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Objective To investigate the phenotype and function of antigen-specific CD8+ cytotoxic T cells (CTL) from HLA-A2+ healthy human donors induced by high-carcinogenic HPV 16 E7 peptide in vitro.Methods Peripheral blood T cells from 26 HLA-A2+ healthy human donors were incubated with HPV 16E711-20 peptide (HLA-A*0201/YMLDLQPETT), recombinant human interleukin 7 (IL-7) and IL-2 for 7 days to yield antigen-specific CD8+ CTL. Then, four-color flow cytometric analysis was performed to detect the percentage of antigen-specific CD8+ CTL expressing different surface markers including CD45 and CD27. The expression of three intracellular cytokines including perforin, granzyme-B and FasL in the antigen-specific CD8+ CTL was also measured by intracellular flow cytometry. A wrong peptide, HBVcoxe18-27 (FLPSDFFPSV),was used as an isotype control. Results A significant increase was observed in the percentage of antigen-specific CD8+ CTL in peripheral T cells stimulated with HPV 16 E7-peptide compared with the non-stimulated T cells (0.73% ± 0.33% vs 0.02% ± 0.03%, P ＜ 0.01). The percentage of CD45RA+CD27- effector T cells,CD45RA-CD27- effector memory T (TEM) cells, CD45RA-CD27+ central memory T (TCM) cells, and CD45RA+CD27+ naive T cells in antigen-specific CD8+ CTL was 26.07% ± 13.46%, 7.97% ± 7.11%, 33.25% ± 19.68%and 32.73% ± 13.89%, respectively in HPV 16 E7-stimulated group, significantly higher than that in nonstimulated group (0.02% ± 0.03%, 0.02% ± 0.03%, 0.02% ± 0.03% and 0.02% ± 0.05%, all P ＜ 0.01 ). Elevated proportions of perforin-, granzyme-B- and FasL-expressing antigen specific CTL were observed in HPV 16 E7-stimulated group compared with non-stimulated group (47.01% ± 18.69% vs 0.38% ± 0.55%, 80.53% ±13.32% vs 0.34% ± 0.22%, 26.48% ± 7.81% vs 0.16% ± 0.16%, all P ＜ 0.01 ). Conclusions HPV 16 E7peptide could induce the clonal proliferation of CD8+ T cells, generation of antigen-specific CD8+ CTL and secretion of toxic cytokines, finally lead to a highly efficient and specific killing of virus-infected target cells through different mechanisms, hence, it might play a crucial role in antiviral immune responses.
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Objective To investigate the possible roles of cellular immunosuppression induced by phenotypic and functional changes of peripheral CD4+CD25+ regulatory T cells (Tregs) in the pathogenesis of condylomata acuminata. Methods Three-color flow cytometry was performed to examine the expression of transcription factor Foxp3 in, along with several inhibitory membrane molecules, i.e. cytotoxic T lympho-cyte associated antigen-4 (CTLA-4), glucocorticoid-induced TNF receptor family-related gene (GITR) and programmed death-1 (PD-1) on peripheral CD4+CD25+ T cells from 46 patients with condylomata acuminata and 43 normal human controls. Meanwhile, high purity of CD4+CD25+ T cells were isolated from peripheral blood using imrnunomagnetic beads, and stimulated to produce intracellular suppressor cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta), which were detected by flow tyro-metric analysis. Results The number of peripheral CD4+CD25+ Foxp3+ Tregs increased significantly in patients with condylomata acuminata than that in the normal controls (7.37% ± 2.43% vs 5.96% ± 2.09%,P ＜ 0.001). The expressions of CTLA-4 and PD-1 were 1.86% ± 1.13% and 2.41% ± 1.12%, respectively,in the patients, which were significantly higher than those in the normal controls(1.36% ± 0.90% and 1.70%± 0.97%, P ＜ 0.05 and 0.01, respectively). The number of TGF-beta-positive CD4+CD25+ T ceils from peripheral blood were increased in patients than that in, the controls (1.57% ± 0.91% vs 0.78% ± 0.24%, P ＜0.001). Conclusions Human papillomavirus infection can induce the activation and proliferation of CD4+CD25+ Tregs, enhance the expression of negative costimulatory molecules and secretion of suppressor cytokines, and inhibit antiviral immune response through multiple mechanisms.
