Antigen-specific memory CD4⁺ T cells are uveitogenic in chronic autoimmune uveitis

Abstract

Purpose : Chronic autoimmune uveitis (CAU) is often a treatment-resistant disease, and the underlying pathogenesis remains poorly understood. Our previous work demonstrates the presence of CD44^hiIL-7R⁺IL-15R⁺IL-17A⁺CD4⁺ memory Th17 cells in the retina, draining lymph nodes, and spleen in a mouse model of CAU. In the present study, we further determined the pathogenicity of these memory T cells in CAU.

Methods : CAU was induced in wild-type C57BL/6 mice by immunization with 150 µg interphotoreceptor retinoid-binding protein (IRBP) peptide 161–180 plus 300 µg IRBP 1–20 emulsified in 0.2ml Complete Freund's Adjuvant (CFA). Mice also received 0.2μg Bordetella pertussis toxin. Establishment of CAU was confirmed by digital fundus imaging at week 12. CAU mice were sacrificed, draining lymph nodes and spleen were collected, and CD44^hiCD4⁺ (memory T cells) and CD44^-/lowCD4⁺ (control T cells) were isolated using CD4⁺ negative MACS sorting combined with FITC-CD44 FACS sorting. The sorted cells were stimulated with 20 µg/mL IRBP in the presence of 1 µg/ml anti-CD28 antibody in vitro. Next, the cultured cells were evaluated for (i) proliferation profile using fluorescence-based dilution assay (Violet CellTrace™ Cell Proliferation Kit), (ii) activation profile using flow cytometry-based PE-CD154 antigen-specific activation assay, and (iii) cytokine profile using ELISA kit for IL-17A detection in cell culture supernatant. Finally, the sorted cells were also adoptively transferred to normal Rag1^-/- mice, and clinical disease in recipients was followed up for 2 weeks using fundoscopy.

Results : The CAU-derived CD44^hiCD4⁺ memory T cells showed 2-fold higher proliferation and 20-fold higher CD154 expression upon IRBP re-stimulation compared to control CD44^-/lowCD4⁺ T cells. The IL-17A expression levels in CD44^hiCD4⁺ memory T cell cultures (15.5 ± 0.6 pg/mL) were significantly higher than that in CD44^-/lowCD4⁺ T cell cultures (8.8 ± 0.4 pg/mL). In addition, Rag1^-/- mice receiving CD44^hiCD4⁺ memory T cells developed multiple retinal lesions by 2 weeks post-transfer; in contrast, those receiving control T cells showed normally appearing retina.

Conclusions : Our data demonstrate that memory CD4⁺ T cells in CAU are antigen-specific pathogenic effector memory T cells.

This is a 2021 ARVO Annual Meeting abstract.