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Epidermal growth factor receptor (EGFR) mutational testing in advanced non-small-cell lung cancer (NSCLC) is usually performed in tumor tissue, although cfDNA (cell-free DNA) could be an alternative. We evaluated EGFR mutations in cfDNA as a complementary tool in patients, who had already known EGFR mutations in tumor tissue and were treated with either EGFR-tyrosine kinase inhibitors (TKIs) or chemotherapy. We obtained plasma samples from 21 advanced NSCLC patients with known EGFR tumor mutations, before and during therapy with EGFR-TKIs and/or chemotherapy. cfDNA was isolated and EGFR mutations were analyzed with the multiple targeted cobas EGFR Mutation Test v2. EGFR mutations were detected at baseline in cfDNA from 57% of patients. The semiquantitative index (SQI) significantly decreased from the baseline (median=11, IQR=9.5-13) to the best response (median=0, IQR=0-0, p<0.01), followed by a significant increase at progression (median=11, IQR=11-15, p<0.01) in patients treated with either EGFR-TKIs or chemotherapy. The SQI obtained with the cobas EGFR Mutation Test v2 did not correlate with the concentration in copies/mL determined by droplet digital PCR. Resistance mutation p.T790M was observed at progression in patients with either type of treatment. In conclusion, cfDNA multiple targeted EGFR mutation analysis is useful for treatment monitoring in tissue of EGFR-positive NSCLC patients independently of the drug received.

Mutation analysis of epidermal growth factor receptor (EGFR) gene is essential for treatment selection in non-small cell lung cancer (NSCLC). Analysis is usually performed in tumor samples. We evaluated the clinical utility of EGFR analysis in plasma cell-free DNA (cfDNA) from patients under treatment with EGFR inhibitors. We selected 36 patients with NSCLC and EGFR-activating mutations. Blood samples were collected at baseline and during treatment with EGFR inhibitors. Wild-type EGFR, L858R, delE746-A750, and T790M mutations were quantified in cfDNA by droplet digital PCR. Stage IV patients had higher total circulating EGFR copy levels than stage I (3523 vs. 1003 copies/mL; p

The nonclassical human leukocyte antigen-G (HLA-G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA-G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA-G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA-G by ELISA. Complexed HLA-G was detected in exosomes, which indicates an intracellular origin of these forms. 2D-PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA-G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA-G complexes. Immunoblot analysis of exudates and exosomes with an anti-ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA-G. HLA-G ubiquitination could be reproduced in vitro in HLA-G1-transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA-G forms in vivo that open a new perspective in the study of HLA-G function and analysis.

Trogocytosis is a rapid transfer between cells of membranes and associated proteins. Trogocytic exchanges have been investigated between different cell types, mainly in two-cell systems, involving one donor and one acceptor cell type. Here, we studied trogocytosis in a more complex system, involving not only several immune cell subsets but also multiple tumor cells. We show that CD4(+) T cells, CD8(+) T cells and monocytes can acquire membrane patches and the intact proteins they contain from different tumor cells by multiple simultaneous trogocytoses. The trogocytic capabilities of CD4(+) and CD8(+) T cells were found to be similar, but inferior to that of autologous monocytes. Activated peripheral-blood mononuclear cells (PBMCs) may also exchange membranes between themselves in an all-autologous system. For this reason, monocytes are capable of acquiring membranes from multiple tumor cell sources, and transfer them again to autologous T cells, along with some of their own membranes (serial trogocytosis). Our data illustrate the extent of membrane exchanges between autologous activated immune effector cells and their environment, and how the cellular content of the local environment, including "bystander" cells, may impact the functions of immune effector cells.

