Dendritic Cells Transduced with SOCS1 Gene Exhibit Regulatory DC Properties and Prolong Allograft Survival

SOCS1 is a key regulator of cytokine signaling and is important for maintaining balance in the immune system. It is thought to participate in negative feedback loops in cytokine signaling and may be an important signal for the regulation of dendritic cell （DC） maturation. However, it remains unclear whether DCs transduced with SOCS1 exhibit characteristics of regulatory DCs and induce allogeneic T-cell hyporesponsiveness. In this study, we constructed adenovirai vector coding SOCS1 （Ad-SOCS1） that can efficiently increase SOCS1 gene expression in bone marrow-derived dendritic cells. DCs transduced with Ad-SOCS1 （DC-SOCS1） expressed low levels of costimulatory and MHC molecules, were resistant to maturation and activation stimulation, induced allogeneic T-cell hyporesponsiveness, and promoted the generation of regulatory-like T cells in vitro. DC-SOCS1 pretreatment significantly prolonged the survival of ailografts and led to a substantial increase in the generation of regulatory T cells. Our data suggest that SOCS1 inhibits DC maturation and induces regulatory DC generation, therefore possessing therapeutic potential to prevent rejection in organ transplantation.     

Failure of Pepsin-Digested Donor Specific Alloantibody to Prolong Renal Allograft Survival in Dogs

Donor specific F(ab')₂ alloantibody fragments, prepared by pepsin digestion of IgG obtained from hyperimmune cytotoxic alloantisera raised in third party dogs, was administered to canine renal allograft recipients in an attempt to allow expression of immunological enhancing properties of alloantibody in a species in which hyperacute rejection occurs. Pepsin digestion did abrogate the ability of donor specific cytotoxic alloantibody to mediate hyperacute rejection, but renal allograft survival in F(ab')₂ treated dogs was not prolonged over graft survival in untreated dogs. Factors which may contribute to the inability of this technique to prolong graft survival include (1) the difficulty, when using outbred animals, of assuring that the alloantibody is directed against all the antigens present in the donor and absent in the recipient, and (2) the decreased potency of F(ab')₂, when compared to an equivalent amount of intact IgG, as an inhibitor of a specific immune response.     

Failure of Pepsin-Digested Donor Specific Alloantibody to Prolong Renal Allograft Survival in Dogs

Donor specific F(ab')₂ alloantibody fragments, prepared by pepsin digestion of IgG obtained from hyperimmune cytotoxic alloantisera raised in third party dogs, was administered to canine renal allograft recipients in an attempt to allow expression of immunological enhancing properties of alloantibody in a species in which hyperacute rejection occurs. Pepsin digestion did abrogate the ability of donor specific cytotoxic alloantibody to mediate hyperacute rejection, but renal allograft survival in F(ab')₂ treated dogs was not prolonged over graft survival in untreated dogs. Factors which may contribute to the inability of this technique to prolong graft survival include (1) the difficulty, when using outbred animals, of assuring that the alloantibody is directed against all the antigens present in the donor and absent in the recipient, and (2) the decreased potency of F(ab')₂, when compared to an equivalent amount of intact IgG, as an inhibitor of a specific immune response.     

Monoclonal Antibody Treatment to Prolong the Secondary Cardiac Allograft Survival in Alloantigen‐primed Mice

