Expression of γδT and CD4 + CD25 + T cells in peripheral blood of HIV-infected patients/AIDS patients and their correlation / 中华微生物学和免疫学杂志

Objective:To understand the expression levels of γδT cells and CD4 + CD25 + regulatory T cells in the peripheral blood of HIV-infected/AIDS patients, and explore the correlation and possible relationship between γδT cells and CD4 + CD25 + regulatory T cells in the progression of HIV infection/AIDS Mechanism. Methods:Immunofluorescent monoclonal antibody labeling technology and flow cytometry were used to detect 12 cases of AIDS, 19 cases of intermediate HIV infection, 15 cases of early HIV infection, and 30 cases of healthy physical examination in peripheral blood CD3 + T cells and CD4 + The expression levels of T cells, CD8 + T cells, γδT cells, CD4 + CD25 + regulatory T cells; Pearson was used to analyze the correlation between γδT cells and CD4 + CD25 + regulatory T cells. Results:The expression (Mean± SD, %) of γδT cells in the peripheral blood of the AIDS group, the mid-stage HIV infection group, the early HIV infection group and the healthy control group were: 4.46±1.37, 3.59±0.67, 3.12±0.33, 1.73±0.36, group The time ratio, F=6.091, P=0.018. The expression (Mean± SD, %) of CD4 + CD25 + regulatory T cells in the peripheral blood of the AIDS group, the mid-stage HIV infection group, the early HIV infection group, and the healthy control group were: 10.28±1.94, 7.37±1.03, 6.68±0.58, 4.03±0.82, ratio between groups, F=13.568, P=0.002. The comparison of γδT and CD4 + CD25 + regulatory T cells among the four groups is statistically significant, AIDS group>HIV mid-infection group>HIV early infection group>control group. In terms of correlation, γδT, CD4 + CD25 + regulatory T cells are negatively correlated with CD4 + T cells ( r value are -0.982, -0.712, P value are 0.001, 0.002, respectively), and positively correlated with CD8 + T cell counts ( r value are 0.873 , 0.809, P value are 0.000 and 0.000 respectively); γδT cells are positively correlated with CD4 + CD25 + regulatory T cells ( r=0.911, P=0.000). Conclusions:HIV infection induces the proliferation of γδT cells and CD4 + CD25 + regulatory T cells, both of which participate in the immune response caused by HIV infection. γδT cells are positively correlated with CD4 + CD25 + regulatory T cells, and the two may have a synergistic effect.

γδ T cells in liver diseases / 医学前沿

γδ T cells display unique developmental, distributional, and functional patterns and can rapidly respond to various insults and contribute to diverse diseases. Different subtypes of γδ T cells are produced in the thymus prior to their migration to peripheral tissues. γδ T cells are enriched in the liver and exhibit liver-specific features. Accumulating evidence reveals that γδ T cells play important roles in liver infection, non-alcoholic fatty liver disease, autoimmune hepatitis, liver fibrosis and cirrhosis, and liver cancer and regeneration. In this study, we review the properties of hepatic γδ T cells and summarize the roles of γδ T cells in liver diseases. We believe that determining the properties and functions of γδ T cells in liver diseases enhances our understanding of the pathogenesis of liver diseases and is useful for the design of novel γδ T cell-based therapeutic regimens for liver diseases.

In vitro expansion of γδT cells and their killing effect on different tumor cells / 国际肿瘤学杂志

