Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (M MSC ), through a prostaglandin E2-dependent mechanism. M MSC were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-b. These cytokines contributed to the immunoregulatory properties of M MSC , which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, M MSC inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNg, while M2 affected only IFNg production. Moreover, M MSC inhibited the proliferation of CD8 1 T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of M MSC to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches. STEM CELLS 2016;34:1909-1921 SIGNIFICANCE STATEMENTMesenchymal stromal cells (MSCs) display various immunoregulatory activities and they are also present in the stroma of different tumors. Emerging evidences revealed that the cellular interactions occurring between tumor cells, stromal cells and immune effectors in the tumor microenvironment play a key role in the establishment of tumor immune escape. In the present study, we show that MSCs induce the polarization of macrophages toward a novel M2-like phenotype (M MSC ). M MSC are able to block natural killer cell activation and induce the expansion of Tregs, thus affecting both innate and adaptive immune responses. These data dissect a novel mechanism through which MSCs may exert their immunoregulatory activity, and may offer new clues for target-based cancer immunotherapy.

Deficiency of glycosylphosphatidylinositol (GPI)-anchored molecules on blood cells accounts for most features of paroxysmal nocturnal hemoglobinuria (PNH) but not for the expansion of PNH (GPI ؊ ) clone(s).A plausible model is that PNH clones expand by escaping negative selection exerted by autoreactive T cells against normal (GPI ؉ ) hematopoiesis. By a systematic analysis of T-cell receptor beta (TCR-␤) clonotypes of the CD8 ؉ CD57 ؉ T-cell population, frequently deranged in PNH, we show recurrent clonotypes in PNH patients but not in healthy controls: 11 of 16 patients shared at least 1 of 5 clonotypes, and a set of closely related clonotypes was present in 9 patients. The presence of T-cell clones bearing a set of highly homologous TCR-␤ molecules in most patients with hemolytic PNH is consistent with an immune process driven by the same (or similar) antigen(s)-probably a nonpeptide antigen, because patients sharing clonotypes do not all share identical HLA alleles. These data confirm that CD8 ؉ CD57 ؉ T cells play a role in PNH pathogenesis and provide strong new support to the hypothesis that the expansion of the GPI ؊ blood cell population in PNH is due to selective damage to normal hematopoiesis mediated by an autoimmune attack against a nonpeptide antigen(s) that could be the GPI anchor itself. IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC) 1 characterized by 3 clinical hallmarks: intravascular hemolysis, tendency to venous thrombosis, and variable degrees of bone marrow failure. [2][3][4] The primary molecular lesion responsible for PNH is a somatic mutation of the X-linked PIGA gene in HSCs, 5,6 resulting in either complete or partial deficiency of all glycosylphosphatidylinositol (GPI)-linked proteins from the cell membrane of the progeny of the mutated HSC (GPI Ϫ ). 7,8 The deficiency of the GPI-linked proteins (including the complement-regulating proteins CD59 and CD55) from the surface of blood cells explains the intravascular hemolysis 9 and probably underlies the increased tendency to venous thrombosis. 10 However, a PIGA gene mutation per se does not explain the bone marrow failure and the expansion of the GPI Ϫ clone. In fact, very rare GPI Ϫ blood cells are present in healthy subjects, 11 but only in PNH patients do the GPI Ϫ cells expand and contribute to hematopoiesis to various degrees, side by side with normal (GPI ϩ ) hematopoiesis. 12,13 Clinical observations, 14,15 in vitro hematopoietic colony studies, 16,17 and data from PNH mouse models [18][19][20] indicate that GPI Ϫ HSCs do not have an absolute growth advantage. The close relationship of PNH to idiopathic aplastic anemia (IAA) has suggested that autoreactive T cells against HSCs believed to be responsible for IAA may be at work also in PNH. Specifically, it has been hypothesized that in PNH autoreactive T cells destroy selectively GPI ϩ (normal) HSCs, whereas GPI Ϫ (PNH)HSCs can escape T-cell-mediated damage, thus being able to survive and expand. 21 In r...

CTLA-4 blockade with monoclonal antibodies can lead to cancer regression in patients with metastatic melanoma (MM). CTLA-4 gene polymorphisms may influence the response to anti-CTLA-4 antibodies although few data are available regarding this issue. We analyzed six CTLA-4 single nucleotide polymorphisms (-1661A > G, -1577G > A, -658C > T, -319C > T, +49A > G, and CT60G > A) in 14 Italian MM patients and 45 healthy subjects. We found a significant association between the -1577G/A and CT60G/A genotypes and improved overall survival (Pc < 0.006, Bonferroni corrected), further confirmed by the diplotype analysis (-1577 & CT60 GG-AA diplotype, p < 0.001). A positive trend toward an association between these genotypes and response to therapy was also observed.

BackgroundHigh-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3.MethodHere, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 106 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel.ResultsNo false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system.ConclusionsOur study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.