Individual myeloma‐specific T‐cell clones eliminate tumour cells and correlate with clinical outcomes in patients with multiple myeloma

Despite novel treatment strategies, multiple myeloma (MM) remains an incurable disease with low immunogenicity and multiple immune defects. We developed an ex vivo strategy for inducing myeloma‐specific cytotoxic T lymphocytes (CTLs) and demonstrate the possibility of identification and long‐term in vivo monitoring of individual myeloma‐specific T‐cell clones using the most sensitive clonotypic assay that is able to detect low frequencies of T‐cell clones (1 clonotypic cell in 10⁶ cells). Ten patients with MM were examined for the presence of tumour‐reactive T cells using dendritic cells loaded with autologous tumour cells. All patients had detectable myeloma‐reactive T cells in vitro. Expanded myeloma‐reactive T cells demonstrated specific cytotoxic effects against autologous tumour cells in vitro (median 39·6% at an effector:target ratio of 40:1). The clonality of myeloma‐specific T cells was studied with a clonotypic assay, which demonstrated both oligoclonal and monoclonal populations of myeloma‐specific T cells. CD8⁺ CTLs were the most immunodominant myeloma‐specific T‐cell clones and clinical responses were closely associated with the in vivo expansion and long‐term persistence of individual CD8⁺ T‐cell clones, usually at very low frequencies (10^?3–10^?6). We conclude that the clonotypic assay is the most sensitive tool for immunomonitoring of low‐frequency T cells.     

Dendritic cells fused with allogeneic hepatocellular carcinoma cell line compared with fused autologous tumor cells as hepatocellular carcinoma vaccines

Purpose: To investigate the specific antitumor responses against autologous hepatocellular carcinoma (HCC) cells of dendritic cells (DCs) fused with allogeneic HCC cell line, and evaluated the feasibility of BEL7402 as an alternative strategy to deliver shared HCC antigens to DCs. Methods: Previous studies demonstrated fusions of patient‐derived DCs and autologous tumor cells could induce T‐cell responses against autologous tumors. These fusion cells require patient‐derived tumor cells, which are not, however, always available. Here, we report the fusing of autologous DCs with allogeneic HCC cell line to induced cytotoxic T‐lymphocyte (CTL) response against autologous HCC cells compare with autologous tumor cells. Results: These DC/ BEL7402 fusion cells co‐expressed tumor‐associated antigens and DC‐derived costimulatory and major histocompatibility complex molecules. Both CD4+ and CD8 T+ cells were activated by the fusion cells as demonstrated by the proliferation of T‐cells, the production of cytokines and the simultaneous induction of specific CTL responses. Significantly, CTL induced by dendritic cell/allogeneic BEL7402 fusion cells were able to kill autologous HCC cells by human leukocyte antigen‐A2 restricted mechanisms. The results did not show significant difference between DC fusion with autologous hepatocellular carcinoma cells and DC fusion with allogeneic hepatocellular carcinoma cell line. Conclusions: The fusion of allogeneic HCC cell line and autologous DCs may have applications in antitumor immunotherapy through cross‐priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

Selective functional deficit in dendritic cell--T cell interaction is a crucial mechanism in chronic hepatitis B virus infection

Summary. A defect in specific T cell immunity has long been assumed to be the central mechanism of persistent Hepatitis B virus (HBV) infection. Recent studies on HBV transgenic mice have suggested, however, that functional deficit of dendritic cells (DC) was an underlying cause for the T cell dysfunction. The functions of monocyte‐derived DC were determined by studying 75 subjects that included chronic hepatitis B patients with low or high HBV load; antibody to hepatitis B surface antigen (anti‐HBs) positive individuals who had recovered completely from previous acute HBV infection; healthy donors who had received hepatitis B vaccination and were anti‐HBs positive; and immunologically naïve to HBV or the vaccine individual. Impaired interactions between monocyte‐derived DC and T cells were shown in chronic HBV infection patients, especially in those with active virus replication. The dysfunctions included: (i) failure of DC to increase human leukocyte antigen (HLA‐II), B7 expression and interleukin‐12 secretion in responses to hepatitis B surface antigen (HBsAg), (ii) defective induction of T cell proliferative response to HBsAg, (iii) failure to activate T cells to produce cytokines and (iv) deficit in the induction of antigen specific cytotoxic T lymphocytes (CTLs). In vitro treatment of DC with tumour necrosis factor‐α improved HLA‐II and B7 expression, as well as Th cell and CTL responses. It is concluded that defective DC‐T cell interactions may account for the specific T cell immune defects in chronic HBV infection. Immunotherapy that aims at restoring DC functions could offer a new opportunity for effectively managing persistent HBV infections.     

