Blood gene expression predicts intensive care unit admission in hospitalised patients with COVID-19.

Background: The COVID-19 pandemic has created pressure on healthcare systems worldwide. Tools that can stratify individuals according to prognosis could allow for more efficient allocation of healthcare resources and thus improved patient outcomes. It is currently unclear if blood gene expression signatures derived from patients at the point of admission to hospital could provide useful prognostic information. Methods: Gene expression of whole blood obtained at the point of admission from a cohort of 78 patients hospitalised with COVID-19 during the first wave was measured by high resolution RNA sequencing. Gene signatures predictive of admission to Intensive Care Unit were identified and tested using machine learning and topological data analysis, TopMD. Results: The best gene expression signature predictive of ICU admission was defined using topological data analysis with an accuracy: 0.72 and ROC AUC: 0.76. The gene signature was primarily based on differentially activated pathways controlling epidermal growth factor receptor (EGFR) presentation, Peroxisome proliferator-activated receptor alpha (PPAR-α) signalling and Transforming growth factor beta (TGF-ß) signalling. Conclusions: Gene expression signatures from blood taken at the point of admission to hospital predicted ICU admission of treatment naïve patients with COVID-19.

Expression Profile of Genes Related to the Th17 Pathway in Macrophages Infected by Leishmania major and Leishmania amazonensis: The Use of Gene Regulatory Networks in Modeling This Pathway.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania-macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4-24 h was observed in the former. However, there was a decrease in basal levels at 48-72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania-macrophage interaction over time that extends beyond the analysis of single-gene expression.

Loss of T cell tolerance in the skin following immunopathology is linked to failed restoration of the dermal niche by recruited macrophages.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1ß and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

Understanding the tuberculosis granuloma: the matrix revolutions.

Mycobacterium tuberculosis (Mtb) causes the human disease tuberculosis (TB) and remains the top global infectious pandemic after coronavirus disease 2019 (COVID-19). Furthermore, TB has killed many more humans than any other pathogen, after prolonged coevolution to optimise its pathogenic strategies. Full understanding of fundamental disease processes in humans is necessary to successfully combat this highly successful pathogen. While the importance of immunodeficiency has been long recognised, biologic therapies and unbiased approaches are providing unprecedented insights into the intricacy of the host-pathogen interaction. The nature of a protective response is more complex than previously hypothesised. Here, we integrate recent evidence from human studies and unbiased approaches to consider how Mtb causes human TB and highlight the recurring theme of extracellular matrix (ECM) turnover.

An IRF1-IRF4 Toggle-Switch Controls Tolerogenic and Immunogenic Transcriptional Programming in Human Langerhans Cells.

Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF-alpha, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF-alpha enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.

Integrated transcriptomic analysis of human tuberculosis granulomas and a biomimetic model identifies therapeutic targets.

Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.

Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors.

BACKGROUND: Natural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the outcome of cancer. In particular, KIR2DS2 is associated with protective responses to several cancers and also direct recognition of cancer targets in vitro. Due to the high homology between activating and inhibitory KIR genes to date, it has been challenging to target individual KIR for therapeutic benefit. METHODS: A novel KIR2DS2-targeting therapeutic peptide:MHC DNA vaccine was designed and used to immunize mice transgenic for KIR genes (KIR-Tg). NK cells were isolated from the livers and spleens of vaccinated mice and then analyzed for activation by flow cytometry, RNA profiling and cytotoxicity assays. In vivo assays of NK cell function using a syngeneic cancer model (B16 melanoma) and an adoptive transfer model for human hepatocellular carcinoma (Huh7) were performed. RESULTS: Injecting KIR-Tg mice with the vaccine construct activated NK cells in both liver and spleens of mice, with preferential activation of KIR2DS2-positive NK cells. KIR-specific activation was most marked on the CD11b+CD27+ mature subset of NK cells. RNA profiling indicated that the DNA vaccine upregulated genes associated with cellular metabolism and downregulated genes related to histone H3 methylation, which are associated with immune cell maturation and NK cell function. Vaccination led to canonical and cross-reactive peptide:MHC-specific NK cell responses. In vivo, DNA vaccination led to enhanced antitumor responses against B16F10 melanoma cells and also enhanced responses against a tumor model expressing the KIR2DS2 ligand HLA-C*0102. CONCLUSION: We show the feasibility of a peptide-based KIR-targeting vaccine strategy to activate NK cells and hence generate functional antitumor responses. This approach does not require detailed knowledge of the tumor peptidomes nor HLA matching with the patient. It therefore offers a novel opportunity for targeting NK cells for cancer immunotherapy.

