Generation of αGal-enhanced bifunctional tumor vaccine

Hepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis and high mortality. In this study, we demonstrated a novel vaccine targeting HCC and tumor neovascular endothelial cells by fusing recombinant MHCC97H cells expressing porcine α-1,3-galactose epitopes (αGal) and endorphin extracellular domains (END) with dendritic cells (DCs) from healthy volunteers. END+/Gal+-MHCC97H/DC fusion cells induced cytotoxic T lymphocytes (CTLs) and secretion of interferon-gamma (IFN-γ). CTLs targeted cells expressing αGal and END and tumor angiogenesis. The fused cell vaccine can effectively inhibit tumor growth and prolong the survival time of human hepatoma mice, indicating the high clinical potential of this new cell based vaccine.

Efficacy of adoptive immunocyte infusion combined with immunodeprivation in the treatment of castration resistant prostate cancer / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To observe the clinical effect of adoptive immunocyte infusion combined with immunodeprivation in the treatment of castration-resistant prostate cancer. Methods: The information of 35 patients with castration resistant prostate cancer, who were treated in the Affiliated Guizhou Provincial Cancer Hospital of Guizhou Medical University from 2011 to 2018 was collected. According to different treatments, these patients were divided into biotherapy group (18 cases) and non-biotherapy group (17 cases). Patients in the non-biotherapy group were treated with abiraterone or docetaxel, while the patients in biotherapy group were treated with cytotoxic T lymphocytes (CTL) in combination with cyclophosphamide (CTX). The treatment efficacy in the biotherapy group and the non-biotherapy group was evaluated by comparing the changes of prostate cancer-specific antigen (PSA), improvement of subjective indicators (bone pain, sleep, physical strength) and clinical efficacy before and after treatment. Results: (1) PSA level: after treatment, PSA was decreased in both groups; the biotherapy group had an obvious decrease (P<0.01),which was more significant than the decrease in non-biotherapy group (P<0.05). (2) Clinical efficacy: The clinical efficacy of patients after CTL treatment was significantly different from that of non-biotherapy group (P<0.01). (3) Subjective indicators: The bone pain, sleep and physical strength of the patients in the biotherapy group were significantly improved after treatment, and there was a significant difference as compared with patients of the non-biological treatment group (P<0.01). (4) Overall survival: The median survival of the patients receiving biotherapy was 4 months longer than patients from non-biological treatment group, but the difference was insignificant (P=0.3935). Conclusion: CTL combined with CTX in the treatment of castration resistant prostate cancer can significantly reduce PSA and improve the quality of life of patients.

Effect of asbestos exposure on differentiation and function of cytotoxic T lymphocytes

Asbestos exposure is known to cause malignant mesothelioma, which is associated with poor prognosis. We focused on and examined the effect of asbestos exposure on the differentiation and function of cytotoxic T lymphocytes (CTLs). CTLs have the ability to specifically attack tumor cells after being differentiated from naïve CD8 T cells following antigen stimulation. Exposure to chrysotile B asbestos suppressed the differentiation of CTLs during the mixed lymphocyte reaction (MLR) and was associated with a decrease in proliferation of CD8 T cells. Additionally, in an effort to investigate the mechanism associated with suppressed CTL differentiation upon exposure to asbestos, we focused on IL-2, a cytokine involved in T cell proliferation. Our findings indicated that insufficient levels of IL-2 are not the main cause for the suppressed induction of CTLs by asbestos exposure, although they suggest potential improvement in the suppressed CTL function. Furthermore, the functional properties of peripheral blood CD8 lymphocytes from asbestos-exposed individuals with pleural plaque (PP) and patients with malignant mesothelioma (MM) were examined. MM patients showed lower perforin levels in CD8 lymphocytes following stimulation compared with PP-positive individuals. The production capacity of IFN-γ in the MM group tended to be lower compared with healthy volunteers or PP-positive individuals. In an effort to determine whether chronic and direct asbestos exposure affected the function of CD8 T cells, cultured human CD8 T cells were employed as an in vitro model and subjected to long-term exposure to chrysotile (CH) asbestos. This resulted in decreased levels of intracellular perforin and secreted IFN-γ. Those findings underlie the possibility that impaired CD8 lymphocyte function is caused by asbestos exposure, which fail to suppress the development of MM. Our studies therefore reveal novel effects of asbestos exposure on CTLs, which might contribute towards the development and implementation of an effective strategy for the prevention and cure of malignant mesothelioma.

