Korean red ginseng slows depletion of CD4 T cells in human immunodeficiency virus type 1-infected patients

We have previously showed that long-term intake of Korean red ginseng (KRG) delayed disease progression in human immunodeficiency virus type 1 (HIV-1)-infected patients. In the present study, to investigate whether this slow progression was affected by KRG intake alone or in combination with HLA factor, we analyzed clinical data in 68 HIV-1-infected patients who lived for more than 5 years without antiretroviral therapy. The average KRG intake over 111.9 +/- 31.3 months was 4,082 +/- 3,928 g, and annual decrease in CD4 T cells was 35.0 +/- 28.7/microl. Data analysis showed that there are significant inverse correlations between the HLA prognostic score (0.29 +/- 1.19) and annual decrease in CD4 T cells (r = -0.347; P < 0.01) as well as between the amount of KRG intake and annual decrease in CD4 T cells (r = -0.379; P < 0.01). In addition, KRG intake significantly slowed the decrease in CD4 T cells even when influence of HLA class I was statistically eliminated (repeated-measure analysis of variance; P < 0.05). We also observed significant correlation between KRG intake and a decrease in serum-soluble CD8 antigen level (r = 0.62; P < 0.001). In conclusion, these data show that KRG intake independently and significantly affected the slow depletion of CD4 T cells irrespective of HLA class I.     

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.     

HTLV-1-infected thymic epithelial cells convey the virus to CD4+ T lymphocytes

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4⁺T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell–cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4⁺ T cell line and to CD4⁺ T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4⁺ T lymphocytes, which in turn will cause disease in the periphery.     

Herpes simplex virus type 1 can either suppress or enhance human immunodeficiency virus type 1 replication in CD4-positive T lymphocytes

It was proposed recently that CEM CD4-positive T cells infected chronically by herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1) (CEM(HSV/HIV)) may be used as a model for studying HIV/HSV interactions. To ascertain whether HSV-HIV coinfection of T lymphocytes has a role in promoting progression of lentiviral infection, T cells infected chronically by either HSV-1 (CEM(HSV)) or HIV-1 (CEM(HIV)) were challenged with a superinfecting dose of HIV-1 or HSV-1. The results show a positive influence on HIV growth when CEM(HIV) cells were superinfected with HSV-1 to an extent that was dependent on the multiplicity of superinfection used. In contrast, HIV superinfection of CEM(HSV) cells resulted in a delay of HIV-1 production and in a lack of HSV-mediated LTR transactivation. These effects were due to cell growth inhibition and apoptosis, resulting from persistent HSV-1 infection. Treatment of CEM(HSV) with acyclovir inhibited completely the HSV-1 cytopathic effects and allowed efficient HIV-1 replication. These data may be relevant in clarifying the role of HIV/HSV interaction in the pathogenesis of AIDS.     

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.     

Lenalidomide enhancement of human T cell functions in human immunodeficiency virus (HIV)-infected and HIV-negative CD4 T lymphocytopenic patients

Suppressed T cell functions in human immunodeficiency virus (HIV) infection were identified and corrected by lenalidomide in middle-aged HIV-infected patients. Chemotaxis of T cells from HIV-infected men (n = 6, mean 43 years) to sphingosine 1-phosphate (S1P) and CCL21 was significantly lower than that of HIV-negative men (n = 6, mean 41 years), and was enhanced significantly up to control levels by 100 and 1000 nM lenalidomide. Generation of interleukin (IL)-2, but not interferon (IFN)-γ, by T cells of middle-aged HIV-infected men was significantly lower than that for controls and was increased significantly by 10-1000 nM lenalidomide up to a maximum of more than 300%. CD4 and CD8 T cells isolated from healthy middle-aged men and reconstituted in vitro at a low CD4 : CD8 ratio typical of HIV infection had depressed chemotaxis to S1P, but not CCL21, and generation of IL-2, but not IFN-γ. Significant enhancement of chemotaxis to S1P and CCL21 was induced by 100-1000 nM lenalidomide only for normal T cells at a low CD4 : CD8 ratio. T cells from HIV-negative middle-aged CD4 T lymphocytopenic patients (n = 3), with a CD4 : CD8 ratio as low as that of HIV-infected patients, had similarly diminished chemotaxis to S1P and CCL21, and depressed generation of IL-2, but not IFN-γ. Lenalidomide at 30-1000 nM significantly enhanced chemotaxis to S1P and IL-2 generation for T cells from HIV-negative CD4 T lymphocytopenic patients as from HIV-infected patients, with less effect on CCL21-elicited chemotaxis and none for IFN-γ generation. Defects in functions of T cells from middle-aged HIV-infected men are partially attributable to CD4 T lymphocytopenia and are corrected by lenalidomide.     