Ileal Transcriptomic Analysis in Paediatric Crohn's Disease Reveals IL17- and NOD-signalling Expression Signatures in Treatment-naïve Patients and Identifies Epithelial Cells Driving Differentially Expressed Genes.

BACKGROUND AND AIMS: Crohn's disease [CD] arises through host-environment interaction. Abnormal gene expression results from disturbed pathway activation or response to bacteria. We aimed to determine activated pathways and driving cell types in paediatric CD. METHODS: We employed contemporary targeted autoimmune RNA sequencing, in parallel to single-cell sequencing, to ileal tissue derived from paediatric CD and controls. Weighted gene co-expression network analysis [WGCNA] was performed and differentially expressed genes [DEGs] were determined. We integrated clinical data to determine co-expression modules associated with outcomes. RESULTS: In all, 27 treatment-naive CD [TN-CD], 26 established CD patients and 17 controls were included. WGCNA revealed a 31-gene signature characterising TN-CD patients, but not established CD, nor controls. The CSF3R gene is a hub within this module and is key in neutrophil expansion and differentiation. Antimicrobial genes, including S100A12 and the calprotectin subunit S100A9, were significantly upregulated in TN CD compared with controls [p = 2.61 x 10-15 and p = 9.13 x 10-14, respectively] and established CD [both p = 0.0055]. Gene-enrichment analysis confirmed upregulation of the IL17-, NOD- and Oncostatin-M-signalling pathways in TN-CD patients, identified in both WGCNA and DEG analyses. An upregulated gene signature was enriched for transcripts promoting Th17-cell differentiation and correlated with prolonged time to relapse [correlation-coefficient-0.36, p = 0.07]. Single-cell sequencing of TN-CD patients identified specialised epithelial cells driving differential expression of S100A9. Cell groups, determined by single-cell gene expression, demonstrated enrichment of IL17-signalling in monocytes and epithelial cells. CONCLUSIONS: Ileal tissue from treatment-naïve paediatric patients is significantly upregulated for genes driving IL17-, NOD- and Oncostatin-M-signalling. This signal is driven by a distinct subset of epithelial cells expressing antimicrobial gene transcripts.

Genomic programming of IRF4-expressing human Langerhans cells.

Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.

OX40+ Regulatory T Cells in Cutaneous Squamous Cell Carcinoma Suppress Effector T-Cell Responses and Associate with Metastatic Potential.

PURPOSE: Cutaneous squamous cell carcinoma (cSCC) is the most common human cancer with metastatic potential. Despite T cells accumulating around cSCCs, these tumors continue to grow and persist. To investigate reasons for failure of T cells to mount a protective response in cSCC, we focused on regulatory T cells (Tregs) as this suppressive population is well represented among the infiltrating lymphocytes. EXPERIMENTAL DESIGN: Flow cytometry was conducted on cSCC lymphocytes and in vitro functional assays were performed using sorted tumoral T cells. Lymphocyte subsets in primary cSCCs were quantified immunohistochemically. RESULTS: FOXP3(+) Tregs were more frequent in cSCCs than in peripheral blood (P < 0.0001, n = 86 tumors). Tumoral Tregs suppressed proliferation of tumoral effector CD4(+) (P = 0.005, n = 10 tumors) and CD8(+) T cells (P = 0.043, n = 9 tumors) and inhibited IFNγ secretion by tumoral effector T cells (P = 0.0186, n = 11 tumors). The costimulatory molecule OX40 was expressed predominantly on tumoral Tregs (P < 0.0001, n = 15 tumors) and triggering OX40 with an agonist anti-OX40 antibody overcame the suppression exerted by Tregs, leading to increased tumoral effector CD4(+) lymphocyte proliferation (P = 0.0098, n = 10 tumors). Tregs and OX40(+) lymphocytes were more abundant in primary cSCCs that metastasized than in primary cSCCs that had not metastasized (n = 48 and n = 49 tumors, respectively). CONCLUSIONS: Tregs in cSCCs suppress effector T-cell responses and are associated with subsequent metastasis, suggesting a key role for Tregs in cSCC development and progression. OX40 agonism reversed the suppressive effects of Tregs in vitro, suggesting that targeting OX40 could benefit the subset of cSCC patients at high risk of metastasis. Clin Cancer Res; 22(16); 4236-48. ©2016 AACR.