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Objective To investigate peripheral blood dendritic cells (DC) responding to herpesvirus infection in the patients with recurrent genital herpes and the expression of immune molecules on mature DC induced by the cytokines in vitro. Methods Using three-color flow cytometry, we studied the phenotype expression of CD11c, CD123, CDla, CD80, CD86, CD40 and CD83 on the two subsets of DC(CD11c+DC and CD123+DC) in peripheral blood. The monocytes isolated from the blood were cultured with recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-?) from 20 patients with recurrent genital herpes (RGH) and 18 healthy controls. Results The number of CD11c+ DC in the peripheral blood increased significantly in the peripheral blood cells of RGH patients (2.54% ? 1.19%) in comparison with that of healthy controls (1.77% ? 0.83%) (P 0.05). Conclusions Peripheral blood CD11c+DC activated by uptaking viral antigens in the patients with RGH results in a high level expression of co-stimulatory molecules on DC and enhance antigen-presenting functions, which is essential for intiating antiviral immune response directly by T lymphocytes.
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Objective To study the role of Th1/Th2 cytokine profile in the pathogenesis of recurrent genital herpes (RGH), and to find out the relationship between Th1/Th2 cytokines. Methods A two－ colour immunofluorescent staining of cell surface antigen and intracellular cytokines (IL－ 2、 IL－ 10、 IL－ 12、 IFN－ γ and TNF－ α ) in CD3＋ T－ lymphocytes of 20 patients with RGH with flow cytometric analysis were performed. Results Compared to controls, patients with RGH showed a decreased number of CD3＋ T cells（ P0.05） . Conclusions RGH patients seem to have a predominance of Th2 cytokine profile, which may play an important role in the pathogenesis of RGH.
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Objective:To investigate the expression of special marker for activation on peripheral blood T lymphocytes in patients with condyloma acuminatum(CA) and its significance.Methods:Immunofluorescent three color flow cytometry was used to study the expression of CD69 and HLA DR on T lymphocytes in 30 patients with CA and 31 normal controls. Results:Expression of CD69 on CD3 + T cells were significantly higher in patients (6 63%?3 13%) than that in controls (5 12%?1 64%, P
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Objective:To study the role of serum chemokines and their receptors expression in pathogenesis of recurrent genital herpes(RGH).Methods:Serum levels of interleukin 8(IL 8),interferon ? inducible protein 10 (IP 10),monocyte chemotactic protein1(MCP 1),macrophage inflammatory protein 1?(MIP 1?),regulated upon activation,normal T cell expressed and secreted factor(RANTES) and macrophage derived chemokine(MDC)were determined by enzyme linked immunosorbent assay in 20 patients with RGH and 30 normal controls, meanwhile, a two color immunofluorescent staining of chemokine receptors CCR2,CCR5,CXCR1 and CXCR3 on peripheral blood CD3 +T lymphocytes of patients with RGH were analyzed by flow cytometer.Results:Serum levels of RANTES were lower in patients with RGH than that in controls(P
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Objective To study the role of Th1/Th2 cytokine profile in the pathogenesis of recurrent genital herpes (RGH), and to find out the relationship between Th1/Th2 cytokines. Methods A two-colour immunofluorescent staining of cell surface antigen and intracellular cytokines (IL-2、 IL-10、 IL-12、 IFN－? and TNF－? ) in CD3＋ T-lymphocytes of 20 patients with RGH with flow cytometric analysis were performed. Results Compared to controls, patients with RGH showed a decreased number of CD3＋ T cells（ P0.05） . Conclusions RGH patients seem to have a predominance of Th2 cytokine profile, which may play an important role in the pathogenesis of RGH.
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Objective To study the roles of phenotype switch of HPV16 antigen-specific CD8~+ cy- totoxic T cells (CTLs) in peripheral blood as well as the cellular immune response of human T lympho- cytes to viral peptides in the pathogenesis of condyloma acuminatum.Methods HPV16E7~(11-20) (HLA-A*0201/YMLDLQPETT) antigen-specific CTLs were stained with fluorescent-labeled recombinant MHC class I-peptide pentamer complex,and co-stained with CD69 (activated CTL) and CD45RO (mem- ory CTL) surface markers,in peripheral blood from 10 patients with condyloma acuminatum and 15 normal controls.The CTLs were quantitated by flow cytometric analysis.Meanwhile,peripheral blood mononuclear cells of 13 healthy donors were incubated for 7 days with HPV16E7_(11-20) peptide,recombinant human inter- leukin 7 and interleukin 2 to yield antigen-specific CD8~+ CTLs and their other surface markers.An irrelevant peptide,HBVcore_(18-27) (FLPSDFFPSV),was used as an isotype control.Results The percentages of periph- eral blood HPV16E7 antigen-specific CD8~+CTLs,activated CTLs and memory CTLs of total CD8~+ T cells were increased significantly in patients with condyloma acuminatum (0.76%?0.43%,0.36%?0.20%,and 0.33%?0.15%,respectively) than those in normal controls (0.24%?0.07%,0.17%?0.05% and 0.15%?0.06%,respectively) (P＜0.01,P＜0.05,and P＜0.01,respectively).For T cells in vitro stimulated with viral peptide for 7 days,the percentages of peptide-specific CD8~+ CTLs,activated CTLs and memory CTLs were significantly higher (0.75%?0.16%,0.35%?0.15% and 0.33%?0.18%,respective- ly) in comparison with those in non-stimulated group (0.24%?0.06%,0.16%?0.03% and 0.13%?0.04%,respectively ) (P＜0.001,P＜0.01 and P＜0.01,respectively ).Conclusions These results,in vivo and in vitro,demonstrate that the proliferation of CD8~+ T cell clones induced by HPV infection could generate antigen-specific CD8~+ CTLs,causing a highly efficient and specific killing of viral-infected target cells directly,which may play an important role in antiviral T cell-mediated immune response.
