T-Lymphocyte Subset Distribution and Activity in Patients With Glaucoma

Abstract

Purpose: Besides glia-driven neuroinflammation, growing evidence from analysis of human blood samples, isolated autoantibodies, and postmortem tissues also support systemic immune responses during neurodegeneration in glaucoma patients. To explore the T-cell-mediated component of systemic immunity, this study analyzed T lymphocytes in patients' blood.

Methods: Blood samples were collected from 32 patients with glaucoma and 21 nonglaucomatous controls, and mononuclear cells were isolated by Histopaque density gradient centrifugation. T-cell subset distribution was analyzed by multicolor flow cytometry after helper (Th) and cytotoxic fractions, and Th subpopulations, were stained with antibodies to CD4, CD8, or distinctive markers, such as IFN-γ (for Th1), IL-4 (for Th2), IL-17A (for Th17), and CD25/FoxP3 (for T regulatory cells [Tregs]). In addition, proliferative activity and cytokine secretion of T cells were analyzed after in vitro stimulation.

Results: Analysis of T-cell subset distribution detected a glaucoma-related shift. Despite similar frequencies of CD4+ or CD8+ T cells, or Th1, Th2, or Th17 subsets in glaucoma and control groups, glaucomatous samples exhibited a trend toward decreased frequency of CD4+ (or CD8+)/CD25+/FoxP3+ Tregs within the entire CD4+ (or CD8+) population (P < 0.001). Furthermore, CD4+ T cells in glaucomatous samples presented a greater stimulation response (∼3-fold) as characterized by increased proliferation and proinflammatory cytokine secretion (P < 0.05).

Conclusions: These findings suggest that the immunity activated in glaucoma may not be counterbalanced by an efficient immune suppression. More work is encouraged to determine whether shifted T-cell homeostasis may contribute to neurodegeneration in glaucoma, and/or whether T-cell subset imbalance may serve as a biomarker of autoimmune susceptibility.

Figure 1

T-lymphocyte subset distribution in glaucoma. Mononuclear cells isolated from peripheral blood samples from glaucoma (n = 32) and control (n = 21) groups were analyzed by multicolor flow cytometry for T-cell subset markers. (A–C) The percentage of CD4+ and CD8+ T cells and the ratio of CD4+ to CD8+ T cells were similar in glaucomatous samples and nonglaucomatous controls (ANOVA, P = 0.96, P = 0.75, P = 0.49, respectively). (D–F) The percentage of T-cell subsets, including CD4+/IFN-γ+ Th1, CD4+/IL-4 Th2, and CD4+/IL-17A+ Th17 subpopulations, was also similar in glaucoma and control groups (ANOVA, P = 0.09, P = 0.06, P = 0.10, respectively). Bars on univariate scatterplots represent the group mean.

Figure 2

T-lymphocyte subset distribution in glaucoma. Mononuclear cells isolated from peripheral blood samples from glaucoma (n = 32) and control (n = 21) groups were analyzed by multicolor flow cytometry for T-cell subset markers. (A) The percentage of CD4+/CD25+/FoxP3+ Tregs (CD4-Tregs) within the entire CD4+ T-cell population was significantly lower in glaucomatous samples than nonglaucomatous controls (ANOVA, P < 0.001). (B, C) The Treg to Th1 or Treg to Th17 ratios were also significantly lower in the glaucoma group than control (ANOVA, P < 0.001). Bars on univariate scatterplots represent the group mean. (D) Shown are representative flow cytometry images after CD4/CD25/FoxP3 immunostaining for Tregs.

Figure 3

T-lymphocyte subset distribution in glaucoma. Mononuclear cells isolated from peripheral blood samples from glaucoma (n = 32) and control (n = 21) groups were analyzed by multicolor flow cytometry for T-cell subset markers. (A) Similar to CD4+ Tregs, the percentage of CD8+/CD25+/FoxP3+ Tregs (CD8-Tregs) was also significantly lower in glaucomatous samples than nonglaucomatous controls (ANOVA, P < 0.001). (B) Shown are representative flow cytometry images after CD8/CD25/FoxP3 immunostaining for Tregs.

Figure 4

In vitro analysis of T lymphocytes in glaucoma. Mononuclear cells isolated from peripheral blood samples from glaucoma (n = 32) and control (n = 21) groups were analyzed for a proliferation marker after in vitro stimulation. (A) The percentage of CD4+/EdU+ cells analyzed by multicolor flow cytometry was significantly higher in glaucomatous samples than nonglaucomatous controls (ANOVA, P < 0.001). Data are presented as mean ± SD in bar graphs, while univariate scatterplots present data distribution. (B) Shown are representative flow cytometry images after triple immunostaining.

Figure 5

In vitro analysis of T lymphocytes in glaucoma. In vitro proliferative activity of T cells was also analyzed after stimulation in the presence of retinal antigens from glaucomatous or nonglaucomatous human donors. Flow cytometry–based analysis indicated that the T cells obtained from glaucomatous patients' blood presented a greater rate of proliferation (as assessed by the percentage of CD4+/EdU+ cells) when incubated with glaucomatous human retinal antigens than when incubated with nonglaucomatous retinal antigens. Data are presented as mean ± SD. P values were obtained by ANOVA.

Figure 6

In vitro analysis of T lymphocytes in glaucoma. T-cell activity was also determined by analysis of cytokine/chemokine secretion in culture supernatant. Cytokine/chemokine secretion of T cells from glaucomatous blood (n = 32) was greater than from controls (n = 21) as determined by a multianalyte ELISArray. Compared to control samples, molecules exhibiting a significantly increased secretion in glaucomatous samples included IL-1β, IL-6, IFN-γ, TNF-α, TGF-β1, MCP1/CCL-2, MIP-1α/CCL-3, and MIP-1β/CCL-4. Data are presented as mean ± SD, and P values are shown when there is statistically significant difference by ANOVA.

Figure 7

In vitro analysis of T lymphocytes in glaucoma. Presented are some of the scatterplots showing a significant relationship between the ratio of CD4+/CD25+/FoxP3+ Tregs (CD4-Tregs) in glaucomatous samples (n = 32) and the rate of in vitro proliferation (as assessed by the percentage of CD4+/EdU+ cells), or ELISA titers of secreted proinflammatory cytokines or chemokines in culture. Data were obtained by linear regression analysis.