The dominant human conjunctival epithelial CD8αβ+ T cell population is maintained with age but the number of CD4+ T cells increases

Abstract

The conjunctiva is a highly specialized ocular mucosal surface that, like other mucosa, houses a number of leukocyte populations. These leukocytes have been implicated in age-related inflammatory diseases such as dry-eye, but their phenotypic characteristics remain largely undetermined. Existing literature provides rudimentary data from predominantly immunohistochemical analyses of tissue sections, prohibiting detailed and longitudinal examination of these cells in health and disease. Using recovered cells from ocular surface impression cytology and flow cytometry, we examined the frequency of leukocyte subsets in human conjunctival epithelium and how this alters with age. Of the total CD45+ leukocyte population within the conjunctival epithelium, 87% [32-99] (median) [range] comprised lymphocytes, with 69% [47-90] identified as CD3 + CD56- T cells. In contrast to peripheral blood, the dominant conjunctival epithelial population was TCRαβ + CD8αβ + (80% [37-100]) with only 10% [0-56%] CD4+ cells. Whilst a significant increase in the CD4+ population was seen with age (r = 0.5; p < 0.01) the CD8+ population remained unchanged, resulting in an increase in the CD4:CD8 ratio (r = 0.5;p < 0.01). IFNγ expression was detectable in 18% [14-48] of conjunctival CD4+ T cells and this was significantly higher among older individuals (<35 years, 7[4-39] vs. >65 years, 43[20-145]; p < 0.05). The elevation of CD4+ cells highlights a potentially important age-related alteration in the conjunctival intra-epithelial leukocyte population, which may account for the vulnerability of the aging ocular surface to disease.

Fig. 1

Lymphocytes are the dominant conjunctival epithelial leukocyte population. Conjunctival OSIC of the superior unexposed bulbar conjunctiva is shown in A. Representative plots of a subject demonstrating the gating strategy used to identify conjunctival leukocytes. B, E The forward and side scatter profiles for peripheral blood and OSIC, respectively. Live leukocytes were identified by gating for CD45+ cells that were negative for the dead cell exclusion dye Sytox blue (lower right box in C and F) and back-gated to show the forward and side scatter profiles of the CD45+ live cells (D and G). Percentages are shown for the representative subject (n = 30)

Fig. 2

TCRαβ + CD8αβ + T cells are the dominant population of lymphocytes in the conjunctival epithelium. Representative scatter profile of leukocyte populations derived from matched peripheral blood and conjunctival OSIC from a healthy subject. Data are shown for a representative subject, gated on CD45+ live cells for peripheral blood (a) and conjunctival OSIC (b). CD45RO staining is shown for CD3 + CD56 − TCRγδ − gated CD4 + CD8β − (top panel) and CD8αβ + (bottom panel) cells. Percentages of CD3 + CD56- lymphocytes are shown for the representative subject (n = 30). Statistical comparisons between peripheral blood and conjunctival impression populations of CD8αβ+, CD4+, CD8αα+ and TCRαβ+CD4 − CD8αβ− (double negative; DN) T cells (c–f) were undertaken by the Mann–Whitney U test (NS not significant [P > 0.05], **P < 0.01, ***P < 0.001)

Fig. 3

Changes in T cell subsets and memory populations in peripheral blood and conjunctiva with age. Changes in the T cell populations in peripheral blood and conjunctival OSIC for CD3 + CD56− lymphocytes (a), as well as CD8αβ+ and CD4+ subsets (b/c, e/f) with their respective CD45RO frequencies (d, g). Statistical analysis was undertaken by Spearman’s correlation (NS not significant [p > 0.05], *P < 0.05, **P < 0.01) (n = 30)

Fig. 4

TCRαβ + CD4+ T cells increase with age but do not alter their cytokine production. Expression of IFNγ, IL-17 and IL-10 in the T cell populations in stimulated peripheral blood (PB) and conjunctival OSIC for CD45 + CD3 + CD56 − CD4+ live lymphocytes (A). Representative figures from a subjects aged <35 and >65 years are shown. The number and percentage of CD4+ T cells are shown for this cohort (B), as well as the number and percentages of cytokine-secreting cells (C). Statistical comparisons were undertaken by the Mann–Whitney U test (NS not significant [p > 0.05], *P < 0.05)