To examine the self-association of CD4 molecules and preliminary studies on its biological function by several indirect methods. A series of CD4 chimeras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutantsD1/D2 devoid of D3 and D4 and D3/D4 devoid of D1 and D2 by PCR techniques, as well as another three CD4 chimeric genes by fused human Fas cytoplasmic death domain to the downstream of the above chimeras respectively. All these molecules were subcloned into pEGFP-N1, forming the corresponding expression vectors. After introducing into HEK293 cells, gene-modified cell morphological changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by transfecting the truncated CD4 recombinant plasmid into the HEK293 cell line and selected by G418. The fluorescene intensity and rosette formation of different clones was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of transfected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7%. Furthermore, both D1/D2-Fas and D3/D4 Fas chimeras could trigger nearly all transfected HEK293 cells to death. Cell adhesion assays showed that neither the D1/D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class II + Raji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluorescence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that the former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class II molecules.
Antigen-Presenting Cells ; immunology ; metabolism ; CD4 Antigens ; chemistry ; genetics ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Cell Line ; Dimerization ; Fas Ligand Protein ; metabolism ; Histocompatibility Antigens Class II ; genetics ; immunology ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Protein Binding ; genetics ; Protein Multimerization

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.
Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; metabolism ; Antigens, Viral ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; CpG Islands ; genetics ; Cytomegalovirus ; immunology ; DNA ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; Lectins, C-Type ; Lentivirus ; genetics ; metabolism ; Mice ; Phosphoproteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism

OBJECTIVETo explore the differential gene expression of peripheral CD4+ among rheumatoid arthritis (RA) patients of cold or heat syndrome type with or without rheumatoid factor (RF).

METHODSDifferential gene expression of peripheral CD4+ lymphocytes purified from fasting venous blood of RA patients and healthy subjects was studied using gene chip technique.

RESULTSThere were 55 differential genes between RA patients with and without RF, mainly involving those related with immune response and signal transduction. In patients with RF, 71 differential genes, mainly related with functional metabolism and immune response, and in those without RF, 70 related with functional metabolism were found between patients of cold and heat syndrome type respectively, all of them were not repetitive with the above-mentioned 55, and only 2 were found repetitive between the 70 and the 71 differential genes, mainly involving functional metabolism.

CONCLUSIONThe differential genes between RA patients with or without RF are different with those between patients of heat and cold syndrome type, suggesting the TCM syndrome classification has its own basis of gene expression profile.

Adult ; Arthritis, Rheumatoid ; blood ; genetics ; CD4 Antigens ; genetics ; CD4-Positive T-Lymphocytes ; metabolism ; Diagnosis, Differential ; Female ; Gene Expression ; Gene Expression Profiling ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Rheumatoid Factor ; blood

The gradual loss of telomeric DNA can contribute to replicative senescence and thus, having longer telomeric DNA is generally considered to provide a longer lifespan. Maintenance and stabilization of telomeric DNA is assisted by binding of multiple DNA-binding proteins, including those involved in double strand break (DSB) repair. We reasoned that declining DSB repair capacity and increased telomere shortening in aged individuals may be associated with decreased expression of DSB repair proteins capable of telomere binding. Our data presented here show that among the DSB repair proteins tested, only the expression of Ku70 and Mre11 showed statistically significant age-dependent changes in human lymphocytes. Furthermore, we found that expressions of Ku70 and Mre11 are statistically correlated, which indicate that the function of Ku70 and Mre11 may be related. All the other DSB repair proteins tested, Sir2, TRF1 and Ku80, did not show any significant differences upon aging. In line with these data, people who live in the regional community (longevity group), which was found to have statistically longer average life span than the rest area, shows higher level of Ku70 expression than those living in the neighboring control community. Taken together, our data show, for the first time, that Ku70 and Mre11 may represent new biomarkers for aging and further suggest that maintenance of higher expression of Ku70 and Mre11 may be responsible for keeping longer life span observed in the longevity group.
Telomere/genetics ; Middle Aged ; Longevity ; Humans ; DNA-Binding Proteins/*metabolism ; DNA Repair/*genetics ; DNA/genetics ; Cell Aging/*physiology ; CD4-Positive T-Lymphocytes/metabolism ; Antigens, Nuclear/*metabolism ; Aging/*physiology ; Aged, 80 and over ; Aged ; Adult

