The role of immune checkpoint ligands in retinal immune homeostasis and inflammation

Abstract

Purpose : The blood-retinal barrier is maintained by both physical barriers (e.g. intercellular tight junctions) and immunomodulatory molecular signals. Infiltrating leukocytes express various immune checkpoint receptors which regulate activation and effector activity upon engagement with their cognate ligand. We characterized the retinal immunosuppressive milieu under physiological and uveitic states with a focus on immune checkpoint ligand expression by retinal cells.

Methods : Experimental autoimmune uveitis (EAU) was induced in 6-10 week-old female C57BL/6J mice by immunization with 400 μg human IRBP1-20 emulsified with 2.5 mg/mL complete Freund’s adjuvant. Age and sex-matched naïve mice were used as controls. Using immunohistochemistry and confocal microscopy (n=4), and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), we profiled the expression of a panel of known immune checkpoint molecules in normal and inflamed retinae.

Results : Immunostaining of mouse retinae revealed expression of HVEM (BTLA ligand), CD200 (CD200R ligand) and PD-L1 (PD-1 ligand), but not PVR (TIGIT ligand) during both homeostasis and inflammation (Figure 1). HVEM was expressed in ganglion cell and bipolar cell bodies, and in the photoreceptor layer. CD200 expression was found to be predominantly axonal in the inner plexiform layer, and in the outer plexiform layer at a lower level. PD-L1 expression in healthy retina was seen in the outer plexiform layer and, to a lesser extent, in the ganglion cell and photoreceptor layers. Interestingly, PD-L1 did not colocalize with Iba1+ microglia, and expression levels appeared reduced in EAU. In contrast, expression levels of the other checkpoint ligands appeared similar in the inflamed retina. CITE-seq was performed to validate these findings at the single-cell level in both retinal and immune cells, revealing distinct immunomodulatory gene expression profiles.

Conclusions : Distinct expression patterns of immune checkpoint ligands by different retinal cell types contribute to immunosuppressive homeostasis within the eye. CITE-seq provides the first simultaneous single-cell gene expression profiling and immunophenotyping of healthy versus inflamed retina.

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

 

Murine retinal expression patterns of immune checkpoint ligands during homeostasis and experimental autoimmune uveitis.