they are not irreversibly silent. Following deletion of the committed Iα promoter by CRISPR/Cas9, other I promoters display a complex transcriptional pattern largely dependent on the initial committing signal.

Figure 1

Deletion of Iα promoter/exon inhibits CSR to IgA. (A) Schematic structure of the IgH locus in CH12F3-2 line. The non-expressed allele is a partially rearranged DJ_H allele that underwent Sµ/Sα recombination, thus deleting all upstream inducible I promoters. The mitogen and cytokines inducing the different I promoters are indicated on top. The sites flanking Iα promoter/exon targeted by the gRNAs are indicated with arrows. The Eµ/Iµ enhancer/promoter between the variable and the constant regions, and the 3′RR super-enhancer downstream of the locus are shown. (B) Flow cytometry analysis of Iα-deleted clones. The 8 clones obtained by CRIPR/Cas9 were analyzed by FACS for IgA surface expression. The parental CH12 line was used as a control prior to (UNS) and following LIT (LPS + IL4 + TGFβ) stimulation. LIT-activated splenic B cells were also included as a control (n = 3). (C) Representative FACS plot obtained with CH12 cells and an Iα-deleted clone (n° 1), before and following LIT stimulation.

Figure 2

Iα-deleted clones fail to undergo CSR following specific stimulation. (A–C) CH12 cells, three Iα-deleted clones, and splenic B cells were activated by LPS (A), LPS + IFNγ (B) or LPS + IL4 (C) and stained for IgG3, IgG2a and IgG1, respectively. Representative plots are shown for unstimulated (UNS) and activated CH12 cells, Iα-deleted clones (clone 5) and primary B cells. (D–F) RT-qPCR quantification of pre-switch transcripts (Sx transcripts) in unstimulated and day 2 activated splenic B cells, CH12 cells or clones 5, 6 and 8 in response to LPS (Sγ3) (D), to LPS + IFNγ (Sγ2a) (E), or to LPS + IL4 (Sγ1 and Sε) (F) (n = 3).

Figure 3

CSR to IgG2b is partially restored in response to TGF-β but not to LPS stimulation. (A,B) CH12 cells, three Iα-deleted clones (2, 3, and 5) and splenic B cells were activated by LPS (A) or LIT (B) for 4 days, and stained for IgG2b. Representative plots are shown for unstimulated (UNS), activated CH12 cells, Iα-deleted clones (clone 5) and primary B cells. (C,D) RT-qPCR quantification of Sγ2b pre-switch transcripts levels in unstimulated clones 5, 6 and 8 and in response to LPS (C) or to LIT (D) (day 2). (E) Comparison of Sγ2b pre-switch transcripts levels in Iα-deleted clones 5, 6 and 8, following LPS and LIT stimulation (n = 3).

Figure 4

Differential induction of CSR in response to LIT and CIT. CH12 cells and three Iα-deleted clones (3, 5 and 8) were activated by LIT or CIT for 4 days, and stained for the indicated isotypes. Representative plots are shown for activated CH12 cells and Iα-deleted clones (clone 5) (n ≥ 3).

Figure 5

Differential induction of switch transcription in response to LIT and CIT. (A–F) RT-qPCR quantification of Sγ2b (A), Sα (B), Sγ2a (C), Sγ3 (D), Sγ1 (E), and Sε (F) pre-switch transcripts levels in CH12 cells and Iα-deleted clones 3, 5 and 8 following LIT or CIT stimulation. Transcripts levels in splenic B cells activated with LIT and CIT for Sγ2b (A) and Sα (B), and with LPS + IL4 (LI), LIT and CIT for Sγ1 and Sε (E,F) are boxed (n ≥ 3).

Figure 6

Stimulus-dependent induction of Aicda gene transcription. CH12 cells, three Iα-deleted clones (3, 5 and 8) and splenic B cells were activated with LPS + IL4 (LI), LIT or CIT for 2 days. Total RNAs were collected from unstimulated and activated cells and Aicda transcripts were quantified by RT-qPCR. Actin transcripts were used for normalization (n ≥ 3).

Figure 7

3′RR transcripts levels upon LIT and CIT stimulations. RT-qPCR quantification of eRNAs levels of hs3a, hs1-2 and hs3b enhancers. Total RNAs were extracted from unstimulated CH12 cells and Iα-deleted clones (3, 5, and 8) at day 2 post-stimulation. Actin transcripts were used for normalization, and (-RT) controls were included throughout (n ≥ 3).

Figure 8

Model of the natural history of the CH12 line. Initial activation of the original B cell clone likely took place in the context of a T-dependent response involving TGF-β and CD40. This led to commitment to Iα promoter and induction of 3′RR transcription on both alleles, and subsequent switching on the non-expressed allele. The CSR machinery retained somehow memory of the initial activating signal (CIT). The Iα promoter and the 3′RR remain active in the committed CH12 line. Upon deletion of the committed Iα promoter, Iγ2b normally induced with either LPS or TGF-β, is only induced in response to TGF-β, but the highest switching levels to IgG2b are preferentially achieved with the initial signal (CIT).