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Raquel Deering, Wen Fury, Sunil K. Srivastava, Se Jeong, Sumit Sharma, Kimberly Baynes, Ashley Lowe, Michael Ramos, Evangelia Malahias, Christina Adler, Min Ni, Yi Wei, Mickey Atwal, Carmelo Romano, Gavin Thurston, jonathan weyne; A novel method to rapidly expand and perform single cell sequencing on T cells from vitreous biopsies. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3463.
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© ARVO (1962-2015); The Authors (2016-present)
Pathogenic T cells in the vitreous of patients are a hallmark of autoimmune uveitis. Characterizing intravitreal phenotypes and T cell receptor (TCR) sequences at the single cell level would enable downstream tests to identify the cognate antigens recognized by intravitreal T cells. Typically, too few viable T cells from vitreous biopsies are available to allow direct single cell sequencing (scSEQ) technologies to be applied. Thus, we developed a novel ex vivo rapid T cell expansion culture method on vitreous biopsies to enable scSEQ for TCR identification.
Vitreous biopsies were collected at the time of steroid-eluting implant replacement. Samples were centrifuged at 4° C to concentrate cells and reduce the volume of vitreous fluid, which inhibits T cell proliferation in culture. The pellets were transferred to tissue culture wells containing irradiated PBMC feeder cells separated from vitreous cells by a well insert that is permeable to growth factors produced by feeder cells while maintaining a physical barrier between feeder and vitreous cells. The media contained supportive T cell cytokines (IL-7, IL-15, IL-2) and the T cell stimulus anti-CD3 antibody (OKT3 clone). Although T cell stimulants were provided, the culture also supported B and NK cells during the 7-day culture period. After 7 days, total cultured vitreous cells were collected and applied to the 10X Genomics Chromium single cell capture and parallel whole genome transcriptome and TCR sequencing.
We first expanded vitreous T cells from a patient with sarcoidosis-related intraocular immune infiltration. Following 7-day expansion and scSEQ analysis, we observed that 675 out of 681 cells expressed CD3. A unique TCR alpha and beta chain was detected in 547 CD3+ cells with a total of 34 clonotypes. Among the top 12 most frequent clonotypes, the number of cells sharing the same clonotype ranged from 5 to 172. These expanded cells were comprised of both CD4 and CD8+ T cells. TCR clonality analysis revealed that T cells from both subsets were clonally expanded.
Using a novel ex vivo rapid expansion protocol for fresh vitreous biopsies, we found that T cells can be analyzed by single cell methods. This approach allows sensitive and in-depth profiling of T cells and provides T cell subtype and clonotype information from auto-immune uveal diseases.
This is a 2021 ARVO Annual Meeting abstract.
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