CD4 is a cell surface glycoprotein that acts as a co-receptor for the T cell antigen receptor by binding to a non-polymorphic portion of MHC molecules. CD4 also functions as a receptor for human immunodeficiency virus type-I (HIV-1) because the viral envelope glycoprotein gp120 binds to CD4 with a high affinity. We have previously demonstrated that introduction of mutations into CD4 abolished the binding of gp120 and prevented HIV-1 from entering cells and spreading. However, whether introduction of such mutations into CD4 causes decreased binding to MHC and loss of function is yet to be determined. We generated transgenic mouse lines by injecting a mutant human CD4 (muthCD4) gene under a murine CD4 enhancer/promoter to ensure tissue and stage specific expression. To exclude the influence of endogenous murine CD4, transgenic mice were crossed with murine CD4-targeted mice to produce muthCD4 transgenic mice lacking endogenous CD4 (muthCD4TG/KO mice). In these mice, T lymphocytes expressing muthCD4 expanded and matured in the thymus and were present in the spleen and lymph nodes. They also activated B cells to mount an antibody response to a T-dependent antigen. The results from this study suggest that a human variant of CD4 modified to be resistant to HIV-1 binding can rescue the signaling for T cell development in the thymus in vivo, having helper T cell functions. Thus, further characterization of muthCD4 molecules should open the way to new HIV treatment modalities.
*Virus Internalization ; T-Lymphocytes, Helper-Inducer/*metabolism/*virology ; Protein Binding ; Mutation/genetics ; Mice, Transgenic ; Mice ; Humans ; HIV-1/*metabolism ; HIV Envelope Protein gp120/metabolism ; Antigens, CD4/*genetics/*metabolism ; Animals

T cell anergy has been successfully induced under different conditions in cloned CD4(+) T cells, but induction of T cell anergy in vivo has been difficult and controversial. Due to the low frequency of naturally occurring T cell population with specificity to a defined antigen, it is very difficult to study anergy of naïve T cells without prior in vivo priming which complicates the interpretation of experimental data. To solve this problem, we adopted the HNT-TCR transgenic mice which have homogeneous antigen specific CD4(+) T cell population. In this study, we generated an influenza virus hemagglutinin (HA) peptide-specific CD4(+) T cell clone from the HNT-TCR transgenic mice and induced anergy using APCs which were treated with the crosslinker, ECDI (1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide). The proliferative response of the cloned or freshly purified naïve CD4(+) transgenic T cells after treatment with ECDI-treated APCs and the HA peptide antigen was monitored as the index of anergy induction. The results showed that anergy was successfully induced in the cloned HNT-TCR transgenic CD4(+) T cells. It was determined that the induced anergy was antigen- and MHC-specific. By contrast, anergy was not observed in freshly purified naïve CD4(+) transgenic T cells under the same conditions. The results suggest that naïve CD4(+) T cells may have different anergy inducing requirements, or that cloned CD4(+) T cells may have certain priming or in vitro cloning artifact which makes them more susceptible to anergy induction. We propose that induction of T cell anergy may depend on the T cell growth, activation and differentiation state or cloning conditions. The results from the present study may have important implications for the study of the mechanism(s) underlying T cell anergy induction in vivo and for applications of immune tolerance based therapy.
Animals ; Antigen-Presenting Cells ; immunology ; metabolism ; Antigens, CD ; genetics ; immunology ; metabolism ; CD4 Antigens ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; Clonal Anergy ; genetics ; immunology ; Clone Cells ; immunology ; Epitopes, T-Lymphocyte ; biosynthesis ; Immune Tolerance ; genetics ; Major Histocompatibility Complex ; immunology ; Mice ; Mice, Transgenic ; Receptors, Antigen, T-Cell ; physiology

