Tumor-infiltrating γδ T cells as targets of immune checkpoint blockade in melanoma.

Melanoma is one of the most sensitive tumors to immune modulation, and the major challenge for melanoma patients' survival is immune checkpoint inhibitor (ICI) therapy. γδ T lymphocytes play an antitumoral role in a broad variety of tumors including melanoma and they are optimal candidates for cellular immunotherapy. Thus, a comprehensive analysis of the correlation between γδ T cells and immune checkpoint receptors in the context of melanoma was conducted, with the aim of devising an innovative combined immunotherapeutic strategy. In this study, using the GEPIA2.0 database, a significant positive correlation was observed between the expression of γδ T cell-related genes (TRGC1, TRGC2, TCRD) and immune checkpoint genes (PDCD1, HAVCR2, LAG3), highlighting the potential role of γδ T cells in the immune response within melanoma. Moreover, flow cytometry analysis unveiled a significant augmentation in the population of γδ T cells within melanoma lesions, which exhibited the expression of immune checkpoint receptors including LAG3, TIM3, and PD1. Analysis of single-cell RNA sequencing data revealed a significant enrichment and functional reprogramming of γδ T cell clusters in response to ICIs. Interestingly, the effects of ICI therapy varied between Vδ1 and Vδ2 γδ T cell subsets, with distinct changes in gene expression patterns. Last, a correlation analysis between γδ T cell abundance, immune checkpoint gene expression, and clinical outcomes in melanoma patients showed that low expression of immune checkpoint genes, including LAG3, HAVCR2, and PDCD1, was associated with improved 1-year overall survival, emphasizing the significance of these genes in predicting patient outcomes, potentially outweighing the impact of γδ T cell abundance. This study offers critical insights into the dynamic interaction between γδ T cells, immune checkpoint receptors, and melanoma, providing valuable perspectives for potential therapeutic avenues and predictive markers in this intricate interplay.

Ferritin Metabolism Reflects Multiple Myeloma Microenvironment and Predicts Patient Outcome.

Multiple myeloma (MM) is a hematologic malignancy with a multistep evolutionary pattern, in which the pro-inflammatory and immunosuppressive microenvironment and genomic instability drive tumor evolution. MM microenvironment is rich in iron, released by pro-inflammatory cells from ferritin macromolecules, which contributes to ROS production and cellular damage. In this study, we showed that ferritin increases from indolent to active gammopathies and that patients with low serum ferritin had longer first line PFS (42.6 vs. 20.7 months and, p = 0.047, respectively) and OS (NR vs. 75.1 months and p = 0.029, respectively). Moreover, ferritin levels correlated with systemic inflammation markers and with the presence of a specific bone marrow cell microenvironment (including increased MM cell infiltration). Finally, we verified by bioinformatic approaches in large transcriptomic and single cell datasets that a gene expression signature associated with ferritin biosynthesis correlated with worse outcome, MM cell proliferation, and specific immune cell profiles. Overall, we provide evidence of the role of ferritin as a predictive/prognostic factor in MM, setting the stage for future translational studies investigating ferritin and iron chelation as new targets for improving MM patient outcome.

γδ T cells and their clinical application in colon cancer.

In recent years, research has focused on colorectal cancer to implement modern treatment approaches to improve patient survival. In this new era, γδ T cells constitute a new and promising candidate to treat many types of cancer because of their potent killing activity and their ability to recognize tumor antigens independently of HLA molecules. Here, we focus on the roles that γδ T cells play in antitumor immunity, especially in colorectal cancer. Furthermore, we provide an overview of small-scale clinical trials in patients with colorectal cancer employing either in vivo activation or adoptive transfer of ex vivo expanded γδ T cells and suggest possible combinatorial approaches to treat colon cancer.

Monoclonal Gammopathies and the Bone Marrow Microenvironment: From Bench to Bedside and Then Back Again.

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by a multistep evolutionary pathway, with an initial phase called monoclonal gammopathy of undetermined significance (MGUS), potentially evolving into the symptomatic disease, often preceded by an intermediate phase called "smoldering" MM (sMM). From a biological point of view, genomic alterations (translocations/deletions/mutations) are already present at the MGUS phase, thus rendering their role in disease evolution questionable. On the other hand, we currently know that changes in the bone marrow microenvironment (TME) could play a key role in MM evolution through a progressive shift towards a pro-inflammatory and immunosuppressive shape, which may drive cancer progression as well as clonal plasma cells migration, proliferation, survival, and drug resistance. Along this line, the major advancement in MM patients' survival has been achieved by the introduction of microenvironment-oriented drugs (including immunomodulatory drugs and monoclonal antibodies). In this review, we summarized the role of the different components of the TME in MM evolution from MGUS as well as potential novel therapeutic targets/opportunities.

Impact of the flame retardant 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles.

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-ß mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells.

Metabolic Changes in Tumor Microenvironment: How Could They Affect γδ T Cells Functions?

The metabolic changes that occur in tumor microenvironment (TME) can influence not only the biological activity of tumor cells, which become more aggressive and auto sustained, but also the immune response against tumor cells, either producing ineffective responses or polarizing the response toward protumor activity. γδ T cells are a subset of T cells characterized by a plasticity that confers them the ability to differentiate towards different cell subsets according to the microenvironment conditions. On this basis, we here review the more recent studies focused on altered tumor metabolism and γδ T cells, considering their already known antitumor role and the possibility of manipulating their effector functions by in vitro and in vivo approaches. γδ T cells, thanks to their unique features, are themselves a valid alternative to overcome the limits associated with the use of conventional T cells, such as major histocompatibility complex (MHC) restriction, costimulatory signal and specific tumor-associated antigen recognition. Lipids, amino acids, hypoxia, prostaglandins and other metabolic changes inside the tumor microenvironment could reduce the efficacy of this important immune population and polarize γδ T cells toward IL17 producing cells that play a pro tumoral role. A deeper knowledge of this phenomenon could be helpful to formulate new immunotherapeutic approaches that target tumor metabolisms.

Cell quality evaluation with gene expression analysis of spheroids (3D) and adherent (2D) adipose stem cells.

Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (SASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by "cell-quality"; for this reason, the involvement of stress signals and cellular aging need to be further investigated. Here, we performed a comparative analysis of genes connected with stemness, aging, telomeric length and oxidative stress, in 3D and 2D primary cultures. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASCs-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). The 3D and 2D cultures also had a different gene expression profile for genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Based on our findings, we propose a "cell quality" model of SASCs, highlighting a precise molecular expression of several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.

Deciphering human γδ T cell response in cancer: Lessons from tumor-infiltrating γδ T cells.

The finding that γδ T cells are present among tumor-infiltrating lymphocytes in humans suggests they participate in tumor immune surveillance, but their relevance is unclear because the relative abundance of tumor-infiltrating γδ T cells correlates with positive or negative, or even do not correlate with prognosis. This likely depends on the fact that tumor-infiltrating γδ T cells may play substantially different effector or regulatory functions, and correlation with patient's prognosis relies on distinct γδ T cell subsets in the context of the tumor. There is interest to exploit γδ T cells in tumor immunotherapy, but to make this approach successful there is urgent need to fully understand the biological functions of γδ T cells and of how they can be manipulated in vivo and ex vivo to safely provide benefit to the host. This review focuses on our previous and ongoing studies of tumor-infiltrating γδ T lymphocytes in different types of human cancer. Moreover, we discuss the interaction of tumor-infiltrating γδ T cells with other cells and molecules present in the tumor microenvironment, and their clinical relevance on the ground, that deep knowledge in this field can be used further for better immunotherapeutic intervention in cancer.

Analysis of colon-infiltrating γδ T cells in chronic inflammatory bowel disease and in colitis-associated cancer.

Inflammatory bowel disease (IBD) remains a global health problem with a significant percentage of patients progressing to chronic inflammation and colitis-associated cancer (CAC). Whether or not γδ T cells contribute to initiation and maintenance of inflammation in IBD and in the development of CAC is not known. We have evaluated the frequency, phenotype, and functions of γδ T cells among tissue-infiltrating lymphocytes in healthy donors and IBD and CAC patients. Results show that Vδ1 T cells are the dominant γδ T-cell population in healthy tissue, whereas Vδ2 T significantly abound in chronic IBD. Vδ2 T cells produce more IFN-γ, TNF-α, and IL-17 than Vδ1 T cells in chronic inflamed IBD. In CAC patients no significant cytokine production was detected in tissue-resident Vδ1 T cells, but Vδ2 T cells produced remarkable amounts of IFN-γ and TNF-α; these data were confirmed by the analysis of an independent cohort of IBD transcriptomes. Moreover, transcriptomes of IBD patients revealed a clear-cut clusterization of genes related with the maintenance of the inflammatory status. In conclusion, our results demonstrating that Vδ2 T cells have a proinflammatory profile in chronic IBD are suggestive of their participation in IBD and CAC pathogenesis.

