Exploring the therapeutic potential of precision T-Cell Receptors (TCRs) in targeting KRAS G12D cancer through in vitro development

Objectives:The Kirsten rat sarcoma virus(KRAS)G12D oncogenic mutation poses a significant challenge in treating solid tumors due to the lack of specific and effective therapeutic interventions.This study aims to explore innovative approaches in T cell receptor(TCR)engineering and characterization to target the KRAS G12D7-16 mutation,providing potential strategies for overcoming this therapeutic challenge.Methods:In this innovative study,we engineered and characterized two T cell receptors(TCRs),KDA11-01 and KDA11-02 with high affinity for the KRAS G12D7-16 mutation.These TCRs were isolated from tumor-infiltrating lymphocytes(TILs)derived from tumor tissues of patients with the KRAS G12D mutation.We assessed their specificity and anti-tumor activity in vitro using various cancer cell lines.Results:KDA11-01 and KDA11-02 demonstrated exceptional specificity for the HLA-A*11:01-restricted KRAS G12D7-16 epitope,significantly inducing IFN-γrelease and eliminating tumor cells without cross-reactivity or alloreactivity.Conclusions:The successful development of KDA11-01 and KDA11-02 introduces a novel and precise TCR-based therapeutic strategy against KRAS G12D mutation,showing potential for significant advancements in cancer immunotherapy.

Milk fat globule membrane supplementation protects againstβ-lactoglobul-ininduced food allergy in mice via upregulation of regulatory T cells and enhancement of intestinal barrier in a microbiota-derived short-chain fatty acids manner

Milk fat globule membrane(MFGM),which contains abundant glycoproteins and phospholipids,exerts beneficial effects on intestinal health and immunomodulation.The aim of this study was to evaluate the protective effects and possible underlying mechanisms of MFGM on cow’s milk allergy(CMA)in aβ-lactoglobulin(BLG)-induced allergic mice model.MFGM was supplemented to allergic mice induced by BLG at a dose of 400 mg/kg body weight.Results demonstrated that MFGM alleviated food allergy symptoms,decreased serum levels of lipopolysaccharide,pro-inflammatory cytokines,immunoglobulin(Ig)E,Ig G1,and Th2 cytokines including interleukin(IL)-4,while increased serum levels of Th1 cytokines including interferon-γand regulatory T cells(Tregs)cytokines including IL-10 and transforming growth factor-β.MFGM modulated gut microbiota and enhanced intestinal barrier of BLG-allergic mice,as evidenced by decreased relative abundance of Desulfobacterota,Rikenellaceae,Lachnospiraceae,and Desulfovibrionaceae,while increased relative abundance of Bacteroidetes,Lactobacillaceae and Muribaculaceae,and enhanced expressions of tight junction proteins including Occludin,Claudin-1 and zonula occludens-1.Furthermore,MFGM increased fecal short-chain fatty acids(SCFAs)levels,which elevated G protein-coupled receptor(GPR)43 and GPR109A expressions.The increased expressions of GPR43 and GPR109A induced CD103+dendritic cells accumulation and promoted Tregs differentiation in mesenteric lymph node to a certain extent.In summary,MFGM alleviated CMA in a BLG-induced allergic mice model through enhancing intestinal barrier and promoting Tregs differentiation,which may be correlated with SCFAs-mediated activation of GPRs.These findings suggest that MFGM may be useful as a promising functional ingredient against CMA.

T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo

T cells modified with chimeric antigen receptor are an attractive strategy to treat Epstein-Barr virus（EBV） associated malignancies.The EBV latent membrane protein 1（LMP1） is a 66-KD integral membrane protein encoded by EBV that consists of transmembrane-spanning loops.Previously,we have identified a functional signal chain variable fragment（scFv） that specifically recognizes LMP1 through phage library screening.Here,we constructed a LMP1 specific chimeric antigen receptor containing anti-LMP1 scFv,the CD28 signalling domain,and the CD3ζchain（HELA/CAR）.We tested its functional ability to target LMP1 positive nasopharyngeal carcinoma cells.HELA/CAR cells were efficiently generated using lentivirus vector encoding the LMP1-specific chimeric antigen receptor to infect activated human CD3＋ T cells.The HELA/CAR T cells displayed LMP1 specific cytolytic action and produced IFN-γ and IL-2 in response to nasopharyngeal carcinoma cells overexpressing LMP1.To demonstrate in vivo anti-tumor activity,we tested the HELA/CAR T cells in a xenograft model using an LMP1 overexpressing tumor.Intratumoral injection of anti-LMP1 HELA/CAR-T cells significantly reduced tumor growth in vivo.These results show that targeting LMP1 using HELA/CAR cells could represent an alternative therapeutic approach for patients with EBV-positive cancers.