We have previously shown that costimulation blockade using a combination of monoclonal antibodies (mAbs) – CTLA4Ig, antibodies to CD154, LFA‐1, and OX40L – can induce tolerance of cardiac allografts in mice with adoptively transferred CD4⁺ memory T cells [ 1 ]. However, the effect of costimulatory blockade in secondary allograft rejection has not been studied. B6 mice that rejected BALB/c skin grafts for more than 4 weeks (defined as alloantigen‐primed mice) were used as recipients. The recipient mice were treated with the mAbs to CD154, LFA‐1, OX40L, and CD122 on days 0, 2, 4, and 6 after the secondary transplantation of BALB/c heart. The mean survival time (MST) of secondary cardiac allografts in rats treated with antibodies to CD154 and LFA‐1 (2‐antibodies approach) and those treated with antibodies to CD154, LFA‐1, OX40L, and CD122 (4‐antibodies approach) was greater than that of the controls (MST = 6.7 days, 22.2 days, and 3.2 days, respectively). The 4‐antibodies approach prevented lymphocytic infiltration in the grafts, inhibited memory T‐cells proliferation in the spleen, increased IL‐10 secretion in the serum, and enhanced the expression of CD4⁺ Foxp3⁺ regulatory T cells (Tregs) in spleen. Expression levels of alloreactive antibodies were high in the recipient mice of experimental and control groups. Inhibiting the memory T cells by costimulation blockade extended allograft survival in secondary transplant models but could not induce tolerance of graft. Alloreactive antibodies may participate in alloresponse and play an important role in secondary cardiac allograft rejection.     

In vitro Assay for Screening of Optimal Targets for Antigen‐Delivery to Murine Dendritic Cells

Targeting of antigen to dendritic cells (DCs) increase the efficiency of immunization procedures and may facilitate the development of more effective vaccines. Several surface molecules on DCs have shown to be useful for antigen targeting, but many more deserves investigation for their efficacy in this respect. With this end in mind, a simple in vitro assay for screening of optimal targets for antigen‐delivery to murine DCs was established. Splenocytes from mice immunized with rat IgG were targeted in vitro with a panel of different rat monoclonal antibodies (mAbs) directed against surface markers on murine DCs. The resulting T‐cell activation was analysed by determining the number of IFN‐γ and IL‐4 secreting cells by ELISPOT. A positive effect of targeting was evident with several of the mAbs. Thus, mAbs against CD11c, CD36, CD205 and Clec7A all induced IFN‐γ responses that were significantly higher than those induced by non‐targeting control mAbs. Anti‐CD36 also induced IL‐4 responses that were significantly higher than the control. The assay described here allows simultaneous analysis of a large number of potential target structures and facilitates direct comparison between the different targets regarding the strength of the T‐cell responses induced by the targeted DCs. The assay could be useful as a first‐line screening of potential target structures on murine DCs.     

静息B细胞免疫诱导小鼠心肌移植物存活时间的延长

本文用小鼠耳后心肌移植作为模型观察静息B细胞在诱导延长移植物存活的作用。用C5 7BL/ 6新生小鼠的心脏移植给Balb/c成年雌性小鼠的耳后 ,受鼠在移植前 1周事先尾静脉注射C5 7BL/ 6静息B细胞 ,同时观察了用静息B细胞免疫后的Balb/c小鼠脾细胞对C5 7BL/ 6小鼠脾细胞的CTL活性。结果显示单独使用静息B细胞免疫组受鼠的同种移植物平均存活时间为 (16± 1 34 )d [空白对照组为 (10± 1 0 5 )d],单独注射阿霉素组心肌平均存活时间为 (14 6± 1 74)d ,静息B细胞预处理同时配合使用阿霉素组 ,心肌平均存活时间延长至 (2 7 8± 3 96 )d (P <0 0 0 1)。而活化B细胞组的小鼠排斥反应强烈 ,在移植后 8d之内全部排斥。静息B细胞免疫鼠对同种细胞的杀伤率 ,明显低于用活化B细胞免疫的对照组。     