Trogocytosis is the uptake of membranes from one cell by another. Trogocytosis has been demonstrated for monocytes, B cells, T cells, and NK cells. The acquisition of the tolerogenic molecule HLA-G by T cells and NK cells makes them behave as regulatory cells. We investigated here whether HLA-G, which is expressed by tumor cells in vivo, could be acquired by monocytes and if this transfer could have functional consequences. We demonstrate that resting, and even more so, activated monocytes efficiently acquire membrane-bound HLA-G from HLA-G tumor cells by trogocytosis. However, we demonstrate that HLA-G quickly disappears from the surface of the monocytes in contrast to the HLA-G acquired by T cells. Consequently, HLA-G(acq+) monocytes do not reliably inhibit the on-going proliferation of autologous activated T cells and do not inhibit their cytokine production. Thus, we show that the acquirer cell may control the functional outcome of trogocytosis.

Ig-like transcript 2 (ILT2) is a suppressive receptor that participates in the control of the autoimmune reactivity. This action is usually carried out in a proinflammatory microenvironment where there is a high production of free radicals and NO. However, little is known regarding whether these conditions modify the protein or affect its suppressive functions. The present study aimed to investigate the suppressive response of the ILT2 receptor under oxidative stress. To address this topic, we treated the ILT2-expressing natural killer cell line, NKL, with the NO donor N-(4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl)propane-1,3-diamine (DETA-NO). We observed that DETA-NO caused ILT2 protein nitration. MS analysis of the chimeric recombinant human ILT2-Fc protein after treatment with the peroxynitrite donor 3-(morpholinosydnonimine hydrochloride) (SIN-1) showed the nitration of Tyr35, Tyr76 and Tyr99, which are involved in human leucocyte antigen-G binding. This modification is selective because other Tyr residues were not modified by SIN-1. Recombinant human ILT2-Fc treated with SIN-1 bound a significantly higher quantity of human leucocyte antigen-G than untreated recombinant human ILT2-Fc. DETA-NO did not modify ILT2 mRNA expression or protein expression at the cell surface. Preincubation of NKL cells with DETA-NO decreased the cytotoxic lysis of K562-human leucocyte antigen-G1 cells compared to untreated NKL cells (P

Human leucocyte antigen G (HLA-G) is a tolerogenic molecule that protects the fetus from maternal immune attack, may favour tumoral immunoescape and is up-regulated in viral and inflammatory diseases. The aim of this work was to discover if nitric oxide (NO) could affect HLA-G expression or function because NO is an important modulator of innate and adaptive immunity. For this purpose HLA-G expression and function were analysed following treatment with a NO donor or a peroxynitrite donor in various cell lines expressing HLA-G either spontaneously or upon transfection. Results showed NO-dependent nitration of both cellular and soluble HLA-G protein, but not all HLA-G moieties underwent nitration. Endogenous biosynthesis of NO by both U-937-HLA-G1 and M8-HLA-G5 stable transfectants also caused HLA-G nitration. The NO decreased total HLA-G cellular protein content and expression on the cell surface, while increasing HLA-G shedding into the culture medium. This effect was post-transcriptional and the result of metalloprotease activity. By contrast, NO pretreatment did not affect HLA-G capability to suppress NK cytotoxicity and lymphocyte proliferation. Our studies show that NO regulates the availability of HLA-G molecules without modifying their biological activities.

Melanoma is an aggressive tumor with increasing incidence worldwide. Biomarkers are valuable tools to minimize the cost and improve efficacy of treatment of this deadly disease. Serological markers have not widely been introduced in routine clinical practice due to their insufficient diagnostic sensitivity and specificity. It is likely that the lack of objective responses with traditional treatment hinder biomarker research and development in melanoma. Recently, new drugs and therapies have, however, emerged in advanced melanoma with noticeable objective response ratio and survival. In this new scenario, serological tumor markers should be revisited. In addition, other potential circulating biomarkers such as cell-free DNA, exosomes, microRNA, and circulating tumor cells have also been identified. In this review, we summarize classical and emerging tumor markers and discuss their possible roles in emerging therapeutics.