GW4064, a farnesoid X receptor agonist, upregulates adipokine expression in preadipocytes and HepG2 cells

AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells.

Distinct toll-like receptor expression in monocytes and T cells in chronic HCV infection

AIM： Hepatitis C virus often establishes chronic infections. Recent studies suggest that viral and bacterial infections are more common in HCV-infected patients compared to controls. Pathogens are recognized by Toll-like receptors （TLRs） to shape adaptive and innate immune responses. METHODS： In this study, to infected host to recognize assess the ability of HCV-infected host to recognize invading pathogens, we investigated Toll-like receptor expression in innate （monocytes） and adaptive （T cells） immune cells by realtime PCR. RESULTS： We determined that RNA levels for TLRs 2, 6. 7, 8, 9 and 10 mRNA levels were upregulated in both monocytes and T cells in HCV-infected patients compared to controls. TLR4 was only upregulated in T lymphocytes, while TLR5 was selectively increased in monocytes of HCV-infected patients. MD-2, a TLR4 coreceptor, was increased in patients＇ monocytes and T cells while CD14 and MyD88 were increased only in monocytes. CONCLUSION： Our data reveal novel details on TLR expression that likely relates to innate recognition of pathogens and immune defense in HCV-infected individuals.

Focused evaluation of the roles of macrophages in chimeric antigen receptor (CAR) T cell therapy associated cytokine release syndrome

Cytokine release syndrome(CRS)is a major obstacle to the widespread clinical application of chimeric antigen receptor(CAR)T cell therapies.CRS can also be induced by infections(such as SARS-CoV-2),drugs(such as therapeutic antibodies),and some autoimmune diseases.Myeloid-derived macrophages play key roles in the pathogenesis of CRS,and participate in the production and release of the core CRS cytokines,including interleukin(IL)-1,IL-6,and interferon-γ.In this review,we summarize the roles of macrophages in CRS and discuss new developments in macrophage activation and the related mechanisms of cytokine regulation in CRS.

Upregulated adenosine 2A receptor accelerates post-infectious irritable bowel syndrome by promoting CD4+T cells’T helper 17 polarization

BACKGROUND Post-infectious irritable bowel syndrome(PI-IBS)is generally regarded as a functional disease.Several recent studies have reported the involvement of lowgrade inflammation and immunological dysfunction in PI-IBS.T helper 17(Th17)polarization occurs in IBS.Adenosine and its receptors participate in intestinal inflammation and immune regulation.AIM To investigate the role of Th17 polarization of CD4+T cells regulated by adenosine 2A receptor(A2AR)in PI-IBS.METHODS A PI-IBS model was established by infecting mice with Trichinella spiralis.The intestinal A2AR and CD4+T lymphocytes were detected by immunohistochemistry,and the inflammatory cytokines were detected by enzyme-linked immunoassay.CD4+T lymphocytes present in the animal’s spleen were separated and cultured with or without A2AR agonist and antagonist.Western blotting and real-time quantitative polymerase chain reaction were performed to determine the effect of A2AR on the cells and intestinal tissue.Cytokine production was determined.The protein and mRNA levels of A2AR associated signaling pathway molecules were also evaluated.Furthermore,A2AR agonist and antagonist were injected into the mouse model and the clinical features were observed.RESULTS The PI-IBS mouse model showed increased expression of ATP and A2AR(P<0.05),and inhibition of A2AR improved the clinical features in PI-IBS,including the abdominal withdrawal reflex and colon transportation test(P<0.05).The number of intestinal CD4+T cells and interleukin-17(IL-17)protein levels increased during PI-IBS,which was reversed by administration of the A2AR antagonist(P<0.05).CD4+T cells expressed A2AR and produced IL-17 in vitro,which was regulated by the A2AR agonist and antagonist.The A2AR antagonist increased the production of IL-17 by CD4+T cells via the Janus kinase-signal transducer and activator of transcriptionreceptor-related orphan receptorγsignaling pathway.CONCLUSION The results of the present study suggested that the upregulation of A2AR increases PI-IBS by promoting the Th17 polarization of CD4+T cells.