Antigen‐presenting Cells in Pregnant and Non‐pregnant Human Myometrium

Citation Ivanisevic M, Segerer S, Rieger L, Kapp M, Dietl J, Kämmerer U, Frambach T. Antigen‐presenting cells in pregnant and non‐pregnant human myometrium. Am J Reprod Immunol 2010; 64: 188–196 Problem Inflammatory cells play a crucial role in human parturition. Different populations of leucocytes invade the reproductive tract. Numerous studies have described the decidual immune cell population in pregnant and non‐pregnant endometrium. However, little is known about the presence of immune cells in human myometrium. Method of study We herein analysed a spectrum of immune cells in human myometrium comparing tissue samples from non‐pregnant (n = 8) and pregnant (n = 10) uteri. Applying immunohistochemistry with a panel of antibodies specific for T cells, monocytes, natural killer cells, B cells and antigen‐presenting cells (CD4, CD8, CD14, CD15, CD16, CD19, CD56, CD68, CD83, HLA‐DR, DC‐Sign, mast cell tryptase), we characterized the immune cell population of human myometrium. Results A significantly higher number of CD14, CD15, CD16, DC‐SIGN as well as CD4‐positive cells were found in myometrium of pregnant compared to non‐pregnant uteri, while mast cells were significantly reduced in pregnant myometrium. Conclusion All markers found increased in pregnant myometrium indicate monocyte/macrophage lineage cells and thus suggest a possible involvement of these cells in healthy pregnancy maintenance. Monocytes/macrophages might produce a microenvironment that permits a controlled invasion of trophoblast cells into the myometrium while preventing a rejection of the semiallogenic conceptus and providing an important barrier against invading pathogenes.     

Generation of Potent Cytotoxic T Lymphocytes Against Castration‐Resistant Prostate Cancer Cells by Dendritic Cells Loaded With Dying Allogeneic Prostate Cancer Cells

To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)‐based immunotherapy against prostate cancer, various tumour antigens should be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for castration‐resistant prostate cancer (CRPC) using prostate cancer–specific CTLs generated in vitro by DCs. Monocyte‐derived DCs from patients with CRPC were induced to mature using a standard cytokine cocktail (in IL‐1β, TNF‐α, IL‐6 and PGE₂: standard DCs, sDCs) or using an α‐type 1‐polarized DC (αDC1) cocktail (in IL‐1β, TNF‐α, IFN‐α, IFN‐γ and polyinosinic:polycytidylic acid) and loaded with the UVB‐irradiated CRPC cell line PC‐3. Antigen‐loaded DCs were evaluated by morphological and functional assays. The αDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the αDC1s were loaded with tumour antigens or not, compared to sDCs. The αDC1s showed a higher production of interleukin‐12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumour antigens, as compared to standard DCs (sDCs). Prostate cancer–specific CTLs against autologous CRPC cells were successfully induced by αDC1s loaded with dying PC‐3 cells. Autologous αDC1s loaded with an allogeneic CRPC cell line can generate greater CRPC‐specific CTL responses as compared to sDCs and may provide a novel source of DC‐based vaccines that can be used for the development of immunotherapy in patients with CRPC.     

EBV LMP2A-specific T Cell Immune Responses Elicited by Dendritic Cells Loaded with LMP2A Protein

Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.     

转化生长因子β1基因修饰树突状细胞延长移植心脏存活时间的研究

为探讨移植前输注人转化生长因子 β1 (TGFβ1 )基因修饰供者树突状细胞 (DC )对移植心脏存活时间的影响。采用重组质粒TGFβ1 pcDNA3转染供者F344大鼠DC ,RT PCR和Westernblot检测转染后DC的TGFβ1基因表达。收集转染各组细胞静脉输注Lewis大鼠。 1周后检测TGFβ1 DC在受者大鼠脾和淋巴结的分布。另一部分输注大鼠接受DC来源的F344同种异体心脏移植 ,观察各组转染DC输注后移植心脏存活时间的变化以及相同时间点移植心脏重量的变化 ,比较移植后各时间点移植排斥反应级别以及输注不同DC各组移植心脏出现排斥反应的时间。结果 :TGFβ1 DC输注受者后 ,1周内可在脾和淋巴结内形成微嵌合 ,输注后DC在受者体内的迁移以及不同时间的存活数量不同。同时发现TGFβ1 DC输注大鼠 ,移植心脏存活时间 (33 1 4± 7 88)d比对照组 (6 5 7± 1 76 )d明显延长 ,移植后排斥反应级别明显低于对照组 ,并且该组移植心脏排斥反应出现时间也明显晚于各对照组。因此 ,TGFβ1 DC能有效抑制移植排斥反应 ,延长移植心脏存活时间。     

