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, 213 (1), 109-22

A Plasma Cell Differentiation Quality Control Ablates B Cell Clones With Biallelic Ig Rearrangements and Truncated Ig Production

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A Plasma Cell Differentiation Quality Control Ablates B Cell Clones With Biallelic Ig Rearrangements and Truncated Ig Production

Nivine Srour et al. J Exp Med.

Abstract

Aberrantly rearranged immunoglobulin (Ig) alleles are frequent. They are usually considered sterile and innocuous as a result of nonsense-mediated mRNA decay. However, alternative splicing can yield internally deleted proteins from such nonproductively V(D)J-rearranged loci. We show that nonsense codons from variable (V) Igκ exons promote exon-skipping and synthesis of V domain-less κ light chains (ΔV-κLCs). Unexpectedly, such ΔV-κLCs inhibit plasma cell (PC) differentiation. Accordingly, in wild-type mice, rearrangements encoding ΔV-κLCs are rare in PCs, but frequent in B cells. Likewise, enforcing expression of ΔV-κLCs impaired PC differentiation and antibody responses without disturbing germinal center reactions. In addition, PCs expressing ΔV-κLCs synthesize low levels of Ig and are mostly found among short-lived plasmablasts. ΔV-κLCs have intrinsic toxic effects in PCs unrelated to Ig assembly, but mediated by ER stress-associated apoptosis, making PCs producing ΔV-κLCs highly sensitive to proteasome inhibitors. Altogether, these findings demonstrate a quality control checkpoint blunting terminal PC differentiation by eliminating those cells expressing nonfunctionally rearranged Igκ alleles. This truncated Ig exclusion (TIE) checkpoint ablates PC clones with ΔV-κLCs production and exacerbated ER stress response. The TIE checkpoint thus mediates selection of long-lived PCs with limited ER stress supporting high Ig secretion, but with a cost in terms of antigen-independent narrowing of the repertoire.