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Objective To study the role of serum chemokines and their receptors in the pathogenesis of atopic dermatitis(AD).Methods Serum levels of interferon ?-inducible protein-10(IP-10),stromal cell-derived factor-1? (SDF-1?),eotaxin,thymus and activation-regulated chemokine(TARC) as well as macrophage-derived chemokine (MDC) were detected by enzyme-linked immunosor bent assay(ELISA) in 39 patients with AD and 39 normal controls.Meanwhile,chem okine receptors CXCR3,CXCR4,CCR3,CCR4 and CCR5 on peripheral blood CD4+T-lymp hocytes were analyzed by flow cytometry with a two-color immunofluorescent sta ining in 39 patients with AD.Results Serum levels of SDF-1?,TARC and MDC we re significantly higher(P 0.05) between the patients[(123.6 ? 110.4) pg/mL a nd (68.7 ? 26.2) pg/mL,respectively]and controls [(100.7 ? 73.7) pg/mL and(66.8 ? 20.5) pg/mL,respectively].The expression of CXCR3,CCR3,CCR4 and CCR5 o n CD4+T cells was increased (P
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Objective To determine the peripheral blood dendritic cell (DC) subsets in patients with condyloma acuminata (CA) and its significance. Methods The CD11c+ peripheral blood DC (DC1) and CD123+ DC(DC2) subsets were analyzed by flow cytometry with three-color immunofluorescent staining in 25 patients with CA and 26 healthy controls. Results The proportion of CD123+ peripheral blood DCs was significantly increased in patients with CA[(0.63 ? 0.34)%]in comparison with that in healthy controls [(0.41 ? 0.17)%](P 0.05). Conclusions The predominance of CD123+ DCs in peripheral blood of CA may favor Th2-dominated immunity, which may be related to recurrence and course of the disease.
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Objective To study the role of serum chemokines and their receptors in the pathogenesis of condyloma acuminatum(CA).Methods Serum levels of interleukin8(IL-8),interferon?-inducible pro-tein10(IP-10),monocyte chemotactic protein1(MCP-1),macrophage inflammatory protein1?(MIP-1?),reg-ulated upon activation,normal T cell expressed and secreted factor(RANTES)and macrophage-derived chemokine(MDC)were determined by enzyme-linked immunosorbent assay(ELISA)in30patients with CA and30normal controls.Meanwhile,chemokine receptors,CXCR1,CXCR3,CCR2and CCR5on peripheral blood CD3 + T-lymphocytes from20patients with CA were analyzed by flow cytometry with two-color im-munofluorescent staining.Results Serum levels of IP-10and MIP-1?were significantly higher in CA patients than those in controls(P
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Objective To detect cytokine levels secreted by CD3 + T cells and monitor the Th cell im-balance in the patients with condyloma acuminatum(CA).Methods The peripheral blood lymphocytes were isolated and stimulated prior to analysis.Cytokine detection was performed using monoclonal antibodies,which were fluorochrome-conjugated for easier direct manipulation in flow cytometry analysis.The results were pre-sented as cytograms showing dual staining of lymphocytes.Results The levels of IL-2,IL-12,IFN-?and TNF-?produced by Th1cells decreased,while the level of IL-10produced by Th2cells had no significant change in CA patients in comparison with healthy controls.Conclusions There is Th1/Th2imbalance in CA patients with a decline of Th1type cytokines.The Th1/Th2imbalance might be the mechanism that HPVs es-cape from immunosurveillance in CA patients.