Combination of CTL‐associated Antigen‐4 Blockade and Depletion of CD25+ Regulatory T Cells Enhance Tumour Immunity of Dendritic Cell‐based Vaccine in a Mouse Model of Colon Cancer

Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via CTL‐associated antigen‐4 (CTLA‐4) blockade or removal of CD4⁺ CD25⁺ regulatory T (Treg) cells appears to be a promising strategy for cancer immunotherapy. In this study, we tested the hypothesis that the combination of CTLA‐4 blockade and depletion of Treg cells would improve the potency of dendritic cell (DC)‐based vaccine in a clinically relevant mouse model, which is transgenic for both carcinoembryonic antigen (CEA) and HLA‐A2 for the treatment of colon carcinoma in a therapeutic setting. We found that administration of anti‐CD25 antibody prior to vaccination or systemic administration of anti‐CTLA‐4 antibody with the vaccine improved tumour‐free survival against CEA‐expressing tumours compared with mice immunized with DC‐based vaccine alone. However, the efficacy of the vaccine proved to be most effective when anti‐CTLA‐4 antibody was combined with Treg inhibition. This vaccination strategy dramatically improved the tumour‐free survival and allowed the development of long‐lasting immune responses. The combined vaccination strategy resulted in increased secretion of IFN‐γ and enhanced HLA‐A2‐restricted CEA‐specific CTL responses. Furthermore, coadministration of anti‐CD25 and anti‐CTLA‐4 antibodies along with the vaccine was effective against more advanced tumours. These results provide evidence that simultaneous blockade of T‐cell regulatory pathways is a promising approach for the induction of therapeutic antitumour immunity against CEA⁺ colon carcinoma.     

Surface‐Mediated Priming During In Vitro Generation of Monocyte‐Derived Dendritic Cells

Ex vivo‐generated human dendritic cells (DC) are most commonly generated from monocytes using standard cell culture dishes. To elucidate the effect of the plastic surface during the differentiation process, we compared a standard adhesive plastic dish with four different mainly non‐adherent surfaces. Untouched monocytes were cultured for 3 days in the presence of IL‐4 and GM‐CSF. Time‐lapse videos were recorded, and the phenotype of the cells was analysed by flow cytometry. The cytokine profiles were analysed using a 25‐plex cytokine assay. The use of non‐adherent surfaces led to a significant reduction in expression of CD14 and CD38, and a significant increase in expression of CD86 compared to standard culture dishes. Expression levels of DC‐SIGN and PD‐L2 were reduced significantly on cells cultured on non‐adherent surfaces. The cytokine production was independent on the surface used. The surface‐mediated priming should therefore be considered when aiming to induce specific immune responses. This is especially important with regard to DC‐based immunotherapy, where an adjustment of the surface during the DC generation process might have highly beneficial effects.     

Development of Dendritic Cells from GM-CSF-/- Mice in vitro : GM-CSF Enhances Production and Survival of Cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF^-/- mice. Multiple cultures from GM-CSF^-/- and wild type mice were established and compared for cell production. GM-CSF^-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF^-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF^-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF^-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

