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 "ALAHGVRAL (core 150-158)". 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.

Effect of Chinese Herbal Medicinal Ingredients on IL-2 mRNA Levels of T Lymphocytes in Mice Measured Using Semiquantification RT-PCR

In this study, the IL-2 mRNA levels of T lymphocytes in normal mice stimulated by nine Chinese herbal medicinal ingredients （CHMIs） were measured using semiquantification reverse transcription polymerase chain reaction. The results showed that astragalus polysaccharide （APS）, epimedium polysaccharide （EPS）, Chinese angelica polysaccharide （CAPS）, propolis flavone （PF）, and astrogalosides （AS） promoted IL-2 mRNA levels in T lymphocytes in vitro and in vivo to differing degrees, and the level of IL-2 mRNA induced by propolis polysaccharide （PPS） in vitro was higher than that induced by the control, which differed from that of PPS in vivo.

The role of apoptosis in the development and function of T lymphocytes

Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work to- gether to regulate T lymphocyte development and function.

Ambiguous nucleus regulates the proliferation and percentage of T lymphocytes in peripheral blood

The aim of this study was to examine the immunomodulatory role of the unilateral ambiguous nucleus （Amb）. We performed electrical stimulation of the unilateral Amb, electrical stimulation of the left parietal cortex and the lateral hypothalamus following unilateral Arab lesion, as well as microinjection of acetylcholine chloride and hemicholine-3 into the unilateral Amb, and electrical stimulation of the unilateral Amb after injection of atropine, mecamylamine, propranolol, and phentolamine. Results showed that the number and proliferation of peripheral blood T lymphocytes were increased after electrical stimulation of the unilateral Arab. The cholinergic neurons in the Amb released choline substances to alter cellular immunity, thus confirming that the Amb mediates the neuro-immunomodulatory process.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective:To predict B cell and T cell epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins.Methods:The sequences of 22-kDa,47-kDa,56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA,DNAstar,Bcepred,ABCpred,NetMHC,NetMHCⅡand IEDB.The 58-kDa tertiary structure model was built by MODELLER9.17.Results:The 22-kDa B-cell epitopes were located at positions 194-200,20-26 and 143-154,whereas the T-cell epitopes were located at positions 154-174,95-107,17-25 and 57-65.The 47-kD a protein B-cell epitopes were at positions 413-434,150-161 and 283-322,whereas the T-cell epitopes were located at positions 129-147,259-267,412-420 and 80-88.The 56-kDa protein B-cell epitopes were at positions 167-173,410-419 and 101-108,whereas the T-cell epitopes were located at positions 88-104,429-439,232-240 and 194-202.The 58-kDa protein B-cell epitopes were at positions 312-317,540-548 and 35-55,whereas the T-cell epitopes were located at positions 415-434,66-84 and 214-230.Conclusions:We identified candidate epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins from Orientia tsutsugamushi.In the case of 58-kDa,the dominant antigen is displayed on tertiary structure by homology modeling.Our findings will help target additional recombinant antigens with strong specificity,high sensitivity,and stable expression and will aid in their isolation and purification.

Cross-talk between hepatic stellate cells and T lymphocytes in liver fibrosis

Background: Fibrosis results from inflammation and healing following injury. The imbalance between extracellular matrix(ECM) secretion and degradation leads to the ECM accumulation and liver fibrosis. This process is regulated by immune cells. T lymphocytes, including alpha beta( αβ) T cells, which have adaptive immune functions, and gamma delta( γδ) T cells, which have innate immune functions, are considered regulators of liver fibrosis. This review aimed to present the current understanding of the cross-talk between T lymphocytes and hepatic stellate cells(HSCs), which are the key cells in liver fibrosis. Data sources: The keywords "liver fibrosis", "immune", and "T cells" were used to retrieve articles published in Pub Med database before January 31, 2020. Results: The ratio of CD8 +(suppressor) T cells to CD4+(helper) T cells is significantly higher in the liver than in the peripheral blood. T cells secrete a series of cytokines and chemokines to regulate the inflammation in the liver and the activation of HSCs to influence the course of liver fibrosis. In addition, HSCs also regulate the differentiation and proliferation of T cells. Conclusions: The cross-talk between T cells and HSCs regulates liver fibrosis progression. The elucidation of this communication process will help us to understand the pathological process of liver fibrosis.