Nucleoprotein vaccine induces cross-protective cytotoxic T lymphocytes against both lineages of influenza B virus

PURPOSE: The influenza B virus diverges into two antigenically distinct lineages: B/Yamagata and B/Victoria. Influenza B is the dominant circulating virus during some influenza seasons, and recent data demonstrated that influenza A and B infection similarly cause severe clinical symptoms in hospitalized patients. Nucleoprotein (NP) is a good target for a universal influenza vaccine. This study investigated whether NP epitope variation within two lineages affects the dominant cytotoxic T lymphocyte (CTL) responses induced by vaccination and the resultant protective immunity. MATERIALS AND METHODS: The NP of B/Yamagata/16/1988, the representative strain of the Yamagata lineage, includes a dominant CTL epitope, FSPIRITFL, while B/Shangdong/7/1997 from the Victoria lineage has one amino acid difference in this sequence, FSPIRVTFL. Two recombinant replication-deficient adenovirus (rAd)-vectored vaccines expressing either NP were prepared (rAd/B-NP(I) and rAd/B-NP(V), respectively) and administered to BALB/c mice intranasally. To examine the efficacy of vaccination, antibody responses, CTL responses, and morbidity/mortality after challenge were measured. RESULTS: Both vaccines induce similar antibody and CD8 T-cell responses cross-reacting to both epitopes, and also confer cross-protection against both lineages regardless of amino acid difference. CONCLUSION: The rAd-vectored vaccine expressing the NP could be developed as universal influenza B vaccine which provides broader protection.

Identification of HLA-A3 restricted cytotoxic T-lymphocyte epitopes from cancer-testis antigen MAGEC2 / 中国病理生理杂志

AIM:To predict and identify an HLA-A3 supertype-restricted cytotoxic T-lymphocyte(CTL) epitope derived from MAGEC2,which is utility in epitope design for the development of HLA-based vaccines and immuno-therapeutics.METHODS:HLA-A3 epitopes from MAGEC2 protein were predicted by BIMAS, SYFPEITHI and IEDB. The binding affinity of the peptides to HLA-A*03 molecule was evaluated by T2A3 cell binding assay.ELISPOT assay was used to investigate the ability of the peptides inducing specific restricted CTLs to release interferon -γ(IFN-γ).The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS:The candidate peptides P147,P167, P196, P229 and P251 showed moderate affinity toward HLA-A3 molecule.ELISPOT assay showed that P167,P196 and P251 were able to induce specific CTLs and higher levels of IFN-γwere released.The CTLs induced by P196 and P251 were able to lyse target cells.CONCLUSION:The peptides P196 and P251 have higher binding affinity with HLA-A3 and retain immunogenicity.They are excellent HLA-A3-restricted CTL epitopes from tumor antigen MA-GEC2,which could serve as new candidates towards antitumor peptide vaccines.

Identification of cytotoxic T-lymphocyte epitopes from hepatocellular car-cinoma antigen MAGEC2 / 中国病理生理杂志

