CTLA4 blockade increases Th17 cells in patients with metastatic melanoma

Abstract

Background: Th17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature.

Methods: Peripheral blood mononuclear cells (PBMC) were collected before and after receiving tremelimumab within two clinical trials, one with tremelimumab alone (21 patients) and another together with autologous dendritic cells (DC) pulsed with the melanoma epitope MART-126-35 (6 patients). Cytokines were quantified directly in plasma from patients and after in vitro stimulation of PBMC. We also quantified IL-17 cytokine-producing cells by intracellular cytokine staining (ICS).

Results: There were no significant changes in 13 assayed cytokines, including IL-17, when analyzing plasma samples obtained from patients before and after administration of tremelimumab. However, when PBMC were activated in vitro, IL-17 cytokine in cell culture supernatant and Th17 cells, detected as IL-17-producing CD4 cells by ICS, significantly increased in post-dosing samples. There were no differences in the levels of Th17 cells between patients with or without an objective tumor response, but samples from patients with inflammatory and autoimmune toxicities during the first cycle of therapy had a significant increase in Th17 cells.

Conclusion: The anti-CTLA4 blocking antibody tremelimumab increases Th17 cells in peripheral blood of patients with metastatic melanoma. The relation between increases in Th17 cells and severe autoimmune toxicity after CTLA4 blockade may provide insights into the pathogenesis of anti-CTLA4-induced toxicities.

Trial registration: ClinicalTrials.gov NCT00471887.

Figure 1

Cytokine quantitation in patient's plasma. A) ELISA analysis of IL-17 in cryopreserved plasma samples taken from patients before and after tremelimumab dosing. B) Multicytokine array quantifying IL-4, IL-5, IL-6, IL-10, IL-13, TNFα,, INF-γ, MCP-1 and RANTES in cryopreserved plasma before and after dosing with tremelimumab.

Figure 2

IL-17 quantification by ELISA. A and B) Pre- and post-dosing IL-17 cytokine determined in culture supernatants of whole PBMC (A) or CD4+-sorted cells (B) after stimulation for 4 days with anti-CD3 and anti-CD28. The supernatant was collected for IL-17 quantitation using an ELISA assay (p values by pairwise t-test). C) Multicytokine array in the same ex vivo stimulated samples quantifying IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12(p70), IL-13, TNFα, and RANTES.

Figure 3

Increase in Th17 cells after tremelimumab-based therapy by intracellular cytokine staining. A) Gating strategy for IL-17 intracellular staining. Starting from either whole PBMC or CD-4 sorted cells (as depicted here), the lymphocyte population was gated on by FSC-H and SSC-H dot plot. Live cells were gated in the same graphic. A second gate was performed in CD3 and SSC-H dot plot. We analyzed for IL-17-producing cells among CD4+ T cells after gating. B) Example of IL-17 intracellular staining. After 4-day activation of CD4-sorted cells with anti-CD3 and anti-CD28, cells were additionally stimulated with PMA and ionomycin while inhibiting protein transport, and the number of Th17 cells was determined by flow cytometry. Depicted are the plots of gated Th17 cells from patient NRA12. The left column is the baseline pre-dosing sample, and the right column the post-dosing sample. C) Th17 quantification by flow cytometry. Pre- and post-dosing whole PBMC (left graph) or CD4+ cells (right graph) analyzed by flow cytometry for Th17 cells as described above (p values by pairwise t-test).

Figure 4

IL-17 Intracellular Staining and IL-17 ELISA According to the Development of Inflammatory or Autoimmune Toxicity. A and B) IL-17 secretion detected by ELISA as described in Figure 1, and Th17 by intracellular staining (ICS) as described in Figure 2, comparing the assay results in whole PBMC cultures from patients with Grade 3 or higher toxicity and the rest of the patients (p values by pairwise t-test). C and D) The same analysis with CD4-sorted cultures.