Antithymocyte globulin induces ex vivo and in vivo depletion of myeloid and plasmacytoid dendritic cells

We investigate the in vivo and ex vivo effect of rabbit antithymocyte globulin (ATG) on myeloid dendritic cells (MDCs) and plasmacytoid dendritic cells (PDCs). After incubation with ATG and complement, the mean number of MDCs and PDCs decreases from 3,168 to 739 x 10/mL (P=0.004) and from 5,314 to 790 x 10/mL (P=0.01), respectively. In vivo ATG given as part of the conditioning regimen before allogeneic stem-cell transplantation induces a stronger reduction of circulating MDCs and PDCs than chemotherapy alone (reduction: 100% vs. 78%-98%). These data show that ATG induces depletion of circulating MDCs and PDCs, which might be in addition to the T-cell depletion a further mechanism to reduce graft-versus-host disease after allogeneic stem-cell transplantation.     

Immunotherapy of NOD mice with bone marrow-derived dendritic cells

We evaluated two bone marrow-derived dendritic cell (DC) populations from NOD mice, the murine model for type 1 human diabetes. DCs derived from GM-CSF [granulocyte/macrophage colony-stimulating factor] + interleukin (IL)-4 cultures expressed high levels of major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 molecules and were efficient stimulators of naive allogeneic T-cells. In contrast, DCs derived from GM-CSF cultures had low levels of MHC class II costimulation/activation molecules, were able to take up mannosylated bovine serum albumin more efficiently than GM + IL-4 DCs, and were poor T-cell stimulators. The two DC populations migrated to the spleen and pancreas after intravenous injection. To determine the ability of the two DC populations to modulate diabetes development, DCs were pulsed with a mixture of three islet antigen-derived peptides or with medium before injection into prediabetic NOD mice. Despite phenotypic and functional differences in vitro, both populations prevented in vivo diabetes development. Pulsing of the DCs with peptide in vitro did not significantly improve the ability of DCs to prevent disease, which suggests that DCs may process and present antigen to T-cells in vivo. In addition, we detected GAD65 peptide-specific IgG1 antibody responses in DC-treated mice. Overall, these results suggest that a Th2 response was generated in DC-treated mice. This response was optimal when using GM + IL-4 DCs, which suggests that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice.     

IL-13 prolongs allograft survival: association with inhibition of macrophage cytokine activation

Th2 cytokines, especially IL-4 and IL-10, may facilitate transplant tolerance induction but the role of IL-13, another Th2 cytokine, is not known. This study examined the effects of rat recombinant IL-13 (rIL-13) on alloimmune responses. In vitro effects of rIL-13 were compared in mixed lymphocyte cultures (MLC) on rat lymphocytes cultured with PVG stimulator cells. DA rats grafted with fully allogeneic PVG neonatal heart grafts were treated with 40,000 units of rIL-13 for 10 days and graft survival monitored by ECG. Cytokine mRNA expression in the graft and lymphoid tissues was studied by RT-PCR and alloantibody levels assayed. rIL-13 had no effect on MLC, unlike rIL-4 which enhanced proliferation and induced Th2 and inhibited Th1 cytokines in MLC. rIL-13 inhibited IL-12p35, IL-12p40 and TNF-alpha mRNA induction in dendritic cell cultures. Treatment with rIL-13 prolonged fully allogeneic PVG neonatal heart graft survival to 18-21 (13-27) days (median (range)); compared to 12 (9-15) days in untreated normal rejection (p<0.05) and 14 (10-24) days in sham treated controls (p<0.05). RT-PCR studies on graft tissue identified reduced mRNA expression for the dendritic cell/macrophage molecules iNOS, TNF-alpha and IL-12 compared to normal rejection. rIL-13 treatment did not increase Th2 cytokines as compared to normal rejection, or the Th2 dependent IgG1 alloantibody response, while IL-4 did. These studies demonstrated that rIL-13 can prolong allograft survival associated with inhibition of IL-12, TNF-alpha and iNOS mRNA induction, and suggest IL-13 could modify graft rejection by inhibition of dendritic cell and/or macrophage function.     

