Acquisition and Presentation of Tumor Antigens by Dendritic Cells.

Dendritic cells (DCs) are master regulators of the immune response and, because of their peculiar features in antigen acquisition, processing, and presentation, they play a critical role in activating an efficient antigenspecific T-lymphocyte response against tumors. However, the DC family is composed of different cell subsets, which may differently contribute to tumor-specific T-cell activation. In addition to the DC subset involved, the induction of a tumor-specific adaptive immune response is also dependent on DC interactions with other innate cell effectors, such as natural killer cells. The different modalities by which DCs can acquire tumor antigens also significantly affect antigen presentation because, in addition to the presentation of tumor antigens on MHC class II upon the classical exogenous antigen processing pathway, DCs are equipped to directly activate cytotoxic T cells via both cross-priming and cross-dressing. Here, the different forms of tumor antigen presentation by DCs are reviewed and discussed. We also discuss the ways in which this novel information could be exploited in the design of DC-based cancer vaccines.

CD56(bright)perforin(low) noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph.

As limited information is available regarding the distribution and trafficking of NK cells among solid organs, we have analyzed a wide array of tissues derived from different human compartments. NK cells were widely distributed in most solid tissues, although their amount varied significantly depending on the tissue/organ analyzed. Interestingly, the distribution appeared to be subset specific, as some tissues were preferentially populated by CD56(bright)perforin(low) NK cells, with others by the CD56(dim)perforin(high) cytotoxic counterpart. Nevertheless, most tissues were highly enriched in CD56(bright)perforin(low) cells, and the distribution of NK subsets appeared in accordance with tissue gene expression of chemotactic factors, for which receptors are differently represented in the two subsets. Remarkably, chemokine expression pattern of tissues was modified after neoplastic transformation. As a result, although the total amount of NK cells infiltrating the tissues did not significantly change upon malignant transformation, the relative proportion of NK subsets infiltrating the tissues was different, with a trend toward a tumor-infiltrating NK population enriched in noncytotoxic cells. Besides solid tissues, CD56(bright)perforin(low) NK cells were also detected in seroma fluids, which represents an accrual of human afferent lymph, indicating that they may leave peripheral solid tissues and recirculate to secondary lymphoid organs via lymphatic vessels. Our results provide a comprehensive mapping of NK cells in human tissues, demonstrating that discrete NK subsets populate and recirculate through most human tissues and that organ-specific chemokine expression patterns might affect their distribution. In this context, chemokine switch upon neoplastic transformation might represent a novel mechanism of tumor immune escape.

Membrane transfer from tumor cells overcomes deficient phagocytic ability of plasmacytoid dendritic cells for the acquisition and presentation of tumor antigens.

The potential contribution of plasmacytoid dendritic cells (pDCs) in the presentation of tumor cell Ags remains unclear, and some controversies exist with regard to the ability of pDCs to phagocytose cell-derived particulate Ags and cross-present them to MHC class I-restricted T lymphocytes. In this study, we show that human pDCs, although inefficient in the internalization of cell membrane fragments by phagocytosis, can efficiently acquire membrane patches and associated molecules from cancer cells of different histotypes. The transfer of membrane patches to pDCs occurred in a very short time and required cell-to-cell contact. Membrane transfer also included intact HLA complexes, and the acquired Ags could be efficiently recognized on pDCs by tumor-specific CD8(+) T cells. Remarkably, pDCs isolated from human colon cancer tissues displayed a strong surface expression of epithelial cell adhesion molecule, indicating that the exchange of exogenous Ags between pDCs and tumor cells also can occur in vivo. These data demonstrate that pDCs are well suited to acquire membrane patches from contiguous tumor cells by a cell-to-cell contact-dependent mechanism that closely resembles "trogocytosis." This phenomenon may allow pDCs to proficiently present tumor cell-derived Ags, despite limited properties of endophagocytosis.

Characterization of human afferent lymph dendritic cells from seroma fluids.