AIM:To observe whether modified epitopes from hepatocellular carcinoma antigen MAGEC 2 have HLA-A2-restricted antitumor ability.METHODS:HLA-A2 epitopes from MAGEC2 protein were predicted by NetCTL 1. 2,SYFPEITHI and IEDB.The change of binding anchor motifs by replacing anchor residues created the modified peptides from MAGEC2.The binding affinity of the peptides to HLA-A*0201 molecule was evaluated by T2 cells binding assay. ELISPOT assay and intracellular cytokine staining were used to investigate the ability of the peptides inducing specific re -stricted cytotoxic T-lymphocytes(CTLs)to release interferon-γ(IFN-γ).The ability of the peptides to induce T-cell re-sponse was investigated by cytotoxicity assay in vitro.RESULTS:The candidate peptides P248, P248-1Y,P356,P356-1Y,P356-2L and P356-1Y2L showed moderate affinity toward HLA-A2 molecule.T2 binding assay showed that P248-1Y and P356-1Y2L showed significantly higher affinity for HLA-A2 than the native peptides.ELISPOT assay and intracellular cytokine staining showed P248, P248-1Y, P356 and P356-1Y2L were able to induce specific CTLs to release IFN-γ. ELISPOT assay showed that significantly higher levels of IFN-γrelease were induced by P248-1Y and P356-1Y2L than the native peptides.The CTLs induced by P248,P248-1Y,P356 and P356-1Y2L lysed HepG2 cells,and P248-1Y and P356-1Y2L peptide-specific CTLs showed higher cytotoxicity against HepG 2 cells than the native peptide-specific CTLs(P<0.05).CONCLUSION: Compared with the native peptides, modified epitopes P248-1Y and P356-1Y2L have higher binding affinity with HLA-A2 and retain immunogenecity.In addition, the antitumor immunity effects of modified epitope P248-1Y and P356-1Y2L are stronger than the native peptides.The peptides P248-1Y and P356-1Y2L are excellent HLA-A2-restricted CTL epitopes from tumor antigen MAGEC 2, which could serve as new candidates towards antitumor peptide vaccines.

Combination of R848 and Poly（I:C）promotes antigen presentation by DC and enhances killing effect of CTL on lung adenocarcinoma A549 cells / 中国病理生理杂志

AIM: To investigate the effect of R848(a Toll-like receptor 7/8 agonist)combined with poly-inosinic:polycytidylic acid [Poly(I:C),a Toll-like receptor 3 agonist] on dendritic cell(DC)maturation,and the killing effect of DC-induced cytotoxic T-lymphocytes(CTL)on human lung adenocarcinoma A549 cells.METHODS:Mononu-clear cells were isolated from human peripheral blood and induced to differentiate into DC.The whole-cell lysate of A549 cells,namely tumor cell lysate(TCL), was used as antigen.R848 combined with Poly(I:C)was used as adjuvant to stimulate the DC.DC surface markers were analyzed by flow cytometry.The DC stimulated by antigen was co-cultured with T-lymphocytes for 7 d to induce CTL.The culture supernatant and CTL were collected.The levels of interleukin-12(IL-12)p70,interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the supernatant were measured by ELISA.The CTL and A549 cells were co-cultured for 16 h,and the cytotoxicity was observed by LDH assay.RESULTS:The expres-sion of CD83 and CD80 on the DC surface,and the secretion of IL-12 p70 in DC-R848+Poly(I:C)group were significant-ly increased compared with DC-TCL group(P<0.01).In addition,the cytotoxicity of CTL for A549 cells in DC-R848+Poly(I:C)group was significantly enhanced compared with DC-TCL group(P<0.01).The secretion levels of IFN-γand TNF-αin DC-R848+Poly(I:C)group were significantly elevated compared with DC-TCL group(P<0.01).CONCLU-SION:R848 combined with Poly(I:C)significantly promotes DC maturation and activation, and enhances the antigen-presenting effect of DC and the cytotoxicity of DC-induced CTL.