Objective To study the killing effect of γδT cells on different tumor cells by inducing and expanding γδT cells in vitro.Methods Collected the peripheral venous blood of 20 healthy volunteers recruited at Xinqiao Hospital of Third Military Medical University from January to March 2018.Peripheral blood mononuclear cells (PBMCs) were induced and expanded using zoledronic acid combined with interleukin-2 (IL-2) to obtain γδT cells.The cell purity was detected on day 0,7,10,14 and 16 using flow cytometry.The killing activity was detected by CCK-8 kit.Cells cultured on the 14th day were used as effector cells,and breast cancer cell BCap-37 and liver cancer cell Bel-7402 were used as target cells,and the cell killing activity was detected at the effective target ratio (E ∶ T) of 5 ∶ 1,10 ∶ 1 and 20 ∶ 1 respectively.Results The purity of γδT cells were respectively (3.35 ± 1.32) ％,(50.76 ± 5.76) ％,(80.43 ± 4.53) ％,(90.56 ± 3.34) ％,(89.54 ± 4.42) ％ on day 0,7,10,14,16,with significant difference (F =18.431,P =0.012).The efficiency of γδT cells killing BCap-37 tumor cells were (31.3 ± 2.0) ％ (E ∶ T =5 ∶ 1),(48.7 ± 1.6) ％(E ∶T=10∶1),(71.3 ±2.4)％ (E ∶T=20 ∶ 1) respectively,with significant difference (F=16.724,P =0.016),further comparison between each two groups:10 ∶ 1 vs.5 ∶ 1 (P =0.013),20 ∶ 1 vs.5 ∶ 1 (P =0.017),20∶1 vs.10 ∶1 (P =0.011).The killing rate increased with the increase of target ratio.The efficiency of γδT cells killing Bel-7402 tumor cells were (34.5 ± 2.0) ％ (E ∶ T =5 ∶ 1),(52.4 ± 1.9) ％(E ∶ T =10 ∶ 1),(74.5 ± 1.6) ％ (E ∶ T =20 ∶ 1) respectively,with significant difference (F =18.253,P=0.013),further comparison between each two groups:10 ∶ 1 vs.5 ∶ 1 (P=0.015),20 ∶ 1 vs.5 ∶ 1 (P=0.012),20 ∶ 1 vs.10 ∶ 1 (P =0.015).The killing rate increased with the increase of target ratio.Conclusion We can obtain high purity γδT cells using zoledronic acid combined with IL-2 induced PBMCs,and the amplified γδT cells have significant killing effect on BCap-37 and Bel-7402 tumor cells.

Dynamic monitoring of γδT cells in murine cornea in vivo using two-photon laser scanning microscopy / 眼科新进展

Objective To compare the removal efficiency of γδT cells between cornea and ear skin and develop an alternative method for dynamic monitoring of γδT cells in mouse cornea in vivo using 2-photon laser scanning microscopy.Methods The γδT cells in mouse ear skin were monitored before and after antibody neutralization,and the mice corneas were excised and stained for counting γδT cells at 6 h,12 h,24 h after antibody neutralization by using 2-photon laser scanning microscopy,followed by comparison of the removal efficiency of γδT cells between the cornea and ear skin.Results The γδT cells in normal mouse cornea were often distributed in the limbal epithelium and superficial stromal layer.The irregular morphology of γδT cells in the epithelial layer was often accompanied by protuberances,while the stromal γδT cells were mostly round or oval and the number of cells was approximately 27 ± 4.After antibody neutralization,the number of γδT cells in the cornea of mice gradually decreased,and the number of cells at 6 h,12 h and 24 h was significantly lower than that of before depletion (P =0.03,0.00,0.00),and the removal efficiencies were 48％,78％,and 96％,respectively.The γδT cells in ear skin of the normal mice were ellipse or stellate with cell processes and they were located in epidermal layer,and the cell number was about 60 ± 9.After antibody neutralization,the number of γδT cells were significantly reduced at 6 h,12 h and 24 h compared with before depletion (P =0.000,0.000,0.000) and the removal efficiency were 43％,72％ and 95％,respectively.Conclusion The number of γδT cells in the cornea and ear skin is gradually decreased after antibody neutralization,and their removal efficiency is consistent with time.Therefore,monitoring the γδT cells in the mouse ear skin is an ideal alternative to dynamically monitoring the changes in the number of γδT cells in the cornea in vivo.

