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In the last 50 years information on the fish immune system has increased importantly, particularly that on species of marked commercial interest (i.e., salmonids, cods, catfish, sea breams), that occupy a key position in the vertebrate phylogenetical tree (i.e., Agnatha, Chondrichtyes, lungfish) or represent consolidated experimental models, such as zebrafish or medaka. However, most obtained information was based on genetic sequence analysis with little or no information on the cellular basis of the immune responses. Although jawed fish contain a thymus and lympho-hematopoietic organs equivalents to mammalian bone marrow, few studies have accounted for the presumptive relationships between the organization of these cell microenvironments and the known immune capabilities of the fish immune system. In the current review, we analyze this topic providing information on: (1) The origins of T and B lymphopoiesis in Agnatha and jawed fish; (2) the remarkable organization of the thymus of teleost fish; (3) the occurrence of numerous, apparently unrelated organs housing lympho-hematopoietic progenitors and, presumably, B lymphopoiesis; (4) the existence of fish immunological memory in the absence of germinal centers.

MSCs products as well as their derived extracellular vesicles, are currently being explored as advanced biologics in cell-based therapies with high expectations for their clinical use in the next few years. In recent years, various strategies designed for improving the therapeutic potential of mesenchymal stromal cells (MSCs), including pre-conditioning for enhanced cytokine production, improved cell homing and strengthening of immunomodulatory properties, have been developed but the manufacture and handling of these cells for their use as advanced therapy medicinal products (ATMPs) remains insufficiently studied, and available data are mainly related to non-industrial processes. In the present article, we will review this topic, analyzing current information on the specific regulations, the selection of living donors as well as MSCs from different sources (bone marrow, adipose tissue, umbilical cord, etc.), in-process quality controls for ensuring cell efficiency and safety during all stages of the manual and automatic (bioreactors) manufacturing process, including cryopreservation, the use of cell banks, handling medicines, transport systems of ATMPs, among other related aspects, according to European and US legislation. Our aim is to provide a guide for a better, homogeneous manufacturing of therapeutic cellular products with special reference to MSCs.

Both EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3 −/− mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2 −/− T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3 −/− lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions.

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.

Eph receptors and their ligands, Ephrins, are involved in the thymocyte-thymic epithelial cell (TEC) interactions, key for the functional maturation of both thymocytes and thymic epithelium. Several years ago, we reported that the lack of EphA4, a Eph of the subfamily A, coursed with reduced proportions of double positive (DP) thymocytes apparently due to an altered thymic epithelial stroma [Munoz et al. in J Immunol 177:804–813, 2006]. In the present study, we reevaluate the lymphoid, epithelial, and extracellular matrix (ECM) phenotype of EphA4 −/− mice grouped into three categories with respect to their proportions of DP thymocytes. Our results demonstrate a profound hypocellularity, specific alterations of T cell differentiation that affected not only DP thymocytes, but also double negative and single positive T cell subsets, as well as the proportions of positively and negatively selected thymocytes. In correlation, thymic histological organization changed markedly, especially in the cortex, as well as the proportions of both Ly51 + UEA-1 − cortical TECs and Ly51 − UEA-1 + medullary TECs. The alterations observed in the expression of ECM components (Fibronectin, Laminin, Collagen IV), integrin receptors (VLA-4, VLA-6), chemokines (CXCL12, CCL25, CCL21) and their receptors (CXCR4, CCR7, CCR9) and in vitro transwell assays on the capacity of migration of WT and mutant thymocytes suggest that the lack of EphA4 alters T-cell differentiation by presumably affecting cell adhesion between TECs and T-TEC interactions rather than by thymocyte migration.