Identification and molecular modification of HLA-A2-restricted cytotoxic T-lymphocyte epitopes from pancreatic tumor antigen MUC4 / 中国病理生理杂志

AIM:To observe whether modified epitopes from pancreatic tumor antigen mucin 4 (MUC4) have HLA-A2-restricted antitumor ability.METHODS:RT-PCR and Western blot were used to identify the expression of MUC4 in the pancreatic tumor cell lines CAPAN-2 and ASPC-1.HLA-A2 epitopes from MUC4 protein were predicted by the software of NetCTL 1.2, BIMAS, SYFPEITHI and IEDB.The modified peptides from MUC4 containing HLA-A2 were obtained by replacing anchor residues of the binding anchor motifs.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecule was evaluated by T2 binding assay.ELISPOT assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T-lymphocytes (CTLs) and release of IFN-γ.The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS:The expression of MUC4 was observed in the CAPAN-2 cells and ASPC-1 cells.The candidate peptides P1944-1Y, P1944-2L, P1944-1Y2L, P2004 and P2004-1Y9V showed moderate affinity toward HLA-A2 molecule.T2 binding assay showed that P1944-1Y2L and P2004-1Y9V had significantly higher affinity for HLA-A2 than the native peptides.ELISPOT assay showed P1944, P1944-1Y2L, P2004 and P2004-1Y9V were able to induce specific CTLs and more amounts of IFN-γ were released.ELISPOT assay showed that significantly more amounts of IFN-γ released by P1944-1Y2L and P2004-1Y9V were observed than the native peptides.The CTLs induced by P1944, P1944-1Y2L, P2004 and P2004-1Y9V lyzed the CAPAN-2 cells.P1944-1Y2L and P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell line CAPAN-2 than the native peptide-specific CTLs.CONCLUSION:Compared with the native peptides, modified epitopes P1944-1Y2L and P2004-1Y9V have higher binding affinity with HLA-A2 and retain immunogenecity.In addition, the anti-tumor immunity of modified epitopes P1944-1Y2L and P2004-1Y9V is stronger than that of the native peptides.The peptides P1944-1Y2L and P2004-1Y9V are excellent HLA-A2-restricted CTL epitopes from tumor antigen MUC4, which could serve as new candidates towards antitumor peptide vaccines.

Research on mechanism of GM-CSF secreting liver cancer vaccine on CTL killing activity of transplanted liver cancer mice / 中国生化药物杂志

Objective To study the effect of granulocyte-macrophage colony-stimulating factor ( GM-CSF) secreting liver cancer vaccine on killing activity of cytotoxic T lymphocytes ( CTL) of transplanted liver cancer mice and its mechanism.Methods There were three groups:liver cancer vaccine group (A group), liver cancer group (B group) and PBS group (C group).The transplanted liver cancer model was builded with injection of H 22 hepatoma cells, while the GM-CSF secreting liver cancer vaccine group and PBS group was builded.GM-CSF secreting liver cancer vaccine group and PBS group were establised.The levels of CD8 +T cell in peripheral blood were detected by flow cytometry.The killing activity of cytotoxic T lymphocytes ( CTL) of spleen cells was detected by MTT method.The expression levels of tumor necrosis factor-α( TNF-α) and interferon-γ(γ-INF) were detected by Western blot.Results The flow cytometry results showed that, compared with B group, the levels of CD8 +T cell of A group significantly increased (P<0.01).MTT results showed that, compared with B group, the killing activity of cytotoxic T lymphocytes (CTL) in A group significantly increased (P<0.01).Western blot results showed that, compared with B group, the expression levels of tumor necrosis factor-α(TNF-α) and interferon-γ(γ-INF) in A group significantly decreased (P<0.01).Conclusion GM-CSF secreting liver cancer vaccine can significantly inhibit the activity of H22 cell, and its possible mechanism of action may be to activated CD8 +T expression, improve cytotoxic activity of CTL of spleen cells, and reduce TNF-αand γ-INF protein expression.

