Abstract Introduction Complement is often viewed as liver-derived and extracellular, yet many non-hepatic cells generate intracellular complement. Proximal tubular epithelial cells (TECs) drive kidney injury and fibrosis and express complement, but how de novo TEC complement controls metabolism, chromatin, and fibrogenesis is unknown. We asked whether a TEC-intrinsic complement axis links inflammatory signaling to metabolic and epigenetic reprogramming after injury. Methods Mouse TEC injury models were combined with confocal microscopy, Seahorse metabolic profiling, single-nucleus and bulk RNA-seq, CUT&Tag, MNase-seq, and EMSA. C3 and CFB were perturbed using conditional C3 deletion, cell-permeable CFB inhibitors, and structure-guided CFB mutants informed by AlphaFold2/GraphSite modeling. Human kidney single-cell and bulk transcriptomes were analyzed for conservation and clinical relevance. Results IL-1β—NF-κB/c-JUN induced C3 and CFB in injured TECs. C3 accumulated in mitochondria and co-localized with CFB, which processed C3, rewired metabolism, and promoted profibrotic programs. CFB also accumulated in nuclei; CUT&Tag/EMSA and modeling defined a DNA-binding motif and pocket in CFB. Nuclear CFB cleaved histones, blocked by CFB inhibitors, and MNase-seq showed increased nucleosome protection with CFB inhibition. Conditional C3 deletion or intracellular CFB inhibition reduced TEC metabolism and attenuated post-injury interstitial fibrosis in mice. In human kidney datasets, TEC C3-CFB co-expression associated with injury signatures and inversely correlated with kidney function. Conclusion A TEC-intrinsic C3—CFB complement axis links inflammatory cytokine signaling to mitochondrial metabolism, chromatin state, and fibrosis after kidney injury. Nuclear CFB acts as a DNA-binding protease that modulates histones and nucleosomes. Targeting intracellular, rather than systemic, complement may limit inflammatory kidney disease while preserving host defense. Funding Source NIH Intramural Research Program Apellis Pharmaceuticals Inc, Waltham, MA, United States Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Abstract Introduction Systemic complement protects the vascular compartment, while cell-intrinsic complement components shape tissue immunity by regulating normal cell physiology and metabolism. In human CD4 T helper cells, intracellular C5a generation engages the intracellular receptor C5aR1, promoting Th1 differentiation. Here, we identify a counter-regulatory role for the alternative C5a receptor, C5aR2, in restraining T cell effector responses by studying the first reported family with a heterozygous C5aR2 mutation. The affected individuals present with an autoinflammatory syndrome, and disease penetrance tracks with the mutation: the affected mother and child carry the variant, whereas the unaffected father and siblings do not. Methods To define the impact of the C5aR2 mutation, we performed single-cell RNA sequencing, in vitro stimulation assays, and flow cytometric profiling of patient and control PBMCs. CPM inhibition or deletion, C5aR2 agonism, and transcriptional profiling of Cpm- or C5ar2-deficient mouse CD4 T cells, along with a T cell transfer colitis model, were used to define mechanistic consequences. Results Patient samples exhibited a profound loss of naïve and central memory CD4 and CD8 T cells, accompanied by expansion of IFN-γ—producing effector memory populations. The C5aR2 mutation abolishes C5aR2 β-arrestin signaling. Additionally, we identified carboxypeptidase M (CPM) as a T cell—intrinsic enzyme generating C5a-desArg, a potent ligand for C5aR2. Loss or inhibition of CPM heightened inflammatory T cell responses, which were normalized by C5aR2 agonism. Mouse Cpm- or C5ar2-deficient CD4 T cells displayed overlapping transcriptional perturbations, and Cpm-knockout CD4 T cells induced more severe colitis. Conclusion Collectively, our data uncover a previously unrecognized CPM-dependent mechanism that balances C5aR1 and C5aR2 signaling to limit pathological T cell activation, revealing an intrinsic complement-driven checkpoint that constrains effector T cell immunity. Funding Source NHLBI/NIH intramural program Topic Categories Immune Mechanisms of Human Disease (HUM)