A subset of CD8+ T cells acquiring selective suppressive properties may play a role in GvHD management

Difficulty in segregating graft-versus-tumor effect (GvT) from graft-versus-host disease (GvHD) remains a major limitation of allogeneic stem cell transplantation (Allo SCT). Naturally occurring regulatory T cells have been suggested to suppress alloreactive T cells involved in GvHD; however, their non-selective suppressive effect raises concern regarding probable attenuation of the GvT effect. Recent studies suggested inducible CD8 (iCD8) cells to be useful in suppressing autoimmune reactions, although their function in the Allo SCT setting has not been fully explored. The current study assessed in-vitro the properties of iCD8 T cells, generated in response to allogeneic dendritic cells (DCs), imitating the Allo SCT conditions. CD25^? peripheral blood mononuclear cells (PBMCs) were stimulated with allogeneic DCs in mixed lymphocyte culture (MLC). The resultant iCD8⁺CD25⁺ population was isolated and assessed for phenotypic markers, cytokine expression profile, cell proliferation, inhibitory capacity and anti-viral response. The generated CD8⁺CD25⁺FOXP3⁺ T cells selectively inhibited the primary allogeneic response, without attenuating T cell response against other stimuli, such as mitogens or a cytomegalovirus (CMV) recall antigen. In conclusion, iCD8⁺CD25⁺ cells suppress allogeneic stimulation, while maintaining the capacity to respond to infectious pathogens. These cells could be potentially efficient in the Allo SCT setting, where GvHD prevention is required.     

Dendritic cells (I): Biological functions

Dendritic cells (DCs) are potent antigen presenting cells (APCs) that possess the ability to stimulate na?ve T cells. They comprise a system of leukocytes widely distributed in all tissues, especially in those that provide an environmental interface. DCs posses a heterogeneous haemopoietic lineage, in that subsets from different tissues have been shown to posses a differential morphology, phenotype and function. The ability to stimulate na?ve T cell proliferation appears to be shared between these various DC subsets. It has been suggested that the so-called myeloid and lymphoid-derived subsets of DCs perform specific stimulatory or tolerogenic function, respectively. DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens (Ags), and their subsequent presentation on the cell surface linked to major histocompatibility (MHC) molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues where they present Ag to T cells and induce an immune response. DCs are receiving increasing scientific and clinical interest due to their key role in anti-cancer host responses and potential use as biological adjuvants in tumour vaccines, as well as their involvement in the immunobiology of tolerance and autoimmunity.     

Involvement of dendritic cells in sarcoidosis

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown aetiology. It has been suggested that T helper type 1 (Th1) polarisation is associated with the pathophysiology of sarcoidosis, but the mechanism of skewing towards Th1 has not been elucidated. Dendritic cells (DCs) are known to regulate immune responses. This study was performed to determine whether DCs are involved in the aetiology of sarcoidosis. METHODS: The numbers of peripheral blood DCs in 24 patients with sarcoidosis were analysed and biopsy specimens from four patients were stained immunohistochemically using monoclonal antibodies. RESULTS: The numbers of both myeloid and lymphoid DC subsets were significantly decreased in the blood and mature DCs were found in the granulomas of patients with sarcoidosis. A number of interferon-gamma (IFN-gamma) producing T cells were also detected in the sarcoid granuloma, as well as many interleukin (IL)-4 producing T cells. Double staining of the biopsy specimen using anti-fascin and anti-CD3 antibodies showed an anatomical interaction between DCs and T cells. CONCLUSIONS: These findings suggest that the blood DC subsets may migrate into the affected tissues, contributing to the formation of the granulomas in sarcoidosis. It is hypothesised that the migrating DCs may regulate the T cell response in sarcoidosis, at least in the granulomatous lesions.     

