Preliminary study on the anti-tumor effect and mechanism of a novel fully human anti-LAG3 monoclonal antibody in vitro / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: The co-culture model of LAG3+Jurkat cells and tumor cells was constructed to investigate the anti-tumor effect and mechanism of a novel fully human anti-LAG3 monoclonal antibody in vitro. Methods: Jurkat cells were stimulated with PHA to simulate TIL, and the secretion of IL-2 was detected by ELISA to evaluate the degree of Jurkat cell activation. Meanwhile, FCM, Immunofluorescence and WB assays were employed to detect the expression of LAG3 in activated Jurkat cells and MHC classⅡmolecule（MHC-Ⅱ）, a LAG3 ligand, in HGC-27, MGC-803 and A549 tumor cells. The co-culture model of activated LAG3+Jurkat cells and tumor cells was constructed, and CCK-8 assays were employed to detect the killing efficiency of LAG3+Jurkat cells against tumor cells at different effector-target ratios and the effect of the anti-LAG3 antibody . The secretion levels of cytokines IL-2, IL-10 and TNF-α in supernatant of co-culture system were detected by ELISA. Results: After 48 h treatment, 2 μg/mL PHA exhibited no obvious cytotoxicity to Jurkat cells (P>0.05), but could significantly induce IL-2 secretion (P<0.01) and LAG3 expression (P<0.01), indicating activated LAG3+Jurkat cells were acquired. MGC-803 and A549 cells significantly expressed MHC-Ⅱ (P<0.01), but HGC-27 cells did not express MHC-Ⅱ (P>0.05). The co-culture model of LAG3+Jurkat cells and tumor cells was constructed at a effector-target ratio of 10∶1. The anti-LAG3 antibody could effectively enhance the killing efficiency of Jurkat cells against MHC-Ⅱ+ tumor cells (P<0.05). Further analysis revealed that the secretion levels of cytokines IL-2, IL-10 and TNF-α were increased in the co-culture supernatant of MHC-Ⅱ+ target cell group (all P<0.01). Conclusion: A co-culture model of LAG3+Jurkat cells and tumor cells was successfully constructed in vitro. The anti-LAG3 antibody might increase the killing effect of Jurkat cells against MGC-803 and A549 tumor cells through blocking LAG3/MHC-Ⅱ interaction, which may be related to the increased secretion levels of cytokines IL-2, IL-10 and TNF-α in the supernatant of co-culture system.

Immunoglobulin-like transcript-4 (ILT4) expression in mononuclear cells on the prognosis of sepsis / 中华急诊医学杂志

Objective To study the biological behaviors and effects of immunoglobulin-like transcript-4 (ILT4) expression in mononuclear cells on the prognosis of sepsis.Methods ILT4 +/+ (WT) and ILT4-knockout mice (ILT4-/-) male BALB/c mice were used for sepsis modeling using cecal ligation puncture (CLP).Flow cytometry was used to measure the levels of expression of ILT4 and major histocompatihility complex class Ⅱ molecules (MHC-Ⅱ) in mononuclear cells of peripheral blood 24 h after CLP.ELISA was used to measure the concentrations of interleukin-6 (IL-6) and serum tumor necrosis factor-alpha (TNF-α) in different groups of mice at 0 h,6 h,12 h,and 24 h after CLP to monitor the survival and prognosis over the course of 168 h.Results ILT4 was highly expressed in mononuclear cells of the peripheral blood of septic mice 24 h after CLP in comparison with that before CLP (1292.00 ± 143.70) vs.(193.50 ± 52.54),P ＜ 0.05.MHC-Ⅱ expression in mononuclear cells of the peripheral blood in ILT4-/-mice was significantly higher than that in WT mice (49.38 ± 5.66)％ vs.(24.25 ± 6.76) ％,P ＜ 0.05).Serum IL-6 was significantly elevated 24 h after CLP compared with that before CLP (470.75 ± 88.03) vs.(54.25 ± 20.04),P ＜ 0.05.The serum IL-6 concentration was much lower in ILT4-/-mice thanthatin MT mice (241.25 ± 45.10)vs.(470.75 ± 88.03),P ＜ 0.05;whereas,there was no significant difference in TNF-α expression between two groups of mice (50.88 ± 6.38) vs.(53.13 ± 5.49),P ＞ 0.05.The survival rate of ILT4-/-mice was significantly higher after CLP compared with WT mice (P ＜ 0.05).Conclusion The high level of ILT4 expression in mononuclear cells were observed in peripheral blood during sepsis and it was found to be associated with high serum IL-6 levels and low MHC-Ⅱ expression in mononuclear cells,leading to increased mortality.

