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.

An analysis of the immunomodulatory properties of human spheroids from adipose-derived stem cells.

AIMS: Current methods to induce tolerance following allotransplantation or in autoimmunity carry significant morbidity, and research is very active in investigating alternative methods which could minimize toxicity. Spheroids from adipose stem cells (SASCs) are increasingly gaining interest, they hold a great proliferative and differentiating potential. An immunomodulatory effect has not been investigated on SASCs yet. In this study, we analysed the immunomodulatory properties of SASCs and compared them to ADSCs. MAIN METHODS: Adipose stem cells (SASCs and ADSCs) and peripheral blood mononuclear cells (PBMCs) were collected from healthy individuals. We analysed the cytokine production and proliferation of T cells co-cultured with adipose samples or conditioned medium. KEY FINDINGS: SASCs modulated cytokines production and proliferation of heterologous and autologous T cells. In the heterologous assays, we observed a reduction of IFNγ and IL-17 production and an increase of IL-9 in γδ T cells. The soluble factors present in SASCs sovranatants were also able to induce a slight reduction of IFNγ and an increase of IL-9, IL-10 and IL-17 while they could not modulate the proliferative ability of γδ T cells. In the autologous assays, we observed a reduction of the proliferative ability of T cells in co-culture at different ratios with SASCs. Analysis of the SASCs secretome showed an increased IL-5, IL-10, IL-4 and IL-13 production compared to the ADSCs one, demonstrating greater anti-inflammatory properties. SIGNIFICANCE: Our preliminary results support the idea that SASCs exert more pronounced biological immune modulation compared to the classical adherent ADSCs, especially in heterologous experimental settings.

κ-Carrageenan and PVA blends as bioinks to 3D print scaffolds for cartilage reconstruction.

3D printing of polymeric scaffolds and autologous stem cells is a promising tool for damaged facial cartilage reconstruction surgeries. To this end, suitable bioinks are needed to generate scaffolds with the required morphological and functional features. We formulated hydrogel bioinks using k-Carrageen (kC) and poly(vinyl alcohol) (PVA) in three different weight ratios. The kC gives the systems the ability to undergo rapid sol-to-gel transitions upon cooling from 60 °C and above to body temperature, while the PVA is used as rheology modifier and porogen. The latter is crosslinked after molding or printing by freeze-thaw cycling for 1 day (FT1) or 5 days (FT5). To select the most suitable formulation for 3D printing, the sol-to-gel transition and the physico-chemical, mechanical and morphological properties of obtained hydrogels were studied. Moreover, the absence of cytotoxic effects of the material on SASCs was assessed in both stemness-preserving or chondro-inductive media. Printing trials were performed to identify optimal process parameters and co-printing and post-printing seeding approaches of SASCs were evaluated. Cells were found to be viable after co-printing and also after the FT1 treatment. Viable adherent cells were also found in the FT5 system, where cells were plated after freezing and thawing treatment.

Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Injectable xyloglucan hydrogels incorporating spheroids of adipose stem cells for bone and cartilage regeneration.

Cartilage or bone regeneration approaches based on the direct injection of mesenchymal stem cells (MSCs) at the lesion site encounter several challenges, related to uncontrolled cell spreading and differentiation, reduced cell viability and poor engrafting. This work presents a simple and versatile strategy based on the synergic combination of in-situ forming hydrogels and spheroids of adipose stem cells (SASCs) with great potential for minimally invasive regenerative interventions aimed to threat bone and cartilage defects. Aqueous dispersions of partially degalactosylated xyloglucan (dXG) are mixed with SASCs derived from liposuction and either a chondroinductive or an osteoinductive medium. The dispersions rapidly set into hydrogels when temperature is brought to 37 °C. The physico-chemical and mechanical properties of the hydrogels are controlled by polymer concentration. The hydrogels, during 21 day incubation at 37 °C, undergo significant structural rearrangements that support cell proliferation and spreading. In formulations containing 1%w dXG cell viability increases up to 300% for SASCs-derived osteoblasts and up to 1000% for SASCs-derived chondrocytes if compared with control 2D cultures. The successful differentiation into the target cells is supported by the expression of lineage-specific genes. Cell-cell and cell-matrix interactions are also investigated. All formulations resulted injectable, and the incorporated cells are fully viable after injection.

