Mesenchymal Stromal Cell (MSC)-Derived Combination of CXCL5 and Anti-CCL24 Is Synergistic and Superior to MSC and Cyclosporine for the Treatment of Graft-versus-Host Disease

The immunosuppressive properties of mesenchymal stromal cells (MSCs) have been clinically proven to be effective in treating graft-versus-host disease (GVHD). However, MSC therapy is limited by the need for laborious and expensive manufacturing processes that are fraught with batch-to-batch variability. Substitution of MSC therapy with key MSC-mediated immunomodulatory factors could be an option for GVHD treatment. Using a simulated in vitro model of the immunosuppressive effects of MSC on allogeneic graft reactions, a synergistic 2-factor combination (2FC) of CXCL5 and anti-CCL24 was identified from a panel of over 100 immunomodulatory factors as being superior to MSCs in the modulation of mixed lymphocyte reactions. This 2FC was superior to cyclosporine in ameliorating both moderate and severe GVHD while being equivalent to MSCs in moderate GVHD and superior to MSCs in severe GVHD. Its immunosuppressive efficacy could be further improved by extended treatment. Mechanistic studies revealed that in vitro the 2FC could only reduce the proliferation of Th 1 and Th 17, whereas in vivo CXCL5 acts in concert with anti-CCL24 antibody to reduce not only transplanted Th 1 and Th 17 but also cytotoxic T lymphocytes and natural killer cells to increase mouse immunosuppressive neutrophils without affecting human hematopoietic stem cell reconstitution. Concurrently, it reduced circulating human proinflammatory cytokines IFN-γ, IL-6, IL-17A, IL-8, macrophage inflammatory protein-1β, and monocyte chemoattractant protein-1. Both in vitro and in vivo data suggest that CXCL5 and anti-CCL24 antibody act in concert to ameliorate GVHD via suppression of Th 1 and Th 17 responses. We propose that this novel 2FC could substitute for MSC therapy in GVHD treatment.     

The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells (BM-MSC) are currently being investigated in preclinical and clinical settings because of their self-renewal and multipotent differentiative capacity or their immunosuppressive function. However, BM may be detrimental because of the highly invasive donation procedure and BM-MSC decline with age. Therefore, MSC derived from other sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. Human umbilical cord blood (UCB) cells are good substitutes for BM-MSC because of the immaturity of newborn cells. In this study, we successfully isolated MSC from UCB. The morphological phenotypes, cell cycle status, surface markers and differentiation potential of these clonally expanded cells are consistent with BM-MSC. Furthermore, UCB-MSC expanded in vitro retain low immunogenicity and an immunomodulatory effect. Flow cytometry analysis showed that UCB-MSC did not express CD40, CD40 ligand, CD80, CD86 and major histocompatibility complex class II molecules. We have demonstrated that UCB-MSC are incapable of inducing allogeneic peripheral blood mononuclear cell (PBMC) proliferation and have a dose-dependent inhibition of PBMC immune responses in mixed lymphocyte reactions (MLR) and phytohaemagglutinin activation assays, even after interferon-gamma treatment. Additionally, we have found that UCB-MSC can suppress the function of mature dendritic cells. Using transwell systems, we have demonstrated an inhibition mechanism that depends on both cell contact and soluble factors. Based on the findings we conclude that banked UCB could serve as a potential alternative source of MSC for allogeneic application in the future.     

Immunomodulatory properties and therapeutic application of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multi-potent progenitor cells that are isolated from the bone marrow and several adult organs and tissues. These cells possess remarkable immunosuppressive properties and can inhibit the proliferation and function of the major immune cell populations, including T cells, B cells and natural killer (NK) cells; modulate the activities of dendritic cells (DCs); and induce regulatory T cells both in vivo and in vitro. These unique properties make MSCs ideal candidates for clinical application as immunosuppressants. The immunomodulatory effect of MSCs is mediated by a non-specific anti-proliferative action of these cells, which is dependent on cell-cell contact or secreted soluble factors such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E(2) (PGE(2) ), nitric oxide (NO), histocompatibility leucocyte antigen-G (HLA-G), transforming growth factor (TGF)-β, interferon (IFN)-γ and interleukin (IL)-1β. Considerable progress has been obtained in preclinical studies on MSCs, including those on their ability to activate allogeneic cells. This review examines the current understanding of the immunomodulatory properties of MSCs and its therapeutic implication for immune-mediated diseases and transplant rejection.     