未成熟树突状细胞在异种移植中的免疫递呈及调节作用

目的　观察树突状细胞 (DC)对异种抗原的免疫递呈作用 ,并探讨未成熟DC对T细胞的免疫调节作用。方法　从BALB/c小鼠骨髓诱生未成熟DC及成熟DC。将DC与小鼠T细胞进行混合淋巴细胞培养 (MLC)作为异种移植体外模型 ,以胰岛细胞作为靶细胞检测细胞毒效应。结果　MLC结果表明保留DC组及体内、外致敏成熟DC组刺激指数 (SI =2 .0 9,3 .91,2 .81)与去除DC组 (SI=1.0 9)相比差异有显著性。体内、外致敏未成熟DC组 (SI =1.0 8,1.16)与体内、外对照组(SI =2 .98,2 .81)相比差异有显著性。细胞毒检测表明成熟DC诱导T细胞最大杀伤活性为 61% ,而未成熟DC诱导T细胞最大杀伤活性为 9%。结论　DC主要以间接方式递呈异种抗原 ,而未成熟型DC可诱导异种抗原特异性T淋巴细胞无能。     

Effects of cyclophilin A on myeloblastic cell line KG-1 derived dendritic like cells (DLC) through p38 MAP kinase activation

BACKGROUND: Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein as well as a secreted protein and has recently been reported to be an immunomodulatory molecule. The objective of this study was to determine the effect of CypA on dendritic cell (DC) differentiation, activation, and functional maturation. The role of p38 MAP kinase in DC functions was also investigated. MATERIALS AND METHODS: KG1 cells (CD34+ human myeloblastic cell line) were treated with cytokines (GM-CSF+IL-4) and/or CypA and expression of cell surface markers was analyzed by FACS analysis. The antigen-uptake capacity of different DCs was determined by FITC-dextran uptake assay. Antigen-presentation capacity of DCs was determined by allogeneic mixed lymphocyte reaction (MLR) by [3H] thymidine incorporation assay. To assess the T cell polarization stimulated by KG1 derived DCs, various Th1 and Th2 cytokines secreted by allostimulated CD4+ and CD8+ T cells were determined by Bioplex cytokine assay. Total and phosphorylated p38 MAPK activity in CypA treated DCs was detected by Bioplex p38 total and phosphoprotein assay. RESULTS: During the differentiation of KG1 cells to immature DCs, cell surface expression of CD11b was increased by 30.6% for CypA alone, 55% for CypA plus cytokines, and 44% for cytokines alone. Similarly, CypA alone increased the cell surface expression of CD11c by 59% as compared to CypA plus cytokines (68%) and cytokines alone (50%). CypA up-regulated the antigen uptake capacity of the immature DCs to a greater extent (5 times) as compared to cytokines alone (2.5 times). Moreover, CypA augmented the capacity of DCs to present antigens to allogenic CD8+ T cells, and also increased the secretion of Th1 type cytokines TNF-alpha and IFN-gamma from the allogenic CD4+ T cells. Furthermore, CypA induced the phosphorylation and hence activation of MAP kinase p38. Pre-treatment with SB-203580, a p38 inhibitor, significantly reduced MLR stimulatory capacity of CypA-induced DCs in both CD8+ and CD4+ T cells (P < 0.05). CONCLUSIONS: CypA enhances DC differentiation and maturation by up-regulating CD11b and CD11c expression. CypA can also augment DC antigen uptake and antigen presentation, which may be mediated by the p38 signaling pathway.     

