Rapid expansion of Treg cells protects from collateral colitis following a viral trigger

Regulatory T cells expressing FOXP3 are critical for immune homeostasis, and defects in Tregs are associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Although genetic predispositions exist, environmental triggers, particularly viral infections, are implicated in initiating disease. Type I interferons, central to antiviral defense, can exacerbate autoimmunity and are linked to disease flares and IFN-based therapies precipitating autoimmune manifestations. How type I IFN-dominated antiviral responses impact Treg stability and function to maintain peripheral tolerance remains unclear. Using LCMV infection, which elicits robust type I IFN responses, the study asks how viral infection alters the Treg compartment, whether induced Tregs compensate for Treg loss, what TCR specificities are involved, and how these dynamics affect antiviral immunity and collateral intestinal immunopathology.

Prior work established that FOXP3+ Tregs prevent autoimmunity and that reduced Treg numbers or function is observed in RA, SLE, and IBD. Viral infections are associated with triggering autoimmune diseases, and gastrointestinal viral infections correlate with later IBD development. Type I IFNs, while protective against viruses, aggravate autoimmune conditions and IFN therapies have precipitated SLE and systemic sclerosis. Reports indicate that type I IFNs can antagonize Treg function during infection and in tumors, and that LCMV triggers potent type I IFN responses that shape immunity. Markers such as Nrp1/Helios distinguish thymic versus peripherally induced Tregs. Microbiota and barrier integrity influence colitis, and some T cell responses may be driven by superantigens. This background motivates examining how IFN-driven viral responses disrupt Treg homeostasis and how the Treg pool adapts to restore tolerance.

• Animal models: C57BL/6 mice and transgenic/reporters including Foxp3-GFP.KI, Nr4a1-GFP (Nur77), Ifnar1−/−, DEREG (for diphtheria toxin-mediated Treg depletion), Tcrb−/−Tcrd−/− (T cell-deficient), and TCR-defined reconstitution models lacking Vβ5+ CD4 T cells. Present address strains and housing per Swiss regulations.
• Viral infections: LCMV strains inducing acute (WE/Armstrong) or chronic (Clone 13) infection; comparative infections with vaccinia virus and murine cytomegalovirus that elicit weaker type I IFN responses. Viral titers measured by foci-forming assays.
• Flow cytometry: Quantified Treg (CD4+Foxp3+) and conventional T cell subsets, CXCR3 and Nrp1 to distinguish iTreg versus nTreg, expression of Treg signature markers (CTLA-4, TIGIT, PD-1, Lag3, Tim-3, CD39), and TCR Vβ usage (Vβ5, Vβ8, etc.). BrdU incorporation assessed proliferation. Nur77-GFP used to quantify TCR signaling; CD5 levels as a proxy for TCR tuning.
• TCR repertoire analysis: RNA-seq-based TCR Vβ transcript usage in Foxp3+ versus Foxp3− T cells; clonal overlap and polyclonality analyses focusing on Vβ5+ Tregs.
• Adoptive transfer/reconstitution: Tcrb−/−Tcrd−/− mice reconstituted with Vβ5− CD4+ T cells plus or minus Vβ5+ CD4+ T cells; selective depletion of Vβ5+ Tregs in DEREG donors via diphtheria toxin to isolate Treg-specific effects; adoptive transfer of sorted Vβ5+Foxp3+ Tregs from infected donors to rescue colitis.
• Type I IFN dependence: Compared WT and Ifnar1−/− mice; assessed whether type I IFN directly drives Vβ5+ iTreg conversion in vitro and via transfer into Ifnar1−/− environments.
• Barrier function and microbiota: FITC–dextran gavage to assess intestinal permeability post-infection; broad-spectrum antibiotics (ampicillin, vancomycin, neomycin, metronidazole) to deplete microbiota; stimulation of splenocytes with gut content/MAMPs to measure CD8+ and CD4+ cytokine responses.
• In vitro iTreg induction: Naive CD4+ T cells stimulated with anti-CD3/anti-CD28, TGF-β, and IL-2 under optimal and suboptimal conditions to compare Vβ5+ versus Vβ5− conversion.
• Human studies: Peripheral blood from healthy donors and IBD patients (active vs inactive disease); flow cytometric quantification of Vβ chains (Vβ2, Vβ5.1, Vβ17) among CD4+CD127lowCD25+ Tregs versus CD4+CD127+CD25− effectors; TCR sequencing datasets referenced. Ethics approvals and informed consent documented.
• Statistics: Mann–Whitney U and one-way ANOVA with appropriate post hoc tests; data presented as mean ± SD.

