Bruno Azzarone

CD8+CD28− T Regulatory Lymphocytes Inhibiting T Cell Proliferative and Cytotoxic Functions Infiltrate Human Cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+CD28− T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+CD28− T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+CD25+ T regulatory lymphocytes associate with CD8+CD28− T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.

Chemokines and CD40 expression in human fibroblasts

Chemokines are cytokines specialized for recruiting leukocytes in inflammatory responses. Recent data indicate that besides macrophages and leukocytes fibroblasts may also be a source of these important immune molecules. We assayed chemokine expression (mRNA /  protein) in cultured fibroblasts isolated from a variety of human tissues and different pathologic states: normal bone marrow vs. myelometaplastic spleen, normal lung vs. metastasis stroma, and normal breast vs. radiation fibrosis and tumor stroma. In all fibroblasts, transcripts for chemokines IL‐8, stromal cell‐derived factor‐1, monocyte chemotactic protein (MCP)‐1 and eotaxin were detected. Although the production of IL‐8 was abundant in most of the fibroblasts studied, fibroblasts from lung and pathologic breast tissue produced significantly less. Conversely, eotaxin production was low in most fibroblasts except in those isolated from myelometaplastic tissue where it was highly produced. Moreover, chemokines MCP‐4, RANTES and macrophage inflammatory protein‐1α were found to be expressed only in fibroblasts from select tissues. When the expression of CD40, an activating surface molecule for immune cells, was investigated, we found that most of the fibroblasts expressed this antigen. Overall these results indicate that cultured human fibroblasts from various tissues and pathologic settings produce a distinct panel of chemokines and express CD 40, suggesting a possible fundamental role of fibroblasts in immune responses and disaese processes.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO2) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1α (HIF-1α) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1α and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1α inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1α resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phopho-STAT3 and HIF-1α are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1α in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Human mesenchymal stem cells derived from induced pluripotent stem cells down-regulate NK-cell cytolytic machinery

A major issue in immunosuppressive biotherapy is the use of mesenchymal stem cells (MSCs) that harbor regulatory capacity. However, currently used bone marrow-derived MSCs (BM-MSCs) are short-lived and cannot assure long lasting immunoregulatory function both in vitro and in vivo. Consequently, we have generated MSCs from human induced pluripotent stem (IPS-MSCs) cells that share similar properties with embryonic stem cells (ES-MSCs). Herein, we compared the immunoregulatory properties of ES/IPS-MSCs with those of BM-MSCs and showed, for the first time, that IPS-derived MSCs display remarkable inhibition of NK-cell proliferation and cytolytic function in a similar way to ES-MSCs. Both MSCs disrupt NK-cell cytolytic machinery in the same fashion that BM-MSCs, by down-regulating the expression of different activation markers and ERK1/2 signaling, leading to an impairment to form immunologic synapses with target cells and, therefore, secretion of cytotoxic granules. In addition, they are more resistant than adult BM-MSCs to preactivated NK cells. IPS-MSCs could represent an attractive alternative source of immunoregulatory cells, and their capacity to impair NK-cell cytotoxicity constitutes a complex mechanism to prevent allograft rejection.

Fibroblasts from Human Spleen Regulate NK Cell Differentiation from Blood CD34+ Progenitors Via Cell Surface IL-15

Besides a structural role in tissue architecture, fibroblasts have been shown to regulate the proliferation and differentiation of other neighboring specialized cell types, but differently according to the anatomic site and pathologic status of their tissue of origin. In this study we report a novel regulatory function of human spleen-derived fibroblasts in the development of NK cells from adult resting blood progenitors. When CD34+ cells were cocultured with spleen-derived fibroblasts in monolayers, nonadherent CD56+CD3− NK cells were predominantly produced after 2–3 wk of culture in the absence of exogenous cytokines. Most NK cells expressed class I-recognizing CD94 and NK p46, p44, and p30 receptors as well as perforin and granzyme lytic granules. Moreover, these cells demonstrated spontaneous killing activity. Cell surface immunophenotyping of spleen-derived fibroblasts revealed a low and consistent expression of IL-15, Flt3 ligand, and c-kit ligand. Additionally, low picogram amounts of the three cytokines were produced extracellularly. Neutralizing Abs to IL-15, but not the other two ligands, blocked NK cell development. Additionally, suppressing direct contacts of CD34+ progenitors and fibroblasts by microporous membrane abrogated NK cell production. We conclude that stromal fibroblasts within the human spleen are involved via constitutive cell surface expression of bioactive IL-15 in the development of functional activated NK cells under physiologic conditions.

