Mesenchymal Stromal Cells Protect Endothelial Cells from Cytotoxic T Lymphocyte‐Induced Lysis

The integrity of the vasculature plays an important role in the success of allogeneic organ and haematopoietic stem cell transplantation. Endothelial cells (EC) have previously been shown to be the target of activated cytotoxic T lymphocytes (CTL) resulting in extensive cell lysis. Mesenchymal stromal cells (MSC) are multipotent cells which can be isolated from multiple sites, each demonstrating immunomodulatory capabilities. They are explored herein for their potential to protect EC from CTL‐targeted lysis. CD8⁺ T cells isolated from human PBMC were stimulated with mitotically inactive cells of a human microvascular endothelial cell line (CDC/EU.HMEC‐1, further referred to as HMEC) for 7 days. Target HMEC were cultured in the presence or absence of MSC for 24 h before exposure to activated allogeneic CTL for 4 h. EC were then analysed for cytotoxic lysis by flow cytometry. Culture of HMEC with MSC in the efferent immune phase (24 h before the assay) led to a decrease in HMEC lysis. This lysis was determined to be MHC Class I restricted linked and further analysis suggested that MSC contact is important in abrogation of lysis, as protection is reduced where MSC are separated in transwell experiments. The efficacy of multiple sources of MSC was also confirmed, and the collaborative effect of MSC and the endothelium protective drug defibrotide were determined, with defibrotide enhancing the protection provided by MSC. These results support the use of MSC as an adjuvant cellular therapeutic in transplant medicine, alone or in conjunction with EC protective agents such as defibrotide.     

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.     

The Cytotoxic Reactivity and Sialic Acid Content of Human Lymphoid Cells

Human lymphocytes treated with neuraminidase show cytotoxicity reactions with a larger number of alloantisera against human leukocyte antigens than do untreated lymphocytes. In general, untreated thymus cells from the lymphocyte donor give reactions comparable to those of the neuraminidase treated lymphocytes with both alloantisera and sera which fail to react with untreated lymphocytes. However, certain sera show clear distinctions between lymphocytes and thymocytes, some reacting only with thymus cells and others only with lymphocytes. The cytotoxic reactivity of lymphocytes and thymocytes correlates with the amount of sialic acid released from the cells by neuraminidase treatment in that thymocytes release less acid than cells from spleen, adenoids, tonsils, or peripheral blood.     

A Suppressor of Cytokine Signaling 1 Antagonist Enhances Antigen-Presenting Capacity and Tumor Cell Antigen-Specific Cytotoxic T Lymphocyte Responses by Human Monocyte-Derived Dendritic Cells

The suppressor of cytokine signaling 1 (SOCS1) has emerged as a critical inhibitory molecule for controlling the cytokine response and antigen presentation by dendritic cells (DCs), thereby regulating the magnitude of both innate and adaptive immunity. The aim of this study was to investigate whether the SOCS1 antagonist pJAK2(1001-1013) peptide can weaken or block the inhibition function of SOCS1 in DCs by evaluating the phenotype and cytokine production, antigen-presenting, and specific T-cell-activating capacities of DCs electroporated with human gastric cancer cell total RNA. Furthermore, STAT1 activation of the JAK/STAT signal pathway mediated by SOCS1 was analyzed by Western blotting. The results demonstrate that the SOCS1 antagonist pJAK2(1001-1013) peptide upregulated the expression of the maturation marker (CD83) and costimulatory molecule (CD86) of RNA-electroporated human monocyte-derived mature DCs (mDCs), potentiated the capacity of mDCs to induce T-cell proliferation, stimulated the secretion of proinflammatory cytokines, and enhanced the cytotoxicity of tumor cell antigen-specific CTLs activated by human gastric cancer cell total RNA-electroporated mDCs. Data from Western blot analysis indicate that STAT1 was further activated in pJAK2(1001-1013) peptide-loaded mDCs. These results imply that the SOCS1 antagonist pJAK2(1001-1013) peptide is an effective reagent for the enhancement of antigen-specific antitumor immunity by DCs.     

The Recognition of Hypothalamo-Neurohypophysial Functions by Developing T Cells

Neuropeptide signals and specific neuropeptide receptors have been described in the thymus supporting the concept of a close dialogue between the neuroendocrine and the immune systems at the level of early T-cell differentiation. In this paper, we review recent data about neurohypophysial (NHP)-related peptides detected in the thymus from different species. We suggest that we are dealing in fact with other member(s) of the NHP hormone family, which seems to exert its activity locally through a novel model of cell-to-cell signaling, that of cryptocrine communication. This model involves exchange of signals between thymic epithelial cells and developing thymocytes. The NHP-related peptides have been shown to trigger thymocyte proliferation and could induce immune tolerance of this highly conserved neuroendocrine family.     