NKp46-expressing human gut-resident intraepithelial Vδ1 T cell subpopulation exhibits high antitumor activity against colorectal cancer.

γδ T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent antitumor activities. However, little is known about their origin, phenotype, and clinical relevance in colorectal cancer (CRC). To determine γδ IEL gut specificity, homing, and functions, γδ T cells were purified from human healthy blood, lymph nodes, liver, skin, and intestine, either disease-free, affected by CRC, or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a subset of cytotoxic Vδ1 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46+/Vδ1 IELs depends both on distinctive features of Vδ1 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident Vδ1 intestinal IELs or its induced expression on IL-2/IL-15-activated Vδ1 thymocytes are associated with antitumor functions. Higher frequencies of NKp46+/Vδ1 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor microenvironment inhibited the expansion of NKp46+/Vδ1 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46+/Vδ1 IELs able to infiltrate CRC, thus providing insights to either follow-up cancer progression or to develop adoptive cellular therapies.

γδ cell-based immunotherapy for cancer.

Introduction: Cancer immunotherapy relies on the development of an efficient and long-lasting anti-tumor response, generally mediated by cytotoxic T cells. γδ T cells possess distinctive features that justify their use in cancer immunotherapy. Areas covered: Here we will review our current knowledge on the functions of human γδ T cells that may be relevant in tumor immunity and the most recent advances in our understanding of how these functions are regulated in the tumor microenvironment. We will also discuss the major achievements and limitations of γδ T cell-based immunotherapy of cancer. Expert opinion: Several small-scale clinical trials have been conducted in cancer patients using either in vivo activation of γδ T cells or adoptive transfer of ex vivo-expanded γδ T cells. Both strategies are safe and give some clinical benefit to patients, thus providing a proof of principle for their utilization in addition to conventional therapies. However, low objective response rates have been obtained in both settings and therefore larger and well-controlled trials are needed. Discovering the factors which influence the success of γδ T cell-based immunotherapy will lead to a better understanding of their mechanism of action and to harness these cells for effective and durable anti-tumor responses.

Single-cell RNA sequencing unveils the shared and the distinct cytotoxic hallmarks of human TCRVδ1 and TCRVδ2 γδ T lymphocytes.

γδ T lymphocytes represent ∼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions.

Characterization of human infiltrating and circulating gamma-delta T cells in prostate cancer.

Purpose: The aim of our study was to prospectively evaluate the distribution of gamma-delta (γδ)1 and γδ2 T cells and their phenotypes in peripheral blood and prostate samples of patients diagnosed with or without prostate cancer (PCa) at prostate biopsy. Materials and Methods: A consecutive series of 43 outpatients underwent trans-rectal echo-guided prostate biopsy for suspected PCa. Flow cytometry analysis was used to identify and characterize the γδ T cells populations in peripheral blood and tissue samples. Patients were stratified according to the presence or not of PCa, and its International Society of Urological Pathology (ISUP) grade (1 vs. ≥2). Results: The distribution of γδ T cells in peripheral blood and prostate tissue showed wide variability and non-significant differences. A slightly higher percentage of δ2 T cells and a slightly lower percentage of δ1 T cells were found in peripheral blood of cancer patients. A non-significantly higher percentage of both Vδ1 and Vδ2 was expressed in cancer tissues, but a trend for lower distribution of δ1 and δ2 T cells was observed in ISUP grade ≥2. The "central memory" and "effector memory" were the most expressed T cells phenotype in peripheral blood and tissue samples. However no substantial differences in T cells subtypes distribution between cancer and healthy tissue were observed. Conclusions: No substantially different percentages of γδ T cells were found in peripheral blood and biopsy samples of healthy and PCa patients. However a non-significant trend for lower infiltrate in higher ISUP grade cancer tissue was observed, suggesting a possible role for the immunosurveillance of PCa.

