Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in Western countries. Today, the role of the host’s immune system in controlling the progression and spread of solid tumors is broadly established. Tumor immunosurveillance escape mechanisms, such as those involving dendritic cells (DCs), the most important antigen-presenting cells, are likewise recognized processes involved in cancer. The present study evaluates the ability of CRC patients to generate DCs in vitro from circulating monocytes at both pre- and post-operative timepoints; the results are correlated with the stage of disease to shed light on the systemic immune statuses of CRC patients. Our data showed that patients’ DCs had lower co-stimulatory molecule expression and were less able to present antigens to allogeneic T cells compared to healthy controls’ (HC) DCs. Furthermore altered cytokine secretion, such as increased IL-10 and reduced IL-12 and TNF-α, was observed. At the post-operative timepoints we observed a recovery of the patients’ ability to generate immature DCs, compared to HCs, but the maturational capacity remained affected. Our study conclusively highlights the persistently impaired in vitro generation of fully mature and functional DCs, which appears to be more altered during advanced stages. This work sheds light on a dendritic cell-based tumor immune escape mechanism that could be useful for the development of more effective immunotherapeutic strategies.

The clinical expression of 22q11.2 deletion syndrome (22q11.2 DS) is extremely variable, as patients can present with recurrent or severe infections, immune dysregulation, atopic diseases, or extra-immunological manifestations. The immunological background underlying the different disease manifestations is not completely elucidated. The aim of this study was to identify the immunophenotypic peculiarities of 22q11.2 DS patients presenting with different disease expressions. This study included 34 patients with 22q11.2 DS, divided into three groups according to the clinical phenotype: isolated extra-immunological manifestations (G1), infectious phenotype with increased/severe infections (G2), and immune dysregulation (G3). The patients underwent extended immunophenotyping of the T and B lymphocytes and analysis of the circulating dendritic cells (DCs). In patients with an infectious phenotype, a significant reduction in CD3+ and CD4+ cells and an expansion of CD8 naïve cells was evidenced. On the other hand, the immunophenotype of the patients with immune dysregulation showed a skewing toward memory T cell populations, and reduced levels of recent thymic emigrants (RTEs), while the highest levels of RTEs were detected in the patients with isolated extra-immunological manifestations. This study integrates the current literature, contributing to elucidating the variability in the immune status of patients with 22q11.2DS with different phenotypic expressions, particularly in those with infectious phenotype and immune dysregulation.

The functional properties of myeloid dendritic cells (DCs) differ, depending on microenvironmental factors as well as on their stage of maturation. The main approaches for the selective enhancement of the tolerogenic properties of DCs include the induction of a pharmacological arrest of the DCs maturation and the genetical engineering of DCs expressing immunosuppressive molecules. Several immunosuppressive/anti-inflammatory agents have been discovered that potentially inhibit DC maturation and immunogenicity. Photopheresis (ECP) is an immunomodulatory therapy in which leucocytes are exposed to 8-methoxypsoralen (8-MOP) and ultraviolet (UV) A radiation (PUVA). The combination of ECP with immunosuppressive agents has demonstrated efficacy in the management of transplanted patients by reducing either the incidence of organ rejection or the pharmacological toxicity. In particular, we have observed in hepatitis C virus (HCV)-positive patients that the same combination has reduced the immunosuppressive burden and improved sustainability and efficacy of pre-emptive antiviral therapy after liver transplantation. Therefore, in our work we investigated the in vitro effects of PUVA, combined with immunosuppressive drugs (IDs), on both in vitro human DC generation and maturation, in order to contribute to understanding the immunological mechanisms underlying this pharmacological combination. Monocyte PUVA-treatment was performed by using an in vitro experimental protocol that we previously described. PUVA-treated or -untreated highly purified CD14+ cells were incubated with the association of the immunosuppressive drugs, used in the management of liver transplantation, at two different concentrations, in the presence of IL-4 and GM-CSF. The treatment with IDs at the highest concentration (corresponding to that used in clinical practice), alone or in association with PUVA, induced an immunosuppressive effect, by impairing both DC generation and maturation. Neither immunosuppressive drugs at the lowest concentration nor their combination with PUVA affected myeloid DC generation, but modified DC functions, strengthening the induction of a tolerogenic pattern. As this ID concentration was arbitrarily chosen, further experiments could highlight whether lower concentrations than those used in clinical practice would elicit the same effect on DCs and potentially improve their functional properties. This work describes an original experimental approach exploring the in vitro mechanism of action of the combined procedure of PUVA with immunosuppressive drugs, used in liver transplantation, on DCs generation and function. Our results contribute to the knowledge of the mechanisms of action of this combined procedure on DCs, suggesting useful therapeutic implications for the in vivo therapy.

The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed.

Chromosome 22q11.2 deletion syndrome (22q11.2DS) is a primary immunodeficiency characterized by a broad and heterogeneous clinical presentation associated with various degrees of T-cell deficiency. We report the clinical, immunologic, and genetic findings of a cohort of eight patients presenting with a clinical phenotype that is highly suggestive of this syndrome but without the 22q11.2 deletion. The cardinal features of 22q11.2DS, such as congenital heart defects, hypoparathyroidism, and facial dysmorphisms, were observed in the majority of the patient cohort. The unusual features are described in detail. The immunologic assessment showed various degrees of immunodeficiency of the T-cell compartment, notably a reduction in the thymic output. Half of the patient cohort exhibited a reduction in total dendritic cells. Array comparative genomic hybridization (CGH) revealed six patients harboring copy number variations (CNVs) never reported in normal subjects. The gene content of these CNVs was carefully analyzed to understand the mechanisms leading to 22q11.2DS phenocopies. According to these results, we suggested that array-CGH should be used as a first-tier tool for patients resembling 22q11.2DS.

22q11.2 is a region prone to chromosomal rearrangements due to the presence of eight large blocks of low-copy repeats (LCR22s). The 3 Mb 22q11.2 “typical deletion”, between LCR22-A and D, causes a fairly well-known clinical picture, while the effects of smaller CNVs harbored in this interval are still to be fully elucidated. Nested deletions, flanked by LCR22B-D, LCR22B-C, or LCR22C-D, are very rare and are collectively described as “central deletions”. The LCR22C-D deletion (CDdel) has never been separately analyzed. In this paper, we focused only on CDdel, evaluating its gene content and reviewing the literature and public databases in order to obtain new insights for the classification of this CNV. At first glance, CDdels are associated with a broad phenotypic spectrum, ranging from clinically normal to quite severe phenotypes. However, the frequency of specific clinical traits highlights that renal/urinary tract abnormalities, cardiac defects, and neurological/behavioral disorders are much more common in CDdel than in the general population. This frequency is too high to be fortuitous, indicating that CDdel is a predisposing factor for these phenotypic traits. Among the genes present in this interval, CRKL is an excellent candidate for cardiac and renal defects. Even if further data are necessary to confirm the role of CDdels, according to our review, this CNV fits into the class of ‘likely pathogenic’ CNVs.