Background Mesenchymal stromal cells (MSC) are components of connective tissues and, in vitro, cell entities characterized by cell adhesion and immunophenotyping, although specific markers for their identification are lacking. Currently, MSC derived from either human bone marrow (BM-MSC) or adipose tissue (Ad-MSC) are considered the main sources of MSC for cell therapy. Eph receptors and their ligands, Ephrins, are molecules involved in cell adhesion and migration in several tissues and organs. In the current study, we analyze the pattern of Eph/Ephrin expression in MSC and evaluate the effects of blockade and stimulation of these receptor/ligand pairs on their biology. Methods Eph/Ephrin expression was analyzed in both BM-MSC and Ad-MSC by qRT-PCR. Then, we supplied BM-MSC cultures with either blocking or activating compounds to evaluate their effects on MSC proliferation, survival, and cell cycle by FACS. Changes in cytoskeleton and integrin α5β1 expression were studied in stimulated BM-MSC by immunofluorescence microscopy and FACS, respectively. Results Higher numbers of Eph/Ephrin transcripts occurred in BM-MSC than in Ad-MSC. In addition, the blocking of Eph/Ephrin signaling correlated with decreased numbers of BM-MSC due to increased proportions of apoptotic cells in the cultures but without variations in the cycling cells. Unexpectedly, activation of Eph/Ephrin signaling by clustered Eph/Ephrin fusion proteins also resulted in increased proportions of apoptotic MSC. In this case, MSC underwent important morphological changes, associated with altered cytoskeleton and integrin α5β1 expression, which did not occur under the blocking conditions. Conclusions Taken together, these results suggest that Eph/Ephrin activation affects cell survival through alterations in cell attachment to culture plates, affecting the biology of BM-MSC.

MSCs exhibit regenerative, anti-inflammatory and immunomodulatory properties due to the large amount of cytokines, chemokines and growth factors they secrete. MSCs have been extensively evaluated in clinical trials, however, in some cases their therapeutic effects are variable. Therefore, strategies to improve their therapeutic potential, such as preconditioning with proinflammatory factors, have been proposed. Several priming approaches have provided non-conclusive results, and the duration of priming effects on MSC properties or their response to a second inflammatory stimulus have not been fully addressed. We have investigated the impact of triple cytokine priming in MSCs on their characterization and viability, their transcriptomic profile, the functionality of innate and acquired immune cells, as well as the maintenance of the response to priming over time, their subsequent responsiveness to a second inflammatory stimulus. Priming MSCs with proinflammatory cytokines (CK-MSCs) do not modify the differentiation capacity of MSCs, nor their immunophenotype and viability. Moreover, cytokine priming enhances the anti-inflammatory and immunomodulatory properties of MSCs against NK and dendritic cells, while maintaining the same T cell immunomodulatory capacity as unstimulated MSCs. Thus, they decrease T-lymphocytes and NK cell proliferation, inhibit the differentiation and allostimulatory capacity of dendritic cells and promote the differentiation of monocytes with an immunosuppressive profile. In addition, we have shown for the first time that proinflammatory priming reduces the variability between different donors and MSC origins. Finally, the effect on CK-MSC is maintained over time and even after a secondary inflammatory stimulus. Cytokine-priming improves the therapeutic potential of MSCs and reduces inter-donor variability.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.

Mesenchymal stem/stromal cells (MSCs) are one of the most widely used cell types in advanced therapies due to their therapeutic potential in the regulation of tissue repair and homeostasis, and immune modulation. However, their use in cancer therapy is controversial: they can inhibit cancer cell proliferation, but also potentially promote tumour growth by supporting angiogenesis, modulation of the immune milieu and increasing cancer stem cell invasiveness. This opposite behaviour highlights the need for careful and nuanced use of MSCs in cancer treatment. To optimize their anti-cancer effects, diverse strategies have bioengineered MSCs to enhance their tumour targeting and therapeutic properties or to deliver anti-cancer drugs. In this review, we highlight the advanced uses of MSCs in cancer therapy, particularly as carriers of targeted treatments due to their natural tumour-homing capabilities. We also discuss the potential of MSC-derived extracellular vesicles to improve the efficiency of drug or molecule delivery to cancer cells. Ongoing clinical trials are evaluating the therapeutic potential of these cells and setting the stage for future advances in MSC-based cancer treatment. It is critical to identify the broad and potent applications of bioengineered MSCs in solid tumour targeting and anti-cancer agent delivery to position them as effective therapeutics in the evolving field of cancer therapy.