Correlation of the percentage of activated,CD3+DR+ lymphocytes to serum neopterin level in HIV-seropositive haemophiliacs

Summary The percentage of activated, CD3+ DR+ and CD8+Leu7+ lymphocytes and the serum neopterin concentration were determined in 17 HIV-seropositive and 10 HIV-seronegative haemophiliacs and in 11 healthy control subjects. All three parameters tested were found to be significantly higher in the seropositive patients than in the seronegative controls. In the seropositive group, a significant positive correlation was found between the neopterin levels and the percentage of CD3+DR+ cells. By contrast, no significant negative or positive correlation was observed between the neopterin levels and the percentage of the CD8+Leu7+ subset. These data suggest that in the HIV-infected patients the activated T cells responsible for the stimulation of macrophages to produce neopterin are those that do not carry CD8.Abbreviations CD cluster designation - AIDS acquired immune deficiency syndrome - HIV human immunodeficiency virus - CDC Centers for Disease Control     

Possession of human leucocyte antigen DQ6 alleles and the rate of CD4 T-cell decline in human immunodeficiency virus-1 infection

Polymorphism amongst the human leucocyte antigen (HLA) class II genes could influence antigen presentation and the ability to control human immunodeficiency virus (HIV)-1 by modulating the virus specific CD4 immune response. To examine the effect of such polymorphisms on disease progression, we studied a cohort of 46 HIV-1 infected long-term non-progressors (LTNPs), 87 intermediate progressors (IPs) and 26 rapid progressors. Kaplan-Meier survival analysis of all patients in the cohort on time to a CD4 count less than 350 cells/ micro l, showed a trend for a slower rate of CD4 decline in patients with, compared to those without, the DRB1*15-DQB1*06 haplotype (hazard ratio (HR) 0.69, 95% CI 0.46-1.01, P = 0.06). A similar effect was not observed with the DRB1*13-DQB1*06 haplotype (HR 1.18, 95% CI 0.75-1.88, P = 0.46), but was observed when DQB1*06 alleles were considered irrespective of their DR association (HR 0.74, 95% CI 0.52-1.05, P = 0.06). Major HLA-DQ6 alleles encode aspartate (Asp) at position 57 on the DQbeta chain, a phenotype associated with protection from other immune disorders. We therefore examined the frequency of all DQbeta57 Asp+ alleles, but could not detect a significant effect on the rate of CD4 decline. To examine whether the genotype associated with slower CD4 decline was over-represented in patients with a slow rate of disease progression, we conducted a categorical analysis of a subset of patients with an extended follow-up of 14+years. We found a higher proportion of LTNPs at 14+ years possessed the DRB1*15-DQB1*06 haplotype compared to IPs at 14+ years (38.46 versus 18.18%), though this difference did not reach statistical significance. When DQB1*06 alleles irrespective of their DR association were considered, the protective effect was greater (76.9% LTNPs versus 18.18% IPs, P = 0.04). Our results highlight the potential protective effect of HLA DQB1*06 alleles on the course of HIV disease.     

Altered distribution of natural killer cell subsets identified by CD56, CD27 and CD70 in primary and chronic human immunodeficiency virus-1 infection

Human natural killer (NK) (CD3- CD56+) cells can be divided into two functionally distinct subsets, CD3- CD56(dim) and CD3- CD56(bright). We analysed the distribution of NK cell subsets in primary and chronic human immunodeficiency virus-1 (HIV-1) infection, to determine if HIV infection stage may influence the subset distribution. In primary infection, contrary to chronic infection, the CD3- CD56(dim) subset was expanded compared to healthy controls. We also studied the effect of antiretroviral therapy administered early in infection and found that NK cell subset distribution was partially restored after 6 months of antiretroviral therapy in primary infection, but not normalized. Recently, NK cells have been divided into CD27- and CD27+ subsets with different migratory and functional capacity and CD27-mediated NK cell activation has been described in mice. We therefore investigated whether CD27 and/or CD70 (CD27 ligand) expression on NK cells, and thus the distribution of these novel NK subsets, was altered in HIV-1-infected patients. We found up-regulated expression of both CD27 and CD70 on NK cells of patients, resulting in higher proportions of CD27(high) and CD70(high) NK cells, and this phenomenon was more pronounced in chronic infection. Experiments conducted in vitro suggest that the high interleukin-7 levels found during HIV-1 infection may participate in up-regulation of CD70 on NK cell subsets. Imbalance of NK cell subsets and up-regulated expression of CD27 and CD70 initiated early in HIV-1 infection may indicate NK cell activation and intrinsic defects initiated by HIV-1 to disarm the innate immune response to the virus.     

