Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells.

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

T cell control of inflammaging.

T cells are a critical component of the immune system, found in abundance in blood, secondary lymphoid organs, and peripheral tissues. As individuals age, T cells are particularly susceptible to changes, making them one of the most affected immune subsets. These changes can have significant implications for age-related dysregulations, including the development of low-grade inflammation - a hallmark of aging known as inflammaging. In this review, we first present age-related changes in the functionality of the T cell compartment, including dysregulation of cytokine and chemokine production and cytotoxicity. Next, we discuss how these changes can contribute to the development and maintenance of inflammaging. Furthermore, we will summarize the mechanisms through which age-related changes in T cells may drive abnormal physiological outcomes.

Interleukin-10 production by B cells is regulated by cytokines, but independently of GATA-3 or FoxP3 expression.

The knowledge of mechanisms of regulation of IL-10 production by B cells remains still very limited. We show here that highly purified mouse B cells stimulated with LPS produce significant levels of IL-10, but Bregs in our model do not express detectable level of either Foxp3 or GATA-3. Nevertheless, IL-10 production by B cells is regulated by cytokines. In activated B cells, IL-10 production was significantly enhanced by IFN-γ and decreased in the presence of IL-4 or TGF-ß. These findings are in sharp contrast with the observations in T cells, where IL-10 production correlates with GATA-3 or FoxP3 expression, and the cytokines regulate IL-10 production in a reverse manner than in activated B cells. These results thus show that the production of IL-10 by Bregs is regulated by cytokines independently of the expression of GATA-3 and FoxP3, which is clearly different from GATA-3-dependent IL-10 production by activated Th2 cells and FoxP3 expression in IL-10-producing Tregs.

Cyclosporine A promotes the therapeutic effect of mesenchymal stem cells on transplantation reaction.

The successful application of mesenchymal stem cells (MSCs) remains a major challenge in stem cell therapy. Currently, several in vitro studies have indicated potentially beneficial interactions of MSCs with immunosuppressive drugs. These interactions can be even more complex in vivo, and it is in this setting that we investigate the effect of MSCs in combination with Cyclosporine A (CsA) on transplantation reaction and allogeneic cell survival. Using an in vivo mouse model, we found that CsA significantly promoted the survival of MSCs in various organs and tissues of the recipients. In addition, compared to treatment with CsA or MSCs alone, the survival of transplanted allogeneic cells was significantly improved after the combined application of MSCs with CsA. We further observed that the combinatory treatment suppressed immune response to the alloantigen challenge and modulated the immune balance by harnessing proinflammatory CD4+T-bet+ and CD4+RORγt+ cell subsets. These changes were accompanied by a significant decrease in IL-17 production along with an elevated level of IL-10. Co-cultivation of purified naive CD4+ cells with peritoneal macrophages isolated from mice treated with MSCs and CsA revealed that MSC-educated macrophages play an important role in the immunomodulatory effect observed on distinct T-cell subpopulations. Taken together, our findings suggest that CsA promotes MSC survival in vivo and that the therapeutic efficacy of the combination of MSCs with CsA is superior to each monotherapy. This combinatory treatment thus represents a promising approach to reducing immunosuppressant dosage while maintaining or even improving the outcome of therapy.

Mesenchymal stem cells and type 1 diabetes treatment.

Type 1 diabetes represents a serious disease which is caused by autoimmune destruction of insulin-producing B cells in the pancreas. Administration of exogenous insulin cannot replace sensitive and gentle regulation of blood glucose levels that is established by B cells in healthy individuals. Pancreas or islet transplantation is limited by a shortage of donor pancreas and by complications associated with transplantations. For those reasons, new approaches of treatment are being searched, the using of mesenchymal stem cells (MSCs) envisions a promising tool for cell-based therapy of type 1 diabetes. MSCs have a significant impact on the regulation of the immune system, are a potent source of various cytokines and growth factors and manifest multilineage differentiation abilities. In context of type 1 diabetes, MSCs can transdifferentiate into insulin-producing cells, support the regeneration of residual B cells by production of trophic and growth factors or participate in the suppression of the autoimmune reaction against B cells. This review is focused on perspectives and mechanisms of MSC-based therapy and its limitations. Key words: autoimmune reaction - differentiation - mesenchymal stem cells - type 1 diabetes.

