Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus / 生物工程学报

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β2m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a SuperdexTM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.

Correlation analysis of serum IL-7 with evaluation of cellular immunity in chronic hepatitis B / 中华微生物学和免疫学杂志

Objective:To explore the possible correlation between serum detection of IL-7, IL-21, HBV-specific cytotoxic T lymphocytes (CTLs), HBV DNA, and the expression of CD127 on the T lymphocytes, and discuss the effect of IL-7 to cellular immune response in patients with chronic hepatitis B (CHB).Methods:Five hundred and sixty serum samples were collected from patients with CHB in Beijing Friendship Hospital from September 2017 to March 2020. The serum IL-7 and IL-21 were detected by enzyme-linked immunosorbent assay (ELISA), and HBV-specific CTLs and the expression of CD127 on the T lymphocytes were determined by flow cytometry. While HBV DNA were tested using quantitative real-time PCR (qRT-PCR). Subjects were divided into groups A, B, and C, according to the IL-7 levels (low: IL-7<20 pg/ml, medium: 20 pg/ml≤IL-7<30 pg/ml, and high: IL-7≥30 pg/ml).Results:The average concentration of serum IL-7 in patients with CHB was significantly lower than that of healthy controls ( P<0.01), and the difference among three groups was statistically significant ( P<0.01). Meanwhile, levels of IL-21, percentages of HBV-specific CTL, and the expression of CD127 on the CD8 + T lymphocytes showed an upward trend among groups, and there were significant differences among three groups ( P<0.01) with a positive correlation between each two variables ( P<0.01). However, HBV DNA showed a downward trend in group A, B and C, and the difference of the three groups were statistically significant ( P<0.01), which were negatively correlated with other variables ( P<0.01). Multiple linear regression analysis showed that HBV-specific CTL was an independent influencing factor for HBV DNA ( P<0.01), and IL-7, the expression of CD127 on the CD8 + T lymphocytes and IL-21 had an independent effect on HBV-specific CTL ( P<0.05). Conclusions:IL-7 could regulate HBV-specific immune response, and might be used as an effective cellular immune indicator to evaluate the cellular immune status of patients with chronic hepatitis B.

Application of quantitative detection of multiple-source cytomegalovirus DNA in diagnosis of cytomegalovirus pneumonia after allogeneic hematopoietic stem cell transplantation / 器官移植

Objective To evaluate the diagnostic value of quantitative detection of cytomegalovirus (CMV) DNA from different sources [plasma, sputum and bronchoalveolar lavage fluid(BALF)] for CMV pneumonia after allogeneic hematopoietic stem cell transplantation. Methods Clinical data of 405 recipients undergoing allogeneic hematopoietic stem cell transplantation were retrospectively analyzed. Among them, 19 recipients diagnosed with CMV pneumonia were assigned into the CMV pneumonia group, and 229 recipients with CMV viremia alone, 11 recipients without CMV pneumonia who received fiberoptic bronchoscopy and 16 recipients diagnosed with bacterial or fungal pneumonia based on pathogenic evidence receiving sputum culture were assigned into the control A, B and C groups, respectively. The incidence of CMV pneumonia was summarized. The CMV DNA load of specimens from different sources (plasma, sputum and BALF) of recipients with CMV pneumonia was analyzed. The clinical prognosis of recipients with CMV pneumonia was evaluated. Results Among 405 recipients undergoing allogeneic hematopoietic stem cell transplantation, 19 cases developed CMV pneumonia, and the overall incidence of CMV pneumonia was 4.7%(19/405). The CMV DNA load in the plasma, sputum and BALF of recipients with CMV pneumonia was higher than those in the control A, B and C groups (all P < 0.05). In the 19 recipients, 12 cases were cured after antiviral treatment and 7 died from treatment failure(3 cases abandoned treatment). The fatality was 37%(7/19). Conclusions Quantitative detection of CMV DNA in the plasma, sputum and BALF may increase the diagnostic rate of CMV pneumonia, thereby improving clinical prognosis of recipients undergoing allogeneic hematopoietic stem cell transplantation.

