BACKGROUNDAmong HIV-infected patients initiating antiretroviral therapy (ART), early changes in CD4+ T-cell subsets are well described. However, HIV-infected late presenters initiating treatment present with a suboptimal CD4+ T-cell reconstitution and remain at a higher risk for AIDS and non-AIDS events. Therefore, factors associated with CD4+ T-cell reconstitution need to be determined in this population, which will allow designing effective immunotherapeutic strategies.

METHODSThirty-one adult patients with baseline CD4+ T-cell count <350 cells/mm3 exhibiting viral suppression after ART initiation were followed in the HIV/AIDS research center of Peking Union Medical College Hospital in Beijing, China, from October 2002 to September 2013. Changes in T-cell subsets and associated determinants were measured.

RESULTSMedian baseline CD4+ T-cell count was 70 cells/mm3. We found a biphasic reconstitution of T-cell subsets and immune activation: a rapid change during the first 6 months followed by a more gradual change over the subsequent 8 years. Baseline CD4+ T-cell count >200 cells/mm3 in comparison to CD4+ T-cell count ≤200 cells/mm3 was associated with more complete immune Reconstitution (77.8% vs. 27.3% respectively; P = 0.017) and normalized CD4/CD8 ratio. We showed that the baseline percentage of naive CD4+ T-cell was a predictive marker for complete immune reconstitution (area under receiver operating characteristic curve 0.907), and 12.4% as cutoff value had a sensitivity of 84.6% and a specificity of 88.2%.

CONCLUSIONSBaseline naive CD4+ T-cell percentage may serve as a predictive marker for optimal immune reconstitution during long-term therapy. Such study findings suggest that increasing thymic output should represent an avenue to improve patients who are diagnosed late in the course of infection.

Adult ; Antiretroviral Therapy, Highly Active ; methods ; CD4 Lymphocyte Count ; CD4-CD8 Ratio ; CD4-Positive T-Lymphocytes ; metabolism ; Female ; HIV Infections ; drug therapy ; immunology ; metabolism ; HIV-1 ; drug effects ; immunology ; pathogenicity ; Humans ; Male ; Prospective Studies ; T-Lymphocyte Subsets ; immunology

BACKGROUNDAlthough CD4(+) T cell apoptosis and CD8(+) T cell responses have been extensively studied during HIV infection, how apoptosis signals being initiated in CD4(+) T cells still need to be elucidated. The present study was designed to characterize the function-unknown gene, C6orf120, and elucidates its primary role in tunicamycin-induced CD4(+) T apoptosis.

METHODSThe C6orf120 coding sequence was amplified from peripheral blood mononuclear cells (PBMCs) total RNA of AIDS patients. The DNA fragment was inserted into the pET-32a expression system, transformed into Escherichia coli, and preparation of C6ORF120 recombinant protein. The magnetic cell separation technology was used to prepare primary CD4(+) T cells and CD8(+) T cells. The primary T cells were cultured at 1 × 10(6) cells/ml, treated with 0, 0.1, 1, 10, 100, and 200 ng/ml of C6orf120 recombinant protein for 48 hours, then harvested for cell cycle and apoptosis analysis. Tunicamycin (0.5 µmol/L) was used to induce endoplasmic reticulum stress in Jurkat cells. The biomarker 78 KDa glucose-regulated protein (GRp78) and growth arrest and DNA damage (GADD) were used to evaluate endoplasmic reticulum stress of Jurkat cells.

RESULTSWe prepared C6ORF120 recombinant protein and its polyclonal antibody. Immunohistochemical analysis showed that C6orf120 mainly expressed in hepatocytes and cells in germinal center of lymph node. At concentration of 0.1, 1, 10, 100, and 200 ng/ml, C6orf120 recombinant protein could induce apoptosis of Jurkat cells and primary CD4(+) T cells, and promoting G2 phase of its cell cycle. Western blotting analysis showed that C6ORF120 recombinant protein increased the expression of GRp78 and GADD in Jurkat cells in vitro.

