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Abstract Treatment of susceptible rats with dopaminergic agonists that reduce prolactin release decreases both severity and duration of clinical signs of experimental allergic encephalomyelitis (EAE). To assess to what extent the presence of an ectopic pituitary (that produces an increase in plasma prolactin levels mainly derived from the ectopic gland) affects EAE, 39 male Lewis rats were submitted to pituitary grafting from littermate donors. Another group of 38 rats was sham-operated by implanting a muscle fragment similar in size to the pituitary graft. All rats received subcutaneous (s.c.) injections of complete Freund's adjuvant (CFA) plus spinal cord homogenate (SCH) and were monitored daily for clinical signs of EAE. Animals were killed by decapitation on days 1, 4, 7, 11 or 15 after immunization and plasma was collected for prolactin RIA. In a second experiment, 48 rats were immunized by s.c. injection of a mixture of SCH and CFA, and then received daily s.c. injections of bromocriptine (1 mg/kg) or saline. Groups of 8 animals were killed on days 8, 11 or 15 after immunization and plasma prolactin was measured. Only sham-operated rats exhibited clinical signs of the disease when assessed on day 15 after immunization. A progressive decrease in plasma prolactin levels was observed in pituitary-grafted rats, attaining a minimum 15 days after immunization, whereas plasma prolactin levels were increased during the course of the disease in sham-operated rats. Plasma prolactin levels were higher in pituitary-grafted rats than in sham-operated rats 1 day after immunization, but lower on days 7, 11 and 15 after immunogen injection. Further supporting a correlation of suppressed prolactin levels with absence of clinical signs of EAE, rats that were administered the dopaminergic agonist bromocriptine showed very low plasma prolactin levels and did not exhibit any clinical sign of EAE. These results indicate that low circulating prolactin levels coincide with absence of clinical signs of EAE in Lewis rats.

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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., (© 2024. The Author(s).)

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gil-Chinchilla, Bueno, Martínez, Ferrández-Múrtula, García-Hernández, Blanquer, Molina-Molina, Zapata, Sackstein, Moraleda and García-Bernal.)

In the present study, we review the structure and function of fish spleen with special emphasis on its condition in Elasmobranchs, Teleosts and Lungfish. Apart from the amount of splenic lymphoid tissue, the histological organization of the organ ensures the existence of areas involved in antigen trapping, the ellipsoids, and exhibit numerous melano-macrophages which appear isolated or forming the so-called melano-macrophage centres. An extensive discussion on the functional significance of these centres conclude that they are mere accumulations of macrophages consequence of tissue homeostasis rather than primitive germinal centres, as proposed by some authors., (Copyright © 2023. Published by Elsevier Ltd.)

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., (© 2022. The Author(s).)

ICAP-1 regulates β1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8 + cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8 + thymocytes of Runx3, a transcription factor required for CD8 + thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1 -/- spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3 + - and CD19 + -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8 + thymocytes and in the control of marginal zone B-cell numbers., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

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., Competing Interests: FS-G has received honoraria and/or research support from Novartis, Kite/Gilead, Celgene/BMS, Pfizer, Takeda and Roche. DG-O is a member of the Advisory Board of Tigenix SAU and received fees from Takeda. ML-P has received honoraria from Novartis, Kite/Gilead and Celgene/BMS. JM has received research support from Roche, Pfizer, Jazz Pharma, Sandoz-Novartis, Gilead, Celgene, and Takeda not related to this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fernández-Santos, Garcia-Arranz, Andreu, García-Hernández, López-Parra, Villarón, Sepúlveda, Fernández-Avilés, García-Olmo, Prosper, Sánchez-Guijo, Moraleda and Zapata.)

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.

During the acute phase of Chagas disease, Trypanosoma cruzi circulation through the bloodstream leads to high tissue parasitism in the host. In primary lymphoid organs, progenitor cell reduction paralleled transient immunosuppression. Herein we showed that acute oral infection in mice promotes diffuse parasitism in bone marrow cells at 14 and 21 days post-infection (dpi), with perivascular regions, intravascular regions, and regions near the bone being target sites of parasite replication. Phenotypic analysis of hematopoietic differentiation in the bone marrow of infected mice showed that the cell number in the tissue is decreased (lineage-negative and lineage-positive cells). Interestingly, analysis of hematopoietic branching points showed that hematopoietic stem and progenitor cells (HSPCs) were significantly increased at 14 dpi. In addition, the pool of progenitors with stem plasticity (HSC-MPP3), as well as multipotent progenitors (MPPs) such as MPP4, also showed this pattern of increase. In contrast, subsequent progenitors that arise from MPPs, such as common lymphoid progenitors (CLPs), lymphoid-primed MPPs (LMPPs), and myeloid progenitors, were not enhanced; conversely, all presented numeric decline. Annexin V staining revealed that cell death increase in the initial hematopoietic branching point probably is not linked to CLPs and that myeloid progenitors decreased at 14 and 21 dpi. In parallel, our investigation provided clues that myeloid progenitor decrease could be associated with an atypical expression of Sca-1 in this population leading to a remarkable increase on LSK-like cells at 14 dpi within the HSPC compartment. Finally, these results led us to investigate HSPC presence in the spleen as a phenomenon triggered during emergency hematopoiesis due to mobilization or expansion of these cells in extramedullary sites. Splenocyte analysis showed a progressive increase in HSPCs between 14 and 21 dpi. Altogether, our study shows that the bone marrow is a target tissue in T. cruzi orally infected mice, leading to a hematopoietic disturbance with LSK-like cell bias accounting on HSPCs possibly affecting myeloid progenitor numbers. The LMPP and CLP reduction converges with defective thymocyte development. Lastly, it is tempting to speculate that the extramedullary hematopoiesis seen in the spleen is a mechanism involved in the hematological maintenance reported during the acute phase of oral T. cruzi infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marins-Dos-Santos, Ayres-Silva, Antunes, Moreira, Pelajo-Machado, Alfaro, Zapata, Bonomo, Savino, de Meis and Farias-de-Oliveira.)

