ISOTOPE: ISOform-guided prediction of epiTOPEs in cancer.

Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE.

Roles of RNA Methylation on Tumor Immunity and Clinical Implications.

RNA methylation is a kind of RNA modification that exists widely in eukaryotes and prokaryotes. RNA methylation occurs not only in mRNA but also in ncRNA. According to the different sites of methylation, RNA methylation includes m6A, m5C, m7G, and 2-O-methylation modifications. Modifications affect the splicing, nucleation, stability and immunogenicity of RNA. RNA methylation is involved in many physiological and pathological processes. In the immune system, especially for tumor immunity, RNA methylation affects the maturation and response function of immune cells. Through the influence of RNA immunogenicity and innate immune components, modifications regulate the innate immunity of the body. Some recent studies verified that RNA methylation can regulate tumor immunity, which also provides a new idea for the future of treating immunological diseases and tumor immunotherapy.

MAVS splicing variants associated with TRAF3 and TRAF6 in NF-κB and IRF3 signaling pathway in large yellow croaker Larimichthys crocea.

Mitochondrial antiviral signaling protein (MAVS) acts as an essential adaptor in host RIG-I-like receptors (RLRs) mediated antiviral signaling pathway. In the present study, two MAVS transcript variants, the typical form and a splicing variant, namely Lc-MAVS_tv1 and Lc-MAVS_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-MAVS_tv1 protein contains 512 aa, with an N-terminal CARD domain, a central proline-rich region, and a C-terminal transmembrane (TM) domain, whereas Lc-MAVS_tv2 contains 302 aa and lacks the C-terminal TM domain due to a premature stop in the 102 bp intron fragment insertion. Lc-MAVS_tv1 was identified as a mitochondrion localized protein whereas Lc-MAVS_tv2 exhibited an entire cytosolic distribution. Quantitative real-time PCR revealed that Lc-MAVS_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-MAVS_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression of Lc-MAVS_tv2 could induce the activation of NF-κB but not IRF3, and Lc-MAVS_tv2 co-transfected with Lc-MAVS_tv1 induced a significantly higher level of NF-κB and IRF3 promoter activity. In addition, Lc-MAVS_tv2 overexpression could enhance TRAF3 and TRAF6 mediated NF-κB activation, but suppress TRAF3 and TRAF6 mediated IRF3 activation, implying that the splicing variant Lc-MAVS_tv2 may function as an important regulator in MAVS mediated signaling pathway.

Splice-Switching Antisense Oligonucleotides as a Targeted Intrinsic Engineering Tool for Generating Armored Redirected T Cells.

Modification of specificity of T cells for the use in adoptive transfer (CAR- or TCR-redirected T cells) has revolutionized the therapy of liquid tumors and some infectious diseases. However, several obstacles are still hampering the efficacy of such potent therapy, hence concurrent modification of the function is also required to obtain successful results. Here we show the use of splice-switching antisense oligonucleotides (SSOs) as a tool to transiently modify T cell function. We demonstrate the possibility to transfect SSOs and an exogenous TCR into primary human T cells in the same electroporation reaction, without affecting viability and function of the transfected T lymphocytes. Moreover, we show that SSOs targeting T cell-specific mRNAs induce the skipping of the targeted exons, and the reduction of the protein and consequent modification of T cell function. This technical work paves the way to the use of SSOs in immune cells, not only for the knockdown of the functional isoform of the targeted proteins, but also for the protein manipulation by elimination of specific domains encoded by targeted exons.

Filgotinib suppresses HIV-1-driven gene transcription by inhibiting HIV-1 splicing and T cell activation.

Despite effective antiretroviral therapy, HIV-1-infected cells continue to produce viral antigens and induce chronic immune exhaustion. We propose to identify HIV-1-suppressing agents that can inhibit HIV-1 reactivation and reduce HIV-1-induced immune activation. Using a newly developed dual-reporter system and a high-throughput drug screen, we identified FDA-approved drugs that can suppress HIV-1 reactivation in both cell line models and CD4+ T cells from virally suppressed HIV-1-infected individuals. We identified 11 cellular pathways required for HIV-1 reactivation as druggable targets. Using differential expression analysis, gene set enrichment analysis, and exon-intron landscape analysis, we examined the impact of drug treatment on the cellular environment at a genome-wide level. We identified what we believe to be a new function of a JAK inhibitor, filgotinib, that suppresses HIV-1 splicing. First, filgotinib preferentially suppresses spliced HIV-1 RNA transcription. Second, filgotinib suppresses HIV-1-driven aberrant cancer-related gene expression at the integration site. Third, we found that filgotinib suppresses HIV-1 transcription by inhibiting T cell activation and by modulating RNA splicing. Finally, we found that filgotinib treatment reduces the proliferation of HIV-1-infected cells. Overall, the combination of a drug screen and transcriptome analysis provides systematic understanding of cellular targets required for HIV-1 reactivation and drug candidates that may reduce HIV-1-related immune activation.

