Influence of interferon-alpha combined with chemo (radio) therapy on immunological parameters in pancreatic adenocarcinoma.

Prognosis of patients with carcinoma of the exocrine pancreas is particularly poor. A combination of chemotherapy with immunotherapy could be an option for treatment of pancreatic cancer. The aim of this study was to perform an immunomonitoring of 17 patients with pancreatic cancer from the CapRI-2 study, and tumor-bearing mice treated with combination of chemo (radio) therapies with interferon-2α. Low doses of interferon-2α led to a decrease in total leukocyte and an increase in monocyte counts. Furthermore, we observed a positive effect of interferon-2α therapy on the dendritic cells and NK (natural killer) cell activation immediately after the first injection. In addition, we recorded an increased amount of interferon-γ and IL-10 in the serum following the interferon-2α therapy. These data clearly demonstrate that pancreatic carcinoma patients also show an immunomodulatory response to interferon-2α therapy. Analysis of immunosuppressive cells in the Panc02 orthotopic mouse model of pancreatic cancer revealed an accumulation of the myeloid-derived suppressor cells in spleens and tumors of the mice treated with interferon-2α and 5-fluorouracil. The direct effect of the drugs on myeloid-derived suppressor cells was also registered in vitro. These data expose the importance of immunosuppressive mechanisms induced by combined chemo-immunotherapy.

Pivotal antitumor role of the immune checkpoint molecule B7-H1 in pancreatic cancer.

Immune checkpoint molecule B7-H1 plays a decisive immune regulatory role in different pathologies including cancer, and manipulation of B7-H1 expression became an attractive approach in cancer immunotherapy. Pancreatic cancer (PDAC) is characterized by pronounced immunosuppressive environment and B7-H1 expression correlates with PDAC prognosis. However, the first attempts to diminish B7-H1 expression in patients were not so successful. This points the complicity of PDAC immunosuppressive network and requires further examinations. We investigated the effect of B7-H1 deficiency in PDAC. Our results clearly show that partial or complete B7-H1 inhibition in vivo let to reduced tumor volume and improved survival of PDAC-bearing mice. This oncological benefit is due to the abrogation of immunosuppression provided by MDSC, macrophages, DC and Treg, which resulted in simultaneous restoration of anti-tumor immune response, namely improved accumulation and functionality of effector-memory CD4 and CD8 T cells. Our results underline the potential of B7-H1 molecule to control immunosuppressive network in PDAC and provide new issues for further clinical investigations.

Interferon-α Up-Regulates the Expression of PD-L1 Molecules on Immune Cells Through STAT3 and p38 Signaling.

Interferon-α (IFNα) has one of the longest histories of use amongst cytokines in clinical oncology and has been applied for the treatment of many types of cancers. Due to its immune-activating properties, IFNα is also an attractive candidate for combinatory anti-cancer therapies. Despite its extensive use in animal tumor models as well as in several clinical trials, the different mechanisms underlying patient responses and affecting desirable clinical benefits are still under investigation. Here we show that in addition to its immune-activating properties, IFNα induces the expression of a key negative regulator, immunosuppressive PD-L1 molecule, in the majority of the specific immune cell populations, particularly in the dendritic cells (DC). DC can modulate immune responses by a variety of mechanisms, including expression of T-cell regulatory molecules and cytokines. Our results showed that treatment of DC with IFNα-2b led to pronounced up-regulation of surface expression of PD-L1 molecules, increased IL-6 and decreased IL-12 production. Moreover, we present evidence that IFNα-treated DC exhibited a reduced capacity to stimulate interferon-γ production in T cells compared to control DC. This T-cell response after treatment of DC with IFNα was recovered by a pre-treatment with an anti-PD-L1 blocking antibody. Further analyses revealed that IFNα regulated PD-L1 expression through the STAT3 and p38 signaling pathways, since blocking of STAT3 and p38 activation with specific inhibitors prevented PD-L1 up-regulation. Our findings underline the important roles of p38 and STAT3 in the regulation of PD-L1 expression and prove that IFNα induces STAT3/p38-mediated expression of PD-L1 and thereby a reduced stimulatory ability of DC. The augmentation of PD-L1 expression in immune cells through IFNα treatment should be considered by use of IFNα in an anti-cancer therapy.

