CD4+ T cell–T cell interactions preserve clonal diversity to recognize antigen variants

Technical Summary: CD4+ T cell–T cell interactions preserve clonal diversity to recognize antigen variants

Problem Statement
Effective CD4+ T cell immunity relies on the recruitment of diverse antigen-specific clones to ensure robustness against antigenic variation. However, a paradox exists in the field: while CD4+ T cells require continuous antigenic stimulation for proliferation, and peptide–MHC-II (pMHC) complexes are long-lived in vivo, the resulting immune responses remain remarkably diverse. Standard models suggest that sustained stimulation and inter-clonal competition should drive the preferential expansion of high-avidity clones, leading to a narrowed, oligoclonal repertoire (analogous to B-cell affinity maturation). Yet, in vivo CD4+ T cell responses often include substantial fractions of lower-avidity clones. The mechanisms preserving this clonal diversity during expansion, and how this diversity impacts the recognition of mutated pathogens, remain poorly understood.

Methodology
The authors employed a multidisciplinary approach combining in vivo immunology, single-cell genomics, mathematical modeling, and machine learning:

1. In Vivo Models: The study utilized a female mouse model immunized with male cells to target the male minor histocompatibility antigen Dby (CD4+ restricted) and Uty/Smcy (CD8+ restricted). This allowed for the comparison of CD4+ and CD8+ responses within the same immunological context.
2. uLIPSTIC System: To directly visualize cell-cell interactions in vivo, the authors used the universal LIPSTIC (uLIPSTIC) proximity-labeling system. This involved transferring donor T cells expressing membrane-bound sortase and acceptor T cells expressing a glycine motif (G5). Upon administration of a biotinylated LPETG substrate, sortase transfers biotin to neighboring acceptor cells only when membranes are within <15 nm, allowing the detection of antigen-specific T cell–T cell (T–T) contacts.
3. Inducible SOCS1 Deletion: To test the role of the proposed inhibitory mechanism, the authors generated bone marrow chimeras with inducible CRISPR-Cas9 deletion of SOCS1 (Suppressor of Cytokine Signaling 1) in mature T cells. This created a system where SOCS1-deficient (KO) cells could be compared against wild-type (WT) cells within the same mouse.
4. High-Throughput Sequencing and Functional Assays:
   • scRNA/TCR-seq: Paired single-cell RNA and TCR sequencing was performed on >32,000 Dby-specific CD4+ T cells from 20 mice (WT and SOCS1 conditions).
   • Avidity Measurements: 74 TCRs were re-expressed in a 58α-β- T hybridoma line to measure functional avidity (F-av) and binding avidity (B-av).
   • APL Screening: 90 TCRs were screened against 54 altered peptide ligands (APLs) of the Dby peptide to map cross-reactivity and promiscuity.
5. Mathematical Modeling: A system of ordinary differential equations was developed to simulate CD4+ T cell expansion. The model incorporated non-cumulative activating signals (from APCs) and cumulative inhibitory signals (from T–T contacts mediated by SOCS1).
6. Machine Learning: The P-TEAM algorithm was trained on the experimental TCR–APL recognition data to predict repertoire-level coverage of antigen variants in WT versus SOCS1-deficient repertoires.

Key Contributions and Results

• Evidence for Antigen-Specific T–T Interactions: Using uLIPSTIC, the authors demonstrated that CD4+ T cells engage in antigen-specific, MHC-II-dependent interactions in vivo. Acceptor T cells of the same specificity were preferentially labeled by donor cells, and this labeling was reduced by anti-MHC-II blockade. This confirms that Ag-experienced CD4+ T cells acquire pMHC from APCs and present it to neighboring T cells.
• SOCS1-Mediated Proliferation Arrest: The study validated that SOCS1 acts as a critical checkpoint. In SOCS1-deficient repertoires, T cells expanded more vigorously but became significantly more oligoclonal. The most expanded clone in SOCS1 KO repertoires accounted for >50% of the repertoire, compared to a median of 16% in WT repertoires. This indicates that SOCS1-dependent inhibition prevents the dominance of a few high-avidity clones.
• Non-Monotonic Avidity–Abundance Relationship: Mathematical modeling and experimental validation revealed a non-monotonic relationship between TCR avidity and clonal expansion in WT responses. While high-avidity clones initially dominate, they rapidly accumulate inhibitory signals (via T–T contacts) and cease proliferation. Consequently, clones with medium-high avidity achieve maximal expansion in WT conditions. In contrast, SOCS1 KO responses showed a strictly positive correlation where the highest-avidity clones dominated.
• Preservation of Repertoire Diversity for Variant Recognition:
  • Cross-Reactivity: TCRs with higher avidity for the original antigen generally recognized a broader range of APLs. However, distinct TCR classes (defined by CDR3 motifs) showed complementary recognition patterns for specific peptide mutations (e.g., mutations at position 6 vs. positions 5/10/11).
  • Coverage: Machine learning predictions showed that WT repertoires, with their broader diversity, covered a significantly higher number of antigen variants than SOCS1 KO repertoires.
  • In Vivo Validation: Upon recall with specific APLs, WT mice mounted robust responses, whereas SOCS1 KO mice often failed to respond to variants that required TCRs not present in their narrowed repertoire. The size of the expanded WT repertoire (median ~20 clones) was predicted to be near-optimal for covering antigenic variation, whereas the smaller SOCS1 KO repertoire (median ~6 clones) was insufficient.

Significance
The paper claims to resolve the paradox of how CD4+ T cell responses maintain diversity despite sustained antigen stimulation. The authors propose that antigen-specific T–T interactions, mediated by the transfer of pMHC complexes, trigger a SOCS1-dependent inhibitory feedback loop. This mechanism acts as a "brake" on the most avid clones, preventing their unchecked expansion and allowing medium-avidity clones to contribute to the response.

The significance of this mechanism is twofold:

1. Regulatory: It establishes a cell-intrinsic regulatory circuit (SOCS1) that shapes the TCR repertoire composition during an ongoing immune response, distinct from the autonomous proliferation seen in CD8+ T cells.
2. Adaptive: By preserving a diverse repertoire with complementary cross-reactivity profiles, this mechanism enhances the immune system's adaptability to mutating pathogens. The study demonstrates that the "braking" mechanism is essential for generating a repertoire capable of recognizing a broad spectrum of antigenic variants, thereby improving the robustness of immunity against viral or bacterial escape mutants.

The authors conclude that antigen-specific T–T interactions and SOCS1 signaling are fundamental controllers of TCR diversity, ensuring that the immune system remains adaptable to antigenic variation.