Dendritic cells (DCs) migrate from peripheral tissues to secondary lymphoid organs (SLOs) through the afferent lymph. Owing to limitations in investigating human lymph, DCs flowing in afferent lymph have not been properly characterized in humans until now. In this study, DCs present in seroma, an accrual of human afferent lymph occurring after lymph node surgical dissection, were isolated and analyzed in detail. Two main DC subsets were identified in seroma that corresponded to the migratory DC subsets present in lymph nodes, that is, CD14(+) and CD1a(+). The latter also included CD1a(bright) Langerhans cells. The two DC subsets appeared to share the same monocytic precursor and to be developmentally related; both of them spontaneously released high levels of TGF-ß and displayed similar T cell-activating and -polarizing properties. In contrast, they differed in the expression of surface molecules, including TLRs; in their phagocytic activity; and in the expression of proteins involved in Ag processing and presentation. It is worth noting that although both subsets were detected in seroma in the postsurgical inflammatory phase, only CD1a(+) DCs migrated via afferent lymph under steady-state conditions. In conclusion, the high numbers of DCs contained in seroma fluids allowed a proper characterization of human DCs migrating via afferent lymph, revealing a continuous stream of DCs from peripheral regions toward SLOs under normal conditions. Moreover, we showed that, in inflammatory conditions, distinct subsets of DCs can migrate to SLOs via afferent lymph.

CD62L expression identifies a unique subset of polyfunctional CD56dim NK cells.

Human natural killer (NK) cells comprise 2 main subsets, CD56(bright) and CD56(dim) cells, that differ in function, phenotype, and tissue localization. To further dissect the heterogeneity of CD56(dim) cells, we have performed transcriptome analysis and functional ex vivo characterization of human NK-cell subsets according to the expression of markers related to differentiation, migration or competence. Here, we show for the first time that the ability to respond to cytokines or to activating receptors is mutually exclusive in almost all NK cells with the exception of CD56(dim) CD62L(+) cells. Indeed, only these cells combine the ability to produce interferon-gamma after cytokines and proliferate in vivo during viral infection with the capacity to kill and produce cytokines upon engagement of activating receptors. Therefore, CD56(dim) CD62L(+) cells represent a unique subset of polyfunctional NK cells. Ex vivo analysis of their function, phenotype, telomere length, frequencies during ageing as well as transfer experiments of NK-cell subsets into immunodeficient mice suggest that CD56(dim) CD62L(+) cells represent an intermediate stage of NK-cell maturation, which after restimulation can accomplish multiple tasks and further develop into terminally differentiated effectors.

NK cells provide helper signal for CD8+ T cells by inducing the expression of membrane-bound IL-15 on DCs.

NK cell recognition of cells that do not express or express low amounts of MHC class I molecules results not only in direct killing of target cells but also in the generation of specific T cell responses consequent to the induction of dendritic cell (DC) activation. While IL-12 production by NK cell-activated DCs is generally thought to play a critical role, a similar DC-mediated NK cell help has been reported also in IL-12-knockout mice. Here, we show that human NK cells can induce on DC surface membrane, via IFN-gamma secretion, the expression of high levels of IL-15. Remarkably, we show that DC expression of this membrane-bound form of IL-15, which is only partially associated with IL-15R molecules, is essential to promote specific CD8(+) T lymphocyte response in the absence of DC-derived IL-12.

Arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression.

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Eosinophil granulocytes account for indoleamine 2,3-dioxygenase-mediated immune escape in human non-small cell lung cancer.

Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non-small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.

A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation.

Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD(+ )T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4(+) T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8(+) T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role.

NCR(+)ILC3 concentrate in human lung cancer and associate with intratumoral lymphoid structures.

Tertiary lymphoid structures (TLSs) are a common finding in non-small cell lung cancer (NSCLC) and are predictors of favourable clinical outcome. Here we show that NCR(+) innate lymphoid cell (ILC)-3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumour-associated TLSs. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, on activation, produces IL-22, TNF-α, IL-8 and IL-2, and activates endothelial cells. Tumour NCR(+)ILC3 may interact with both lung tumour cells and tumour-associated fibroblasts, resulting in the release of cytokines primarily on engagement of the NKp44-activating receptor. In patients, NCR(+)ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumour stages and their presence correlate with the density of intratumoral TLSs. Our results indicate that NCR(+)ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumour-associated TLSs.

The anti-tumor activity of bacillus Calmette-Guerin in bladder cancer is associated with an increase in the circulating level of interleukin-2.

Bacillus Calmette-Guerin (BCG) is currently employed in the treatment of superficial bladder cancer but, despite its recognized effectiveness in preventing recurrences and progression, the immune mechanisms behind its antitumor activity remain to be delineated. In this study we provide evidence that a prolonged increase in the plasma levels of IL-2, but not IL-1beta, IL-4, IL-10, IL-2R or TNF-alpha occured in patients affected by bladder cancer following effective BCG treatment. Conversely, a drop in circulating IL-2 was consistently associated with tumor relapse. The level of IL-2 was elevated even further 15 days after the last BCG administration in patients who did not experience tumor recurrence, suggesting a prolonged T cell-mediated response against antigens other than BCG. Our results indicate that a specific type 1 immune response plays a major role in the anti-cancer activity of BCG. In addition, monitoring IL-2 plasma levels may offer a useful tool for predicting tumor recurrences.