Effect of IL-15 addition on asbestos-induced suppression of human cytotoxic T lymphocyte induction

BACKGROUND@#Asbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8@*METHODS@#For MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8@*RESULTS@#IL-15 addition partially reversed the decrease in CD3@*CONCLUSION@#These findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.

Salidroside regulates DC through TLR4 to increase the lethality of T cells to lung cancer 3LLcells / 中国肿瘤生物治疗杂志

@#Objective： ：To investigatetheeffectofsalidroside(SAL)onthephenotype of dendritic cells (DCs) and the antitumor ability of cytotoxic T lymphocytes (CTL). Methods： ：Lewis lung cancer cell line 3LL, wild type (WT) C57BL/6 mice and TLR4-/- C57BL/6 mice were chosen for this study. Mice bone marrow derived DC precursor cells were obtained to differentiate into immature DCs, which were harvested on the sixth day of culture. CD11c+ DCs were obtained by magnetic beads screening, and further divided into PBS group, SAL group and lipopolysaccharide (LPS) group.After being cultured for 48 h, the effects of SAL on surface molecules and phagocytosis of DCs as well as the efffect of TLR4 pathway on the killing effect of T cells were detected by Fow cytometry. Results： ： Compared with PBS group, expressions of DC surface molecules CD80, CD86 and MHC Ⅱ significantly increased (all P<0.05), phagocytosis significantly decreased (P<0.05), and TLR4 expression level significantly increased (P<0.01) in SAL group; Compared with WT group, after being treated with SAL or LPS, the expressions of DC surface molecules CD80, CD86 and MHC Ⅱ decreased significantly in TLR4-/- group (all P<0.05); ComparedwithPBSgroup,theactivatedCTLinSALgroupexhibited a significantly elevated killing effect against lung cancer 3LLcells (P<0.05). Conclusion：SAL can induce DC maturation by regulating TLR4, thus improving the killing ability of T cells.

Cytotoxic activity of mammaglobin A-specific CD8+ cytotoxic T lymphocytes and cytokine-induced killers against breast cancer cells in vitro / 西安交通大学学报(医学版)

Objective: To compare the cytotoxic activity of mammaglobin A-specific CD8+ cytotoxic T lymphocytes (CTLs) and cytokine-induced killers (CIKs) against breast cancer cells in vitro. Methods: PBMCs were isolated in vitro from the peripheral blood of healthy volunteers. Then DCs, CTLs and CIKs were isolated, induced and cultured from PBMCs in vitro. CD8+ CTLs were purified with immunomagnetic beads from CTLs. DCs were infected with recombinant adenovirus encoding mammaglobin A(Ad-MGBA). CTLs and CIKs were co-cultured with DCs being infected with Ad-MGBA. The cytotoxic activity of mammaglobin A-specific CD8+ CTLs and CIKs against breast cancer cells was compared by flow cytometry. Results: The apoptosis rate of breast cancer cell MDA-MB-415, which expressed MGBA was 63.07% by CD8+ CTL killing and 48.35% by CIK killing (P<0.05). The apoptosis rate of breast cancer cell MDA-MB-231, which could express MGBA was 14.62% by CD8+CTL killing and 29.29% by CIK killing (P<0.05). Conclusion: The cytotoxic activity of antigen-specific CD8+ CTLs for the same antigen-expressing oncology cells is higher than CIKs. However, for different antigen-expressing oncology cells, the cytotoxic activity of CD8+CTLs is lower than that of CIKs.