骨髓间质干细胞吲哚胺2，3-双加氧酶活性对T淋巴细胞的影响(英文)

目的: 探讨骨髓间质干细胞（MSCs）吲哚胺2，3-双加氧酶（IDO）活性对抑制T淋巴细胞应答反应的影响。方法: 从人骨髓中分离培养间质干细胞,通过其形态特点、表面标志及多向分化能力检测进行鉴定。以浓度为2×105 U/L的 IFN-γ对分离的MSCs诱导18 h，检测MSCs上IDO mRNA和IDO蛋白表达。将经过IFN-γ 2×105 U/L诱导的MSCs预先接种在培养板中，再建立混合淋巴细胞培养(MLR)体系，利用MTT法检测T淋巴细胞增殖率，并用反相高效液相色谱法检测IDO活性。结果: IFN-γ能诱导MSCs上IDO mRNA和IDO蛋白的表达；MSCs的IDO活性抑制MLR体系中T淋巴细胞增殖率。结论： 经IFN-γ刺激后的MSCs在体外可抑制异体T淋巴细胞的免疫应答，IDO活性参与了这种免疫抑制作用的发挥。     

胎盘源间充质干细胞对T淋巴细胞体外增殖的影响

目的从人胎盘组织中分离培养获得间充质干细胞（mesenchymal stem cells,MSCs）,并进一步研究其对T淋巴细胞体外增殖的影响。方法首先从胎盘组织中分离培养胎盘间充质干细胞（PMSCs）,在体外观测其形态,并通过细胞表面抗原表达、分化潜能等特征进行鉴定;然后将体外分离培养扩增的PMSCs,按照不同比例加入双向混合淋巴细胞培养体系（mixed lymphocyte reaction,MLR）中,共同培养6d后,测定T淋巴细胞的增殖。结果从人胎盘组织中分离培养获得问充质干细胞,具有与骨髓间充质于细胞（BMSCs）极为相似的细胞形态及细胞表面标志,表达CD29、CD44和CD105,不表达CD34、CD45、CD106和HLA—DR,并且还具有与BMSCs相似的跨胚层分化能力,能在一定条件下被诱导分化出神经元样细胞。PMSCs与同种异体混合淋巴细胞共同培养能抑制T淋巴细胞的体外增殖。结论胎盘组织是MSCs的有效来源,PMSCs具有与BMSCs相似的生物学特性及免疫调节机制。它为问充质干细胞的研究提供了又一重要的细胞来源。     

Interferon-gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells

The ability of mesenchymal stem cells (MSC) to suppress alloresponsiveness is poorly understood. Herein, an allogeneic mixed lymphocyte response was used as a model to investigate the mechanisms of MSC-mediated immunomodulation. Human MSC are demonstrated to express the immunosuppressive cytokines hepatocyte growth factor (HGF), interleukin (IL)-10 and transforming growth factor (TGF)-beta1 at concentrations that suppress alloresponses in vitro. MSC also express cyclooxygenase 1 and 2 and produce prostaglandin E2 constitutively. Blocking studies with indomethacin confirmed that prostaglandins contribute to MSC-mediated allosuppression. The proinflammatory cytokine interferon (IFN)-gamma did not ablate MSC inhibition of alloantigen-driven proliferation but up-regulated HGF and TGF-beta1. IFN-gamma also induced expression of indoleamine 2,3, dioxygenase (IDO), involved in tryptophan catabolism. Use of an antagonist, 1-methyl-L-tryptophan, restored alloresponsiveness and confirmed an IDO contribution to IFN-gamma-induced immunomodulation by MSC. Addition of the tryptophan catabolite kynurenine to mixed lymphocyte reactions (MLR), blocked alloproliferation. These findings support a model where IDO exerts its effect through the local accumulation of tryptophan metabolites rather than through tryptophan depletion. Taken together, these data demonstrate that soluble factors, or products derived from MSC, modulate immune responses and suggest that MSC create an immunosuppressive microenvironment capable of modulating alloresponsiveness even in the presence of IFN-gamma.     

Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation.

