Hypoxia-driven transcriptional programming in uveitogenic memory CD4⁺ T cells

Abstract

Purpose : We have recently demonstrated the pathogenicity of memory CD4⁺ T cells in a well-characterized murine model of chronic autoimmune uveitis (CAU). Herein, we aim to determine whether chronic inflammation in CAU is associated with tissue and cellular hypoxia and if so, whether hypoxia regulates transcriptional programming of the uveitogenic memory T cells.

Methods : CAU was induced in wild-type C57BL/6 mice by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptides emulsified in complete Freund’s adjuvant (CFA), along with Bordetella pertussis toxin injection. Establishment of CAU was confirmed by digital fundus imaging at week 12. CAU mice were then intraperitoneally injected with pimonidazole (Hypoxyprobe^TM). After 24 hours, mice were sacrificed, and eyeballs, draining lymph nodes (dLN) and spleen were collected. Tissue cross sections were stained with Red549-anti-pimonidazole, AlexaFluor488-anti-CD3, and DAPI to detect tissue and cellular hypoxia. In addition, 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 subjected to qPCR analysis of mRNA genes. Flow cytometric analysis was also performed by staining BV421-CD4, PerCP/Cy5.5-CD44 and PE-hypoxia-inducible factor-1α (HIF-1α).

Results : Local hypoxia was demonstrated in the inflamed retina in CAU, most prominent in the destructed photoreceptor layer (PRL). In addition, significantly more T cells showing hypoxic state were present in the dLN (18.3 ± 0.9 vs 4.3 ± 0.5 per section) and spleen (29.3 ± 2.2 vs 3.5 ± 0.3 per section) in CAU as compared to normal controls. The sorted memory T cells showed ~ 2-fold up-regulation of HIF-1α mRNA expression (no difference in HIF-2α). Consistently, expression of HIF-1α protein in CAU-memory T cells was significantly increased (mean fluorescent intensity: 1496 ± 44 vs 964 ± 34). Finally, genes of HIF-1-associated transcriptional pathways including glycolysis (Hk2, Ldha, Slc2a1, and Slc16a3) and canonical Wnt/β-catenin activity (Ctnnbl1, Axin2, and GSK3b) were up-regulated in memory T cells.

Conclusions : Our data demonstrate that the uveitogenic memory CD4⁺ T cells in CAU are in hypoxic state and exhibit hypoxia-driven transcriptional activity that is potentially essential for their long-term survival and function.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.