The immune microenvironment: a major player in human cancers.

Cancer is a major public health issue and figures among the leading causes of death in the world. Cancer development is a long process, involving the mutation, amplification or deletion of genes and chromosomal rearrangements. The transformed cells change morphologically, enlarge, become invasive and finally detach from the primary tumor to metastasize in other organs through the blood and/or lymph. During this process, the tumor cells interact with their microenvironment, which is complex and composed of stromal and immune cells that penetrate the tumor site via blood vessels and lymphoid capillaries. All subsets of immune cells can be found in tumors, but their respective density, functionality and organization vary from one type of tumor to another. Whereas inflammatory cells play a protumoral role, there is a large body of evidence of effector memory T cells controlling tumor invasion and metastasis. Thus, high densities of memory Th1/CD8 cytotoxic T cells in the primary tumors correlate with good prognosis in most tumor types. Tertiary lymphoid structures, which contain mature dendritic cells (DC) in a T cell zone, proliferating B cells and follicular DC, are found in the tumor stroma and they correlate with intratumoral Th1/CD8 T cell and B cell infiltration. Eventually, tumors undergo genetic and epigenetic modifications that allow them to escape being controlled by the immune system. This comprehensive review describes the immune contexture of human primary and metastatic tumors, how it impacts on patient outcomes and how it could be used as a predictive biomarker and guide immunotherapies.

Immune infiltration in human tumors: a prognostic factor that should not be ignored.

The natural history of a tumor includes phases of 'in situ' growth, invasion, extravasation and metastasis. During these phases, tumor cells interact with their microenvironment and are influenced by signals coming from stromal, endothelial, inflammatory and immune cells. Indeed, tumors are often infiltrated by various numbers of lymphocytes, macrophages or mast cells. It is generally believed that the latter produce factors that maintain chronic inflammation and promote tumor growth, whereas lymphocytes may control cancer outcome, as evidenced in mouse models. In this study, we analyze data from large cohorts of human tumors, clearly establishing that infiltration of the primary tumor by memory T cells, particularly of the Th1 and cytotoxic types, is the strongest prognostic factor in terms of freedom from disease and overall survival at all stages of clinical disease. We review data suggesting that tertiary lymphoid structures adjacent to tumors and composed of mature dendritic cells (T and B cells organized as germinal centers) may be the site of an antitumor reaction. We propose an immune scoring based on the type, density and location of lymphocyte infiltrates as a novel prognostic factor for use in addition to tumor node metastasis staging to predict disease-free survival and to aid in decisions regarding adjuvant therapies in early stage human cancers.

Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic skin disease in which environmental factors play a great role. A widely used murine model for AD has provided a useful tool to study the disease. OBJECTIVE: The purpose of this study is to investigate kinetically the induction of this AD model and the processes involved in the development of AD due to extrinsic allergen exposures. METHODS: BALB/c mice were epicutaneously exposed to ovalbumin (OVA) for 3 weeks; each week was separated by a 2-week resting period. Mice were killed after each exposure week. Skin biopsies and blood were obtained for histological study, RNA isolation and antibody analysis. RESULTS: There was a progressive and significant thickening of the epidermis and dermis in OVA-exposed mice. Significantly increased dermal cell infiltration of eosinophils, mast cells and total inflammatory cells, including CD3 and CD4 cells, was found after each OVA exposure week. Total IgE, IgG2a and OVA-specific IgE were significantly increased after the second and third exposure week, while OVA-specific IgG2a was significantly induced after the third exposure week. Gradual and/or significant increases in mRNA expression of IL-1beta, TNF-alpha, IL-4, IL-10, IL-13, IFN-gamma and IL-12p35 were found after each exposure week. Chemokines and their receptors involved in both T-helper type 1 (Th1)- and Th2-type cell recruitment (CCL1, CCL8, CCL11, CCL24, CXCL9, CXCL10, CCR1, CCR3, CCR5, CCR8 and CXCR3) were up-regulated significantly at different time-points. CONCLUSION: This study provides an insight into the dynamic nature and time-dependent transition of skin inflammation and systemic immune responses in a murine AD model induced by repeated epicutaneous exposures to OVA.

Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin.

The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery. However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo. Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207(+) LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC-lysosomal-associated membrane protein (LAMP)/CD208. Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-beta1, we further found that tumor necrosis factor (TNF)-alpha, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC-LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21. This indicates that LC migration and maturation can be independently regulated events. We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation. Immature LCs might regulate immune responses during chronic inflammation.

IL-10 induces CCR6 expression during Langerhans cell development while IL-4 and IFN-gamma suppress it.

Immune responses are initiated by dendritic cells (DC) that form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells colonizing epithelial surfaces. We have recently shown that macrophage-inflammatory protein-3alpha/CCL20, a chemokine secreted by epithelial cells, induces the selective migration of LC among DC populations. In this study, we investigated the effects of cytokines on the expression of the CCL20 receptor, CCR6, during differentiation of LC. We found that both IL-4 and IFN-gamma blocked the expression of CCR6 and CCL20 responsiveness at different stages of LC development. The effect of IL-4 was reversible and most likely due to the transient blockade of LC differentiation. In contrast, IFN-gamma-induced CCR6 loss was irreversible and was concomitant to the induction of DC maturation. When other cytokines involved in DC and T cell differentiation were tested, we found that IL-10, unlike IL-4 and IFN-gamma, maintained CCR6 expression. The effect of IL-10 was reversible and upon IL-10 withdrawn, CCR6 was lost concomitantly to final LC differentiation. In addition, IL-10 induced the expression of CCR6 and responsiveness to CCL20 in differentiated monocytes that preserve their ability to differentiate into mature DC. Finally, TGF-beta, which induces LC differentiation, did not alter early CCR6 expression, but triggered its irreversible down-regulation, in parallel to terminal LC differentiation. Taken together, these results suggest that the recruitment of LC at epithelial surface might be suppressed during Th1 and Th2 immune responses, and amplified during regulatory immune responses involving IL-10 and TGF-beta.

Expression of macrophage inflammatory protein-3alpha, stromal cell-derived factor-1, and B-cell-attracting chemokine-1 identifies the tonsil crypt as an attractive site for B cells.

The expression of 3 lymphoid chemokines-macrophage inflammatory protein-3alpha (MIP-3alpha), stromal cell-derived factor-1 (SDF-1), and B-cell-attracting chemokine-1 (BCA-1)-in the tonsil and the possible correlation between their sites of expression and B-cell localization within this tissue were studied. The results show that all 3 chemokines are produced in the crypts but differ by the nature of the cells that produce them and their location within the crypt. SDF-1 and MIP-3alpha are produced by epithelial cells, but their secretion is mutually exclusive. Both MIP-3alpha- and SDF-1-expressing cells are in close contact with memory B cells. By contrast, BCA-1-producing cells in the crypt are not epithelial and form clusters colocalized with plasma cells. Altogether, these data suggest that the chemokines produced in the tonsillar crypt may (1) attract memory B cells to antigen and (2) recruit and retain plasma cells and memory B cells within the supportive epithelial microenvironment of the crypt. (Blood. 2001;97:3992-3994)

Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors.

Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3alpha plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3alpha was the most potent chemokine inducing the selective migration of in vitro-generated CD34(+) hematopoietic progenitor cell-derived LC precursors and skin LCs in accordance with the restricted MIP-3alpha receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3alpha was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3alpha. (c) In vivo, MIP-3alpha was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3alpha upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3alpha was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1beta plus tumor necrosis factor alpha) or T cell signals. Results of this study suggest a major role of MIP-3alpha in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis.

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.

Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC).

