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In a previous study, lack of IL-12 signaling in il12rb2 knock-out mice was found to predispose to lung adenocarcinoma (LAC). We asked whether specific polymorphisms of the human IL12RB2 gene may confer susceptibility to LAC. We studied IL12RB2 single nucleotide polymorphisms (SNPs) spanning from the promoter to the first untranslated exon of the gene. Genotypes of 49 individuals with LAC were compared with those of 93 healthy subjects. Two allele variants were found to be associated with increased susceptibility to LAC. One haplotype (hap), hap18, was more frequent in patients (18%) versus controls (6%) and significantly associated with increased probability of disease occurrence. Furthermore, IL-12 driven STAT4 phosphorylation in T cell blasts from healthy individuals was found to correlate with both single allele variants and haplotypes. In conclusion, genetically determined low signaling activity of IL-12R predisposes to the development of LAC., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αβ(+) T cells and CD19(+) B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19(+) B-cell and αβ(+) T-cell-depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #NCT01810120., (© 2015 by The American Society of Hematology.)

Interleukin (IL)-23 is a proinflammatory cytokine belonging to the IL-12 superfamily. The antitumor activity of IL-23 is controversial, and it is unknown whether or not the cytokine can act directly on tumor cells. The aim of this study was to investigate the potential direct antitumor activity of IL-23 in pediatric B-acute lymphoblastic leukemia (B-ALL) cells and to unravel the molecular mechanisms involved. Here, we show, for the first time, that IL-23R is up-regulated in primary B-ALL cells, compared with normal early B lymphocytes, and that IL-23 dampens directly tumor growth in vitro and in vivo through the inhibition of tumor cell proliferation and induction of apoptosis. The latter finding is related to IL-23-induced up-regulation of miR15a expression and the consequent down-regulation of BCL-2 protein expression in pediatric B-ALL cells. This study demonstrates that IL-23 possesses antileukemic activity and unravels the underlying mechanisms. Thus, IL-23 may be a candidate novel drug for the treatment of B-ALL patients unresponsive to current therapeutic standards.

Polyalanine expansions in the PHOX2B gene have been detected in the vast majority of patients affected with congenital central hypoventilation syndrome, a neurocristopathy characterized by absence of adequate control of breathing, especially during sleep, with decreased sensitivity to hypoxia and hypercapnia. The correlation between length of the alanine expanded tracts and severity of congenital central hypoventilation syndrome respiratory phenotype has been confirmed by length-dependent cytoplasmic PHOX2B retention with formation of aggregates. To deepen into the molecular mechanisms mediating the effects of PHOX2B polyalanine expansions, we have set up experiments aimed at assessing the fate of cells characterized by PHOX2B polyalanine aggregates. In particular, we have observed that activation of the heat shock response by the drug geldanamycin is efficient both in preventing formation and in inducing clearance of PHOX2B pre-formed polyalanine aggregates in COS-7 cells expressing PHOX2B-GFP fused proteins, and ultimately also in rescuing the PHOX2B ability to transactivate the Dopamine-beta-Hydroxilase promoter. In addition, we have demonstrated elimination of PHOX2B mutant proteins by the proteasome and autophagy, two cellular mechanisms already been involved in the clearance of proteins containing expanded polyglutamine and polyalanine tracts. Moreover, our data suggest that geldanamycin effects on PHOX2B aggregates may be also mediated by the proteasome pathway. Finally, analysis of cellular toxicity due to polyalanine aggregates has confirmed the occurrence of cell apoptosis consequent to expression of PHOX2B carrying the longest expanded alanine tract and shown that geldanamycin can delay cell progression toward the most advanced apoptotic stages.