CONCLUSIONOur results suggested that C6ORF120 could induce apoptosis of CD4(+) T cells, at least in part, mediated with endoplasmic reticulum stress.

Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; CD8-Positive T-Lymphocytes ; Cell Cycle ; Cells, Cultured ; Endoplasmic Reticulum Stress ; Female ; HIV Infections ; immunology ; Humans ; Immunohistochemistry ; Male ; Microscopy, Confocal ; Proteins ; genetics ; metabolism ; Tunicamycin ; pharmacology

CD4+ T cells mainly interact with Sphingosine 1-phosphate (S1P) to regulate immune function through Sphingosine 1-phosphate receptor 1 (S1P1). This study was aimed to investigate the effects of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) mobilization on S1P1 expression in CD4+ T cells of donor's peripheral blood. The CD4+T cells of peripheral blood were isolated by magnetic beads from 17 allo-hematopoietic stem cell transplantation (allo-HSCT) donors before and at fourth day of mobilization with rhG-CSF. The S1P1 expression was detected by real time quantitative PCR in the RNA extracted from CD4+ T cells collected before and after rhG-CSF mobilization. The results showed that the expression of S1P1 was found in CD4+ cells before and after rhG-CSF mobilization, but the expression level of SIP1 in CD4+ cells after rhG-CSF mobilization was significantly lower than that before rhG-CSF mobilization (p<0.01). It is concluded that the mobilization with rhG-CSF obviously down-regulates the expression of S1P1 in CD4+ T cells of donor's peripheral blood.
CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Hematopoietic Stem Cell Transplantation ; Humans ; Receptors, Lysosphingolipid ; metabolism ; Recombinant Proteins

BACKGROUNDArthritogenic T lymphocytes with common T cell receptor (TCR) Vbeta clonotypes, infiltrating in the articulars of rheumatoid arthritis (RA) patients, play a central role in the pathogenesis of RA. TCR Vbeta5.2 and TCR Vbeta8.2 are the main pathogenic T cell clonotypes in the course of collagen-induced arthritis (CIA) progression in Lewis rats. To investigate a TCR-based immunotherapy for RA, we constructed recombinant DNA vaccines encoding TCR Vbeta5.2 and TCR Vbeta8.2, and evaluated the inhibitive effects of the two vaccines on CIA rats.

METHODSGenes encoding TCR Vbeta5.2 and TCR Vbeta8.2 were amplified by RT-PCR from spleen lymphocytes of Lewis rats and cloned into the eukaryotic expression vector pTargeT. The expression of vaccines was confirmed by RT-PCR and immunohistochemistry. The inhibitive effects of the vaccines on articulars of CIA rats were assessed with arthritis index evaluation and histology. Interferon gamma (IFN-gamma) and interleukin (IL)-4 production by spleen lymphocytes were tested with enzyme-linked immunospot assay (ELISPOT) technique, the changes in peripheral CD4(+) and CD8(+) lymphocyte populations were tested by flow cytometry, and the level of anti-CII antibody in serum was assayed by enzyme-linked immunosorbent assay (ELISA).

RESULTSRecombinant DNA vaccines pTargeT-TCR Vbeta5.2 and pTargeT-pTCR Vbeta8.2 were successfully constructed. Both vaccines inhibited CIA, which alleviated the arthritis index score (P < 0.05), decreased the level of IFN-gamma (P < 0.05), and reduced the ratio of CD4(+)/CD8(+) lymphocytes (P < 0.05) and the anti-CII antibody in serum (P < 0.05). In addition, the histological change in DNA-vaccinated rats was less serious than CIA rats. Compared to pTCR Vbeta 8.2 and pTCR Vbeta 5.2 groups, the group that was injected with a combination of the two vaccines showed stronger inhibitive effects on CIA than either individual vaccine.