• LCMV infection causes a transient loss of Treg cells at the peak response (day 5–7), with recovery dominated by CXCR3+Nrp1− induced Tregs (iTregs). Chronic LCMV leads to especially pronounced iTreg expansion, accounting for over half of the Treg pool by day 14.
• TCR usage shifts: All tested LCMV strains selectively enrich Vβ5+ Tregs; RNA-seq confirmed Vβ5.2 transcripts specifically enriched in Tregs during infection. Vaccinia and MCMV, which induce weaker type I IFN responses, did not elicit similar Vβ5+ Treg enrichment.
• Type I IFN dependence: Ifnar1−/− mice showed strongly reduced frequencies and numbers of Vβ5+ iTregs, indicating that the Vβ5+ enrichment arises indirectly from type I IFN–mediated Treg niche loss rather than a direct effect on conversion. In vitro, type I IFN did not enhance CD4+Foxp3− to iTreg conversion.
• Function of Vβ5+ Tregs: Infection-induced Vβ5+ Tregs expressed Treg signature markers and IL-10 and suppressed effectively in vitro. However, in vivo, mice lacking Vβ5+ CD4+ T cells mounted largely normal LCMV-specific CD4+ and CD8+ IFNγ responses and controlled virus similarly to controls; only slight increases in IFNγ+ CD8+ T cells were observed, indicating Vβ5+ Tregs are dispensable for antiviral control.
• Protection from colitis: In the absence of Vβ5+ Tregs, LCMV-infected mice developed severe lower GI tract pathology (colitis). Selective depletion of Vβ5+ Tregs reproduced pathology, while adoptive transfer of Vβ5+Foxp3+ Tregs rescued disease; transfer of Vβ5+ non-Tregs did not. Antibiotic treatment prevented colitis, implicating microbiota. Increased gut permeability after infection was observed (elevated FITC–dextran in serum). CD8+ T cells, but not CD4+ T cells, responded to gut content stimulation with pro-inflammatory cytokines, indicating microbiota-driven, CD8-mediated colitogenic responses restrained by Vβ5+ Tregs.
• Polyclonality and priming: Vβ5+ CXCR3+ Tregs were highly polyclonal with many public clones, consistent with broad antigen or superantigen-driven activation. Depletion of microbiota did not reduce Vβ5+ iTreg induction, suggesting non-microbial antigens drive their expansion.
• Intrinsic properties of Vβ5+ T cells: Vβ5+ Tregs exhibited nearly twofold higher proliferation (BrdU incorporation) than other Treg subsets and sustained higher TCR signaling (Nur77-GFP) at steady-state and during infection, with elevated CD5 expression. Under suboptimal TGF-β/IL-2, Vβ5+ conventional CD4+ T cells converted to iTregs more efficiently than Vβ5− cells, enabling rapid Treg niche replenishment.
• Human correlate: Human Vβ2+ T cells were overrepresented in the Treg pool compared to effector T cells, and Vβ2+ Tregs were increased in IBD patients with inactive disease versus active disease and healthy donors, suggesting a compensatory expansion analogous to murine Vβ5+ Tregs.

The study links antiviral type I IFN responses to transient impairment of the Treg compartment, necessitating rapid replenishment by iTregs to maintain tolerance. A specific TCR β subset (murine Vβ5+, human Vβ2+) exhibits intrinsic properties—heightened tonic TCR signaling, elevated CD5, superior proliferation, and preferential conversion under limiting cues—that enable rapid restoration of the Treg niche. While these Vβ5+ Tregs are not essential for antiviral immunity, they are critical to prevent collateral, microbiota-driven colitis arising from increased intestinal permeability during infection and consequent activation of CD8+ T cells. The highly polyclonal nature and public clonotypes of Vβ5+ Tregs suggest activation by broad antigenic stimuli, potentially including endogenous superantigens, facilitating their rapid, repertoire-wide expansion. The findings support a division of labor: thymus-derived nTregs maintain steady-state tolerance, whereas peripherally induced iTregs—here enriched for Vβ5/Vβ2—restore regulation after IFN-driven insults. Clinically, the expansion of Vβ2+ Tregs in IBD patients with inactive disease aligns with a compensatory mechanism restoring Treg competence after inflammatory flares. These insights underscore how antiviral IFN responses can serve as environmental triggers for autoimmunity if Treg replenishment fails or is delayed.

Antiviral type I IFN responses transiently deplete Tregs, and a rapid, preferential conversion/expansion of Vβ5+ conventional CD4+ T cells into iTregs replenishes the regulatory niche in mice, preventing microbiota-driven, CD8-mediated colitis. Vβ5+ iTregs are fully suppressive but not required for antiviral control, highlighting their specialized role in maintaining barrier tolerance post-infection. Human Vβ2+ Tregs likely fulfill an analogous function and are increased in IBD patients with inactive disease, suggesting translational relevance. Future work should elucidate the molecular drivers of Vβ-biased iTreg priming (including potential superantigens and antigen-presenting cell pathways), define how IFN signaling dynamics modulate Treg niche vulnerability, and explore therapeutic strategies to enhance selective iTreg replenishment or preserve barrier tolerance during antiviral responses and IFN-based therapies.

• The mechanistic basis for the Vβ5 bias in iTreg induction and expansion remains incompletely defined; superantigen involvement is suggested but not directly proven.
• Findings are derived primarily from LCMV mouse models and may not fully generalize to other infections or to human disease.
• Human data are correlative (Vβ2+ Treg enrichment in inactive IBD) without direct demonstration of causality in preventing flares.
• The exact antigenic sources and antigen-presenting pathways driving Vβ5+ iTreg priming (e.g., roles of specific dendritic cell autophagy pathways) require further clarification.