Characterization of γδ T Cells in Intestinal Mucosa From Patients With Early-Onset or Long-Standing Inflammatory Bowel Disease and Their Correlation With Clinical Status.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is a complex chronic inflammatory disease of the human gut with no clear aetiology. Traditionally, dysregulated adaptive immune responses play an important role even though accumulating evidence suggests a role also for innate immunity. Because of the well-known plasticity of γδ T cells, we investigated their percentage occurrence, phenotypic features and effector functions in the intestinal mucosa of early-onset and long-standing IBD patients, as compared to healthy subjects. METHODS: Fresh biopsies from 30 Crohn's disease and ulcerative colitis patients were obtained and digested, and cells were analysed by flow cytometry. RESULTS: We found a reduced frequency of Vδ1 T cells in tissue from early and late IBD patients (2.24% and 1.95%, respectively, vs 5.44% in healthy tissue) but an increased frequency of Vδ2 T cells in the gut of late IBD patients (3.19% in late patients vs 1.5% in early patients and 1.65% in healthy tissue). The infiltrating Vδ2 T cells had predominant effector memory and terminally differentiated phenotypes and produced elevated levels of tumour necrosis factor-α [TNF-α] and interleukin-17 [IL-17]. The frequency of tissue Vδ2 T cells correlated with the extent of the inflammatory response and the severity of IBD. CONCLUSION: Our study shows that tissue Vδ1 T cells are decreased in IBD patients while Vδ2 T cells are increased in the gut of IBD patients and contribute to TNF-α production. Moreover, we identify an as yet unappreciated role of Vδ2 T cells in IL-17 production in the gut of long-standing IBD patients, suggesting that they also participate in the chronic inflammatory process.

Immunomodulation in Vascularized Composite Allotransplantation: What Is the Role for Adipose-Derived Stem Cells?

Hand and face transplants are becoming increasingly common, recording progressively more penile, uterus, abdominal wall, and allotransplantation cases reported worldwide. Despite current protocols allow long-term survival of the allografts, the ultimate goal of donor-specific tolerance has not been achieved yet. In fact, the harmful adverse effects related to the lifelong administration of immunosuppressive agents are the main drawbacks for vascularized composite allotransplantations. Research is very active in investigating alternative methods to induce greater tolerance while minimizing toxicity. Adipose-derived stem cells (ASCs) represent promising cell therapies for immunomodulation in preclinical and clinical settings. Their clinical appeal is due to their easy harvest in large quantities through a noninvasive and well-accepted approach; they may well promote donor-specific tolerance and potentially reduce immunosuppression. Several experimental studies exist, but lacking review articles reporting current evidence. This work proposes a literature review on the immunomodulatory role of ASCs in vascularized composite allotransplantations. In vitro and in vivo evidence will be summarized. The role that cell passaging and upstream progenitors-the so-called spheroid ASCs-may play in modulating the immune response will also be discussed. Finally, this article will summarize current knowledge on biodistribution, migration, and homing of injected stem cells. This review may well provide useful information for preclinical and clinical studies, aiming at a breakthrough for donor-specific tolerance.

γδ T Cells and Tumor Microenvironment: From Immunosurveillance to Tumor Evasion.

γδ T cells possess cytotoxic antitumor activity mediated by production of proinflammatory cytokines, direct cytotoxic activity, and regulation of the biological functions of other cell types. Hence, these features have prompted the development of therapeutic strategies in which γδ T cells agonists or ex vivo-expanded γδ T cells are administered to tumor patients. Several studies have shown that γδ T cells are an important component of tumor-infiltrating lymphocytes in patients affected by different types of cancer and a recent analysis of ~18,000 transcriptomes from 39 human tumors identified tumor-infiltrating γδ T cells as the most significant favorable cancer-wide prognostic signature. However, the complex and intricate interactions between tumor cells, tumor microenvironment (TME), and tumor-infiltrating immune cells results in a balance between tumor-promoting and tumor-controlling effects, and γδ T cells functions are often diverted or impaired by immunosuppressive signals originating from the TME. This review focuses on the dangerous liason between γδ T cells and tumoral microenvironment and raises the possibility that strategies capable to reduce the immunosuppressive environment and increase the cytotoxic ability of γδ T cells may be the key factor to improve their utilization in tumor immunotherapy.