Generation of cellular immune responses to HCV NS5 protein through in vivo activation of dendritic cells

Summary. Chronic hepatitis C (HCV) infection is a substantial medical problem that leads to progressive liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The aim of this study was to achieve sustained cellular immune responses in vivo to a HCV nonstructural protein using dendritic cell (DC)‐based immunization approach. We targeted the HCV NS5 protein to DCs in vivo by injecting microparticles loaded with this antigen. The DC population was expanded in BALB/C mice (H‐2^d) by hydrodynamic injection of a plasmid pUMVC3‐hFLex expressing the secreted portion of the human Fms‐like tyrosine kinase receptor‐3 ligand (hFlt3). Mice were subsequently injected with microparticles coated with HCV NS5 protein via the tail vein. Cellular immune responses were determined with respect to secretion of INFγ and IL2 by CD4⁺ cells and cytotoxic T‐lymphocyte (CTL) assays in vitro; inhibition of tumour cell growth was employed for the assessment of CD8⁺ generated activity in vivo. We found that Flt3L treatment expanded the DC population in the spleen to 43%, and such cells displayed a striking upregulation of CD86 as well as CD80 and CD40 co‐stimulating molecules. Viral antigen‐specific T_H1 cytokine secretion by splenocytes was generated, and CTL activity against syngeneic NS5 expressing myeloma target cells was observed. In addition, these cells inhibited tumour growth indicating that NS5‐specific robust CTL activity was operative in vivo. Thus, the capability of activating DCs in vivo using the methods described is valuable as a therapeutic vaccine strategy for chronic HCV infection.     

树突细胞负载骨髓瘤抗原介导的免疫反应

目的 研究负载肿瘤抗原的树突细胞能否诱导特异性细胞霉T淋巴细胞反应。方法 用多发性骨髓瘤患者骨髓CD34^ 细胞诱生树突细胞，并将骨髓瘤冻融物冲击致敏树突细胞。采用MTT法检测骨髓瘤抗原致敏及未致敏树突细胞诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞株)的杀伤率。结果 骨髓瘤冻融物致敏树突细胞诱导的自身T细胞对患者骨髓瘤细胞的杀伤远大于对K562细胞的杀伤。结论 患者骨髓瘤冻融物致敏的树突细胞能有效诱导自身T细胞发生特异性抗肿瘤免疫。     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

Tumour cell/dendritic cell fusions as a vaccination strategy for multiple myeloma

Multiple myeloma (MM) cells express certain tumour-associated antigens (TAAs) that could serve as targets for active-specific immunotherapy. The aim of the present study was to test the MM/dendritic cell (DC) fusion as a vaccination strategy. We fused MM cells with DC to generate fusion cells (FCs) and tested their antigen presenting cell (APC) function in mixed lymphocyte reactions and cytotoxicity assays. First, the HS Sultan and SK0-007 HAT sensitive human MM cell lines and DCs generated from peripheral blood of normal donors were fused in the presence of 50% polyethylene glycol to form FCs. Next, tumour cells freshly isolated from patients were similarly fused with autologous DCs to generate FCs. The FCs demonstrated a biphenotypic profile, confirmed both by flow-cytometry and dual immunofluorescence microscopy. These FCs induced MM-specific cytotoxicity. FCs, but not MM cells or DCs alone, were potent stimulators of autologous patient T cells. More importantly, FC-primed autologous peripheral blood mononuclear cells demonstrated major histocompatibility complex-restricted MM-specific cytolysis. These studies therefore demonstrated that MM/DC FC can trigger an autologous immune response to MM cells and formed the framework for a clinical trial currently underway.     

Hepatitis C virus NS4 protein impairs the Th1 polarization of immature dendritic cells

Summary. Dendritic cells (DCs) in chronic hepatitis C patients display impaired function, although the details remain unclear. To investigate the hepatitis C virus (HCV) protein that has the most impact on DC function, we compared five recombinant proteins and seven HCV protein genes in modulating DC phenotype and function. Immature DCs (iDCs) were established from healthy donor peripheral blood monocytes with granulocyte–macrophage colony stimulating factor (GM‐CSF) and IL‐4. Lipopolysaccharide was used to establish mature DCs (mDCs). Cells were then pulsed with HCV recombinant proteins or transfected with HCV plasmids and subsequently assayed for cell surface marker expression by flow cytometry. For cytokine and proliferative T‐cell response analysis, DCs were cultured with autologous CD4 T cells and tuberculin purified protein derivative (PPD). Mean fluorescent intensity of CD86 was reduced in HCV protein‐pulsed iDCs. Proliferative T‐cell responses and Th1 cytokine concentrations were reduced with HCV nonstructural proteins (NS), particularly with HCV NS4. HCV nonstructural proteins, particularly NS4, change the iDC phenotype and reduce antigen‐specific T‐cell stimulatory function with Th1 cytokine reductions.     

