Effect of IFNα-2a on Fas expression and apoptosis rate of peripheral blood cytotoxic T cells in patients with hepatitis B

Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNα-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells (CTLs) in patients with hepatitis B. METHODS:Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNα-2a treatment. RESULTS:Before IFNα-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNα-2a treatment,Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNα-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS:Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNα-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.

In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）. METHODS： Lymphotactin gene modified DCs （DCLptn） were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor （GM- CSF）, interleukin-4 （IL-4） and tumor necrosis factor alpha （TNF-α）. DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTl_s by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay. RESULTS： Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-α, and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn＋H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn ＋ H22, DC/H22 groups. CONCLUSION： Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

Dendritic cells engineered to secrete anti-Dc R3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro

AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

Frequencies and Characterization of HBV-specific Cytotoxic T Lymphocytes in Self-limited and Chronic Hepatitis B Viral Infection in China

Hepatitis B virus （HBV）-specific cytotoxic T lymphocytes （CTLs） are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A＊0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononuclear cells （PBMCs） from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8＋T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8＋T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8^＋ T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immunotherapeutic approaches should be aimed at not only boosting a HBV-specific CD8^＋T response but also improving its function.

Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

AIM:To understand how interactions between hepatitis C virus(HCV) and the host's immune system might lead to viral persistence or effective elimination of HCV.METHODS:Nucleotides 3519-3935 of the non-structural 3(NS3) region were amplified by using reverse transcription polymerase chain reaction(PCR).PCR products of the HCV NS3 regions were integrated into a PCR T7TOPO TA vector and then sequenced in both directions using an automated DNA sequencer.Relative major histocompatibility complex binding levels of wild-type and variant peptides were performed by fluorescence polarization-based peptide competition assays.Peptides with wild type and variant sequences of NS3 were synthesized locally using F-moc chemistry and purified by high-performance liquid chromatography.Specific cytotoxic T lymphocytes(CTLs) clones toward HCV NS3 wild-type peptides were generated through limiting dilution cloning.The CTL clones specifically recognizing HCV NS3 wild-type peptides were tested by tetramer staining and flow cytometry.Cytolytic activity of CTL clones was measured using target cells labeled with the fluorescence enhancing ligand,DELFIA EuTDA.RESULTS:The pattern of natural variants within three human leukocyte antigen(HLA)-A2-restricted NS3 epitopes has been examined in one patient with chronic HCV infection at 12,28 and 63 mo post-infection.Results obtained may provide convincing evidence of immune selection pressure for all epitopes investigated.Statistical analysis of the extensive sequence variation found within these NS3 epitopes favors a Darwinian selection model of variant viruses.Mutations within the epitopes coincided with the decline of CTL responses,and peptide-binding studies suggested a signif icant impact of the mutation on T cell recognition rather than peptide presentation by HLA molecules.While most variants were either not recognized or elicited low responses,such could antagonize CTL responses to target cells pulsed with wild-type peptides.CONCLUSION:Cross-recognition of CTL epitopes from wild-type and naturally-occurring HCV variants may lead to impaired immune responses and ultimately contribute to viral persistence.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide &quot;ALAHGVRAL (core 150-158)&quot;. The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Association of Graves’ disease and Graves’ ophthalmopathy with the polymorphisms in promoter and exon 1 of cytotoxic T lymphocyte associated antigen-4 gene

Objective: To investigate the association of Graves’ disease and Graves’ ophthalmopathy with the C/T transition polymorphism at position –318 of promoter and the A/G transition polymorphism at position 49 of exon 1 within cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene. Methods: Thirty-three patients with ophthalmopathy of Graves’ disease, fifty-six Graves’ patients without ophthalmopathy and sixty normal subjects as control were involved in the present case-control study. The polymorphisms were evaluated by polymerase chain reaction fragment length polymorphism (PCR-RFLP). Com-parisons were made of gene frequencies and allele frequencies between the groups. Results: The gene frequencies of CT and allele frequencies of T were much higher in Graves’ patients with ophthalmopathy than that in the group without ophthalmopathy (P=0.020, P=0.019). The gene frequencies of GG and allele frequencies of G in patients with Graves’ disease were significantly increased as compared with control group (P=0.008, P=0.007). The data suggest that smokers with Graves’ disease seemed to be more predisposed to ophthalmopathy than non-smokers (P=0.018). Conclusion: Our results suggest that an allele of T at position –318 of promoter is associated with genetic susceptibility to Graves’ ophthalmopathy while an allele of G at position 49 of exon 1 is associated with genetic susceptibility to Graves’ disease instead. Smoking is believed to be a major risk factor for ophthalmo-pathy.