Research on PR1 specific cytotoxic T lymphocytes of HLA-A0201 positive chronic myelogenous leukemia / 白血病·淋巴瘤

Objective To evaluate the relationship between proportion of PR1 specific cytotoxic T lymphocytes (CTLs) in the peripheral blood and prognosis and curative effect in patients with HLA-A0201 positive chronic myelogenous leukemia (CML),and to discuss whether PR1 peptide could be used as the following immune therapeutic method for patients who had achieved the standard of stop treatment.Methods The soluble HLA-A0201/PR1 tetramer and flow cytometry were applied to determine the proportion and the frequency of PR1 specific CTLs in peripheral blood from 28 HLA-A0201 positive CML patients.The proportions were compared among different phases of patients.The correlations between the proportion of PR1 specific CTLs and clinical parameters were analyzed.Results There was a negative correlation between PR1 specific CTLs and PCR (bcr-abl/abl)Is (r =-0.658,P ＜ 0.001).The frequencies of PR1 specific CTLs at 3-month,6-month,9-month,12-month,2-year,3-year,4-year,5-year,6-year were (0.06±0.02) ％,(0.10± 0.02) ％,(0.14±0.02) ％,(0.16±0.02) ％,(0.20±0.03) ％,(0.18±0.03) ％,(0.18±0.01) ％,(0.17±0.05) ％ and (0.18±0.03) ％,respectively.The frequency of PR1 specific CTLs at 3-month,6-month or 9-month was statistically different compared with that of the other time spots (P ＜ 0.05),and there were no statistical differencies among the frequencies at 1-year,2-year,3-year,4-year,5-year,6-year (P ＞ 0.05).For patients treated with IM 400 mg qd,the frequency of PR1 specific CTLs in high-risk group was lower than that in low-risk or intermediate-risk groups.Conclusion PR1 specific CTLs can be detected in patients who achieved good curative effect,and is correlated with tumor burden,which indicates that PR1 specific CTLs may be related to the action of resisting leukemia and provide the evidence for PR1 peptide as a potential immune therapeutic schedule in patients who have achieved stable MR45 and MR50.

Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2 / 中国病理生理杂志

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2.METHODS:RT-PCR and Western blot was used to determine the expres-sion of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29.HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay.ELISPOT as-say was used to investigate the levels of IFN-γ.The cytotoxicity assay in vitro was also used to determine the ability of indu-cing T cell response by the peptides.RESULTS:The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule.ELISPOT assay showed P485 and P965 induced CTLs of IFN-γrelease form CTLs.The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION:The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.

Application of HLA-A*0201/CAP-1 tetramer in colorectal cancer / 中国免疫学杂志

Objective:To analyze the frequencies of HLA-A*0201 restricted CEA-specific CD8+T cells, HLA-A*0201/FLUmp tetramer and HLA-A*0201/CAP-1 tetramer were applied in patients with colorectal cancer.Methods: Lymphocytes from peripheral blood and lymph node,1×106 cells/ml,were incubated with 1μg HLA-A*0201/peptide tetramers and anti-CD8 for 1 h at 25 coseperately.The cells were then washed in PBS.Next,the cells were illuminated by detecting frequencies of FLUmp-specific CD8+T cells and CAP-1-specific CD8+T cells with flow cytometry.Results: HLA-A*0201/peptide were used to detect CAP-1 or FLUmp-specific CD8+T cells,which were analyzed either healthy individuals or patients with colorectal cancer.We did not find differences in average frequencies of FLUmp-specific CD8+T cells between 11 HLA-A*0201+patients with colorectal cancer and 14 HLA-A*0201+healthy individuals [ ( 0.671 ±0.421 )%, ( 0.564 ±0.408 )%].But the frequencies of CAP-1-specific CD8+T cells of HLA-A*0201+patients with colorectal cancer showed higher than HLA-A*0201+healthy individuals [ ( 2.409 ± 2.385 )%, ( 0.020 ± 0.021)%respectively],which was statistically significant(P=0.008).Conclusion:The frequencies of CAP-1-specific CD8+T cells in PBMC from peripheral blood and lymph node of HLA-A*0201+patients were increased,showed CEA-specific CTs has a vital role in colorectal cancer.

