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  4. Th17 differentiation assay - % Th17 & IL-17A production

Th17 differentiation assay - % Th17 & IL-17A production

Ankylosing Spondylitis

Ankylosing Spondylitis

Contact:
Lynn Williams
lynn.williams@kennedy.ox.ac.uk
www.ndorms.ox.ac.uk/team/lynn-williams
Rationale

In support of genetic data genetics (which implicates type 17 related genes), the strongest biologic factor reported to be associated with AS is IL-17 and type 3 immunity. IL-17A was found to be elevated in serum, synovial fluid, joints and CD4+ (TH17) cells from patients with AS(1, 2).

Aim

To identify probes and pathways that regulate generation of Th17 cells or downregulate production of IL-17A from CD4 T cells from AS patient peripheral blood

Conclusions

Aside from BET & CREBBP/EP300 bromodomain inhibitors already demonstrated to reduce IL-17A and Th17 axis (3), the BRPF inhibitor OF-1 shows strongest potential for inhibiting IL-17A and % Th17 cells (CD4+ T cells producing IL-17A), an established therapeutic target in AS & PsA (4, 5).

Experimental Protocol
  1. PBMCs were isolated from whole blood from SpA patients using Histopaque and rested overnight at 37oC/5% CO2. 
  2. CD4+ T cells were then isolated by Miltenyi Biotec negative selection kit and plated in a U bottom 96 well plate in 200 µl RPMI 1640 containing 10% FBS and glutamine. 
  3. IL-2, Th17-skewing cytokines and T cell expander beads were added. 
  4. 1 µM probes or DMSO as a negative control were also added (d0). 
  5. At day 3, 100 µl of culture supernatant was removed and 100 µl of fresh probes in culture media with cytokines was added. 
  6. On day 6, cells were spun down and supernatants collected and frozen for quantification of IL-17A by ELISA. 
  7. For intracellular cytokine staining, the remaining cells were stimulated with PMA/ionomycin plus brefeldin A and monensin for 4 hrs, then surface staining for CD3, CD4, CD8 and live/dead staining was carried out. 
  8. Cells were washed, fixed and permeabilised. Permeabilised cells were then labeled with antibodies to IL-17A and IFNg, then analysed on BD Calibur Fortessa.
  1. Collect 20ml of blood from SpA patient
  2. Isolate whole PBMCs, rest overnight
  3. CD4+ T cell negative isolation
  4. Culture of CD4+ T cells under Th17-skewing condition for 6 days plus expander beads
  5. Probes added from day 0, and replenished at day 3
  6. At day 6, harvest cells for FACS (Th1, Th2, Th17) and collect supernatant for ELISA (IL-17A).

Patient T cells were cultured as described and cells were analysed by flow cytometry and elisa to assess cytotoxicity induced by the probes (A), Th17 frequency (B), and levels of IL-17A production (C). Cytotoxicity was detected by positive live/dead staining, with dead cells excluded from analysis for % Th17 cells.

IL-17A production from probe treated SpA patient T cells

AS (AS prefix) or PsA (PS prefix) patient T cells were cultured according to the described protocol and IL-17A production was measured in the culture supernatants. Results are normalized to the negative DMSO control for each donor. The final column is the relative mean of all donors.

% Th17 cells from probe treated SpA patient T cells

AS (AS prefix) or PsA (PS prefix) patient T cells were cultured according to the described protocol and %Th17 cells (CD4+, IL-7A+) was enumerated by flow cytometry. Results are normalized to the negative DMSO control for each donor. The final column is the relative mean of all donors.

 

  1. Mei, Y., Pan, F., Gao, J., Ge, R., Duan, Z., Zeng, Z., Liao, F., Xia, G., Wang, S., Xu, S., Xu, J., Zhang, L., and Ye, D. (2011) Increased serum IL-17 and IL-23 in the patient with ankylosing spondylitis, Clin. Rheumatol. 30, 269-273.
  2. Shen, H., Goodall, J. C., and Hill Gaston, J. S. (2009) Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis, Arthritis Rheum. 60, 1647-1656.
  3. Hammitzsch, A., Tallant, C., Fedorov, O., O'Mahony, A., Brennan, P. E., Hay, D. A., Martinez, F. O., Al-Mossawi, M. H., de Wit, J., Vecellio, M., Wells, C., Wordsworth, P., Muller, S., Knapp, S., and Bowness, P. (2015) CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses, Proc. Natl. Acad. Sci. U. S. A. 112, 10768-10773.
  4. Koenders, M. I., and van den Berg, W. B. (2016) Secukinumab for rheumatology: development and its potential place in therapy, Drug design, development and therapy 10, 2069-2080.
  5. Paine, A., and Ritchlin, C. T. (2016) Targeting the interleukin-23/17 axis in axial spondyloarthritis, Curr. Opin. Rheumatol. 28, 359-367.