Cytotoxicity study of ethanol extract of the stem bark of asam kandis (Garcinia cowa Roxb.) on T47D breast cancer cell line

Objective:To investigate the cytotoxic effect of ethanol extract of the stem bark of asam kandis[Garcinia cowa Roxb.(G.cowa)]on T47 D breast cancer cell line.Methods:The cytotoxicity of ethanol extract was carried out against human breast cancer cell line(T47D) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay.The extract was added at various concentrations(0.1.1,10 and 100 μg/mL).The level of cytotoxicity was determined by calculating the level of IC_(50),that was based on the percentage of the cell death after 24 h treatment with the extract.Cell morphological changes were observed by using inverted microscope.Results:The 3-(4.5-dimelhylthiazol-2-yl)-2.5-diphenyltelrazolium bromide assay showed that ethanol extract of G.cowa exhibited significant cytotoxic effect on T47 D with IC_(50) value of(5.10+1.68) μg/mL.Morphological alteration of the cell lines after exposure to ethanol extract of G.cowa was observed under phase contrast microscope in a dosc-dependent manner.ConclusionsThe results suggest the possible use of ethanol extract of asam kandis for preparing herbal medicine for cancer-related ailments.

Cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells

Today it is generally accepted that most bonding agents are cytotoxic. In this study the relative cytotoxicity of seven recent dentine bonding agents on mouse 3T3 fibroblast cells were investigated. Materials and Methods. Near-confluent mouse 3T3 fibroblast cells were exposed to Dulbecco Modified Eagle’s Medium containing extractions from the seven different bonding agents. The cell survival rate was then determined using the standard MTT assay. Results. The cell survival rate ranking is: iBond (94%) < Gbond (78%) < Xeno V (71%) < Adper Easy Bond (63%) < Xeno V+ (61%) < Adper Scotchbond SE (33%) < XP Bond (32%). Part A of Adper Scotchbond SE had a survival rate of 35% and part B 38%. These two parts did not differ significantly. Adper Scotchbond SE and XP Bond do not differ significantly. While Xeno V+, Xeno V and Adper Easy Bond do not differ. (p < 5%;Tukey-Kramer Multiple-Comparison Test). Conclusion. All of the tested adhesive bonding agents were cytotoxic with survival rate of 3T3 cells between 94% to 31%. Of the 7 bonding agents tested iBond was found to be only slightly toxic and by far the least toxic. The two bonding agents (XP Bond and Adper Scotchbond SE) containing UDMA plus TEGDMA plus HEMA plus camphorquinone were found to be the most toxic.

Effect of cytotoxic T-lymphocyte antigen-4,TNF-alpha polymorphisms on osteosarcoma: evidences from a meta-analysis

Objective： Previous studies have investigated the role of cytotoxic T-lymphocyte antigen-4 （CTLA-4） and tumor necrosis factor-alpha （TNF-a） in carcinogenesis of osteosarcoma, but their results were inconsistent. We aimed to clarify the associations between CTLA-4, TNF-a polymorphism and osteosarcoma risk by using meta-analysis. Methods： We searched relevant studies without language restriction in PubMed, EMbase, Cochrane Library, Google Scholar databases, Chinese National Knowledge Infrastructure （CNKI） and conference literature in humans published prior to March 2013. The strengths of the associations between genetic variants and osteosarcoma risk were estimated by odds ratio （OR） with 95% confidence interval （95% CI）. Results： A total of seven studies with 1,198 osteosarcoma patients and 1,493 controls were selected. Four studies were eligible for CTLA-4 （1,003 osteosarcoma and 1,162 controls）, and three studies for TNF-a （195 osteosarcoma and 331 controls）. Pooled results showed that rs231775 polymorphism of CTLA-4 was associated with osteosarcoma risk （GG vs. AA： OR=1.63, 95% CI=1.24-2.13; GG ＋ GA vs. AA： OR=1.56, 95% CI=1.21-2.01; AA ＋ GA vs. GG： OR=0.83, 95% CI=0.71-0.97; G vs. A： OR=1.21, 95% CI=1.08-1.36）. No significant heterogeneity was observed across the studies. No significant associations were found between rs5742909 polymorphism of CTLA-4 or rs1800629 polymorphism of TNF-a and osteosarcoma risk. Conclusions： These results suggest that the rs231775 polymorphism of CTLA-4 may play an important role in carcinogenesis of osteosarcoma.

