Evidence of clonal expansion among vitreous-derived T-cells in birdshot chorioretinopathy: A single cell profiling-based molecular case study

Abstract

Purpose : Birdshot chorioretinopathy (BSCR) is a rare autoimmune posterior uveitis with a significant association with the HLA-A29 genotype. T cell infiltration has been observed in the vitreous of BSCR patients. However, no expanded T cell receptor (TCR) clones with paired TCR alpha chain (TRA) and TCR beta chain (TRB) information and associated T cell phenotype have been reported. Here we report single cell-based profiling results from a BSCR patient.

Methods : A single patient with history of BSCR, diagnosed by clinical presentation and positive HLA-A29 testing was included in this IRB approved prospective study. The vitreous sample was collected during fluocinolone acetonide implantation with pars plana vitrectomy. 10X Genomics scSEQ platform and Illumina NextSeq500 were used for 5’ mRNA and V(D)J sequencing using the standard protocol, except that TRA and TRB were further enriched using primers designed in-house. Cell Ranger (v2.2.0) and Seurat (v4) were used for data processing and analysis. GRCh38 and germline segment V(D)J from IMGT were used as references.

Results : Clustering analysis revealed 107 droplets in the CD3+ cluster and 313 in the CD68+ cluster. Among the 63 CD3+ droplets with both TRA and TRB information, highly skewed TCR clonalities were found: 1 clonotype shared by 16 droplets, 2 shared by 3 droplets each, 5 shared by 2 droplets each, and 31 singletons. Two of the topmost expanded clonotypes were CD8A+, none were CD4+, and all were granzyme positive and/or NKG7+, suggesting a cytotoxic phenotype.

Conclusions : Using single cell mRNA and V(D)J sequencing, we have identified expanded TCR clones with paired TRA and TRB information from the vitreous of a BSCR patient. The TCR clonotypes were highly skewed towards a cytotoxic T cell phenotype. Otherwise, ~75% were CD68+ myeloid cells. This suggests a role for CD8+ T cells in the pathogenesis of BSCR. Whether a similar CD8+ T cell repertoire can be identified in the systemic circulation, engages peptide-bound HLA-A29, and/or if their presence in the vitreous correlate with disease activity will all be relevant to address in future studies.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.