Different doses of bone morphogenetic protein 4 promote the expression of early germ cell-specific gene in bone marrow mesenchymal stem cells

Bone morphogenetic protein (BMP)-4 has a crucial role on primordial germ cells (PGCs) development in vivo which can promote stem cell differentiation to PG-like cells. In this study, we investigated the expression of Mvh as one of the specific genes in primordial germ cells after treatment with different doses of BMP4 on bone mesenchymal stem cells (BMSCs)-derived PGCs. Following isolation of BMSCs from male mouse femur and tibia, cells were cultured in medium for 72 h. Passage 4 murine BMSCs were characterized by CD90, CD105, CD34, and CD45 markers and osteo-adipogenic differentiation. Different doses of BMP4 (0, 0.01, 0.1, 1, 5, 25, 50, and 100 ng/ml) were added to BMSCs for PGCs differentiation during 4-days culture. Viability percent, proliferation rates, and expression of Mvh gene were analyzed by RT-qPCR. Data analysis was done with ANOVA test. CD90⁺, CD105⁺, CD34⁻, and CD45⁻ BMSCs were able to differentiate to osteo-adipogenic lineages. The results revealed that proliferation rate and viability percent were raised significantly (p ≤ 0.05) by adding 1, 5, 25 ng/ml of BMP4 and there were decreased to the lowest rate after adding 100 ng/ml BMP4 (p ≤ 0.05). There were significant up regulation (p ≤ 0.05) in Mvh expression between 25, 50, and 100 ng/ml BMP4 with other doses. So the selective dose of BMP-4 for treatment during 4-day culture was 25 ng/ml. The results suggest that addition of 25 ng/ml BMP4 had the best effects based on gene-specific marker expression.     

A regulatory effect of IL-21 on T follicular helper-like cell and B cell in rheumatoid arthritis

Introduction

Interleukin (IL)-21 is a member of type I cytokine family. Recent studies indicate that IL-21 can promote T follicular helper (Tfh) cell differentiation and survival, a specialized T cell subset which provides help for B cell. It can also regulate the activation, proliferation and differentiation of human B cell and immunoglobulin (Ig) production as well as isotype switching of plasma cell. Rheumatoid arthritis (RA) is characterized by auto-antibodies overproduction such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody, suggesting a pivotal role of Tfh cell and B cell in the pathogenesis of RA. This study aimed to investigate whether IL-21 had a regulatory effect on Tfh cell and B cell in RA.

Methods

Serum IL-21 concentrations were measured by ELISA. The correlations between serum IL-21 levels and clinical features of RA patients were analyzed by Spearman''s rank test. The percentages of Tfh-like cells, IL-21 receptor (R) expression on Tfh-like cells and B cells in peripheral blood (PB) were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated by rIL-21 (100 ng/ml) in the presence or absence of anti-CD40 and/or anti-IgM, and changes of IL-21R, activation-associated surface markers (CD25, CD69 and CD40), the proliferation, apoptosis and differentiation of B cells were analyzed by flow cytometry. Production of IgG and IgM in the culture supernatants was determined by ELISA.

Results

The results showed that the serum IL-21 levels in RA patients were significantly higher than that of healthy controls (HC). IL-21 concentrations were positively correlated with 28-joint count disease activity score (DAS28) and anti-CCP antibody in RA patients with high IL-21 levels. Furthermore, the frequencies of peripheral CXCR5⁺PD-1⁺CD4⁺ Tfh-like cells markedly increased in RA patients and the percentages of Tfh-like cells were positively correlated with DAS28 and anti-CCP antibody levels. Moreover, elevated IL-21 levels were also correlated with the frequencies of Tfh-like cells. IL-21R expression on both Tfh-like cells and B cells were significantly enhanced in RA patients. In cultures vitro, exogenous IL-21 upregulated IL-21R expression and activation-associated surface markers on B cells and promoted more B cell proliferation in RA than in HC. This IL-21-mediated effect could be reversed by IL-21R-specific neutralizing antibody. Importantly, IL-21 promoted more differentiation of B cell into plasmablast and higher levels of IgG and IgM production in RA than in HC.

Conclusions

Increased serum IL-21 levels in RA patients correlate with DAS28, anti-CCP antibody and frequencies of Tfh-like cells. IL-21 supports B cell activation, proliferation and antibody secretion via IL-21R pathway. Thus, IL-21 may be involved in the pathogenesis of RA and antagonizing IL-21 could be a novel strategy for the therapy of RA.     