Prediction of B cell Epitopes and HLA I Restricted CTL Epitopes Derived from Tumor Antigen SCCAg / 现代医院

Ojbective To predicte the HLAⅠrestricted CTL epitopes and B cell antigen epitopes derived from tumor antigen SCCAg. Methods The linear B cell epitopes and conformational B cell epitopes of tumor antigen SCCAg were predicted by Ellipro program. In addition, the HLAⅠrestricted CTL epitopes of SCCAg were predicted by NetCTL, Prot-Param and so on. Results B cell epitopes analysis revealed that SCCAg had 10 potential linear B cell epitopes and 5 conformational B cell epitopes; Combined with peptide HLAⅠbinding, proteasomal C - terminal cleavage and TAP transport efficiency, the NetCTL predicts that multiple HLAⅠrestricted CTL epitopes were present in the tumor antigen SCCAg. Conclusion The B cell epitopes and HLAⅠrestricted CTL epitopes can be predicted by multiple methods, which may lay the foundation for the further research on immunotherapy for targeting SCCAg.

Experimental study on ovarian cancer immunotherapy by EpCAM activated dendritic cells induce antigen-specific CD8+ cytotoxic T lymphocytes / 中国免疫学杂志

Objective:To observe the effect on ovarian cancer immunotherapy by dendritic cells (DC) which activated by Epithelial cell adhesion molecule (EpCAM) induce antigen-specific CD8+ cytotoxic T lymphocytes (CTL) and to provide some help to ovarian cancer immunotherapy.Methods: Interleukin (IL)-12,and IL-10 of DC were tested after inducing by EpCAM.Subsequently,EpCAM specific CTL CD8+ was induced by EpCAM-DC.The therapeutic effect and interferon (IFN)-γ of EpCAM-DC-CD8+ CTL on normal ovarian epithelial cells IOSE80 and ovarian cancer cell SKVO3 was detected.After treatment of EpCAM-DC-CD8+ CTL,the volume of ovarian tumor of bearing BALB/c-nu/nu mice was detected.Meanwhile,the morphology changes of tumor tissue were observed by HE staining.Results: Compared with PBS,EpCAM stimulation significantly inceased surface markers DC80,DC83,DC86 and HLA-DR levels,and added up to 4.79,4.85,4.60 and 10.91 times (P0.05).However,the killing rate of EpCAM-DC-CD8+ CTL on SKVO3 cells was 6.82-folds as much as that of DC-CD8+ CTL.Animal experiments showed that ovarian cancer transplantation tumor volume ratio after EpCAM-DC-CD8+ CTL treatment,was significantly lower than PBS group and DC-CD8+ CTL group,which reached 0.27 and 0.28 times,respectively (P<0.05).HE staining showed that EpCAM-DC-CD8+ CTL treatment resulted in significant changes of tumor tissues in pathology.Conclusion: EpCAM protein stimulated the maturation of DC that induced the production of EpCAM specific CD8+ CTL.EpCAM-DC-CD8+ CTL can effectively kill ovarian tumor cells and delay the growth of tumor,which is of great significance for the immunotherapy of ovarian cancer.

Experimental study of tumor-specific CTL induced by rmIL-18 treated on hepatocellular carcinoma / 中国免疫学杂志

Objective:To research rmIL-18 in vitro culture system CCs induce tumor-specific cytotoxic T lymphocytes CTL and anti-tumor effect in mice.Methods:Used Stem SepTM immune magnetic cells separation method to culture mouse spleen NK cells,T cells and DCs,established culture systems in vitro;used of different approaches,different doses rmIL-18 to immunize HCC tumor-bearing mice,researched the effect of rmIL-18 on tumor growth rate and survival time.Results:rmIL-18 could induce and promote tumor-specific CTL-mediated killing effects in vitro culture system;tumor-specific CTL could significantly inhibit tumor growth(P<0.01) of and prolong the survival time of liver cancer tumor-bearing mice(P<0.01),and the effect was increased with rmIL-18 concentration increased(P<0.01),and intratumoral injection was superior to intraperitoneal injection(P<0.01).Conclusion:rmIL-18 can induce tumor-specific CTL in vitro and play a role in anti-liver cancer in mice.