Chaperonin containing T-complex protein 1 subunit epsilon (CCT5) serves as γδT cell-related autoantigen / 基础医学与临床

Objective To examine the relationship between CCT 5 ,γδ T cell and autoimmune diseases .Methods Recombinant CCT5 protein was cloned , expressed and purified in E.coli.Three peptides of CCT5 protein were used to prepare for anti-CCT5 monoclonal antibodies .Purified CCT5 protein was used to expand γδT cells from pe-ripheral blood mononuclear cells (PBMC).Plasma level of CCT5 in healthy donors, patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) were detected by ELISA assays .The correlation analysis between plasma CCT5 concentration and the percentage of different subtypes of γδT cells measured by flow cytometry was made . Results The CCT5 gene was amplified by PCR and the length of the target fragment was 1 750 bp.The expressed 65 ku CCT5 protein was purified and validated by SDS-PAGE.Two paired monoclonal anti-CCT5 antibodies were screened to detect CCT5 protein in plasma.Immobilized recombinant CCT5 protein was able to induce specific sig-nificant amplification of peripheral γδT cells.Correlation analysis of 10 healthy donors indicated significant corre-lation between the plasma CCT 5 concentration and the proportion of Vγ9 and Vδ2 γδ T cells.The plasma CCT5 concentration significantly decreased in autoimmune diseases patients , including RA and SLE .Conclusions These data suggest that CCT 5 could be a novel Vγ9δ2 γδT cell-related factor in autoimmune diseases , which deepen the understanding of Vγ9δ2 γδT cell function in autoimmune diseases .

Vγ1+γδT Cells Are Correlated With Increasing Expression of Eosinophil Cationic Protein and Metalloproteinase-7 in Chronic Rhinosinusitis With Nasal Polyps Inducing the Formation of Edema

PURPOSE: We have found that expression of γδT cells is increased in pathological mucosa of chronic rhinosinusitis with nasal polyps (CRSwNP) compared with normal nasal mucosa. This increase is correlated with the infiltration of eosinophils in CRSwNP. Here, we investigated the expression of γδT cells, inflammation and tissue remodeling factors as well as their probable relationships in different types of chronic rhinosinusitis (CRS) in China. METHODS: A total of 76 surgical tissue samples that included 43 CRSwNP samples (15 eosinophilic and 28 non-eosinophilic), 17 CRS samples without nasal polyps (CRSsNP), and 16 controls were obtained. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression levels of Vγ1⁺γδT cells, Vγ4⁺γδT cells, eosinophil cationic protein (ECP), interleukin (IL)-8, transforming growth factor (TGF)-β2, metalloproteinase (MMP)-7, tissue inhibitor of metalloproteinase (TIMP)-4 and hypoxia-inducible factor (HIF)-1α. Enzyme linked immunosorbent assay (ELISA) was used to measure the protein level of ECP and MMP-7 in CRSwNP. The eosinophils were counted and the level of edema was analyzed with HE staining. RESULTS: The mRNA expression levels of the Vγ1 subset, ECP and MMP-7 were significantly increased in CRSwNP with histological characteristics of eosinophilic infiltration and edema. The expression of the Vγ1 gene in CRSwNP correlated positively with the expression of both ECP and MMP-7. No significant decreases in the mRNA expression levels of TGF-β2, TIMP-4 or HIF-1α were observed in the CRSwNP samples. The expression levels of Vγ1 gene, ECP and MMP-7 were significantly increased in eosinophilic CRSwNP compared to non-eosinophilic CRSwNP. CONCLUSIONS: Our results suggest the associations between Vγ1⁺γδT cells, ECP and MMP-7 in CRSwNP, indicating that Vγ1⁺γδT cells can induce the eosinophilic inflammation, which has a further effect on the formation of edema.

Distinct distributions of mouse γδ T ce lls in various tissues and changes after infection / 中国免疫学杂志

Objective:The study focuses on the distinct distributions of γδT cells in various tissues and the changes after Sal-monella typhimurium infection,and attempts to explore the physiological significance of γδT cell distribution and the role of γδT cells in infectious diseases .Methods:Flow cytometry and PCR technique were used to detect the proportion of different γδ T cell subsets among thymus,spleen,lymph nodes,liver,skin,and intestinal intraepithelial lymphocytes .Flow cytometry was applied to detect the secretion of IFN-γand IL-17a.The changes of various γδT cell subsets in liver and intestinal intraepithelial lymphocytes were analyze after Slamonella typhimurium infection.Res ults: γδ T cells were rich in the intestinal epithelium , skin and liver, but poor in the thymus,spleen and lymph nodes .The distribution of different subsets was quite dissimilar .Vγ5+γδT cells chiefly existed in skin ,and Vγ1+,Vγ4+,Vγ7+γδ T cells largely existed in small intestine.γδ T cells in liver mainly secreted IL-17a;however,γδ T cells in intestinal intraepithelial secreted IFN-γ.After infection by Salmonella typhimurium , the proportion of γδ T cells in intestinal intraepithelial increased significantly ,particularly Vγ1 +γδT cells.In Liver,there was no significant change of total γδT cell ratio,but the ratio of Vγ1 +γδT cells reduced ,Vγ4 +γδT cells raised.Conclusi on:γδT cells are rich in the intestinal epithelium ,skin and liv-er.The distribution of different subgroups has specificity .There are large differences in the ability of cytokine secretion among various subgroups of γδT cells.The distribution of γδT cell subgroups in small intestine and liver changes during Salmonella typhimurium in-fection.

TWS119 upregulates CCR5 expression of γδT cells by inhibiting STAT3 phos-phorylation / 中国免疫学杂志

Objective:To investigate the mechanisms of TWS119 induced CCR5 expression in hunman γδT cells. Methods:After treatment with various concentrations of TWS119 for 48h, the expression of CCR5 in γδT cells were detected by flow cytometry. The p-STAT3 and GAPDH expression were examined by Western blot analysis. Results: TWS119 could upregulate the expression of CCR5 in dose dependent manner. Western blot analysis revealed that TWS119 inhibit phosphorylation of STAT3,but had no significant impact on GAPDH. In addition, pretreatment of γδT cells with 0. 5 μmol/L STAT3 specific phosphorylation inhibitor Stattic could upregulate the expression of CCR5 and enhance the TWS119 induced CCR5 expression. Conclusion: TWS119 could upregulate CCR5 expression of γδT cells by inhibiting STAT3 phosphorylation in vitro.

Study on phenotype and function of intestinal gamma delta T lymphocytes in mice with post-infectious irritable bowel syndrome / 中国免疫学杂志

Objective:To investigate the phenotype and function of the intestinal γδT lymphocytes in post-infectious irritable bowel syndrome mouse model. Methods:The mouse model for post-infectious irritable bowel syndrome was established by the infection with trichinella spiralis. The intestinal inflammation,abdominal withdrawal reflex( AWR) and colon transportation test were observed. 2 and 8 weeks later,the animals were sacrificed and the lymphocytes in the intestinal lymph nodes and spleen were collected,from which the γδT lymphocytes were isolated and purified by monoclonal antibody-immuno-microbeads method. The functions of the purified γδT lymphocytes were evaluated,including proliferation by 3 HTdR;CD69,CD62L molecule staining by flow cytometry. Furthermore,the con-centration of cytokine IL-17 and IFN-γ in the supernatant of the cultured γδT lymphocytes were detected by ELISA. Results: At 2nd weeks after infection,significant intestinal inflammation was observed,with increasingγδT lymphocytes,proliferating and activating with increasing production of IL-17. At 8th weeks after infection, the intestinal inflammation disappeared, whereas the number of γδT lymphocytes remained increasing,also with proliferating and activating with increasing production of IL-17. Meanwhile,the mice show higher AWR score and Bristol score. Conclusion: γδT lymphocytes could participate in the pathogenesis of PI-IBS via their proliferation,activation and production of IL-17.

Enhancement of γδT cells proliferation and cytotoxicity by Hyperoside / 中国免疫学杂志

Objective:To investigate the anti-tumor effect of Hyperoside.Methods: Human γδT cells were amplified by isopentenyl pyrophosphate from peripheral blood cells.The proliferation capacity of γδT cells was measured with CCK-8 assay after treated with different concentrations of Hyperoside.Cytotoxicity of γδT cells was detected with LDH assay , and the expression of granzyme,perforin CD107a and IFN-γonγδT cells were measured by flow cytometry before and after treatment.Results: Hyperoside could significantly stimulate the proliferation of γδT cells at the concentration of 3.13-12.5 μg/ml.Cytotoxicity and expression of granzyme,perforin and IFN-γofγδT cells were increased after treatment.Conclusion:Hyperoside could enhance cytotoxicity of humanγδT cells through up-regulation of granzyme ,perforin CD107 a and IFN-γexpression.

Inhibitory effect ofγδT cells on proliferation of ovarian cancer SKOV3 cells / 吉林大学学报(医学版)

Objective:To study the inhibitory effect of γδT cells on the proliferation of ovarian cancer SKOV3 cells,and to clarify its possible mechanism of inducing apoptosis. Methods:The human ovarian cancer SKOV3 cells cultured in vitro were used as control group,and theγδT and SKOV3 cells were co-cultured for 72 h as γδT cells treatment group.Laser scanning confocal microscope was used to obeserve the morphological changes of nucleus SKOV3 cells,and the inhibitory rate of proliferation of SKOV3 cells in two groups were detected by MTT method;Transwell Chambers was used to detect the cell migration ability,then the apoptotic rates of SKOV3 cells were tested by flow cytometry (FCM).Results:The apoptotic morphology of nucleus of SKOV3 cells in γδT cells treatment group were found under microscope,such as nuclear shrinkage.The MTT resultes displayed that the inhibitory rate of proliferation of SKOV3 cells in γδT cells treatment group was higher than that in control group (P <0.05).The Transwell Chambers results showed that the number of transmembrane cells in γδT cells treatment group was lower than that in control group,and the migration rate was decreased compared with control group (P <0.05).The FCM results showed that the apoptotic rate of SKOV3 cells in γδT cells treatment group was higher than that in control group (P < 0.05 ).Conclusion:γδT cells can inhibit the proliferation and the migration abilities of ovarian cancer SKOV3 cells,and promote the apoptosis.

Effect of TWS119 on the activities of human SGC-7901 and γδ T cells in vitro and its mechanism / 中国药学杂志

OBJECTIVE: To study the antitumor activity of TWS119 and its effect on the killing activity of γδT cells in vitro. METHODS: CCK-8 assay was used for detecting the influence of TWS119 on the proliferation of SGC-7901 and γδT cells and the cytotoxic activity of γδT cells to SGC-7901 cells. The cell apoptosis was measured by flow cytometric analysis. The protein expression was examined by Western blot analysis. RESULTS: It was found that TWS119 could inhibit the growth and induce the apoptosis of SGC-7901 cells. The expression of Bcl-2 in SGC-7901 cells treated with TWS119 was downregulated, while p-ERK1/2 and p-STAT3 were up-regulated. In addition, TWS119 at 0.5-4.0μmol · L-1 could promote the growth of γδT cells and enhance the cytotoxic activity of γδT cells to SGC-7901 cells especially at 4.0 μmol · L-1 for 72 h. Furthermore, TWS119 could upregulate the expression of Bcl-2 and inhibit the expression of p-ERK1/2 and p-STAT3. CONCLUSION: TWS119 in some concentrations can inhibit the growth of SGC-7901 cells, promote the 78T cells growth and enhance the cytotoxic activity of γδT cells to SGC-7901 cells, and the mechanism may be involve in Wnt, ERK1/2, Bcl-2 and p-STAT3 signaling pathways.

Proliferation activating effects of Mtb-Ag on γδT cells and its effects on expression of CD69 molecules inγδT cells / 国际生物医学工程杂志

Objective To activate and amplify γδT cells with low molecular peptide antigen of Mycobacterium tuberculosis low molecular peptide antigen (Mtb-Ag),and to investigate the expression of CD69 molecules on γδT cellular surface.Methods Healthy human peripheral blood mononuclear cells (PBMC) were obtained and separated,then positive cells were isolated by immuno-magnetic beads selection,and the proportion of γδT cells in the PBMCs was detected by fluorescent monoclonal TCR γδT-PE staining and flow cytometry.Expression of CD69 molecules in γδT cells was detected by γδ-PE/CD69 FITC double staining.Results The proportion of γδT cells was (4.9±1.85)％ in freshly obtained PBMC,(69.2±6.57)％ after 10 d of Mtb-Ag activation,and (99.3±8.92)％ after immuno-magnetic beads selection.The expression of CD69 molecules in γδT cells reached the peak (75.2％) at 24 h after initial Mtb-Ag stimulation,and reached the peak (72.0％) at 6 h after second stimulation.Conclusions MtbAg can specifically stimulate the proliferation of γδT cells in the PBMC.Both its initial and the second stimulation can specifically activate γδT cells.

The expression of γδT cells and other lymphocytes in patients with acute and subacute brucellosis and its significance / 中华传染病杂志

Objective To observe the variation of the levels of γδT cells,CD4+ and CD8+ T lymphocytes in patients with acute and subacute brucellosis before and after treatment.Methods A total of 159 patients with brucellosis were enrolled from the Second People's Hospital of Tianjin between September 2009 and August 2014,of which 122 were with acute phase and 37 with subacute stage.γδT cells,CD4+ and CD8+ T lymphocytes in peripheral blood of all patients before and after treatment were detected by flow cytometry.Thirty healthy controls were also enrolled.Measurement data were analyzed using t test and nonparametric Kruskal-Wallis test.Results The medians of γδT cells,CD4+ and CD8+ T lymphocyte counts of brucellosis patients with acute stage before treatment were 84.0/μL,780.5/μL and 774.0/μL,respectively,and those with subacute stage were 76.0/μL,887.0/μL and 890.0/μL,respectively.Those in control group were 49.0/μL,772.5/μL and 592.5/μL,respectively.γδT cells (U=114.5,P =0.001) and CD8+ T lymphocyte counts (U=1 185.5,P =0.003) in acute phase group were elevated than control group.γδT cells (U=359.0,P=0.013),CD4+ T lymphocyte counts (U=325.5,P=0.004) and CD8+ T lymphocyte counts (U=316.5,P=0.003) in subacute phase group were increased than control group.CD4+ T lymphocyte counts in subacute phase group were elevated than acute phase group (U=1 619.0,P=0.009).There were 31 patients with acute phase and 30 patients with subacute phase who completed 6-week treatment.γδT cells ([120.7 ± 104.9]/μL vs [82.2 ±85.3]/μL;t=3.568,P=0.001),CD4+ T lymphocyte ([802.1±300.6]/μL vs [663.0±280.6]/μL;t=2.904,P=0.007) and CD8+ T lymphocytes counts ([908.1±422.7]/μL vs [651.1±241.7]/μL;t=3.885,P=0.001) of brucellosis patients with acute stage before and after treatment were significantly different.γδT cells ([67.1± 38.4]/μL vs [48.4±32.1]/μL;t=2.695,P=0.012),CD4+ T lymphocytes ([817.1± 147.4]/μL vs [650.8± 196.5]/μL;t=5.301,P＜0.01) and CD8+ T lymphocytes counts ([703.7±191.5]/μL vs [537.4±186.0]/μL;t=5.152,P＜0.01) of brucellosis patients with subacute stage after treatment were significantly decreased compared with those of patients before treatment.Conclusions γδT cells,CD4+ and CD8+ T lymphocytes in patients with acute and subacute brucellosis are all in a state of immune activation.The levels of activation are highly correlated with disease progression.And detection of the variation of these cells contributes to understand the immune state of the acute and subacute brucellosis.

Effect of glycogen synthase kinase-3β inhibitor TWS119 on proliferation and phenotypic characteristics of human γδT cells in vitro / 中国免疫学杂志

Objective:To investigate the effect of glycogen synthase kinase-3β inhibitor TWS119 on hunman γδT cells growth and phenotypic characteristics. Methods:Using γδT medium to cultivate human peripheral blood γδT cells in vitro. After co-cultured with different concentrations of glycogen synthase kinase-3β( GSK-3β) inhibitor 4, 6-disubstituted pyrrolopyrimidine ( TWS119 ) for indicated time,growth curve and Wnt/β-catenin activation of in each group were determined by CCK-8 and Western blot assays. The CD62L and CD45RA expression theγδT cells were detected using flow cytometry. Results:Wnt/β-catenin pathway ofγδT cells could activate by TWS119. In the first group,TWS119 could upregulate the expression of CD62L and CD45RA in dose dependent manner while inhibit the growth and ratio of γδT cells. In the second group,TWS119 could promote the growth and ratio of γδT cells with the increase in concentration from 0 μmol/L to 4. 0 μmol/L and decreased thereafter. Besides,TWS119 could promote the expression of CD62L in a dose-dependent and had no effect on the CD45RA. Conclusion: Human γδT cells isolated from peripheral blood treated with TWS119 gave rise to two subsets of CD45RA+CD62L+γδT cells and CD62L+γδT cells.

Chlam ydia mu ridra um respiratory tract infection induces the proliferation ofγδT cells and the secre-t ion of IFN-γand IL-17 / 中华微生物学和免疫学杂志

Objective To investigate the proliferation, activation and cytokine production of γδT cells during different periods of Chlamydia muridarum ( Cm) respiratory tract infection. Methods C57BL/6 mice were inoculated intranasally with 3×103 inclusion-forming units(IFU)of Cm strains to induce the murine model of chlamydial pneumonitis.Mononuclear cells were isolated from lung tissue samples col-lected from mice at different time points after infection.Flow cytometry analysis was used to detect the per-centages of CD3+TCRγδ+T cells in lung tissues and the expression of CD69 molecule.Intracellular cytokine staining was performed to analyze the secretion of IL-17 and IFN-γbyγδT cells.Results The mouse model of chlamydial pneumonitis was successfully established by intranasal inoculation of C57BL/6 mice with 3×103 IFU of Cm strains.Compared with the mice without Cm infection, the percentage and the absolute number of CD3+TCRγδ+T cells in mice lung tissues were significantly increased after Cm infection, the peak of which was reached on the 7th day, followed by a decline.The expression of CD69 molecule onγδT cells isolated from mice lung tissues reached to the highest level on the third day after Cm infection and declined slightly afterwards.Moreover, enhanced secretion of IL-17 and IFN-γby γδT cells were observed in mice with Cm infection and the highest levels of the two cytokines were detected on the third day after infection. Most of the cytokines ( IL-17 and IFN-γ) were secreted by γδT cells rather than by αβT cells in the early stage of Cm infection.However, the CD3+CD4+T cells were more capable of producing IFN-γand IL-17 thanγδT cells after three days of Cm infection.Conclusion Cm respiratory tract infection could induce the aggregation and activation ofγδT cells at the infection site.TheγδT cells were the predominant cells produ-cing IL-17 and IFN-γin the early stage of host anti-chlamydia response.

ESAT-6 enhances the expression of immune substances ofγδT cells through upregulating the expression of TLR-4 / 重庆医学

Objective To observe the expression of immune substances secreted by peripheral bloodγδT cells after stimulated by early secreted antigenic target-6(ESAT-6),and explore its mechanism of signaling pathway.Methods Peripheral blood mononucle-ar cells(PBMC)were collected from health human blood with the ficoll density gradient centrifugation method,and theγδT cells were separated from the PBMC with flow cytometry;the inhibitor of TLR-4 signaling pathway(E5564)was used to cocultured withγδT cells to inhibit the function of TLR-4,and the change of TLR-4 was analyzed by the methods of PCR and Western blot.ESAT-6 were used to stimulate theγδT cells,and the control group without any stimulating factor was established,then the expression levels of IL-17,TNF-α,IFN-γwere determined by ELISA method after 0、1、3、6、9 and 12 days.Results The results of PCR and Western blot showed that ESAT-6 could increase the expression of TLR-4(P<0.01);The results of ELISA showed that ESAT-6 could enhance the expression of IL-17,TNF-αand IFN-γ(P<0.01),and the inhibitor of TLR-4(E5564)could decrease the expres-sion of IL-17,INF-α,IFN-γ(P<0.01).Conclusion ESAT-6 can induceγδT cells to produce more IL-17,TNF-α,IFN-γ,and the mechanism of which maybe concerned with TLR-4 signaling pathway.

Research advances in the role of γδT cells in the pathogenesis of rheumatoid arthritis / 中华实用儿科临床杂志

Rheumatoid arthritis (RA) is a common systemic autoimmune disease characterized by chronic inflammation of joints,bone and cartilage erosion,synovial hyperplasia,and the pathogenesis of RA is not clear.γδT cells are a new kind of phenotype and function T lymphocyte subsets,which mainly distribute in the mucosal and epithelial tissue and account for 1％-10％ of the total T cells in the peripheral blood,and bridge innate and adaptive immunity.γδT cells play an important role in the pathogenesis of RA by the functions of antigen-presenting capacity,secretion of proinflammatory cytokines,immunomodulatory effects,and auxiliary function for B cells.

Research of mechanism about enhancing the activity of γδδT cells treated by resveratrol on colonic cancer cell lines SW-1116 / 中华微生物学和免疫学杂志

Objective To explore the effect of γδT cells on colonic cancer cell lines SW-1116 treated by resveratrol. MethodsAmplification γδT cells of human peripheral blood in vitro by using isopentenylpyrophosphate. Different density resveratrol induced γδT cells and colonic cancer cell SW-1 116, methyl thiazolyl tetrazolium(MTT) detectde the effect of growth and proliferation when expression veratrol action to γδT cells and colonic cancer cell lines SW-1116. Flow cytometer( FCM )detectde the expression of porforin, granzymeB and CD107a on γδT cells before it treated by resveratrol and after that. γδT cells against colonic cancer cell lines SW-1116 were detectde by lactate dehydrogenase(LDH) releasing assay before and after treated by resveratrol.Western blot analyzed the liveness of extracellular signal-regulated kinase1/2 of γδT cells before it treated by resveratrol and after that. ResultsγδTcells would be proliferation when the density of resveratrol in 0.39-3.125 μmol/L, while the effect on colonic cancer cell lines SW-1116 was not significance. There was significant difference of the expression of porforin, ranzymeB and CD107a on γδT cells before it treated by resveratrol and after that ( P＜0.05 ). γδT cells against colonic cancer cell lines SW-1116 treated by resveratrol was enhanced compared with control group( P＜0.05 ) . The p-ERK 1/2expression of γδT cells enhanced when the density of resveratrol treated to γδT cells in 0.1-10 μmol/L( P＜0.05 ). Conclusion Resveratrol could promote γδT cells, proliferation and strengthen the cytotoxicity of γδT cells against colonic cancer cell SW-1116, the mechanism might concerned with activating p-ERK1/2 and enhancing the expression of porforin, granzymeB and CD107a on γδT cells.