Introduction

Modulation of the immune response has formed the basis of most recently developed therapies for inflammatory disease, organ transplantation, and strikingly for some cancers. The rational development of new immunotherapies that can be successfully translated into clinical practice is facilitated by a detailed understanding of the underlying molecular immunology.

Inosine pranobex (IP) has been demonstrated to have therapeutic benefit in human papillomavirus‐induced warts 1, measles virus infection resulting in subacute sclerosing panencephalitis (SSPE) 2 and alopecia 3. It is currently licensed for use in the treatment of genital papilloma virus‐induced warts, HSV infections and SSPE in many countries.

Despite this clinical use, the molecular mechanisms underlying IP‐induced immunomodulation remain unclear. Existing studies provide mechanistic insights. IP enhances the proliferative response of lymphocytes exposed to conventional mitogens, including phytohaemagglutinin 4, 5, Con A 6, and anti‐CD3 4, but lacks intrinsic lymphocyte mitogenic capability 7. IP‐treated lymphocytes are both more responsive to, and more productive of, IL‐2 5, 8. While some studies demonstrate IP‐induced enhanced NK cell cytotoxicity 6, other studies have not observed this effect 9. These findings were largely published before the discovery of the NKG2D receptor and its ligands, and do not address the possibility that the effects observed may be mediated through increased triggering of NKG2D by activating ligands on target cells. In most of these studies, the duration of exposure of target cells to IP during the experiments is unclear.

NKG2D is an activating receptor expressed predominantly on NK cells, CD8+ T cells, γδ T cells, NKT cells, and some CD4+ T cell populations 10. A series of eight activating ligands for NKG2D have been identified in humans and these include the MICA, MICB and the RAET1 or ULBP molecules. While the NKG2D ligands are generally not expressed in healthy quiescent cells, expression can be induced in response to several stimuli, including viral infection 11, DNA damage 12, inflammatory cytokines 13, loss of cell adhesion 14, and proliferative cell activation 15. We have previously demonstrated that enhanced purine nucleoside synthesis controls NKG2D ligand upregulation 16. The transcriptional control of NKG2D ligands is incompletely understood, but in the best studied ligand, MICA, it involves intragenic transcriptional interference between tandem promoters 17. Cells expressing NKG2D ligands become targets for immune cells that express the NKG2D receptor 18. Binding between the NKG2D receptor on immune cells and NKG2D ligands on potential target cells can lead to cytotoxicity against these target cells 19, cytokine secretion 20, or co‐stimulation 21 depending on the immune context (Fig. 1A).

Figure 1 Open in figure viewer PowerPoint IP induces dose‐dependent cell surface NKG2D ligand expression. (A) NKG2D ligands are not typically expressed on healthy quiescent cells. Stimuli including malignant transformation, viral infection, and proliferative lymphocyte activation are associated with NKG2D ligand induction. Expression can cause cytotoxicity, cytokine secretion, or costimulation through binding to the activating receptor, NKG2D. (B) HEK293T cells were cultured in 5 mM glucose with 0.25, 1, or 2 mM IP for 48 h, and cell surface expression of MICA (2C10) was measured by flow cytometry. A strong dose‐dependent increase in MICA expression was observed. Isotype controls (dotted histogram), cells cultured in 5 mM glucose only (light grey shaded histogram) or in 25 mM glucose (dark grey shaded histogram) are also shown. (C) Cells were cultured in 5 or 25 mM glucose with IP in biological triplicates and MICA expression was measured by flow cytometry. In 5 mM glucose, IP produced a significant increase in cell surface MICA expression compared to untreated cells. In 25 mM glucose, a significant increase in MICA expression was observed at higher IP concentrations. (D) HEK293T cells, (E) HT1080 cells (human fibrosarcoma), and (F) HeLa cells (human cervical carcinoma) demonstrate dose‐dependent MICA (2C10) expression when cultured with IP. (G) We tested whether IP influenced total cellular MICA levels by staining permeabilized and non‐permeabilized cells in parallel. Permeabilized cells displayed the same dose‐dependent IP‐induced MICA expression as non‐permeabilized cells. (H) We tested for adequate cell permeabilization by measuring the expression of PCNA (14‐9910‐80) in both non‐permeabilized and permeabilized cells by flow cytometry. PCNA was only detected in permeabilized cells and did not increase in an IP‐dependent manner. (I) The effect of IP on the induction of multiple NKG2D ligands including MICB (MAB1599), ULBP1 (MAB1380), ULBP2 (MAB1298), ULBP3 (MAB1517), ULBP4 (6E6), and ULBP5 (6D10) was tested by flow cytometry. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. Histograms represent mean and 95% confidence interval. The data shown is from a single experiment that contained three biological replicates. Experiments were performed independently three times with consistent results. Means were compared using t‐tests. MFI, mean fluorescence intensity.

We noted an overlap between the reported consequences of IP treatment and the consequences of NKG2D activation, including enhanced lymphocyte proliferation, cytokine secretion, and NK cell cytotoxicity. Therefore, we hypothesized that IP acts by inducing NKG2D ligand expression in metabolically susceptible target cells, as distinct from a direct action on immune cells alone. To test this hypothesis, we measured the impact of IP treatment on NKG2D ligand induction and assessed the functional effect of this ligand induction on NK cell cytotoxicity.