Intracellular IL-4, IL-5, and IFN-γ as the main characteristic of CD4+CD30+ T cells after allergen stimulation in patients with vernal keratoconjunctivitis

Patients

Seventeen consecutive patients from the Department of Immunology at Institute of Ophthalmology (9 males and 8 females, mean age 13.11, range 8–25 years) with active forms of VKC were included in the study. VKC diagnosis was based on clinical ophthalmological history and eye examination. All patients were classified as having active forms of vernal keratoconjunctivitis characterized by limbal, tarsal, or mixed varieties of VKC. The clinical ophthalmological characteristics of the patients were described according to [6], and are depicted in Appendix 1. The specific allergic reaction to Dermatophagoides pteronyssinus (D. pteronyssinus) was confirmed with a skin-prick test positive for D. pteronyssinus (wheal, >3 mm diameter). Healthy age- and sex-matched volunteers were used as controls. All participants gave informed consent or their assent consent for blood sampling after written information was provided, and patient anonymity was preserved during the study. The study adhered to the ethical principles of the Declaration of Helsinki and the E11 Statements of International Conference of Harmonization (E11-ICH). The Institutional Ethics Committee Board of the Institute of Ophthalmology Fundación Conde de Valenciana, Mexico City, approved this study.

Monoclonal antibodies and reagents

Phycoerythrin (PE) labeled mouse monoclonal antibodies (mAbs) against human CD30, IL-5, and IL-4; PECy5-labeled mAbs anti-human CD4 and CD8; and fluorescein isothiocyanate (FITC)-labeled antibodies against human IL-4, IFN-γ, and CD30 were purchased from BD Biosciences (San Jose, CA). Lymphoprep (Ficoll 1.077 density) was obtained from Nycomed Pharma (Nyegaard, Oslo, Norway). RPMI-1640 culture medium, Concanavalin A (Con A), Phorbol myristate acetate (PMA), ionomycin, saponin, brefeldin-A, and salts were from Sigma Chemical Co. (St. Louis, MO). Sodium pyruvate, L-glutamine, and 2-mercaptoethanol were purchased from Gibco BRL (Rockville, MD). Fetal calf serum was from HyClone Labs (Logan, UT). D. pteronyssinus was purchased from Allerstand Co. (Mexico City, Mexico).

Peripheral blood mononuclear cells

Blood samples were collected by venipuncture. Heparinized peripheral blood was diluted 1:2 in PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂P0₄, pH 7.4). Peripheral blood mononuclear cells (PBMCs) were separated on a Ficoll density gradient with centrifugation at 500 ×g for 30 min at room temperature. Then, cells in the interface were collected, washed twice, and counted using a handheld automated cell counter (Millipore Co., Billerica, MA); viability was assessed with eosin dye exclusion.

Cell cultures

The PBMCs were cultured in 96-well flat-bottomed cell culture plates (Costar, Cambridge, MA) at 2×10⁵ cells/well in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 50 μg/ml gentamicin, and 0.5% heat-inactivated fetal calf serum and incubated at 37 °C in a 5% CO₂ humidified chamber. After 24 h, the culture medium was removed. Fresh culture medium supplemented with 10% heat-inactivated fetal calf serum and D. pteronyssinus (7.5 μg/ml) was added. After 7 days of culture, the cells were harvested and processed to measure intracellular cytokine expression and CD30 on the cell surface with flow cytometry. Con A mitogen (2 μg/ml) and/or PMA/ionomycin (5 ng/ml and 0.2 μg/ml, respectively) was used as the cell stimulation positive control. Supernatants were collected from the culture and stored at −70 °C to determine soluble CD30. To assess intracellular IL-4, IL-5, and IFN-γ synthesis, 4 h before the antigen or polyclonal cultures ended, brefeldin-A was added (10 µg/ml). At the end of the incubation period, the cells were harvested and then processed for immunofluorescence staining.

Immunofluorescence staining of cell surface markers

Tri- or four-color staining was performed on the PBMCs with direct immunofluorescence. Briefly, 2×10⁵ cells were suspended in 20 μl PBS supplemented with 0.2% BSA and 0.2% sodium azide (PBA), and incubated with fluorochrome-labeled mAb for 30 min at 4 °C. After incubation, the cells were washed twice with PBA, fixed with 1% p-formaldehyde, and analyzed with flow cytometry.

Immunofluorescence staining of intracellular markers

Stimulated or non-stimulated PBMCs were washed with PBA and stained with PECy5-labeled mAbs against CD4 or FITC for 30 min. After washing, the cells were fixed with 4% p-formaldehyde in PBS for 10 min at 4 °C. The cells were washed twice with PBS and permeabilized with the saponin buffer (0.1% saponin and 10% BSA in PBS) by shaking gently for 10 min at room temperature. Then, the cells were incubated with FITC-labeled anti-human IFN-γ or IL-4 and/or PE-labeled anti-human IL-5 or IL-4. In all cases, isotype-matched controls were used.

Blockade of cytokines

Capture mAbs of anti-human IL-4 (10 μg/ml) were added at the beginning of the cell culture according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN). Then, 5 μg/ml of anti-IL-4 were added at day 3, and on day 5 as a blocking maintenance dose. Isotype-matched negative antibodies were used as controls at the same concentrations. After 7 days of culture, cells were harvested and then processed for immunofluorescence staining as described.