Human Spheroids from Adipose-Derived Stem Cells Induce Calvarial Bone Production in a Xenogeneic Rabbit Model.

ABSTRACT: Calvarial defects can result from several causes. Tissue engineering hold the potential to restore native form and protective function. We have recently shown that stemness and differentiation ability of spheroids from adipose-derived stem cells (S-ASCs) promotes osteoblasts growth within Integra in a small vertebral lesion. In our study, we aimed to test osteogenic potential of S-ASCs in aiding regeneration of a calvarial defect. Groups containing Integra showed increased bone regeneration at the calvarial defect-Integra interface compared with the control group. In particular, S-ASC-derived osteoblasts group showed a superior calvarial remodeling than undifferentiated S-ASCs group. Clusters of ossification were observed in these both groups with enhanced microvasculature density and fibrosis. In conclusion, seeding of S-ASCs in dermal regeneration templates enhanced bone healing in a rabbit calvarial defect model. These findings could prompt the elective use of S-ASCs with enhanced multilineage differentiation potential for tissue engineering purposes.

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.

MicroRNAs in solid organ and vascularized composite allotransplantation: Potential biomarkers for diagnosis and therapeutic use.

Nowadays, solid organ transplantation (SOT) is an established treatment for patients with end-organ dysfunction, which dramatically improves the quality-of-life. Vascularized composite allotransplants (VCAs) including hand and face have been reported worldwide over the last 20 years. However, VCAs, differently to SOT, are life-enhancing instead of life-saving and are not routinely performed due to the risk of immune rejection and the adverse effects of immunosuppression. Over the past decade, although considerable improvements in short-term outcomes after allotransplantation have been registered, these results have not been translated into major progress in long-term allograft acceptance and patient survival. Recently active researches in the field of biomarker discovery have been conducted to develop individualized therapies for allograft recipients. MicroRNAs (miRNAs) are a small noncoding RNAs functioning as critical regulators of gene and protein expression by RNA interference. They have been connected in numerous biological processes and diseases. Due to their immunomodulatory functions, miRNAs have been amended as potential diagnostic and prognostic biomarker for the detection of rejection in allotransplantation. Due to their specific circulating expression profile, they could act as noninvasive predictive tools for rejection that may help clinicians in an early adjustment of the immunosuppression protocol during acute rejections episodes. Indeed, specific anti-sense oligonucleotides suppressing miRNAs expressed in rejection could reduce the rejection rate in allografts and decrease the use of immunosuppressants. We present a literature review of the immunomodulatory properties and characteristics of miRNAs. We will summarize the current knowledge on miRNAs as potential biomarkers for allograft rejection and possible application in allotransplantation monitoring. Finally, we will discuss the advances in preclinical miRNA-based therapies for immunosuppression.

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.

Adipose tissue, angiogenesis and angio-MIR under physiological and pathological conditions.

Angiogenesis is a crucial process for the maintenance of normal tissue physiology and it is involved in tissue remodeling and regeneration. This process is essential for adipose tissue maintenance. The adipose tissue is composed by different cell types including stromal vascular cells as well as adipose stem cells (ASCs). In particular, ASCs are multipotent somatic stem cells that are able to differentiate and secrete several growth factors; they are recently emerging as a new cell reservoir for novel therapies and strategies in many diseases. Several studies suggest that ASCs have peculiar properties and participate in different disease-related processes such as angiogenesis. Furthermore, pathological expansion of adipose tissue brings to hypoxia, a major condition of unhealthy angiogenesis. Recent evidences have shown that microRNAs (miRNAs) play a crucial role also on ASCs as they take part in stemness maintenance, proliferation, and differentiation. It has been suggested that some miRNAs (MIR126, MIR31, MIR221 MIR222, MIR17-92 cluster, MIR30, MIR100 and MIR486) are directly involved in the angiogenic process by controlling multiple genes involved in this pathway. With the present review, we aim at providing an updated summary of the importance of adipose tissue under physiological and pathological conditions and of its relationship with neovascularization process. In particular, we report an overview of the most important miRNAs involved in angiogenesis focusing on ASCs. Hopefully the data presented will bring benefit in developing new therapeutic strategies.