Characterization of myeloid leukocytes and soluble mediators in pancreatic cancer: importance of myeloid-derived suppressor cells.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. PDAC cells activate tumor-specific immune responses but simultaneously trigger a strong immunosuppression. We showed that PDAC cells produce high amount of chronic inflammatory mediators and PDAC tumors build an immunosuppressive cytokine milieu, which correlates with tumor progression. We observed a low frequency of dendritic cells (DC) and a pronounced accumulation of macrophages and myeloid-derived suppressor cells (MDSC) in murine PDAC tumors. A strong accumulation of MDSC has also been demonstrated in the peripheral blood of resected PDAC patients. While DC and macrophages seem not to play a significant role in this PDAC model in the context of immunosuppression, MDSC are highly suppressive, and their accumulation is associated with an increase in intratumoral VEGF concentration during the PDAC progression. Application of the phosphodiesterase-5 inhibitor sildenafil led to a prolonged survival of PDAC-bearing female mice, which was due to the decrease in MDSC frequencies and in the systemic VEGF level. This led to a restoration of anticancer immune responses, manifested in the recovery of T lymphocyte functions and in an increase in the frequency of conventional CD4+ T cells in tumors and IFNγ level in serum of PDAC-bearing mice. Thus, MDSC are strongly involved in the PDAC-associated immunosuppression and that their depletion could create new approaches for therapy of PDAC.

Immunological in vivo effects of B7-H1 deficiency.

B7-H1 regulatory protein, a member of the B7-H family, plays a crucial role in the modulation of immune response in healthy steady-state conditions as well as in different pathologies. B7-H1 knockout mice represent an important model to elucidate further molecular and cellular mechanisms involved, among others, in autoimmunity development and cancer progression. However, a deep immunologic characterization of this model is not complete yet. This study examined the role of B7-H1 in vivo further by direct comparison of specifically phenotyped spleen immune-cell subpopulations and their activation and naïve/memory state as well as cytokine profile in wild-type and B7-H1 knockout mice. Our results demonstrated that B7-H1 deficiency in vivo modulates several immunological parameters, including the amount and composition of Gr1(+)CD11b(+) myeloid population, the composition and activation state of the DC compartment, the frequency and status of NK and NKT cells, B-cells, naïve/memory state of CD8 T-cells and production of IL-2 and IL-10 cytokines. Moreover, we observed an increase in the PD-1 expression in the immune cells in B7-H1 knockout mice compared to the wild-type animals. Valuing the importance of B7-H1 knockout mice for their use in disease models, these data underline the role of B7-H1 in vivo also in healthy state and should be taken into account in future studies on this immunosuppressive molecule.

Gender-specific immunological effects of the phosphodiesterase 5 inhibitor sildenafil in healthy mice.

Phosphodiesterase 5 (PDE5) is a pharmacological target in erectile dysfunction, pulmonary hypertension and in other indications. In tumor-bearing mice an inhibition of PDE5 with sildenafil prolongs survival of the animals through the augmentation of antitumor immunity, indicating the immunomodulatory properties of this drug. Effects of sildenafil on the immune system in healthy organisms are poorly investigated. In this work we showed that chronic application of sildenafil in healthy mice leads to opposite gender-dependent effects on NK cells, subpopulations of CD4(+) and CD8(+) T cells, activated conventional T cells, and to a decrease in Gr-1(+)CD11b(+) immature myeloid cells. Besides, sildenafil treatment decreases the serum concentration of interleukin-6. Ex vivo cultivation of isolated splenocytes with sildenafil results in an increase in CD4(+) T cells and a concomitant decrease in B cells and central memory CD8(+) T cells. Ex vivo modulatory properties of sildenafil are not gender-specific, indicating the importance of sildenafil's pharmacokinetics for it immunomodulatory activity in vivo. While the PDE5 expression is equal in the splenocytes from both genders, splenocytes from female mice possess higher basal level of cGMP compared to the male ones. Moreover, cultivation of splenocytes obtained from female but not male mice with sildenafil leads to an increase in cGMP concentration, making sildenafil's pharmacodynamics also responsible for gender-specific effects of the drug. Thus, this work secures conclusive evidence that the PDE5 inhibitor sildenafil possesses immunomodulatory properties and these effects are gender-specific. Immunological clinical trials are needed to prove the potential immunomodulatory effects of sildenafil in humans.