Relevant nursing measures for the adverse reactions associated with chimeric antigen receptor T cells(CAR-T) immunotherapy: a systematic review of case reports

Objective: Cytokine release syndrome (CRS) and tumor lysis syndrome (TLS) that occur after chimeric antigen receptor T (CAR-T) cells are reinfused, which severely affect the survival and prognosis of patients. Although several articles have reported on the care of CAR-T cell immunotherapy, the quality of the study and the effectiveness of holistic nursing interventions have not been systematically reviewed. The purpose of this study was to systematically evaluate the existing holistic nursing interventions of CAR-T cell immunotherapy. Methods: A literature search for keywords was performed in PubMed, EMBASE, the Cochrane Library, CNKI, CBM, and Wanfang Data from its inception until January 2018. Studies were deemed eligible if they comprised patients with tumor receiving CAR-T cell immunotherapy, described the holistic nursing process, and were published in Chinese and English. Results: A total of 6 articles on holistic nursing interventions of CAR-T cell immunotherapy are reported, and the nursing methods and results of each article are analyzed. The quality of the studies included was medium. All nursing measures were considered effective. Conclusions: Holistic nursing programs reduce the incidence of CRS and TLS and improve the quality of life of cancer patients.

Chimeric antigen receptor-engineered T-cell therapy for liver cancer

Background: Chimeric antigen receptor-engineered T-cell(CAR-T) therapy is a newly developed immunotherapy used in the treatment of cancers. Because CAR-T therapy has shown great success in treating CD19-positive hematological malignancies, its application has been explored in the treatment of solid tumors, such as liver cancer. In this review, we discuss the immune characteristics of liver cancer, the obstacles encountered during the application of CAR-T therapy, and preclinical and clinical progress in the use of CAR-T therapy in patients with liver cancer.Data sources: The data on CAR-T therapy related to liver cancers were collected by searching Pub Med and the Web of Science databases prior to December 2017 with the keywords "chimeric antigen receptor","CAR-T", "liver cancer", "hepatocellular carcinoma", and "solid tumor". Additional articles were identified by manual search of references found in the primary articles. The data for clinical trials were collected by searching Clinical Trials.gov.Results: The liver has a tolerogenic nature in the intrahepatic milieu and its tumor microenvironment significantly affects tumor progression. The obstacles that reduce the efficacy of CAR-T therapy in solid tumors include a lack of specific tumor antigens, limited trafficking and penetration of CAR-T cells to tumor sites, and an immunosuppressive tumor microenvironment. To overcome these obstacles, several strategies have emerged. In addition, several strategies have been developed to manage the side effects of CAR-T, including enhancing the selectivity of CARs and controlling CAR-T activity. To date, no clinical trials of CAR-T therapy against HCC have been completed. However, preclinical studies in vitro and in vivo have shown potent antitumor efficacy. Glypican-3, mucin-1, epithelial cell adhesion molecule, carcinoembryonic antigen, and other targets are currently being studied.Conclusions: The application of CAR-T therapy for liver cancer is just beginning to be explored and more research is needed. However, we are optimistic that CAR-T therapy will offer a new approach for the treatment of liver cancers in the future.

Activating interactions of sulfanilamides with T cell receptors

Activation and expansion of drug reactive T cells are key features in drug hypersensitivity reactions. Drugs may interact directly with immune receptors such as the human leukocyte antigens (HLA) or the T-cell receptors (TCR) itself, the pharmacological interaction [p-i] concept. To analyze whether the drug sulfamethoxazole (SMX) interacts directly with the TCR and thereby contributing to signaling and T cell activation, we analyze two SMX specific T cell clones (TCC “1.3”and “H13”). Proliferation to SMX and 11 related sulfanilamides, Ca++ influx in drug stimulated T-cells and the inhibitory effect of non-reactive sulfanilamides on SMX stimulation were analyzed. In silico docking of SMX and related sulfanilamide to the TCR were used to identify possible drug binding sites, and correlated to in vitro data to find the correct docking. In Ca++ influx assays, reactions occurred as early as 14 sec after adding SMX to TCC and APC. The broadly reactive clone (“H13”) was stimulated by 5 additional sulfanilamide, while one TCC (“1.3”) was reactive exclusively with SMX but not other sulfanilamides. Competition experiments with sulfanilamide inhibited SMX induced Ca++ influx and proliferation of the TCC1.3 ina dose dependent way. Docking experiments with SMX and related sulfanilamides confirmed and explained the in vitro data as docking localized binding sites for SMX and the 5 stimulating sulfanilamides on the CDR2β domain of the clone H13, while the 6 non-stimulatory SA failed to bind. In TCC 1.3, SMX could be docked on the CDR3α of the TCR. The other, non-stimulatory but inhibitory SA could also be docked to the same site. The combined analysis of in vitro and in silico data show that sulfanilamide can bind directly to TCRs. It shows that TCR, like other receptors, appear to be reamenable to manipulations by small molecules.