Level,phenotype and activation status of CD4+FoxP3+ regulatory T cells in patients chronically infected with human immunodeficiency virus and/or hepatitis C virus

CD4⁺ regulatory T (T_reg) cells have been involved in impaired immunity and persistence of viral infections. Herein, we report the level, phenotype and activation status of T_reg cells in patients chronically infected with human immunodeficiency virus (HIV) and/or hepatitis C virus (HCV). Expression of CD25, CD45RA, CD27, CD127 and CD38 was assessed on these cells using polychromatic flow cytometry in 20 healthy controls, 20 HIV‐monoinfected, 20 HCV‐monoinfected and 31 HIV/HCV‐co‐infected patients. T_reg cells were defined as CD4⁺forkhead box P3 (FoxP3)⁺. The percentage of T_reg cells was increased significantly in HIV patients compared with controls. Moreover, there was a significant inverse correlation between CD4 counts and T_reg cell levels. Fewer than 50% of T_reg cells expressed CD25, with differences in terms of CD127 expression between CD25⁺ and CD25(^–) T_reg cells. CD4⁺Foxp3⁺ T_reg cells displayed predominantly a central memory phenotype (CD45RA^–CD27⁺), without differences between patients and healthy controls. Activated T_reg cells were increased in HIV patients, particularly considering the central memory subset. In summary, HIV infection, but not HCV, induces an up‐regulation of highly activated T_reg cells, which increases in parallel with CD4 depletion. Hypothetically, this might contribute to the accelerated course of HCV‐related liver disease in HIV‐immunosuppressed patients.     

Co-localization of Fyn with CD3 complex,CD45 or CD28 depends on different mechanisms

The Src family protein tyrosine kinase Fyn (p59^fyn) plays an important role in thymocyte development and T cell receptor (TCR) signal transduction. Fyn has been shown to associate with the TCR-CD3 complex, the protein tyrosine phosphatase CD45 and several co-receptors such as CD28 which are crucial for initiating T cell activation and proliferation. The molecular basis of how Fyn is associated with these transmembrane proteins is largely unknown. To investigate the Fyn association with the TCR-CD3 complex, CD45 and CD28 at the molecular level, various Fyn/β-galactosidase fusion proteins were constructed and expressed in Jurkat cells. Co-localization experiments applying antibody-induced co-capping and double immunofluorescence staining techniques were used to study the association of these fusion proteins with the TCR-CD3 complex, CD45 and CD28. Our results revealed that co-localization of Fyn with the TCR-CD3 complex requires the unique N terminus whereas co-localization with CD45 depends on the unique N terminus, the Src homology (SH)3- and a functional SH2 domain. CD28 co-localizes with Fyn molecules that contain the N terminus and a functional SH2 domain. These results suggest that Fyn association with the TCR-CD3 complex, CD45 and CD28 is mediated by different molecular mechanisms.     

CD4+ T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4(+) and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.     

中国汉族人HIV-1协同受体CXCR4基因编码区新的突变位点研究

目的 对中国汉族人群HIV—1协同受体CXCR4编码区的基因多态性进行研究，为中国的获得性免疫缺陷综合征(AIDS)防治提供依据。方法 CXCR4(cDNA编号AFl47204)编码区用2对引物进行PCR扩增，然后分别测序，测序样本数为48例。用DNAstar分析测序结果，寻找SNP位点。结果 在编码区发现了7个SNP位点，其中3个同义突变(129位C→T、426位C→T，968C→T)，3个有义突变(38位C→T、90位A→T、712位A→C)，1个终止突变(106位C→T使谷氨酸密码子变成终止密码子)。其中38位C→T、90位A→T、712位A→C、106位C→T，基因突变频率为4．2％、4．2％、9、4％和3．1％。结论 在CXCR4编码区找到的7个SNP位点中，4个引起氨基酸改变，1个已有报道，对HIV—1感染和AIDS病程的影响值得研究。     

Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection

Human immunodeficiency virus (HIV) has been isolated from plasma in 6 of 7 patients showing clinical symptoms of a primary HIV infection. Parallel cultures from peripheral blood mononuclear cells (PBMC) yielded virus in 5 patients. In one case, virus could only be isolated from the cerebrospinal fluid but not from peripheral blood. Detectable viremia was transient and preceded the appearance of HIV specific antibodies. After cessation of acute symptoms, the frequency of HIV isolations was similar to that of asymptomatic carriers (23 and 26%, respectively). The role of the immune response in terminating detectable viremia remains to be established.     

Inducible-costimulator-mediated suppression of human immunodeficiency virus type 1 replication in CD4(+) T lymphocytes

We investigated the effects of signaling through CD28 family molecules on human immunodeficiency virus type 1 (HIV-1) replication in vitro. A monoclonal antibody (mAb) specific for inducible costimulator (ICOS) suppressed both X4 and R5 HIV-1 replication in CD4(+) peripheral blood mononuclear cells (PBMC). This suppression was not attributable to reduced cell growth or viability. CD28 mAb showed variable effects and also suppressed HIV-1 replication when immobilized. Replication of pseudotype viruses with HIV-1-but not with vesicular stomatitis virus G-envelope was efficiently suppressed in CD4(+) PBMC treated with ICOS or CD28 mAbs. However, CD4, CXCR4, and CCR5 expression on the surface was not down-regulated. Moreover, HIV-1 replication in CD4(+) PBMC was suppressed by a soluble form of human B7-H2, a ligand of ICOS, but was enhanced by soluble B7-1, a ligand for CD28. These findings suggest that natural or artificial ligands for ICOS potentially suppress HIV-1 replication mainly at the entry stages.     

CD5 (OKT1) augments CD3-mediated intracellular signaling events in human T lymphocytes

CD5 is expressed on thymocytes, all mature T cells, and a subset of mature B cells, and probably contributes to T-cell–B-cell adhesion. We assessed whether CD5-crosslinking by mAb augments T-cell stimulation. Plate-bound anti-CD5 or anti-CD3 mAb alone had no effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD5 and CD3 by plate-bound antibodies resulted in marked increases in T-cell surface CD69 expression and T-cell metabolism, as assessed by the T cell's ability to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD5 and CD3 caused a significant (p < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone or anti-CD3 mAb plus anti-CD5 isotype control antibody. These results indicate that CD5 augments signaling through CD3 and consequently functions as a costimulatory molecule for resting T cells.     

Combined Env- and Gag-specific T cell responses in relation to programmed death-1 receptor and CD4+ T cell loss rates in human immunodeficiency virus-1 infection

Additional progression markers for human immunodeficiency virus (HIV) infection are warranted. In this study we related antigen-specific responses in CD4⁺ and CD8⁺ T cells to CD38, reflecting chronic immune activation, and to CD4⁺ T cell loss rates. Clones transiently expressing CD107a (CD8⁺) or CD154 (CD4⁺) in response to Gag, Env and Nef overlapping peptide pools were identified, along with their expression of the inhibitory programmed death-1 receptor (PD-1) in fresh peripheral blood mononuclear cells (PBMC) from 31 patients off antiretroviral treatment (ART). HIV-specific CD8⁺ T cell responses dominated over CD4⁺ T cell responses, and among CD8⁺ responses, Gag and Nef responses were higher than Env-responses (P < 0·01). PD-1 on CD8⁺ HIV-specific subsets was higher than CMV-specific CD8⁺ cells (P < 0·01), whereas PD-1 on HIV-specific CD4⁺ cells was similar to PD-1 on CMV-specific CD4⁺ cells. Gag and Env CD8⁺ responses correlated oppositely to the CD4 loss rate. Env/Gag CD8⁺ response ratios, independently of PD-1 levels, correlated more strongly to CD4 change rates (r = −0·50 to −0·77, P < 0·01) than the total number of Gag-specific CD8⁺ cells (r = 0·44–0·85, P ≤ 0·02). The Env/Gag ratio performed better than CD38 and HIV-RNA in logistic regression analysis predicting CD4 change rate as a measure of progression. In conclusion, HIV-specific CD8⁺CD107a⁺ Env/Gag response ratio was a stronger predictor for progression than CD38 and HIV-RNA. The Env/Gag ratio may reflect the balance between possibly beneficial (Gag) and detrimental (Env) CD8⁺ T cell responses and should be explored further as a progression marker.     