CONCLUSIONThe recombinant plasmids pTargeT-TCR Vbeta5.2 and pTargeT-TCR Vbeta8.2 have obvious inhibatory effects on CIA rats and better effects could be achieved when the vaccines were used in combination.

Animals ; Arthritis, Experimental ; metabolism ; prevention & control ; CD4-Positive T-Lymphocytes ; drug effects ; CD8-Positive T-Lymphocytes ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Immunohistochemistry ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Muscles ; drug effects ; metabolism ; Peptide Fragments ; antagonists & inhibitors ; Rats ; Rats, Inbred Lew ; Receptors, Antigen, T-Cell, alpha-beta ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, DNA ; pharmacology

OBJECTIVETo investigate the mechanism of trichostatin A(TSA), a histone deacetylase (HDAC) inhibitor, in inhibiting the activation of CD(4)(+) T cells in mice.

METHODSThe CD(4)(+) T cells isolated from the spleen of C57BL mice were treated with different concentrations of TSA (2, 20, and 200 nmol/L) for 24 h, and CD(3), CD(28) and interleukin-2 (IL-2) mRNA levels were measured with reverse transcription-polymerase chain reaction. The protein expressions of CD(3), CD(28) and IL-2 were measured by fluorescence-activated cell sorting and ELISA analysis. ZAP70 and PI3K protein expression in CD(4)(+) T cells activated by CD(3) and CD(28) monoclonal antibody were analyzed by Western blotting.

RESULTSTSA dose-dependently inhibited the transcription and protein expression of CD28 in CD(4)(+) T cells and reduced the expression of PI3K protein in activated CD(4)(+) T cells, without showing significant effect on the expression of ZAP70. TSA treatment of the cells also resulted in significantly decreased mRNA and protein expressions of IL-2 (P<0.01).

CONCLUSIONTSA can regulate the immunological activity of CD(4)(+) T cells by inducing mRNA and protein expressions of CD(28), which inhibits the activation of the co-stimulatory signal transduction in CD(4)(+) T cells and decreases the secretion of IL-2.

ADP-ribosyl Cyclase 1 ; antagonists & inhibitors ; Animals ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Cell Line ; Female ; Histone Deacetylase Inhibitors ; pharmacology ; Hydroxamic Acids ; pharmacology ; Interleukin-2 ; metabolism ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Inbred C57BL ; Signal Transduction ; drug effects

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.
4-1BB Ligand/genetics/*metabolism ; Animals ; Antigens, CD137/genetics/*metabolism ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CD8-Positive T-Lymphocytes/cytology/metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Line ; Cells, Cultured ; Cyclophosphamide/pharmacology ; Epithelial Cells/cytology ; Gene Expression Regulation ; Immunosuppressive Agents/pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Messenger/genetics ; *Regeneration ; T-Lymphocytes/*cytology/metabolism ; Thymus Gland/*cytology/drug effects/*physiology

OBJECTIVE: To investigate the effect of total glucosides of peony (TGP) on expression and DNA methylation status of ITGAL gene (CD11a) in CD4(+) T cells from patients with systemic lupus erythematosus (SLE). METHODS: CD4(+) T cells were isolated by positive selection using CD4 beads. CD4(+) T cells were treated by TGP at 0, 62.5, 312.5 and 1562.5 mg/L for 48 h. The MTT method was used to assess cell viability; mRNA expression level was measured by realtime-PCR; protein level of CD11a was measured by flow cytometric analysis; DNA methylation status was assayed by bisulfite sequencing. RESULTS: No significant change in cell viability was found in CD4(+) T cells among the different concentration groups (P>0.05). Compared with control, the mRNA and protein levels of ITGAL were down-regulated significantly in SLE CD4(+) T cells treated with TGP (1562.5 mg/L) (P< 0.01). Furthermore, the extent of DNA methylation of ITGAL promoter was increased in TGP (1562.5 mg/L) treated CD4(+) T cells compared with control group (P<0.01). CONCLUSION TGP can repress CD11a gene expression through enhancing DNA methylation of ITGAL promoter in CD4(+) T cells from patients with SLE. This observation represents a preliminary step in understanding the mechanism of TGP in SLE therapy.
CD11a Antigen ; genetics ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; DNA Methylation ; drug effects ; Down-Regulation ; drug effects ; Glucosides ; pharmacology ; Humans ; Lupus Erythematosus, Systemic ; genetics ; immunology ; Paeonia ; chemistry ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; genetics ; metabolism