While inputs regulating CD4+ T helper (Th) cell differentiation are well defined, the integration of downstream signaling with transcriptional and epigenetic programs that define Th lineage identity remains incompletely resolved. PI3K signaling is a critical regulator of T cell function; activating mutations affecting PI3Kδ result in an immunodeficiency with multiple T cell defects. Using mice expressing activated PI3Kδ, we found aberrant expression of proinflammatory Th1 signature genes under Th2-inducing conditions, both in vivo and in vitro. This dysregulation was driven by a PI3Kδ-IL-2-Foxo1 signaling amplification loop, fueling Foxo1 inactivation, loss of Th2 lineage restriction, and extensive epigenetic reprogramming. Surprisingly, ablation of Fasl, a Foxo1-repressed gene, normalized both Th2 differentiation and TCR signaling. BioID and imaging revealed Fas interactions with TCR signaling components, which were supported by Fas-mediated potentiation of TCR signaling that could occur in the absence of FADD. Our results highlight Fas-FasL signaling as a critical intermediate in phenotypes driven by activated PI3Kδ, thereby linking two key pathways of immune dysregulation.

Abstract Introduction IL-2 is central to CD8 T cell responses and signals via JAK-STAT5, RAF-ERK-MAPK, and PI3K-AKT pathways, but how STAT5-dominant output is enforced from this shared receptor is unclear. We hypothesized that uncharacterized IL-2-induced, STAT5-regulated E3 ubiquitin ligases sculpt this signaling hierarchy to optimize antiviral CD8 T cell function. Methods RNA-seq of human CD8 T cells stimulated with IL-2 ± JAK inhibition plus STAT5 ChIP-seq identified IL-2/STAT5-regulated ubiquitin genes. Rnf144a-/- mice and mixed bone marrow chimeras were used to define CD8-intrinsic roles during influenza infection. RNF144A localization and substrates were mapped by biochemical and imaging assays, and whole-blood transcriptomes from patients with moderate or severe influenza were analyzed to relate RNF144A expression and gene signatures to clinical severity. Results IL-2 broadly remodeled the T cell ubiquitin program, with RNF144A emerging as the most strongly induced STAT5-bound E3 ligase. RNF144A localized to the plasma membrane, associated with IL-2Rβ and STAT5, and enhanced STAT5 recruitment and phosphorylation, sustaining STAT5-dependent transcription. In parallel, RNF144A acted as a bona fide E3 ligase that directly polyubiquitinated RAF1 for degradation, reducing ERK activation and thereby preserving JAK-STAT5 dominance over RAF-ERK-MAPK output. CD8 T cells from Rnf144a-/- mice showed impaired IL-2-induced effector gene expression, degranulation, and cytokine production, and CD8-intrinsic deficiency in mixed chimeras reduced antigen-specific responses and worsened weight loss and lung inflammation after influenza infection. In human influenza, RNF144A expression was reduced in severe disease, inversely correlated with an ERK-MAPK target gene signature, and discriminated severe from moderate cases with performance comparable to established severity markers. Conclusion RNF144A is an IL-2-STAT5-induced E3 ligase that enforces STAT5-dominant signaling and limits viral immunopathology. Funding Source N/A Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Abstract Introduction 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. Methods 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. Results 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. Conclusion 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. Funding Source N/A Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Abstract Introduction IL-2-STAT5 signaling is vital for CD4+ T cell function, whereas BACH2 restrains activation. How these transcription factors co-operate at the genome remains unclear. Intracellular C3 supports T cell survival and effector programming, but how it is transcriptionally controlled and whether it matches serum C3 is unknown. We hypothesized that a BACH2-STAT5 module coordinates C3 expression and licenses a distinct C3 proteoform in activated CD4+ T cells. Methods We profiled wild-type and Bach2—/— CD4+ T cells by RNA-seq and mapped STAT5 and BACH2 binding using ChIP-seq and H3K27ac Hi-ChIP. STAT5 and BACH2 were re-expressed in Stat5—/— T cells, and shared elements at the C3 locus were deleted by CRISPR. IL-2/STAT5 inhibition, C3 overexpression or knockdown, and complement activation assays defined C3 expression and function. AlphaFold2 modeling and domain analyses characterized novel C3 proteoforms. Results IL-2/STAT5 target genes were enriched in Bach2—/— CD4+ T cells, with overlapping STAT5 and BACH2 binding at differentially expressed loci. Combined activating or repressive control was confirmed by re-expression in Stat5—/— cells. At the C3 locus, BACH2-STAT5 co-occupancy and IL-2/STAT5 signaling were required for C3 transcription; C3 was lost in Bach2—/— cells, after IL-2/STAT5 blockade, or upon CRISPR deletion of shared elements. Resting CD4+ T cells expressed canonical, signal peptide-containing C3, whereas activation induced a shorter, signal peptide-deficient transcript predicted to encode an intracellular proteoform. Purified short C3 failed to seed classical C3 convertase or support hemolysis, indicating altered function. Conclusion We identify a BACH2—STAT5 transcriptional module that is essential for C3 expression in activated CD4+ T cells and redirects C3 output toward a short, intracellular proteoform with noncanonical complement activity. Proteoform-specific, T cell-intrinsic complement suggests new opportunities to target BACH2-STAT5 and C3 in autoimmunity and immunotherapy. Funding Source NA Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Background: Complement is classically viewed as a liver-derived, extracellular defense cascade, but many components are also transcribed by parenchymal cells, where they regulate metabolism and injury responses. The kidneys, which are highly susceptible to complement-mediated damage, commonly exhibit local complement deposition, yet the extent, distribution, and function of de novo complement produced by renal cells remain poorly defined. We asked whether tubular epithelial cell (TEC)-derived complement shapes the metabolic and fibrotic sequelae of acute kidney injury (AKI). Methods: We analyzed existing and de novo bulk and single-cell high-throughput data from multiple acute kidney injury (AKI) models, followed by CUT&RUN, CRISPR editing, and regulon analysis to map transcription factor occupancy at complement loci. Mechanistic studies used proximal TEC specific C3 knockout mice, a cell permeable factor B (CFB) inhibitor, metabolic flux (Seahorse) profiling, confocal and histological imaging, and mass spectrometric mapping of C3. Human relevance was explored in transcriptomic cohorts of human disease. Results: Across AKI models, folic-acid nephropathy (FAN) showed the strongest enrichment of complement genes. Single-nucleus RNA-seq revealed injury-induced Vcam1+ TECs co-expressing C3 and Factor B (Cfb), which signalled to infiltrating immune cells via C3a-C3AR1 and integrins. An inflammatory cytokine was the dominant upstream inducer: deletion of its receptor reduced C3 and Cfb transcripts in response to injury. Two downstream transcriptional regulators were confirmed as transactivators at both genes, as confirmed by CUT&RUN and CRISPR disruption, and their regulon activity correlated with C3/Cfb expression. Confocal imaging tracked C3 trafficking from ER/Golgi to endolysosomes and mitochondria, co-localizing with CFB. Conditional C3 deletion or intracellular CFB inhibition lowered glycolytic and oxidative phosphorylation and attenuated interstitial fibrosis in FAN mice. In human kidney datasets, C3 and CFB co-expression inversely correlated with eGFR. Conclusion: Inflammatory-cytokine–driven transcription of C3 and factor B in proximal TECs couples intracellular complement activation to metabolic reprogramming and fibrogenesis after kidney injury. Targeting this intrinsic complement axis offers a therapeutic avenue beyond systemic complement blockade for inflammatory kidney diseases. References: This abstract has been submitted by the PI on behalf of an ECR.