The study of heat shock protein 70 as a adjuvant carrier on HBV-specific immune response in vitro / 重庆医学

Objective To investigate the effect of mycobacterium tuberculosis heat shock protein 70(TB .HSP70) as an adjuvant carrier on stimulating hepatitis B virus (HBV) core antigen(HBcAg)specific immune response to an accompanying cytotoxic T lym-phocytes epitope peptide from HBV core antigen in vitro .Methods Recombinant proteins HSP70(P1)、HSP70-HBcAg(18-27) (P2)、HSP70-PreS2B (18-24)-PreS2Th(37-53)-HBcAg(18-27)(P3) were expressed in methylotropic yeast Pichia pastoris GS115 . The expression of recombinant proteins was identified by SDS-PAGE and Western blot .The effect of recombinant proteins on den-dritic cell and lymphocytes of chronic HBV infection volunteers was investigated in vitro .The maturation of dendritic cell was meas-ured by flow cytometry ;the secretion of Th1 cytokines such as IL-12p70 ,IL-1β,TNF-αand IFN-γ was measured by ELISA ;the proliferation of lymphocytes was measured by TdR-3H ;the HBV-spesific cytotoxic activity was measured by the classic 51 Cr .Re-sults The recombinant proteins (P1 ,P2 ,P3) were constructed successfully .P1 ,P2 ,P3 could activate dendritic cell from chronic HBV infection volunteers by upregulation CD1a ,CD40 ,CD86 and production Th1 cytokines such as IL-12p70 ,IL-1β and TNF-α. Especially P3 could better induce autologous T cells to generate HBV specific cytotoxic T lymphocytes response ,activate the prolif-eration of lymphocytes and release IFN-γeffectively .However ,the recombinant HSP70 showed no target cell killing and could not induce immune response effectively .Conclusion TB .HSP70 can be used as an adjuvant carrier to stimulate HBV specific immune response to an accompanying cytotoxic T lymphocytes epitope peptide from HBV core antigen ,and enhance immunogenicity of the cytotoxic T lymphocytes epitope peptide .The P3 with B-and T-epitope can activate the HBV specific immune response effectively .

Antígenos leucocitarios humanos y su asociación con resistencia/susceptibilidad a la infección por el VIH-1 / Human leukocyte antigens associated to resistance/susceptibility to HIV infection

El curso clínico de la infección por el virus de inmunodeficiencia humana tipo 1 es un proceso variable y complejo que depende de componentes virales y del hospedero. En la mayoría de los individuos infectados, la respuesta inmune generada en las fases iniciales de la infección logra controlar la replicación viral por mecanismos efectores innatos, de anticuerpos neutralizantes específicos y particularmente de la actividad de los linfocitos T CD8+ (LT CD8+). A pesar de generarse una respuesta inmune específica, esta se vuelve ineficaz en las etapas crónicas de la infección debido a cambios en los péptidos virales blanco, los cuales conducen a una pérdida del reconocimiento del antígeno presentado; dichos cambios son dados por la baja fidelidad de la transcriptasa reversa y la selección de cuasi-especies por la presión inmunológica. Durante la activación de los LT CD8+ es importante la señal ejercida por el péptido viral, el cual se presenta en el contexto de una molécula del complejo mayor de histocompatibilidad clase I (CMH-I). Estudios de correlación entre el CMH-I y la resistencia/susceptibilidad (R/S) al VIH se han centrado en cuatro aspectos: 1) la expresión de alelos específicos; 2) el grado de homocigocidad/heterocigocidad; 3) la exposición a diversos aloantígenos; 4) la relación con receptores KIR. En esta revisión se aborda el fenómeno de resistencia/susceptibilidad a la infección por el VIH-I relacionado con el CMH-I, cuyo entendimiento favorecerá el desarrollo de herramientas novedosas de intervención terapéutica.

