Immunoglobulin light-chain toxicity in a mouse model of monoclonal immunoglobulin light-chain deposition disease.

Light chain (LC) deposition disease (LCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a monoclonal immunoglobulin LC, leading to nodular glomerulosclerosis and nephrotic syndrome. We developed a transgenic model using site-directed insertion of the variable domain of a pathogenic human LC gene into the mouse immunoglobulin κ locus, ensuring its production by all plasma cells (PCs). High free LC levels were achieved after backcrossing with mice presenting increased PC differentiation and no immunoglobulin heavy chain production. Our mouse model recapitulates the characteristic features of LCDD, including progressive glomerulosclerosis, nephrotic-range proteinuria, and finally kidney failure. The variable domain of the LC bears alone the structural properties involved in its pathogenicity. RNA sequencing conducted on PCs demonstrated that LCDD LC induces endoplasmic reticulum stress, likely accounting for the high efficiency of proteasome inhibitor-based therapy. Accordingly, reduction of circulating pathogenic LC was efficiently achieved and not only preserved renal function but also partially reversed kidney lesions. Finally, transcriptome analysis of presclerotic glomeruli revealed that proliferation and extracellular matrix remodeling represented the first steps of glomerulosclerosis, paving the way for future therapeutic strategies in LCDD and other kidney diseases featuring diffuse glomerulosclerosis, particularly diabetic nephropathy.

Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphocytes.

The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid the synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Thus, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphocytes.

Mutant and non-mutant neoantigen-based cancer vaccines: recent advances and future promises.

Major advances in cancer treatment have emerged with the introduction of immunotherapies using blocking antibodies that target T-cell inhibitory receptors, such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), known as immune checkpoints. However, most cancer patients do not respond to immune checkpoint blockade (ICB) therapies, suggesting the development of resistance mechanisms associated with either an insufficient number of preexisting tumor-specific T-cell precursors and/or inappropriate T-cell reactivation. To broaden clinical benefit, anti-PD-1/PD-1 ligand (PD-L1) neutralizing antibodies have been combined with therapeutic cancer vaccines based on non-mutant and/or mutant tumor antigens, to stimulate and expand tumor-specific T lymphocytes. Although these combination treatments achieve the expected goal in some patients, relapse linked to alterations in antigen presentation machinery (APM) of cancer cells often occurs leading to tumor escape from CD8 T-cell immunity. Remarkably, an alternative antigenic peptide repertoire, referred to as T-cell epitopes associated with impaired peptide processing (TEIPP), arises on these malignant cells with altered APM. TEIPP are derived from ubiquitous non-mutant self-proteins and represent a unique resource to target immune-edited tumors that have acquired resistance to cytotoxic T lymphocytes (CTLs) related to defects in transporter associated with antigen processing (TAP) and possibly also to ICB. The present review discusses tumor-associated antigens (TAAs) and mutant neoantigens and their use as targets in peptide- and RNA-based therapeutic cancer vaccines. Finally, this paper highlights TEIPP as a promising immunogenic non-mutant neoantigen candidates for active cancer immunotherapy and combination with TAA and mutant neoantigens. Combining these polyepitope cancer vaccines with ICB would broaden T-cell specificity and reinvigorate exhausted antitumor CTL, resulting in the eradication of all types of neoplastic cells, including immune-escaped subtypes.

Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching.

Class switch recombination (CSR) changes antibody isotype by replacing Cµ constant exons with different constant exons located downstream on the immunoglobulin heavy (IgH) locus. During CSR, transcription through specific switch (S) regions and processing of non-coding germline transcripts (GLTs) are essential for the targeting of activation-induced cytidine deaminase (AID). While CSR to IgG1 is abolished in mice lacking an Iγ1 exon donor splice site (dss), many questions remain regarding the importance of I exon dss recognition in CSR. To further clarify the role of I exon dss in CSR, we first evaluated RNA polymerase II (RNA pol II) loading and chromatin accessibility in S regions after activation of mouse B cells lacking Iγ1 dss. We found that deletion of Iγ1 dss markedly reduced RNA pol II pausing and active chromatin marks in the Sγ1 region. We then challenged the post-transcriptional function of I exon dss in CSR by using antisense oligonucleotides (ASOs) masking I exon dss on GLTs. Treatment of stimulated B cells with an ASO targeting Iγ1 dss, in the acceptor Sγ1 region, or Iµ dss, in the donor Sµ region, did not decrease germline transcription but strongly inhibited constitutive splicing and CSR to IgG1. Supporting a global effect on CSR, we also observed that the targeting of Iµ dss reduced CSR to IgG3 and, to a lesser extent, IgG2b isotypes. Altogether, this study reveals that the recognition of I exon dss first supports RNA pol II pausing and the opening of chromatin in targeted S regions and that GLT splicing events using constitutive I exon dss appear mandatory for the later steps of CSR, most likely by guiding AID to S regions.

Physiological and druggable skipping of immunoglobulin variable exons in plasma cells.

The error-prone V(D)J recombination process generates considerable amounts of nonproductive immunoglobulin (Ig) pre-mRNAs. We recently demonstrated that aberrant Ig chains lacking variable (V) domains can be produced after nonsense-associated altered splicing (NAS) events. Remarkably, the expression of these truncated Ig polypeptides heightens endoplasmic reticulum stress and shortens plasma cell (PC) lifespan. Many questions remain regarding the molecular mechanisms underlying this new truncated Ig exclusion (TIE-) checkpoint and its restriction to the ultimate stage of B-cell differentiation. To address these issues, we evaluated the extent of NAS of Ig pre-mRNAs using an Ig heavy chain (IgH) knock-in model that allows for uncoupling of V exon skipping from TIE-induced apoptosis. We found high levels of V exon skipping in PCs compared with B cells, and this skipping was correlated with a biallelic boost in IgH transcription during PC differentiation. Chromatin analysis further revealed that the skipped V exon turned into a pseudo-intron. Finally, we showed that hypertranscription of Ig genes facilitated V exon skipping upon passive administration of splice-switching antisense oligonucleotides (ASOs). Thus, V exon skipping is coupled to transcription and increases as PC differentiation proceeds, likely explaining the late occurrence of the TIE-checkpoint and opening new avenues for ASO-mediated strategies in PC disorders.

A plasma cell differentiation quality control ablates B cell clones with biallelic Ig rearrangements and truncated Ig production.

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.