USP12 promotes CD4+ T cell responses through deubiquitinating and stabilizing BCL10

Deubiquitinases (DUBs) regulate diverse biological processes and represent a novel class of drug targets. However, the biological function of only a small fraction of DUBs, especially in adaptive immune response regulation, is well-defined. In this study, we identified DUB ubiquitin-specific peptidase 12 (USP12) as a critical regulator of CD4⁺ T cell activation. USP12 plays an intrinsic role in promoting the CD4⁺ T cell phenotype, including differentiation, activation, and proliferation. Although USP12-deficient CD4⁺ T cells protected mice from autoimmune diseases, the immune response against bacterial infection was subdued. USP12 stabilized B cell lymphoma/leukemia 10 (BCL10) by deubiquitinating, and thereby activated the NF-κB signaling pathway. Interestingly, this USP12 regulatory mechanism was identified in CD4⁺ T cells, but not in CD8⁺ T cells. Our study results showed that USP12 activated CD4⁺ T cell signaling, and targeting USP12 might help develop therapeutic interventions for treating inflammatory diseases or pathogen infections.Subject terms: Ubiquitins, T cells     

In vitro activation and differentiation of naïve CD4 and CD8 T cells into HCV Core- and NS3-specific armed effector cells: A new role for CD4 T cells

Viral clearance in hepatitis C virus (HCV) infection has been correlated with strong, multi-specific and sustained T cell responses. The number of functionally active effector T cells determines the outcome of infection. Only a small number of antigen-specific naïve T cells are originally present. Upon infection, they undergo activation, clonal expansion and differentiation to become effector cells. In this study, we determined the ability of dendritic cells (DCs) to prime T cells in vitro to become effector cells upon stimulation with various TLR ligands or IFNα. T cell priming and activation was determined by proliferation and production of effector molecules, IFN-γ and Granzyme B (GrB). HCV Core-specific T cells showed significant increase in proliferation, and the number of HCV Core-specific CD4⁺ and CD8⁺ T cells producing IFN-γ and GrB was higher than control or NS3-specific T cells. These in vitro-primed CD4⁺ and CD8⁺ T cells exhibit the phenotype of just-activated and/or armed effector lymphocytes confirming the transition of naïve T cells to effector cells. This is the first study demonstrating the activation of GrB⁺CD4⁺ T cells against antigen(s) derived from HCV. Our study suggests a novel role of CD4⁺ T cells in immunity against HCV.     

Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL⁺ killer B cells, showing they are enriched in the IgM^highCD5⁺CD1d^high B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL⁺ B cells, but most FasL⁺ B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL⁺ B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL⁺ cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4⁺ T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL⁺ B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders.     

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34⁺ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34⁺ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5–50 ng/ml had not effect on the expansion of CD34⁺ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34⁺ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.     

Activated Human T Cells Secrete Exosomes That Participate in IL-2 Mediated Immune Response Signaling

It has previously been shown that nano-meter sized vesicles (30–100 nm), exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3⁺ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3⁺ T cells have on resting CD3⁺ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3⁺ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3⁺ T cells together with IL-2 were able to generate proliferation in autologous resting CD3⁺ T cells. The CD3⁺ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8⁺ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3⁺ T cells communicate with resting autologous T cells via exosomes.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

Glioma-Derived ADAM10 Induces Regulatory B Cells to Suppress CD8+ T Cells

CD8⁺ T cells play an important role in the anti-tumor activities of the body. The dysfunction of CD8⁺ T cells in glioma is unclear. This study aims to elucidate the glioma cell-derived ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in the suppression of CD8⁺ effector T cells by the induction of regulatory B cells. In this study, glioma cells were isolated from surgically removed glioma tissue and stimulated by Phorbol myristate acetage (PMA) in the culture. The levels of ADAM10 in the culture were determined by enzyme-linked immunosorbent assay. Immune cells were assessed by flow cytometry. The results showed that the isolated glioma cells express ADAM10, which was markedly up regulated after stimulated with PMA. The glioma-derived ADAM10 induced activated B cells to differentiate into regulatory B cells, the later suppressed CD8⁺ T cell proliferation as well as the induced regulatory T cells, which also showed the immune suppressor effect on CD8⁺ effector T cell proliferation. In conclusion, glioma cells produce ADAM10 to induce Bregs; the latter suppresses CD8⁺ T cells and induces Tregs.     

