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)
Dhaneshwar Kumar, S. Sahoo, B. Yan et al.· Journal of Immunology· 0 citations
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.
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.
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.
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.
NHLBI/NIH intramural program
Immune Mechanisms of Human Disease (HUM)
Erin E. West, Nicolas S. Merle, Ayden Case et al.· Journal of Immunology· 0 citations