Mesenchymal stem cells (MSCs) may be derived from adult bone marrow, fat, and several fetal tissues. In vitro, MSCs can be expanded and have the capacity to differentiate into several mesenchymal tissues, such as bone, cartilage, and fat. They escape the immune system in vitro, and this may make them candidates for cellular therapy in an allogeneic setting. They also have immunomodulatory effects, inhibit T-cell proliferation in mixed lymphocyte cultures, prolong skin allograft survival, and may decrease graft-versus-host disease (GVHD) when cotransplanted with hematopoietic stem cells. MSCs induce their immunosuppressive effect via a soluble factor. Some candidates have been suggested, and various mechanisms have also been suggested, although contradictory data exist; this may be due to differences in the cells and systems tested. A major problem has been that it has been difficult to identify and isolate MSCs after transplantation in vivo. However, MSCs seem to enhance hematopoietic engraftment in recipients of autologous and allogeneic grafts. Recently, they were found to reverse grade IV acute GVHD of the gut and liver. No tolerance was induced, however. Controlled studies are warranted. Thus, in allogeneic stem cell transplantation, MSCs may be used for hematopoiesis enhancement, as GVHD prophylaxis, and for the treatment of severe acute GVHD. They are also of potential use in the treatment of organ transplant rejection and in autoimmune inflammatory bowel disorders where immunomodulation and tissue repair are needed.     

Induction of immunotolerance via mPEG grafting to allogeneic leukocytes

The induction of anergy or tolerance to prevent allorecognition is of clinical interest. To this end, the effects of methoxypoly(ethylene glycol) [mPEG] grafting to allogeneic lymphocytes on proliferation and phenotype (Th17 and Treg) was examined in?vitro and in?vivo. Control studies demonstrated that PEGylation did not affect cells viability or proliferation (mitogen) potential. Conditioned media (1° MLR) collected at 72?h from resting PBMC demonstrated no immunomodulatory effects whereas the control MLR demonstrated significant (p?相似文献   

AMLR and MLR stimulating activity of human T lymphocytes activated in vitro by soluble HLA-DR antigens.

We have examined the allogeneic mixed lymphocyte reaction (MLR) and autologous mixed lymphocyte reaction (AMLR) stimulating activities of T cells precultured in vitro with soluble allogeneic or autologous HLA-DR antigens. These cells (Ts) are known to suppress the human MLR: this suppression is specific in that it occurs only when stimulator cells have the same HLA-DR antigen as that used to induce differentiation of suppressor cells. Ts cells express new membrane specificities; they can be separated by immunoabsorption into two populations: Ts enriched (Tx+; with suppressive activity) and Ts depleted (Ts-; with helper function). In the present study, we have demonstrated that both Ts cell subsets activated by soluble HLA-DR alloantigens are able to stimulate both MLR and AMLR. Ts cells activated by soluble autologous HLA-DR antigens are able to stimulate MLR, but not AMLR.     

Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA-unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell-activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact-dependent and required the presence of interferon (IFN)-gamma produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN-gamma. The suppressive effect of IFN-gamma was related to its ability to stimulate the production by MSCs of indoleamine 2,3-dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft-versus-host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell- derived IFN-gamma.     

Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells: a critical role for nitric oxide

We examined the mechanisms involved in interleukin (IL)-12-mediated suppression of cellular immunity in mice using allogeneic mixed leukocyte reaction (MLR) stimulated by dendritic cells (DCs) in vitro and compared the effect of IL-12 on MLR in mice and humans. Although IL-12 stimulated human MLR, the addition of IL-12 or interferon-gamma (IFN-gamma) resulted in a dose-dependent suppression of MLR in mice. The treatment with N(G)-monomethyl-L-arginine (L-NMMA) completely abrogated IL-12- and IFN-gamma-mediated suppression of MLR in mice. Furthermore, IL-12 enhanced the alloreactive cytolytic T lymphocyte (CTL) induction in human MLR, whereas the addition of L-NMMA was required to generate alloreactive CTLs in the presence of IL-12 in mice. Nitric oxide (NO) was detected only in mouse MLR. Murine DCs could produce NO, but neither human CD34(+) cell- nor monocyte-derived DCs produced a detectable amount of NO. These results suggest that NO produced by DCs might play an important role in IL-12-mediated immune suppression in mice but not in humans.     

Canine and Equine Mesenchymal Stem Cells Grown in Serum Free Media Have Altered Immunophenotype

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE₂, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE₂. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.     