Roles of antigen receptors and CA215 in the innate immunity of cancer cells

Antigen receptors, including immunoglobulins and T-cell receptors, are known to be widely expressed by cancer cells through unconfirmed mechanisms and for unknown purposes. Recently, a monoclonal antibody, designated as RP215, was generated against the ovarian cancer cell line, OC-3-VGH, and was shown to react with CA215, which consisted mainly of these cancer cell-expressed antigen receptors. Experimental evidence has clearly indicated that cancerous immunoglobulins play significant roles in the growth and proliferation of cancer cells in vitro and in vivo. RP215 and anti-antigen receptor antibodies were equally effective in inducing apoptosis and complement-dependent cytotoxicity reactions to cultured cancer cells. Through gene regulation studies, both RP215 and antibodies against antigen-receptors were shown to affect more than a dozen of genes involved in cell proliferation (such as NFκB-1, IgG, P21, cyclin D1, ribosomal P1, and c-fos). Furthermore, selected toll-like receptor genes (TLR- 2, -3, -4, and -9) were also found to be highly regulated by both RP215 and anti-antigen receptor antibodies. For example, RP215 and anti-antigen receptor antibodies were found to both up-regulate TLR-2 and/or TLR-3 and down- regulate TLR-4 and TLR-9 intwo types of cancer cells. Based on these studies, it is reasonable to postulate that cancerous immunoglobulins play important roles in the modulation of the innate immune system to allow the growth and survival of cancer cells within the human body. Consequently, RP215 inits humanized forms may be utilized to target cancer cells for potential therapeutic purposes.

Association between genetic variations in tumor necrosis factor receptor genes and survival of patients with T-cell lymphoma

The prognosis of T-cell lymphoma (TCL) has been shown to be associated with the clinical characteristics of patients. However, there is little knowledge of whether genetic variations also affect the prognosis of TCL. This study investigated the associations between single nucleotide polymorphisms(SNPs) in tumor necrosis factor receptor superfamily(TNFRSF) genes and the survival of patients with TCL. A total of 38 tag SNPs in 18 TNFRSF genes were genotyped using Sequenom platform in 150 patients with TCL. Kaplan-Meier survival estimates were plotted and significance was assessed using log-rank tests. Cox proportional hazard models were used to analyze each of these 38 SNPs with adjustment for covariates that might influence patient survival, including sex and international prognostic Index score. Hazard ratios (HRs) and their 95% confidence intervals(CIs) were calculated. Among the 38 SNPs tested, 3 were significantly associated with the survival of patients with TCL. These SNPs were located at LTβR (rs3759333C>T) and TNFRSF17(rs2017662C>T and rs2071336C>T). The 5-year survival rates were significantly different among patients carrying different genotypes and the HRs for death between the different genotypes ranged from 0.45 to 2.46. These findings suggest that the SNPs in TNFRSF genes might be important determinants for the survival of TCL patients.

The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors(TCRs) or chimeric antigen receptors(CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cells to target B-cell malignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin's lymphoma.

A facile,branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

Glucocorticoid(GC) steroid hormones are used to treat acute lymphoblastic leukemia(ALL) because of their pro-apoptotic effects in hematopoietic cells.However,not all leukemia cells are sensitive to GC,and no assay to stratify patients is available.In the GC-sensitive T-cell ALL cell line CEM-C7,auto-up-regulation of RNA transcripts for the glucocorticoid receptor(GR) correlates with increased apoptotic response.This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations.The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA(bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay.There were significant correlations between both assay platforms when measuring total GR(exon 5/6) transcripts in various cell lines and patient samples,but not for a probe set that detects a specific,low abundance GR transcript(exon 1A3).Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and,with further development,may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma

AIM:To study the diagnostic value of immunoglobulin heavy chain(IgH)and T-cell receptorγ (TCR-γ)gene monoclonal rearrangements in primary gastric lymphoma(PGL).METHODS:A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011.The patients were divided into three groups(a PGL group,a gastric linitis plastica group,and a benign gastric ulcer group)based on the pathological results(gastric mucosal specimens obtained by endoscopy or surgery)and follow-up.Endoscopic ultrasonography(EUS)and EUSguided biopsy were performed in all the patients.The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.RESULTS:EUS and EUS-guided biopsy were successfully performed in all 48 patients.In the PGL group(n=21),monoclonal IgH gene rearrangements were detected in 14(66.7%)patients.A positive result for each set of primers was found in 12(57.1%),8(38.1%),and 4(19.0%)cases using FR1/JH,FR2/JH,and FR3/JH primers,respectively.Overall,12(75%)patients with mucosal-associated lymphoid tissue lymphoma(n=16)and 2(40%)patients with diffuse large B-cell lymphoma(n=5)were positive for monoclonal IgH gene rearrangements.No patients in the gastric linitis plastica group(n=17)and only one(10%)patient in the benign gastric ulcer group(n=10)were positive for a monoclonal IgH gene rearrangement.No TCRgene monoclonal rearrangements were detected.The sensitivity of monoclonal IgH gene rearrangements was 66.7%for a PGL diagnosis,and the specificity was96.4%.In the PGL group,8(100%)patients with stage IIE PGL(n=8)and 6(46.1%)patients with stage IE PGL(n=13)were positive for monoclonal IgH gene rearrangements.CONCLUSION:IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.

ANALYSIS OF T CELL CLONALITY BY CDR3 SIZE OF T-CELL ANTIGEN RECEPTOR Vβ REPERTOIRE IN HCL AND c-ALL

Objective: To analyze the distribution and clonality of TCR Vβ subfamily T cells in hairy cell leukemia (HCL) and common-acute lymphoblastic leukemia (c-ALL). Methods: Peripheral blood mononuclear cell samples from 3 cases of HCL and 1 case of c-ALL were investigated for analysis of complementarity determining region 3 (CDR3) size of T cell receptor Vβ repertoire using reverse transcriptase-polymerase chain reaction (RT-PCR). The products were further analyzed by genescan to identify T cell clonality. Results: Some Vβ subfamily PCR products from 4 patients contained monopeak (monoclone) or a dominant peak (oligoclone). In contrast, multipeak (polyclone) distributions were found in all Vβ subfamily PCR products from normal control cases. Conclusion: T cell clonal expansion may be found in HCL and c-ALL cases that may indicate a host response directed against leukemia related antigen. In addition, it may be useful to detect the minimal residual disease.

T-CELL RECEPTOR GENE REARRANGEMENT ANALYSIS IN THE PRIMARY CUTANEOUS T-CELL LYMPHOMA

Object: The present paper is to evaluate the significance of T cell receptor (TCR) gene rearrange ments in primary cutaneous T cell lymphomas (PCTCL) as detected by analysis of Southern Blot (SBA) and polymerase chain reaction (PCR). Patients and Methods: Skin specimens and peripheral blood samples were taken from 44 patients with PCTCL, including 30 patients with mycosis fungoides (MF), 2 patients with Sezary's syndrome (SS), and 12 patients with PCTCL other than MF and SS (PNCTCL). 11 patients with a presumptive diagnosis of MF, 23 patients with lymphoproliferative dermatoses including lymphomatoid papulosis (LyP) and 8 patients with benign cutaneous lymphoid infiltrates were simultaneously studied by the amplification of junctional V (variable) J (joining) sequences of the rearranged TCRγ genes by PCR(TCRγPCR) and the analysis of TCRb chain genes by SBA(TCRβSBA) for detection of clonal gene rearrangements (GR). One lymph node specimen of a case with MF IIA was also detected by TCRγ PCR and TCRβSBA. Results: In MF, GR were detected by TCRγPCR and TCRβSBAb in 83.3 85.7% and 66.7% 71.4% of skin specimens of cases IIA IIB and in 57.1% 70.0% and 14.3% 10.0% of those of cases IA IB, respectively. GR were seen in 66.7% 71.4% and 33.3% 43.0.% of blood samples of cases IIA IIB, and 42.9% 40.0% and 0 10.0% of those of cases IA IB, respectively. GR was confirmed by TCRγ PCR and TCRβSBA in one lymph node showing dermato pathic lymphadenopathy of a case with MF IIA. In 11 patients of clinically suspected MF, GR were present in skin specimens of 5 cases (45.4%) and in blood samples of 3 cases ( 27.3% ) by TCRγ PCR. In PNCTCL, GR were found in 9 skin specimens (90.0%) from 10 patients detected by TCRγ PCR and in 6 skin specimens (75.0%) from 8 patients detected by TCRβSBA. GR were also seen in 6 blood samples (72.8%) from 11 patients detected by TCRγ PCR, and in 7 blood samples (70.0%) from 10 patients by TCRβSBA. In SS and LyP, GR were detected by TCRγ PCR and TCRβSBA in each of the two skin specimens of two cases with LyP and in each of the two blood samples of two cases with SS. GR were seen in one skin specimen of one case with SS and one blood sample of one case with LyP detected by TCRγPCR. Conclusions: This study demonstrated that TCRγ PCR is a rapid, more sensitive tool than TCRβSBA, can be used in the analysis of T cell clonality in skin, lymph node and blood samples of patients with PCTCL and indicated that this method forms a useful supplement to other methods for diagnosis of early and suspected MF, confirmation of PNCTCL and determination of extracutaneous involvement of lymph node and blood.

