Distribution of Interleukin-22-secreting Immune Cells in Conjunctival Associated Lymphoid Tissue

Abstract

Purpose: Interleukin (IL)-22 is a cytokine involved in epithelial cell regeneration. Currently, no research studies have analyzed the distribution of the three distinct IL-22-secreting cell populations in human or mouse conjunctiva. This study investigated the distribution of the three main populations of IL-22-secreting immune cells, αβ Th cells, γδ T cells, or innate cells (innate lymphoid cells [ILCs] or natural killer cells), in conjunctival associated lymphoid tissues (CALTs) in human and mouse models.

Methods: We collected discarded cadaveric bulbar conjunctival tissue specimens after preservation of the corneo-limbal tissue for keratoplasty from four enucleated eyes of the domestic donor. The bulbar conjunctiva tissue, including the cornea from normal (n = 27) or abraded (n = 4) B6 mice, were excised and pooled in RPMI 1640 media. After the lymphoid cells were gated in forward and side scattering, the αβ Th cells, γδ T cells, or innate lymphoid cells were positively or negatively gated using anti-CD3, anti-γδ TCR, and anti-IL-22 antibodies, with a FACSCanto flow cytometer.

Results: In normal human conjunctiva, the percentage and number of cells were highest in αβ Th cells, followed by γδ T cells and CD3- γδ TCR- IL-22+ innate cells (presumed ILCs, pILCs) (Kruskal-Wallis test, p = 0.012). In normal mice keratoconjunctiva, the percentage and total number were highest in γδ T cells, followed by αβ Th cells and pILCs (Kruskal-Wallis test, p = 0.0004); in corneal abraded mice, the population of αβ Th cells and pILCs tended to increase.

Conclusions: This study suggests that three distinctive populations of IL-22-secreting immune cells are present in CALTs of both humans and mice, and the proportions of IL-22+αβ Th cells, γδ T cells, and pILCs in CALTs in humans might be differently distributed from those in normal mice.

Keywords: Conjunctiva; Conjunctival associated lymphoid tissues; Innate lymphoid cell; Interleukin-22; Th22 cell.

Fig. 1. Distribution of the interleukin (IL)-22–secreting cells in human conjunctiva. (A) A representative photo of the gating strategy for αβ Th cells (CD3⁺ γδ TCR ⁻ IL-22⁺), γδ T cells (CD3⁺ γδ TCR ⁺ IL-22⁺), and pILCs (CD3⁻ γδ TCR ⁻ IL-22⁺). (B) The percentage was highest in αβ Th cells, followed by γδ T cells and pILCs. (C) The total number was highest in αβ Th cells, followed by γδ T cells and presumed innate lymphoid cells, which was statistically significant (Kruskal-Wallis test, p = 0.012). ^*p < 0.05.

Fig. 2. Distribution of interleukin (IL)-22–secreting cells in mouse keratoconjunctiva. (A) A representative photo of gating strategy for αβ Th cells, γδ T cells, and presumed innate lymphoid cells (pILCs). (B) The percentage was highest in γδ T cells, followed by αβ Th cells and pILCs (Kruskal-Wallis test, p = 0.0004). (C) The number was highest in γδ T cells, followed by αβ Th cells and pILCs. (D,E) In corneal abraded mice, the populations of αβ Th cells and pILCs were markedly increased, although this result was not statistically significant. ^**p < 0.01.