Glypican-3-specific cytotoxic T lymphocytes induced by human leucocyte antigen-A*0201-restricted peptide effectively kill hepatocellular carcinoma cells in vitro

Objective: To investigate potential human leucocyte antigen(HLA)-A2-restricted epitope peptides of glypican-3(GPC3) and determine the cytotoxicity of peptidespecific cytotoxic T lymphocytes(CTLs) against hepatocellular carcinoma(HCC) cells.Methods: The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3+SMMC 7721 and Hep G2 cells was detected using IFN-g based enzymelinked immunospot and lactate dehydrogenase release assays in vitro.Results: A total of six peptides were identified for bindings to HAL-A2 and the GPC3522–530 and GPC3 229–237 peptides with HLA-A*0201 molecules displayed high binding affinity and stability. The CTLs induced by the GPC3 522–530 or positive control GPC3 144–152 peptide responded to the peptide by producing IFN-g, which were abrogated by treatment with anti-HLA-A2 antibody. The GPC3 522–530-specific CTLs responded to and killed SMMC 7721 and Hep G2 cells, instead of GPC3-silenced SMMC7721 or Hep G2 cells. GPC3 522–530-specific CTLs response to HCC cells was blocked by anti-HLA-A2 antibody.Conclusions: The GPC3 522–530 peptide contains antigen-determinant and its specific CTLs can effectively kill HCC in a HLA-A2-restricted and peptide-dependent manner. Our findings suggest that this peptide may be valuable for development of therapeutic vaccine.

INVESTIGATION OF INDUCING EFFECT OF SPECIFIC CYTOTOXICITY OF CTLS BY ANTIGEN PEPTIDES FROM T LYMPHOCYTIC LEUKEMIA CELLS

Objective: To investigate the characteristics of specific antitumor immunity induced by antigen peptides mixture from T lymphocytic leukemia cells. Method: Antigen peptides mixtures were prepared from different leukemia cell lines and then bound with Hsp70 in vitro. Human peripheral blood mononuclear cells (PBMC) were cultured in vitro, and activated with Hsp70-antigen peptides. The activated PBMC was cultured continuously in vitro, and used as effector cells in vitro test of cytotoxicity to different target cells. Results: The antigen peptides from different leukemia cell lines were peptides mixture and could activate PBMC effectively if they were presented by Hsp70. The activated PBMC could proliferate in the presence of IL-2 and Hsp70-antigen peptides. The proliferative PBMC had specific cytotoxicity to leukemia cells corresponding to the antigen peptides. PBMC activated by antigen peptides from T lymphocytic leukemia cell lines could effectively kill T lymphocytic leukemia cells, and the cytotoxicity of these PBMC to T lymphocytic leukemia cells was significantly stronger than that of PBMC activated by antigen peptides from other leukemia cells (P < 0.05). PBMC activated by either Hut78-peptides or Molt 4-peptides could effectively kill Jurkat cells. And the cytotoxicity of PBMC activated by Hut78/Molt-4-peptides to Jurkat cells was significantly stronger than that of PBMC activated by either Hut78-peptides or Molt-4-peptides alone (P<0.05). Conclusion: Antigen peptides mixture from T lymphocytic leukemia cell lines can induce specific cytotoxic effect to T lymphocytic leukemia cells. There exists cross-reactivity among antigen peptides mixture from different T lymphocytic leukemia cell lines. The cross-reactivity could be amplified by blending of different antigen peptides from different T lymphocytic leukemia cell lines, suggesting that it is possible to prepare broad-spectrum antigen peptide vaccine against T lymphocytic leukemia by using multiple leukemia cell lines.

Perforin Expression in <i>Theileria parva</i>Specific Cytotoxic T Cells Correlates with Cytotoxicity

Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.

Research progress of follicular cytotoxic T cells in human immunodeficiency virus infection

Recently, a new type of CD8^+ T cell subset, namely, the CXCR5^+ CD8^+ T cell subset(also called the follicular cytotoxic T cell(TFC) subgroup), has been discovered around B cell follicles. The discovery has aroused widespread interest.However, the processes and mechanisms of TFCs taking part in the immune response of the germinal center and their specific roles must still be clearly identified. This paper reviews domestic and foreign studies on factors regulating the phenotype,physiological functions, maturity, and differentiation of TFCs and roles and clinical significance these cells in human immunodeficiency virus infection. Our review has shown good application prospects for TFCs. We believe that further studies on TFCs can provide another tool for cytotherapy of controlling or curing chronic viral infections or tumors.