Objective: Certain diseases such as obesity and cancer can cause impaired wound healing. Adipose tissue derived stem cells (ASCs) are a novel field of research. Many studies have evidenced their high degree of safety and potential for wound repair due to their immunomodulatory and tissue-regeneration properties. The purpose of this study is to determine the impact of obesity and cancer on the therapeutic potential of ASCs., Materials and Methods: We isolated and characterized the phenotype, differentiation capacities, secretome, and in vitro migration capacities of ASCs. Furthermore, we analyze their capacity of in vitro migration associated with the plasma of the different patients., Results: We observed that ASCs isolated from obese and cancer patients have the same phenotype, cell proliferation, and migration capacities as ASCs derived from healthy donors. However, they do not have the same differentiation potential and exhibit distinct profiles of both pro-inflammatory and regulatory secreted cytokines, which, together with the signals received from the bloodstream, could account for the impaired healing in patients with these diseases., Conclusions: We consider the ASCs from patients with either obesity or cancer are slightly altered, and this may be the cause of worse wound healing in these patients., (Copyright: © 2022 Permanyer.)

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 + MHCII hi 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 TCRαβ hi thymocytes and peripheral T lymphocytes., (© 2021. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 García-Bernal, García-Arranz, Yáñez, Hervás-Salcedo, Cortés, Fernández-García, Hernando-Rodríguez, Quintana-Bustamante, Bueren, García-Olmo, Moraleda, Segovia and Zapata.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo . Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases., Competing Interests: According to National Institutes of Health policies and procedures, the Brigham and Women’s Hospital has assigned intellectual property rights regarding cell surface glycan engineering to RS, and RS has licensed portions of this technology to an entity he has founded (Warrior Therapeutics, LLC), to BioTechne, Inc., and to Mesoblast LTD. RS’s ownership interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policy. DG-O is member of the Advisory Board of TiGenix S.A.U. MG-A and DG-O have applied for a patent related to Cx401 and Cx601 titled “Identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue” (WO 2006/057649). DG-O and MG-A are shareholders of Biosurgery, an educational company providing services to Takeda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 García-Bernal, García-Arranz, García-Guillén, García-Hernández, Blanquer, García-Olmo, Sackstein, Moraleda and Zapata.)

Intimate interactions between thymic epithelial cells (TECs) and thymocytes (T) have been repeatedly reported as essential for performing intrathymic T-cell education. Nevertheless, it has been described that animals exhibiting defects in these interactions were capable of a proper positive and negative T-cell selection. In the current review, we first examined distinct types of TECs and their possible role in the immune surveillance. However, EphB-deficient thymi that exhibit profound thymic epithelial (TE) alterations do not exhibit important immunological defects. Eph and their ligands, the ephrins, are implicated in cell attachment/detachment and govern, therefore, TEC-T interactions. On this basis, we hypothesized that a few normal TE areas could be enough for a proper phenotypical and functional maturation of T lymphocytes. Then, we evaluated in vivo how many TECs would be necessary for supporting a normal T-cell differentiation, concluding that a significantly low number of TEC are still capable of supporting normal T lymphocyte maturation, whereas with fewer numbers, T-cell maturation is not possible.

Background: The epithelial microenvironment is involved in thymus aging, but the possible role of EphB receptors that govern the thymic epithelium development has not been investigated. Herein, we study the changes undergone by the thymus of EphB-deficient mice throughout their life., Results: Immune alterations occurring throughout life were more severe in mutant than in wild-type (WT) mice. Mutant thymuses exhibit lower cellularity than WT ones, as well as lower proportions of early thymic progenitors cells and double-positive (CD4 + CD8 + ) thymocytes, but higher of double-negative (CD4 - CD8 - ) and single-positive (CD4 + CD8 - , CD4 - CD8 + ) cells. Throughout life, CD4 + naïve cells decreased particularly in mutant mice. In correlation, memory T cells, largely CD8 + cells, increased. Aged thymic epithelium undergoes changes including appearance of big epithelial free areas, decrease of K8 + K5 - areas, which, however, contain higher proportions of Ly51 + UEA1 - cortical epithelial cells, in correlation with reduced Aire + medullary epithelial cells. Also, aged thymuses particularly those derived from mutant mice exhibited increased collagen IV, fat-storing cells, and connective cells., Conclusions: The absence of EphB accelerates the alterations undergone throughout life by both thymic epithelium and thymocytes, and the proportions of peripheral naïve and memory T cells, all of which are hallmarks of immune aging., (© 2020 Wiley Periodicals LLC.)