The clinical course of infection with human immunodeficiency virus type-1 (HIV-1) is a variable and complex process that depends on viral and host components. In the majority of infected individuals, the immune response is generated from the initial phases of infection, achieving the control of the viral replication through innate effector mechanisms, neutralizing specific antibodies and particularly through cytotoxic CD8+T cell activity. Despite the generation of these specific cellular and humoral responses, it becomes ineffective in chronic stages of infection because of changes in viral peptide targets, the low fidelity of the reverse transcriptase and the immune pressure. During the activation of CD8+ T cells, the signal delivered by the viral peptide presented in the context of the class I major histocompatibility complex (MHC-I) molecules, is essential. Correlation studies between the MHC-I and the resistance/ susceptibility (R/S) to HIV infection have focused on four aspects, namely: 1) the expression of specific alleles; 4) the degree of homozygosity/heterozygosity; 3) the degree of exposure to different alloantigens; 4) the relation with KIR receptors. In this review, we focus on resistance/susceptibility to HIV-1 infection, particularly related to the MHC, hoping to have a better understanding of this phenomenon that may allow the development of novel therapeutic intervention tools.

Human dendritic cells sequentially matured with CD4+ T cells as a secondary signal favor CTL and long-term T memory cell responses

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

Morphologic Evidence of Anti-Tumor Specificity of T Cells Activated by Denritic Cells Derived from Peripheral Blood Mononuclear Cells of Thyroid Cancer Patients

Recent studies suggest that immunization with autologous dendritic cells (DCs) results in protective immunity and rejection of established tumors in various human malignancies. The purpose of this study is to determine whether DCs are generated from peripheral blood mononuclear cells (PBMNs) by using cytokines such as F1t-3 ligand (FL), granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4, and TNF-alpha, and whether cytotoxic T cells activated against the thyroid cancer tissues by the DCs. Peripheral blood was obtained from 2 patients with thyroid cancer. DCs were established from PBMNs by culturing in the presence of FL, GM-CSF, IL-4, and TNF-alpha for 14 days. At day 14, the differentiated DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDla, CD83, and CD86 were analyzed by immunofluorelescence microscopy. At day 18, DCs and T cells were incubated with thyroid cancer tissues or normal thyroid tissues for additional 4 days, respectively. DCs generated from the PBMNs showed the typical morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed also. DCs and the CTLs were attached to the cancer tissues on scanning electron microscope. The DCs activated the CTLs, which able to specifically attack the thyroid cancer. This study provides morphologic evidence that the coculture of T cells/cancer tissues activated the T cells and differentiated CTLs. The CTLs tightly adhered to cancer tissues and lysed cancer tissues vigorously. Therefore DCs could be used as potential vaccines in the immunotherapy.

Research of dendritic cells pulsed with HPV peptide inducing specific CTL in vitro / 中华微生物学和免疫学杂志

Objective To explore the potential of autologous dendritic cells (DC) pulsed with HLA-A201-binding peptide E613-21(KLPDLCTEL) and E786-94(TLGIVCPI)in inducing specific T cells respouse in vitro.Methods Cervical carcinoma patients with positive HLA-A201 were enrolled and their monocytes isolated and induced into dendritic cells and pulsed with HLA-A201-binding peptide E613-21 and E786-94.PBLs were primed by DCs every week for thee times.The cytokine level of supernatant of CTLs was tested by ELISA.The percentage of special CTLs was tested by flow cytometry.The specific killing effect of CTLs was tested by MTT.Results the numbers of DCs of eleven cervical carcinoma patients were (10.79±0.88) ×106(100 ml peripheral blood).CDllc+HLA-DR+(97.15±2.41)％,CD80+(84.28+5.39)％,CD83 +(85.17±5.06) ％,CD86 + (97.74+0.87) ％.Proliferation index of PBLs primed by DCs three times was 15.4± 1.5.Cytokine levels including IL-2,IL-12,IFN-γ and TNF-α were obviously higher than nonpriming PBLs[(2551.9+195.3) pg/ml,(554.9±64.0) pg/ml,(2416.9±281.7) pg/ml,(632.4 +71.1)pg/ml,respectively] (P＜0.05),but IL-10 was no significant difference between priming CTLs and nonpriming CTLs.The average percentage of special CTLs was obviously higher than control group[(6.32±1.54)％,P＜0.05].The killing effect of CTLs was obviously higher than control group(P＜0.05).Conclusion Dendritic cells pulsed with peptide E613-21 and E786-94 can induce special CTLs in vitro and stimulate CTLs secret cytokines.This will provide science basis for research of therapeutic HPV vaccine.