High titre of antiglutamic acid decarboxylase autoantibody is a strong predictor of the development of thyroid autoimmunity in patients with type 1 diabetes and latent autoimmune diabetes in adults

Objective Type 1 diabetes mellitus (T1DM) is frequently associated with autoimmune thyroid diseases (AITD), but little is known about the risk of AITD in latent autoimmune diabetes in adults (LADA). We evaluated the genetic and immunological factors involved in the development of thyroid autoimmunity in patients with LADA and T1DM. Patients and measurements One hundred and ninety T1DM and 135 LADA patients were recruited in the study. Thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TGAb), glutamic acid decarboxylase antibody (GADA) and thyroid function were measured. The cytotoxic‐lymphocyte‐associated antigen‐4 (CTLA‐4) +49A/G and CT60 polymorphisms and the human leucocyte antigen (HLA)‐DQA1‐DQB1 genotype were determined. Results The prevalence of thyroid antibodies (TGAb and/or TPOAb) and thyroid dysfunction was 27·4% and 9·5% in patients with T1DM, and 21·5% and 11·1% in patients with LADA. Thyroid‐antibody‐positive T1DM patients had higher frequencies of GADA and HLA‐DQA1*03‐DQB1*0401 haplotypes than thyroid‐antibody negatives (P < 0·05). Thyroid‐antibody‐positive LADA patients had higher GADA titre, lower C‐peptide levels and higher frequencies of HLA‐DQA1*03‐DQB1*0401 haplotypes (P < 0·05). The CTLA‐4 +49A/G and CT60 polymorphism was associated with T1DM complicated with thyroid autoimmunity (OR = 2·33 and 2·54). Logistic regression revealed that only high‐titre GADA was associated with development of thyroid autoimmunity in patients with T1DM and LADA (OR = 3·50 and 3·10, respectively), and the presence of thyroid antibody predicted high risk for thyroid dysfunction in patients with T1DM and LADA (OR = 9·25 and 10·70, respectively). Conclusion Regular screening of thyroid antibody and function are recommended, especially in patients with T1DM and LADA with high GADA titre.     

Immunotherapy by autologous dendritic cell vaccine in patients with advanced HCC

Background

Dendritic cells (DCs) could be used as potential cellular adjuvant for the production of specific tumor vaccines.

Objectives

Our study was aimed to evaluate the safety and efficacy of autologous pulsed DC vaccine in advanced hepatocellular carcinoma (HCC) patients in comparison with supportive treatment.

Methods

Thirty patients with advanced HCC not suitable for radical or loco-regional therapies were enrolled. Patients were divided into 2 groups, group I consisted of 15 patients received I.D vaccination with mature autologous DCs pulsed ex vivo with a liver tumor cell line lysate. Group II (control group, no. 15) received supportive treatment. One hundred and 4 ml of venous blood were obtained from each patient to generate DCs. DCs were identified by CD80, CD83, CD86 and HLA-DR expressions using flow cytometry. Follow up at 3, and 6 months post injection by clinical, radiological and laboratory assessment was done.

Results

Improvement in overall survival was observed. Partial radiological response was obtained in 2 patients (13.3 %), stable course in 9 patients (60 %) and 4 patients (26.7 %) showed progressive disease (died at 4 months post-injection). Both CD8⁺ T cells and serum interferon gamma were elevated after DCs injection.

Conclusion

Autologous DC vaccination in advanced HCC patients is safe and well tolerate.     