Molecular mechanism of CD8+ T cell activation and differentiation / 中国免疫学杂志

CD8+ T cells play an essential role in defending against viruses,intracellular bacteria,protozoal infections and clearance of tumors since almost all the nucleated cells express MHCⅠ molecule.Following antigen recognition,CD8+ T cells are activated and differentiated to different subsets of effector or memory cells,which could clear the pathogen and form long-term protection.Phenotypic markers,functional properties and anatomical locations are different among these CD8+ T cell subsets.They also show variation in surviving time,proliferation and effector functions when re-challenged with the pathogen or tumor.Multiple signaling pathways and transcriptional factors are involved in CD8+ T cells activation and differentiation,and will be discussed in this review.We will also briefly summary the clinical applications of T cells against tumor or pathogens.

CD133 epitope vaccine with gp96 as adjuvant elicits an antitumor T cell response against leukemia / 生物工程学报

Cancer stem cells are currently under intensive investigation due to their capabilities for tumor initiation, self-renewal, and resistance to chemotherapy. CD133 is implicated in stemness and the malignancy of tumor cells. Here, we explored heat shock protein gp96 adjuvanted CD133 epitope vaccine against leukemia. We screened and identified three H2-Kd-restricted cytotoxic T lymphocyte (CTL) epitopes derived from CD133, CD133₄₁₉₋₄₂₈, CD133₇₀₂₋₇₁₀ and CD133₇₆₀₋₇₆₉. The immunogenicity and antitumor activity of the epitope vaccine using heat shock protein gp96 as adjuvant were further determined in CD133⁺ leukemia xenograft mice. Finally, we demonstrate that adoptive transfer of epitope-specific CTLs led to suppression of leukemia growth. Our data therefore provide the basis for designing a CD133 epitope vaccine to activate specific CTLs against CD133⁺ leukemia and other cancers.

Restoration of Declined Immune Responses and Hyperlipidemia by Rubus occidenalis in Diet-Induced Obese Mice

Hyperlipidemia, which is closely associated with a fatty diet and aging, is commonly observed in the western and aged society. Therefore, a novel therapeutic approach for this disease is critical, and an immunological view has been suggested as a novel strategy, because hyperlipidemia is closely associated with inflammation and immune dysfunction. In this study, the effects of an aqueous extract of Rubus occidentalis (RO) in obese mice were investigated using immunological indexes. The mice were fed a high-fat diet (HFD) to induce hyperlipidemia, which was confirmed by biochemical analysis and examination of the mouse physiology. Two different doses of RO and rosuvastatin, a cholesterol synthesis inhibitor used as a control, were orally administered. Disturbances in immune cellularity as well as lymphocyte proliferation and cytokine production were significantly normalized by oral administration of RO, which also decreased the elevated serum tumor necrosis factor (TNF)-α level and total cholesterol. The specific immune-related actions of RO comprised considerable improvement in cytotoxic T cell killing functions and regulation of antibody production to within the normal range. The immunological evidence confirms the significant cholesterol-lowering effect of RO, suggesting its potential as a novel therapeutic agent for hyperlipidemia and associated immune decline.

The inlfuence of different concentrations of IL-2 on the cultivation system for peptide-speciifc CTL inductionin vitro / 中国癌症杂志