The Inability of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells to Survive in Newborn Mice and to Induce Transplantation Tolerance.

An encounter of the developing immune system with an antigen results in the induction of immunological areactivity to this antigen. In the case of transplantation antigens, the application of allogeneic hematopoietic cells induces a state of neonatal transplantation tolerance. This tolerance depends on the establishment of cellular chimerism, when allogeneic cells survive in the neonatally treated recipient. Since mesenchymal stem/stromal cells (MSCs) have been shown to have low immunogenicity and often survive in allogeneic recipients, we attempted to use these cells for induction of transplantation tolerance. Newborn (less than 24 h old) C57BL/6 mice were injected intraperitoneally with 5 × 106 adipose tissue-derived MSCs isolated from allogeneic donors and the fate and survival of these cells were monitored. The impact of MSC application on the proportion of cell populations of the immune system and immunological reactivity was assessed. In addition, the survival of skin allografts in neonatally treated recipients was tested. We found that in vitro expanded MSCs did not survive in neonatal recipients, and the living MSCs were not detected few days after their application. Furthermore, there were no significant changes in the proportion of individual immune cell populations including CD4+ cell lineages, but we detected an apparent shift to the production of Th1 cytokines IL-2 and IFN-γ in neonatally treated mice. However, skin allografts in the MSC-treated recipients were promptly rejected. These results therefore show that in vitro expanded MSCs do not survive in neonatal recipients, but induce a cytokine imbalance without induction of transplantation tolerance.

Difference between mitogen-stimulated B and T cells in nonspecific binding of R-phycoerythrin-conjugated antibodies.

Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of R-phycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con A-stimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE+ B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.

Antiapoptotic Properties of Mesenchymal Stem Cells in a Mouse Model of Corneal Inflammation.

Mesenchymal stem cells (MSCs) represent a population of adult stem cells that have potent immunoregulatory, anti-inflammatory, and antiapoptotic properties. In addition, they have ability to migrate to the site of inflammation or injury, where they contribute to the regeneration and healing process. For these properties, MSCs have been used as therapeutic cells in several models, including treatment of damages or disorders of the ocular surface. If the damage of the ocular surface is extensive and involves a limbal region where limbal stem cell reside, MSC therapy has been proved as the effective treatment approach. Although the anti-inflammatory properties of MSCs have been well characterized, mechanisms of antiapoptotic action of MSCs are not well recognized. Using a chemically damaged cornea in a mouse model, we showed that the injury decreases expression of the gene for antiapoptotic molecule Bcl-2 and increases the expression of proapoptotic genes Bax and p53. These changes were attenuated by local transplantation of MSCs after corneal damage. The antiapoptotic effect of MSCs was tested in an in vitro model of co-cultivation of corneal explants with MSCs. The apoptosis of corneal cells in the explants was induced by proinflammatory cytokines and was significantly inhibited in the presence of MSCs. The antiapoptotic effect of MSCs was mediated by paracrine action, as confirmed by separation of the explants in inserts or by supernatants from MSCs. In addition, MSCs decreased the expression of genes for the molecules associated with endoplasmic reticulum stress Atf4, Bip, and p21, which are associated with apoptosis. The results show that MSCs inhibit the expression of proapoptotic genes and decrease the number of apoptotic cells in the damaged corneas, and this action might be one of the mechanisms of the therapeutic action of MSCs.

The Altered Migration and Distribution of Systemically Administered Mesenchymal Stem Cells in Morphine-Treated Recipients.

