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. 2009 Aug;50(8):3802-7.
doi: 10.1167/iovs.08-2417. Epub 2009 Apr 1.

Characterization of effector T cells in dry eye disease

Jaafar El Annan  1 , Sunil K Chauhan, Tatiana Ecoiffier, Qiang Zhang, Daniel R Saban, Reza Dana
Affiliations

Characterization of effector T cells in dry eye disease

Jaafar El Annan et al. Invest Ophthalmol Vis Sci. 2009 Aug.

Abstract

Purpose: Dry eye disease (DED) is associated with ocular surface inflammation that is thought to be mediated primarily by CD4 T cells. The purpose of this study was to investigate whether this T cell-mediated immune response is generated in the lymphoid compartment and to characterize the functional phenotype of the T cells activated in DED.

Methods: DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled environment chamber and to systemic scopolamine. T cells from regional draining lymph nodes (LNs) of DED mice and normally sighted mice were analyzed for surface activation markers (CD69 and CD154), chemokine and cytokine receptors, and proliferation potential.

Results: Draining LNs of DED mice showed increased frequencies of CD69- and CD154-expressing T cells with higher proliferative capacity. In addition, these LN T cells primarily showed a helper T-cell (Th)1 phenotype, expressing significantly higher levels of IFN-gamma and IL-12Rbeta2 but not IL-4R. Similarly, the LNs of DED mice showed significantly increased frequencies of T cells expressing CXCR3 and CCR5, but not CCR4, suggesting a bias toward a Th1 phenotype.

Conclusions: These data demonstrate that a Th1-type immune response is induced in the regional LNs of DED mice. The identification of specific cytokine/chemokine receptors overexpressed by these T cells may signify potential novel targets/strategies for the treatment of DED.

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Figures

Figure 1
Figure 1. Analysis of T cell surface activation markers expression and its proliferative capacity
Draining LN were harvested from DE vs. normal (NL) mice; single-cell suspensions were prepared and dual stained with anti-CD4 FITC (A) or anti-CD8 FITC (B) and either one of the activation marker antibodies anti-CD69 PE, or anti-CD154 PE. Data shown are representative of five independent experiments. (C) CD4+ T cells were sorted from the draining LN of DE vs. NL mice using the MACS cell isolation kit. CD4+ T effector cells were cocultured with T cell-depleted syngeneic splenocytes and anti-CD3 antibody for three days. Proliferative response was assessed by BrdU incorporation assay. Results are presented as optical density(OD) reading ± SEM. Data shown are representative of three independent experiments.
Figure 2
Figure 2. Analysis of T cell cytokine secretion
The frequencies of reactive T cells upon CD3 stimulation from draining LN of DE vs. NL mice were evaluated using the ELISPOT assay for(A) IFN-γ and (B) IL-4. The results are depicted as the mean number of spots per 0.5 million responder T cells loaded ± SEM. Data shown are representative of three independent experiments.
Figure 3
Figure 3. Flow cytometric analysis of cytokine receptors by T cells
Draining LN were harvested from DE vs. NL mice, single-cell suspensions were prepared and dual stained with anti-CD4 FITC (A) or anti-CD8 FITC (B) and either one of the cytokine receptor antibodies anti-IL-12Rβ2 PE, or anti-IL-4R PE. Data shown are representative of four independent experiments.
Figure 4
Figure 4. Flow cytometric analysis of chemokine receptors by T cells
Draining LN were harvested from DE vs. NL mice, single-cell suspensions were prepared and dual stained with anti-CD4 FITC (A) or anti-CD8 FITC (B) and either one of the chemokine receptor antibodies anti-CCR5 PE, anti-CXCR3 PE, or anti-CCR4 PE. Data shown are representative of four independent experiments.
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