Cross-Talks between Natural Killer Cells and Distinct Subsets of Dendritic Cells.

In recent years, the essential role of bi-directional cross-talk between natural killer (NK) and dendritic cells (DC) during immune responses has been clearly elucidated. In particular, this cross-talk results in the development of an efficient innate response, through DC-mediated NK cell activation, and a potent adaptive immune response, through NK-mediate DC editing and maturation. Recently, some novel human DC subsets have been identified: migratory DCs in afferent lymph and draining lymph nodes; CLEC9A(+)/BDCA3(+) (CD141) DCs in interstitial dermis, liver, lung; inflammatory DCs in several inflammatory fluids. At the same time, it has been shown that also human NK cells are present in these compartments. Here, we will review the most recent findings on NK/DC cross-talk and we will discuss the necessity of acquiring more complete knowledge about these interactions in view of the new information available on both DC and NK cell subsets.

Drag cells in immunity: Plasmacytoid DCs dress up as cancer cells.

Cross-dressing, in immunology, is a term originally coined to indicate the transfer of peptide-MHC complexes belonging to neighboring cells on antigen presenting cells. We have recently shown that plasmacytoid dendritic cells (pDCs) are particularly suited to be cross-dressed by tumor cells and that this phenomenon provides a unique pathway for abundant presentation of tumor antigens by pDCs.

Novel perspectives on dendritic cell-based immunotherapy of cancer.

Advances in immunobiology knowledge as well as in cell culture processes that generate large numbers of purified and functionally mature dendritic cells (DCs) have raised the possibility that DCs might represent promising clinical agents to generate effective immune responses against cancer. Here, we discuss the present pitfalls of dendritic cell vaccines for the treatment of human cancer with regard to the most recent knowledge in the biology of DCs. In particular, we highlight the relevance of improving our current understanding of DC trafficking, functions and interactions with other cells of innate immunity for the development of more effective cancer vaccines.

Dendritic cell editing by activated natural killer cells results in a more protective cancer-specific immune response.

Over the last decade, several studies have extensively reported that activated natural killer (NK) cells can kill autologous immature dendritic cells (DCs) in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c(+) DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.

A mixture of bacterial mechanical lysates is more efficient than single strain lysate and of bacterial-derived soluble products for the induction of an activating phenotype in human dendritic cells.

Dendritic cells (DCs), following an optimal maturation, are able to drive an efficient immune-response. For this, both co-stimulatory molecules (CD80 and CD86), activation molecules (CD83) and peptide presenting molecules (HLA) are over-expressed. The in vitro treatment of immature DC with fragments of bacterial strains, obtained by using a mechanical lysis as well as with bacterial-derived molecules (such as lipopolysaccharide and protido-glycan), induced the maturation of DCs and the secretion of a panel of cytokines and chemokines. Of note, ex vivo treated circulating DCs and plasmacytoid DCs were also activated by these bacterial bodies. However, while the particulate fraction of single bacterial strains or soluble bacterial-derived molecules induced a sub-optimal maturation (as evaluated by the expression of an activating phenotype on DCs and the amount of cytokine secretion), the addition of the mixture of the particulate fractions of the different bacterial strains was able to mediate an optimal maturation. These results were also confirmed by using the secretion of both cytokines and chemokines as markers of DC activation. All these findings suggest that the particulate fraction of bacterial lysate mixtures, because of their ability to interact with different surface structures, might be exploited not only as an immunogen, but also as an adjuvant treatment to boost an immune-response to poorly "antigenic" proteins, such as cancer antigens or allergens.

Seroma fluid subsequent to axillary lymph node dissection for breast cancer derives from an accumulation of afferent lymph.

