T-Cell Repertoire Analysis in the Conjunctiva of Murine Dry Eye Model

**Background:** Dry eye is a chronic inflammatory disease involving T-cell infiltration in the conjunctiva. The T-cell receptor (TCR) repertoire determines T-cell specificity and diversity, but its characteristics in dry eye are unknown. This study aimed to analyze the αβ TCR repertoire in the conjunctiva of a murine dry eye model and assess the effect of glucocorticoid treatment.

**Methods:** A desiccating stress (DS) model was established in female C57BL/6 mice (8–12 weeks) by housing them in low humidity (<25%) with subcutaneous scopolamine injections (0.5 mg/0.2 mL three times daily) for 7 days. Control mice were kept in normal environment. A glucocorticoid (GC) group received topical dexamethasone three times daily for 7 days. Ocular surface damage was evaluated by slit-lamp imaging, Oregon-green-dextran (OGD) staining, tear secretion test (phenol red thread), and periodic acid–Schiff (PAS) staining for goblet cells. T-cell infiltration and activation were assessed by immunohistochemistry and flow cytometry (CD3, CD4, CD69, TCRβ, TCRγδ). RNA was extracted from conjunctiva, and αβ TCR repertoire was analyzed by next-generation sequencing of CDR3 regions of TCRα and TCRβ chains. Diversity was measured using Gini coefficient, Shannon–Wiener index, and rank-abundance curves. V and J gene usage, V(D)J recombination, and CDR3 amino acid (aa) motifs were compared between groups.

**Key Results:** DS model successfully induced dry eye: tear production decreased (phenol red thread length reduced), corneal permeability increased (OGD staining intensity higher), goblet cell count decreased (PAS staining), and MMP-3/MMP-9 expression increased. T-cell infiltration (CD3+ cells) in conjunctiva was significantly higher in dry eye group (p<0.05). Flow cytometry showed increased CD3+CD45+ T cells in conjunctiva and cervical lymph nodes, predominantly αβ T cells, with CD4+ T-cell activation. TCR sequencing revealed significantly higher αβ TCR diversity in dry eye group: CDR3 aa length was longer (Gaussian distribution centered at 11–17 aa), Gini coefficient was higher, and clonotypes were more numerous. On TCRα chain, TRAV6-6, TRAV7-4, TRAV6-4, TRAV4-3, TRAJ15, TRAJ6, TRAJ11, TRAJ9, TRAJ45, TRAJ35 were increased; TRAV6N-6, TRAV8-1, TRAJ23, TRAJ18 were decreased. On TCRβ chain, TRBV13-3, TRBV15, TRBV4 were increased; TRBV19 decreased. Unique V(D)J pairings were found only in dry eye group (e.g., TRAV8N-2-J37, TRAV12D-2-J13, TRAV12N-3-J40, TRAV16N-J40; TRBV13-Dx-Jy combinations). CDR3 aa motifs differed between groups. Glucocorticoid treatment reversed many changes: tear production improved, corneal permeability reduced, goblet cell count increased, TCR clonotypes and diversity decreased (Shannon–Wiener index), and specific V(D)J combinations (e.g., TRBV13-1-J1-4, TRBV13-3-D1-J1-2, TRBV13-3-D2-J2-3, TRBV13-3-D2-J2-4) declined. Rank-abundance curves showed dry eye group had longest and steepest curve, indicating high diversity, which was reduced by GC.

**Clinical Implications:** This study provides the first comprehensive analysis of αβ TCR repertoire in dry eye conjunctiva, demonstrating increased diversity and disease-specific clonotypes. The identified TCR signatures (e.g., specific V/J gene usage, CDR3 motifs) may serve as potential biomarkers for early diagnosis or monitoring of dry eye. The reversal of TCR abnormalities by glucocorticoids suggests that targeting T-cell clonotypes could be a therapeutic strategy. However, limitations include the animal model (not human), small sample size, and lack of comparison across dry eye subtypes. Future studies should validate these findings in patients and explore the functional role of identified T-cell clones.