ABSTRACT
CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune-mediated reactions. The mechanisms of this suppression in animals and patients with autoimmune, allergic or oncogenic diseases have been investigated under various conditions. However, the precise mode of suppression by CD4(+)CD25(+) Tregs is still not clear. In this report, Tim-3-Galectin-9 pathway was explored as one of the mechanisms for the suppression and cytotoxicity induced by Tregs. Here, we demonstrated that Galectin-9 was expressed on CD4(+)CD25(+) Tregs by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Interestingly, blocking Tim-3-Galectin-9 pathway resulted in an obvious decreased suppression activity of Tregs and enhanced Th1 cytokine level in vitro. Furthermore, blocking Tim-3-Galectin-9 pathway negated prolonged survival of allogeneic skin grafts induced by CD4(+)CD25(+) Tregs in vivo. Our results suggest that Tim-3-Galectin-9 pathway involves the function of CD4(+)CD25(+) Tregs.
Subject(s)
Galectins/immunology , Galectins/metabolism , Graft Rejection/immunology , Membrane Proteins/immunology , Receptors, Virus/immunology , Receptors, Virus/metabolism , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , Antibodies, Blocking , Galectins/genetics , Hepatitis A Virus Cellular Receptor 2 , Immune Tolerance/immunology , Interferon-gamma/metabolism , Interleukin-2/metabolism , Interleukin-4/metabolism , Lymphocyte Activation , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Virus/genetics , Signal Transduction , Skin Transplantation , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Th1 Cells/immunology , Th1 Cells/metabolism , Th1 Cells/pathology , Transplantation ImmunologyABSTRACT
OBJECTIVE: To construct a novel 10-23 deoxyribozyme (10-23DZ) expression vector, identify the intracellular production of specific 10-23DZ and its inhibitory effect upon the expression of TACO gene in macrophage. METHODS: An oligonucleotide containing the primer binding sequence of mouse Moloney leukemia viral reverse transcriptase (MLV-RT), a multiple cloning site (MCS) and a stem-loop structure for termination of reverse transcription was designed and inserted into one MCS of plasmid pBUDCE4.1, downstream of the cytomegalovirus promoter (P(CMV)). The gene fragment encoding MLV-RT was inserted into another MCS of pBUDCE4.1, downstream of elongation factor 1alpha promoter (P(EF-1alpha)). The resulting plasmid was named pSDE01. Then pSDE01 was transfected into RAW264.7 cell and the expression of MLV-RT and the reverse transcriptase activity of expression product was identified by RT-PCR. To identify the cellular expression of 10-23DZ by pSDE01, a 10-23DZ targeting the TACO mRNA of macrophage was designed according to the predicted secondary structure of TACO mRNA. The expression sequence of designed 10-23DZ, DZ1, was synthesized and inserted into the MCS of ODN-PSL in pSDE01. The resulting plasmid, pSDE01-DZ1, was transfected into RAW264.7 cell and the expression of DZ1 was identified by PCR and dot-blot respectively. At 48 h after transfection of pSDE01 into RAW264.7 cells, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and Western blotting respectively. RESULTS: Restrictive analysis and sequencing data showed that the 10-23DZ expression vector, pSDE01, was successfully constructed and could express MLV-RT with reverse transcriptase activity in cell. When transfected into RAW264.7 cells, pSDE01-DZ1 expressed DZ1 effectively and inhibited the expression of TACO gene. And TACO mRNA decreased by 77.7% (0.193 +/- 0.008 vs 0.864 +/- 0.005, P < 0.05) and TACO protein decreased by 73.3% (0.114 +/- 0.051 vs 0.427 +/- 0.043, P < 0.05). CONCLUSIONS: A novel expression vector capable of generating specific 10-23DZ in cells has been successfully constructed. And the intracellular product 10-23DZ may inhibit the expression of any target gene of interest.
Subject(s)
DNA, Catalytic/genetics , DNA, Single-Stranded/genetics , Genetic Vectors , Animals , Cell Line , Humans , Macrophages/metabolism , Microfilament Proteins/genetics , Plasmids , RNA, Messenger/genetics , TransfectionABSTRACT
OBJECTIVE: To investigate two single nucleotide polymorphism sites of the promoter region in T cells immunoglobulin domain and mucin domain protein-3 (TIM-3) and detect their relationship with allergic asthma in a population of adult Hans from Hubei province of China. METHODS: The polymorphisms were detected with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allelic specific polymerase chain reaction (ASPCR). The genotype and allele frequencies were calculated and analyzed. RESULTS: The genotype frequencies of CC, CT and TT in -1541 C/T polymorphism were 0.961, 0.039 and 0 respectively in the healthy population and were 0.935, 0.065 and 0 respectively in the allergic asthma population. No significant difference in genotype and alleles frequencies was found between the allergic asthma patients and control subjects (P=0.314, P=0.321). The genotype frequencies of GG, GT and TT in -574 T/G polymorphism were 0.992, 0.008 and 0 respectively in the healthy population and were 0.941, 0.059 and 0 respectively in the allergic asthma population. There was significant difference in genotype and allele frequencies between the allergic asthma patients and control subjects (P=0.046, P=0.048). CONCLUSION: There are polymorphism sites of the promoter region in TIM-3 , and one of these sites, the -574 G/T polymorphism site, may be associated with allergic asthma in the population of adult Hans from Hubei province of China.