Effects of immunoglobulin D on expression of IgD receptor and protein tyrosine kinase signaling in human CD4^+ T cells

OBJECTIVE To observe whether human CD4^+T cells could be activated by immuno-globulin D(IgD) via IgD receptor(IgDR)-Lck.METHODS Human CD4^+T cells were purified from peripheral blood mononuclear cells(PBMCs) with microbeads.The viability of T cells were detected by CCK-8.The binding affinity and expression of IgDR on T cells were detected by flow cytometry.The protein expression of IgDR,Lck and P-Lck were analyzed by western blot.RESULTS IgD could concentration-dependent bind to IgDR on CD4^+T cells.The expression of IgDR was increased in response to treatment with IgD in a time-dependent and concentration-dependent manner.Stimulating by IgD resulted in enhanced phosphorylation of Lck compared with that in the medium control sample.The expression of Lck was not changed.As inhibitor of PTK,Herbimycin A or A770041,which combined with IgD could significantly inhibit phosphorylation of Lck(Tyr^(394)).The proliferation promoting effect of IgD was blocked by Herbimycin A or A770041.IgD could stimulate CD4^+T cell activation and proliferation through upregulating activating tyrosine residue of Lck(Tyr^(394)) phosphorylation.CONCLUSION These results demonstrate that IgD exaggerates CD4^+T cell activities,which may be through promoting Lck phosphorylation.

Effect of radiation on TCR，CD_3，CD_（18），and CD_（25） expressionof human T cells

Using a flow cytometry (FCM), we detected and enumerated the positive cell percentage of T cell receptor (TCRα/β), CD3, CD18, and CD25, in normal human peripheral blood mononuclear cells (PBMC) irradiated by γ ray in vitro and PBMC from persons who suffered acute radiation symptoms 3 years earlier. The results showed that after PBMC from the normal persons were irradiated with 1～6 Gy dose the positive cell percentage of TCR, CD3, CD18, and CD25, did not change significantly when analysis was conducted immediately after PBMC were irradiated, while the positive cell percentage of TCR, CD3 and CD25, decreased with the increase in irradiation dose and increased with prolongation of culture time significantly at different levels. The change in positive cell percentage of CD18 was relatively smaller than that of the others after irradiated PBMC were cultured in vitro for 8～14 d. Immunocytochemical analysis showed that more TCR and CD3 positive stains presented in cytoplasm 8 ～12d after irradiation. These results implied that the decrement of positive TCR and CD3 T cells induced by irradiation might be related to abnormal expression and disabled assembly of TCR/CD3 complex.

Recombinant E.coli LLO/OVA Induces Murine BMDCs Maturation via TLR4 and NOD1 Receptor and Promotes Specific Cytotoxic T Cell Immunity

Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor （TLR） and nucleotide-binding oligomerization domain （NOD） receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4＋T and CD8＋T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8＋T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-？ increased also.The mature BMDCs stimulated the allergic CD4＋T and CD8＋T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8＋T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.