T cell antigenicity and immunogenicity of allogeneic exosomes

Extracellular vesicles, including exosomes, are regularly released by allogeneic cells after transplantation. Recipient antigen-presenting cells (APCs) capture these vesicles and subsequently display donor MHC molecules on their surface. Recent evidence suggests that activation of alloreactive T cells by the so-called cross-dressed APCs plays an important role in initiating the alloresponse associated with allograft rejection. On the other hand, whether allogeneic exosomes can bind to T cells on their own and activate them remains unclear. In this study, we showed that allogeneic exosomes can bind to T cells but do not stimulate them in vitro unless they are cultured with APCs. On the other hand, allogeneic exosomes activate T cells in vivo and sensitize mice to alloantigens but only when delivered in an inflammatory environment.     

Fibronectin restores defective in vitro proliferation of patients' lymphocytes after marrow grafting

In search for means of improving the impaired lymphocyte function of recipients after marrow grafting, we investigated the effect of fibronectin (FN) on patients' lymphocytes in allogeneic mixed lymphocyte cultures (MLC) and in cell-mediated lympholysis (CML) assays, since these tests are usually defective in transplanted patients. Four subgroups of marrow recipients were tested: patients within the first 100 days of transplantation (short-term) with (n = 16) or without (n = 14) acute graft-versus-host disease (GVHD), and long-term recipients with (n = 23) or without (n = 15) chronic GVHD. Exogenous FN (25 micrograms/ml) increased the proliferative response in the allogeneic mixed lymphocyte culture (MLC) significantly in cells from short-term patients without acute GVHD (+42%) and in those from long-term recipients with (+117%) and without chronic GVHD (+48%). In cells from patients with chronic GVHD, 3H-thymidine uptake after the addition of FN was enhanced to the level of that in lymphocytes of the corresponding marrow donor without exogenous FN. Fibronectin was effective only if added at the beginning of the MLC. In contrast to the results in MLC, exogenous FN failed to enhance phytohemagglutinin or OKT-3-induced lymphocyte proliferation and had no effect on CML activity. Moreover, FN did not show mitogenic activity in 3-6-day cultures. Our results demonstrate that FN in vitro is capable of restoring defective lymphocyte proliferation in marrow grafted patients, and circumstantial evidence suggests that this effect is mediated by an interaction between FN and monocytes.     

Failure of rat kidney nephron components to induce allogeneic lymphocytes to proliferate in mixed lymphocyte kidney cell culture

Rat kidney glomerular mesangial, glomerular epithelial, and tubular epithelial cells were isolated in virtually pure form and studied in mixed lymphocyte kidney cell culture (MLKC) for their ability to induce allogeneic spleen lymphocyte proliferation. The responses were compared with proliferative responses induced by allogeneic endothelial cells or spleen lymphocytes in the same strain combination. Stimulator cells were treated or left untreated with gamma-interferon, known to increase the major histocompatibility complex class II (and class I) expression of the stimulator cells. The results demonstrate that the nephron components are not able to induce lymphoproliferation in the MLKC. In contrast, the endothelial cells of rat heart were potent inducers of lymphoproliferation in mixed lymphocyte endothelial cell cultures (MLEC), as were allogeneic spleen cells. Although the kidney parenchymal cells have been shown to be immunogenic in vivo, the present finding suggests that they are unable to function as antigen-presenting cells on their own.     

Melanoma skews dendritic cells to facilitate a T helper 2 profile

BACKGROUND: Patients with progressing melanoma have a circulating cytokine profile reflecting a T helper cell type 2 (Th2) imbalance, while patients responding to therapy favor a Th1 profile. The aim of this study was to determine the role of circulating dendritic cells (DCs) in mediating this imbalance. METHODS: Isolated human peripheral blood mononuclear cells (PBMCs) were exposed to cell-free melanoma-conditioned medium (MCM) or control fibroblast-conditioned medium before stimulation. In separate experiments, isolated circulating DCs were exposed to MCM before addition of T cells. DC maturation and function were determined. Mixed leukocyte response T-cell proliferation was quantified and supernatants were assayed for Th1 (interleukin [IL]-2 and interferon gamma) and Th2 (IL-4, IL-5, and IL-10) cytokines. RESULTS: PBMCs exposed to MCM produced significantly more Th2-type cytokines (IL-4, IL-5, and IL-10) over time than those exposed to control medium. DCs exposed to MCM before addition of T cells, produced a similar pattern of a sustained longer term Th2 response after an initial burst of IL-2. Exposure to MCM did not significantly affect DC maturation or IL-12 production. T-cell proliferation did not change significantly in the mixed leukocyte response, however, the percentage of viable CD4+ T cells in the MCM-treated group was significantly less than control (37 vs 50%, P < .05). CONCLUSIONS: Exposure of PBMCs to melanoma produces a Th2-type cytokine profile, which may be, in part, facilitated by DCs.     