Loss of MBNL1 induces RNA misprocessing in the thymus and peripheral blood.

The thymus is a primary lymphoid organ that plays an essential role in T lymphocyte maturation and selection during development of one arm of the mammalian adaptive immune response. Although transcriptional mechanisms have been well documented in thymocyte development, co-/post-transcriptional modifications are also important but have received less attention. Here we demonstrate that the RNA alternative splicing factor MBNL1, which is sequestered in nuclear RNA foci by C(C)UG microsatellite expansions in myotonic dystrophy (DM), is essential for normal thymus development and function. Mbnl1 129S1 knockout mice develop postnatal thymic hyperplasia with thymocyte accumulation. Transcriptome analysis indicates numerous gene expression and RNA mis-splicing events, including transcription factors from the TCF/LEF family. CNBP, the gene containing an intronic CCTG microsatellite expansion in DM type 2 (DM2), is coordinately expressed with MBNL1 in the developing thymus and DM2 CCTG expansions induce similar transcriptome alterations in DM2 blood, which thus serve as disease-specific biomarkers.

Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8+ T cells in HIV-1 infection.

Peptides generated by proteasome-catalyzed splicing of noncontiguous amino acid sequences have been shown to constitute a source of nontemplated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8+ T cells are key mediators of HIV type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I-bound peptides on HIV-infected cells. We demonstrate that HIV-1-derived spliced peptides comprise a relatively minor component of the HLA-I-bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8+ T cell responses relatively infrequently during infection, CD8+ T cells primed by partially overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically underutilized during natural infection.

The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages.

While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.

Coordinated host-pathogen transcriptional dynamics revealed using sorted subpopulations and single macrophages infected with Candida albicans.

The outcome of fungal infections depends on interactions with innate immune cells. Within a population of macrophages encountering Candida albicans, there are distinct host-pathogen trajectories; however, little is known about the molecular heterogeneity that governs these fates. Here we developed an experimental system to separate interaction stages and single macrophage cells infected with C. albicans from uninfected cells and assessed transcriptional variability in the host and fungus. Macrophages displayed an initial up-regulation of pathways involved in phagocytosis and proinflammatory response after C. albicans exposure that declined during later time points. Phagocytosed C. albicans shifted expression programs to survive the nutrient poor phagosome and remodeled the cell wall. The transcriptomes of single infected macrophages and phagocytosed C. albicans displayed a tightly coordinated shift in gene expression co-stages and revealed expression bimodality and differential splicing that may drive infection outcome. This work establishes an approach for studying host-pathogen trajectories to resolve heterogeneity in dynamic populations.

A synonymous splice site mutation in IL2RG gene causes late-onset combined immunodeficiency.

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.

Production of spliced peptides by the proteasome.

CD8+ cytolytic T lymphocytes are essential players of anti-tumor immune responses. On tumors, they recognize peptides of about 8-to-10 amino acids that generally result from the degradation of cellular proteins by the proteasome. Until a decade ago, these peptides were thought to solely correspond to linear fragments of proteins that were liberated after the hydrolysis of the peptide bonds located at their extremities. However, several examples of peptides containing two fragments originally distant in the protein sequence challenged this concept and demonstrated that proteasome could also splice peptides together by creating a new peptide bond between two distant fragments. Unexpectedly, peptide splicing emerges as an essential way to increase the peptide repertoire diversity as these spliced peptides were shown to represent up to 25% of the peptides presented on a cell by MHC class I. Here, we review the different steps that led to the discovery of peptide splicing by the proteasome as well as the lightening offered by the recent progresses of mass spectrometry and bioinformatics in the analysis of the spliced peptide repertoire.

RNA Splicing in the Transition from B Cells to Antibody-Secreting Cells: The Influences of ELL2, Small Nuclear RNA, and Endoplasmic Reticulum Stress.