While inputs regulating CD4+ T helper cell (Th) differentiation are well-defined, the integration of downstream signaling with transcriptional and epigenetic programs that define Th-lineage identity remain unresolved. PI3K signaling is a critical regulator of T cell function; activated PI3Kδ syndrome (APDS) is an inborn error of immunity caused by heterozygous activating mutations in PI3Kδ. APDS patients have recurrent respiratory infections (Th1-type) and develop allergic (Th2-type) diseases; we therefore hypothesized that activated-PI3Kδ (Pik3cdE1020K) signaling may alter CD4 T cell differentiation. Using mice expressing activated-PI3Kδ, we found aberrant expression of proinflammatory Th1-signature genes under Th2-inducing conditions, following both house dust mite induced asthma in vivo and CD4 T cell polarization in vitro. This dysregulation was driven by a robust PI3Kδ-IL-2-Foxo1 signaling loop, fueling Foxo1-inactivation, loss of Th2-lineage restriction, altered chromatin accessibility and global impairment of CTCF-DNA interactions. Surprisingly, ablation of Fasl, a Foxo1-repressed gene, restored normal Th2 differentiation, TCR signaling and CTCF expression. BioID proximity labeling revealed Fas interactions with TCR-signaling components, which were supported by Fas-mediated potentiation of TCR signaling. Our results highlight Fas-FasL signaling as a critical intermediate in phenotypes driven by activated-PI3Kδ, thereby linking two key pathways of immune dysregulation. This work was supported by the Division of Intramural Research of NIAID, NIH. Lymphocyte Differentiation and Peripheral Maintenance (LYM)