Efficient expansion of tumor-infiltrating lymphocytes from solid tumors by stimulation with combined CD3 and CD28 monoclonal antibodies

Combined CD3 and CD28 monoclonal antibodies (mAb) may initiate efficient activation and expansion of tumor-infiltrating lymphocytes (TIL). In this study we compared phenotypical and functional characteristics of TIL from a group of 17 solid human tumors, stimulated either by high-dose recombinant interleukin 2 (rIL-2, 1000 IU/ml) or by a combination of anti-CD3 and anti-CD28 monoclonal antibodies in the presence of low-dose rIL-2 (10 IU/ml). Compared to activation with high-dose rIL-2, stimulation of TIL with CD3/CD28 mAb induced significantly stronger proliferation and yielded higher levels of cell recovery on day 14. Following the CD3/CD28 protocol, expansion of an almost pure population of CD3⁺ cells was obtained. Whereas CD4⁺ cells dominated in the first week of culturing, within 4 weeks the CD8⁺ population increased to over 90%. The specific capacity to kill autologous tumor cells was not increased as compared to the high-dose rIL-2 protocol, but all cultures showed high cytotoxic T cell activity as measured in a CD3-mAb-mediated redirected kill assay. These studies show that combined CD3 and CD28 mAb are superior to rIL-2 with respect to the initiation of expansion of CD8⁺ cytolytic TIL from solid tumors. Stimulation with specific tumor antigens at a later stage of culturing may further augment the expansion of tumor-specific cytolytic T cells.     

Role of Reactive Oxygen Species in the Radiation Response of Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34⁺ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34⁺ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34⁺ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34⁺ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34⁺ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34⁺ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.     

Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10⁶ pre-B cells had readily detectable serum levels of IgM (54 ± 26 m?g/ml) and IgG (60 ± 95 m?g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley-Liss, Inc.     

The Use of MAGE C1 and Flow Cytometry to Determine the Malignant Cell Type in Multiple Myeloma

The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34⁺) and early pro-B to pre-B cells (CD34^+/−/CD19⁺), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34⁺/MAGE C1⁺ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells.     

T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (αCD3/αCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or αCD3/αCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or αCD3/αCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3⁺CD4⁺ or CD3⁺CD8⁺ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time.In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or αCD3/αCD28 as positive control.     

CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon-γ Production In Vivo

Upon their recognition of antigens presented by the MHC, T cell proliferation is vital for clonal expansion and the acquisition of effector functions, which are essential for mounting adaptive immune responses. The CD98 heavy chain (CD98hc, Slc3a2) plays a crucial role in the proliferation of both CD4⁺ and CD8⁺ T cells, although it is unclear if CD98hc directly regulates the T cell effector functions that are not linked with T cell proliferation in vivo. Here, we demonstrate that CD98hc is required for both CD4⁺ T cell proliferation and Th1 functional differentiation. T cell-specific deletion of CD98hc did not affect T cell development in the thymus. CD98hc-deficient CD4⁺ T cells proliferated in vivo more slowly as compared with control T cells. C57BL/6 mice lacking CD98hc in their CD4⁺ T cells could not control Leishmania major infections due to lowered IFN-γ production, even with massive CD4⁺ T cell proliferation. CD98hc-deficient CD4⁺ T cells exhibited lower IFN-γ production compared with wild-type T cells, even when comparing IFN-γ expression in cells that underwent the same number of cell divisions. Therefore, these data indicate that CD98hc is required for CD4⁺ T cell expansion and functional Th1 differentiation in vivo, and suggest that CD98hc might be a good target for treating Th1-mediated immune disorders.     

The effects of 1,25-dihydroxyvitamin D3 on markers related to the differentiation and maturation of bone marrow-derived dendritic cells from control and obese mice

Vitamin D has been reported to regulate the maturation and function of dendritic cells (DCs). Obesity was shown to be associated with the dysregulation of vitamin D metabolism and malfunction of DCs. We investigated the effects of in vitro 1,25(OH)₂D₃ treatment (0, 1, or 10 nM) on phenotype and expression of genes related to function of bone marrow-derived DCs (BMDCs) from control and obese mice. C57BL/6 N mice were fed a control or high-fat (10% or 45% kcal fat: CON or HFD) diets for 15 weeks. Differentiation toward DCs was induced with GM-CSF (20 ng/ml) and maturation was induced by LPS (50 ng/ml); 10 nM 1,25(OH)₂D₃ treatment inhibited BMDC differentiation (CD11c⁺) and decreased the percentage of mature DCs (MHCII^highCD11c⁺ and CD86^highCD11c⁺) in both CON and HFD groups. The Il10 expression in stimulated BMDCs from the CON group increased with the 10 nM 1,25(OH)₂D₃ treatment, but not in those from the HFD group. The Il12b mRNA levels in stimulated BMDCs were lower in the HFD group than in the CON group. In conclusion, lower levels of Cd 40, Cd83 and Il12 mRNA in LPS-stimulated BMDCs from obese mice suggest malfunction of DCs as antigen presenting cells. 1,25(OH)₂D₃ treatment inhibited the differentiation and maturation of BMDCs in both control and obese mice. Differential effects of 1,25(OH)₂D₃ on the expression of Il10 between control and obese mice suggest that regulation of immune response by vitamin D could be influenced by obesity.     