Successful cross-presentation of allogeneic myeloma cells by autologous alpha-type 1-polarized dendritic cells as an effective tumor antigen in myeloma patients with matched monoclonal immunoglobulins

For wide application of a dendritic cell (DC) vaccination in myeloma patients, easily available tumor antigens should be developed. We investigated the feasibility of cellular immunotherapy using autologous alpha-type 1-polarized dendritic cells (αDC1s) loaded with apoptotic allogeneic myeloma cells, which could generate myeloma-specific cytotoxic T lymphocytes (CTLs) against autologous myeloma cells in myeloma patients. Monocyte-derived DCs were matured by adding the αDC1-polarizing cocktail (TNFα/IL-1β/IFN-α/IFN-γ/poly-I:C) and loaded with apoptotic allogeneic CD138(+) myeloma cells from other patients with matched monoclonal immunoglobulins as a tumor antigen. There were no differences in the phenotypic expression between αDC1s loaded with apoptotic autologous and allogeneic myeloma cells. Autologous αDC1s effectively took up apoptotic allogeneic myeloma cells from other patients with matched subtype. Myeloma-specific CTLs against autologous target cells were successfully induced by αDC1s loaded with allogeneic tumor antigen. The cross-presentation of apoptotic allogeneic myeloma cells to αDC1s could generate CTL responses between myeloma patients with individual matched monoclonal immunoglobulins. There was no difference in CTL responses between αDC1s loaded with autologous tumor antigen and allogeneic tumor antigen against targeting patient's myeloma cells. Our data indicate that autologous DCs loaded with allogeneic myeloma cells with matched immunoglobulin can generate potent myeloma-specific CTL responses against autologous myeloma cells and can be a highly feasible and effective method for cellular immunotherapy in myeloma patients.     

Mechanism of restoration of immune responses of patients with chronic hepatitis B during lamivudine therapy: increased antigen processing and presentation by dendritic cells

Summary. Restoration of host immunity has been reported in patients with chronic hepatitis B (CHB) after treatment with lamivudine; however, the underlying mechanisms of this treatment have not been determined. This study examined the role of antigen‐presenting dendritic cells (DC) in restoration of host immunity. Circulating DC were isolated from peripheral blood of 23 patients with CHB before and 1, 3, and 12 months after starting lamivudine therapy. The non‐antigen‐specific proliferation of DC was assessed in allogenic mixed leucocyte reaction. Dendritic cells were cultured with hepatitis B surface antigen (HBsAg) to prepare HBsAg‐pulsed DC. Proliferative capacity and production of interleukin (IL)‐12 and interferon (IFN)‐γ of HBsAg‐pulsed DC were evaluated. Circulating unpulsed DC and HBsAg‐pulsed DC showed significantly higher levels of T‐cell proliferation capacities 1 month after lamivudine therapy compared to proliferation levels before therapy (P < 0.05). HBsAg‐pulsed DC also produced significantly higher levels of IL‐12 and IFN‐γ with lamivudine therapy compared to levels before therapy (P < 0.05). HBsAg‐pulsed DC from lamivudine‐treated patients induced proliferation of T cells of patients with CHB in an antigen‐specific manner (P < 0.05). However, T‐cell stimulatory capacity of DC did not increase significantly 3 and 12 months after lamivudine therapy compared to 1 month after lamivudine therapy. Immune restoration as a result of lamivudine therapy is regulated at least in part by activation of DC. However, progressive activation of DC was not seen as treatment duration progressed, indicating the limitations of this mechanism of viral clearance.     

Optimizing dendritic cell‐based immunotherapy in multiple myeloma: intranodal injections of idiotype‐pulsed CD40 ligand‐matured vaccines led to induction of type‐1 and cytotoxic T‐cell immune responses in patients

Vaccination with idiotype (Id) protein‐pulsed dendritic cells (DCs) has been explored in multiple myeloma and the results have been disappointing. To improve the efficacy of DC vaccination in myeloma, we investigated the use of Id‐ and keyhole limpet haemocyanin (KLH)‐pulsed, CD40 ligand‐matured DCs administered intranodally. Nine patients with smouldering or stable myeloma without treatment were enrolled and DC vaccines were administered at weekly intervals for a total of four doses. Following vaccination, all patients mounted Id‐specific γ‐interferon T‐cell response. Interleukin‐4 response was elicited in two, and skin delayed‐type hypersensitivity reaction occurred in seven patients. More importantly, Id‐specific cytotoxic T‐cell responses were also detected in five patients. Most if not all patients mounted a positive T‐cell response to KLH following vaccination. At 1‐year follow‐up, six of the nine patients had stable disease, while three patients had slowly progressive disease even during the vaccination period. At 5‐year follow‐up, four of the six patients continued with stable disease. No major side effects were noted. In summary, intranodal administration of Id‐pulsed CD40 ligand‐matured DCs was able to induce Id‐specific T and B‐cell responses in patients. Current efforts are geared towards breaking tumour‐mediated immune suppression and improving clinical efficacy of this immunotherapy.     

