IRX-2, a novel immunotherapeutic, enhances and protects NK-cell functions in cancer patients

Abstract

Background: IRX-2 is a primary biologic which has been used for the therapy of head and neck squamous cell cancer (HNSCC) with promising clinical results. Since NK-cell function is compromised in HNSCC patients, we tested the effects of IRX-2 on the restoration of human NK-cell functions in vitro.

Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 23 HNSCC patients and 10 normal controls (NC). The NK-cell phenotype and functions were compared before and after culture ± IRX-2 or ± 50 IU/ml rhIL-2. Flow cytometry was used to study the NK-cell phenotype, cytotoxic activity and cytokine expression.

Results: Impaired NK-cell cytotoxicity in HNSCC patients was related to lower expression of NKG2D, NKp30 and NKp46 receptors (P < 0.05) and not to a decreased frequency of NK cells. Incubation of patients' NK cells with IRX-2 up-regulated the percentage of receptor-positive NK cells (P < 0.05). It also up-regulated cytotoxicity of patients' NK cells (P < 0.01) more effectively than rhIL-2 (P < 0.01). IRX-2, but not rhIL-2, protected NK cells from suppression mediated by TGF-β, and it restored (P < 0.05) expression of activating NK-cell receptors and NK-cell cytotoxicity suppressed by TGF-β. Expression of pSMAD was decreased in NK cells treated with IRX-2 but not in those treated with rhIL-2.

Conclusions: IRX-2 was more effective than IL-2 in enhancing NK-cell cytotoxicity and protecting NK-cell function of HNSCC patients in vitro, emphasizing the potential advantage of IRX-2 as a component of future therapies for HNSCC.

Fig. 1

NK-cell frequency in HNSCC patients and NC: a percentages of CD3⁻CD56⁺ NK cells in PBMC of HNSCC patients (n = 23) and NC (n = 10) were determined by flow cytometry. Box plots show median values, boxes are upper and lower quartile, and whiskers show the maximum and the minimum values. b The distribution of CD3⁻CD56^dimCD16⁺ (white) and CD3⁻CD56^brightCD16⁻ (black) NK-cell subsets among total CD3⁻CD56⁺ NK-cell fraction in HNSCC patients and NC. The frequency was determined by flow cytometry gating on lymphocytes. The data for 23 HNSCC patients and 10 NC are expressed as medians

Fig. 2

NK-cell phenotype and cytotoxicity in HNSCC patients and NC: a expression of activating and inhibitory receptors on NK cells in the peripheral blood of HNSCC patients (n = 12) and NC (n = 10). Phenotype was determined by flow cytometry, and percentages of positive cells in the CD3⁻CD56⁺ lymphocytes gate are shown. Bars indicate medians. b Box plots show NK cytotoxicity in 16 HNSCC patients (10 patients with T1/T2 tumors and 6 patients with T3/T4 tumors) and 10 NC. Bars indicate medians. In this and all other figures, cytotoxicity is expressed in LU₂₀/10⁵ NK cells. c Representative histograms of NK-cell marker expression on CD3⁻CD56⁺ lymphocytes obtained from NC and HNSCC patients. Isotype controls are shown in gray

Fig. 3

Effects of IRX-2 on the NK-cell phenotype and cytotoxicity: a expression of activating and inhibitory receptors on NK cells of 12 HNSCC patients after culture in the presence of IRX-2, rhIL-2 or medium. Median percentages of positive cells are shown. *P < 0.05. b Box plots showing cytotoxicity in 12 HNSCC patients after culture in the presence of IRX-2, IL-2 or medium. Bars indicate medians. c Cytotoxicity was determined in 5 HNSCC patients after a short-term culture in the presence of IRX-2 and the effects of NCR blocking. Blocking antibodies against NKp30 (second column) and NKp46 (third column) as well as a nonspecific isotype IgG (last column) do not significantly decrease cytotoxicity. In contrast, blocking of NKG2D decreases cytotoxicity (P < 0.01). Bars indicate medians

Fig. 4

Effects of IRX-2 on NK-cell cytokine expression: isolated NK cells from HNSCC patients were restimulated with PMA/Ionomycin in the presence of Brefeldin A after 16-h culture in the presence of IRX-2, rhIL-2 or medium, and intracellular cytokine expression was determined by flow cytometry. Results from 6 different HNSCC patients shown, and bars indicate medians

Fig. 5

Protective effects of IRX-2 against TGF-β-mediated suppression: a surface expression of NKG2D and activating NCRs on NK cells of HNSCC patients after culture in the presence of IRX-2, IL-2 or medium ± TGF-β. The phenotype was determined by flow cytometry, and percentages of positive cells gated on CD3⁻CD56⁺ lymphocytes from 6 HNSCC patients are shown. Bars indicate medians. b Cytotoxicity was measured with NK cells of 6 HNSCC patients after culture in the presence of IRX-2, IL-2 or medium ± TGF-β. Bars indicate medians

Fig. 6

IRX-2 prevents SMAD2/3 phosphorylation: representative data for pSMAD2/3 expression as determined by flow cytometry with NK cells obtained from a HNSCC patient after culture ± TGF-β. One set of histograms out of 5 independent experiments is shown