A systematic catalog of eRNAs in human adaptive immune cells is defined and a distal transcribed enhancer is identified that fine-tunes KRAS signaling in Tfh cells to support effective vaccine responses, establishing eRNAs as noncoding regulators of T cell circuits controlling antibody production.
Abstract
Enhancer RNAs (eRNAs) are noncoding transcripts from active enhancers whose functions in adaptive immunity are poorly defined. Because small changes in signaling strength can alter T cell fate and B cell help, we hypothesized that eRNAs act as rheostats for key fate decisions and signaling modules shaping antigen-induced immune responses.
We integrated rRNA-depleted RNA-seq, ATAC-seq, and ChIP-seq to map transcribed enhancers in human B, CD4, and CD8 T cells. We then focused on a conserved eRNA ∼140 kb upstream of KRAS (eKRAS) and tested its function using si/shRNA, CRISPR perturbations, and phospho-signaling assays in human T cells, together with eKras—/— mice, mixed bone marrow chimeras, influenza infection, and SARS-CoV-2 mRNA vaccination with downstream cellular and serologic analyses.
We catalogued and characterized ∼2,000 eRNAs in human adaptive immune cells; eKRAS was among the most highly expressed and conserved and functioned as a cis-acting enhancer of KRAS. Disruption of eKRAS reduced KRAS mRNA and attenuated RAS-ERK activation. Although eKras—/— mice developed normally, immunized mixed chimeras revealed a cell-intrinsic defect in T follicular helper (Tfh) differentiation, with impaired germinal center formation, reduced Tfh effector programs, and defective neutralizing antibody responses to protein antigens and influenza. Following SARS-CoV-2 mRNA vaccination, eKras—/— mice showed reduced class-switched anti-spike antibodies. An eKRAS-dependent Tfh transcriptional program was conserved in human blood and associated with neutralizing antibody titers after COVID-19 vaccination.
We define a systematic catalog of eRNAs in human adaptive immune cells and identify a distal transcribed enhancer that fine-tunes KRAS signaling in Tfh cells to support effective vaccine responses. These findings establish eRNAs as noncoding regulators of T cell circuits controlling antibody production and nominate the eKRAS-KRAS axis as a target to optimize humoral immunity.
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Immune Response Regulation: Molecular Mechanisms (IRM)
Findings highlight a HuR-SerpinB9 axis that regulates T-cell senescence and persistence, offering a potential therapeutic target in cancer and autoimmune diseases.
P. Chakraborty, Mrinmoyee Majumder, W. Wofford et al.· Journal of Immunology· 0 citations
CD8 T cells respond to viral infection and generate memory populations that protect against subsequent encounters with the same antigen. The transcription factor (TF) RUNX3 is required for the formation of circulating and tissue-resident memory CD8 T cells; however, the roles of the co-expressed paralogs RUNX1 and RUNX2 in this process remain poorly defined.
We used P14 TCR-transgenic CD8 T cells which recognize LCMV-Armstrong, a virus that produces an acutely resolving infection in mice. Chromatin accessibility at RUNT motifs was assessed by ATAC-seq in RUNX3-depleted P14 cells at day eight post-infection. The three RUNT TFs, or their obligate co-factor CBFβ, were individually depleted by retrovirally delivered shRNAmirs in Tcf7-reporter P14 cells. These experiments were integrated with previously generated scRNA-seq data from day 8 P14 cells. De novo transcriptome assembly of bulk RNA-sequencing data was used to identify expressed RUNX TF splice isoforms.
Depletion of RUNX3 resulted in an incomplete loss of chromatin accessibility at RUNT motifs, suggesting compensation by RUNX1 or RUNX2. RUNX1 directed formation of CX3CR1hiKLRG1hi terminal effector cells, whereas RUNX3 counteracted this program to generate a stable CX3CR1intKLRG1int precursor population that gives rise to tissue-resident and central memory CD8 T cells. Depletion of RUNX2 had minimal effects on most effector populations but was required for the formation of Tcf7hiCX3CR1loKLRG1lo central memory precursors. These findings were corroborated by single-cell RNA-sequencing, which revealed variation in RUNX TF activity correlated to distinct transcriptional states. De novo transcriptome assembly further identified differential usage of Runx1 and Runx2 splice isoforms that differed by inclusion of an ETS interaction motif.
The three RUNX TFs, and their distinct splice isoforms, likely compete at RUNT motifs, resulting in variation of RUNX activity that directs formation of specific CD8 T cell states.
NIH U19AI109976, R01AI095634 , P01AI145815 (M.E.P.) AHA 24PRE1191318 (D.S.A.)
Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Dominic S. Albao, Matthew Pipkin· Journal of Immunology· 0 citations
Ribosome Sequencing (RiboSeq) has identified translated open reading frames (ORFs) in “noncoding” elements of the transcriptome, including long noncoding RNAs (lncRNAs) and 5’ untranslated regions (5’UTRs). To date, targeted studies scratch the surface relative to the number of predicted ORFs, and the few high-throughput screens have focused on proliferation in cancer cell lines. To link novel translation events to immune cell function, we performed proliferation and NFkB reporter screens in murine macrophages, linking hundreds of novel ORFs to immune phenotype for the first time.
RiboSeq reads from 21 immune cell datasets were collected and processed. Input datasets included macrophages, dendritic cells, CD4/8 T cells, and B cells, with both resting and activated samples. Novel translation events were called against a custom transcriptome. RiboSeq predicted ORFs were validated using mass spectrometry data from immune cells and MHC I peptidomics. 3,000 high confidence novel ORFs on lncRNAs and in 5’UTRs were knocked out in a CRISPR dropout screen and a Tlr2-NFkB sorting screen. A top hit from each screen was characterized in follow-up studies.