We recently reported the identification of a chemokine (CTACK), which has been renamed CCL27 according to a new systematic chemokine nomenclature. We report that CCL27 binds the previously orphan chemokine receptor GPR-2, as detected by calcium flux and chemotactic responses of GPR-2 transfectants. We renamed this receptor CCR10. Because of the skin-associated expression pattern of CCL27, we focused on the expression of CCL27 and CCR10 in normal skin compared with inflammatory and autoimmune skin diseases. CCL27 is constitutively produced by keratinocytes but can also be induced upon stimulation with TNF-alpha and IL-1beta. CCR10 is not expressed by keratinocytes and is instead expressed by melanocytes, dermal fibroblasts, and dermal microvascular endothelial cells. CCR10 was also detected in T cells as well as in skin-derived Langerhans cells. Taken together, these observations suggest a role for this novel ligand/receptor pair in both skin homeostasis as well as a potential role in inflammatory responses.

Dendritic cell biology and regulation of dendritic cell trafficking by chemokines.

DC (dendritic cells) represent an heterogeneous family of cells which function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. Then, following inflammatory stimuli, they leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. The key role of DC migration in their sentinel function led to the investigation of the chemokine responsiveness of DC populations during their development and maturation. These studies have shown that immature DC respond to many CC and CXC chemokines (MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-5, MCP-3, MCP-4, RANTES, TECK and SDF-1) which are inducible upon inflammatory stimuli. Importantly, each immature DC population displays a unique spectrum of chemokine responsiveness. For examples, Langerhans cells migrate selectively to MIP-3 alpha (via CCR6), blood CD11c+ DC to MCP chemokines (via CCR2), monocytes derived-DC respond to MIP-1 alpha/beta (via CCR1 and CCR5), while blood CD11c- DC precursors do not respond to any of these chemokines. All these chemokines are inducible upon inflammatory stimuli, in particular MIP-3 alpha, which is only detected within inflamed epithelium, a site of antigen entry known to be infiltrated by immature DC. In contrast to immature DC, mature DC lose their responsiveness to most of these inflammatory chemokines through receptor down-regulation or desensitization, but acquire responsiveness to ELC/MIP-3 beta and SLC/6Ckine as a consequence of CCR7 up-regulation. ELC/MIP-3 beta and SLC/6Ckine are specifically expressed in the T-cell-rich areas where mature DC home to become interdigitating DC. Altogether, these observations suggest that the inflammatory chemokines secreted at the site of pathogen invasion will determine the DC subset recruited and will influence the class of the immune response initiated. In contrast, MIP-3 beta/6Ckine have a determinant role in the accumulation of antigenloaded mature DC in T cell-rich areas of the draining lymph node, as illustrated by recent observations in mice deficient for CCR7 or SLC/6Ckine. A better understanding of the regulation of DC trafficking might offer new opportunities of therapeutic interventions to suppress, stimulate or deviate the immune response.

Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines.

DC function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led to the investigation of the chemokine responsiveness of DC during their development and maturation. These studies have shown that immature and mature DC are not recruited by the same chemokines. Immature DC respond to many CC- and CXC-chemokines (MIP-1alpha, MIP-1beta, MIP-5, MCP-3, MCP-4, RANTES, TECK, and SDF-1) and in particular to MIP-3alpha/LARC, which acts through CCR6, a receptor mainly expressed in DC and lymphocytes. Like most other chemokines acting on immature DC, MIP-3alpha is inducible on inflammatory stimuli. In contrast, mature DC have lost their responsiveness to most of these chemokines through receptor down-regulation or desensitization, but acquired responsiveness to MIP-3beta/ELC and 6Ckine/SLC as a consequence of CCR7 up-regulation. MIP-3alpha mRNA is only detected within inflamed epithelial crypts of tonsils, the site of antigen entry known to be infiltrated by immature DC, whereas MIP-3alpha and 6Ckine are specifically expressed in the T cell-rich areas where mature IDC home. These observations suggest a role for chemokines induced on inflammation such as MIP-3alpha in recruitment of immature DC at the site of injury and a role for MIP-3beta/6Ckine in accumulation of antigen-loaded mature DC in T cell-rich areas of the draining lymph node. A better understanding of the regulation of DC trafficking might offer new opportunities of therapeutic interventions to suppress or stimulate the immune response.