BACKGROUNDChemokines and their receptors have been a research focus in transplantation immunology. Chemokines and their receptors play a role in lymphocyte recruitment and differentiation process. This study aimed to observe whether IL-4 and IL-10 may regulate the expression of chemokine receptors CCR3, CCR5 and CXCR3 on CD4(+) T cells in CBA/JxDBA/2 mouse model and to explore the role of CCR3, CCR5, CXCR3 in immune tolerance in pregnancy.

METHODSThe mouse model of spontaneous abortion (CBA/JxDBA/2) and the normal pregnant mouse model (CBA/JxBALB/c) were used. CBA/JxDBA/2 mice were injected with IL-4 (CBA/JxDBA/2-IL-4), IL-4 and IL-10 (CBA/JxDBA/2-IL-4+IL-10), or normal saline (CBA/JxDBA/2-NS) as a control. The expression of CCR3, CCR5 and CXCR3 on CD4(+) T cells from mouse peripheral blood was measured by the double-labelled FCM method, and the embryo resorption rate was also examined.

RESULTSThe embryo resorption rate in the CBA/JxDBA/2 group without any treatment was significantly higher than that in the CBA/JxBALB/c group (17.9% vs 3.7%, P < 0.01). The embryo resorption rate in the CBA/JxDBA/2 group immunized with IL-4 or IL-4 together with IL-10 was significantly decreased, compared with that in the control and NS groups respectively. CCR3 expression on CD4(+) T cells in the CBA/JxDBA/2 group without any treatment was significantly lower than that in the CBA/JxBALB/c group (0.3738 +/- 0.3575 vs 1.2190 +/- 0.2772, P < 0.01); both CCR5 (3.0900 +/- 1.5603 vs 1.2390 +/- 0.6361, P < 0.01) and CXCR3 (2.4715 +/- 0.9074 vs 0.9200 +/- 0.5585, P < 0.01) expressions on CD4(+) T cells of the CBA/JxDBA/2 group without any treatment were significantly higher than those of the CBA/JxBALB/c group. Significant up-regulation of CCR3 and down-regulation of CXCR3 were found in the CBA/JxDBA/2 group treated with IL-4 (CCR3: 2.0360 +/- 0.6944, CXCR3: 1.3510 +/- 0.5263, P < 0.01) or IL-4 and IL-10 (CCR3: 1.8160 +/- 1.0947, CXCR3:1.0940 +/- 0.7168, P < 0.01). Because of the CCR5, IL-4 and IL-10 (1.9400 +/- 0.8504 vs 3.0900 +/- 1.5603, P < 0.05), but IL-4 alone (2.5310 +/- 1.3595 vs 3.0900 +/- 1.5603, P > 0.05) treatment significantly decreased the expression of CCR5 in CBA/JxDBA/2.

CONCLUSIONSThe abnormal expression of CCR3, CCR5 and CXCR3 on CD4(+) T cells may play an important role in the pathogenesis of spontaneous abortion. The pregnancy immune tolerance may be induced through selective induction of CCR3, CCR5 and CXCR3 expressions by IL-4 together with IL-10.