Complete HIV-1 env genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) DNA of 60 HIV-1 positive paid blood donors in Henan province, and the amplified full-length genes were sequenced. Twenty one full-length env genes were obtained, sequence analysis found that 15 of them had intact open reading frame (ORF). Fourteen sequences conformed to subtype B', their average genetic distance with the international reference sequence RL42 was 4.87% +/- 0.31%. One was subtype B, its genetic distance with the international reference sequence HXB2 was 5.43%. The amino acid sequences of these env genes were deduced according to their nucleotide sequences and extensive analysis and comparison of important structural motifs were performed. The results indicated that there was no drastic alteration in the number and position of potential N-linked glycosylation sites among these 15 sequences. And the residues involved in forming the CD4 binding site were highly conserved. Genotype prediction of coreceptor usage based on V3 sequence and net charge suggested that most samples use CCR5 coreceptor. GPGR motif at the tetrapeptide crown in the V3 loop was most common in these samples and it was detected in 40% sequences. The cleavage site of gp120/gp41 was highly conserved, so Gp160 precursor of all isolates would be efficiently cleaved into the Gp120 and Gp41 subunits. The known neutralizing antibody binding sites for 2G12, IgG1b12, 4E10 and 2F5 were also highly conserved, it is expected that most of these isolates will be sensitive to neutralization by these antibodies. Further study to elucidate the correlation of the env genotype to functionally relevant motifs is necessary and that will aid vaccine and novel drug design.
Base Sequence ; Blood Donors ; supply & distribution ; CD4 Antigens ; metabolism ; China ; Clinical Laboratory Techniques ; Conserved Sequence ; HIV Envelope Protein gp120 ; genetics ; HIV-1 ; genetics ; Humans ; Receptors, CCR5 ; chemistry ; genetics ; env Gene Products, Human Immunodeficiency Virus ; chemistry ; genetics

OBJECTIVETo study the activating effect of protein transduction domain (PTD) mediated BCR/ABL protein on T cells from CML patients.

METHODSThe plasmid containing PTD and b3a2 bcr/abl of CML was constructed by genetic engineering and expressed in E. coli. The peripheral blood mononuclear cells from CML patients were stimulated in vitro with purified PTD-BCR/ABL protein and the expression of the early activation antigen CD(69) on CD(8)(+) and CD(4)(+) T cells was detected by flow cytometry (FCM).

RESULTSThe optimal concentration of PTD-BCR/ABL protein for activating CD(8)(+) T cells in vitro was 100 micro g/ml, CD(69) expression peaked in three days stimulation. CD(8)(+) T cells were activated in 10 of 15 CML patients, the expression rate of CD(69) was (15.01 +/- 3.75)%. CD(4)(+) T cells were activated in 4 of 15 patients, the expression rate of CD(69) was (10.32 +/- 3.08)%. Both CD(8)(+) and CD(4)(+) T cells were activated simultaneously in 3 of them. However, neither CD(4)(+) nor CD(8)(+) T cells was activated by stimulation with BCR/ABL protein in all 15 specimens, the expression rate of CD(69) on CD(8)(+) and CD(4)(+) T cells was (1.36 +/- 0.31)% and (1.41 +/- 0.43)%, respectively. There was no difference compared with that of PBS control group (P > 0.05).

CONCLUSIONBy using a PTD-mediated antigen delivering system, exogenous BCR/ABL protein can be delivered into APC, processed and presented onto surface of APC to activate Ag-specific CD(8)(+) and CD(4)(+) T cells in vitro.