Differential intracellular trafficking, secretion and endosomal localization of two IL-15 isoforms

To analyze the intracellular trafficking of two IL‐15 isoforms bearing 48‐ or 21‐amino acid leader peptides (L), we have generated cDNA encoding the two proteins fused at the C terminus with green fluorescent protein (GFP). Confocal microscopy analyses showed that, when transfected in CHO cells, 48L IL‐15/GFP was localized in the Golgi apparatus and in early endosomes, while 21L IL‐15/GFP was detectable only in the cytosol. The presence of 48L IL‐15/GFP in endosomes was confirmed by enzyme‐linked immunosorbent assay on endosome‐enriched subcellular fractions. Exogenous IL‐15 was bound and taken up in endosomes by untransfected CHO cells, indicating that endosomal localization was, at least in part, related to a receptor‐mediated uptake. The 48L IL‐15/GFP fusion protein was efficiently secreted by COS‐7 or CHO cell transfectants, while IL‐15 secretion was less efficient in transfectants expressing 21L IL‐15/GFP or untagged 48L or 21L IL‐15. Treatment with brefeldin A or with inhibitors of N‐linked glycosylation further indicated that the 48L IL‐15/GFP is secreted through the endoplasmic reticulum/Golgi pathway. Our data suggest a different trafficking of the two IL‐15 isoforms and multiple mechanisms controlling IL‐15 secretion.

Role of common-gamma chain cytokines in NK cell development and function: perspectives for immunotherapy.

NK cells are components of the innate immunity system and play an important role as a first-line defense mechanism against viral infections and in tumor immune surveillance. Their development and their functional activities are controlled by several factors among which cytokines sharing the usage of the common cytokine-receptor gamma chain play a pivotal role. In particular, IL-2, IL-7, IL-15, and IL-21 are the members of this family predominantly involved in NK cell biology. In this paper, we will address their role in NK cell ontogeny, regulation of functional activities, development of specialized cell subsets, and acquisition of memory-like functions. Finally, the potential application of these cytokines as recombinant molecules to NK cell-based immunotherapy approaches will be discussed.

Differentiation Therapy: Targeting Human Renal Cancer Stem Cells with Interleukin 15

BACKGROUND Many renal cancer patients experience disease recurrence after immunotherapy or combined treatments due to persistence of cancer stem cells (CSCs). The identification of reliable inducers of CSC differentiation may facilitate the development of efficient strategies for eliminating CSCs. We investigated whether interleukin 15 (IL-15), a regulator of kidney homeostasis, induces the differentiation of CD105-positive (CD105(+)) CSCs from human renal cancers. METHODS CD105(+) CSCs were cultured to preserve their stem cell properties and treated with recombinant human IL-15 (rhIL-15) to evaluate their ability to differentiate, to acquire sensitivity to chemotherapeutic drugs, and to form spheroids in vitro and tumors in vivo. Expression of stem cell and epithelial markers were studied by flow cytometry, immunocytochemistry, and immunoblotting. Identification of a CSC side population fraction and its sensitivity to chemotherapy drugs and expression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) activities were determined by flow cytometry. Spheroid formation was determined in limiting dilution assay. Xenograft tumors were generated in severe combined immunodeficient mice (n = 12-18 mice per group). All statistical tests were two-sided. RESULTS CD105(+) CSCs treated with rhIL-15 at 10 pg/mL differentiated into cells expressing epithelial markers. rhIL-15 induced epithelial differentiation of all CD105(+) CSCs subsets and blocked CSC self-renewal (sphere-forming ability) and their tumorigenic properties in severe combined immunodeficient mice. Vinblastine and paclitaxel induced statistically significant higher levels of apoptosis in rhIL-15-differentiated epithelial cells compared with CD105(+) CSCs (mean percentage of apoptotic cells, vinblastine: 33% vs 16.5%, difference = 16.5%, 95% confidence interval = 12.25% to 20.74%, P = .0025; paclitaxel: 35% vs 11.6%, difference = 23.4%, 95% confidence interval = 22.5% to 24.24%, P = .0015). The higher sensitivity of rhIL-15-differentiated epithelial cells to chemotherapeutic drugs was associated with loss of detoxifying mechanisms such as ALDH and ABC transporter activities. CONCLUSION IL-15 directs the epithelial differentiation of renal CSCs and meets the criteria for a treatment strategy: CSC pool depletion and generation of differentiated nontumorigenic cells that are sensitive to chemotherapeutic agents.