The complement system is an important component of the innate immune system involved in host defense and maintaining homeostasis. While the liver is the main source of complement proteins in the bloodstream, recent research has shown that various tissues, including the kidneys, can produce complement components locally in response to both acute and chronic inflammation. This Review highlights evidence from animal models of glomerular and tubulointerstitial kidney disease showing increased expression of intracellular complement in the kidneys. Studies using knockout mice for complement and complement receptors, along with complement inhibitors, have demonstrated that reduced complement activation in animal models of kidney fibrosis led to reduced inflammation and fibrosis, thereby supporting the pathogenic role of complement activation. Data from single-cell RNA-sequencing, spatial transcriptomics, and proteomics studies further demonstrate that alterations in local complement levels contribute to the fibrotic microenvironment observed in these models. Additionally, kidney biopsy results from patients with acute kidney injury and chronic kidney disease (CKD) indicate an increased expression of intracellular complement components as disease progresses. Developing drugs aimed at diminishing the expression and activation of local complement in glomerular and tubulointerstitial kidney disease could provide a novel approach to managing CKD.

Background: Understanding the mechanisms that drive (acute kidney injury (AKI) and subsequent tissue repair is crucial for developing novel therapeutic strategies to halt inflammation and prevent tissue scarring. Intracellular and locally secreted complement components are increasingly recognised as modulators of tissue injury and repair, yet the spatial niches in which they operate within the kidney remain undefined. Standard single-cell RNA-seq detects extra-hepatic complement transcription but loses positional context, obscuring micro-domains where complement mediates crosstalk among parenchymal, stromal, and immune cells during acute kidney injury (AKI). Methods: Formalin-fixed paraffin-embedded kidney sections from mouse models of ischaemia–reperfusion injury (IRI), unilateral ureteral obstruction (UUO), and sham surgery underwent 10x Visium HD and Xenium in-situ–hybridisation spatial transcriptomics. Data were pre-processed, normalised, and clustered, followed by differential-expression, pathway-enrichment, cell-neighbourhood, and ligand–receptor interaction analyses. Key transcripts and proteins were validated by RNAscope, RT-qPCR, immunoblotting, and confocal microscopy. Results: Spatial maps revealed a loss of healthy S1/S2 proximal tubules and expansion of VCAM1+ injured tubules in both IRI and UUO compared with shams. Injured tubules showed robust up-regulation of C3 and Cfb, while stromal cells and macrophage clusters selectively increased other complement components. Cell-neighbourhood analysis identified discrete loci where C3-high tubules and C3ar1-high macrophages were spatially adjacent. Ligand-receptor modelling confirmed significant C3-C3AR1 and integrin-mediated signalling within these niches. RNAscope and confocal imaging validated C3 protein localisation to injured tubules and C3AR1 to nearby macrophages. Conclusion: High-resolution spatial transcriptomics uncovers compartmentalised complement expression and signalling hotspots in AKI, linking injured proximal tubules with infiltrating macrophages through local C3 production. These findings provide a tissue atlas for targeting intrinsic complement pathways and will guide ongoing translation to human kidney biopsies. References: This abstract has been submitted by the PI on behalf of an ECR.