High mobility group box 1 protein(HMGB1)as an immune-modulating factor for polarization of human T lymphocytes

Objective This study was performed to investigate the effect of high mobility group box-1 protein(HMGB1)on immune function of human T lymphocytes in vitro and explore its potential role in cell-mediated immune dysfunction.Methods Fresh blood was obtained from healthy adult volunteers and peripheral blood mononuclear cells(PBMCs)were isolated,then rhHMGB1 was added to PBMCs.Four-color flow cytometric(FCM)analysis was used for the measurement of intracellular cytokine including interleukin IL-4 and interferon IFN-?ELISA kits were employed for the determination of IL-2 and sIL-2R protein levels in cell culture supernatants.Results(1)Different stimulating time and dosage of rhHMGB1 did not alter the number of IFN-αpositive cells(Th1).rhHMGB1 stimulation provoked a dose-dependent and time-dependent increase in Th2 subset and decrease in ratio of Th1 to Th2.(2)Compared with the untreated cells,when the cells were coincubated with rhHMGB1(10-100ng/ml)for 12 hrs,protein release of IL-2 and sIL-2R were significantly up-regulated.At 48 hrs,in contrast,protein production was relatively lower in cells after exposure to 100-1000 ng/ml rhHMGB1.Conclusions These findings demonstrated that HMGB1 has a dual influence on immune functions of human T lymphocytes.

Duodenal intraepithelial T lymphocytes in patients with functional dyspepsia

AIM： To quantify the intraepithelial lymphocytes （IELs） and to document the membrane expression of CD4, CD8, TCRγδ and adhesion and/or activation-associated molecules （CD103, CD28, CD44, CD69, HLA-DR, CD95/ Fas） in the duodenal mucosa of patients with functional dyspepsia （FD） in order to provide arguments for an immunological process in FD. METHODS： Twenty-six FD patients according to Rome Ⅱ criteria （20 were H pylori negative） were studied and compared to 12 healthy adults. IELs were isolated from five duodenal biopsy samples, then quantified by microscopy and flow cytometry while the membrane phenotypes were determined by cytofluorometry. RESULTS： Duodenal histological examination was normal. In H pylori negative patients, the number of IELs was not different from that in healthy controls. Median percentage expression of CD4, CD8, or TCRy8 and CD103, CD44, CD28, CD69 on CD3＋ IELs, among the adhesion/activation associated molecules tested, was not different from that in healthy controls. In contrast, the median percentage expression of CD95/ Fas [22 （9-65） vs 45 （19-88）, P = 0.03] and HLADR expressing CD3＋ IELs [4 （0-30） vs 13 （4-42）, P = 0.04] was significantly lower in the H pylori negative FD group than in healthy controls, respectively. The number of IELs was significantly greater in H pylori positive FD patients than in healthy controls [median ratio for 100 enterocytes 27.5 （6.7-62.5） vs 10.8 （3-33.3）, P = 0.02] due to a higher number of CD8＋ CD3＋ IELs. CONCLUSION： In H pylori negative FD patients, the phenotypic characterization of IELs suggests that we cannot exclude a role of IELs in FD.