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients., Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 10 6 (IQR 0.50 × 10 6 ) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters., Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement., Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects., Funding: None., Competing Interests: Dr. Sanchez-Guijo reports grants and personal fees from Novartis, personal fees from BMS, Pfizer, Incyte, Gilead, Roche and Amgen, outside the submitted work; Dr. López-Parra reports personal fees from Gilead, Novartis, BMS and Janssen, outside the submitted work. Dr. JL del Pozo reports grants from Novartis and personal fees from Novartis, Pfizer, Gilead and Roche, outside the submitted work; Dr. Moraleda reports personal fees and other from Gilead, grants and personal fees from Jazz Pharma, and personal fees from Novartis and from Sandoz, outside the submitted work. Dr. García-Olmo has received personal fees from Takeda, outside the submitted work; Dr. Prosper reports personal fees from Oryzon Genomics, and from Janssen, outside the submitted work; Dr. García-Arranz and Dr. García-Olmo have applied for two patents related with this study entitled “Identification and isolation of multipotent cells from nonosteochondral mesenchymal tissue” (WO 2006/057649) and “Use of adipose tissue-derived stromal stem cells in treating fistula” (WO 2006/136244), and both are shareholders of Biosurgery, an educational company providing services to Takeda. All other authors declare no conflict of interest., (© 2020 The Authors.)

Mesenchymal stromal/stem cells (MSCs) have emerged as important therapeutic agents, owing to their easy isolation and culture, and their remarkable immunomodulatory and anti-inflammatory properties. However, MSCs constitute a heterogeneous cell population which does not express specific cell markers and has important problems for in vivo homing, and factors regulating their survival, proliferation, and differentiation are largely unknown. Accordingly, in the present article, we review the current evidence on the relationships between Eph kinase receptors, their ephrin ligands, and MSCs. These molecules are involved in the adult homeostasis of numerous tissues, and we and other authors have demonstrated their expression in human and murine MSCs derived from both bone marrow and adipose tissue, as well as their involvement in the MSC biology. We extend these studies providing new results on the effects of Eph/ephrins in the differentiation and immunomodulatory properties of MSCs., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The mechanisms that determine the commitment of thymic epithelial precursors to the two major thymic epithelial cell lineages, cTECs and mTECs, remain unknown. Here we show that FoxN1 nu mutation, which abolishes thymic epithelium differentiation, results in the formation of a tubular branched structure according to a typical branching morphogenesis and tubulogenesis developmental pattern. In the presence of FoxN1, in alymphoid NSG and fetal Ikaros-/- thymi, there is no lumen formation and only partial apical differentiation. This initiates cortex-medulla differentiation inducing expression of medullary genes in the apically differentiating cells and of cortical genes in the non-apically differentiating cells, which will definitely differentiate in wt and postnatal Ikaros-/- mice. Therefore, the thymus development is based on a branching morphogenesis and tubulogenesis developmental pattern: FoxN1 expression in the thymic primordium inhibits tubulogenesis and induces the expression of genes involved in TEC differentiation, which culminates with the expression of functional cell markers, i.e., MHCII, CD80, Aire in both postnatal Ikaros-/- and WT thymi after arrival of lymphoid progenitor cells.

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.

In the present study, the relevance of EphB2 and EphB3 tyrosine kinase receptors for the maturation of medullary thymic epithelial cells (TECs) is analyzed. The absence of both molecules, but particularly that of EphB2, courses with altered maturation of medullary Cld3,4 hi SSEA1 + epithelial progenitor cells, mature medulla epithelial cells, defined by the expression of specific cell markers, including UEA1, MHCII, CD40, CD80, and AIRE, and reduced expansion of medullary islets. In vivo assays demonstrate that these changes are a consequence of the absence of EphBs in both TECs and thymocytes. On the other hand, the changes, that remains in the adult thymus, correlated well with reduced proportions of E15.5 Vγ5 + RANKL + cells in EphB-deficient thymi that could result in decreased stimulation of RANK + medullary TECs to mature, a fact that was confirmed by recovering of proportions of both CD40 hi CD80 + and MHCII hi UEA1 + mature medullary TECs of mutant E14.5 alymphoid thymic lobes by agonist anti-RANK antibody treatment. Accordingly, the effects of EphB deficiency on medullary TECs maturation are recovered by RANK stimulation.

