Profiling Secreted Cytokines from Human Th2 and Th17 Cells using Antibody Arrays

D. Finkel, A. James, K. Brumbaugh, S. Dhawan, C. Goetz, J. Bonnevier and G. Wegner

ABSTRACT

Naïve T Helper cells can differentiate into Th1, Th2, and Th17 lineages each of which secrete a characteristic profile of molecules. In this study, we demonstrate the utility of antibody arrays to monitor T cell differentiation processes. Naïve T cells were isolated from PBMC preparations obtained from different donors. Following activation, the generation of each T cell subtype was confirmed by flow cytometry. Secreted proteins present in the cell culture supernatants were analyzed with a Proteome Profiler™ Human XL Cytokine Array capable of recognizing 102 analytes. More than 30 proteins were identified, including cytokines specific to each Th subtype. ELISA data were used to quantitatively confirm the increases and decreases of multiple Th-specific cytokine levels observed using the array. The data demonstrate that antibody arrays provide an efficient tool to simultaneously study multiple parameters in complex biological processes where alternative differentiation protocols are being tested along with evaluating the impact of donor-to-donor variability.

Proteome Profiler Array Assay Principle

Arrays are composed of capture and control antibodies spotted in duplicate on nitrocellulose membranes. Cell culture supernatants or extracts were diluted, mixed with a cocktail of biotinylated detection antibodies, and incubated overnight with the Proteome Profiler Human XL Cytokine Antibody Array (Catalog #ARY022). Streptavidin-HRP and chemiluminescent detection reagents were applied to the arrays, and the signal produced at each capture spot corresponded to the amount of protein bound.

Proteome Profiler Human XL Cytokine Array Content

Naïve T cell Enrichment.

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Figure 1. Human PBMCs were processed on a Ficoll density gradient and analyzed using a CELL-DYN Sapphire (A). Naïve T cells were isolated from these PBMCs using the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115). A portion these cells, pre- and post-enrichment, were stained with Fluorescein-conjugated Mouse Anti-Human CD4 Antibody (Catalog # FAB3791F) and either Phycoerythrin-conjugated anti-human CD45RO antibody (for memory T cells) or Phycoerythrin-conjugated anti-CD45RA Antibody (for naïve T cells). Data shown (B) were gated on lymphocytes using forward and side scatter parameters. Quadrant markers were set based on staining of isotype control antibodies.

Th1 Differentiation Profile.

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Figure 2. Naïve T Cells were isolated from PBMCs using the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115). These cells were either untreated (Th0) or treated with Human Th1-inducing reagents; Mouse Anti-Human CD3 epsilon Antibody (Catalog # MAB100), Recombinant Human IL-2 (Catalog # 202-IL) and Recombinant Human IL-12 (Catalog # 219-IL), for 5 days. Supernatants were collected and run on the Proteome Profiler Human XL Cytokine Array Kit (Catalog # ARY022). Array data show the presence of Th0 and Th1 secreted cytokines (A). The Human IFN-gamma Quantikine® ELISA (Catalog # DIF50), Human IL-10 Quantikine ELISA (Catalog # D1000B) and Human TNF-alpha Quantikine ELISA Kits (Catalog # DTA00C) were run to confirm the presence (or absence, Th0) of these cytokines (B). The profile of other selected cytokines in the supernatants of Th0 and Th1 cultures are shown (C).

Th2 Differentiation Profile.

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Figure 3. Naïve T Cells were isolated from PBMCs using the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115). These cells were either untreated (Th0) or treated with Human Th2-inducing reagents; Mouse Anti-Human CD3 epsilon Antibody (Catalog # MAB100), Recombinant Human IL-2 (Catalog # 202-IL), Recombinant Human IL-4 (Catalog # 204- IL), Mouse Anti-Human IFN-gamma Antibody (Catalog # MAB285) and Mouse Anti-Human CD28 Antibody for 5 days. Supernatants were collected and run on the Proteome Profiler Human XL Cytokine Array Kit (Catalog # ARY022). Array data show the presence of Th0 and Th2 secreted cytokines (A). The Human IL-3 Quantikine ELISA (Catalog # D3000), Human IL-5 Quantikine ELISA (Catalog # D5000B) and Human TNF-alpha Quantikine ELISA Kits (Catalog # DTA00C) were run to confirm the presence (or absence, Th0) of these cytokines (B). The profile of other selected cytokines in the supernatants of Th0 and Th2 cultures are shown (C).