A CPLs vector-based RNAi technology to inhibit the expression of rat CⅡTA and MHCⅡ genes / 中华微生物学和免疫学杂志

Objective To investigate the inhibitory effects of cationic polymeric liposomes (CPLs) vector-based RNA interference (RNAi) technology on the expression of rat MHCⅡ transactivator ( CⅡTA) and MHCⅡgenes .Methods According to the genetic information of CⅡTA downloaded from GenBank, three short hairpin RNA (shRNA) sequences targeting CⅡTA sequences were designed .CPLs vectors were constructed and coupled to shRNA plasmid vectors to form pCⅡTA-CPLs vectors .Six groups including control group , CPLs control group , pHK-CⅡTA control group and three pCⅡTA-CPLs groups were set up.Rat dendritic cells (DCs) were transfected in vitro.Real time PCR and flow cytometry analysis were used to detect the expression of CⅡTA and MHCⅡat mRNA and protein levels in DCs after transfec-tion.Results The pCⅡTA-CPLs vectors were successfully constructed .Compared with control groups ,the transcription level of CⅡTA and MHCⅡand the expression of MHCⅡat protein level were significantly in-hibited in all pCⅡ TA-CPLs groups ( P<0 .01 ) .The strongest inhibitory effects of pCⅡTA-CPLs on the ex-pression of CⅡTA and MHCⅡgenes were observed in the second pCⅡTA-CPLs group.There was a positive correlation between the expression of CⅡTA and MHCⅡ.Conclusion CPLs vectors were effective gene carriers.The constructed pCⅡTA-CPLs vectors significantly inhibited the in vitro expression of rat CⅡTA and MHCⅡ, which provided evidences for further investigation on pCPLs-CⅡTA vectors in vivo.

Effect of IL-18 on expression of molecule on surface of 9 L in vitro and in vivo / 中国免疫学杂志

Objective:To explore the influence of IL-18 on the expression of MHCⅠ,MHCⅡ,CD80,CD86 on the surface of 9L cells,to identify the effect of IL-18 on the immunogenicity and the efficiency of tumor antigen presentation of 9L.Methods:Retroviruses were used to transduce the mIL-18 gene into rat glioma cells and the cell clones (9L/IL-18) which steadily expressing mIL-18 gene were obtained ,and got the control cells of 9L/LXSN by the same method.The expression of MHCⅠ,MHCⅡ,CD80,CD86 on the cell surface were assessed by flow cytometry.The cell suspension of 9L/IL-18,9L/LXSN and 9L cells were inoculated into the brain of F344 rat to establish the animal models by the stereotactic technique ,got the tumor tissues to analyze the expression of MHCⅠ, MHCⅡ,CD80 and CD86 on the surface of tumor cells after 14 days.Results:The expression of MHCⅠ,MHCⅡ,CD80 and CD86 on the surface of 9L/IL-18 were (10.9±1.44)%,(0.61±0.14)%,(1.01±0.14)%,(0.57±0.11)% ; had no significant differences with the others in vitro (P>0.05);while the expression of MHCⅠ,CD80 and CD86 on the surface of 9L/IL-18 were(67.51± 1.40)%,(12.51±1.57)%,(6.95±0.56)%which were higher than 9L/LXSN and 9L cells (P0.05) in vivo.Conclusion: IL-18 did not affect the immunogenicity of 9L in vitro,but improve the immunogenicity and tumor antigen presentation in vivo.

The role of MHCⅡ + T lymphocytes in immune tolerance induction / 中华肝胆外科杂志

Traditionally,antigen presenting cells were shown to express MHC Ⅱ antigens.However,some researchers have demonstrated that a subset of T lymphocytes could express MHCⅡ antigens in some situation.Its regulatory mechanism and biological effects remains challenging to researchers.This review provides an overview of the results of these MHC Ⅱ + T lymphocytes regarding to the generation,mechanisms,and the role in immune tolerance induction,aim to provide some insights in clinical immune tolerance induction.