EphB and their ligands ephrin-B are an important family of protein tyrosine kinase receptors involved in thymocyte-thymic epithelial cell interactions known to be key for the maturation of both thymic cell components. In the present study, we have analyzed the maturation of cortical thymic epithelium in EphB-deficient thymuses evaluating the relative relevance of EphB2 and EphB3 in the process. Results support a relationship between the epithelial hypocellularity of mutant thymuses and altered development of thymocytes, lower proportions of cycling thymic epithelial cells and increased epithelial cell apoptosis. Together, these factors induce delayed development of mutant cortical TECs, defined by the expression of different cell markers, i.e. Ly51, CD205, MHCII, CD40 and β5t. Furthermore, although both EphB2 and EphB3 are necessary for cortical thymic epithelial maturation, the relevance of EphB3 is greater since EphB3-/- thymic cortex exhibits a more severe phenotype than that of EphB2-deficient thymuses.

Epithelial-free areas, present in both thymic cortex and medulla, have been studied in WT and EphB-deficient mice that have important alterations in the development of thymic epithelium due to the lack of proper thymocyte-thymic epithelial cell interactions. In both WT and mutant thymuses, the number and size of epithelial-free areas are significantly larger in the medulla than in the cortex. The two parameters show a reverse correlation: low numbers of these areas course with large epithelial-free areas and vice versa. However, their structure and cell content are similar in mutant and WT thymuses. Cortical epithelial-free areas just contain DP thymocytes, while the medullary ones consist of SP cells, blood vessels, mesenchyme-derived ER-TR7 + cells and components of the extracellular matrix (i.e., collagen IV, fibronectin, laminin). Other components, such as desmin, αSMA, PDGFRβ and Ng2, frequently associated with blood vessel walls, also appear. Vimentin, although present in medullary epithelial-free areas, does not co-express with epithelial cells. Other markers related to epithelial-mesenchymal transitions, such as Snail, Slug or FSP1, are not expressed. These results suggest that alterations in the cell interactions between distinct thymic cell components that induce both increased proportions of apoptotic thymic epithelial cells and altered behavior of the mesenchyme associated with the medullary vasculature could explain the appearance of these areas and their differences in the cortex and medulla.

Background Aims: The immunomodulatory properties of mesenchymal stromal cells (MSCs), together with their tissue regenerative potential, make them interesting candidates for clinical application., Methods: In the current study, we analyzed the in vitro immunomodulatory effects of MSCs derived from bone marrow (BM-MSCs) and from adipose tissue (AT-MSCs) obtained from the same donor on both innate and acquired immunity cells. BM-MSCs and AT-MSCs were expanded to fourth or fifth passage and co-cultured with T cells, monocytes or natural killer (NK) cells isolated from human peripheral blood and stimulated in vitro. The possible differing impact of MSCs obtained from distinct sources on phenotype, cell proliferation and differentiation, cytokine production and function of these immune cells was comparatively analyzed., Results: BM-MSCs and AT-MSCs induced a similar decrease in NK-cell proliferation, cytokine secretion and expression of both activating receptors and cytotoxic molecules. However, only BM-MSCs significantly reduced NK-cell cytotoxic activity, although both MSC populations showed the same susceptibility to NK-cell-mediated lysis. AT-MSCs were more potent in inhibiting dendritic-cell (DC) differentiation than BM-MSC, but both MSC populations similarly reduced the ability of DCs to induce CD4(+) T-cell proliferation and cytokine production. BM-MSCs and AT-MSCs induced a similar decrease in T-cell proliferation and production of inflammatory cytokines after activation., Conclusions: AT-MSCs and BM-MSCs from the same donor had similar immunomodulatory capacity on both innate and acquired immunity cells. Thus, other variables, such as accessibility of samples or the frequency of MSCs in the tissue should be considered to select the source of MSC for cell therapy., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Thymus development is a complex process in which cell-to-cell interactions between thymocytes and thymic epithelial cells (TECs) are essential to allow a proper maturation of both thymic cell components. Although signals that control thymocyte development are well known, mechanisms governing TEC maturation are poorly understood, especially those that regulate the maturation of immature TEC populations during early fetal thymus development. In this study, we show that EphB2-deficient, EphB2LacZ and EphB3-deficient fetal thymuses present a lower number of cells and delayed maturation of DN cell subsets compared to WT values. Moreover, deficits in the production of chemokines, known to be involved in the lymphoid seeding into the thymus, contribute in decreased proportions of intrathymic T cell progenitors (PIRA/B(+)) in the mutant thymuses from early stages of development. These features correlate with increased proportions of MTS20(+) cells but fewer MTS20(-) cells from E13.5 onward in the deficient thymuses, suggesting a delayed development of the first epithelial cells. In addition, in vitro the lack of thymocytes or the blockade of Eph/ephrin-B-mediated cell-to-cell interactions between either thymocytes-TECs or TECs-TECs in E13.5 fetal thymic lobes coursed with increased proportions of MTS20(+) TECs. This confirms, for the first time, that the presence of CD45(+) cells, corresponding at these stages to DN1 and DN2 cells, and Eph/ephrin-B-mediated heterotypic or homotypic cell interactions between thymocytes and TECs, or between TECs and themselves, contribute to the early maturation of MTS20(+) TECs.