In the transition from B cells to Ab-secreting cells (ASCs) many genes are induced, such as ELL2, Irf4, Prdm1, Xbp1, whereas other mRNAs do not change in abundance. Nonetheless, using splicing array technology and mouse splenic B cells plus or minus LPS, we found that induced and "uninduced" genes can show large differences in splicing patterns between the cell stages, which could influence ASC development. We found that ∼55% of these splicing changes depend on ELL2, a transcription elongation factor that influences expression levels and splicing patterns of ASC signature genes, genes in the cell-cycle and N-glycan biosynthesis and processing pathways, and the secretory versus membrane forms of the IgH mRNA. Some of these changes occur when ELL2 binds directly to the genes encoding those mRNAs, whereas some of the changes are indirect. To attempt to account for the changes that occur in RNA splicing before or without ELL2 induction, we examined the amount of the small nuclear RNA molecules and found that they were significantly decreased within 18 h of LPS stimulation and stayed low until 72 h. Correlating with this, at 18 h after LPS, endoplasmic reticulum stress and Ire1 phosphorylation are induced. Inhibiting the regulated Ire1-dependent mRNA decay with 4u8C correlates with the reduction in small nuclear RNA and changes in the normal splicing patterns at 18 h. Thus, we conclude that the RNA splicing patterns in ASCs are shaped early by endoplasmic reticulum stress and Ire1 phosphorylation and later by ELL2 induction.

Uncoupling of mucosal gene regulation, mRNA splicing and adherent microbiota signatures in inflammatory bowel disease.

OBJECTIVE: An inadequate host response to the intestinal microbiota likely contributes to the manifestation and progression of human inflammatory bowel disease (IBD). However, molecular approaches to unravelling the nature of the defective crosstalk and its consequences for intestinal metabolic and immunological networks are lacking. We assessed the mucosal transcript levels, splicing architecture and mucosa-attached microbial communities of patients with IBD to obtain a comprehensive view of the underlying, hitherto poorly characterised interactions, and how these are altered in IBD. DESIGN: Mucosal biopsies from Crohn's disease and patients with UC, disease controls and healthy individuals (n=63) were subjected to microbiome, transcriptome and splicing analysis, employing next-generation sequencing. The three data levels were integrated by different bioinformatic approaches, including systems biology-inspired network and pathway analysis. RESULTS: Microbiota, host transcript levels and host splicing patterns were influenced most strongly by tissue differences, followed by the effect of inflammation. Both factors point towards a substantial disease-related alteration of metabolic processes. We also observed a strong enrichment of splicing events in inflamed tissues, accompanied by an alteration of the mucosa-attached bacterial taxa. Finally, we noted a striking uncoupling of the three molecular entities when moving from healthy individuals via disease controls to patients with IBD. CONCLUSIONS: Our results provide strong evidence that the interplay between microbiome and host transcriptome, which normally characterises a state of intestinal homeostasis, is drastically perturbed in Crohn's disease and UC. Consequently, integrating multiple OMICs levels appears to be a promising approach to further disentangle the complexity of IBD.

Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus.

The thymus has spatially distinct microenvironments, the cortex and the medulla, where the developing T-cells are selected to mature or die through the interaction with thymic stromal cells. To establish the immunological self in the thymus, medullary thymic epithelial cells (mTECs) express diverse sets of tissue-specific self-antigens (TSAs). This ectopic expression of TSAs largely depends on the transcriptional regulator Aire, yet the mechanism controlling Aire expression itself remains unknown. Here, we show that Jmjd6, a dioxygenase that catalyses lysyl hydroxylation of splicing regulatory proteins, is critical for Aire expression. Although Jmjd6 deficiency does not affect abundance of Aire transcript, the intron 2 of Aire gene is not effectively spliced out in the absence of Jmjd6, resulting in marked reduction of mature Aire protein in mTECs and spontaneous development of multi-organ autoimmunity in mice. These results highlight the importance of intronic regulation in controlling Aire protein expression.

Pioneer translation products as an alternative source for MHC-I antigenic peptides.