Figures

Figure 1.
Figure 1.
Alternative splicing of nonproductive Igκ transcripts in B cell and plasma cell lines. (A, left) Mouse Igκ locus showing Vκ segments (in normal and inverse orientations), the four functional Jκ segments, the intronic κ enhancer (Eiκ), and the constant Cκ exon. (right) In-frame (F) and out-of-frame (VPTC and CPTC) VκJκ5 junctions are depicted, and the position of normal stop codons (stop) and PTCs are shown. Primers used are indicated (black arrows). (B) Representation of in-frame (F), VPTC, and CPTC mini-gene constructs. Out-of-frame junctions were created by inserting 5 nt (VPTC) or 10 nt (CPTC) at VκJκ5 junctions. (C) RT-PCR was performed using L1-135-for/Cκ-rev primers to identify full-length (κLC) and alternatively spliced (ΔV-κLC) mRNAs simultaneously. (D) Semiquantitative RT-PCR analysis of S194 cell lines transfected with F, VPTC, and CPTC constructs. (E) A20 (mature B) and S194 (PC) cell lines were cotransfected with F and either VPTC or CPTC constructs (A20-F+VPTC, n = 9; A20-F+CPTC, n = 5; S194-F+VPTC, n = 6; S194-F+CPTC, n = 5). Relative ΔV-κLC mRNA levels were quantified using capillary electrophoresis by dividing areas of peaks corresponding to ΔV-κLC mRNAs to those of control functional (F) κLC mRNAs (ΔV-κLC/F ratios) as previously described (Chemin et al., 2010); part of these data (A20-F+VPTC, n = 4; S194-F+VPTC, n = 3) comes from this previous study. Data are representative of at least three independent experiments (n = 5–9). Bars represent mean expression ± SEM. Unpaired two-tailed Student’s test was performed to determine significance. ***, P < 0.001.
Figure 2.
Figure 2.
Production of truncated Ig and exacerbated ER stress response in VPTC-expressing cells lines. Sp2/0 (hybridoma) clones isolated after transfection with F (n = 5), VPTC (n = 6), or CPTC (n = 5) constructs were treated or not with MG132 (1 µM, 8 h). Complete κLCs (25 kD) and truncated ΔV-κLCs (12 kD) were assessed by Western blot (A) and ELISA (B) on cell pellets and culture supernatants, respectively. (A) A representative experiment from a pool of four clones per group is shown. (B) ELISA assays were performed in all transfected cells using anti-Igκ Abs. (C) Annexin V staining was performed in S194 cells either nontransfected (NT) or transfected with VPTC (n = 9) and CPTC (n = 6) constructs, and treated or not with MG132 (1 µM, 5 h). (D) In those cells, Chop mRNA levels were assessed by Q-PCR. Data are representative of two (A and B) or three (C and D) independent experiments (n = 5–9). Bars represent mean expression ± SEM. Unpaired two-tailed Student’s test was performed to determine significance. *, P < 0.05; **, P < 0.01.
Figure 3.
Figure 3.
Exon skipping of Igκ transcripts occurs in normal B cells but not in PCs. (A) Primary B and plasma cells were isolated from bone marrow and sorted after staining with anti-B220, anti-IgL (κ and λ), and anti-CD138 mAbs. Representative dot plots and gates used for cell sorting are depicted. The purity of sorted populations was always >90%. (B) RT-PCR was performed using L1-135 forward and constant Cκ reverse primers to analyze both full-length κLC and ΔV-κLC mRNA levels in sorted cell populations. One representative experiment is shown. (C) Quantification of amplification products was done using an Agilent Bioanalyzer (left). ΔV-κLC/κLC mRNA ratios were determined for each sorted populations (n = 3; right). FU, fluorescence unit. Bars represent mean expression ± SEM. Data are representative of three independent experiments using a pool of two B6 mice per experiment.
Figure 4.
Figure 4.
Altered plasma cell differentiation and antibody responses in model mice reproducing the TIE checkpoint. (A) Gene targeting strategy. Map of the Igκ locus in germline configuration (top), after Jκ replacement (middle), and Cre-mediated recombination of the loxP-flanked hsvTK-NeoR cassette (bottom). (B) Western blot analysis of complete κLCs and truncated ΔV-κLCs performed on spleen cells from WT (+/+) and iTIE/+ mice 6 d after IP injection of SRBCs. At day 4 and 5, mice received additional IP injections of PBS (−) or bortezomib (+Bz). A representative experiment using four mice per genotype is shown. (C) FACS analysis was performed on spleen cells isolated 6 to 7 d after SRBC injection. (D) The percentage (left) and absolute numbers (right) of PCs (B220neg/CD138pos; top), B cells (B220pos; middle), and GC B cells (B220pos/GL7pos; bottom) are shown for Crepos (gray bars) and Creneg iTIE/+ (empty bars; n = 16 mice/group). (E and F) Crepos (gray) and Creneg (empty) iTIE/+ mice (n = 8/group) were immunized with two IP injections (day 0 and 14) of DNP-Ficoll (E) and OVA (F). Sera were collected at day 7 and 24 and ELISA assays were performed to determine DNP- (E) and OVA- (F) specific IgM (left), IgG3 (E, right), and IgG1 (F, right) titers. Data are from two (B) and at least three (C–F) independent experiments. n = 8–16. Bars represent mean expression ± SEM. Unpaired two-tailed Student’s test was performed to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5.