A role of endothelial cells in the survival of CLL cells during extravasation is presently unknown. Herein we show that CLL cells but not normal B cells can receive apoptotic signals through physical contact with TNF-α activated endothelium impairing survival in transendothelial migration (TEM) assays. In addition, the CLL cells of patients having lymphadenopathy (LApos) show a survival advantage during TEM that can be linked to increased expression of α4 and αL integrin chains. Within this context, ephrinA4 expressed on the surface of CLL cells sequestrates integrins and inactivates them resulting in reduced adhesion and inhibition of apoptotic/survival signals through them. In agreement, ephrinA4 silencing resulted in increased survival of CLL cells of LApos patients but not LA neg patients. Similarly was observed when a soluble ephrinA4 isoform was added to TEM assays strongly suggesting that accumulation of this isoform in the serum of LApos patients could contribute to CLL cells dissemination and survival in vivo. In supporting, CLL lymphadenopathies showed a preferential accumulation of apoptotic CLL cells around high endothelial venules lacking ephrinA4. Moreover, soluble ephrinA4 isolated from sera of patients increased the number and viability of CLL cells recovered from the lymph nodes of adoptively transferred mice. Finally, we present evidence suggesting that soluble ephrinA4 mediated survival during TEM could enhance a transcellular TEM route of the CLL cells. Together these findings point to an important role of ephrinA4 in the nodal dissemination of CLL cells governing extravasation and survival., Competing Interests: The authors declare no conflict of interest.

Mesenchymal stem cells (MSCs) are key components of the bone marrow microenvironment which contribute to the maintenance of the hematopoietic stem cell niche and exert immunoregulatory functions in innate and adaptive immunity. We analyze the immunobiology of MSCs derived from acute lymphoblastic leukemia (ALL) patients and their impact on NK cell function. In contrast to the inhibitory effects on the immune response exerted by MSCs from healthy donors (Healthy-MSCs), we demonstrate that MSCs derived from low/intermediate risk ALL patients at diagnosis (ALL-MSCs) promote an efficient NK cell response including cytokine production, phenotypic activation and most importantly, cytotoxicity. Longitudinal studies indicate that these immunostimulatory effects of ALL-MSCs are progressively attenuated. Healthy-MSCs adopt ALL-MSC-like immunomodulatory features when exposed to leukemia cells, acquiring the ability to stimulate NK cell antitumor function. The mechanisms underlying to these functional changes of ALL-MSCs include reduced production of soluble inhibitory factors, differential expression of costimulatory and coinhibitory molecules, increased expression of specific TLRs and Notch pathway activation. Collectively our findings indicate that, in response to leukemia cells, ALL-MSCs could mediate a host beneficial immunomodulatory effect by stimulating the antitumor innate immune response., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Numerous studies emphasize the relevance of thymocyte-thymic epithelial cell (TECs) interactions for the functional maturation of intrathymic T lymphocytes. The tyrosine kinase receptors, Ephs (erythropoietin-producing hepatocyte kinases) and their ligands, ephrins (Eph receptor interaction proteins), are molecules known to be involved in the regulation of numerous biological systems in which cell-to-cell interactions are particularly relevant. In the last years, we and other authors have demonstrated the importance of these molecules in the thymic functions and the T-cell development. In the present report, we review data on the effects of Ephs and ephrins in the functional maturation of both thymic epithelial microenvironment and thymocyte maturation as well as on their role in the lymphoid progenitor recruitment into the thymus.

The thymus represents an epithelial microenvironment specialized in the generation of T-cells. The mechanisms or signals that determine the initial differentiation of the two well distinguished histological compartments of the thymus, cortex and medulla, remain unknown. Here, we report a three-dimensional analysis of the distribution of some established thymic epithelial markers in relation to thymic anatomical development during the first steps of thymus organogenesis. In the thymic primordium, initial lumen is lined by claudin (Cld)3/4+K5+ cells, after thymus growth and lobulation they form a continuous branched structure that increases its length and branching degree. Within it, the presence of luminal structures can be distinguished, even at E13.5. The medullary marker mouse thymic stroma 10 (MTS10) is upregulated in these Cld3/4+ lumen forming cells in a proximal-distal sequence. This structural organisation is histologically similar to that described in other epithelial organs undergoing a branching morphogenesis process. These results indicate that the thymic medulla can be evidenced as a continuous branched structure from early stages and suggest a thymic developmental program based on or containing elements of a branching morphogenesis program modified by the presence of lymphoid cells, in which medullary epithelial cell commitment is initially determined by lumen formation.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors regulating differentiation and proliferation in numerous systems including the immune system. Previously, we described that the BMP signaling pathway is functional in human monocyte-derived dendritic cells (MoDCs), which were found to express both the specific receptors and the Smad proteins required for signal transduction. In this study, we provide evidence that human MoDCs produce BMP-4 and that this production is increased over the maturation process as is BMP signal transduction. When DCs are matured in the presence of an inhibitor of the BMP pathway, the expression of the maturation markers PD-L1 and PD-L2 is reduced, while cytokine production is not affected. As a result, these mature DCs present an augmented ability to stimulate both T cells and NK cells. Eventually, the inhibition of BMP signaling during maturation causes a reduced expression of IRF-1, a transcription factor that positively regulates the expression of PD-L1 and PD-L2. The present study indicates that the BMP signaling pathway regulates PD-L1 and PD-L2 expression in human MoDCs during the maturation process, probably through the IRF-1 transcription factor, and also points out that the manipulation of BMP signaling might considerably improve the immunogenicity of MoDCs used in immunotherapy., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe the structure of the lympho-granulocytic tissue associated with the wall of the spiral valve of the African lungfish Protopterus annectens. The study was performed under freshwater conditions and after 6 months of aestivation. The lympho-granulocytic tissue consists of nodes surrounded by reticular tissue. The nodes are formed by an outer and an inner component separated by a thin collagenous layer. The outer component is a reticular-like tissue that contains two types of granulocytes, developing and mature plasma cells and melanomacrophage centres (MMCs). The inner component, the parenchyma, contains a meshwork of trabeculae and vascular sinusoids and shows dark and pale areas. The dark areas contain diffuse lymphoid tissue, with a large number of mitoses and plasma cell clusters. The pale areas contain a small number of macrophages and lymphocytes. Macrophages and sinus endothelial cells are filled with haemosiderin granules and appear to form part of the reticuloendothelial system of the lungfish. The reticular tissue houses granulocytes, plasma cells and MMCs and might serve for the housing and maturation of cells of the white series. After aestivation, the nodes undergo lymphocyte depletion, the suppression of mitosis, granulocyte invasion and the occurrence of cell death. By contrast, few histological changes occur in the reticular tissue. Whereas the nodes appear to be involved in lymphocyte proliferation and plasma cell maturation, the function of the reticular tissue remains obscure.