Background and purpose:Cytotoxic T lymphocyte (CTL) plays a vital role in the process of anti-tumor immunology. The aim of this study was to investigate whether changes in concentration of IL-2 (50, 200 and 1 000 U/mL) would affect the sub-population and cytotoxic function of cells cultivated by peptide-specific CTL induction systemin vitro and also observe whether using the concentration of IL-2 at a range of 50-1 000 U/mL isbeneifcial to regulatory cells (Tregs) enrichment.Methods:Peripheral blood from 10 healthy donors and 10 cancer patients that were HLA-A2 positive, were collected in the study. HLA-A2 restricted CTL epitope P321 (ILIGETIKI) derived from COX-2 pulsed with different concentrations of IL-2 were used to induce peptides-speciifc CTLin vitro. Flow cytometry was performed to analyze the proliferative capability, the proportion of different T-cell subsets, and secretion of perforin, granzyme B and IFN-γ. IFN-γ secretion was assessed by ELISpot assay.Results:High concentration of IL-2 increased the proliferative activity. The percentage of CD4+ T cells of cancer patient group was signiifcantly higher than that of healthy donor group, while the percentage of CD8+ T cells of cancer patient group was signiifcantly lower than that of healthy donor group. And there was no signiifcant difference in the percentages of CD4+ T cells, CD8+ T cells and Tregs among groups with different IL-2 concentrations. No difference was seen in cytokine (perforin, granzyme B, IFN-γ) secretion capacity of CD8+ T cells. ELISpot study revealed that high-dose IL-2 resulted in the increasing of IFN-γ secretion.Conclusion:The sub-population and the function of cells cultured by peptide-speciifc CTL induction systemin vitro are not affected by different concentrations of IL-2. Furthermore, high concentrations of IL-2 (50-1 000 U/mL) do not provide the enrichment for Tregs. Higher concentration of IL-2 is likely to cause high secretion of IFN-γ in ELISpot assay. In order to exclude the distraction of NK cells or NKT cells, the concentration of 50 U/mL is better choice.

Study on immunological effect of dendritic cell transfected by recombinant plasmid ofΔNp73αon breast cancer / 中国免疫学杂志

Objective:To research the imumunological effect of dendritic cell transfected by recombinant plasmid of ΔNp73αon breast cancer.Methods:Cultivate dendritic cells by cytokines( GM-CSF,IL-4,TNF-α) from human umbilical cord blood in vitro, CD1a,CD83 were detected by flow cytometry.Recombinant plasmid pcDNA-HA/ΔNp73αwas used to transfected DCs,the transfection was detected by Western blot.Transfected DCs cocultivate with T cell induce specific cytotoxic lymphocytes( CTL).Proliferation ability of T cell was detected by MTT methods.IFN-γin the culture supernatant of the transfected DCs were detected by ELISA.The killing effect on breast cancer MDA-MB-231 was detected by LDH method.Results:The expression of CD1a(56%),CD83(74%)in mature DC was higher than immature DC(CD1a 19%,CD83 13%)(P<0.01).ΔNp73αtransfected group had a special band.The killing effect of specific CTL induced byΔNp73α-DC and T cells cocultivation on breast cancer cell MDA-MB-231 was statistic stronger than DC group(P<0.05),the proliferation of T cell and secretion level of IFN-γcompared with DC and pcDNA group were statistically significant(P<0.01).Conclusion:The CTL induced byΔNp73αtransfected DC vaccine has significant effect on killing breast cancer.

Effects of HIV infection on the disease progression of hepatitis C in patients with HIV/HCV coinfec-tion / 中华微生物学和免疫学杂志

