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

Abstract

Purpose: Dry eye is closely related to the activation and proliferation of immune cells, especially T cells. However, the determination of the preferential T-cell clonotypes is technically challenging. This study aimed to investigate the characterization of T-cell receptor (TCR) repertoire in the conjunctiva during dry eye.

Methods: A desiccating stress animal model was established using C57/BL6 mice (8-10 weeks, female). After 7 days of stress stimulation, the slit-lamp image and Oregon-green-dextran staining were used to evaluate the ocular surface injury. Periodic acid-Schiff staining was used to measure the number of goblet cells. Flow cytometry was used to detect the activation and proliferation of T cells in the conjunctiva and cervical lymph nodes. Next-generation sequencing was used to detect the αβ TCR repertoire of the conjunctiva.

Results: The αβ TCR diversity increased significantly in the dry eye group, including the higher CDR3 amino acid length, marked gene usage on TCR V and J gene segments, extensive V(D)J recombination, and distinct CDR3 aa motifs. More important, several T-cell clonotypes were uniquely identified in dry eye. Furthermore, these perturbed rearrangements were reversed after glucocorticoid administration.

Conclusions: A comprehensive analysis of the αβ TCR repertoire in the conjunctiva of the dry eye mouse model was performed. Data in this study contributed significantly to the research on dry eye pathogenesis by demonstrating the TCR gene distribution and disease-specific TCR signatures. This study further provided some potential predictive T-cell biomarkers for future studies.

Figure 1.

Change in the ocular surface of the desiccating stress model. (A) Representative slit-lamp image and OGD staining image of the two groups. (B) Phenol red thread test indicating tear secretion (n = 6). (C) Statistical intensity analysis of OGD fluorescence (n = 6). (D) PAS staining shows goblet cell reduction in the conjunctiva. (E) Number of goblet cells in the conjunctiva (sum of superior and inferior conjunctiva, n = 6). (F, G) Representative immunofluorescent images of MMP-3 (green), MMP-9 (green), and DAPI (blue) in the corneal epithelium. The data are represented as mean ± SD. The P values were analyzed using the Student’s t-test. Scale bar: 200 µm (D) and 50 µm (F, G).

Figure 2.

TCR β⁺ T cells were expanded in the DS model. (A) Representative immunohistochemistry staining images of CD3 in the conjunctiva. (B) Statistical results of CD3⁺ cells (n = 6). (C) Gating strategy for the T-cell population in the mice conjunctiva by flow cytometry. (D, E) Representative plots show frequency of CD45⁺ and CD3⁺ cells in the conjunctiva. The data are expressed as mean ± SD. The P values were calculated using the Student’s t-test. Scale bar: 20 µm (A).

Figure 3.

Diversity of TCR repertoires increased during desiccating stress. (A, B) TCR clonotypes of the TRA and TRB in the control and dry eye groups. (C) Comparison of CDR3 aa diversity using the Gini–Simpson index. (D, E) Distribution of CDR3 aa length in the TRA and TRB chains. (F, G) Top clonal clonotypes with the specific proportion between the two groups. The data are expressed as mean ± SD. The P values were calculated with the Student’s t-test (A–C) and multiple t-tests (F, G); n = 4 for each group.

Figure 4.

Comparison of TRA gene distributions and alterations in the two groups. (A) Proposed model of V–J recombination of the α subunit. (B, C) Heatmap and volcano plot, respectively, reveal the altered V genes on the α subunit under DS. (D, E) Parts-of-whole graphs show the frequency of TRAV and TRAJ gene usage in each group. (F, G) Heatmap and volcano plot, respectively, showing the altered V gene on the α subunit under DS. The P values were calculated with multiple t-tests; n = 4 for each group.

Figure 5.

Comparison of TRB gene distributions and alterations in the two groups. (A) Proposed model of V(D)J recombination of the β subunit. (B, C) Comparison of the altered V gene on the β subunit under DS. (D, E) Parts-of-whole graphs reveal the frequency of TRBV and TRBJ gene usage in each group. (F, G) Comparison of the altered J gene on the β subunit under DS; the P values are shown in the heatmap and volcano plot. The P values were calculated using multiple t-tests; n = 4 for each group.

Figure 6.

Usage patterns of TRB V–D–J combinations in the two groups. (A) Altered differences of V–D–J gene combinations between the two groups. The data are expressed as mean with SD. (C) Volcano plot demonstrates relevant P values among the groups. (B, D) Chord diagram exhibits abundant V–D–J pairings in the two groups. (E) TCR CDR3 aa motifs of all TRBV13-related combinations in the dry eye group. The P values were calculated using multiple t-tests; n = 4 for each group.

Figure 7.

Abundance of TCR repertoires improved after using glucocorticoids. (A) Quantification of tear production using the phenol red cotton test (n = 6). (B) Representative images of OGD staining. (C) Representative images of PAS staining in the conjunctiva. (D) Number of goblet cells in the conjunctiva (sum of superior and inferior conjunctiva, n = 6). (E) Clonotypes on TRA and clones on TRB of three groups. (F) Shannon–Wiener index demonstrates the diversity in the three groups on both TRA and TRB. (G) Rank-abundance curve of TRA and TRB repertories in the three groups. The data are expressed as mean ± SD. The P values were calculated using a one-way analysis of variance; n = 4 for each group (E–G). Scale bar: 200 µm (C).

Figure 8.

Distribution of TRB repertoire declined in the glucocorticoid group. (A) Count, frequency, and diversity compared with the chord diagram in the three groups. A, B, and C represent the control group, the dry eye group, and the glucocorticoid group, respectively. (B) Comparison of specific V(D)J combinations in the three groups. (C) CDR3 motif analyses on TCRβ in the three groups (control, 11; dry eye, 37; and GC, 11). The data are expressed as mean ± SD. The P values were calculated using one-way analysis of variance; n = 4 for each group.