IL-15/IL-15Rα intracellular trafficking in human melanoma cells and signal transduction through the IL-15Rα

There are two IL-15 isoforms and eight isoforms for the IL-15Rα chain whose biological role is poorly understood. Here, we have analysed the intracellular trafficking of IL-15 and IL-15Rα and tried to shed some light on their function(s). In IL-15/GFP CHO transfectants both IL-15 isoforms show nuclear localization. Two melanoma cell lines (MELP and MELREO) spontaneously expressing the IL-15 isoforms, display different intracellular trafficking of the IL-15/IL-15Rα complex. In MELP cells only IL-15Rα is detected inside the nucleus, whereas IL-15 and IL-15Rα assemble at the cell surface and are internalized. Moreover, the transducing molecule TRAF2 co-immunoprecipitates with IL-15Rα and may be deflected to TNFRI using anti-IL-15 blocking mAbs and TNF-α. By contrast, MELREO cells display IL-15Rα and IL-15 nuclear localization but only a partial co-localization of these molecules on the cell surface. In these cells, TRAF2 is strongly associated with IL-15Rα and cannot be deflected by any treatment. Since TRAF2 activates the transcription factor NF-κB, IL-15 through IL-15Rα, could have a role in the control of this pathway. Indeed, anti-IL-15 MaB inhibit the constitutive nuclear localization of NFκB and the phosphorylation of its inhibitor Iκ-Bα. Thus, IL-15Rα controls NF-κB activation, however differences in the intracellular trafficking of the IL-15 and/or IL-15Rα suggest a different biological role for this complex in MELP versus MELREO cells.

Long-Lasting Inhibitory Effects of Fetal Liver Mesenchymal Stem Cells on T-Lymphocyte Proliferation

Human bone marrow mesenchymal stem cells (BM-MSC) are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK) cells, Dendritic Cells (DC), and T cells. This study compared the use of MSC isolated from bone marrow and fetal liver (FL-MSC) to determine which displayed the most efficient immunosuppressive effects on T cell activation. Although both types of MSC exhibit similar phenotype profile, FL-MSC displays a much more extended in vitro life-span and immunomodulatory properties. When co-cultured with CD3/CD28-stimulated T cells, both BM-MSC and FL-MSC affected T cell proliferation by inhibiting their entry into the cell cycle, by inducing the down-regulation of phospho-retinoblastoma (pRb), cyclins A and D1, as well as up-regulating p27kip1expression. The T cell inhibition by MSC was not due to the soluble HLA-G5 isoform, but to the surface expression of HLA-G1, as shown by the need of cell-cell contact and by the use of neutralizing anti-HLA-G antibodies. To note, in a HLA-G-mediated fashion, MSC facilitated the expansion of a CD4low/CD8low T subset that had decreased secretion of IFN-γ, and an induced secretion of the immunomodulatory cytokine IL-10. Because of their longer lasting in vitro immunosuppressive properties, mainly mediated by HLA-G, and their more efficient induction of IL-10 production and T cell apoptosis, fetal liver MSC could be considered a new tool for MSC therapy to prevent allograft rejection.