Experimental Study on Double Blocking of T Lymphocytes Apoptosis Induced by Fas/FasL

Objective： To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes （CTL）. Methods： Liver cancer cells and tumor infiltrating lymphocytes （TIL） were isolated from FasL positive fresh specimens, and co-cultured. Specific CTL were activated and prepared in the presence of the co-stimulation of monoclonal antibody CD28. Then the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder test simultaneously. The apoptosis-blocking effect was compared with the control group. Furthermore, the changes of T cell proliferation and killing activity were detected by the method of ^3H thymidine incorporation and ^51Cr release test. Results： There was a significant increase in apoptosis rate in unblocking group compared with blocking group and quiescent group, with the unblocking group of 47.82%±0.13%, quiescent group of 3.76%±0.25%, and the blocking group of 8.22%±0.26% respectively （P〈0.01）. T cell-ladder appeared in unblocking group by DNA ladder test. Both the killing ability and proliferation rate of T cells were significantly increased after blocking. There was significant difference among blocking group, unblocking group and quiescent group （P〈0.01）. Conclusion： With this method we obtained large amounts of tumor-specific T lymphocytes, which was able to kill liver cancer cells effectively.

Prediction of promiscuous T cell epitopes in RNA dependent RNA polymerase of Chikungunya virus

Objective: To explore RNA dependent RNA polymerase of Chikungunya virus(CHIKV) and develop T cell based epitopes with high antigenicity and good binding affinity for the human leukocyte antigen(HLA) classes as targets for epitopes based CHIKV vaccine. Methods: In this study we downloaded 371 non-structural protein 4 protein sequences of CHIKV belonging to different regions of the world from the US National Institute of Allergy and Infectious Diseases(NIAID) virus pathogen resource database. All the sequences were aligned by using CLUSTALW software and a consensus sequence was developed by using Uni Pro U Gene Software version 1.2.1. PropredⅠand Propred software were used to predict HLAⅠ and HLAⅡ binding promiscuous epitopes from the consensus sequence of non-structural protein 4 protein. The predicted epitopes were analyzed to determine their antigenicity through Vaxijen server version 2.0. All the HLAⅠ binding epitopes were scanned to determine their immunogenic potential through the Immune Epitope Database(IEDB). All the predicted epitopes of our study were fed to IEDB database to determine whether they had been tested earlier. Results: Twenty two HLA class Ⅱ epitopes and eight HLA classⅠepitopes were predicted. The promiscuous epitopes WMNMEVKII at position 486–494 and VRRLNAVLL at 331–339 were found to bind with 37 and 36 of the 51 HLA class Ⅱ alleles respectively. Epitope MANRSRYQS at position 58–66 and epitopes YQSRKVENM at positions 64–72 were predicted to bind with 12 and 9 HLAⅠI alleles with antigenicity scores of 0.754 9 and 1.013 0 respectively. Epitope YSPPINVRL was predicted to bind 18 HLAⅠ alleles and its antigenicity score was 1.425 9 and immunogenicity score was 0.173 83. This epitope is very useful in the preparation of a universal vaccine against CHIKV infection. Conclusions: Epitopes reported in this study showed promiscuity, antigenicity as well as good binding affinity for the HLA classes. These epitopes will provide the baseline for development of efficacious vaccine for CHIKV.

THE ADAPTABLE CHANGE OF THE FUNCTION OF T LYMPHOCYTES FOR PHYSICAL EXERCISE WITH OXYGEN

Objective To find out the possible regularity and mechanism of the adaptable change of human be- ing T lymphocytes for physical exercise with oxygen and bring the original data for the Movement of All People Im- proving their Health. Methods We selected 16 untrained female students in university and let them had the same amount of exercise for 8 weeks. After that, we collected the cycle blood at the time point or before exercise, the end of exercise and 1 hour after exercise at the end or the 0,first, 2 nd, 4 nd, 6 nd and 8 nd week respectively, so as to de- termine its stimuli index (SI) by MTT method. Results In the different time sect, such as the early stage or exer- cise, quiet condition,as soon as the end of exercise and 1 hour after exercise, we found that the SI were obviousIy low- er than that or normal (P<0.05),especially in the time sect of the end or exercise. Continuing to 4 weeks,the func- tion of T lymphocytes restored gradualy and it lasted to the 8th week, the SI in quiet condition and 1 hour after exer- cise had restored to normal (P>0.05),but in the end or exercise, it still was low,however. the extent of the cases se- lected was in a condition or acute excitability. Conclusion As the bodies adapting to the exercise, the function of T lymphocytes restored slowly and the rate increased faster and faster.