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that depletes l -tryptophan, which provokes a decreased T cell response. This enzyme is expressed in human placenta, and can be also induced in many cell types such as monocytes, where endothelial (eNOS) and inducible (iNOS) nitric oxide synthases are also expressed. Previous studies have shown that nitric oxide (NO) inhibits IDO activity, which could cause a suppression of the biological function of IDO when both enzymes are coexpressed. As NO can exert different effects depending on several factors such as its concentration, we studied the effect of low concentrations of NO in the IDO activity in the U-937 and THP-1 monocytic cell lines. Results demonstrated that NO caused a bimodal effect in IDO function in IFN-γ-stimulated monocytic cells: while high micromolar concentrations of the NO donors SIN-1 and DETA-NO decreased IDO activity, low micromolar concentrations of these NO donors increased IDO activity. Related to this, the NOS inhibitors L-NMMA and aminoguanidine, and the calmodulin antagonist W7 also decreased IDO activity. The effect of NO in IDO activity was not through cGMP production. Immunoprecipitation analysis showed a nitration of the IDO protein in unstimulated and stimulated U-937 and THP-1 cells. However, in monocyte-derived macrophages, with a higher NO production, aminoguanidine increased IDO activity, but the NOS substrate arginine decreased IDO activity. Considering the role of IDO in suppression, these results suggest a function in tolerance of the NOS enzymes depending on the NO production.

Nonclassical human leukocyte antigen (HLA) class I molecule HLA-G and indoleamine 2,3 dioxygenase (INDO) in humans and mice, respectively, have been shown to play crucial immunosuppressive roles in fetal-maternal tolerance. HLA-G inhibits natural killer and T cell function by high-affinity interaction with inhibitory receptors, and INDO acts by depleting the surrounding microenvironment of the essential amino acid tryptophan, thus inhibiting T cell proliferation. We investigated whether HLA-G expression and INDO function were linked. Working with antigen-presenting cell (APC) lines and monocytes, we found that functional inhibition of INDO by 1-methyl-tryptophan induced cell surface expression of HLA-G1 by HLA-G1- negative APCs that were originally cell-surface negative, and that in reverse, the functional boost of INDO by high concentrations of tryptophan induced a complete loss of HLA-G1 cell surface expression by APCs that were originally cell-surface HLA-G1-positive. This mechanism was shown to be posttranslational because HLA-G protein cell contents remained unaffected by the treatments used. Furthermore, HLA-G cell surface expression regulation by INDO seems to relate to INDO function, but not to tryptophan catabolism itself. Potential

In pregnancy there occurs maternal tolerance to the foetus. Several mechanisms have been proposed to explain this phenomenon. The main immune population in the decidua are macrophages and natural killer cells, but with some "special" suppressor characteristics. There is also a predominant TH2 response. The non classical MCH type I HLA-G is expressed by trophoblasts and can suppress lymphomononuclear cytotoxicity. Other system to avoid the immune system is the expression of indoleamine-2,3-dioxygenase, that suppresses T cell activation by degrading tryptophan. Even though in the placenta there is a high production of nitric oxide, a well-known immune modulator, low attention has been paid to its role in maternal tolerance. There are many data showing that NO affects the IDO, CD95/CD95-L and the balance between TH1/TH2. Maybe NO could interact with several mechanisms at the same time, which could modify the tolerogenic activity depending on the concentration and the presence of other factors in the medium.

Human leukocyte antigen-G (HLA-G) is a low polymorphic nonclassical HLA-I molecule restrictively expressed and with suppressive functions. HLA-G gene products are quite complex, with seven HLA-G isoforms, four membrane bound, and other three soluble isoforms that can suffer different posttranslational modifications or even complex formations. In addition, HLA-G has been described included in exosomes. In this review we will focus on HLA-G biochemistry with special emphasis to the mechanisms that regulate its expression and how the protein modifications affect the quantification in biological fluids.

Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) class I molecule that, through interaction with its receptors, exerts important tolerogenic functions. Its main physiological expression occurs in placenta where it seems to participate in the maternal tolerance toward the fetus. HLA-G has been studied as a marker of pregnancy complications such as abortion or pre-eclapmsia. Although HLA-G is not expressed in most adult tissues, its ectopic expression has been observed in some diseases such as viral infections, autoimmune disorders, and especially cancer. HLA-G neo-expression in cancer is associated with the capability of tumor cells to evade the immune control. In this review, we will summarize HLA-G biology and how it participates in these physiopathological processes. Special attention will be paid to its role as a diagnostic tool and also as a therapeutic target.

Human leukocyte antigen (HLA)-G is a suppressive molecule that can be shed to the medium by a metalloprotease-dependent mechanism. Nitric oxide increases HLA-G shedding and also causes tyrosine nitration. As the presence of nitrated HLA-G has not been demonstrated in vivo, the aim of this work was to investigate whether this post-translational modification in the HLA-G molecule could also occur in vivo. Exudates and blood samples were collected during an analytical routine. HLA-G was analyzed by enzyme-linked immunoabsorbent assay and nitrites plus nitrates by colorimetry. Samples were immunoprecipitated with anti-nitrotyrosine antibody. After nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot with anti-HLA-G antibody, a band at 45 kDa was visualized and assigned to nitrated HLA-G. In addition, there were several bands at higher molecular weight that disappeared in the immunoblot performed after electrophoresis under reducing conditions, where only the band at 45 kDa remained. This indicates that nitrated HLA-G was also present as multimers. The degree of HLA-G nitration did not correlate with the concentrations of nitrites plus nitrates or HLA-G. These results indicate that HLA-G could circulate as nitrotyrosine modified protein, both as monomer and as multimer.

In the past years, it has been shown that kynurenines pathway is a regulator of both the innate and the adaptive immune responses. Particularly, the initial enzyme of this pathway, indoleamine 2,3-dioxygenase (IDO), is implicated in maintaining tolerance during pregnancy, and also can be expressed in tumors to avoid the immune attack. In this chapter, we will describe how the kynurenine pathway affects the immune system with important implications both in physiology and in pathology. The incorrect activation or blockade suppressive properties of the kynurenine pathway are also implicated in a number of other diseases such as AIDS or autoimmune diseases.

Dendritic cells (DC) are strong inducers of immunity but they can also be tolerogenic. During monocyte differentiation to DC the immunosuppressive indoleamine-2,3-dioxygenase (IDO) is induced. IDO degrades Trp to kynurenine, which is further metabolized to 3-hydroxyanthranilic acid. DC can also express mRNA and protein of the tolerogenic molecule HLA-G, but there is no surface expression. We studied the effect of the Trp degrading pathway on HLA-G expression by DC. When monocytes were differentiated to immature DC in presence of either Trp or its metabolites kynurenine or 3-hydroxyanthranilic acid they expressed cell surface HLA-G, and Trp also increased shedding of HLA-G1. Trp induced HLA-G cell surface expression when present during maturation with IFN-gamma+LPS, but not with TNF-alpha. Kynurenine increased HLA-G expression in both TNF-alpha and IFN-gamma+LPS matured DC, and 3-hydroxyanthranilic acid had a very weak effect on HLA-G cell surface expression when present during maturation. Shedding of HLA-G1 was more pronounced in IFN-gamma+LPS-matured DC than in immatured DC. Maturation with IFN-gamma+LPS in presence of kynurenine also increased HLA-G5 secretion. The mechanism involved seems to be post-translational as mRNA and cellular HLA-G protein content was not increased with Trp, kynurenine or 3-hydroxyanthranilic acid treatments. Finally, immature DC preincubated with Trp, kynurenine and 3-hydroxyanthranilic acid have after a decreased capacity to stimulate T cells in mixed lymphocyte reaction. In IFN-gamma+LPS-matured DC this decreased capacity was obtained with kynurenine and 3-hydroxyanthranilic acid. These results suggest that IDO can induce HLA-G cell surface expression in DC, and that these two molecules can cooperate in the immune suppression.