The notion that alternative peptide substrates can be processed and presented to the MHC class I pathway has opened for new aspects on how the immune system detects infected or damaged cells. Recent works show that antigenic peptides are derived from intron sequences in pre-mRNAs target for the nonsense-mediated degradation pathway. Introns are spliced out co-transcriptionally suggesting that such pioneer translation products (PTPs) are synthesized on the nascent RNAs in the nuclear compartment to ensure that the first peptides to emerge from an mRNA are destined for the class I pathway. This illustrates an independent translation event during mRNA maturation that give rise to specific peptide products with a specific function in the immune system. The characterization of the translation apparatus responsible for PTP synthesis will pave the way for understanding how PTP production is regulated in different tissues under different conditions and will help designing new vaccine strategies.

Long noncoding RNAs in innate and adaptive immunity.

The differentiation and activation of both innate and adaptive immune cells is highly dependent on a coordinated set of transcriptional and post-transcriptional events. Chromatin-modifiers and transcription factors regulate the accessibility and transcription of immune genes, respectively. Immune cells also express miRNA and RNA-binding proteins that provide an additional layer of regulation at the mRNA level. However, long noncoding RNAs (lncRNAs), which have been primarily studied in the context of genomic imprinting, cancer, and cell differentiation, are now emerging as important regulators of immune cell differentiation and activation. In this review, we provide a brief overview of lncRNAs, their known functions in immunity, and discuss their potential to be more broadly involved in other aspects of the immune response.

Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.

Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output, with important functional consequences. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs or proteins simultaneously, because genomic profiling methods could not be applied to single cells until very recently. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.

Osteopontin splice variants expressed by breast tumors regulate monocyte activation via MCP-1 and TGF-ß1.

Osteopontin (OPN), a multifunctional glycoprotein, has three transcripts that have distinct roles in tumors in vitro. Whether OPN transcripts have different functions in tumor processes in vivo is unclear. It has been reported that immune cell-derived OPN can promote tumor formation. We propose a hypothesis that tumor-derived OPN may facilitate tumor immune escape by affecting immune cell differentiation and function. In this study, we constructed lentiviral expression vectors of OPN transcripts and transfected them into the MCF-7 cell line. MCF-7 cells transfected with OPN transcripts were injected into the armpit of nude mice, and tumor growth was monitored. The results showed that all OPN transcripts promoted local tumor formation, but that there was no significant difference among transcripts. We also investigated the effect of the OPN expressed by tumor cells on monocyte differentiation by coculturing monocytes with tumor supernatant. We found OPN-c upregulated CD163 levels compared with OPN-a and OPN-b; however, none of the transcripts affected HLA-DR and CD206 levels. All OPN transcripts significantly inhibited TNF-α and enhanced IL-10 production by monocytes. Furthermore, we found that the overexpression of OPN transcripts significantly upregulated TGF-ß1 and MCP-1 production by tumor cells. Using neutralizing antibody and recombinant cytokines, we found that OPN overexpressed by tumor cells regulates the production of TNF-α and IL-10 by monocytes partly via MCP-1 and TGF-ß1, respectively. Collectively, our results show that OPN transcripts have no distinct role in breast cancer formation in vivo. We also demonstrate that OPN regulates the alternative activation of monocytes via TGF-ß1 and MCP-1, which may represent an additional mechanism for tumor immune escape.

A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins.

We developed a simple T4 DNA ligase mediated strategy for inframe splicing of two or more cohesive genes generated by hetero-staggered PCR and directionally cloning the spliced product bearing sticky overhangs in to a correspondingly cut vector. For this, two pairs of primers are used in two different parallel PCRs, for generation of each cohesive gene product. We exemplified this strategy by splicing two major super-antigen genes of Staphylococcus aureus, namely, staphylococcal enterotoxin A (sea), and toxic shock syndrome toxin (tsst-1) followed by its directional cloning into pre-digested pRSET A vector. The fusion gene encoding chimeric recombinant SEA-TSST protein (32kDa) was expressed in E. coli BL21(DE3) host strain. The recombinant chimeric protein retained the antigenicity of both toxins as observed by the strong immunoreactivity with commercial antibodies against both SEA and TSST-1 toxin components by Western blot analysis. We observed that the present method for gene splicing with cohesive ends is simple since it does not require elaborate standardization and a single fusion product is obtained consistently during nested PCR with forward primer of first gene and reverse primer of second gene. For comparison, we fused the same genes using splicing by overlap extension PCR (SOE-PCR) and consistently obtained DNA smearing and multiple non-specific bands even after several rounds of PCRs from gel excised product. Moreover, the newly described method requires only two to six complimentary sticky ends between the genes to be spliced, in contrast to long stretch of overlapping nucleotides in case of SOE-PCR.