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein:An in silico approach

Objective:To predict immunogenic promiscuous T-cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools.To date,no epitope data are available for the Zika virus in the IEDB database.Methods:We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks.A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using Propred1 and Propred immunoinformatic algorithms respectively.The antigencity predicted score was also calculated for each predicted epitope using the Vaxi Jen 2.0 tool.Results:By using Pro Pred1,23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus.The greatest number of MHC class I binding epitopes were projected within the NS5(21%),followed by Envelope(17%).For MHC class II,greatest number of predicted epitopes were in NS5(19%) followed by the Envelope,NS1 and NS2(17% each).A variety of epitopes with good binding affinity,promiscuity and antigenicity were predicted for both the HLA classes.Conclusion:The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates,which will be able to induce a broad range of immune responses in a heterogeneous HLA population.However,our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.

Correlation analysis of TCM syndrome types with T lymphocytes and biochemical indices in patients with HBV-related primary liver cancer

Objective:To investigate the correlation between T lymphocytes and biochemical indices in patients with Primary liver cancer(PLC)associated with hepatitis B virus(HBV)and TCM syndrome differentiation.Methods:263 HBV-related PLC patients who were admitted to the First Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2018 to December 2019 were retrospectively collected.There were 127 cases of liver depression and spleen deficiency syndrome(48.3%),48 cases of spleen deficiency and dampness syndrome(18.3%),31 cases of liver and gallbladder dampness and heat syndrome(11.8%),35 cases of liver and blood stasis syndrome(13.3%),and 22 cases of liver and kidney Yin deficiency syndrome(8.4%).The general data,T cell subsets,oncology and virology indicators,oncology characteristics,biochemical indicators and other data were counted.Epidata and Excel were used to collect and summarize the data,and SPSS26.0 software was used for statistical analysis.Results:There was no significant difference in gender and age distribution among the five syndrome types(χ^(2)=5.462,F=1.979,ALL P>0.05).The differences among T lymphocyte count(χ^(2)=57.785,P<0.001),CD4(+)T cell count(χ^(2)=47.103,P<0.001)and CD8(+)T lymphocyte count(F=12.760,P<0.001)were statistically significant.The T lymphocyte count,CD4(+)T lymphocyte count and CD8(+)T lymphocyte explicit count in patients with liver and kidney Yin deficiency syndrome were significantly lower than those in the other four syndrome types.AFP(χ^(2)=89.986,P<0.001),CEA(χ^(2)=95.501,P<0.001),CA199(χ^(2)=30.044,P<0.001)of the five syndrome types increased successively from the syndrome of liver depression and spleen deficiency to the syndrome of liver and kidney Yin deficiency,and the difference was statistically significant.There were statistically significant differences in the inner diameter of main portal vein,portal vein cancer thrombin and extrahepatic metastasis among the five syndrome types(ALL P<0.001).The main symptoms of portal vein cancer thrombin and extrahepatic metastasis were liver-gallbladder dampness-heat syndrome and liver-blood stasis syndrome.The differences among PLT(χ^(2)=39.234,P<0.001),Alb(χ^(2)=75.171,P<0.001),TBil(χ^(2)=51.140,P<0.001),AST(χ^(2)=55.881,P<0.001),PT(χ^(2)=21.515,P<0.001)were statistically significant.PLT and Alb decreased successively from the syndrome of liver depression and spleen deficiency to the syndrome of liver and kidney Yin deficiency.PLT and Alb of the syndrome of liver depression and spleen deficiency were significantly higher than those of the other four groups,and TBil and AST of the syndrome of liver and gallbladder dampness and heat were significantly higher than those of the other four groups.PT of liver and kidney Yin deficiency was significantly higher than that of the other four groups.The lymphocyte count,CD4(+) lymphocyte count and CD8(+) lymphocyte count were negatively correlated with AFP,PT and TBil(ALL P<0.05),and positively correlated with PLT(P<0.05).T lymphocyte count was positively correlated with AIb(P<0.05).Conclusion:This study found that patients with liver depression and spleen deficiency syndrome have better cellular immune function,liver function and prognosis.Patients with liver and kidney Yin deficiency have lower cellular immunity,worse liver function,and worse prognosis.Portal vein carcinoma embolus and extrahepatic metastasis were mainly characterized by dampness and heat of liver and gallbladder and blood stasis of liver.Patients with lower lymphocyte counts have poorer blood clotting,worse the liver reserve,and the higher the risk of further cancer.