Mesenchymal stem cells (MSCs) have the ability to migrate to the site of injury or inflammation, and to contribute to the healing process. Since patients treated with MSCs are often users of analgesic drugs, to relieve their uncomfortable pain associated with the tissue disorder, there is a possibility of negative effects of these drugs on the migration of endogenous and exogenous MSCs. Therefore, we tested the impact of acute and chronic treatment with morphine on the migration and organ distribution of exogenous adipose tissue-derived MSCs in mouse models. Firstly, we showed that the incubation of MSCs with morphine significantly reduced the expression of adhesive molecules CD44 (HCAM), CD54 (ICAM-1) and CD106 (VCAM-1) on MSCs. Using a model of systemic administration of MSCs labeled with vital dye PKH26 and by the application of flow cytometry to detect living CD45-PKH26+ cells, we found a decreased number of labeled MSCs in the lung, spleen and bone marrow, and a significantly increased number of MSCs in the liver of morphine-treated recipients. A skin allograft model was used to study the effects of morphine on the migration of exogenous MSCs to the superficial wound. Intraperitoneally administered MSCs migrated preferentially to the wound site, and this migration was significantly decreased in the morphine-treated recipients. The present results showed that morphine significantly influences the distribution of exogenous MSCs in the body, and decreases their migration to the site of injury.

The Immunomodulatory Potential of Mesenchymal Stem Cells in a Retinal Inflammatory Environment.

Retinal degenerative disorders are characterized by a local upregulation of inflammatory factors, infiltration with cells of the immune system, a vascular dysfunction and by the damage of retinal cells. There is still a lack of treatment protocols for these diseases. Mesenchymal stem cell (MSC)-based therapy using immunoregulatory, regenerative and differentiating properties of MSCs offers a promising treatment option. In this study, we analyzed the immunomodulatory properties of mouse bone marrow-derived MSCs after their intravitreal delivery to the inflammatory environment in the eye, caused by the application of pro-inflammatory cytokines IL-1ß, TNF-α and IFN-γ. The intravitreal administration of these cytokines induces an increased expression of pro-inflammatory molecules such as IL-1α, IL-6, inducible nitric oxide synthase, TNF-α and vascular endothelial growth factor in the retina. However, a significant decrease in the expression of genes for all these pro-inflammatory molecules was observed after the intravitreal injection of MSCs. We further showed that an increased infiltration of the retina with immune cells, mainly with macrophages, which was observed after pro-inflammatory cytokine application, was significantly reduced after the intravitreal application of MSCs. The similar immunosuppressive effects of MSCs were also demonstrated in vitro in cultures of cytokine-stimulated retinal explants and MSCs. Overall, the results show that intravitreal application of MSCs inhibits the early retinal inflammation caused by pro-inflammatory cytokines, and propose MSCs as a promising candidate for stem cell-based therapy of retinal degenerative diseases.

Immunomodulatory Properties of Bone Marrow Mesenchymal Stem Cells from Patients with Amyotrophic Lateral Sclerosis and Healthy Donors.

Pathogenesis of amyotrophic lateral sclerosis (ALS) involves several mechanisms resulting in a shift from a neuroprotective to a neurotoxic immune reaction. A promising tool for ALS treatment is represented by mesenchymal stem cells (MSCs), which possess both regenerative potential and immunomodulatory properties. In this study, we aimed to compare the immunomodulatory properties of MSCs isolated from the bone marrow of patients suffering from ALS and healthy donors. Moreover, the influence of proinflammatory cytokines on the immunoregulatory functions of MSCs was also evaluated. We found that MSCs from ALS patients and healthy donors comparably affected mitogen-stimulated peripheral blood mononuclear cells and reduced the percentage of T helper (Th)1, Th17 and CD8+CD25+ lymphocytes. These MSCs also equally increased the percentage of Th2 and CD4+FOXP3+ T lymphocytes. On the other hand, MSCs from ALS patients decreased more strongly the production of tumour necrosis factor-α than MSCs from healthy donors, but this difference was abrogated in the case of MSCs stimulated with cytokines. Significant differences between cytokine-treated MSCs from ALS patients and healthy donors were detected in the effects on the percentage of CD8+CD25+ and CD4+FOXP3+ T lymphocytes. In general, treatment of MSCs with cytokines results in a potentiation of their effects, but in the case of MSCs from ALS patients, it causes stagnation or even restriction of some of their immunomodulatory properties. We conclude that MSCs from ALS patients exert comparable immunomodulatory effects to MSCs from healthy donors, but respond differently to stimulation with proinflammatory cytokines. Graphical Abstract Treatment of mesenchymal stem cells (MSCs) with cytokines results in a potentiation of their effects, but in the case of MSCs from amyotrophic lateral sclerosis (ALS) patients, it causes stagnation (an equal reduction of the percentage of CD8+CD25+ T lymphocytes) or even restriction (no increase of proportion of CD4+FOXP3+ T lymphocytes) of some of their immunomodulatory properties. It means that MSCs from ALS patients exert comparable immunomodulatory effects to MSCs from healthy donors, but respond differently to stimulation with proinflammatory cytokines.