Anisakis pegreffii impacts differentiation and function of human dendritic cells

Human dendritic cells (DCs) show remarkable phenotypic changes when matured in the presence of helminth‐derived products. These modifications frequently elicited a polarization towards Th2 cells and regulatory T cells thus contributing to immunological tolerance against these pathogens. In this study, the interaction between DCs and larvae of the zoonotic anisakid nematode Anisakis pegreffii was investigated. A. pegreffii larvae were collected from fish hosts, and monocyte‐derived DCs were cocultured in the presence of the live larvae (L) or its crude extracts (CE). In both experimental conditions, A. pegreffii impacted DC viability, hampered DC maturation by reducing the expression of molecules involved in antigen presentation and migration (ie HLA‐DR, CD86, CD83 and CCR7), increased the phagosomal radical oxygen species (ROS) levels and modulated the phosphorylation of ERK1,2 pathway. These biological changes were accompanied by the impairment of DCs to activate a T‐cell‐mediated IFNγ. Interestingly, live larvae appeared to differently modulate DC secretion of cytokines and chemokines as compared to CE. These results demonstrate, for the first time, the immunomodulatory role of A. pegreffii on DCs biology and functions. In addition, they suggest a dynamic contribution of DCs to the induction and maintenance of the inflammatory response against A. pegreffii.     

Phase I/II study exploring ImMucin,a pan‐major histocompatibility complex,anti‐MUC1 signal peptide vaccine,in multiple myeloma patients

ImMucin, a 21‐mer cancer vaccine encoding the signal peptide domain of the MUC1 tumour‐associated antigen, possesses a high density of T‐ and B‐cell epitopes but preserves MUC1 specificity. This phase I/II study assessed the safety, immunity and clinical response to 6 or 12 bi‐weekly intradermal ImMucin vaccines, co‐administered with human granulocyte‐macrophage colony‐stimulating factor to 15 MUC1‐positive multiple myeloma (MM) patients, with residual or biochemically progressive disease following autologous stem cell transplantation. Vaccination was well tolerated; all adverse events were temporal grade 1 2 and spontaneously resolved. ImMucin vaccination induced a robust increase in γ‐interferon (IFN‐γ‐producing CD4+ and CD8+ T‐cells (≤80‐fold), a pronounced population of ImMucin multimer CD8+ T‐cells (>2%), a 9·4‐fold increase in peripheral blood mononuclear cells proliferation and 6·8‐fold increase in anti‐ImMucin antibodies, accompanied with T‐cell and antibody‐dependent cell‐mediated cytotoxicity. A significant decrease in soluble MUC1 levels was observed in 9/10 patients. Stable disease or improvement, persisting for 17·5‐41·3 months (ongoing) was achieved in 11/15 patients and appeared to be associated with low‐intermediate PDL1 (CD274) bone marrow levels pre‐ and post‐vaccination. In summary, ImMucin, a highly tolerable cancerous vaccine, induces robust, diversified T‐ and B‐cell ImMucin‐specific immunity in MM patients, across major histocompatibility complex‐barrier, resulting in at least disease stabilization in most patients.     

Low antigen-dependent activity of T cells after repeated stimulation using dendritic cells and expansion with interleukin-2

Both CD8+ and CD4+ T cells with specific activity against tumor antigens are needed for an efficient antitumor immune response. Activation and proliferation of T cells require cellular interactions including adhesion, recognition of peptides presented by MHC molecules to the T cells receptor, and costimulation. In a series of experiments we attempted to generate and expand specific T cells by repeated stimulation using antigen-loaded autologous dendritic cells (DCs). DCs were obtained from peripheral blood mononuclear cells (PBMC) in the presence of IL-4 and GM-CSF. TNF-a was added to induce maturation. A conjugate of myeloma idiotypic protein with keyhole limpet hemocyanin was used as antigen. Nonadherent peripheral blood mononuclear cells were cultured in the presence of Il-2 and IL-7. Autologous DCs were added to the lymphocyte cultures on days 3, 10, and 17. The lymphocytes were stimulated by high concentration of IL-2 between days 21 and 27. Lymphocytes harvested on day 27 proliferated in response to antigen-loaded DC but failed to do so if less than 0.3 x 10(6) DCs were added for stimulation during culture. However, no cytotoxic activity against autologous DCs was detected and IFN-g production in the T cell cultures was low at the end of culture. In conclusion, the generation and expansion of T cells using repeated stimulation by autologous DCs is feasible but defective cytotoxic response of these cells occurs, possibly as a consequence of repeated frequent exposure to antigen.     

