IL-21 Modulates Activation of NKT Cells in Patients with Stage IV Malignant Melanoma

Jonathan M Coquet, Kresten Skak, Ian D Davis, Mark J Smyth, Dale I Godfrey, Jonathan M Coquet, Kresten Skak, Ian D Davis, Mark J Smyth, Dale I Godfrey

Abstract

Interleukin-21 (IL-21) is a common γ-chain cytokine produced by T helper and natural killer T (NKT) cells. It has been shown to regulate the response of various lymphocyte subsets including NK, NKT, T and B cells. Owing to its potent anti-tumor function in preclinical studies and its ability to induce cytotoxicity and interferon-γ (IFN-γ) production in NK and CD8 T cells, recombinant IL-21 (rIL-21) was fast-tracked into early-phase clinical trials of patients with various malignancies. In a phase 2a trial of patients with metastatic melanoma, we analyzed the frequency and function of NKT cells in patients receiving rIL-21. NKT cells were present at a low frequency, but their levels were relatively stable in patients administered rIL-21. Unlike our observations in NK and CD8 T cells, rIL-21 appeared to reduce IFN-γ and TNF production by NKT cells, whereas it enhanced IL-4 production. It also modulated the expression of cell surface markers, specifically on CD4(-) NKT cells. In addition, an increase in CD3(+)CD56(+) NKT-like cells was observed over the course of rIL-21 administration. These results highlight that IL-21 is a potent regulator of NKT cell function in vivo.

Keywords: Cancer; IL-21; NKT; T helper.

Figures

Figure 1
Figure 1
Natural killer T (NKT) cell frequency and CD4 expression in the peripheral blood remain stable following rIL-21 administration. Blood samples from patients receiving rIL-21 were collected on days 1, 2, and 5 and analyzed for NKT cells (α-GC/CD1d tetramer+ CD3+) and CD4 and CD8 expression on NKT cells. (a) Contour and dot plots of peripheral blood NKT cells (left-hand column) and CD4 vs. CD8 expression (middle) on gated NKT cells from one patient. Numbers depict the percentage of cells in the gate/quadrant. (b) The percentage of NKT cells among total lymphocytes and the percentage of CD4+CD8−, CD4−CD8+, and CD4−CD8− (DN) cells among gated NKT cells is depicted over time in patients receiving rIL-21. (c) Contour plots of Forward scatter-height (FSC-H) vs. Side scatter-height (SSC-H) on gated NKT cells from one patient over time. Numbers depict the mean fluorescence intensity (MFI) of SSC-H. The graph shows the relative MFI of SSC-H on gated peripheral blood NKT cells from patients receiving rIL-21. Results were analyzed using the Wilcoxon signed rank test on the raw data with a Bonferroni correction to account for the number of comparisons being made (**P<0.01).
Figure 2
Figure 2
CD25 expression on CD4+ and CD4− natural killer T (NKT) cells following rIL-21 administration. Blood samples were collected and CD25 expression was analyzed on gated NKT cells. (a) Dot plots of gated NKT cells from one patient depicting CD25 vs. CD4 expression. Numbers show the MFI of cells in the gated region. (b) The relative MFI of CD25 expression on gated peripheral blood CD4+ and CD4− NKT cells from patients receiving rIL-21. Note that for some patients, NKT cell proportions were too low to reliably depict CD25 expression, and hence, these patients were omitted from the analysis. Results were analyzed using the Wilcoxon signed rank test on the raw data with a Bonferroni correction to account for the number of comparisons being made (*P<0.05).
Figure 3
Figure 3
CD161 and CD56 expression on CD4+ and CD4− natural killer T (NKT) cells following rIL-21 administration. Blood samples were collected and CD161 and CD56 expression was analyzed on gated NKT cells. (a, c) Dot plots of gated NKT cells from one patient depicting CD161 and CD56 expression. Numbers show the MFI of cells in the gated region. (b, d) The relative MFI of CD161 and CD56 expression on gated peripheral blood CD4+ and CD4− NKT cells from patients receiving rIL-21. Note that for some patients, NKT cell proportions were too low to reliably depict CD161 or CD56 expression, and hence, these patients were omitted from the analysis. Results were analyzed using the Wilcoxon signed rank test on the raw data with a Bonferroni correction to account for the number of comparisons being made (*P<0.05, **P<0.01).
Figure 4
Figure 4
RIL-21 administration alters the production of T helper 1 (Th1) and Th2 cytokines by natural killer T (NKT) cells. On the days of sample acquisition, an aliquot of cells was frozen for subsequent analysis at a later time. At this time, peripheral blood cells from patients in which NKT cells were most prevalent were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 2.5 h in the presence of GolgiStop. Subsequently, cells were stained for α-GC/CD1d tetramer, CD3, CD4, intracellular interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-4 (IL-4) and analyzed by flow cytometry. Vehicle-loaded tetramer was also added to the staining cocktail to gate out nonspecific events. (a) Shown are contour plots of NKT cells from one patient over the course of rIL-21 administration. Plots represent IFN-γ vs. IL-4 or TNF on gated NKT cells. (b–d) Graphs depict the total frequency of cytokine-producing cells among NKT cells (b), the ratio of IL-4/IFN-γ production in NKT cells (c) and the ratio of IL-4/IFN-γ production in conventional CD4 T cells (d) from five individual patients.
Figure 5
Figure 5
Natural killer T (NKT)-like (CD56+βTCR+) cells become more prevalent over the course of rIL-21 administration. (a) Representative contour plots of β T-cell receptor (βTCR) vs. CD56 expression on non-α-GC/CD1d-restricted cells. Numbers represent the percentage of cells in the gated region. (b) The frequency of NKT-like cells in patients administered rIL-21. (c) The frequency of mucosal-associated invariant T (MAIT) cells (CD161+ among βTCR+CD8+CD4−α-GC/CD1d tetramer−) was analyzed over the course of rIL-21 administration. Results were analyzed using the Wilcoxon signed rank test with a Bonferroni correction to account for the number of comparisons being made (*P<0.05).

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Source: PubMed

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