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.     

Functional dichotomy of CD4+ T helper lymphocytes in asymptomatic human immunodeficiency virus infection.

The majority of asymptomatic, human immune deficiency virus seropositive (HIV+) individuals exhibit a defect in CD4+ T helper cell (Th) function that is selective for responses to recall antigens, but not to HLA alloantigens. The CD4-dependent Th response to HLA alloantigens (Allo) can be mediated by two distinct Th pathways: self-restricted CD4+ Th that recognize allogeneic determinants processed and presented by autologous or self accessory or antigen-presenting cells (sAC); and allo-restricted, CD4+ Th that recognize allogeneic determinants directly on allogeneic accessory or antigen-presenting cells (aAC). In contrast, the Th response to recall antigens requires CD4+ Th and sAC and is therefore limited to the major histocompatibility complex (MHC) self-restricted pathway. Peripheral blood leukocytes from 56 asymptomatic HIV+ patients that exhibited a selective defect in CD4+ Th function were analyzed to determine whether the Th response to Allo was entirely functional, or whether one of the CD4-mediated components of the Allo Th response was also defective. By depletion of AC and/or CD8+ Th subsets (to analyze CD4+ Th function), we demonstrated that HIV+ patients who were selectively deficient in Th function to recall antigens were also unresponsive to Allo presented by autologous AC (HLA self-restricted Th pathway), but retained Allo Th activity presented by allogeneic AC (allo-restricted CD4+ Th pathway). These findings indicate that the CD4+ Th defect seen in the majority of asymptomatic, HIV+ individuals is not limited to recall antigens, but also extends to the component of the response to HLA alloantigens that involves the self-restricted, CD4+ Th pathway. Thus, the Th defect observed in asymptomatic, HIV+ patients does not involve a CD4+ Th defect per se, but is limited to the HLA self-restricted component of Th function.     

CD69 expression reliably predicts the anti-CD3-induced proliferative response of lymphocytes from human immunodeficiency virus type 1-infected patients.

Published studies suggest that mitogenic responses of lymphocytes can be reliably assessed by monitoring the expression of lymphocyte surface CD69 after 24 h of culture with the stimulant. We tested this hypothesis by determining the ability of lymphocyte CD69 expression to predict the outcome (normal or abnormal) of lymphocyte proliferative responses to anti-CD3 in a group of human immunodeficiency virus type 1 (HIV-1)-infected patients (n = 47). Cutoff values for defining normal and abnormal CD69 expression and proliferative ([3H]thymidine incorporation) responses were established with lymphocytes from healthy uninfected controls (n = 20). Lymphocytes from 29 HIV-infected patients exhibited an abnormal proliferative response, and those from 25 of the 29 also exhibited abnormal CD69 expression (sensitivity, 86.2%). Similarly, lymphocytes from 18 HIV-infected patients exhibited a normal proliferative response, and those from 16 of the 18 also exhibited normal CD69 expression (specificity, 88.9%). The predictive value of a normal CD69 result was 80%, and the predictive value of an abnormal CD69 result was 92.6%. These findings demonstrate that HIV-1-associated impairments in lymphocyte activation can be reliably detected by the rapid and nonradioactive CD69 expression assay.     