The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated.

The signals that determine the survival/death of the thymic epithelial cells (TECs) component during embryonic development of the thymus are largely unknown. In this study, we combine different in vivo and in vitro experimental approaches to define the role played by the tyrosine kinase receptors EphB2 and EphB3 and their ligands, ephrinsB, in the survival of embryonic and newborn (NB) TECs. Our results conclude that EphB2 and EphB3 are involved in the control of TEC survival and that the absence of these molecules causes increased apoptotic TEC proportions that result in decreased numbers of thymic cells and a smaller-sized gland. Furthermore, in vitro studies using either EphB2-Fc or ephrinB1-Fc fusion proteins demonstrate that the blockade of Eph/ephrinB signalling increases TEC apoptosis, whereas its activation rescues TECs from cell death. In these assays, both heterotypic thymocyte-TEC and homotypic TEC-TEC interactions are important for Eph/ephrinB-mediated TEC survival.

Cell migration is a crucial event for normal T-cell development, and various ligand/receptor pairs have been implicated. Most of them, including chemokines and extracellular matrix proteins, have attractant properties on thymocytes. We discuss herein two further groups of ligand/receptor pairs, semaphorins/neuropilins and ephs/ephrins, which are constitutively expressed by thymocytes and thymic microenvironmental cells. Evidence shows that the corresponding interactions are relevant for developing T-cell migration, including the entry of bone marrow progenitor cells, migration of CD4/CD8-defined thymocyte subpopulations triggered by chemokines and/or extracellular matrix proteins, and thymocyte export. Conceptually, the data summarized here show that thymocyte migration results from a complex network of molecular interactions, which generate not only attraction, but also repulsion of migrating T-cell precursors.

The bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34(+)CD1a(-)BMPRIA(+) precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34(+) cells mostly differentiate into functional CD56(+) natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15-responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.

From the discovery of the first line of human embryonic stem cells, thousands of studies have been published concerning adult stem cells and their possible alleged therapeutic potential. However, very little real progress has been made in the application of cell therapy to patients. We can conclude that there remains a great deal for us to learn about the biology of stem cells, and especially, the mechanisms that regulate their differentiation and use under conditions of biosafety. In this chapter, we are going to review some of the mechanisms that seem to control the biology of stem cells, in particular the microenvironments, also called niches, where they house and which exert a strong influence over them. The regulation, survival, proliferation and differentiation of stem cells is ultimately determined by a combination of factors intrinsic to the stem cells themselves and extrinsic signals received from the microenvironment. A better understanding of the cellular components of microenvironments and their cellular and molecular interactions with the other components of the niche, including the stem cells themselves, will be key to make progress in this field.

In order to carry out an in-depth study of the roles of EphB receptors in T-cell development and to determine the specific relevance of forward and reverse signals in the process, we established severe combined immunodeficient (SCID) mice chimeras with wild-type (WT) or EphB-deficient bone marrow cells. The obtained results demonstrate that EphB2 contributes more significantly than EphB3 in the control of CD4(-)CD8(-) (DN)-CD4(+)CD8(+) (DP) progression, and that reverse signals generated in SCID mice receiving EphB2LacZ precursors, which express the EphB2 extracellular domain, partially rescue the blockade of DN cell maturation observed in EphB2-null chimeras. In addition, increased apoptotic DP thymocytes occurring in EphB2 and/or EphB3 SCID chimeras also contribute to the reduced proportions of DP cells. However, EphB2LacZ chimeras do not show any changes in the proportions of apoptotic DP cells, thus suggesting that there is a role for ephrinB reverse signaling in thymocyte survival. The maturation of DP to CD4(+)CD8(-) or CD4(-)CD8(+) seems to need EphB2 forward signaling and EphB3; a fact that was confirmed in reaggregates formed with either EphB2- or EphB3-deficient DP thymocytes and WT thymic epithelial cells (TECs). The DP thymocyte-TEC conjugate formation was also affected by the absence of EphB receptors. Finally, EphB-deficient SCID chimeras show profoundly altered thymic epithelial organization that confirms a significant role for EphB2 and EphB3 receptors in the thymocyte-TEC crosstalk.

