T cells found in cornea for the first time! Protect eyes from viral infection

All organs or tissues in the body have immune protection against pathogens, and the eye is no exception.

The eye consists of the retina, iris, and the front layer, the cornea. The corneal microenvironment is anti-inflammatory and immunosuppressive. Several mechanisms protect against invading pathogens, including during ocular infections with herpes simplex virus (HSV), where T cells are recruited to the tissue and can induce immunopathological changes such as stromal keratitis. During the clinical stages of HSV infection, CD4+ T cells orchestrate many aspects of the disease. CD8+ T cells also infiltrate the cornea and contribute to viral control in the cornea.

It is currently unknown whether memory T cells are formed in the cornea after infection and whether these cells differentiate into TRM cells (tissue-resident memory T cells) that can promote local immunity.

In a new study, researchers found that memory T cells that patrol and fight viral infections exist in the cornea, overturning the current view that T cells do not exist in healthy corneas, which expands our understanding of the eye's immune response to infections. The relevant research results were published in the journal on May 24, 2022.

To study the T cell response to corneal herpes simplex virus (HSV) infection, the researchers used well-characterized T cell receptor transgenic mice. gDT-II CD4+ T cells entered the cornea 5 days after infection. Virus-specific T cell numbers peaked in the cornea 10 days later, then contracted on day 14 and persisted for at least 4 weeks.

Corneas were then isolated from HSV-infected mice and the tissue was examined by confocal microscopy. By day 6, CD8+ and CD4+ T cells entered the limbal zone adjacent to the cornea. These cells were then observed to gradually migrate toward the central cornea. By day 10, large numbers of CD8+ and CD4+ T cells were observed within the central cornea.

To better define T cell infiltration of the cornea during infection, the researchers used intraocular two-photon microscopy to visualize T cells in live mice. On day 5 of infection, we observed both CD4+ and CD8+ T cells in the cornea. By days 7-10, large numbers of effector T cells were observed in the cornea. T cells in both the stromal and epithelial tissue zones of the cornea were motile. T cells that entered the corneal epithelium exhibited a dendritic morphology. Therefore, corneal infiltrating T cells are a highly dynamic population recruited during infection that accumulate in large numbers during the effector phase of the response.

T cell responses and memory in the cornea during ocular HSV infection

The researchers then used in vivo confocal microscopy (IVCM) to perform time-lapse imaging of the wheel cells in the inner nasal, paracorneal areas of six healthy volunteers, taking images of the same corneal area every 6-8 minutes for 30-46 minutes. Imaging showed that in 4/6 corneas, there were cells with sparse, elongated dendrites and little movement; these cells resembled DCs (macrophages and dendritic cells) that have also been described in mice. Strikingly, the corneas of 6/6 volunteers had smaller, less elongated dendritic immune cells that were highly motile. These human immune cells were very similar to the corneal T cells observed in mice. Although further work is needed to confirm the identity of these cells, this suggests that motile immune cells patrol the healthy cornea of ​​humans and that they may contribute to local immunity.

Four weeks after mice were infected with HSV, high numbers of T cells persisted in the cornea, prompting the researchers to investigate whether populations of memory T cells form and persist in this location.

The researchers imaged corneal full-face preparations from mice that had been infected with HSV at least 30 days previously. Memory T cells imaged with two-photon microscopy were observed in both the epithelial and corneal stroma layers, with T cells in the epithelial layer consistently displaying reticular cell morphology. Cells exhibited sustained slow motility with an average speed of 6.06 μm/min. The study found that almost 50% (44% ± 10%) of memory gBT-I cells observed in the central cornea were located within the epithelial layer.

(A) Whole mount confocal imaging of the cornea of ​​mT/mG mice; (B) Two-photon microscopic imaging of the cornea of ​​mice 32 days after HSV infection. The upper image is in the xy direction, and the lower image is in the xz direction. Scale bar, 20 μm.

Tissue-resident memory T cells express the typical marker CD69, and TRM cells in epithelial tissue (i.e., skin epidermis) express the integrin CD103. Analysis showed that TRM cells form on the cornea after resolution of viral infection and indicated that the heterogeneity of CD103 expression may be due to the localization of cells in the corneal epithelium.

To determine the retention of memory T cells in the cornea, the researchers treated mice with anti-Thy1 antibodies to deplete circulating T cells. To further investigate whether corneal memory CD8+ T cells recirculate, memory mice were treated with the immunomodulatory drug FTY720, a sphingosine 1-phosphate receptor agonist that inhibits lymphocyte egress from LNs and induces lymphopenia. After 4 weeks of treatment, CD8+ T cells were reduced in the spleen and blood. However, the number of T cells in the cornea did not decrease after treatment. Together, these data support the conclusion that memory CD8+ T cells reside in the cornea.

In summary, the study shows that the formation of corneal TRM requires CD103 expression for local antigen presentation. The researchers do not know which cells present antigens to T cells in the cornea, and infected epithelial cells in the cornea may present antigens to T cells. In the absence of antigen stimulation, inflammatory signals induced by LPS or CpG or viruses lacking cognitive antigens are not sufficient to induce the formation of a large number of corneal TRM cells. This may be influenced by the generally anti-inflammatory corneal microenvironment. Nevertheless, the study found that corneal TRM cells can respond rapidly to antigens in situ, and circulating antigen-specific and bystander memory T cells are rapidly recruited to the cornea during secondary challenges. It is worth noting that the formation of secondary CD103+TRM cells by recruited memory cells also requires local antigen recognition.

Image from Cell Reports, 2022, doi:10.1016/j.celrep.2022.110852.

Taken together, the study's images show that long-lived memory T cells are generated in the mouse eyes to fight infection. After the virus is eliminated, these memory T cells remain on the cornea to protect against any future HSV reinfection. Advanced imaging of healthy human eyes also showed immune cells patrolling the cornea.

"These findings have important implications for understanding how the eye defends itself against dangerous infections," said study author Scott Mueller. "They could also help us understand chronic diseases such as eye allergies, which may be caused by excess T cells."

References:

Joon Keit Loi et al. Corneal tissue-resident memory T cells form a unique immune compartment at the ocular surface. Cell Reports, 2022, doi:10.1016/j.celrep.2022.110852.