Intranasal Immunization with Cytotoxic T-Lymphocyte Epitope Peptide and Mucosal Adjuvant Cholera Toxin: Selective Augmentation of Peptide-Presenting Dendritic Cells in Nasal Mucosa-Associated Lymphoid Tissue

We previously reported that cholera toxin (CT) was required as a mucosal adjuvant for the induction of peptide-specific cytotoxic T lymphocytes (CTL) following intranasal immunization with CTL epitope peptides (A. Porgador et al., J. Immunol. 158:834–841, 1997). The present study was performed to identify the site and the antigen-presenting cell (APC) population responsible for the presentation of intranasally administered CTL epitope peptide immunogens and to determine whether CT directly affects antigen presentation by these APCs. For these experiments, C57BL/6 mice were intranasally immunized with the ovalbumin H-2K^b-restricted CTL epitope SIINFEKL with or without CT. Cells were then isolated from the cervical lymph nodes (CLN) and the nasal mucosa-associated lymphoid tissue (NALT) and tested for the ability to stimulate the B3Z T-cell hybridoma, which recognizes SIINFEKL in association with H-2K^b. Dendritic cell (DC)-enriched CLN cells from mice immunized with peptide and CT or peptide only could stimulate B3Z cells, while DC-depleted CLN cells from either group were unable to stimulate B3Z cells. NALT cells of mice immunized with peptide and CT, but not with peptide alone, were able to efficiently stimulate B3Z hybridomas. Depletion of N418-positive DC from these NALT cells resulted in significant reduction of B3Z activation. Our results indicate that DC are the APC responsible for the presentation of CTL epitope peptides following intranasal immunization and that CT augments the ability of dendritic cells in the NALT, but not in the draining CLN, to present CLT epitope peptides. This finding suggests that CT acts locally as a mucosal adjuvant and that NALT DC are the predominant APC involved with the induction of immunity after intranasal immunization with peptide immunogens and CT.     

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.     

Changes in the Surface of Virus-Infected Cells Recognized by Cytotoxic T Cells

Infection of cells with either ectromelia or lymphocytic choriomeningitis (LCM) virus in the presence of 2-deoxy-D-glucose (2-DOG) inhibited by up to 70% the extent to which the infected cells become susceptible to virus-specific cell-mediated lysis. The concentration of 2-DOG used had little effect on the extent of total protein synthesis (incorporation of [35S] methionine) but inhibited (up to 25%) glycoprotein synthesis, as measured by incorporation of [3H] fucose. This suggested that glycoprotein synthesis was a necessary event for infected cells to become susceptible to T-cell mediated lysis. The profiles (polyacrylamide gel electrophoresis) of newly synthesized, cellular glycoproteins from unifected and ectromelia-infected cells in the presence and absence of 2-DOG were compared and found to be very complex, with only minor changes. However, when convalescent serum from infected mice was used to isolate newly synthesized components from the cell surface shortly after infection, it showed four main species ranging in size from 25,000 to 70,000 daltons. 2-DOG inhibited production of these by 70%, thus corresponding to the biological data. The nature of the new glycoproteins seen in infected cells and whether they are in fact the structures recognized by effector T cells remain to be determined.     

Changes in the Surface of Virus-Infected Cells Recognized by Cytotoxic T Cells

P-815 mastocytoma cells developed susceptibility to immune T-cell-mediated cytolysis shortly after infection by ectromelia virus. Intracellular viral replication and late protein synthesis seem to bu unnecessary events. Interference with early protein synthesis, however, inhibits the development of susceptibility to lysis. The important intracellular events necessary for subsequent cytolysis appear to occur within 1 hour of infection. Virus rendered non-infectious by ultraviolet irradiation but not by gamma irradiation is able to induce these changes. By determining the minimum and essential events of the infectious process which result in T-cell-mediated cytolysis, the task of establishing the molecular changes occurring in the target cell surface membrane necessary for immune T-cell recognition should be simplified.     