Seroma is a frequent complication of breast cancer surgery, the etiology of which remains indefinite. It represents a subcutaneous accumulation of fluid frequently reported after surgical procedures such as axillary lymph node dissection. Despite previous studies have associated seroma fluid to an inflammatory exudate, the surgical removal of draining lymph nodes may indicate that seroma might not represent a mere exudate but rather an accrual of lymph drained from tributary tissues. To verify this hypothesis, seromas were collected at different intervals of time in patients operated upon for axillary lymph node removal. Fluids were analyzed in details by flow cytometry and biochemical assays for their cellular content and for their molecular features and relevant cytokine content. Lymphocytes and other peculiar blood mononuclear cells were present, while erythrocytes, platelets and granulocytes were absent or extremely rare. The protein concentration resulted lower (median 64%) than in peripheral blood. However, specific proteins related to locoregional tissues resulted highly concentrated (e.g. up to 500% for ferritin and 300% for lactate deydrogenase and exclusive presence of interleukin-6) whereas all enzymes and proteins synthesized in the liver or other organs (e.g. alkaline phosphatase, ALT, gammaGT, prealbumin, transferrin, ceruloplasmin, C3 and C4, alpha2 macroglobulin from liver; apolipoproteins from liver and gut; amylase and lipase from pancreas) were represented in reduced concentrations, thus ruling out that seroma proteins derive directly from blood serum. As a whole, this comprehensive cytological and molecular analysis provided evidences that seroma is constituted by serum ultrafiltrated-derived extracellular fluid of regions located upstream of removed lymph nodes. This fluid is then enriched by proteins and cells collected in the drained regions. Remarkably, seroma fluids collected in the same patient at different time points (up to 50 days following surgery) displayed similar biochemical features, clearly indicating that fluid composition was not significantly affected by post-surgical locoregional flogosis. Finally, the period of seroma formation indicates that lymph accumulates in the axillary region during the interval of time needed for afferent lymphatic vessels to re-anastomose with the efferent ducts. Therefore, seroma fluid represents a font of biological material suitable for investigating the biology of breast cancer, healing tissues and lymph.

Role of natural killer cells in the pathogenesis and progression of multiple sclerosis.

Natural killer (NK) cells are a subset of lymphocytes which have long been alleged to play an immunoregulatory role in the prevention of autoimmune diseases. Here, we briefly review NK cell features and the major findings from studies on NK cells in human and animals susceptible to multiple sclerosis (MS). Although most studies in human seem to suggest an association between disease and deficiencies in NK cells, it is also clear that NK cells can be both protective and pathogenic in MS models. These contrasting observations could result from differences in experimental procedures as well as from differences in NK cell subset targeted. Whatever the case, the functional features of these cells and their potential role in regulation of autoimmunity suggest that NK cell-based therapies might be an interesting approach for the treatment of multiple sclerosis.

Natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in CD56 bright CD16(-) cells and display an impaired capability to kill tumor cells.

BACKGROUND: Despite natural killer (NK) cells being originally identified and named because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltrating malignant tumors, especially in humans. METHODS: NK cells infiltrating human nonsmall cell lung cancers (NSCLC) were analyzed with the aim of identifying their potential protective role in an antitumor immune response. Both relevant molecule expression and functions of NK cells infiltrating NSCLC were analyzed in comparison with autologous NK cells isolated from either peritumoral normal lung tissues or peripheral blood. RESULTS: The CD56 bright CD16(-) NK cell subset was consistently observed as being highly enriched in tumor infiltrate and displayed activation markers, including NKp44, CD69, and HLA-DR. Remarkably, the cytolytic potential of NK cells isolated from cancer tissues was lower than that of NK cells from peripheral blood or normal lung tissue, whereas no difference was observed regarding their capability of producing cytokines. With regard to their localization within tumor, NK cells were found in tumor stroma, whereas they were not in direct contact with cancer cells. CONCLUSIONS: For the first time NK cells infiltrating NSCLC have been characterized and it is shown that they are mainly capable of producing relevant cytokines rather than exerting direct cancer cell killing.

Distinct gut-derived lactic acid bacteria elicit divergent dendritic cell-mediated NK cell responses.

Lactic acid bacteria (LAB) are abundant in the gastrointestinal tract where they continuously regulate the immune system. NK cells are potently activated by dendritic cells (DCs) matured by inflammatory stimuli, and NK cells are present in the gut epithelium and in mesenteric lymph nodes, but it is not known how NK-DC interactions are affected by the predominantly non-pathogenic LAB. We demonstrate that human DCs exposed to different strains of gut-derived LAB consistently induce proliferation, cytotoxicity and activation markers in autologous NK cells. On the contrary, strains of LAB differ greatly in their ability to induce DC-dependent IFN-gamma production by NK cells. This suggests that DCs stimulated by gut LAB may expand the pool of NK cells and increase their cytotoxic potential. Specific LAB, inducing high levels of IL-12 in DCs, may promote amplification of a type-1 response via potent stimulation of IFN-gamma production in NK cells. Combining IFN-gamma-inducing and non-inducing LAB completely abrogates DC-mediated IFN-gamma production by NK cells, and therefore LAB modulating IFN-gamma production in NK cells may be important regulators of the immune response.