Objective To analyze the differentiation status of CTL and to evaluate its clinical val-ue in patients with HIV/HCV coinfection .Methods Twenty-eight patients with HIV/HCV coinfection and twelve patients with single HCV infection were enrolled in this study .The technique of Fibro-Scan was used to evaluate liver fibrosis .The viral load of HCV was detected by real-time quantitative PCR .Flow cytometry analysis was performed to measure the differentiation status of CTL .Results Both of the levels of alanine transaminase ( ALT) and alkaline phosphatase ( ALP) in patients with HIV/HCV coinfection were signifi-cantly higher than those in patients with single HCV infection [(53.7464±48.1180) U/L vs (27.4750± 13.9850) U/L, P=0.012;(24.5071±8.1940) g/L vs (16.9667±7.1890) g/L, P=0.009].The liver stiffness of patients with HIV/HCV coinfection was higher than that of patients with single HIV infection [(5.9500, 5.8250) Kpa vs (5.1500, 1.0500) Kpa, P=0.117].Compared with the patients with single HCV infection, the patients with HIV/HCV coinfection showed higher viral loads of HCV [( 6.4768, 5.3434) lg copy/ml vs (2.6815, 1.6990) lg copy/ml, P=0.012], but lower clearance rate of HCV [32.14%vs 75%, P=0.032].Compared with the patients with single HCV infection , the patients with HIV/HCV coinfection showed lower percentages of CD 27+CD28+CTL [(28.265±15.095)%vs (18.068±10.263)%, P=0.017), but higher percentages of CD27+/-CD28+CTL [(62.449 ±14.561)% vs (71.111±12.681)%, P=0.066].A trend toward negative correlation was observed between the percent -age of CD27+CD28+CTL and the degree of liver stiffness (r=-0.310, P=0.058).Conclusion HIV in-fection could accelerate the progression of liver disease in patients with HIV /HCV coinfection by affecting the differentiation of CTL .

Relationship between HBV DNA level, expression of PD-1 on CTL and liver functions in patients with hepatitis B virus cirrhosis / 实用医学杂志

Objective To explore the relationship between serum HBV DNA level , expression of PD-1 on CTL and liver functions in patients with hepatitis B virus cirrhosis. Methods 109 HBV hepatic cirrhosis patients with , HBV DNA positive , HBeAg positive and HLA-A2 positive were divided into two groups according to their HBV DNA levels,52 cases in group A, HBV DNA 2-4 log10 copies/mL, 57cases in group B, HBV DNA 5-7 log10 copies/mL , differences of HBV specific CTL surface PD-1 expressions , HBV specific CTL levels and liver functions were compared between two groups. Results HBV specific CTL surface PD-1 expressions of group A were lower than that of group B (t = 11.101, P < 0.01), HBV specific CTL levels of group A were higher than that of group B (t = 24.424, P < 0.01), ALT of group A were lower than that of group B (t = 2.652, P < 0.01), ALB of group A were higher than that of group B (t = 2.347, P < 0.05). Child-pugh rating, grade C of group A was lower than that of group B (χ2= 4.262,P < 0.05). Conclusions HBV specific CTL surface PD-1 expression levels were higher in those hepatic cirrhosis patients with , high level of serum HBV DNA, and HBV specific CTL levels were lower, liver functions damage were much serious. So, lower HBV DNA levels , lower HBV specific CTL surface PD-1 expression levels and higher HBV specific CTL levels can product relatively good effect of anti-viral treatment.

Avaliação fenotípica e funcional dos linfócitos T citotóxicos de indivíduos infectados pelo HTLV-1 com diagnóstico de HAM/TSP / Phenotypic and functional assessment of cytotoxic T lymphocytes from individuals infected with HTLV-1 diagnosed with HAM / TSP

O Brasil representa uma das áreas endêmicas para o vírus linfotrópico de células T humanas do tipo 1 (HTLV-1) e a cidade de Salvador, Bahia, possui a maior prevalência nacional da infecção por este retrovírus (1,8%), com cerca de 50.000 pessoas infectadas. O HTLV-1 foi o primeiro retrovírus humano descrito e está classicamente associado à leucemia/linfoma de células T do adulto (ATLL) e à mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). A HAM/TSP é uma doença inflamatória do sistema nervoso central, cujos mecanismos imunopatogênicos não estão completamente elucidados. O papel dos linfócitos T citotóxicos na patogênese desta doença ainda não está bem definido. Neste estudo, foram avaliados o fenótipo e a função de linfócitos T citotóxicos de pacientes infectados pelo HTLV-1 com HAM/TSP...

