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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.

IntroductionMesenchymal stem cells (MSCs) are important tools in treating immune disorders and in tissue repair by their multipotency, immunosuppressive properties, and production of cytokines or growth factors, Many sources of MSCs have been described and the main candidates for clinical application are bone marrow and adipocyte tissues widely available and easy to collect by standardized procedures. However, as they have in vitro and in vivo time-limited functions, 1,2 several research groups are searching for MSCs with prolonged lifetime and immunoregulatory properties. In the last decade, MSCs have been isolated from fetal or neonatal tissues [3][4][5][6][7] and embryonic tissues (ES-MSCs). [8][9][10] At present, no specific markers for the origin of the MSCs have been identified and all types of MSCs are defined by their CD105, CD90, CD73, CD44, CD29, CD146, and CD166 expression. Adult BM-MSCs exhibit immunomodulatory functions on immune cells by both cell-cell contact and soluble factors. 2,[11][12][13] Recently, human somatic cells have been successfully reprogrammed into induced pluripotent cells (iPSC) [14][15][16][17][18][19] that exhibit characteristics similar to human ES. 20 IPSC hold enormous promise for personalized cellreplacement therapy 21 and for research into various human diseases. 22,23 Therefore, MSCs derived from iPSC may be a novel source of tolerance induction, though their immunosuppressive activity remains to be explored.We report that MSCs isolated from diverse human iPS Cell lines (iPS-MSCs) can strongly inhibit the cytotoxic functions of natural killer (NK) cells. Most of these MSC-mediated inhibitory effects are because of a general impairment of NK activation and disruption of the secretory machinery on NK cells. Interestingly, iPS-MSCs and ES-MSCs are more resistant than BM-MSCs to preactivated NK cells. Our current data indicate that iPS-MSCs could represent a promising alternative strategy for the treatment of various immune-mediated diseases. Methods ReagentsThe antibodies used to assess NK and MSC phenotypes are described in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Recombinant human IL-2 was purchased from Immunotools, whereas Mitomycin, Monensin, and Brefaldin were purchased from Sigma-Aldrich. 1-Methyl-tryptophan (1-MT) and NS-398, specific inhibitors for Indoleamine 2,3-dioxygenase (IDO) and Prostaglandin (PGE)-2, respectively, were also purchased from Sigma-Aldrich. 87G antagonist antibody anti-HLA-G was purchased from Exbio. Pluripotent stem-cell linesWe used 3 human iPS cell lines PB3, PB10, and PB11 provided from the Stem cell Core-Facility (ES Team Paris Sud, University Paris 11, Villejuif, France) that were derived from amniotic fluid cells (AFC-iPS) after amniocentesis (Antoine Béclère Hospital, Clamart France). Their pluripotency was validated by teratoma assay, flow cytometry and RT-PCR and registered into the European Registry Web site (http://www.hescreg.eu). In addition, we de...

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.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-β1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

Interleukin-21 (IL-21

Subepithelial fibrosis in the bronchi of asthmatics is the result of an irreversible lung fibroblast activation, triggered by cytokines secreted by IL-4- and IL-5-activated inflammatory cells. Here, we provide evidence that human lung fibroblasts (ICIG7 cells) express a single class of high-affinity IL-4 receptor (IL-4R). This receptor is functional and composed of at least the IL-4Ralpha and IL-13Ralpha1 chains in the absence of the IL-2Rgamma chain. The IL-4Ralpha is efficiently internalized at 37 degrees C within 15 min in the presence of IL-4, whereas this process is slower with IL-13. In ICIG7 cells, IL-4 triggers the tyrosine phosphorylation of at least two proteins (110 and 180 kDa), and up-regulates the transcription of c-fos, c-jun and c-myc proto-oncogenes. In addition, the secretion of several cytokines [IL-6, granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor (GM-CSF)] as well as the expression of beta1 integrin and VCAM-1 adhesion molecules are augmented by IL-4. IL-13 displays similar biological activities, but less effectively than IL-4. On the other hand, ICIG7 cells could constitute a lung fibroblast population defined by the spontaneous release of several pro-inflammatory cytokines (IL-6, IL-11 and GM-CSF) and cell surface phenotype (CD4 and Thy-1). Through this peculiar cytokine pattern and the IL-4/IL-13-dependent activities, these cells could act as effector cells in the pathogenesis of asthma, triggering and maintaining the recruitment, homing and activation of bone marrow-derived inflammatory cells, and playing a role in the remodeling process of the airways.

The clonal expansion of chronic lymphocytic leukemia (CLL) cells requires the interaction with the microenvironment and is under the control of several cytokines. Here, we investigated the effect of IL-15 and IL-21, which are closely related to IL-2 and share the usage of the common ␥ chain and of its JAK3-associated pathway. We found remarkable differences in the signal transduction pathways activated by these cytokines, which determined different responses in CLL cells. IL-15 caused cell proliferation and pre- IntroductionChronic lymphocytic leukemia (CLL), the most common leukemia in adults, is characterized by the progressive accumulation of CD5 ϩ neoplastic B cells, 1-3 which results from a dynamic balance between cell death and proliferation. 3,4 The finding that purified CLL cells do not proliferate and tend to undergo spontaneous apoptosis in vitro suggests that factors inducing CLL cell survival and proliferation might be present in the microenvironment of lymphoid organs or of the bone marrow. 5,6 Several cytokines such as the TNF family members BAFF and APRIL, 7,8 the chemokine SDF-1, 9,10 and interleukin-2 (IL-2) 11,12 and IL-15 13 stimulate CLL cell proliferation and/or survival in vitro. IL-2 and IL-15 share the usage of the IL-2R␤ (CD122) and of the common ␥ chain (␥ c ; CD132) 14,15 and thus have similar functional effects on IL-2R␤/␥ c ϩ cells. 16,17 The IL-2R␤ and ␥ c chains signal through the JAK1 and JAK3 tyrosine kinases, which activate downstream STAT pathways. 18,19 In addition to the IL-2R␤/␥ c complex, specific 21 molecules are required for high-affinity binding of each cytokine. Unlike IL-2R␣, IL-15R␣ is a high-affinity receptor also when expressed as an isolated chain, and is present on both lymphoid and nonlymphoid cell types. 20,21 IL-15R␣ is crucial for IL-15 activity, since both IL-15-and IL-15R␣-deficient mice display similar defects in the development of natural killer (NK) cells, of intraepithelial T lymphocytes, and of CD8 ϩ memory-type T cells. 22,23 IL-15 gene is expressed in several tissues, and IL-15 can be either secreted in low amounts or displayed as a membrane-bound cytokine by several cell types. [24][25][26][27][28] Therefore IL-15 is considered a microenvironmental factor, which supports lymphoid cell homeostasis and survival. 29-31 IL-15 is a growth or survival factor not only for CLL cells 13 but also for cells of other lymphoid neoplasias, 32-34 at least in vitro. Moreover, in IL-15-transgenic mice, IL-15 overexpression induces fatal T or NK lymphoproliferative disorders. 35 We and others recently demonstrated that IL-21, the last identified member of the IL-2 family, 36-38 is unable to trigger CLL cell proliferation, but rather induces their apoptosis. 39,40 This effect is potentiated by cell activation with CD40L, 39 with CpG synthetic oligonucleotides, or by BCR cross-linking. 40 IL-21 induces the JAK1 and JAK3 and the STAT1, STAT3, and STAT5 tyrosine phosphorylation in CLL B cells. 39 Similar early signaling events are also activated by IL-21, IL-2, or IL-1...