Construction of TCR gene modified cytotoxic T lymphocytes and its application / 国际肿瘤学杂志

Cytotoxic T lymphocytes (CTLs) are critical effectors which play important roles both in anti-tumor and anti-virus immune responses.Through T cell receptors(TCRs),CTLs can specifically recognize MHC- Ⅰ -peptides complexes presented on the surface of target cells,and then release biological substances such as perforin and granzymes into the target cells and dissolve them.Since the significant potential value of CTLs,more and more people are focusing on its basic and appliedstudies.Most researches are about the transformation of the TCR gene.With the development of molecular biology,cloning and transduction of TCR gene have been more mature.Researchers are finding ways to ensure that TCR gene expresses efficiently and assemble correctly.

Alpha-Type 1 Polarized Dendritic Cells Loaded with Apoptotic Allogeneic Breast Cancer Cells Can Induce Potent Cytotoxic T Lymphocytes against Breast Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Various tumor antigens can be loaded onto dendritic cells (DCs) to induce a potent cytotoxic T lymphocyte (CTL) response in DC-based immunotherapy against breast cancer. However, in the clinical setting, obtaining a sufficient number of autologous tumor cells as a source of tumor antigens is a laborious process. We therefore investigated the feasibility of immunotherapy using breast-cancer-specific CTLs generated in vitro by use of alpha-type 1 polarized DCs (alpha DC1s) loaded with ultraviolet B-irradiated cells of the breast cancer cell line MCF-7. MATERIALS AND METHODS: alphaDC1s were induced by loading allogeneic tumor antigen generated from the MCF-7 UVB-irradiated breast cancer cell line. Antigen-pulsed alphaDC1s were evaluated by morphological and functional assays, and the breast-cancer-specific CTL response was analyzed by cytotoxic assay. RESULTS: The alphaDC1s significantly increased the expression of several molecules related to DC maturation without differences according to whether the alphaDC1s were loaded with tumor antigens. The alphaDC1s showed a high production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens. Breast-cancer-specific CTLs against autologous breast cancer cells were successfully induced by alphaDC1s loaded with apoptotic MCF-7 cells. CONCLUSION: Autologous DCs loaded with an allogeneic breast cancer cell line can generate potent breast-cancer-specific CTL responses. This may be a practical method for cellular immunotherapy in patients with breast cancer.

Virosome vaccines and their application in cancer immunotherapy

Background: An increasing number of tumor associated antigens (TAA) capable of inducing immune responses have been identified in the last two decades. Unfortunately, they are weak immunogens and require potent adjuvants to promote their immunogenicity. Virosomes, nano-lipid vesicles containing viral protein spikes, have been proven amenable for transferring antigen (Ag) to the major histocompatibility complex (MHC) class I processing pathway with the aim to prime cytotoxic T-ymphocytes (CTL) responses. Objective: This mini-review outlines the virosomes platform, ranging from virosomal preparation, their intracellular trafficking and mechanism of action. Methods: The review is directed toward an application of virosomes as a novel and potential vaccine adjuvant in active cancer immunotherapy. Results and conclusion: Virosomes have been proven to be a suitable TAA delivery carrier. Ag encapsulated inside the lumen of virosomes could be prevented from serum- and cell-associated peptidases. Virosomes also facilitated the intracellular trafficking of encapsulated Ag, in which Ag could reach the cytosol and be presented by the MHC-Class I machinery. In addition, virosomes with TAA encapsulated are amenable to be a novel tumor immunotherapeutic strategy.