The inhibiting effect on rat C Ⅱ TA and MHC Ⅱ gene expression by plasmid vector-based RNAi technulogy / 中华微生物学和免疫学杂志

Objective To investigate the inhibitory effect on rat MHC Ⅱ transactivator (C Ⅱ TA) and MHC Ⅱ gene expression by plasmid vector-based RNAi technology. Methods According to C Ⅱ TA ge-netic information, three short hairpin RNA (shRNA) sequences were designed and the corresponding plas- mid vectors were constructed. Rat dendritic cell (DC) was transfected in vitro and rat spleen was transfected in vivo. Real time RT-PCR and flow cytometry were used to detect the expression of C Ⅱ TA and MHC Ⅱ in DC and spleen after transfection. Results C Ⅱ TA-shRNA plasmids were successfully constructed. Com-pared with control groups, the mRNA transcription levels of C Ⅱ TA and MHC Ⅱ and the expression level of MHC H were significantly inhibited in all three pC Ⅱ TA-shRNA experimental groups ( P < 0.01 ). There was positive correlation between the expression of C Ⅱ TA and MHC Ⅱ. Among the three shRNA groups, the first one showed the strongest inhibitory effect. Conclusion The expression of rat C Ⅱ TA and MHC Ⅱ can be obviously inhibited by plasmid vector of shRNA targeting C Ⅱ TA gene, which can be used for further investi-gation of gene therapy.

The Importance of Helper T-cell Epitope in Vaccine Development / 热带医学杂志

The major challenge in vaccine development a gainst various disease-causing organisms is to use defined antigen to stimulate appropriate immune responses that lead to resistance. The use of peptide-based vaccine is gaining greater attention asits flexibility in the incorporation of multiple defined and different epitopes into a single construct for eliciting d esirable arms of the immune system. It is generally safer than the use of live a ttenuated vaccine while it is relative ease of manufacture than subunit vaccine. However, development of peptide-based vaccine faces significant challenges. This approach has limited ability to elicit immune responses in a genetically dive rse outbred population due to the Major Histocompatibility Complexes (MHC) polym orphism. For the same reason, peptide immunizations often elicit inadequate cyto toxic T lymphocyte (CTL) and antibody (Ab) responses due to the lack of appropri ate helper Tlymphocyte (HTL) activity. Another possible disadvantage of using linear peptide construct is that, for eliciting appropriate antibody responses, s urface immunoglobulin (Ig) receptor clustering is needed in order to activate th e resting B cells. Problems caused by MHC polymorphism may be circumvented by the use of promiscuous T-cell epitopes. Promiscuous T-cell epitopes from the mea sles virus F protein (amino acid [aa] 288 to 302) and a murine defined T-helper cell epitope (V1E8, aa 191-209) that bind to multiple MHC molecules have bee n identified and have been used in highly immunogenic constructs to overcome hap lotype-restricted immune responses. Synthetic non-natural Pan DR Epitope (PADR E) which have degenerate binding capacity to several common HLA-DR can enhance immunogenicity of short-peptide immunogen, both in terms of absolute titers and quality of antibody responses. Besides, a number of so called "promiscuous" T -cell epitopes from Influenza virus hemagglutinin (HA), Plasmodium falciparum pre-erythrocytic stage antigens and mycobacterial proteins have been reporte d to be universally immunogenic. For promiscuous binding to several isotypic and allotypic forms of MHC class Ⅱ molecules, these peptides should display overla pping MHC-binding motifs or should use anchors that are conserved among ligands and should lack allele-specific anchor residues that would prevent binding wit h the other class Ⅱ molecules. Understanding the biophysical basis for both the promiscuity and the specificity of peptide recognition by MHC Ⅱ molecules will provide a molecular rationale for strategies to overcome genetic restriction in the context of vaccine design.

Expression of MHC Ⅱ on rat corneal keratocytes is inhibited by special siRNA targeting CⅡTA / 中国病理生理杂志

AIM: To investigate the feasibility to inhibit the expression of MHCⅡ by special siRNA targeting class Ⅱ major histocompatibility complex (MHC Ⅱ) transactivator (CⅡTA), which might regulate MHC Ⅱexpression for suppressing immune rejection. METHODS: Five different siRNA were designed, synthesized and transfected into freshly isolated rat corneal keratocytes. At 24 hours posttransfection, the changes of MHC Ⅱexpression were detected by flowcytometry, and the mRNA abundance of CⅡTA and MHC Ⅱ was measured by FQ-PCR after inducing with recombinant rat interferon-gamma (IFN-?). RESULTS: Different siRNA showed different reduction in MHC Ⅱ and CⅡTA expression compared with the control (P