IL-21 induces in vivo immune activation of NK cells and CD8(+) T cells in patients with metastatic melanoma and renal cell carcinoma

Abstract

Purpose: Human interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine.

Experimental design: Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 microg/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8(+) T cells and CD56(+) NK cells by quantitative RT-PCR, and gene array profiling of CD8(+) T cells.

Results: Effects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8(+) T cells and CD56(+) NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8(+) T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo.

Conclusions: IL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8(+) T cell activation.

Fig. 1

IL-21 induced STAT3 phosphorylation at all dose levels. Whole blood was drawn immediately before the first IL-21 dose and 15 min after dosing. Half of the pre-dose sample was stimulated for 15 min ex vivo with 10 ng/mL IL-21 to provide a positive control. Samples were analyzed by flow cytometry for pSTAT3 in CD3⁺ T cells. a Representative dot plots of pre-dosing, post-dosing and ex vivo positive control samples. b For each dose level the average pSTAT3 mean fluorescence intensity in CD3⁺ T cells in percent of the ex vivo positive control samples is shown

Fig. 2

IL-21 induced transient lymphopenia and upregulation of cell adhesion molecules. Absolute number of lymphocytes were measured in the US trial by standard complete blood count analysis and depicted per dose level in cycle 1 (a) or per dose cycle at 30 μg/kg (b) (mean ± SEM). c, d Lymphocyte subsets were measured in the US trial by flow cytometry. c Absolute numbers of T helper cell (CD4⁺), T effector cell (CD8⁺), NK cells (CD16/56⁺), B cells (CD19⁺) and monocytes (CD14⁺) at 30 μg/kg cycle 1 (mean ± SEM). d Percent of naïve B cells (CD19⁺CD38⁻) and plasma cells (CD19⁺CD38^HI) at 30 μg/kg cycle 1 (mean ± SEM). In the Australian trial CD8⁺ T cell adhesion markers were measured by flow cytometry. Percentage of CD8⁺ T cells that express CD62L (e) CCR7 (f) or CD45RA (g) is shown for individual patients in the 5 + 9 regimen. All samples were drawn on the indicated dosing day prior to dosing. As IL-21 administration was initiated on day 1, samples drawn on day 1 represent pre-dose samples

Fig. 3

a Gene ontology (GO-) tree of significantly overrepresented GO-terms (Biological Process “BP” terms). Each circle in the tree corresponds to a particular GO term, and the size of the circle indicates the degree of overrepresentation (see also ESM Table 2). Circles close to the root of the tree corresponds to very generic terms whereas circles closer to the end of the branches represent increasingly specific terms. The ten most significantly overrepresented GO terms are: 1 cell cycle, 2 mitotic cell cycle, 3 cell division, 4 M phase, 5 mitosis, 6 M phase of mitotic cell cycle, 7 DNA replication, 8 DNA metabolism, 9 spindle organization and biogenesis, 10 microtubule-based process. b Heatmap of clustered gene regulations (immunorelated genes). Log₂-fold regulations (day 5 versus day 1 (pre-dose)) for individual patients enrolled in the “5 + 9” regimen at six different dose levels are shown. Some genes are represented by two or three individual probesets. Average fold regulations (dose levels 30, 50, and 100 μg/kg) are listed for the individual probesets

Fig. 4

Quantitative RT-PCR data. a Analysis of granzyme B and perforin mRNA levels in CD8⁺ and CD56⁺ cells from patients dosed three times weekly (“3/week”) or five consecutive days of dosing followed by 9 days with no dosing (“5 + 9”). b Analysis of granzyme A (GZMA), interferon-γ (IFN-γ), hyaluronan-mediated motility receptor (HMMR), and CXCR3 mRNA levels in CD8⁺ cells from patients in the 5 + 9 regimen. Data from day 1 (pre-dose) and day 5 samples are shown. Part of Fig. 4a has previously been published [9]

Fig. 5

Enhanced NK cytotoxicity towards K562. PBMC isolated at screening, day 1 (pre-dose) and day 5 were frozen immediately and stored in liquid nitrogen. After thawing the PBMC were incubated for 4 h with ⁵¹Cr-labeled K562 erythroleukemia cells. For each subject PBMC from all time points were tested in the same assay