Th17 Differentiation Profile.

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Figure 4. Naïve T Cells were isolated from PBMCs using the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115). These cells were either untreated (Th0) or treated with Human Th17-inducing reagents; Mouse Anti-Human CD3 epsilon Antibody (Catalog # MAB100), Recombinant Human IL-2 (Catalog # 202-IL), Recombinant Human IL-23 (Catalog # 1290-IL), Recombinant Human IL-1 beta (Catalog # 201-LB), Recombinant Human IL-6 (Catalog # 206-IL) and Mouse Anti-Human CD28 antibody for 5 days. Supernatants were collected and run on the Proteome Profiler Human XL Cytokine Array Kit (Catalog # ARY022). Array data show the presence of Th0 and Th17 secreted cytokines (A). The Human IL-17 Quantikine ELISA (Catalog # D1700), Human IL-22 Quantikine ELISA (Catalog # D2200), Human CCL20/MIP3-alpha Quantikine ELISA (Catalog # DM3A00) and Human TNF-alpha Quantikine ELISA Kits (Catalog # DTA00C) were run to confirm the presence (or absence, Th0) of these cytokines (B). The profile of other selected cytokines in the supernatants of Th0 and Th17 cultures are shown (C).

Th17 Differentiation Profiles - Multiple Donors.

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Figure 5. Naïve T Cells from eight different donors were treated with Human Th17-inducing reagents. Cultures on the left (A) were treated with Mouse Anti-Human CD3 epsilon Antibody (Catalog # MAB100), Recombinant Human IL-23 (Catalog # 1290-IL), Recombinant human IL-6 (Catalog # 206-IL), Recombinant Human TGF-beta (Catalog # 240-B), Mouse Anti-Human IFN-gamma Antibody (Catalog # MAB285) and mouse anti-human CD28 antibody for 5 days. Cultures on the right (B) were treated with Mouse Anti-Human CD3 epsilon Antibody (Catalog # MAB100), Recombinant Human IL-2 (Catalog # 202-IL), Recombinant Human IL-23 (Catalog # 1290-IL), Recombinant Human IL-6 (Catalog # 206-IL), Recombinant Human IL-1 beta (Catalog # 201-LB), and mouse anti-human CD28 antibody for 5 days. Supernatants were collected and run on the Proteome Profiler Human XL Cytokine Array Kit (Catalog # ARY022). Array images are shown.

Th2 Differentiation Profiles - Time Course.

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Figure 6. Naïve T Cells were treated with Human Th2-inducing reagents; Mouse Anti-Human CD3 epsilon Antibody (Catalog # MAB100), Recombinant Human IL-2 (Catalog # 202-IL), Recombinant Human IL-4 (Catalog # 204-IL), Mouse Anti-Human IFN-gamma Antibody (Catalog # MAB285) and mouse anti-human CD28 antibody for 3, 5 and 7 days. Supernatants were collected and run on the Proteome Profiler Human XL Cytokine Array Kit (Catalog # ARY022). Spot images of Proteome Profiler membrane arrays are shown (A) and the corresponding histogram profiles of select analytes are shown (B).

CONCLUSION

The Proteome Profiler Human XL Cytokine Array is an economical alternative to traditional methods for screening differences in soluble protein expression. This membrane-based array assay is easy to perform and can be completed in approximately 4.5 hours. In addition, this assay employs a chemiluminescence detection method, thus, no specialized equipment beyond what is typically used to collect Western blot data is required. This array can profile the changes of up to 102 cytokines from a single sample. This allows the researcher to screen for and monitor the levels of many analytes in one experiment.

For research use only. Not for use in diagnostic procedures.