Flow cytometric analysis

All cells were analyzed for the expression of phenotypic markers on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software, and 10,000 events were counted. To analyze the staining of the cell-surface markers, the lymphocytes were first gated by their physical properties (forward and side scatter). Then a second gate was drawn based on the immunofluorescence characteristics of the gated cells, and fluorescence intensity was assessed with histograms. To determine CD30+ T cells, the cells were first gated on an Forward Scattered (FSC)-Side Scattered (SSC) dot plot. Then the lymphocytes were gated on CD4+ or CD8+ T cells in an SSC-CD4 or SSC-CD8 dot plot, CD4+ or CD8+ cells were selected, and a dot plot was created to select CD30+ or CD30- on CD4+ and CD8+ T cells. Finally, to analyze the intracellular cytokine staining on helper and cytotoxic CD30+ or CD30- T cells, a histogram was created to analyze the percentage of cytokine positive cells. Data are presented as dot plots or histograms. Control stains were performed using isotype-matched mAbs of unrelated specificity. Background staining was <1% and was subtracted from the experimental values.

Determination of sCD30

Soluble CD30 (human sCD30, Kamiya Biomedical Company, Seattle, WA) was measured with enzyme-linked immunosorbent assay (ELISA) in the supernatants of the cultures according to the manufacturer’s instructions and read at 620 nm in a microplate reader (ThermoLabsystems, Multiskan Ascent, Helsinki, Finland).

Statistical analysis

Results are described in this work using Media and standard deviation (SD), or Median (MD) and interquartile ranges (IQR), according with data distribution. The Mann–Whitney U test and the Wilcoxon signed-rank test were used to detect significant differences. The analysis was performed with GraphPad Prism software v.6.0. Differences were considered statistically significant when p value was less than 0.05.

CD30 is expressed after D. pteronyssinus stimulation on CD4+ and CD8+ T cells from patients with VKC

We began by determining the percentage of CD4+CD30+ T cells and CD8+CD30+ T cells in the peripheral blood of patients with vernal keratoconjunctivitis and healthy controls. No significant differences were observed in the frequency of circulating CD4+CD30+ T cells between patients with VKC and healthy controls (HCs; MD 3%, IQR 1.3–3.3 versus MD 1.7%, IQR 1–1.7, respectively; p=0.08). Similarly, we did not find differences in CD8+CD30+ T cells between patients with VKC and the HCs (MD 1.5%, IQR 0.5–1.8 versus MD 1% IQR 0.4–1.8, p=0.1). However, after allergen stimulation, we observed a significant increase in the percentage of CD4+CD30+ T cells from patients with VKC compared with the HCs (MD 22.2%, IQR 14.6–41.7 versus MD 9.3%, IQR 6–11, respectively; p=0.004). Likewise, when we compared the frequency of the CD8+CD30+ T cells after the D. pteronyssinus stimulation, we observed a significant increase cytotoxic CD30+ cells in patients with VKC compared with the HCs (MD 19.9%, IQR 8–7-30.8 versus MD 7.2%, IQR 6.4–8.2, p=0.03).

As shown in Table 1, the frequency of CD4+CD30+ T cells from patients with VKC increased 16.8-fold compared with non-stimulated cells (p<0.0001), and was 2.6 times increased compared with PMA/ionomycin (p<0.0001; Figure 1). We also observed that the helper CD30+ T cells increased 1.18-fold compared with the frequency of the CD8+CD30+ T cells after D. pteronyssinus stimulation (p=0.03; Table 1).

CD30+ helper T cells express IL-4, IL-5, and IFN-γ after D. pteronyssinus stimulation

To establish the potential involvement of the specific antigenic-stimulation in the expression of Th1/Th2 cytokines in helper CD30+ or CD30-T helper cells, we assessed the percentage of IL-4, IL-5, and IFN-γ after D. pteronyssinus stimulation in patients with active VKC. Upon allergen stimulation, IFN-γ was expressed 11.4-fold more in CD4+CD30+ T cells than in CD4+CD30- T cells (p<0.0001). Similarly, IL-4 was expressed 4.3-fold more in CD4+CD30+ T cells than in CD4+CD30- T cells (p<0.0001). Likewise, IL-5 was expressed 4.1-fold more in CD30+ helper T cells than in CD30- helper T cells (p=0.009; Figure 2).

sCD30 increased after polyclonal stimulation in patients with VKC

To understand whether polyclonal stimulation (Con A) induces secretion of sCD30, we evaluated soluble CD30 at 24, 48, 72, and 96 h in the supernatant of cells from patients with VKC and the HCs. We observed 4.5 times more sCD30 in the supernatant of the cultures from patients with VKC since 72 h after Con A stimulation, compared with the HCs (p=0.03), and 7.4 times more sCD30 after Con A stimulation at 96 h in the supernatant of the cultures from patients with VKC compared with the HCs (p=0.01; Figure 3).

Blockage of IL-4 decreased CD30 expression on CD4+ T cells

To see whether IL-4 is involved in CD30 expression on helper T cells from patients with VKC, we blockaded IL-4 function by adding neutralizing mAbs against IL-4 during the D. pteronyssinus stimulation. We observed 3.9-fold less frequency of CD4+CD30+ T cells when we added anti-IL-4 (p=0.03) (Figure 4).