肝癌细胞裂解物致敏的树突状细胞瘤苗体外诱导特异性抗肝癌免疫

目的 探讨肝癌患者肿瘤细胞裂解物致敏的树突状细胞(DC)瘤苗体外诱导自体T淋巴细胞特异性抗肝癌免疫效应。 方法 从肝癌患者外周血单个核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏。用流式细胞仪检测D C细胞表面分子的表达,酶联免疫吸附法检测T淋巴细胞培养上清液中干扰素(I F N)γ和白细胞介索-12(IL-12)的含量,液体闪烁计数仪测定肝癌细胞裂解物致敏的D C刺激自体T淋巴细胞增殖效应,四甲基偶氮唑盐法检测肝癌细胞裂解物致敏D C诱导的细胞毒T淋巴细胞对自体肝癌细胞的特异性杀伤作用。 结果 肝癌细胞裂解物致敏的DC瘤苗可上调DC表面CD1 a、CD40、CD86和人类白细胞抗原-DR分子表达水平,其与T淋巴细胞共培养产生的IFN γ、IL-12的浓度明显高于未致敏的D C组(t值分别为2.30、2.14,P<0.05),肝癌细胞裂解物组(t值分别为14.01、15.40,P<0.01)和对照组(t值分别为14.85、16.87,P<0.01)。同时肝癌细胞裂解物致敏的瘤苗可明显诱导T淋巴细胞的增殖,其诱导的细胞毒性T淋巴细胞对自体肝癌细胞的杀伤率(81.72％±9.49％)显著高于对HepG2的杀伤率(49.37％±11.21％)和人鼻咽癌肿瘤细胞的杀伤率(17.14％±5.65％),P<0.01。 结论 肝癌细胞     

Telomere length and somatic mutations in correlation with response to immunosuppressive treatment in aplastic anaemia

We investigated the frequencies of cytogenetic aberrations and somatic mutations of prognostic relevance in 393 patients with aplastic anaemia (AA). Clonality was determined by G‐banding/fluorescence in situ hybridization (FISH) (n = 245), and targeted capture sequencing was performed for 88 haematopoiesis‐related genes (n = 70). The telomere length (TL) of bone marrow nucleated cells was measured at the single cell level by FISH (n = 135). Eighteen (4·6%) patients showed disease progression, and monosomy 7 (50·0%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (32·9%) presented at least 1 mutation; the most frequently mutated genes were NOTCH1, NF1, SCRIB, BCOR and DNMT3A. The patient group with clonal changes (30·7%) showed an adverse response to immunosuppressive treatment (IST), compared to the non‐clonal group, but this finding did not show statistical significance. The TL of AA patients was significantly shorter than normal control and patients with clonal changes showed significantly shorter TLs. Patients with TL>5·9 showed a higher response rate to IST (P = 0·048). In conclusion, the patients with clonal changes or TL attrition showed a poor response to IST. Shorter TL can be used not only as a biomarker, but also as a predictive marker for treatment response to IST.     

Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus

Summary. Studies assessing the function of monocyte derived dendritic cells (MD‐DC) in individuals with hepatitis C virus (HCV) infection have shown conflicting results. Impaired MD‐DC function in chronic HCV infection would have important implications both for understanding the pathogenesis of HCV infection and in the use of autologous MD‐DC in vaccination strategies. We determined the allostimulatory capacity of MD‐DC in the same patient before and after HCV infection. Next, the phenotype, cytokine production and allostimulatory function of immature and mature MD‐DC in individuals with persistent HCV infection were compared directly with MD‐DC from healthy individuals. Finally, we assessed the ability of MD‐DC to prime autologous naïve peptide specific CD8+ T cells using HLA‐A2 class‐I tetramers. DCs retained the same allostimulatory capacity before and following the establishment of persistent HCV infection. The surface phenotype and the amount of interleukin (IL)‐10 and IL‐12(p70) produced during DC maturation did not differ between HCV‐infected individuals and healthy controls. Mature DCs from HCV‐infected individuals performed comparably in an allogeneic MLR compared with healthy individuals. Mature MD‐DC from HCV‐infected individuals stimulated the expansion of peptide specific naïve CD8+ T cells. MD‐DC from HCV‐infected and healthy individuals are phenotypically indistinguishable and perform comparably in functional assays.