Several observations suggest a potential role of T-cell-mediated immunity in the control of neuroblastoma (NB). However, the generation of NB-specific cytotoxic T lymphocytes (CTL) on T-cell priming with tumor mRNA-transfected dendritic cells (DC) has never been investigated before. In the present study, the feasibility of this strategy has been analyzed, both in healthy donors and in NB patients. Monocyte-derived DC were raised from three human leukocyte antigen (HLA) A2+ NB patients and seven HLA-A1+ or HLA-A2+ healthy donors transfected with mRNA from four NB cell lines and cocultured with autologous CD8+ lymphocytes. Expanded CTL expressed an effector/memory phenotype and a T cytotoxic 1-like profile of cytokine secretion. CTL specificity was demonstrated by interferon-gamma release on incubation with HLA-matched NB cell lines. The latter cell lines, but not autologous T-cell blasts, were lysed by CTL in an HLA-restricted manner. Cytotoxicity was found to involve the release of granzyme B. When tested for reactivity against NB-associated antigens, CTL from normal individuals recognized anaplastic lymphoma-associated kinase (ALK) and preferentially expressed antigen of melanoma (PRAME) peptides only, whereas patients' CTL reacted also to survivin, telomerase, and tyrosine hydroxylase peptides. This study demonstrates that DC transfected with NB mRNA induce the generation of patients' CTL specific for different NB-associated antigens, supporting the feasibility of NB T-cell immunotherapy.

The aim of this study was to investigate T cell immunity to Toxoplasma gondii (Tg) in pregnant women with primary toxoplasmosis. This issue has never been addressed before in humans and available information derives from murine models. Peripheral blood mononuclear cells (PBMC) from pregnant women with primary Tg infection were stimulated with Tg tachyzoites, excretory-secretory antigens (ESA) or recombinant surface antigen-1 (rSAG-1), and tested for proliferation, immunophenotype, cytokine production and antigen specific cytotoxic activity. Pregnant women with primary toxoplasmosis displayed a significant decrease of the CD4/CD8 T cell ratio and a significant increase of circulating T cell receptor (TCR) gammadelta+ cells as compared to their uninfected counterparts. T cells from Tg infected pregnant women proliferated to Tg tachyzoites, ESA or rSAG-1. Most tachyzoite and ESA specific T cell blasts were CD4+, whereas SAG-1 specific blasts were CD4+ and CD8+. ESA and tachyzoite specific T cell blasts displayed a Th1 or Th0 cytokine profile with overexpression of IFN-gamma. This pattern was unchanged upon in vitro exposure of T cells to progesterone, tested at a concentration close to that reached in vivo at the maternal-fetal interface. Finally, tachyzoite or ESA specific T cell blasts lysed, through a granule exocytosis dependent mechanism, autologous lymphoblastoid cell lines presenting Tg antigens. In conclusion, pregnant women with primary toxoplasmosis mounted in vitro Tg-specific Th1/Th0 responses whose impact on neonatal infection warrants further investigation.

Neuroblastoma (NB) is a pediatric extracranial tumor characterized by downregulation of human leukocyte antigen class I and defects of the antigen processing machinery, two features that make it an appropriate target for natural killer (NK)-mediated lysis. NKG2D is an activating immunoreceptor expressed by cytotoxic T lymphocytes and NK cells. The ligands for NKG2D are the major histocompatibility complex class I-related chain (MIC)A and MICB glycoproteins, and the UL-16-binding proteins (ULBPs). Here, the expression of NKG2D ligands was investigated in human primary NB tumors and cell lines because scanty information is available on this issue. MICA, MICB, and ULBP transcripts were found in most tumors and cell lines. MICA protein was detected in some NB cell lines but not in primary tumors. A soluble form of MICA (sMICA) was identified in most patient sera and in some cell line supernatants. sMICA downregulated surface NKG2D in normal peripheral blood CD8(+) cells and decreased NK-mediated killing of MICA(+) NB cells. MICB was detected exclusively in the cytosol of primary tumors and cell lines. Approximately 50% of primary tumors expressed ULBP-2, but not ULBP-1 or -3. ULBP-3 was expressed in 5 of 9 cell lines, ULBP-2 in 2 of 9, whereas ULBP-1 was never detected. These studies delineate novel potential pathways of tumor escape and immunodeficiency in NB.