Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

OBJECTIVE: To investigate the level of H2-Ab1 in the nasal mucosa of mice with allergic rhinitis. METHOD: Twenty-four female 129/sv mice were divided into 2 groups: ovalbumin (OVA) group and control. The allergic rhinitis models were induced by classical method with OVA. After the last challenge, the pathological differences between the two groups were surveyed. The levels of H2-AB1 were measured by ELISA and quantitative real time PCR. RESULT: The expression of H2-Ab1 is higher in subjects with AR than that in controls (P < 0.05). CONCLUSION The levels of H2-Ab1 were highly increased in allergic rhinitis group, which might be associated with the pathogenesis of allergic rhinitis.
Animals ; Female ; Gene Expression ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Mice ; Nasal Mucosa ; metabolism ; Rhinitis, Allergic ; metabolism

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.
Animals ; *Bandages ; Base Sequence ; DNA, Complementary/analysis ; Dogs/*genetics/metabolism ; Gene Expression Profiling/veterinary ; Gene Expression Regulation ; Histocompatibility Antigens Class II/*genetics/metabolism ; Molecular Sequence Data ; Omentum/*metabolism ; Proteasome Endopeptidase Complex/*genetics/metabolism ; RNA, Messenger/analysis ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; *Wound Healing

Antigens, Differentiation, B-Lymphocyte ; genetics ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; metabolism ; Gene Fusion ; Gene Rearrangement ; Histocompatibility Antigens Class II ; genetics ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; Protein Kinase Inhibitors ; therapeutic use ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Sodium-Phosphate Cotransporter Proteins, Type IIb ; genetics

OBJECTIVETo investigate the expressions of different forms of ROS fusions in Chinese patients with cholangiocarcinoma (CCA).

METHODSRT-PCR was employed to examine formalin-fixed and paraffin-embedded CCA samples from stage I-IV patients for detection of ROS fusions involving Fused in Glioblastoma (FIG), solute carrier protein (SLC34A2) and major histocompatibility complex class II invariant chain (CD74). Serpin peptidase inhibitor clade A member 1 (SERPINA1) was detected as the reference gene.

RESULTSIn all the 56 CCA samples, 80.4% (45/56) were positive for SERPINA1 expression as evaluable samples. Of these evaluable samples, none expressed the ROS fusions.

CONCLUSIONROS fusions are not common in Chinese CCA patients.

Antigens, Differentiation, B-Lymphocyte ; genetics ; metabolism ; Bile Duct Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; metabolism ; Cholangiocarcinoma ; metabolism ; pathology ; Female ; Gene Expression ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Paraffin Embedding ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Sodium-Phosphate Cotransporter Proteins, Type IIb ; genetics ; metabolism

OBJECTIVETo study the ex vivo effect of sirolimus on capacity of splenic dendritic cells (DC) from traumatized mice in inducing T cell responses.

METHODSTwenty-four BALB/c mice were divided into control group and trauma group according to the random number table, with 12 mice in each group. Mice in trauma group were bled followed by closed femur fracture after anaesthesia, while mice in control group were only anaesthetized without injury. Twenty-four hours later DC were isolated from spleens and divided into 4 subgroups: sirolimus devoid control (trauma) groups [consisted of cells from control (trauma) groups, without sirolimus treatment] and sirolimus treated control (trauma) groups [consisted of cells from control (trauma) groups, treated with 10 microg/L sirolimus for 6 hours]. Then their autophagic activity, DC-induced mixed lymphocyte reaction (MLR) were measured and recorded as fluorescence intensity (FI) value and absorbance value respectively. The expression of major histocompatibility complex class (MHC) II and costimulatory molecules CD40, CD80, and CD86 on DC surface were measured with flow cytometry. IL-12p40, IL-12p70 and IL-10 levels in lipopolysaccharide-stimulated DC supernatants were determined by ELISA. Data were processed with one-way analysis of variance.