Evaluation of a flow cytometry method for CD4 T cell enumeration based on volumetric primary CD4 gating using thermoresistant reagents

Laboratory follow-up of HIV patients in resource-limited settings requires appropriate instruments for CD4 T cell enumeration. In this study, we evaluated the application of a simplified, mobile and robust flow cytometry system, the Apogee Auto 40 analyzer (Auto40) using thermoresistant reagents, for CD4 T cell enumeration. We measured the absolute CD4 counts in fresh whole blood samples from 170 Senegalese subjects, including 129 HIV-positive (HIV+) patients and 41 HIV-negative (HIV-) controls. Based on volumetric primary CD4 gating, cells were stained with commercially available reagents (Easy MoAb CD4;Bio-D, Valenzano, Italy) and analyzed on the Auto40. The results were compared with those from the FACSCount system (Becton Dickinson, San Jose, USA). Repeatability analysis was performed on duplicate testing of 49 samples on both FACSCount and Auto40. The intra-run precision was measured by 10 replicates using 3 clinical blood samples with low, intermediate and high CD4 concentrations. The results from the two instruments were in good agreement. The percent similarity between the results of both instruments was 99%±relative standard deviation of 12.7%. The concordance correlation coefficient was 0.99. The absolute bias and limits of agreement (LOA) between the two instruments, calculated by Bland-Altman analysis, were clinically acceptable (bias: +4 cells/μl; LOA: -111 to +120 cells/μl). The clinical agreement between the two instruments at a cutoff of 200 CD4 cells/μl was 94%. The repeatability of measurements on the Auto40 was also similar to that observed with FACSCount system (bias +0.1 cells/μl, coefficient of variation 2.5% vs bias -1.1cells/μl, coefficient of variation 2.9% respectively). In conclusion, our results indicate that the Auto 40 system, using thermoresistant reagents, is suitable for CD4 T cell enumeration and will be a helpful tool to improve HIV laboratory monitoring in resource-limited settings.     

Increased CD4+ T-lymphocyte senescence fraction in advanced human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) infection is accompanied by peripheral CD4+ T-cell losses. CD4+ T-cell numbers often increase during antiviral treatment of acquired immune deficiency syndrome (AIDS), however, alterations in the CD4+ T-cell repertoire have not been completely corrected for these patients. Such individuals remain at increased risk of infection. Although senescence of the CD4+ T cells has not been adequately evaluated for advanced HIV-1 infection, hypothetically, replicative senescence could complicate therapeutic reconstitution of the CD4+ T cells in AIDS. In this study, correlates of replicative senescence, terminal restriction fragment (TRF) length and percentage short (< 5.0 kb) telomeric DNA (senescence fraction), were measured for the CD4+ T cells of HIV-1-infected patients with peripheral CD4+ T-cell counts of < 200/mm3. The results show that for advanced HIV-1 infection the TRF length of the CD4+ T cells is decreased (P < 0.01), and the senescence fraction increased (P < 0.05), when compared with uninfected controls. These findings suggest that cellular senescence may contribute to disruption of CD4+ T-cell diversity observed following the therapeutic, immunologic reconstitution of AIDS.     

Levels of soluble CD40 ligand (CD154) in serum are increased in human immunodeficiency virus type 1-infected patients and correlate with CD4(+) T-cell counts

CD40 ligand (CD40L or CD154) is a costimulatory molecule expressed mainly on activated CD4(+) T cells. Concentrations of the soluble form of CD40L (sCD40L) in serum were determined for a cohort of 77 human immunodeficiency virus type 1 (HIV-1)-infected patients before and after initiation of highly active antiretroviral treatment (HAART) by a quantitative enzyme-linked immunosorbent assay. Circulating sCD40L levels were higher by twofold in untreated patients than in healthy controls (means +/- standard deviations [SD]: 1.41 +/- 1.48 versus 0.69 +/- 0.59 ng/ml; P < 0.001). HIV-1-infected patients classified as CD4 T-cell category 1 had significantly higher sCD40L levels than patients classified as CD4 categories 2 and 3 (mean +/- SD: 2.08 +/- 1.46 ng/ml versus 1.57 +/- 1.58 [category 2] and 0.94 +/- 1.25 ng/ml [category 3]; P = 0.046), while no correlation with clinical categories A, B, and C was found. Individual serum sCD40L levels correlated with CD4(+) T-cell counts (P = 0.039) but not with viral load, gamma globulin levels, or acute-inflammatory-response markers. After 8 to 12 months of HAART, a further threefold increase of serum sCD40L levels, which paralleled the increase of CD4(+) T-cell counts, was observed. These novel findings suggest that sCD40L measurement in HIV-1-infected patients could serve as a new surrogate marker useful in the assessment of treatment efficacy, especially in settings where well-equipped laboratories and funding required for CD4(+) T-cell count and viral load measurements are not available.     