Activation of blood T lymphocytes down-regulates CXCR4 expression and interferes with propagation of X4 HIV strains

The chemokine receptor CXCR4 serves as a coreceptor for HIV-1 entry into CD4⁺ cells, in particular for strains emerging late in the infection. Cell surface expression of CXCR4 has, therefore, important implications for HIV-1 pathogenesis. Using blood lymphocytes cultured under various conditions, we studied the expression and regulation of CXCR4. Flow cytometry showed that only about 20 % of freshly isolated lymphocytes expressed CXCR4 on the cell surface whereas in 80 % of resting blood lymphocytes CXCR4 was located intracellularly. Within a few hours in culture, the intracellular CXCR4 was translocated to the surface and was expressed in the large majority of both naive and memory lymphocytes. A decrease in surface expression of CXCR4 was found when lymphocytes cultured overnight for maximal receptor expression were stimulated with phytohemagglutinin, anti-CD3 antibodies, phorbol 12-myristate 13-acetate and stromal cell-derived factor-1. The superantigen staphylococcal enterotoxin A, a more selective stimulus, induced a marked decrease in CXCR4 expression preferentially in cells positive for the CD25 activation marker. Confocal laser scanning microscopy demonstrated the presence of CXCR4 in the cytosol and on the surface of resting lymphocytes and also showed CXCR4 redistribution after activation. The number of cells infected by the X4 HIV strain NL4.3 paralleled the expression of CXCR4 in CD4⁺ T lymphocytes. Sustained reduction of CXCR4 cell surface expression upon activation with phytohemagglutinin correlated with a low number of CD4⁺ T lymphocytes expressing HIV p24 gag antigen. Our results indicate that activation of CD4⁺ T lymphocytes reduces surface expression of CXCR4 in part by receptor internalization and that cell activation-dependent CXCR4 down-regulation limits spread of infection by X4 viruses.     

Changes in the levels of some acute-phase proteins in human immunodeficiency virus-1 infected patients, following interleukin-2 treatment

Intermittent interleukin (IL)‐2 administration to human immunodeficiency virus (HIV)‐1 infected patients is well documented and generally used, but there is limited information about the changes of acute‐phase protein (APP) levels in response to this treatment. Fifteen patients undergoing highly active anti‐retroviral therapy (HAART) treatment, with undetectable viral load, but low CD4⁺ cell count (<300/µl), have been treated with 3·6 M IU Proleukine® administered twice daily by subcutaneous injection over 5 days. C‐reactive protein (CRP), d ‐dimer, C3, C9, C1‐inh and alpha‐2HS glycoprotein levels were measured immediately before IL‐2 administration, as well as on day 5 and 2–3 weeks thereafter. After IL‐2 administration, both mean d ‐dimer and CRP levels increased significantly (P < 0·001), but returned (P < 0·001) to baseline within the subsequent 2–3 weeks. Alpha‐2HS glycoprotein decreased immediately after IL‐2 administration. No significant differences were detected in the levels of C3, C9 and C1‐inh. A significant, positive correlation (r = 0·5178, P = 0·0008) was ascertained between the changes of CRP level, measured immediately before as well as 5 days after IL‐2 administration, and changes in CD4 T cell counts measured 2–3 weeks before and after treatment, respectively. IL‐2 administration induces rapid elevation of two major APPs (CRP, d ‐dimer). The positive correlation observed between the changes of CRP levels and CD4⁺ cell counts after IL‐2 administration may indicate that the abrupt, but transitory overproduction of CRP might contribute to the CD4⁺ cell count‐increasing effect of the drug and/ or may be associated with serious side effects.     