Animals ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Cells, Cultured ; Embryo Loss ; metabolism ; Embryo, Mammalian ; Female ; Interleukin-10 ; pharmacology ; Interleukin-4 ; pharmacology ; Mice ; Mice, Inbred BALB C ; Pregnancy ; Receptors, CCR3 ; metabolism ; Receptors, CCR5 ; metabolism ; Receptors, CXCR3 ; metabolism

Mounting evidence has demonstrated that CD4(+) T cells play an important role in anti-tumor immune responses. Thus, adoptive transfer of these cells may have great potential for anti-cancer therapy. However, due to the difficulty to generate sufficient tumor-specific CD4(+) T cells, the use of CD4(+) T cells in tumor therapy is limited. It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8(+) T cells, but the effect of IL-15 transfection on CD4(+) T cells remains unknown. Here, the effects of retrovirus-mediated IL-15 expression in Ova-specific CD4(+) T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4(+) T cells expressed both soluble and membrane-bound IL-15. Retrovirus-mediated IL-15 expression led to a selective expansion of antigen-specific CD4(+) T cells by inhibiting their apoptosis. In vivo IL-15 transfected CD4(+) T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones. To ensure the safety of the method, the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4(+) T cells following ganciclovir treatment. Together, we show that IL-15 transfection induced a selective expansion of antigen-specific CD4(+) T cells ex vivo and enhanced their tumor-suppression effects in vivo. This has an important significance for improving the efficacy of adoptive T cell therapy.
Animals ; Antineoplastic Agents ; pharmacology ; CD4-Positive T-Lymphocytes ; cytology ; drug effects ; immunology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Humans ; Interleukin-15 ; metabolism ; Mice ; Mice, Transgenic ; Neoplasms ; drug therapy ; Recombinant Fusion Proteins ; genetics ; Retroviridae ; genetics

Inducible costimulatory molecule (ICOS), a CD28 family member expressed on activated T cells, plays an important roles in T cell activation and effector function. This study was purposed to investigate the effect of blocking ICOS-B7h signal pathway by ICOS-Ig fusion protein on allogeneic reactive T cells and its mechanism. CHO cells stably and highly expressing ICOS-Ig were established, while the human ICOS-Ig fusion protein was harvested and purified from supernatant of CHO cells transfected with pSecTag2/Hygro A-ICOS-Ig. The CD4(+) cells from spleen of C57BL/6 mice were used as reactive cells, the bone marrow derived dendritic cells (DCs) from BALB/C mice were used as stimulatory cells, these cells were treated with different concentrations of ICOS-Ig or human Ig (h-Ig) as control. The results showed that the target protein with molecular weigh 54 kD and endotoxin level < 10 EU/ml was gained. The ICOS-Ig (> or = 10 microg/ml) could significantly inhibited the proliferative effect of allogeneic reactive T cells resulting from stimulation of DCs (p < 0.01). ICOS-Ig did not influence the activation of CD4(+) T cells. ICOS-Ig concentration positively related to the apoptosis of CD4(+) T cells. The percentages of CD4(+) Annexin V(+)PI(-) cells in simple stimulated group, ICOS-Ig 10 microg/ml group and ICOS-Ig 20 microg/ml group were 15.1%, 26.4% and 33.6% respectively. ICOS-Ig decreased secretion of TNFalpha and increased secretion of IL-4. It is concluded that the ICOS-Ig fusion protein has bioactivity of inhibiting T cell proliferation and altering the polarization of T helper cells to Th2 cells which promotes the apoptosis of allogeneic reactive T cells but had no effect on the activation of allo-reactive CD4(+) T cells.
Animals ; Antigens, Differentiation, T-Lymphocyte ; pharmacology ; Apoptosis ; drug effects ; CD4-Positive T-Lymphocytes ; drug effects ; immunology ; metabolism ; CHO Cells ; Cell Proliferation ; drug effects ; Cricetinae ; Cricetulus ; Inducible T-Cell Co-Stimulator Protein ; Interleukin-4 ; secretion ; Lymphocyte Activation ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Recombinant Fusion Proteins ; pharmacology ; Signal Transduction ; Th1 Cells ; drug effects ; immunology ; metabolism ; Th2 Cells ; drug effects ; immunology ; metabolism