The Novel Association of the Chemokine CCL22 with Abdominal Aortic Aneurysm

The aim of this study was to examine aortic biopsies with a cytokine array to identify new cytokines associated with abdominal aortic aneurysm (AAA). We assessed the relative expression of 79 cytokines using antibody-based cytokine arrays in a total of 12 AAA and 12 control aortic biopsies. Based on these findings we validated the findings for one cytokine by examining a further 11 AAA and 11 atherothrombosis biopsies and serum from 1028 men, 315 of whom had an AAA. Three cytokines (interleukins 1B and 8, and Chemokine CC motif ligand 22 [CCL22]) were consistently up-regulated in AAA biopsies. Since CCL22 had not previously been associated with aortic dilatation, we confirmed the upregulation of this cytokine in further tissue biopsies and serum using enzyme-linked immunosorbent assay. Median serum concentrations of CCL22 were greater in men with AAA (0.69 ng/ml) than controls (0.56 ng/ml, P < 0.01). Serum CCL22 was independently associated with both small (OR 1.51, 95% CI 1.21–1.88) and large AAA (OR 1.33, 95% CI 1.08–1.62) after adjusting for other risk factors. The association between CCL22 and AAA was also confirmed using immunohistochemistry. The results presented in this study demonstrate a novel association between CCL22 and AAA as well as illustrate how a protein array can be used to identify novel markers of potential pathogenic and diagnostic significance for AAA.Abdominal aortic aneurysm (AAA) is recognized as an important cause of mortality.¹ Marked accumulation of leukocytes, macrophages, B and T cells is a consistent finding in biopsies of human AAA.^2,3 The influx, migration, and effects of these cells are controlled by an array of pro-inflammatory cytokines, chemokines, and growth factors (referred to collectively as cytokines in this article).^4,5 High concentrations of various cytokines have been demonstrated in both AAA biopsies and within the circulation of patients with AAA.^6,7,8 Some cytokines, such as interleukin (IL) 6, have been shown to be present within the circulation at increased concentrations distal to AAAs, suggesting they are released directly from the aortic wall.⁸ At present, however, which cytokines are most relevant to AAA is not clear.We hypothesized that cytokines up-regulated within human AAA biopsies would also be present in increased concentrations within the circulation of patients. The aim of this study was to identify cytokines consistently up-regulated within human AAA biopsies using antibody arrays to measure relative expression of 79 proteins. The up-regulation of one cytokine in human AAA, not previously associated with aortic dilatation, was validated in additional aortic biopsies using alternative outcome techniques and further examined within the blood of 1028 men to assess any association between circulating levels and AAA.     

激活的脐血树突状细胞对肿瘤细胞的直接杀伤作用

探索脐血树突状细胞 (DC)在γ干扰素 (IFN γ )及细菌脂多糖 (LPS )激活前后对肿瘤细胞杀伤活性的差异。分离健康脐血单核细胞 ,用重组粒单细胞集落刺激因子 (GM CSF )、白介素 4 (IL 4 )和α肿瘤坏死因子α (TNF α )诱导为DC。于诱导第11天在合成培养基中加入LPS或IFN γ继续培养 12h ,将其激活。用流式细胞仪检测DC表面共刺激分子的改变 ,以明确LPS或IFN γ对DC的不同刺激作用 ;同时 ,以恶性血液病细胞株Jurkat及Daudi为靶细胞 ,以不同效靶比 (E∶T )与DC共同培养 18h ,采用51Cr释放试验检测DC激活前后抗肿瘤活性的差异。结果 (1)LPS及IFN γ可不同程度地上调DC表面CD86、CD80、CD83及CD1a的表达 ,尤以LPS刺激组明显 ;(2 )DC在未加刺激因子前能有效杀伤Jurkat细胞 ,在效靶比为 2 0∶1时 ,LPS或IFN γ可使其杀伤活性进一步提高至 5 0 0 %、 36 9% ,均与未加刺激因子前有显著差异 (P <0 0 0 1,P <0 0 2 5 ) ;(3)在效靶比为 2 0∶1时 ,LPS可使DC对Daudi的杀伤率提高至 19 8% (P <0 0 2 5 ) ,而未加刺激因子组及IFN γ刺激组DC对Daudi在任何效靶比均未显示明显的杀伤作用。LPS或IFN γ激活的脐血DC对Jurkat及Daudi细胞具有选择性杀伤作用     

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.     

Induction of Bcl‐xL‐Specific Cytotoxic T Lymphocytes in Mice

The induction of active immunity against tumour‐associated antigens to prevent relapse of cancer is a promising approach but has so far shown only low efficacy. This low efficacy may in part be due to clonal escape of tumour cell variants by the downregulation of antigen expression or inflammation‐induced dedifferentiation. Identification of novel tumour‐associated antigens that at the same time are essential for continued tumour cell survival is thus critical for the development of active cancer vaccinations. At the same time, identification of novel endogenous murine tumour antigens will help improve preclinical development of cancer immunotherapy. The anti‐apoptotic protein Bcl‐xL has been suggested to be such an essential tumour antigen, but the lack of well‐defined murine epitopes have delayed preclinical studies of Bcl‐xL‐targeting cancer vaccines. Here, we report the identification of two novel murine tumour‐associated epitopes TAYQSFEQV and AFFSFGGAL derived from mouse Bcl‐xL. Dendritic cell (DC)‐based vaccination induced CD8⁺ T cells capable of producing IFN‐γ upon restimulation with these epitopes. Thus, our data may benefit the design of future immunotherapy strategies by providing a preclinical model for cancer vaccination with an endogenous tumour antigen that can be combined with other cancer treatments.     

小鼠脾脏树突状细胞免疫功能的体外研究

本实验通过对小鼠脾脏分离到的树突状细胞免疫功能的研究表明,在ConA,PHA刺激的淋巴细胞增殖反应和混合淋巴细胞反应(MLR)中树突状细胞具有明显强于巨噬细胞的抗原递呈作用(P<0.001或P<0.005)。树突状细胞的这种作用可被特异性的单克隆抗体所抑制(抑制率>95％)。少量的巨噬细胞可促进其作用。