Changes in mixed lymphocyte culture-reactive lymphocytes following alloimmunization of single lymph nodes in sheep.

When an "isolated" single lymph node is challenged with irradiated allogeneic lymphocytes, there is a change in the reactivity of the lymphocytes flowing out of the node when they are cultured in vitro in unidirectional mixed lymphocyte cultures (MLC) against the immunizing lymphocytes. These changes in the reactivity of the recipient lymphocytes are shortlived, follow a set time sequence in relation to the cell traffic changes accompanying the immune response, are the property of small lymphocytes and not blast cells, are exhibited by surface Ig-negative cells, and they are specific for the donor lymphocytes. It is suggested that antigen causes the selective retention of antigen-specific lymphocytes within the stimulated node followed by the proliferation and differentiation of large numbers of antigen-specific cells, which then leave the lymph node as small lymphocytes.     

Alemtuzumab (Campath-1H) induction therapy and dendritic cells: Impact on peripheral dendritic cell repertoire in renal allograft recipients

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) and are pivotal for initiating allograft immunity. Recently, particular DC subsets have been implicated also in allogeneic T cell hyporesponsiveness. Alemtuzumab (anti-CD52, Campath-1H) is a novel T cell depleting antibody that is currently under investigation for the use in allogeneic organ transplantation. While recent studies demonstrated a conspicuous effect of alemtuzumab on peripheral DC in clinical graft-versus-host disease, its efficiency in patients receiving allogeneic organ transplants is still undefined. In the present study we assessed the peripheral DC repertoire in kidney transplant recipients after either alemtuzumab induction therapy followed by FK506 monotherapy or after conventional immunosuppression (FK506, mycophenolate mofetil and steroids) without any induction agent. Induction with alemtuzumab caused a strong and sustained reduction of the total number of peripheral DC and a significant shift from myeloid to plasmacytoid DC subsets (mDC/pDC ratio) as early as 1 month post-transplantation. These data show that alemtuzumab induction targets the peripheral DC repertoire, which might add another mechanism allowing immunosuppressive drug minimization. Further studies are warranted to further elucidate the functional significance of these finding in the setting of allogeneic organ transplantation.     

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.     

Induction of tolerance-inducing antigen-presenting cells in bone marrow cultures in vitro using monoclonal antibodies to CD200R

CD200 to CD200R interactions produce immunoregulation. We investigated whether the expression of CD200R on dendritic cell (DC) precursors affects their developmental fate. C57BL/6 bone marrow (BM) cells were cultured in vitro in the presence of (interleukin-4 + granulocyte-macrophage colony-stimulating activity) to generate allostimulatory DCs, which were in turn used to induce cytotoxic T-lymphocyte and cytokine production after culture with C3H responder spleen cells. Some marrow cultures included anti-CD200R antibodies. The inclusion of monoclonal antibodies in different isoforms of CD200R in the BM culture led to a generation of cells (tolerogenic DCs) that were unable to produce allostimulation in vitro with responder cells. Cells taken from these latter mixed leukocyte cultures (MLCs) now contained CD4(+)CD25(+) cells able to inhibit the antigen-specific MLC response of fresh C3H responder cells to stimulation with C57BL/6 cells, but not stimulation with BALB/c cells. Tolerogenic DCs, infused in vivo into mice receiving C57BL/6 skin grafts, produced antigen-specific decreased rejection of BL/6 allografts, not BALB/c allografts, compared with mice receiving control DCs (generated from BM in the absence of anti-CD200R). The induction of CD4(+)CD25(+) suppressor cells in MLCs using tolerogenic DCs from the initial BM cultures could be overcome by using limiting numbers of tolerogenic DCs and an excess of allostimulatory DCs derived from BM cultures maintained in the absence of anti-CD200R. These data indicate that anti-CD200R biases stem cells in BM toward the development of suppressive antigen-presenting cells, which can induce CD4(+)CD25(+) regulatory T cells. Tolerogenic DCs have the potential to modify graft acceptance in vivo.     

