Interleukin- (IL-) 21 is a pleiotropic cytokine that regulates the activity of both innate and specific immunity. Indeed, it costimulates T and natural killer (NK) cell proliferation and function and regulates B cell survival and differentiation and the function of dendritic cells. In addition, IL-21 exerts divergent effects on different lymphoid cell leukemia and lymphomas, as it may support cell proliferation or on the contrary induce growth arrest or apoptosis of the neoplastic lymphoid cells. Several preclinical studies showed that IL-21 has antitumor activity in different tumor models, through mechanism involving the activation of NK and T or B cell responses. Moreover, IL-21's antitumor activity can be potentiated by its combination with other immune-enhancing molecules, monoclonal antibodies recognizing tumor antigens, chemotherapy, or molecular targeted agents. Clinical phase I-II studies of IL-21 in cancer patients showed immune stimulatory properties, acceptable toxicity profile, and antitumor effects in a fraction of patients. In view of its tolerability, IL-21 is also suitable for combinational therapeutic regimens with other agents. This review will summarize the biological functions of IL-21, and address its role in lymphoid malignancies and preclinical and clinical studies of cancer immunotherapy.

IL-27, a member of the IL-12-family of cytokines, has shown anti-tumor activity in several pre-clinical models due to anti-proliferative, anti-angiogenic and immune-enhancing effects. On the other hand, IL-27 demonstrated immune regulatory activities and inhibition of auto-immunity in mouse models. Also, we reported that IL-27, similar to IFN-γ, induces the expression of IL-18BP, IDO and PD-L1 immune regulatory molecules in human cancer cells. Here, a proteomic analysis reveals that IL-27 and IFN-γ display a broad overlap of functions on human ovarian cancer cells. Indeed, among 990 proteins modulated by either cytokine treatment in SKOV3 cells, 814 showed a concordant modulation by both cytokines, while a smaller number (176) were differentially modulated. The most up-regulated proteins were common to both IFN-γ and IL-27. In addition, functional analysis of IL-27-regulated protein networks highlighted pathways of interferon signaling and regulation, antigen presentation, protection from natural killer cell-mediated cytotoxicity, regulation of protein polyubiquitination and proteasome, aminoacid catabolism and regulation of viral protein levels.Importantly, we found that IL-27 induced HLA class I molecule expression in human cancer cells of different histotypes, including tumor cells showing very low expression. IL-27 failed only in a cancer cell line bearing a homozygous deletion in the B2M gene. Altogether, these data point out to a broad set of activities shared by IL-27 and IFN-γ, which are dependent on the common activation of the STAT1 pathway. These data add further explanation to the anti-tumor activity of IL-27 and also to its dual role in immune regulation.

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immuneenhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4 + CD25 + Treg cells and other CD4 + CD25 − regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4 + T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.Immune checkpoints are fundamental for the physiological maintenance of tolerance and protection of tissues from the damage that an unregulated immune response may cause. It is now clear that immune checkpoint dysregulation represents an important mechanism through which tumors escape immune system recognition and progress 1 . Studies in preclinical tumor models and cancer patients have proven that the targeting of immune checkpoints restores a silenced anti-tumor immune response, which then becomes effective in eliminating tumor cells. Indeed, the monoclonal antibodies (mAbs) blocking immune checkpoints such as Cytotoxic T-Lymphocyte Antigen (CTLA)-4 and Programed Death-Ligand1 (PD-L1)/Programed Death-1 (PD-1) showed extraordinary anti-tumor effects in clinical trials leading to their approval as anti-cancer drugs in different metastatic cancers 2 . In particular, anti-PD-1 mAbs have shown unprecedented clinical activity in patients with metastatic melanoma 3-8 , non-small-cell lung carcinoma (NSCLC) 9,10 and renal cell carcinoma 11 , with a significant fraction of patients showing prolonged clinical benefit. The combination of anti-PD-1 and anti-CTLA-4 leads to a further increase in clinical responses in metastatic melanoma 12 . Synergistic therapeutic activity may relate to the different site of action of CTLA4 and PD-L1/PD-1-blocking agents, i.e. the secondary lymphoid organs and the peripheral tissues, respectively. In addition, CTLA-4 blockade induces a proliferative gene expression signature

IL-21 is a member of the IL-2 cytokine family, produced by CD41 T cells. We previously showed that immunotherapy (IT) with IL-21-transduced neuroblastoma cells (Neuro2a/IL-21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL-21 vaccine. The administration of anti-CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti-CD4 mAb had a more potent effect leading to 80% cure rate. Anti-CD25 mAb, indeed, only partially depleted CD41CD251FoxP31 Treg cells, whereas anti-CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine1anti-CD4 mAb, which developed systemic immunity to NB, CD41 T cells counts completely recovered in 90 days. Depletion of CD81 T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD81 T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD-SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL-21 vaccination showed increased expression of IFN-alpha2, -beta1 and -gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine1anti-CD4 mAb showed increased IFN-gamma serum levels and IFN-gamma-producing CD81 T cells were found in spleen cells. In conclusion, anti-CD4 mAb potentiated IL-21-based IT by removing Treg cells and/or their precursors and other potentially immune-suppressive CD41 cell subsets, thus allowing the development of an IL-21-driven CD81 T cell response, which mediates NB rejection.