Insights into chitosan hydrogels on dentine bond strength and cytotoxicity

Contemporary dental adhesives show favorable immediate results in terms of bonding effectiveness. However, the durability of resin-dentin bonds is their major problem. Materials and Methods: Preparation of 3 chitosan-antioxidant hydrogels was achieved using modified hydrogel preparation method. Their effect on the bond strength to dentine both short term (after 24 hours) and long term (after 6 months) were evaluated using shear bond strength measurements using Instron Universal Testing Mascine). The SEM was used to study the surface of the hydrogels. The cell survival rate (cytotoxicity) of the antioxidants resveratrol, β-carotene and propolis towards Balb/c 3T3 mouse fibroblast cells was also assessed using the standard MTT assay. Results: It was found that chitosan-H treated dentine gives significantly (p β-carotene (92%) > propolis (68%) > resveratrol (33%). Conclusion: the antioxidant-chitosan hydrogels significantly improved bonding to dentine with or without phosphoric acid treatment. The pH of the growth medium had a high influence on the cell survival rate of Balb/c mouse 3T3 fibroblast cells. The release of the antioxidant β-carotene would not have an influence on the pulp cells. These materials might address the current perspectives for improving bond durability.

Effects of the Cytotoxic T-Cells on the Dynamics of Co-Infection of HIV-1 and Mycobacterium tuberculosis

Enhancement of the Human Immunodeficiency Virus (HIV) specific cytotoxic T-cells mechanisms in an HIV-1 and Mycobacterium tuberculosis (Mtb) co-infected individual seems to improve the clinical picture of an individual by reducing Acquired Immuno Deficiency Syndrome (AIDS) state progression rate. In this paper, we develop a system of deterministic differential equations representing the immune cells involved in an HIV-1 and Mtb co-infected individual. Results show that although the non-lytic arm of the HIV-1 cytotoxic T-cells affects the co-infection dynamics more than the lytic factors, a combination of both factors results in a more positive reduced progression to the AIDS state. This is due to the increased protection of the CD4+ T-cells by the CTL mechanisms by further reducing infections and replications by the HIV. Thus, HIV-1 specific CTLs mechanisms’ involvement is here recommended to be part of a solution to the HIV and Mtb co-infection problems.

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4^(+)T cells

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency(PAD),analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects.CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation.Indeed,while circulating CD57^(+)CD4^(+)T cells are normally rare,we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade.We performed single-cell RNA-seq analysis of matched CD57^(+)CD4^(+)T cells from blood and tonsil samples.Circulating CD57^(+)CD4^(+)T cells(CD4cyt)exhibited a cytotoxic transcriptome similar to that of CD8^(+)effector cells,could kill B cells,and inhibited B-cell responses.CTLA4 restrained the formation of CD4cyt.While CD57 also marked an abundant subset of follicular helper T cells,which is consistent with their antigen-driven differentiation,this subset had a preexhaustion transcriptomic signature marked by TCF7,TOX,and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition.Thus,CD57^(+)CD4^(+)T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers.CTLA4 is a key modifier of CD4^(+)T-cell cytotoxicity,and the pathological CD4cyt phenotype is accentuated by infection.

Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

Tumor immunotherapy has achieved breakthroughs in a variety of tumors. However, the systemic absence of T cells in tumors and immunosuppressive tumor microenvironment so far limits the efficacy of immunotherapy to a small population of patients. Therefore, novel agents to increase T-cell tumor infiltration are urgently needed in the clinic. We recently found that inhibition of the ADP-ribosylation factor 1 (Arf1)-mediated lipid metabolism not only kills cancer stem cells (CSCs) but also elicits an anti-tumor immune response. In this study, we revealed a mechanism that targeting Arf1 promotes the infiltration of cytotoxic T lymphocytes (CTLs) into tumors through the C-C chemokine ligand 5 (CCL5)- C-C chemokine receptor type 5 (CCR5) pathway. We found that blockage of Arf1 induces the production of the unsaturated fatty acid (PE 18:1) that binds and sequestrates peroxisome proliferator- activated receptor-γ (PPARγ) from the PPARγ-nuclear factor-κB (NF-κB) cytoplasmic complex. The released NF-κB was then phospho-rylated and translocated into the nucleus to regulate the transcription of chemokine CCL5. CCL5 promoted infiltration of CTLs for tumor regression. Furthermore, the combination of the Arf1 inhibitor and programmed cell death protein 1 (PD-1) blockade induced an even stronger anti-tumor immunity. Therefore, targeting Arf1 represents a novel anti-tumor immune approach by provoking T-cell tumor infiltration and may provide a new strategy for tumor immunotherapy.