Dendritic cells (DCs) are critical regulators of immune responses that integrate signals from the innate and adaptive immune system and orchestrate T cell responses toward either immunity or tolerance. Growing evidence points to the Wnt signaling pathway as a pivotal piece in the immune balance and focuses on DCs as a direct target for their immunoregulatory role. Our results show that the increase in Wnt5a signaling during the differentiation of human DCs from monocytes alters their phenotype and compromises their subsequent capacity to mature in response to TLR-dependent stimuli. These Wnt5a-DCs produce scant amounts of IL-12p70 and TNF-α but increased levels of IL-10. Consequently, these Wnt5a-DCs have a reduced capacity to induce Th1 responses that promote IL-10 secretion by CD4 T cells. Changes in the transcriptional profile of Wnt5a-DCs correlate with their unconventional phenotype caused presumably by increased IL-6/IL-10 signaling during the process of DC differentiation. The effect of Wnt5a is not a consequence of β-catenin accumulation but is dependent on noncanonical Ca(2+)/calmodulin-dependent protein kinase II/NF-κB signaling. Our results therefore suggest that under high levels of Wnt5a, typical of the inflammatory state and sepsis, monocytes could differentiate into unconventional DCs with tolerogenic features.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β superfamily, are multifunctional polypeptides regulating a broad spectrum of functions in embryonic and adult tissues. Recent reports have demonstrated that BMPs regulate the survival, proliferation and differentiation of several cell types in the immune system. In this study, we investigate the effects of BMP signaling activation on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human DCs express type I and type II BMP receptors (BMPRIA, BMPRIB, type IA activin receptor, BMPRII) and BMP signal transduction molecules (Smad1, 5, and 8, as well as Smad4). On BMP stimulation, Id1-3 (inhibitor of differentiation 1-3/DNA binding) mRNA expression is upregulated and this effect can be blocked with the inhibitor dorsomorphin, showing that the canonical BMP signal transduction pathway is functionally active in DCs. BMP signaling activation promotes the phenotypic maturation of human DCs by increasing the expression of co-stimulatory molecules and also CD83, programmed cell death ligand 1 (PD-L1) and PD-L2, and stimulates cytokine secretion, mainly interleukin-8 and tumor necrosis factor-α. Accordingly, BMP-treated DCs exhibit an enhanced T-cell stimulatory capacity. BMP signaling also enhances the survival of human DCs increasing the Bcl-2/Bax ratio. Finally, the expression of Runx transcription factors is increased in mature DCs, and the mRNA levels of Runx1-3 are upregulated in response to BMP stimulation, indicating that Runx transcription factor family may mediate the effects of BMP signaling in human DC maturation.

Importance of the Field: In the last decade, knowledge of mesenchymal stem cells (MSCs) has evolved rapidly; their immunomodulatory properties and paracrine interactions with specific cell types in damaged tissues and promising results in some clinical applications have made these cells an attractive option for the treatment of certain diseases., Areas Covered in This Review: We present some relevant methodological issues and biological properties of MSCs, as well as clinical applications of MSC therapies with particular emphasis in the treatment of graft versus host disease (GVHD), complex perianal fistula and refractory metastatic neuroblastoma. Other topical aspects relevant to the application of cellular therapies such as biosafety studies and cellular production of MSCs are also discussed in this review., What the Reader Will Gain: The growing optimism regarding MSCs research is based on the promising results obtained in in vitro and in vivo studies. The rapid translational research with MSCs necessitated standardization of methodology and terminology and greater focus on other aspects such as biosafety and cellular production, especially for clinical use of MSCs., Take Home Message: Much has been learned about the biology and applications of MSCs and much remains to be learned.

A better knowledge of the molecular mechanisms that govern leukocyte trafficking is of major relevance for the clinics. Both normal and pathologic extravasation of lymphocytes are a fine-tuned spatio-temporal event of migratory path-finding likely regulated by molecular guidance cues underlying cell movements in other systems. We have recently reported that members of the Eph family of receptor tyrosine kinases, namely EphA2 and one of its ligands, ephrin-A4 (EFNA4) can mediate in the traffic of chronic lymphocytic leukemia (CLL) cells and presumably of normal B cells between the blood and the tissues. The importance of EphA2-EFNA4 interactions at the endothelium-lymphocyte interface during TEM could rely on their attractive/repulsive properties. In the present work, we expand on those results by including additional insights and new suggestions for future studies that discuss the relevance of these molecules in overall cell adhesion dynamic events.