The effect of human immunodeficiency virus-1 on monocyte-derived dendritic cell maturation and function

Dendritic cells (DC) are mediators of the adaptive immune response responsible for antigen presentation to naive T cells in secondary lymph organs. Human immunodeficiency virus (HIV-1) has been reported to inhibit the maturation of DC, but a clear link between maturation and function has not been elucidated. To understand further the effects of HIV-1 on DC maturation and function, we expanded upon previous investigations and assessed the effects of HIV-1 infection on the expression of surface molecules, carbohydrate endocytosis, antigen presentation and lipopolysaccharide (LPS) responsiveness over the course of maturation. In vitro infection with HIV-1 resulted in an increase in the expression of DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as well as decreases in maturation-induced CCR7 and major histocompatibility complex (MHC)-II expression. Retention of endocytosis that normally occurs with DC maturation as well as inhibition of antigen presentation to CD8(+) T cells was also observed. Mitogen-activated protein kinase (MAPK) responsiveness to LPS as measured by phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK)1/2 was not affected by HIV-1 infection. In summary, in-vitro HIV-1 impairs DC maturation, as defined by cell surface protein expression, with selective alterations in mature DC function. Understanding the mechanisms of DC dysfunction in HIV infection will provide further insight into HIV immune pathogenesis.     

The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value.

The objective of this prospective cohort study was to evaluate the expression of activation markers on CD8 lymphocytes at various clinical stages of HIV infection and to determine the value of these markers in identifying patients likely to have rapidly progressive disease. One hundred and three HIV+ patients, divided into four disease stages, and 34 seronegative controls were evaluated at study entry using flow cytometric immunophenotyping. The HIV patients were followed clinically for disease progression during the following 2 years. CD8 cell numbers and percentage of lymphocytes are increased after HIV infection. Expression of the CD38, HLA-DR and CD57 markers on CD8 cells was significantly increased in asymptomatic HIV-infected patients when compared with controls, as was the CD8 cell population which did not coexpress Leu-8. These activation markers were observed to be further increased in patient groups with more clinically advanced infection. The percentage of CD38 on CD8 cells emerged not only as a discriminator of disease severity, but was a strong predictor of progression in asymptomatic, lymphadenopathy and ARC patients. Given the utility of activation markers on CD8 lymphocytes in staging disease and predicting clinical outcome, the measurement of these parameters should be considered in the monitoring and management of HIV patients.     

In vitro susceptibility of Thai seronegative donor CD8+ T lymphocytes to human immunodeficiency virus-1 (HIV-1) infection

To determine whether CD8+ T lymphocytes from Thai donor cells are susceptible to HIV-1 infection, undepleted peripheral blood mononuclear cells (PBMC) and CD8-enriched PBMC were infected with HIV-1 Thai subtype B and CRF01_AE (E) primary isolates. Virus kinetics in HIV-1 infection of CD4+ and CD8+ T lymphocytes peaked at day 7 or 10 post infection (pi); the TCID50 used for cell infection was proportional to the level of p24 production in the cultures. We also found that the level of p24 antigen in the supernatants of infected undepleted PBMC was significantly higher than that of infected CD8-enriched PBMC. Interestingly, both single positive T lymphocytes (CD4+ and CD8+ T lymphocytes) as well as double positive CD4+/CD8+ T lymphocytes were infected with HIV-1. The double positive T lymphocytes in PBMC were found only in the presence of both CD4+ and CD8+ T lymphocytes. The majority of p24+/CD4-/CD8- T lymphocytes were HIV-1 infected CD4 down-modulated PBMC. This report provides direct evidence that single positive CD8+ T lymphocytes and double positive CD4+/ CD8+ T lymphocytes from Thai donors can be infected with HIV-1 subtypes B and E in vitro.