Brazil represents one of the largest endemic areas for human T-lymphotropic virus cells type 1 (HTLV-1) infection and associated diseases. Salvador, Bahia, is considered as the Brazilian city with the highest national HTLV-1prevalence (around 1.8% in the general population). HTLV -1 was the first human retrovirus described and is classically associated with adult Tcell leukemia/lymphoma (ATLL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic and progressive inflammatory disease of the central nervous system and your immunopathogenic mechanisms are not completely understood. The role of cytotoxic T-lymphocytes (CTLs) in the pathogenesis of this disease is still undefined. In this study we evaluated the phenotype and function of cytotoxic Tlymphocytes from HTLV-1-infected patients with HAM/TSP...

Induction of Potent Antigen-specific Cytotoxic T Cell Response by PLGA-nanoparticles Containing Antigen and TLR Agonist

Previously we showed that biodegradable nanoparticles containing poly-IC or CpG oligodeoxynucleotide (ODN) together with ovalbumin (OVA) were efficient at inducing MHC-restricted presentation of OVA peptides in dendritic cells. The CTL-inducing activities of the nanoparticles were examined in the present study. Nanoparticles containing poly-IC or CpG ODN together with OVA were prepared using biodegradable polymer poly(D,L-lactic acid-co-glycolic acid), and then were opsonized with mouse IgG. The nanoparticles were injected into the tail vein of mice, and 7 days later the OVA-specific CTL activities were measured using an in vivo CTL assay. Immunization of mice with the nanoparticles containing poly-IC or CpG ODN together with OVA elicited potent OVA-specific CTL activity compared to those containing OVA only. In accordance with these results, nanoparticles containing poly-IC or CpG ODN together with OVA exerted potent antitumor activity in mice that were subcutaneously implanted with EG7.OVA tumor cells. These results show that encapsulation of poly-IC or CpG ODN together with antigen in biodegradable nanoparticles is an effective approach for the induction of potent antigen-specific CTL responses in vivo.

Deoxypodophyllotoxin Induces a Th1 Response and Enhances the Antitumor Efficacy of a Dendritic Cell-based Vaccine

BACKGROUND: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-kappaB. METHODS: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. RESULTS: DPT promoted the activation of CD8+ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-gamma production and induction of cytotoxic T lymphocyte activity. CONCLUSION: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

Characterization of CTL Clones Specific for Single Antigen, H60 Minor Histocompatibility Antigen

BACKGROUND: Disparities of Minor H antigens can induce graft rejection after MHC-matched transplantation. H60 has been characterized as a dominant antigen expressed on hematopoietic cells and considered to be an ideal model antigen for study on graft-versus-leukemia effect. METHODS: Splenocytes from C57BL/6 mice immunized with H60 congenic splenocytes were used for establishment of H60-specific CTL clones. Then the clones were characterized for proliferation capacity and cytotoxicity after stimulation with H60. Clone #14, #15, and #23 were tested for the TCR binding avidity to H60-peptide/H-2Kb and analyzed for TCR sequences. RESULTS: H60-specific CTL clones showed different levels of proliferation capacity and cytotoxic activity to H60-stimulation. Clones #14, #15, and #23 showed high proliferation activity, high cytotoxicity, and low activities on both aspects, respectively, and have TCRs with different binding avidities to H60-peptide/H-2Kb with t 1/2 values of 4.87, 6.92, and 13.03 minutes, respectively. The TCR usages were Valpha12D-3-01+Jalpha11-01 and Vbeta12-1-01+Dbeta1-01+J2-7-01 for clone #14, Valpha13D-1-02+Jalpha34-02 and Vbeta13-1-02+Dbeta2-01+Jbeta2-7-01 for clone #15, and Valpha16D+Jalpha45-01 and Vbeta12-1-01+Dbeta1-01+Jbeta2-5-01 for clone #23. CONCLUSION: The results will be useful for modeling GVL and generation TCR transgenic mouse.