Adolescent ; Adult ; Aged ; Amino Acid Sequence ; Antigens, CD ; analysis ; Antigens, Differentiation, T-Lymphocyte ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; Dose-Response Relationship, Drug ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Products, tat ; genetics ; metabolism ; Humans ; Lectins, C-Type ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; Male ; Middle Aged ; Recombinant Fusion Proteins ; immunology ; metabolism ; pharmacology ; T-Lymphocytes ; drug effects ; immunology

OBJECTIVETo investigate the significance of detecting TCR gene clonal rearrangement in the diagnosis of mycosis fungoides (MF) and to optimize the primers used for detecting the TCR gene clonal rearrangement with PCR in paraffin embedded tissues of MF.

METHODSNineteen cases of MF were enrolled into the study. A panel of 10 antibodies were used for immunophenotypic analysis and polymerase chain reaction for TCR-γ and TCR-β gene rearrangement detection in this study.

RESULTSTCR gene clonal rearrangements were detected in all 19 cases, in which 84.2% cases (16/19) had TCR-γ gene clonal rearrangements. The positive rates of the primers T(VG)/T(JX), V(2-5)/V(8-12)/JGT(1) and BIOMED-2-TCR-γ were 47.4%, 78.9% and 31.6%, respectively. The positive rate of V(2-5)/V(8-12)/JGT(1) was statistically significantly higher than that of T(VG)/T(JX) and BIOMED-2-TCR-γ (P < 0.05). No TCR gene clonal rearrangement was detected using the primers V(γ11)/V(γ101)/Jγ12 and V(γ11)/V(γ101)/J(p12). TCR-β gene clonal rearrangement was detected in 31.6% (6/19) cases.

CONCLUSIONSTCR gene clonal rearrangement analysis is a useful tool in the diagnosis of MF and TCR-γ gene is a good target gene for the detection. The primers T(VG)/T(JX), V(2-5)/V(8-12)/JGT(1) and BIOMED-2-TCR-γ can be used in clinicopathologic detection for TCR gene clonal rearrangement and V(2-5)/V(8-12)/JGT(1) may be the first choice.

Adolescent ; Adult ; Aged ; Antigens, CD7 ; metabolism ; Base Sequence ; CD2 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD4 Antigens ; metabolism ; Child ; Child, Preschool ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Humans ; Leukocyte Common Antigens ; metabolism ; Male ; Middle Aged ; Molecular Sequence Data ; Mycosis Fungoides ; diagnosis ; genetics ; metabolism ; pathology ; Paraffin Embedding ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Receptors, Antigen, T-Cell, gamma-delta ; genetics ; Skin Neoplasms ; diagnosis ; genetics ; metabolism ; pathology ; Young Adult

OBJECTIVESTo study the relationship between the mutation of Leu60Val in HBV core region and the cellular immunity in patients with chronic hepatitis B (CHB).

METHODSHBV DNA C gene mutation was confirmed by polymerase chain reaction (PCR) and sequencing the products directly. The cytokines (IFN-gamma, TNF-alpha and IL-2) levels in serum were measured by enzyme linked immunosorbent assay (ELISA). The distribution of T-lymphocyte subpopulations in peripheral blood was detected by flow cytometry (FCM).

RESULTSThe mutation of Leu60Val was found in 19 out of the 91 CHB patients. With the CHB severity, the mutation rate was getting higher, especially in the severe hepatitis group. The IFN-gamma and TNF-alpha levels were much higher in mutant strain group than those in wild strain group (t=2.584, 4.766, P<0.01), so was the ratio of CD4+/CD8+ (t=2.275, P<0.05).

CONCLUSIONThe mutant strain of 60Val may increase affinity to HLA-I molecule, or up-regulate the expression of HLA-I molecule, resulting in the activation of CTL to release the cytokines and cause immune response in liver.

Adult ; Aged ; CD4-CD8 Ratio ; Flow Cytometry ; Hepatitis B Core Antigens ; genetics ; Hepatitis B, Chronic ; immunology ; virology ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Interferon-gamma ; biosynthesis ; Middle Aged ; Mutation ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis