Gong Feili

Vα24-Invariant NKT Cells from Patients with Allergic Asthma Express CCR9 at High Frequency and Induce Th2 Bias of CD3+ T Cells upon CD226 Engagement

We have demonstrated that Vα24+Vβ11+ invariant (Vα24+i) NKT cells from patients with allergic asthma express CCR9 at high frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic Vα24+i NKT cells but not the normal cells. A large number of CCR9-positive Vα24+i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected. Asthmatic Vα24+i NKT cells, themselves Th1 biased, induce CD3+ T cells into an expression of Th2 cytokines (IL-4 and IL-13) in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic Vα24+i NKT cells. CCL25/CCR9 ligation causes directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate Vα24+i NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic Vα24+i NKT cells to induce Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits Vα24+i NKT cells to induce Th2-cytokine production by CD3+ T cells, indicating that CD226 engagement is necessary for Vα24+i NKT cells to induce Th2 bias of CD3+ T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic Vα24+i NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing Vα24+i NKT cells into the lung to promote other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal asthmatic Vα24+i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the pathogenesis of allergic asthma.

Peptide Nucleic Acid Antisense Prolongs Skin Allograft Survival by Means of Blockade of CXCR3 Expression Directing T Cells into Graft

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-γ-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-γ/CXCL9. It was reported that CXC chemokines IFN-γ-inducible protein 10/CXCL10 and monokine induced by IFN-γ/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 ± 2.4 days) compared with physiological saline treatment (7.5 ± 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 ± 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.

Different Neurotropic Pathogens Elicit Neurotoxic CCR9- or Neurosupportive CXCR3-Expressing Microglia

What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-α; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+Irg1− microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-β. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1− microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1− cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer’s disease and AIDS dementia.

Studies on Activity of NK Cells in Preeclampsia Patients

SummaryThe activity of the NK cells in patients with preeclampsia was studied to investigate the pathogenesis of preeclampsia. By using MTT and21Cr releasing technique, the proliferation and killing ability of the NK cells in maternal and umbilical blood from preeclampsia patients (n=18) and normal third trimester pregnant women (n=18) were detected. The NK-92 cell line was as the positive control. The results showed that the NK cell counts of umbilical blood in preeclampsia patients and normal third trimester pregnant women were significantly greater than those of maternal blood (bothP<0.05). Compared with that in normal third trimester pregnant women, the proliferative ability of the NK cells in, preeclampsia patients was apparently increased (P<0.05). Compared with that in maternal blood, the proliferative ability of the NK cells in umbilical blood from both preeclampsia patients and normal third trimester pregnant women was dramatically increased. The killing ability of the NK cells in preeclampsia patients was significantly higher than that in normal third trimester pregnant women (P<0.05). It was suggested that both number and function of the NK cells in preeclampsia women were increased, and that in umbilical blood was greater than that in maternal blood, speculating that the function of the NK cells may affect the maintenance of the maternal and fetal immune tolerance during pregnancy.

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2.

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426–434) or a self-peptide (Tyr369–377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426–434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369–377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77–85) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±1.01%, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.

Establishment of stable high expression cell line with green fluorescent protein and resistance genes

SummaryIn order to establish state high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percent-age of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percent-ages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones.

SummaryTo observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found thatin vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn’t change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. Thet tests showed the difference between experimental and control groups was statistically significant (P<0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclonesin vitro but also restrain the growth of those cell subclonesin vivo.

Regulatory Effects of X-linked Inhibitor of Apoptosis Protein and Pro-apoptotic Protein Smac on Apoptosis Resistance to Chemotherapy in Pancreatic Cancer Cells~*

AbstractObjective: To investigate the relation of X-linked inhibitor of apoptosis (XIAP) and second mitochondria-derived activator of caspase (Smac) signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods: Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil (FU) were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Panc-1 cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results: Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion: Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.

Immuno-detection of exfoliated cells in sputum with a monoclonal antibody

ObjectiveTo develop a prompt and specific method for diagnosis of lung cancer.MethodsA murine monoclonal antibody against lung cancer cells was developed and characterized with the techniques of ELISA, immunohistochemistry and immunocytochemical detection of sputum.ResultsThe antibody selectively bound to lung cancer tissues and exfoliated cells in the sputum from the patients with lung cancer, but did not bind to normal lung tissues and the cells in sputum either from the patients with pneumonia or from normal individuals.ConclusionThe antibody 2C25 has potential application for immunocytological detection of lung cancer cells in sputum.

Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones

SummaryTo evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression inneo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.