Figure 5.
Altered plasma cell differentiation after inducible production of truncated Ig in germinal center B cells. FACS analysis was performed on spleen cells after gating on B220neg/CD138pos PCs (A) or B220pos B cells (C), as depicted in Fig. 4 C. The frequency and absolute numbers of EYFPpos PCs (B) and EYFPpos B cells (D) were determined 7 d after IP injections of SRBCs in tamoxifen-treated WT (+/+; n = 6) and iTIE/+ (n = 6) mice on AID-Cre-EYFP background. Data are from two independent experiments. Bars represent mean expression ± SEM. Unpaired two-tailed Student’s test was performed to determine significance. **, P < 0.01; ***, P < 0.001.
Figure 6.
Figure 6.
PCs expressing ΔV-κLCs synthesize low amounts of Ig and preferentially accumulate as short-lived plasmablasts. (A) Representative FACS analysis of spleen cells isolated from Crepos and Creneg homozygous iTIE/iTIE mice after staining with anti-B220 and anti-CD138 mAbs. The percentage and absolute numbers of B cells (B220pos/CD138neg) and PCs (B220neg/CD138pos) were determined on spleen cells (n = 6/group) isolated 7 d after IP injection of SRBCs. (B) ELISA assays were performed in sera from 8-wk-old Creneg (n = 9) and Crepos (n = 9) iTIE/iTIE mice to determine the amounts of Ig isotypes (IgM, Igλ, IgG1 IgG2b, IgG3, and total IgG). (C) Relative ΔV-κLC mRNA levels were determined in purified B cells (B220pos/CD138neg; n = 3) and PCs (B220neg/CD138pos; n = 3) sorted from spleens of iTIE/iTIE Crepos mice, 7 d after SRBC injection. RT-qPCR was performed using constant Cκ primers, after normalization to Gapdh transcripts. (D) Intracellular contents of Igλ were assessed by measuring the mean fluorescence intensity (MFI) in splenic Crepos (gray) and Creneg (empty) iTIE/iTIE B220negCD138pos PCs (n = 6/group), 7 d after SRBC injection. (E) Crepos and Creneg iTIE/iTIE mice (n = 4/group) were injected with BrdU as described in experimental procedures. At day 10, the frequency of BrdUpos (cycling) and BrdUneg (noncycling) cells was analyzed in B cells (B220posCD138neg; left) and PCs (B220negCD138pos; right) isolated from bone marrow. (F) Relative mRNA levels of ER stress markers were assessed in purified PCs (B220neg/CD138pos) sorted from spleens of Crepos (gray; n = 3) and Creneg (empty; n = 3) iTIE/iTIE mice, 7 d after SRBC injection. Data are representative of at least two independent experiments. n = 3–9. Bars represent mean expression ± SEM. Unpaired two-tailed Student’s test was performed to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 7.
Figure 7.
Intrinsic toxicity of truncated Ig encoded by ΔV-κLC mRNAs. Experiments were performed on the DH-LMP2A background lacking IgH expression to analyze intrinsic effects of ΔV-κLCs in terminally differentiated B lineage cells. (A) The frequency of (B220pos/CD138pos) plasmablasts (PBs) was determined in purified splenic B cells from iTIE/iTIE::DH-LMP2A (gray bars; n = 4) and wt/wt::DH-LMP2A (empty bars; n = 4) mice 4 d after LPS stimulation (1 µg/ml). The gates used are depicted in dot plots. (B and C) In those purified B cells, relative mRNA levels of ER stress markers were assessed at day 0 (B) or 4 d after LPS-stimulation (C). (D and E) The expression of ΔV-κLCs (D) and ER stress markers (E) was assessed in activated B cells (B-act: B220pos/CD138neg) and PBs (B220pos/CD138pos) sorted 4 d after LPS stimulation of B cells. (D) ΔV-κLC mRNA levels were assessed in the iTIE/iTIE::DH-LMP2A model by qPCR using Cκ primers, as described in Fig. 6 C. (E) Chop, BiP, Herp, and Xbp1s mRNA levels were assessed in purified PBs from iTIE/iTIE::DH-LMP2A mice (gray; n = 3), and compared with DH-LMP2A controls (empty; n = 3). Relative mRNA levels were determined after normalization to Gapdh transcripts. (F) To analyze CHOP, BIP, HERP, and IRE1α at the protein level, Western blots were performed in total LPS-stimulated B cells (day 4). A representative experiment is shown. Mean fold-change between iTIE/iTIE::DH-LMP2A (n = 3) and DH-LMP2A (n = 3) was calculated after normalization to GAPDH, using ImageJ software (National Institutes of Health). Data are representative of at least two independent experiments. n = 3–4. Bars represent mean expression ± SEM. Unpaired two-tailed Student’s test was performed to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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