Background: Intracellular production of complement component 3 (C3) has emerged as indispensable for T-cell survival, proliferation, and the metabolic reprogramming that fuels Th1 differentiation. Cytokine-responsive transcriptional networks govern CD4+ T-cell fate and effector function and are vital for dictating effective versus aberrant responses. Yet how cytokine-activated transcription factors (TFs) are integrated in the context of other broadly modulatory TFs and cooperate at chromatin to govern C3 and other metabolism-linked genes remains incompletely understood. Methods: Here, we studied a cytokine-responsive transcription factor (TF-A) and a broadly modulatory transcription factor (TF-B). We combined bulk RNA-seq from wild-type and TF-A-deficient CD4+ T cells with TF-specific ChIP-seq and H3K27ac Hi-ChIP to map binding and enhancer-promoter contacts. Functional relevance was tested using TF over-expression, competition assays, CRISPR-Cas9 editing of cis-elements, pharmacological blockade of cytokine signalling, and C3 reporter mice. Results: We found pronounced enrichment of cytokine-responsive and complement gene signatures in TF-B-deficient CD4+ T cells. There was ~40% overlap between TF-A and TF-B-bound loci genome-wide and ~70% of differentially expressed genes displayed overlapping TF-A/TF-B binding, indicating extensive genomic co-occupancy. TF-A and TF-B single and co-expression experiments, followed by RNAseq, in TF-A-deficient T cells resolved distinct clusters where the factors acted synergistically or antagonistically. This was epitomized at the C3 locus, where TF-A/B co-occupancy was essential for expression, evidenced by lost C3 transcription in TF-B–/– cells, inhibition of cytokine signalling or deletion of bound loci. Hi-ChIP showed activation-dependent looping between these elements and the C3 promoter. Conclusion: Co-ordinated action of a cytokine-responsive factor (TF-A) and a broadly modulatory factor (TF-B) is essential for complement C3 induction during CD4+ T-cell activation. This mechanistic framework links cytokine signalling to intracellular complement regulation and offers a tractable axis for modulating T-cell-driven inflammation. References: 1.Liszewski, Kolev et al. Immunity 2013; 39(6): 1143–1157.2.Kolev et al. Immunity 2020; 52(3): 513–527. References: This abstract has been submitted by the PI on behalf of an ECR

While inputs regulating CD4+ T helper cell (Th) differentiation are well-defined, the integration of downstream signaling with transcriptional and epigenetic programs that define Th-lineage identity remain unresolved. PI3K signaling is a critical regulator of T cell function; activating mutations affecting PI3Kδ result in an immunodeficiency with multiple T cell defects. Using mice expressing activated-PI3Kδ, we found aberrant expression of proinflammatory Th1-signature genes under Th2-inducing conditions, both in vivo and in vitro. This dysregulation was driven by a robust PI3Kδ-IL-2-Foxo1 signaling loop, fueling Foxo1-inactivation, loss of Th2-lineage restriction, altered chromatin accessibility and global impairment of CTCF-DNA interactions. Surprisingly, ablation of Fasl, a Foxo1-repressed gene, restored normal Th2 differentiation, TCR signaling and CTCF expression. BioID revealed Fas interactions with TCR-signaling components, which were supported by Fas-mediated potentiation of TCR signaling. Our results highlight Fas-FasL signaling as a critical intermediate in phenotypes driven by activated-PI3Kδ, thereby linking two key pathways of immune dysregulation.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendric cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

Immune cell function is critically dependent on precise control over transcriptional output from the genome. In this respect, integration of environmental signals that regulate gene expression, specifically by transcription factors, enhancer DNA elements, genome topography and non-coding RNAs (ncRNAs), are key components. The first three have been extensively investigated. Even though non-coding RNAs represent the vast majority of cellular RNA species, this class of RNA remains historically understudied. This is partly because of a lag in technological and bioinformatic innovations specifically capable of identifying and accurately measuring their expression. Nevertheless, recent progress in this domain has enabled a profusion of publications identifying novel sub-types of ncRNAs and studies directly addressing the function of ncRNAs in human health and disease. Many ncRNAs, including circular and enhancer RNAs, have now been demonstrated to play key functions in the regulation of immune cells and to show associations with immune-mediated diseases. Some ncRNAs may function as biomarkers of disease, aiding in diagnostics and in estimating response to treatment, while others may play a direct role in the pathogenesis of disease. Importantly, some are relatively stable and are amenable to therapeutic targeting, for example through gene therapy. Here, we provide an overview of ncRNAs and review technological advances that enable their study and hold substantial promise for the future. We provide context-specific examples by examining the associations of ncRNAs with four prototypical human autoimmune diseases, specifically rheumatoid arthritis, psoriasis, inflammatory bowel disease and multiple sclerosis. We anticipate that the utility and mechanistic roles of these ncRNAs in autoimmunity will be further elucidated in the near future.

Mutational detail in EBV (S3) and host samples(S4). Full legend is provided in Supplementary methods.

Table S5J-part3, expression values (transcripts per million; T.P.M.) of all genes (rows) in each sample (columns).