小鼠不成熟CD8α+树突状细胞的免疫抑制功能的体外实验

目的 观察不成熟CD8α+树突状细胞(DC)的体外免疫抑制功能.方法 取C57BL/6(H-2b)小鼠和Balb/c(H-2d)小鼠的骨髓和脾脏,制备不成熟CD8α+DC和脾淋巴细胞,并经丝裂霉素处理.采用甲基噻唑基四唑(MTT)法,实验设混合淋巴细胞反应(MLR)组(阳性对照组)、MLR加不同密度的同系CD8α+DC组、MLR加不同密度同种异型CD8α+DC组、MLR加不同密度CD8α+DC的培养上清组、CD8α+DC加同系T淋巴细胞组及阴性对照组.MLR组按刺激细胞/反应细胞10∶1建立.CD8α+DC按与反应细胞比例0.2∶1、0.5∶1、0.8∶1和1∶1的梯度加入MLR中分别建立同系CD8α+D组和同种异型CD8α+D组;MLR中加入不同密度(1×105/ml～5×106/ml)CD8α+DC的培养上清液建立CD8α+DC上清组;CD8α+DC与同系淋巴细胞共培养建立CD8α+DC加同系T淋巴细胞组;以反应细胞2×105/孔作为阴性对照.采用酶联免疫吸附试验法检测MLR加同系CD8α+DC组(1∶1)上清液中γ干扰素(IF-γ)和白细胞介素10(IL-10)的浓度.结果 同系和同种异型不成熟CD8α+DC对MLR均有抑制作用(P＜0.05),二者间差异无统计学意义(P＞0.05).抑制作用随DC比例的增加而增强,当CD8α+DC/反应细胞比例大于0.2时显示抑制作用(P＜0.05),比例为1时抑制作用最强.CD8α+DC体外刺激同系淋巴细胞增殖的能力较弱,当DC与T淋巴细胞的比值大于2时,显示出一定的刺激作用(P＜0.05),其培养上清液对MLR也有抑制作用(P＜0.05),其中密度5×105/ml的细胞培养上清液抑制作用最强.MLR加同系CD8α+DC组(1∶1)上清液中IL-10的含量为(451.9±12.2)pg/ml,IFN-γ的含量为(1.0±1.2)pg/ml.结论 不成熟CD8α+DC体外具有免疫抑制或诱导免疫耐受的功能,可产生较高水平的IL-10,CD8α+DC及其培养上清液均可抑制MLR.