Characterisation of mesenchymal stem cells from patients with amyotrophic lateral sclerosis.

AIMS: Mesenchymal stem cells (MSCs) have recently been tested in clinical trials to treat severe diseases, including amyotrophic lateral sclerosis (ALS). Since autologous MSCs are frequently used for therapy, we aimed to evaluate the possible influence of the disease on characteristics and function of these cells. METHODS: MSCs were isolated from the bone marrow of patients with ALS and compared with MSCs from healthy controls (HC). The cells were tested for phenotype, growth properties, differentiation ability, metabolic activity, secretory potential, expression of genes for immunomodulatory molecules and for the ability to regulate proliferation of mitogen-stimulated peripheral blood leucocytes. MSCs from patients with ALS and HC were either unstimulated or treated with proinflammatory cytokines for 24 hours before testing. RESULTS: MSCs isolated from patients with ALS have a higher differentiation potential into adipocytes, express elevated levels of mRNA for interleukin-6, but produce less hepatocyte growth factor than MSCs from HC. On the other hand, there were no significant differences between MSCs from patients with ALS and HC in the expression of phenotypic markers, growth properties, metabolic activity, osteogenic differentiation potential and immunoregulatory properties. CONCLUSIONS: The results suggest that, in spite of some differences in cytokine production, MSCs from patients with ALS can be useful as autologous cells in therapy of ALS.

The Impact of Morphine on the Characteristics and Function Properties of Human Mesenchymal Stem Cells.

Morphine is an analgesic drug therapeutically administered to relieve pain. However, this drug has numerous side effects, which include impaired healing and regeneration after injuries or tissue damages. It suggests negative effects of morphine on stem cells which are responsible for tissue regeneration. Therefore, we studied the impact of morphine on the properties and functional characteristics of human bone marrow-derived mesenchymal stem cells (MSCs). The presence of µ-, δ- and κ-opioid receptors (OR) in untreated MSCs, and the enhanced expression of OR in MSCs pretreated with proinflammatory cytokines, was demonstrated using immunoblotting and by flow cytometry. Morphine modified in a dose-dependent manner the MSC phenotype, inhibited MSC proliferation and altered the ability of MSCs to differentiate into adipocytes or osteoblasts. Furthermore, morphine rather enhanced the expression of genes for various immunoregulatory molecules in activated MSCs, but significantly inhibited the production of the vascular endothelial growth factor, hepatocyte growth factor or leukemia inhibitory factor. All of these observations are underlying the selective impact of morphine on stem cells, and offer an explanation for the mechanisms of the negative effects of opioid drugs on stem cells and regenerative processes after morphine administration or in opioid addicts.

The Identification of Interferon-γ as a Key Supportive Factor for Retinal Differentiation of Murine Mesenchymal Stem Cells.