Effects of human mesenchymal stem cells on the differentiation of dendritic cells from CD34+ cells

Mesenchymal stem cells (MSCs) have profound immunomodulatory functions both in vitro and in vivo. However, their effects on the differentiation of dendritic cells (DCs) are unknown. In this study, we employed an in vitro model to investigate the effects of human MSCs on the development of DCs. CD34(+) cells isolated from cord blood were cultured under conventional DC(cDC) or plasmacytoid DC (pDC) differentiation conditions, in the presence or absence of MSCs or their conditioned medium. Here we show that both MSCs and their conditioned medium dramatically increased the numbers of cells generated under either condition. The percentage of cells with the cDC phenotype is significantly reduced in the presence of MSCs or their conditioned medium, whereas the percentage of pDC increased. The capacity of cDCs from MSCs or their conditioned medium-treated CD34(+) cells to stimulate allogeneic T cells was weakened. Furthermore, MSCs can skew the DC function from cDC to pDC, thus biasing the immune system toward Th2 and away from Th1 responses. Blocking the prostaglandin E(2) (PGE(2)) synthesis of MSCs can reverse most of these influences of MSCs on DCs differentiation and function. Therefore, MSCs can significantly influence DC development through PGE(2) production.     

Immunosuppressive activity of bromocriptine on human T lymphocyte function in vitro.

Bromocriptine (BRC), a dopamine type 2 agonist, prevents secretion of pituitary prolactin (PRL). BRC has been shown to impair lymphocyte responsiveness toward antigenic stimulation by decreasing serum PRL levels. Hypoprolactinaemia induced by BRC produces a similar immunosuppressive effect, as observed in hypophysectomized rats, which is restored by the administration of PRL. Therefore, the immunosuppression induced by BRC has been interpreted as the result of hypoprolactinaemia. However, the direct mechanism of BRC in immune response has never been evoked. We recently reported that BRC has an immunosuppressive activity on human B lymphocyte function in vitro. In the present study we demonstrate that BRC suppresses T cell proliferation by means of blocking IL-2 production by T cells as well as mixed lymphocyte reaction (MLR) in a dose-dependent manner. We could not detect the immunoreactive PRL activity in the conditioned medium from polyclonal T cell mitogen-stimulated T cell cultures. Then, the immunosuppressive activity of BRC on human T cell function appeared to be independent of its hypoprolactinaemic effect. Treatment with low-dose cyclosporin A (CsA) or FK506 in combination with BRC has proved more effective than either drug alone in suppression of T cell proliferation and CD25 antigen expression. Thus, the therapeutic application of BRC in combination with immunosuppressants may enhance the immunosuppressive effect, while at the same time decreasing the toxicity.     

Cyclic AMP inhibits macrophage suppressor function and enhances lymphocyte proliferation.

The effects of increasing the level of cyclic AMP (cAMP) activity on lymphocyte proliferation in the rat mixed lymphocyte reaction (MLR) were investigated. Dibutyryl cAMP (dbcAMP) and prostaglandin E2 (PGE2) produced a dose-dependent reduction in proliferation in the lymph node (LN) MLR, but produced a substantial increase in proliferation in the spleen MLR at the lower concentrations used (10(-5)-10(-4) M dbcAMP; 10(-7)-10(-6) M PGE2). Enhancement of proliferation was dependent on the presence of macrophages and was probably due to inhibition of macrophage activation. This was based on the following findings: (1) spleen MLR proliferation was lower than that in the LN MLR; (2) depletion of spleen macrophages increased proliferation in the spleen MLR and addition of these macrophages to the LN MLR reduced proliferation; (3) macrophage depletion from the spleen MLR abolished the proliferation-enhancing effect of dbcAMP. In conclusion, cAMP enhances lymphocyte proliferation in this system, apparently as a consequence of suppressing the inhibitory influence of macrophages.     

Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex

We aimed to study the effects of mesenchymal stem cells (MSCs) on alloreactivity and effects of T-cell activation on human peripheral blood lymphocytes (PBLs) in vitro. MSCs were expanded from the bone marrow of healthy subjects. MSCs isolated from second to third passage were positive for CD166, CD105, CD44, CD29, SH-3 and SH-4, but negative for CD34 and CD45. MSCs cultured in osteogenic, adipogenic or chondrogenic media differentiated, respectively, into osteocytes, adipocytes or chondrocytes. MSC added to PBL cultures had various effects, ranging from slight inhibition to stimulation of DNA synthesis. The stimulation index (SI = (PBL + MSC)/PBL) varied between 0.2 and 7.3. The SI was not affected by the MSC dose or by the addition of allogeneic or autologous MSCs to the lymphocytes. Suppression of proliferative activity was observed in all experiments after the addition of 10,000-40,000 MSCs to mixed lymphocyte cultures (MLCs). Lymphocyte proliferation was 10-90%, compared with a control MLC run in parallel without MSCs. In contrast, the addition of fewer MSCs (10-1000 cells) led to a less consistent suppression or a marked lymphocyte proliferation in several experiments, ranging from 40 to 190% of the maximal lymphocyte proliferation in control MLCs. The ability to inhibit or stimulate T-cell alloresponses appeared to be independent of the major histocompatibility complex, as results were similar using 'third party' MSCs or MSCs that were autologous to the responder or stimulating PBLs. The strongest inhibitory effect was seen if MSCs were added at the beginning of the 6 day culture, and the effect declined if MSCs were added on day 3 or 5. Marked inhibitory effects of allogeneic and autologous MSCs (15,000) were also noted after mitogenic lymphocyte stimulation by phytohaemagglutinin (median lymphocyte proliferation of 30% of controls), Concanavalin A (56%) and protein A (65%). Little, if any, inhibition occurred after stimulation with pokeweed mitogen. Low numbers of MSCs (150 cells) were unable to inhibit mitogen-induced T-cell responses. MSCs have significant immune modulatory effects on MLCs and after mitogenic stimulation of PBL. High numbers of MSCs suppress alloreactive T cells, whereas very low numbers clearly stimulated lymphocyte proliferation in some experiments. The effect of a larger number of MSCs on MLCs seems more dependent on cell dose than histocompatibility and could result from an 'overload' of a stimulatory mechanism.     

小鼠脂肪干细胞的免疫原性及移植安全性*

背景：多组实验证实脂肪干细胞体外能够大量扩增,经一定诱导条件作用后能够向3个胚层分化,并且能够加以一定的基因修饰。 目的：观察小鼠脂肪干细胞的免疫原性和经静脉移植后的安全性。 方法：体外分离培养小鼠脂肪干细胞,流式细胞仪鉴定细胞表面的免疫标记物;体外淋巴细胞增殖试验检测脂肪干细胞对淋巴细胞的活化能力;经小鼠尾静脉移植脂肪干细胞,观察细胞在体内对小鼠T、B淋巴细胞的刺激作用;仔细解剖移植后小鼠,观察细胞移植后的成瘤性。 结果与结论：脂肪干细胞在体外容易扩增培养,表达间质干细胞表面标记物;体外与淋巴细胞共培养时,不能有效刺激淋巴细胞增殖;经静脉移植后,不能激活机体细胞免疫反应,但是能够活化B淋巴细胞,激活机体体液免疫反应;细胞移植后未观察到肿瘤形成和组织畸变。提示脂肪干细胞总体免疫原性较低,移植后不会激活宿主细胞免疫系统;脂肪干细胞移植后无肿瘤样生长,安全性较好。     

Allogeneic and xenogeneic transplantation of adipose-derived stem cells in immunocompetent recipients without immunosuppressants

Mesenchymal stem cells (MSCs) are well known for their immunomodulatory capabilities. In particular, their immunosuppressive property is believed to permit their allogeneic or even xenogeneic transplantation into immunocompetent recipients without the use of immunosuppressants. Adipose-derived stem cell (ADSC), owing to its ease of isolation from an abundant tissue source, is a promising MSC for the treatment of a wide range of diseases. ADSC has been shown to lack major histocompatibility complex-II expression, and its immunosuppressive effects mediated by prostaglandin E2. Both preclinical and clinical studies have shown that allogeneic transplantation of ADSCs was able to control graft-versus-host disease. In regard to xenotransplantation a total of 27 preclinical studies have been published, with 20 of them performed with the investigators' intent. All 27 studies used ADSCs isolated from humans, possibly due to the wide availability of lipoaspirates. On the other hand, the recipients were mouse in 13 studies, rat in 11, rabbit in 2, and dog in 1. The targeted diseases varied greatly but all showed significant improvements after ADSC xenotransplantation. For clinical application in human medicine, ADSC xenotransplantation offers no obvious advantage over autotransplantation. But in veterinary medicine, xenotransplantation with porcine ADSC is a practical alternative to the costly and inconvenient autotransplantation.