Over 20,000 novel translation events were predicted across all cell types (adj p < 0.01), and hundreds were found to have supporting proteomics data (adj p < 0.01). In follow-up CRISPR screens 200 novel ORFs impacted cell proliferation, notably ORFs in the 5’UTR of Tlr1, Tlr2, and Irf2. 15 novel ORFs were implicated in Tlr2-NFkB signaling. We followed up on two hits which were derived from retroviral envelope proteins, one acting as a secreted cytokine and one as a transmembrane protein.
We have generated a resource of novel translation events in immune cells and demonstrated that 100s of novel ORFs play a role in macrophage biology. Of the predicted ORFs, 8 were found to be derived from retroviral proteins, two of which were top hits in our proliferation and NFkB screens, indicating novel repurposing of retroviral elements.
NIH NIAID F31 Grant
Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Eric Malekos, Susan Carpenter, Zach Rugee et al.· Journal of Immunology· 0 citations
PRMT5 is expressed at high levels in many cancers, where it regulates diverse cellular pathways that contribute to oncogenesis. Here, we have defined a new role for PRMT5 in regulating and coordinating the interplay between the innate and adaptive immune response. This occurs, in part, through the influence of PRMT5 and E2F1 on RNA splicing and the presence of retained introns (RIs). We found that RIs have a propensity to form double-stranded RNAs that contribute to the innate response. Furthermore, many RIs contain non-canonical open-reading frames (ncORFs), which can be translated and then processed into small peptides that assemble with the MHC class I complex. Significantly, RI-derived peptides are highly immunogenic and, as a murine cancer vaccine, carrying a string of antigenic RI peptides, delayed tumour growth and enhanced survival. RIs are present in human tumour cells, and we identified T lymphocytes in human cancer patients, with antigen specificity for RI-derived peptides, that killed human tumour cells in vitro. Regulating intron retention thus offers a new therapeutic approach to enhance tumour immunogenicity. PRMT5-regulated intron retention generates non-canonical MHC class I antigens that drive anti-tumour immunity and can be exploited for cancer vaccine development. PRMT5 was found to regulate retained intron (RI)-derived transcripts with the propensity to form double-stranded RNA that contribute to the innate interferon response. The identified retained introns were shown to contain non-canonical open reading frames that are translated and processed into MHC class I-presented peptides. A vaccine containing RI-derived peptides (PepMInt) was demonstrated to delay tumour growth and prolong survival in a syngeneic mouse model. T lymphocytes reactive to RI-derived peptides were detected in colorectal cancer patients and were shown to kill tumour cells in vitro. PRMT5 was found to regulate retained intron (RI)-derived transcripts with the propensity to form double-stranded RNA that contribute to the innate interferon response. The identified retained introns were shown to contain non-canonical open reading frames that are translated and processed into MHC class I-presented peptides. A vaccine containing RI-derived peptides (PepMInt) was demonstrated to delay tumour growth and prolong survival in a syngeneic mouse model. T lymphocytes reactive to RI-derived peptides were detected in colorectal cancer patients and were shown to kill tumour cells in vitro. PRMT5-regulated intron retention generates non-canonical MHC class I antigens that drive anti-tumour immunity and can be exploited for cancer vaccine development.
Wiktoria Blaszczak, Wojciech Barczak, Chu-Yue Zhang et al.· EMBO Molecular Medicine· 0 citations
The B cell receptor (BCR) dictates B lymphocyte identity and function, allowing for a robust adaptive immune response through antigen recognition. Strict regulation of BCR surface density dictates proper B cell signaling, immune regulation, and the prevention of malignancy, yet the factors regulating this density remain undefined. To systematically identify these regulators, we performed a genome-wide CRISPR interference (CRISPRi) screen in Ramos B cells, a human non-Hodgkin’s lymphoma cell line that undergoes constitutive somatic hypermutation (SHM) and identified Exportin-5 (XPO5) as a central regulator of BCR surface expression.
We transduced dCas9-KRAB Ramos cells with a genome-wide guide RNA library and isolated BCR-positive and -negative populations by flow cytometry. Hits were validated with individual guide RNAs. Mechanisms were investigated using bulk and small RNA sequencing, and BCR mutation frequencies were quantified via the TRUST4 pipeline.
XPO5 deficient cells exhibited an accelerated loss of surface BCR with no change in transcript levels, suggesting a potential post-transcriptional regulatory mechanism. Further analysis revealed XPO5 depletion led to increased mutation rates specifically within the BCR light chain, implicating XPO5 in regulating the rate and fidelity of somatic hypermutation (SHM). Transcriptomic and small RNA sequencing revealed a global reduction in miRNA levels and enrichment of target gene sets indicative of cell cycle arrest and increased DNA damage response.
We identify XPO5 as a multi-faceted regulator of B cell identity. By maintaining the global miRNA landscape, XPO5 supports cell cycle progression and regulates the fidelity of somatic hypermutation. Loss of XPO5 uncouples these processes, driving light chain biased hypermutation and the loss of functional BCR surface expression. This highlights a novel link between miRNA nuclear export machinery and the preservation of BCR integrity.
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Immune Response Regulation: Molecular Mechanisms (IRM)
Tylar Kirch, Zachary D. Miller, Jake Dearborn et al.· Journal of Immunology· 0 citations
It is demonstrated that reconfiguring existing protein domains may uncover non-evolved genes that program therapeutically relevant cell states that program therapeutically relevant cell states.
Oliver Takacsi-Nagy, Sivakanthan Kasinathan, Austin Hartman et al.· Cell· 0 citations
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