Increasing evidence includes Wnt proteins inside the group of master-signaling pathways that govern immune and nonimmune differentiation systems, fundamental for normal development and homeostasis. Although their precise functions in bone marrow and thymus are still controversial, numerous studies have shown that Wnt signaling is able to control the proliferation of hematopoietic stem cells and thymic progenitors and might also affect their cell-fate decisions and subsequent maturation. In the present work, we analyze the effect of transient stimulation of the canonical Wnt pathway in the differentiation potential of Lin(-)CD34(+) CD1a(-) human thymic progenitors, a multipotent and heterogeneous cell population that has the capacity to develop into T cells, NK cells, monocytes, cDC, and pDC. Our results demonstrate that giving a boost to canonical Wnt signaling, triggered by transient exposure to Wnt3a or LiCl, the differentiation capacity of thymic progenitors changes, enhancing NK cell production. On the contrary, Wnt3a- or LiCl-pretreated thymic progenitors generate a significantly lower number of myeloid lineage cells, monocytes, and cDC and exhibit a reduced capacity to differentiate into pDC lineage. As a possible mechanism for this effect, we show that Wnt3a- and LiCl-pretreated progenitors change their membrane levels of receptors for cytokines pivotal for their expansion and differentiation, such as Flt3L. Moreover, canonical Wnt pathway stimulation modifies the transcription factor profile of CD34(+)CD1(-) thymocytes, increasing Hes-1 and ID3 expression levels.

CXCL12 is an important CXC chemokine involved in numerous biological processes. We had previously demonstrated the synergistic participation of CXCL12 and IL-7 in the control of both survival and proliferation of CD34(+) human thymic lymphoid progenitors. On this basis, we hypothesize a presumptive role for CXCL12 and its receptor, CXCR4, in the thymus involution. In this respect, in the current report we describe the expression of both molecules in the human thymus during aging. Our results demonstrate that, despite the profound alterations observed in the thymic epithelial microenvironment of aged thymuses, the proportions of different CD4/CD8 thymocyte subsets do not undergo significant variations. Remarkably, a strong CXCL12 expression was found in older thymuses, which appeared in the same locations as in younger thymuses: the subcapsulary and medullary areas. The proportions of CXCR4(+) cells, most of them belonging to the CD3(-) compartment, showed no important variations in the older thymuses. However, within the CD34(+) cell population, a significant reduction in the expression of CXCR4 molecules was observed., (Copyright 2010 S. Karger AG, Basel.)

The role of EphB2 and EphB3 in the organization of thymic epithelial cells has been studied in EphB-deficient fetal thymus lobes grafted under the kidney capsule of WT mice. The deficient lobes, as compared with WT ones, showed altered distribution of medullary areas, shortening of medullary epithelial cell processes and presence of K5(-)K8(-) areas. EphB2 and EphB3 expressed on thymic epithelial cells play an autonomous role in their organization. The relevance of Eph/ephrinB forward and reverse signals for this process was evaluated in grafted fetal thymus lobes from mice expressing a truncated EphB2 receptor capable of activating reverse, but not forward, signaling. These deficient lobes showed important alterations of the thymic epithelial organization as compared with the grafted WT lobes, but a less severe phenotype than the grafted EphB2-deficient thymus lobes, which confirms the relevance of EphB2 forward signal for the thymic epithelial organization but, also, a role of the reverse signaling in determining the final epithelial phenotype.

Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.

Eph receptors and their ligands, ephrins, are molecules involved in the morphogenesis of numerous tissues, including the central nervous system in which they play a key role in determining cell positioning and tissue domains containing or excluding nerve fibers. Because common features have been suggested to occur in the microenvironmental organization of brain and thymus, a highly compartmentalized organ central for T cell differentiation, we examined the expression and possible role of Eph/ephrins in the biology of the thymus gland. We reviewed numerous in vivo and in vitro results that confirm a role for Eph and ephrins in the maturation of the thymic epithelial cell (TEC) network and T cell differentiation. Their possible involvement in different steps of early thymus organogenesis, including thymus primordium branching, lymphoid colonization, and thymocyte-TEC interactions, that determine the organization of a mature three-dimensional thymic epithelial network is also analyzed.

In the current study, we extend our own previous results on the thymocyte phenotype of EphB2 and/or EphB3 deficient mice by analyzing the phenotype and the histological organization of their thymic epithelial stroma. All studied adult EphB-deficient thymi showed profound alterations with respect to the wild-type (WT) ones. Each mutant exhibited a specific phenotype, but also showed common features including occurrence of K5+K8+MTS10+ immature medullary epithelial cells, numerous K5-K8-MTS20+ cells and K5+K8+ cells in the thymic cortex and cortical and medullary K5-K8- areas devoid of epithelial cell markers. In addition, comparative analysis of WT and EphB-deficient embryonic and newborn thymi demonstrated that the observed adult phenotype was a consequence of the gradual accumulation of early phenotypic and morphological defects, becoming more severe at the end of embryonic life and in newborn animals. Together, these results confirm a role for EphB2 and EphB3 in thymus morphogenesis. The obtained data are discussed from the point of view of the recognized role played by these two Ephs in the homeostasis of other epithelia and their possible relationships with molecules known to be involved in thymic epithelial cell development.