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.

The injury progression of T lymphocytes in a mouse model with subcutaneous injection of a high dose of sulfur mustard

Background: In clinical studies, the findings on sulfur mustard(SM) toxicity for CD3+CD4+ and CD3+CD8+ T lymphocyte subsets are contradictory. In animal experiments, the effect of SM on the T cell number and proliferation is incompatible and is even the opposite of the results in human studies. In this study, we observed the dynamic changes of T lymphocytes in the first week in a high-dose SM-induced model.Methods: Mice were exposed to SM by subcutaneous injection(20 mg/kg) and were sacrificed 4 h, 24 h, 72 h and 168 h later. Spleen T lymphocyte proliferation was evaluated by 3H-Td R. Flow cytometric analysis was used to observe the percentage of CD3+CD4+ and CD3+CD8+ T lymphocyte subsets. The IL-1e assayed using the Luminex method. DNA damage in bone marrow ceβ, IL-6, IL-10 and TNF-lls was observed with α levels in plasma werthe single cell gel electrophoresis technique(SCGE).Results: SM continuously inhibited the proliferation of lymphocytes for 7 days, and there was a significant rebound of Con A-induced T lymphocyte proliferation only at 24 h. The percentage of CD3+CD4+ and CD3+CD8+ lymphocytes was upregulated, which was accompanied by increased IL-1β and TNF-creased in the PG group at 4 h. The peak of lymphocytic apoptα and decreased IL-10. The IL-6 level was gradually deosis and DNA damage occurred at 24 h and 72 h, respectively. Conclusion: Our results show that SM significantly inhibited T lymphocyte proliferation as well as induced CD3+CD4+ and CD3+CD8+ upregulation. SM intoxication also significantly increased the levels of pro-inflammatory cytokines(IL-1β, IL-6 and TNF-α) and inhibited the level of anti-inflammatory cytokine IL-10. Our results may partly be due to the significant SM induced significant apoptosis and necrosis of lymphocytes as well as DNA damage of bone marrow cells. The results provided a favorable evaluation of SM immune toxicity in an animal model.

Influence of REV and ALV-J Co-Infection on Immunologic Function of T Lymphocytes and Histopathology in Broiler Chickens

The aim of present investigation is to study the effect of single- and co-infection with REV and ALV-J on T lymphocytes bioactivities and histopathology in broiler chickens. The bioactivities of blood and spleen T lymphocytes including lymphoproliferation responses, cytotoxicitic responses, and histopathology of spleen were detected in broiler chickens singly- or co-infected with REV and ALV-J at different days post inoculation and the virus expressions in spleen of infected broiler chickens were detected with immunofluorescence assay （IFA）. The results indicated that blood and spleen T lymphocytes proliferation responses and cytotoxicity in broilers infected with REV or/and ALV-J were inhibited in the whole observed period compared with controls. In the co-infected chickens they were highly inhibited than in the single-infected. The histopathology of spleen in infected chickens at 17 and 37 d post inoculation （dpi） indicated that cell interium increased, the numbers of lymphocytes decreased, and the regrowth were destroyed or decreased, especially more significantly at 17 than at 37 dpi. The different numbers of virus were detected in spleen lymphocytes in REV- infected and/or ALV-J-infected chickens. In the spleen of co-infected chicken, both REV and ALV-J were detected and the total numbers of viruses were more than in chickens singly-infected with REV or ALV-J. Thus, the co-effect of REV and ALV-J caused more immunosuppression on T lymphocytes bioactivities in broiler chickens than single-effect of ALV-J or REV, which contributed to the sever histopathology and the product of tumor cells. This study will be helpful for understanding the effect of co-infection with many viruses and control them in poultry.