RESULTS(1) Compared with those of sirolimus devoid control group (FI value = 22 +/- 6), DC autophagic activity (FI value = 13 +/- 2) and DC-induced MLR in mice from sirolimus devoid trauma group were significantly weakened (F = 212.836, P < 0.05). Compared with those of sirolimus devoid control (trauma) groups, DC autophagic activity in mice from sirolimus treated control (trauma) groups (FI = 45 +/- 8, 44 +/- 8 respectively) were significantly strengthened (F = 212.836, P < 0.05 or P < 0.01). MLR in mice from sirolimus treated trauma group was stronger than that from sirolimus devoid trauma group (with F value respectively 101.426, 86.533, P values all below 0.05). (2) Compared with those of sirolimus devoid control group [MHC II (85 +/- 6)%, CD40 (8 +/- 1)%], the expressions of MHCII [(60 +/- 9)%] and CD40 [(4 +/- 1)%] on DC surface from sirolimus devoid trauma group were significantly reduced (with F value respectively 37.918, 40.426, P values all below 0.05). The expression of MHCII from sirolimus treated trauma group [(78 +/- 7)%] was higher than that from sirolimus devoid trauma group (F = 37.918, P < 0.05). (3) IL-12p40, IL-12p70 secretion by DC from sirolimus devoid trauma group [(120 +/- 13), (10 +/- 3) pg/mL] were significantly reduced as compared with those from sirolimus devoid control group [(200 +/- 25), (20 +/- 6) pg/mL, with F value respectively 218.646, 310.253, P values all below 0.05]. Compared with those from sirolimus devoid control (trauma) groups, IL-12p40 [(560 +/- 34), (540 +/- 29) pg/mL], IL-12p70 [(55 +/- 8), (60 +/- 11) pg/mL] secretion by DC from sirolimus treated control (trauma) groups were obviously enhanced (with F value respectively 218.646, 310.253, P values all below 0.01), while IL-10 secretion levels were significantly decreased (F = 246.108, P < 0.01).

CONCLUSIONSSirolimus can partially ameliorate DC functions ex vivo in traumatized mice, and further enhance the capacity of DC in inducing T cell responses.

Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Histocompatibility Antigens Class II ; immunology ; Interleukin-12 Subunit p40 ; metabolism ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; Sirolimus ; pharmacology ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology ; Wounds and Injuries ; immunology

OBJECTIVETo investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.

METHODSMice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.

RESULTSB220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].

CONCLUSIONAfter GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Stomach Neoplasms ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

BACKGROUNDGenetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed.

METHODSThe silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(®) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used.

RESULTSThe Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P < 0.05). Furthermore, tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4(+) and CD8(+) T cells were significantly activated in the Ii siRNA group (P < 0.05).

CONCLUSIONSilencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.

Animals ; Antigens, Differentiation, B-Lymphocyte ; genetics ; metabolism ; Blotting, Western ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Female ; Flow Cytometry ; Gene Silencing ; physiology ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Mice ; Neoplasms ; immunology ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction

Animals ; Antigens, Differentiation, B-Lymphocyte ; immunology ; Gene Expression ; Hepacivirus ; immunology ; Histocompatibility Antigens Class II ; immunology ; Mice ; Mice, Inbred BALB C ; Th1 Cells ; immunology ; metabolism ; Viral Hepatitis Vaccines ; genetics ; immunology

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.
Animals ; Antigens, CD/immunology/metabolism ; Bacterial Outer Membrane Proteins/*pharmacology ; Cells, Cultured ; Female ; Histocompatibility Antigens Class II/immunology/metabolism ; Host-Pathogen Interactions ; Interleukin-12/biosynthesis ; Interleukin-6/biosynthesis ; Legionella pneumophila/*immunology/metabolism ; Legionnaires' Disease/immunology/metabolism ; Lipoproteins/*pharmacology ; Macrophage Activation/drug effects/immunology ; Macrophages, Peritoneal/drug effects/immunology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Toll-Like Receptor 2/*metabolism ; Tumor Necrosis Factor-alpha/biosynthesis