Retinal disorders represent the main cause of decreased quality of vision and even blindness worldwide. The loss of retinal cells causes irreversible damage of the retina, and there are currently no effective treatment protocols for most retinal degenerative diseases. A promising approach for the treatment of retinal disorders is represented by stem cell-based therapy. The perspective candidates are mesenchymal stem cells (MSCs), which can differentiate into multiple cell types and produce a number of trophic and growth factors. In this study, we show the potential of murine bone marrow-derived MSCs to differentiate into cells expressing retinal markers and we identify the key supportive role of interferon-γ (IFN-γ) in the differentiation process. MSCs were cultured for 7 days with retinal extract and supernatant from T-cell mitogen concanavalin A-stimulated splenocytes, simulating the inflammatory site of retinal damage. MSCs cultured in such conditions differentiated to the cells expressing retinal cell markers such as rhodopsin, S antigen, retinaldehyde-binding protein, calbindin 2, recoverin, and retinal pigment epithelium 65. To identify a supportive molecule in the supernatants from activated spleen cells, MSCs were cultured with retinal extract in the presence of various T-cell cytokines. The expression of retinal markers was enhanced only in the presence of IFN-γ, and the supportive role of spleen cell supernatants was abrogated with the neutralization antibody anti-IFN-γ. In addition, differentiated MSCs were able to express a number of neurotrophic factors, which are important for retinal regeneration. Taken together, the results show that MSCs can differentiate into cells expressing retinal markers and that this differentiation process is supported by IFN-γ.

Mesenchymal Stem Cells Attenuate the Adverse Effects of Immunosuppressive Drugs on Distinct T Cell Subopulations.

Immunosuppressive drugs are widely used to treat undesirable immune reaction, however their clinical use is often limited by harmful side effects. The combined application of immunosuppressive agents with mesenchymal stem cells (MSCs) offers a promising alternative approach that enables the reduction of immunosuppressive agent doses and simultaneously maintains or improves the outcome of therapy. The present study aimed to determinate the effects of immunosuppressants on individual T cell subpopulations and to investigate the efficacy of MSC-based treatment combined with immunosuppressive drugs. We tested the effect of five widely used immunosuppressants with different action mechanisms: cyclosporine A, mycophenolate mofetil, rapamycin, and two glucocorticoids - prednisone and dexamethasone in combination with MSCs on mouse CD4+ and CD8+ lymphocyte viability and activation, Th17 (RORγt+), Th1 (T-bet+), Th2 (GATA-3+) and Treg (Foxp3+) cell proportion and on the production of corresponding key cytokines (IL-17, IFNγ, IL-4 and IL-10). We showed that MSCs modulate the actions of immunosuppressants and in combination with immunosuppressive drugs display distinct effect on cell activation and balance among different T lymphocytes subpopulations and exert a suppressive effect on proinflammatory T cell subsets while promoting the functions of anti-inflammatory Treg lymphocytes. The results indicated that MSC-based therapy could be a powerful strategy to attenuate the negative effects of immunosuppressive drugs on the immune system.

Distinct Immunoregulatory Mechanisms in Mesenchymal Stem Cells: Role of the Cytokine Environment.

Mesenchymal stem cells (MSCs) represent a population of cells which have the ability to regulate reactivity of T and B lymphocytes by multiple mechanisms. The immunoregulatory activities of MSCs are strictly influenced by the cytokine environment. Here we show that two functionally distinct cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), significantly potentiate the ability of MSCs to inhibit IL-10 production by activated regulatory B cells (Bregs). However, MSCs in the presence of IL-4 or IFN-γ inhibit the IL-10 production by different mechanisms. Preincubation of MSCs with IFN-γ led to the suppression, but pretreatment with IL-4 of neither MSCs nor B cells resulted in the suppression of IL-10 production. The search for candidate regulatory molecules expressed in cytokine-treated MSCs revealed different patterns of the gene expression. Pretreatment of MSCs with IFN-γ, but not with IL-4, induced expression of indoleamine-2,3-dioxygenase, cyclooxygenase-2 and programmed cell death-ligand 1. To identify the molecule(s) responsible for the suppression of IL-10 production, we used specific inhibitors of the putative regulatory molecules. We found that indomethacine, an inhibitor of cyclooxygenase-2 (Cox-2) activity, completely abrogated the inhibition of IL-10 production in cultures containing MSCs and IFN-γ, but had no effect on the suppression in cell cultures containing MSCs and IL-4. The results show that MSCs can inhibit the response of B cells to one stimulus by different mechanisms in dependence on the cytokine environment and thus support the idea of the complexity of immunoregulatory action of MSCs.