Conformation of the human immunoglobulin G2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies.

Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.

Distinct molecular signature of human skin Langerhans cells denotes critical differences in cutaneous dendritic cell immune regulation.

Langerhans cells (LCs) are professional antigen-presenting cells (APCs) residing in the epidermis. Despite their high potential to activate T lymphocytes, current understanding of human LC biology is limited. Genome-wide comparison of the transcriptional profiles of human skin migratory CD1a+ LCs and CD11c+ dermal dendritic cells (DDCs) demonstrated significant differences between these "dendritic cell (DC)" types, including preferential expression of 625 genes (P<0.05) in LC and 914 genes (P<0.05) in DDC. Analysis of the temporal regulation of molecular networks activated after stimulation with tumor necrosis factor-α (TNF-α) confirmed the unique molecular signature of LCs. Although LCs conformed to the phenotype of professional APC, inflammatory signaling activated primarily genes associated with cellular metabolism and mitochondrial activation (e.g., CYB561 and MRPS35), cell membrane re-organization, and antigen acquisition and degradation (CAV1 and PSMD14; P<0.05-P<0.0001). Conversely, TNF-α induced classical activation in DDCs with early downregulation of surface receptors (mannose receptor-1 (MRC1) and C-type lectins), and subsequent upregulation of cytokines, chemokines (IL1a, IL1b, and CCL18), and matrix metalloproteinases (MMP1, MMP3, and MMP9; P<0.05-P<0.0001). Functional interference of caveolin abrogated LCs superior ability to cross-present antigens to CD8+ T lymphocytes, highlighting the importance of these networks to biological function. Taken together, these observations support the idea of distinct biological roles of cutaneous DC types.

CD70-CD27 interaction augments CD8+ T-cell activation by human epidermal Langerhans cells.

Human cutaneous dendritic cells (DCs) from epidermal and dermal compartments exhibit functional differences in their induction of CD4+ T-cell and humoral immune responses; however, differences in the regulation of memory CD8+ T-cell responses by human skin DCs remain poorly characterized. We tested the capacity of human Langerhans cells (LCs) and dermal dendritic cells (DDCs) to induce antigen-specific cytokine production and proliferation of memory CD8+ cells. Although tumor necrosis factor-α-matured human DCs from both epidermal and dermal compartments showed efficient potential to activate CD8+ cells, LCs were constitutively more efficient than DDCs in cross-presenting CD8+ epitopes, as well as direct presentation of viral antigen to Epstein-Barr virus-specific CD8+ T cells. LCs showed greater expression of CD70, and blockade of CD70-CD27 signaling demonstrated that superiority of CD8+ activation by epidermal LC is CD70 dependent. This CD70-related activation of CD8+ cells by LCs denotes a central role of LCs in CD8+ immunity in skin, and suggests that regulation of LC CD70 expression is important in enhancing immunity against cutaneous epithelial pathogens and cancer.

Isolation of inflammatory cells from human tumours.

Inflammatory cells are present in many tumours, and understanding their function is of increasing importance, particularly to studies of tumour immunology. The tumour-infiltrating leukocytes encompass a variety of cell types, e.g. T lymphocytes, macrophages, dendritic cells, NK cells, and mast cells. Choice of the isolation method greatly depends on the tumour type and the leukocyte subset of interest, but the protocol usually includes tissue disaggregation and cell enrichment. We recommend density centrifugation for initial enrichment, followed by specific magnetic bead negative or positive panning with leukocyte and tumour cell selective antibodies.

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC).

BACKGROUND: NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. METHODS: The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. CONCLUSION: Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer.

Melanoma vaccines: The problems of local immunosuppression.

The incidence of cutaneous melanoma in Europe is rising, and the disease is incurable once metastases occur. Because melanoma expresses antigens that can be specifically recognized by the immune system, and because this disease occasionally undergoes spontaneous regression mediated by anti-tumor immunity, a number of different melanoma vaccines have been developed and tested clinically. Although most such vaccines show efficacy in vitro and an ability to stimulate anti-melanoma immune responses in blood, they have proved disappointing in clinical practice. It has become increasingly clear that the interaction between melanoma and the immune system is determined locally, within the tumor or draining lymph nodes. It is now clear that melanoma cells have the ability to anergize the immune system by inducing an immunosuppressive microenvironment that may explain the inability of systemic vaccines to alter patient outcomes. This subversion of the immune system involves alteration of dendritic cell (DC) function by tumor-derived cytokines, leading to the generation of suppressive and regulatory T lymphocytes. Successful melanoma vaccination probably requires therapeutic neutralization of the immunosuppressive microenvironment, which will require greater understanding of the molecular mechanisms used by the tumor to promote immunosuppression. Nevertheless, if these problems can be overcome, it seems likely that the efficacy of melanoma vaccines could be greatly enhanced.

