A Hierarchy of Intestinal Antigens Instructs the CD4+ T Cell Receptor Repertoire

Rheumatology and Immunology Infectious Diseases Gastroenterology Life Sciences Medicine Immunology
intestinal immunity T cell receptor microbiota dietary antigens inflammatory bowel disease

I. Research Background

The intestinal immune system must balance tolerance and defense against dietary antigens, microbiota-derived antigens, and self-antigens. Although CD4+ T cells are known to play a central role in gut immunity, how different antigen sources shape the composition of the T cell receptor (TCR) repertoire remains unclear. The traditional view holds that the small intestine (SI) is the primary site for dietary antigen tolerance, while the colon regulates responses to microbial antigens, but this “biogeographic determinism” lacks genome-wide validation. Furthermore, the complex interactions between diet and microbiota in influencing T cell differentiation (e.g., the balance between regulatory T cells [Treg] and effector T cells [Teff]) are poorly understood. This study establishes a hierarchical TCR classification framework to systematically dissect the regulatory hierarchy of intestinal antigens on the TCR repertoire and reveals the dynamic interplay between diet, microbiota, and immunity in homeostasis and colitis.

II. Paper Source

This study was co-authored by Jaeu Yi, Jisun Jung, et al., with corresponding author Chyi-Song Hsieh (Division of Rheumatology, Washington University School of Medicine). Collaborating institutions include Pohang University of Science and Technology (Korea) and the University of Missouri. The paper was published in May 2025 in the top-tier immunology journal Immunity (DOI: 10.1016/j.immuni.2025.04.011).

III. Research Process and Results

1. Establishment of the Hierarchical TCR Classification Framework

Experimental Design:
- Study Subjects: Transgenic mice with a fixed TCRβ chain (TCLIβ TCRβ tg) were used to limit TCR diversity and track α-chain variations.
- Layered Conditions:
- Antigen-Free (AF) Group: Germ-free (GF) mice fed a peptide-free amino acid diet (AAD), exposed only to self-antigens.
- Dietary Antigen (GF) Group: GF mice fed normal chow diet (NCD), adding dietary antigens.
- Microbiota Antigen (SPF) Group: Specific-pathogen-free (SPF) mice fed NCD, introducing complex microbiota antigens.
- Methods:
- Flow-sorted Foxp3+ Treg and Foxp3− Teff cells from SI and colon were subjected to TCRα sequencing.
- Two classification strategies: Reproducible TCR analysis (TCRs present in >30% of samples) and Total TCR analysis (based on >25-fold frequency differences).

Key Results:
- Microbiota Dominates TCR Repertoire Remodeling: NMDS analysis showed that TCR repertoire differences between SPF and GF mice far exceeded those between GF and AF mice (Figure 1B), indicating microbiota’s greater impact than diet.
- Asymmetric T Cell Differentiation: In SPF mice, microbiota-dependent TCRs were enriched in Teff cells, while Treg cells contained self-, diet-, and microbiota-dependent TCRs (Figures 1D-E).

2. Micro-Immunological Features of Dietary Antigens

Findings:
- Diet-Reactive T Cells in the Colon: Contrary to the belief that dietary antigens induce tolerance only in the SI, some diet-dependent TCRs (e.g., D1) were enriched in the colon (Figure 3B).
- Microbiota Regulates Dietary Antigen Presentation: In SPF mice, diet-dependent TCR frequencies were generally lower than in GF mice, but specific TCRs (e.g., D1) were significantly enhanced by microbiota (Figures 3G-H).

Validation Experiments:
- In Vitro Hybridoma Assay: D1 TCR recognized plant components like corn, while D2 TCR reacted only to corn (Figure 3D).
- In Vivo Proliferation Assay: Retrovirally transduced (RV) D1 TCR cells proliferated in NCD-fed mice but not in AAD-fed mice (Figure 3E).

3. Antigen-Free Diet (AAD) Modulates T Cell Responses via Microbiota

Mechanistic Insights:
- AAD Alters Microbiota Composition: 16S rRNA sequencing revealed that AAD feeding significantly reduced segmented filamentous bacteria (SFB) in the colon (Figures 4J-K).
- Identification of SFB-Specific TCRs: Correlation analysis linked Teff TCR M3 to SFB abundance (Figure 5A). Monocolonization experiments confirmed that M3 and M4 TCRs were activated only in the presence of SFB (Figures 5D-E).

Clinical Implications:
- Colitis Treatment: In a DSS/anti-IL-10R-induced colitis model, switching to AAD alleviated inflammation, dependent on adaptive immunity (Figure 6A). TCR analysis showed that 50% of colitis-induced Teff TCRs originated from the steady-state Treg pool (e.g., D1), suggesting inflammation disrupts tolerance (Figure 6F).

4. Network Analysis Reveals T Cell-Microbiota Interactome

Methodological Innovation:
- TCR-ASV (amplicon sequence variant) networks were constructed based on Pearson correlation coefficients (r > 0.8) (Figure 7A).
- Key Discoveries:
- Inflammation-Driving Microbes: Lachnospiraceae family ASVs correlated strongly with weight loss and connected multiple de novo TCRs (Figure 7G).
- Immunologically Silent Microbes: Akkermansiaceae expanded during colitis but failed to trigger TCR responses (Figures 7C-D).

IV. Research Conclusions and Impact

  1. Scientific Significance:
    • Proposes the “antigen hierarchy” concept, quantifying contributions of self, diet, and microbiota to the TCR repertoire.
    • Challenges the “SI-exclusive dietary tolerance” hypothesis, revealing the colon’s role in food antigen responses.
  2. Practical Value:
    • Offers new therapeutic targets for food allergies (e.g., FPIES) and inflammatory bowel disease (IBD) (e.g., targeting SFB or dietary antigens).
    • Clarifies how elemental diets exert therapeutic effects by modulating microbiota rather than merely reducing antigen exposure.

V. Research Highlights

  • Methodological Innovation: Pioneers hierarchical TCR classification, combining macro/micro-immunological analyses.
  • Paradigm-Shifting Discovery: Microbiota suppresses diet-reactive Teff cells by competing for antigen presentation, explaining the “hygiene hypothesis.”
  • Integrated Technologies: Single-cell RNA-seq (Figures 5H-J) and network analysis (Figure 7) multi-dimensionally validate hypotheses.

VI. Additional Information

  • Data Availability: TCR and 16S data are deposited in the European Nucleotide Archive (ENA: PRJEB82363/PRJEB82415).
  • Limitations: Fixed TCRβ models may overlook complexities of polyclonal TCR repertoires, requiring future high-throughput αβ paired sequencing for validation.