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.

Prediction of HLA-A 2.1-restricted CTL epitopes from IGFBP7 antigen of lung carcinoma

Objective： With the development of peptide-based cancer specific immunotherapy, the prediction of CTL epitopes from insulin-like growth factor-binding protein 7 （IGFBP7） is very important for some research about tumor metastasis. Because HLA-A2.1-expressing individuals cover 〉50% in the population of China, we aimed at identifying IGFBPT-encoded peptide presented by HLA-A2.1. Methods： In our study, a HLA-A2.1 restricted CTL epitope was identified by using the following two-step procedure： （a） computer-based epitope prediction from the amino acid sequence of IGFBP7 antigen; （b） Validation with epitope molecular modeling. Results： We obtained four epitopes with high immunogenicity scores by all of the three algorithms, i.e., BIMAS, SYFPEITH1 and IMTECH. Each of the four candidates satisfied the criteria of the HLA-A2.1- restricted CTL epitopes in molecular modeling analysis. Conclusion： The combination of BIMAS, SYFPEITHI and IMTECH method can improve the prediction efficiency and accuracy. Due to this research herein, this four epitopes have potential value for further studied, also have potential application in peptide-mediated immunotherapy. These epitopes may be useful in the design of therapeutic peptide vaccine for lung carcinoma and as immunotherapeutic strategies against lung carcinoma after identified by immunology experiment.

A dysfunction of CD4^+ T lymphocytes in peripheral immune system of Parkinson's disease model mice

Objective Parkinson's disease(PD),a neurodegenerative disorder,has been reported to be associated with brain neuroinflammation in its pathogenesis.Herein,changes in peripheral immune system were determined to better understand PD pathogenesis and provide possible target for treatment of PD through improvement of immune disorder.Methods l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine(MPTP) was intraperitoneally injected into mice to prepare PD model.Expression levels of pro-inflammatory and anti-inflammatory cytokines and transcription factors of CD4^+ T lymphocyte subsets in spleen and mesenteric lymph nodes and concentrations of the cytokines in serum were examined on day 7 after MPTP injection.Percentages of CD4^+ T lymphocyte subsets were measured by flow cytometry.Results MPTP induced PD-like changes such as motor and behavioral deficits and nigrostriatal impairment.Expression levels of the pro-inflammatory cytokines including interferon(IFN)-γ,interleukin(IL)-2,IL-17 and IL-22,in spleen and mesenteric lymph nodes were upregulated and their concentrations in serum were elevated in PD progression.But,the concentrations of the anti-inflammatory cytokines including IL-4,IL-10 and transforming growth factor(TGF)-β were not altered in the two lymphoid tissues or serum of PD mice.In addition,expression of T-box in T cells(T-bet),the specific transcription factor of helper T(Th) 1 cells,was downregulated,but expression of transcription factor forkhead box p3(Foxp3),the transcription factor of regulatory T(Treg) cells,was upregulated.In support of the results,the numbers of IFN-γ^+-producing CD4^+cells(Th1 cells) were reduced but CD4^+CD25^+ cells(Treg cells) were elevated in both the lymphoid tissues of PD mice.Conclusion PD has a dysfunction of peripheral immune system.It manifests enhancement of proinflammatory response and CD4^+T cell differentiation bias towards Treg cells away from Thl cells.

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.