Activation of tonsil dendritic cells with immuno-adjuvants.

BACKGROUND: Dendritic cells (DC) play the key role in directing antigen-specific immune responses and manipulating their function may be a useful tool for immunotherapy. The balance between immune stimulation and tolerance is particularly important at mucosal interfaces, where discrimination between dangerous pathogens and innocuous antigens takes place. In humans, although much is known about the responses of monocyte derived DC, relatively little is known about effect of immuno-stimulatory adjuvants on DC found in tonsil. RESULTS: To examine this, tonsil DC were isolated and cultured with potent DC activators; IFNgamma, anti-CD40 antibody, LPS and Poly I:C either singly or in combination. To measure maturation and activation, DC were examined for changes in the expression of HLA-DR, HLA- class I, CD83, CD40, CD80 and CD86 and the release of IL12p70. The DC isolated from tonsil were a mixed population containing both myeloid and plasmacytoid DC, but all showed similar responses. Tonsil DC released IL12p70 upon stimulation with IFNgamma , anti-CD40 antibody, and LPS, but unlike monocyte-derived DC, they did not increase the expression of cell surface activation molecules above those induced by culture alone. Poly I:C, a potent stimulator of laboratory generated DC inhibited the activation of tonsil DC by other adjuvants. CONCLUSION: As the response of this mixed population of DC does not mirror that of DC generated in vitro, this may have implications for other tissue residing DC and might be an important consideration for immunotherapy.

Presence and phenotype of dendritic cells in uveal melanoma.

BACKGROUND: Uveal melanoma arises in an immune-privileged site and can itself add to the immunosuppressive environment. Previous studies on cutaneous melanoma have shown the presence of tolerogenic dendritic cells (DCs), which could play an important role in the progression of the tumour. AIM: To examine the presence and functional status of DCs in a small series of uveal melanomas. METHODS: 10 cases of uveal melanoma were examined for the expression of FXIIIa, CD68, human leucocyte antigen (HLA)-DR, CD40, CD83, transforming growth factor betaR1 and indolamine 2,3 dioxygenase by immunohistochemical analysis on sections embedded in paraffin wax. RESULTS: CD68-positive macrophages were present in all of the tumours and were evenly distributed throughout. DCs expressing FXIIIa-positive were seen in 7 cases, and were often found concentrated in foci within the tumour mass. These cells were dendritic and expressed high levels of HLA-DR. The DCs did not express the maturation markers CD83 or CD40. In one case, concentration of DCs around the area of tumour necrosis was observed, and some of these cells expressed CD83. CONCLUSION: Numerous tolerising antigen-presenting cells may play a role in melanoma-related immunosuppression in the eye, although activation of DCs may be associated with tumour necrosis.

Presence and maturity of dendritic cells in melanoma lymph node metastases.

Immune avoidance mechanisms play a key role in the successful dissemination of melanoma. One mechanism whereby this could be achieved is by interfering with dendritic cell (DC) presentation of tumour-associated antigens to naïve T cells. In particular, immature DCs characterized by the absence of accessory molecules are known to be immunosuppressive and to be involved in the induction of tolerance. The present study has investigated the presence and activation status of DCs within melanoma metastases in the regional lymph nodes. Using image analysis techniques, the expression of Factor XIIIa (FXIIIa), CD40, CD83 and HLA-DR and the morphological features of DCs were examined in paraffin sections from 26 lymph nodes containing melanoma metastases. DCs expressing FXIIIa were found in 70% of the lymph nodes. The number of DCs identified was generally small but there were more concentrated areas of DCs designated as hotspots. In these areas of high FXIIIa staining, the percentage area occupied by DCs varied between 0.1% and 10%. The majority of FXIIIa-positive cells did not express the DC maturation markers CD83 or CD40 and morphologically were rounded with few dendrites, indicating that they were immature. The cells did, however, express high levels of HLA-DR, suggesting that they have the ability to present antigen but lack the accessory molecules required to initiate an immune response. Immature DCs, characterized by phenotypic and morphological features, are therefore present within the tumour deposits in lymph nodes infiltrated by melanoma and may specifically modulate the anti-melanoma immune response.