GPC3刺激对DC-CTL靶向杀伤肝细胞癌的影响

目的本研究将磷脂酰肌醇蛋白聚糖3(GPC3)作为肝细胞癌(LIHC)的靶分子,研究其在激活树突状细胞(DC)-细胞毒性T淋巴细胞(CTL)和增强肿瘤杀伤能力中的作用。方法为更系统、更全面地鉴定GPC3在多种恶性肿瘤中的意义,对GPC3在泛癌中的基因表达水平进行评估,并从LIHC与临床特征的相关性角度进一步研究GPC3。分别通过生存预后分析(Kaplan-Meier图)和受试者工作特征(ROC)曲线分析评估GPC3在LIHC中的预后价值和诊断价值。重组GPC3蛋白用于刺激DC,然后与CTL细胞共培养,经酶联免疫吸附试验(ELISA)分析以检测DC-CTL细胞中细胞因子的表达。通过CCK8测定法测定在Huh7细胞上用GPC3培养刺激的DC-CTL细胞的细胞毒性。结果GPC3的基因表达水平在大多数癌症中显著不同,并且在LIHC中与正常组织相比更高。GPC3在血清中与年龄和甲胎蛋白(AFP)水平相关(均P<0.001),而与LIHC的TNM分期、病理分期和组织学分级无关。ROC曲线分析表明,GPC3可用于准确预测LIHC。GPC3的表达水平与LIHC的总生存期无关。联合治疗显著上调了IFN-γ和TNF-α的分泌。用150 ng/ml和200 ng/ml GPC3刺激的DC-CTL细胞杀死Huh7细胞的能力强于不使用GPC3刺激的DC-CTL细胞(均P<0.05)。结论用GPC3刺激DC-CTL细胞通过促进细胞毒性在体外抑制肿瘤生长。GPC3可能是一种潜在的肿瘤标志物,可用于提高DC-CTL细胞的治疗效率。

Clinical evaluation of sintilimab in conjunction with bevacizumab for advanced colorectal cancer with microsatellite stable-type after failure of first-line therapy

BACKGROUND At present,immune checkpoint inhibitors(ICIs)remain the 1st-line therapy me-thod for patients suffering from high microsatellite instability/deficient misma-tch repair metastatic colorectal cancer(mCRC).However,ICI treatments demon-strate minimal therapeutic efficacy against microsatellite stable(MSS)/proficient mismatch repair(pMMR)CRC.This is mainly because this type of tumor is a“cold tumor”with almost no lymphocyte infiltration.Anti-angiogenic drugs have been found to improve the immune microenvironment by promoting many immune cells to enter the immune microenvironment,thereby exerting anti-tumor effects.AIM To investigate the effects of ICIs combined with bevacizumab monoclonal anti-body on tumor immune cells in MSS/pMMR advanced CRC patients with first-line treatment failure.METHODS A total of 110 MSS/pMMR patients with advanced CRC after first-line treatment failure in the Affiliated Hospital of Qinghai University were enrolled for a ran-domized controlled trial.In short,patients in the experimental group(n=60)were given sintilimab plus bevacizumab for 4 cycles,and those in the control group(n=50)patients were treated with FOLFIRI combined with bevacizumab for 4 cycles.The expression levels of cluster of differentiation(CD)8(+)T cells,tumor-associated macrophages(TAMs),and cancer-associated fibroblasts(CAFs)were comprehensively evaluated to assess the effects of sintilimab combined with bevacizumab on MSS/pMMR advanced CRC sufferers following failure of 1st-line therapy.RESULTS The positive expression rates of CD8(+)T lymphocytes(30%vs 50%),TAMs(23.30%vs 60%),and CAFs(23.30%vs 50%)before and after treatment in both groups exhibited statistical significance(P<0.05).Additionally,the therapeutic effects of both groups(partial remission:26.67%vs 10%;objective response rate:26.70%vs 10%)were significantly different(P<0.05).Although the experimental group showed a higher progression-free survival,median progression-free survival,and disease control rate than the control group,the difference was not statist-ically significant.Moreover,no significant difference in the occurrence rate of drug-related adverse reactions after treatment between the two groups was found(P>0.05).CONCLUSION ICIs in combination with bevacizumab can not only improve the patient’s prognosis but also yield safe and controllable adverse drug reactions in patients suffering from MSS/pMMR advanced CRC after failure to a 1st-line therapy.