Abstract：

Objective To observe the function of immature CD8α+ dentritic cells (DCs) in vitro. Methods The bone marrow and spleen of C57BL/6(H-2b) and Balb/c (H-2d) mice were got to prepare immature CD8α+ DCs and spleen lymphocytes,and treated by mytomycin. MTT test was used.MLR group, MLR plus variable density syngeneic CD8α+ DC group, MLR plus variable density allogeneic CD8α+ DC group,MLR plus variable density CD8α+ DC supernatant group,CD8α+ DC plus syngeneic T cell group and negative control group were established. MLR group was set up by responder cell ratio of 0.2,0.5,0.8,1.0,to build the MLR plus syngeneic and allogeneic CD8α+ DC experimental groups. Culture supernatant from different density (1 × 105/ml - 5 × 106/ml) of CD8α+DCs was added into MLR to build CD8α+ DC supernatant group. CD8α+ DCs were co-cultured with syngeneic T cells to build CD8α+ DCs plus syngeneic T cells group. 2 × 105/well responder cells served as the negative control group. ELISA was used to detect the concentrations of IFN-γ and IL-10 in the DCs could both suppress MLR (P＜0. 05), and the difference was not statistically significant (P＞0. 05). When CD8α+ DCs were increased, the suppressive effect was enhanced. When CD8α+ DC/responder cell ratio ＞0. 2, the inhibitory effect could be observed, and this effect reached the peak when the ratio was 1.0. The CD8α+ DCs had weak ability to stimulate syngeneic lymphocyte proliferation in vitro, and certain stimulating effect could be seen only when CD8α+ DC/responder cell ratio ＞2 (P＜0. 05). Its culture supernatant also showed suppressive effect (P＜0. 05), and the supernatant with a cell density of 5 × 105/ml showed the maximum effect. IL-10 concentration in the concentration was 1.0 ± 1.2 pg/ml. Conclusion The in vitro function of immature CD8α+ DCs was immunosuppression/tolerance,and they could secret high level of IL-10. The CD8α+ DCs and their culture supernatant could suppress MLR in vitro.     

In vitro immunogenicity of cadaver donor bone marrow cells used for the induction of allograft acceptance in clinical transplantation.

BACKGROUND: The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions to induce allograft acceptance in clinical transplantation is not fully understood. Aside from acting as immune responding and regulatory cells, the infused DBMC also may sensitize the recipient to the donor antigens. METHODS: To analyze this stimulatory activity of DBMC, in vitro mixed lymphocyte cultures (MLC) and cell-mediated lymphocytotoxicity (CML) culture systems analogous to the transplant model with DBMC infusion were used. RESULTS: When responding peripheral blood lymphocytes (PBL) from normal volunteers were placed in culture with suspensions of Ficoll-purified, T cell-depleted, un-irradiated allogeneic DBMC (NT-DBMC), a reaction was seen in both MLC and CML. However, when compared to allogeneic spleen cells as stimulating cells, the responses to NT-DBMC were of markedly lower magnitude and were not seen when the NT-DBMC was irradiated (3000 R). When responding PBL were stimulated with either NT-DBMC that had been previously cultured with irradiated cells from the responders for 1 week (activated NT-DBMC), NT-DBMC further depleted of CD15+ and glycophorin A-positive cells (NT-LP/DBMC), or purified CD34+ and CD2+ DBMC subsets, stronger lymphoproliferative and cytotoxic responses were observed. Moreover, these responses were not abrogated by irradiation of the stimulating DBMC subpopulations. Depletion of antigen-presenting cells by positive selection of CD3+ cells from the responding PBL abrogated MLC and CML reactivity, even when purified NT-LP/DBMC, the most stimulatory cells, were used. This latter observation was in contrast to the responses seen with cultures containing allogeneic stimulating spleen cell populations. This indicated the requirement for indirect alloantigen presentation, i.e., the failure of these DBMC to stimulate by direct alloantigen presentation. NT-DBMC was able to stimulate responding PBL in secondary MLC and CML responses with an equivalent magnitude, irrespective of whether the stimulators were spleen cells or NT-DBMC. Finally, the MLC and CML responses were inhibited by tacrolimus (FK506), mycophenolic acid (MPA), and cyclosporine (CsA) in a dose dependent manner, in contrast to previously observed refractoriness of DBMC preparations to these agents if DBMC was tested as responder cells or in modulatory assays. CONCLUSIONS: These results indicate that DBMC are able to function as effective in vitro stimulators, but only by indirect antigen presentation, and that the immune responses mediated by them can be down-regulated by their own inherent suppressive nature, an effect that can be enhanced by the presence of immunosuppressive drugs.