PD-1 blockade enhances cytokine-induced killer cell-mediated cytotoxicity in B-cell non-Hodgkin lymphoma cell lines.

The clinical utility of immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors used alone or in combination with other therapies, is currently gaining attention. In this particular scenario, the inclusion of cytokine-induced killer (CIK) cells has proven to be a novel therapeutic approach. CIK cells have shown anticancer activity in various hematopoietic malignancies, but their defined cytotoxicity in B-cell non-Hodgkin lymphoma (B-NHL) remains to be fully elucidated. The present study investigated the role of PD-1/PD-L1 blockades on the cytotoxic efficacy of CIK cells primarily in B-NHL cell lines. The current analysis revealed that CIK cells prompted cytotoxicity against B-NHL cell lines (DAUDI and SU-DHL-4), and a significant increase in PD-L1 expression was observed when CIK cells were co-cultured with B-NHL cells. Additionally, a combination of PD-1 and PD-L1 antibodies with CIK cells significantly decreased cell viability only in DAUDI cells. Furthermore, IFN-γ elevation was observed in both cell lines treated with CIK alone or with PD-1 antibody, but this tendency was not observed for PD-L1. Since PD-1 can suppress immune inactivation, whereas CD40L can promote it, the effects of CD40L blockade were also examined; however, no significant changes in cell viability were observed. Overall, the present in vitro data suggested that CIK cells exerted a cytotoxic function in B-NHL cells, and a combination of PD-1 inhibitors with CIK cells may provide a potential therapeutic option for this type of lymphoma. Nevertheless, in vivo experiments are warranted to undermine the extent to which PD-1 inhibitors may be used to enhance the antitumor activity of CIK cells in B-NHL.

Interaction Between CCL18 and GPR30 Differs from the Interaction Between Estradiol and GPR30.

BACKGROUND/AIM: C-C motif chemokine ligand 18 (CCL18) is overexpressed in the microenvironment of tumors, promotes invasion and metastasis and is thus important for the therapeutic outcome of many tumor entities. The Gs-coupled seven-transmembrane receptor GPR30 is known as both a CCL18 and an estrogen receptor; its activation by estradiol leads to a transactivation of membrane-tethered pro-heparin-binding EGF-like growth factor and the MAPK/ERK pathway. We examined whether this signaling pathway remains the same under CCL18 stimulation, as opposed to estradiol stimulation. MATERIALS AND METHODS: We investigated the effects of CCL18 on the lung cancer cell line A549, that show low GPR30 expression and the breast cancer cell lines MCF-7, that has high GPR30 expression and MDA-MB-231. These cells were stimulated in different media with CCL18 and then analyzed by qPCR, In-Cell Western®, western blot and ELISA. RESULTS: Many similarities on the effect of CCL18 on the already known estradiol-activated signaling pathway via the G protein-coupled estrogen receptor GPR30 were identified. GPR30 is involved in the expression of matrix metalloproteinases (MMPs), which may play a role in the transactivation of ERK-1/-2 via the cleavage of membrane-bound HB-EGF, via Src-related tyrosine kinases and Gßγ-subunits. With increasing CCL18 concentration, the expression of MMP7 decreased in A549 cells. With decreasing estrogen content of the medium, there was an increasing effect of CCL18 on the inhibition of the relative expression of MMP7. Inhibition of GPR30 with G15 also resulted in a decrease in the relative expression of MMP7, irrespective of the subsequent stimulation with CCL18. This is a rather unexpected result, because the estrogen estradiol and CCL18 both activate GPR30. MCF-7 cells which express more GPR30 did not show any dependence of the relative MMP7 expression on CCL18 except in estrogen-free FCS medium. CCL18 induced an increased relative ERK activation in In-Cell western (ICW) at A549 cells. Stimulation with CCL18 caused decreased ERK activation with simultaneous inhibition of adenylate cyclase in MCF-7. However, stimulation with CCL18 and simultaneous inhibition of cyclooxygenase in MCF-7 resulted in increased ERK activation. In A549, stimulation with CCL18 and co-incubation with dbcAMP resulted in decreased ERK activation in both ICW and Western blot. CONCLUSION: In summary, the Gs-coupled receptor GPR30 plays an important role in the signaling pathway of CCL18. CCL18 and estradiol may not lead to the same signaling pathway after activating GPR30.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.

Clinical Trials with Combination of Cytokine-Induced Killer Cells and Dendritic Cells for Cancer Therapy.

Adoptive cellular immunotherapy (ACI) is a promising treatment for a number of cancers. Cytokine-induced killer cells (CIKs) are considered to be major cytotoxic immunologic effector cells. Usually cancer cells are able to suppress antitumor responses by secreting immunosuppressive factors. CIKs have significant antitumor activity and are capable of eradicating tumors with few side effects. They are a very encouraging cell population used against hematological and solid tumors, with an inexpensive expansion protocol which could yield to superior clinical outcome in clinical trials employing adoptive cellular therapy combination. In the last decade, clinical protocols have been modified by enriching lymphocytes with CIK cells. They are a subpopulation of lymphocytes characterized by the expression of CD3+ and CD56+ wich are surface markers common to T lymphocytes and natural killer NK cells. CIK cells are mainly used in two diseases: in hematological patients who suffer relapse after allogeneic transplantation and in patients with hepatic carcinoma after surgical ablation to eliminate residual tumor cells. Dendritic cells DCs could play a pivotal role in enhancing the antitumor efficacy of CIKs.