Tomalka, J. | Search bioRxiv

bioRxiv2026

Microbiome-Derived Metabolites Shape CD4⁺ T-Cell Differentiations and Immune Aging in Chronic HIV-1 Infection

The role of aromatic gut-derived bacterial metabolites (GDBMs) in shaping immune cell metabolism and function remains poorly explored. Using ex vivo metabolomic profiling of paired plasma and CD4 T-cells from people living with HIV-1 (PLWH), we identified a network of aromatic GDBMs whose cell-associated abundance, rather than systemic levels, was linked to broad alterations in CD4 T-cell metabolic and functional states. Among these metabolites, p-cresol sulfate (PCS) emerged as a mechanistic prototype investigated in depth. Ex vivo flow cytometry and single-cell RNA sequencing of CD4 T-cells stratified by cell-associated PCS levels revealed dose-dependent enrichment of transcriptional programs associated with impaired differentiation capacity, regulatory-like identity, and cellular senescence. Consistently, in vitro transcriptomic and proteomic analyses of PCS-exposed CD4 T cells demonstrated induction of cell-cycle arrest, mitochondrial dysfunction, and senescence-associated programs, including upregulation of p16 and p21. Integration of these immunometabolic features with measurements of HIV-1 reservoir size in PLWH revealed that CD4 T-cell states defined by cell-associated GDBMs track with intact proviral DNA levels in vivo. Together, these findings define a microbiome-derived axis that reshapes CD4 T-cell metabolism and fate and promotes immune aging-associated states in PLWH. Our data suggest that cell-associated GDBMs may foster immunometabolic CD4 T-cell states previously linked to long-term HIV-1 reservoir persistence in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/699280v1_figa1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@faf44aorg.highwire.dtl.DTLVardef@1bc590aorg.highwire.dtl.DTLVardef@79c557org.highwire.dtl.DTLVardef@8b0a64_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO PCS-driven metabolic reprogramming and senescence promoting CD4+ T-cell immune cell aging.Dietary proteins are metabolized by proteolytic gut microbiota into p-cresol, which is absorbed and converted in the liver to PCS. Circulating PCS accumulates in CD4 T-cells, where it activates the aryl hydrocarbon receptor (AhR). AhR signaling reduces glycolysis and mTOR activity, while enhancing TGF-, Wnt/-catenin, and TCF7 pathways, driving a regulatory-like and stem-like transcriptional program. These changes are associated with increased expression of p16 and p21, leading to cell cycle arrest and cellular senescence promoting CD4+ T-cell immune cell aging. C_FIG

systems biology↗

bioRxiv2025

Innate antiviral readiness drives the expansion of protective T stem cell memory against influenza

The development of T-cell-based influenza vaccines relies on eliciting broad CD8+ T-cell immunity, wherein T stem cell-like memory (TSCM) cells serve as the ultimate long-lived reservoir for immune memory, thereby unlocking the potential for durable protection against viral drift and shift. However, the specific immunological cues that drive the robust expansion and functional preservation of this self-renewing, multipotent subset remain unknown. Here, utilizing multi-omic systems immunology in a pediatric cohort immunized with live attenuated influenza vaccine, we identified the determinants governing the expansion of influenza virus-reactive TSCM cells. We show that a pre-existing state of innate antiviral readiness, defined by a plasmacytoid dendritic cell-associated type I interferon signature, is the requisite condition for a robust TSCM expansion. Mechanistically, this baseline innate state enhances antigen priming and enforces a qualitative divergence in T-cell fate, driving responders toward a functionally poised, Th1-dominant phenotype while non-responders default to a dysfunctional, hyper-proliferative state. To determine the clinical relevance of this cellular subset, we analyzed an independent controlled human influenza challenge study. This validation revealed a critical functional division of labor in host defense: whereas pre-existing antibodies primarily mitigated symptom severity, the baseline frequency of influenza virus-reactive TSCM cells was the strongest predictor of rapid viral load clearance. These findings establish that the expansion of durable cellular memory is not stochastic but is predetermined by the innate cytokine environment, providing a predictive biomarker for patient stratification and a validated target for adjuvants designed to expand the TSCM reservoir deliberately.

immunology↗