Lymphatic tissue fibrosis is associated with reduced numbers of naive CD4+ T cells in human immunodeficiency virus type 1 infection

The organized structure of lymphatic tissues (LTs) constitutes a microenvironment referred to as a niche that plays a critical role in immune system homeostasis by promoting cellular interactions and providing access to cytokines and growth factors on which cells are dependent for survival, proliferation, and differentiation. In chronic human immunodeficiency virus type 1 (HIV-1) infection, immune activation and inflammation result in collagen deposition and disruption of this LT niche. We have previously shown that these fibrotic changes correlate with a reduction in the size of the total population of CD4+ T cells. We now show that this reduction is most substantial within the na?ve CD4+ T-cell population and is in proportion to the extent of LT collagen deposition in HIV-1 infection. Thus, the previously documented depletion of na?ve CD4+ T cells in LTs in HIV-1 infection may be a consequence not only of a decreased supply of thymic emigrants or chronic immune activation but also of the decreased ability of those cells to survive in a scarred LT niche. We speculate that LT collagen deposition might therefore limit repopulation of na?ve CD4+ T cells with highly active antiretroviral therapy, and thus, additional treatments directed to limiting or reversing inflammatory damage to the LT niche could potentially improve immune reconstitution.     

Isolation and characterization of HIV-1-infected resting CD4+ T lymphocytes in breast milk.

An HIV-1 reservoir comprised primarily of latently infected resting CD4+ T lymphocytes that can be stimulated in vivo to produce virus may play a critical role in mother-to-child postnatal transmission of HIV-1 by breastfeeding. Here, we describe an experimental protocol for the detection of resting CD4+ T cell HIV-1 reservoir from breast milk. We adapted a method for the purification of resting CD4+ T lymphocytes in blood to isolate resting CD4+ T cells in breast milk from HIV-1-infected-lactating women (n=18) and from controls (n=3). Purified resting CD4+ T cells from blood and breast milk samples of HIV-1-infected-lactating women were polyclonally stimulated to characterize and enumerate HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) by an enzyme-linked immunospot (ELISpot) assay. Resting CD4+ T cells represented more than 90% of purified viable breast milk cells. CD4+ T cell polyclonal stimulation combined with the ELISpot assay led to the characterization of a breast milk T cell HIV-1 reservoir greater than the blood reservoir (median 400 and 57.14 HIV-1-Ag-SCs/10(6) resting CD4+ T cells, respectively, p<0.001). Our strategy could be adapted to other body fluids and be useful for characterizing new HIV-1 reservoirs.     

CD27 and CD57 expression reveals atypical differentiation of human immunodeficiency virus type 1-specific memory CD8+ T cells

The failure of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells to control chronic HIV-1 infection could be due to the progressive loss of their capacities to undergo normal memory effector differentiation. We characterized and compared the expressions of CD27, CD28, CD57, and CD62L by Epstein-Barr virus (EBV)-, cytomegalovirus (CMV)-, and HIV-1-specific CD8+ T cells by six-color, eight-parameter flow cytometry. In contrast to the maturation of EBV- and CMV-specific memory CD8+ T cells, we found that HIV-1-specific CD8+ T cells did not display coordinated down-regulation of CD27 and up-regulation of CD57 and accumulated in an atypical CD27(high) CD57(low) subset. Moreover, the accumulation of CD27(high) CD57(low) HIV-1-specific CD8+ T cells was positively correlated with HIV-1 plasma viremia. The differentiation of HIV-1-specific CD8+ T cells to an effector subset is therefore impaired during chronic HIV-1 infection. This lack of normal CD8+ T-cell differentiation could contribute to the failure of cellular immune control of HIV-1 infection.