The Antioxidative Effect of Heat‐Shock Protein 70 in Dendritic Cells

Reactive oxygen species (ROS) are produced by dendritic cells (DCs) during antigen presentation in contact hypersensitivity (CHS). ROS cause a number of non‐enzymatic protein modifications, such as carbonylation. Carbonylated proteins in DCs in response to hapten have not been fully identified yet. To identify the proteins carbonylated by ROS, murine epidermis‐derived DC line XS106 was challenged with a hapten, 2,4,6‐trinitrobenzene sulphonic acid (TNBS). MALDI‐TOF analysis revealed that heat‐shock protein 70 (HSP70) was one of the carbonylated proteins induced by TNBS. To verify the role of HSP70 in TNBS‐treated XS106 cell, we fused protein transduction domain (PTD) with HSP70 to facilitate protein delivery into the cell. The transfected fusion protein HSP70 within the cell caused transient increase of the cellular level of HSP70. Transient increase of HSP70 level in XS‐106 DCs resulted in inhibition of ROS production, carbonylation of HSP70, p38 MAPK activation and subsequently IL‐12 secretion. To investigate the effects of PTD–HSP70 in vivo, ear‐swelling experiments with 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) were performed in BALB/c mice. Pretreatment of PTD–HSP70 reduced the CHS response to TNCB in vivo. We report here that carbonylation of HSP70 by ROS is associated with the pathogenesis of CHS, suggesting possibility of HSP70‐targeting therapy in CHS.     

Gene‐based SNP genetic association study of the corticotropin‐releasing hormone receptor‐2 (CRHR2) in major depression

An increasing amount of data suggests that affective disorders are related to dysregulation of the hypothalamic‐pituitary‐adrenal (HPA) axis, the stress‐response system. Corticotropin‐releasing hormone receptor‐2 (CRHR2)‐deficient mice display a stress‐sensitive and anxiety‐like phenotype suggesting that the CRHR2 is a plausible functional candidate gene influencing the reactivity of the HPA axis and therefore the liability to develop affective disorders. In this study, a gene‐based single nucleotide polymorphism (SNP) map of the corticotropin‐releasing hormone receptor 2 (CRHR2) was constructed containing one synonymous cSNP in exon 10, two intronic SNPs, and two SNPs in the 5′ upstream regulatory region. No significant difference in allele or genotype frequency was found for four out of the five SNPs between Belgian unipolar (UP) patients and age‐, gender‐, and ethnicity‐matched controls. The cSNP did show allelic and genotypic association with borderline significance (P = 0.04). However, a replication study of this cSNP in a bipolar sample of Belgian origin and a Swedish UP sample did not show significant differences in allele and genotype frequencies. © 2002 Wiley‐Liss, Inc.     

Efficient Generation of Gene‐Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9‐Induced Homology‐Directed Repair in Zygotes

Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9)‐induced homology‐directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non‐homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR‐derived mutation, in this work, without inhibition of NHEJ, we first generated gene‐modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9‐induced HDR in zygotes using single‐strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9‐induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR‐derived mutation in pig zygotes, suggesting a possible balance for optimal HDR‐derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR‐derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off‐targeting, suggesting CRISPR/Cas9‐induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.     

Mannan Derivatives Instruct Dendritic Cells to Induce Th1/Th2 Cells Polarization via Differential Mitogen‐Activated Protein Kinase Activation

Mannan derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Immunization with antigen conjugated to oxidized mannan (OM) or reduced mannan (RM) have induced differential immune responses in mice. Yet, the adjuvant effect and differences in molecular profiles of OM and RM on APCs is unresolved. Here, we investigated the response of mouse bone marrow‐derived DCs to OM and RM. OM and RM stimulated DCs to produce differential Th1/Th2‐inducing cytokines in vitro. OM and RM‐activated DCs stimulated allogeneic T‐cell Th1 and Th2 polarization reaction. OM instruct DCs to stimulate Th1 responses via IL‐12p70 production, which depends on the phosphorylation of p38, RM barely induce IL‐12p70, but IL‐10 and IL‐4, and magnitude of ERK phosphorylation, which results in a Th2 bias. These findings indicate that OM and RM were potent adjuvant capable of directly initiating DC activation Th1 and Th2 polarization respectively.