Interleukin-2-induced lymphoproliferative responses

Summary Interleukin-2 (IL-2) is capable of both stimulating an in vitro lymphoproliferative response and augmenting non-major-histocompatibility-complex-(MHC)-restricted cytotoxicity. However, there are conflicting reports about the phenotypes of responding cells. In the present studies, we determined phenotypes of Ficoll/Hypaque-separated peripheral blood mononuclear cells stimulated with 50, 100 or 1000 U/ml IL-2; analyses were performed after 1, 3 and 5 weeks. With all concentrations, there was a progressive increase in CD3⁺ cells; after 3–5 weeks more than 90% of the cells reacted with this antibody. However, the proportions of CD4⁺ and CD8⁺ cells proved to be a function of the IL-2 concentration. Cultures containing 50 U/ml or 100 U/ml favored the expansion of the CD4⁺ subset. By contrast, in cultures stimulated with 1000 U/ml, CD8⁺ cells predominated. At baseline, CD8⁺ cells comprised 28±2%; after 3 weeks, this value increased to 51±5%. In addition, the proportion of CD56⁺ (Leu19, NKH-1) cells depended on the amount of IL-2. At 50 U/ml, there was no appreciable change in CD56⁺ cells. However, at 1000 U/ml, CD56⁺ cells increased from 17±1% (day 0) to 39±4% (3 weeks). This increase was primarily due to an expansion of the CD3⁺ CD56⁺ subset (non-NMC restricted cytotoxic T cells). By contrast, natural killer (NK) cells, as measured by the CD16 antibody, steadily declined at all IL-2 concentrations.These studies were supported by a grant from the National Cancer Institute, NIH (RO1 CA24429-15)     

Mesenchymal Stem Cells Derived from Human Exfoliated Deciduous Teeth (SHEDs) Induce Immune Modulatory Profile in Monocyte-Derived Dendritic Cells

Background

Mesenchymal stem cells have prominent immune modulatory properties, which may have clinical applications; however their major source, bone marrow, is of limited availability. On the other hand, mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs) are readily accessible, but their immune regulatory properties have not been completely investigated. This study was designed, therefore, to evaluate the SHEDs influence on DCs differentiation, maturation, ability to activate T cells and to expand CD4⁺Foxp3⁺ T cells.

Methodology/Principal Findings

The experiments were based in cellular co-culture during differentiation and maturation of monocyte derived-DCs (moDCs), with, or not, presence of SHEDs. After co-culture with SHEDs, (moDCs) presented lower expression of BDCA-1 and CD11c, in comparison to DC cultivated without SHEDs. CD40, CD80, CD83 and CD86 levels were also decreased in mature DCs (mDCs) after co-cultivation with SHEDs. To assess the ability of SHEDs-exposed moDCs to modulate T cell responses, the former were separated from SHEDs, and co-cultured with peripheral blood lymphocytes. After 5 days, the proliferation of CD4⁺ and CD8⁺ T cells was evaluated and found to be lower than that induced by moDCs cultivated without SHEDs. In addition, an increase in the proportion of CD4⁺Foxp3⁺IL-10⁺ T cells was observed among cells stimulated by mature moDCs that were previously cultivated with SHEDs. Soluble factors released during co-cultures also showed a reduction in the pro-inflammatory cytokines (IL-2, TNF-α and IFN-γ), and an increase in the anti-inflammatory molecule IL-10.

Conclusion/Significance

This study shows that SHEDs induce an immune regulatory phenotype in moDCs cells, evidenced by changes in maturation and differentiation rates, inhibition of lymphocyte stimulation and ability to expand CD4⁺Foxp3⁺ T cells. Further characterization and validation of this phenomenon could support the use of SHEDs, directly or indirectly for immune modulation in the clinical practice.