Sara Lozano

Differential Upregulation of Cd38 on Different T-cell Subsets May Influence the Ability to Reconstitute Cd4+ T Cells Under Successful Highly Active Antiretroviral Therapy

Background:Immune activation is an independent surrogate marker of CD4 T-cell depletion in HIV-infected patients. Highly active antiretroviral therapy (HAART) reduces disease progression as a direct consequence of suppressing HIV replication. Immune function does not normalize completely in most subjects on HAART, however, perhaps reflecting residual HIV replication. So far, it is unclear to what extent immune activation may influence the evolution of CD4 T-cell counts in patients on HAART. Patients and Methods:The expression of CD38 on naive and memory subsets of CD4+ and CD8+ T cells was measured quantitatively by flow cytometry in 62 drug-naive HIV-positive and 30 HIV-uninfected controls. In addition, the evolution of this marker as well as that of some virologic parameters (plasma viremia and proviral load) and CD4 counts were assessed in 25 HIV-infected individuals who initiated HAART and were followed for 12 months. Results:The mean level of CD38 on memory CD4+ and CD8+ T cells as well as in naive CD8+ cells was significantly higher in drug-naive HIV-positive subjects than in HIV-negative controls. Moreover, it was highly correlated with viral load titers. In patients on successful HAART, immune activation declined in all T-cell subsets, particularly among memory CD8+ cells. It remained elevated with respect to HIV-negative controls, however, even after 12 months of HAART. There was a significant correlation between the CD8+ T-cell activation decay and the increase of CD4+ T cells on HAART. Patients with the highest decline in CD8 activation were those showing the highest CD4 T-cell gains after 12 months of therapy. Conclusions:The level of CD38 expression on different T-cell subsets is differentially upregulated in drug-naive HIV-infected patients. After successful HAART, immune activation decreases in all T-cell subsets, although it still remains elevated in most cases after 12 months of HAART. The extent of immune deactivation under successful HAART correlates with the ability to reconstitute CD4 counts.

CD38 Expression on CD8 T Lymphocytes as a Marker of Residual Virus Replication in Chronically HIV-Infected Patients Receiving Antiretroviral Therapy

The level of CD8+ CD38+ T lymphocytes in blood correlates with disease progression in HIV-infected individuals, independently of the CD4 count. Effective antiretroviral therapy reduces this lymphocyte subset in parallel with plasma viremia, although CD38 expression on CD8+ cells does not normalize completely in most subjects, and might reflect residual HIV replication. The expression of CD38 on CD8+ cells (as number of CD38 molecules per CD8+ cell) was measured quantitatively by flow cytometry in 200 individuals, of whom 170 were HIV positive and 30 were HIV-uninfected controls. Forty-six HIV-infected subjects were on antiretroviral therapy and had undetectable viral load. The remaining 124 HIV-positive persons were not on therapy and had detectable plasma viremia. The mean level of CD38 on CD8+ cells was higher in HIV-positive, untreated patients than in subjects on antiviral therapy and controls (5023, 2029, and 1978 molecules per CD8+ cell, respectively, p < 0.01). In HIV-positive, untreated subjects, the higher CD38 expression mainly occurred on CD45RO+ CD8+ cells. The level of CD38 strongly correlated with plasma HIV-RNA (r = 0.63, p < 0.001). The levels of CD38 on CD8+ cells declined steadily in HIV-positive subjects after beginning antiretroviral therapy. A few individuals presented viral blips whereas being on antiviral treatment, levels of CD38 on CD8+ cells increased transiently in parallel with episodes of viral replication. Levels of CD38 on CD8+ cells are increased in chronic HIV infection, and strongly correlate with plasma viremia. The slow decline of CD38 expression on CD8+ cells over time in subjects with undetectable plasma viremia while being on antiretroviral therapy suggests that CD38 expression on CD8+ cells could be used as a marker of residual virus replication.

Down-Regulation of Interleukin-7 Receptor (CD127) in HIV Infection Is Associated with T Cell Activation and Is a Main Factor Influencing Restoration of CD4+ Cells after Antiretroviral Therapy

BACKGROUND Factors influencing the depletion of CD4(+) cells and the restoration of CD4(+) cells after antiretroviral therapy are not completely understood. Recently, attention has been paid to interleukin (IL)-7 and its receptor (CD127). We analyzed the influence of T cell activation and of suppression of viremia with antiretroviral therapy on this system, as well as its role in CD4(+) cell restoration after long-term antiretroviral therapy. METHODS IL-7 levels and CD127 expression on several subsets of CD4(+) and CD8(+) T lymphocytes and the activation status (CD38) of these cells were examined at baseline and during 24 months of complete viral suppression under highly active antiretroviral therapy (HAART). RESULTS A total of 42 individuals with human immunodeficiency virus (HIV) infection and 10 age-matched, uninfected control subjects were examined. Before HAART, IL-7 levels were increased and CD127 expression was decreased. Down-regulation of CD127 was mainly associated with T cell activation and reverted only partially after suppression of detectable plasma HIV RNA with HAART. In a multivariate analysis, CD127 expression on CD8(+) T cells was the main determinant of the extent of CD4(+) cell gains after successful HAART. CONCLUSIONS The IL-7-CD127 system is impaired in HIV-infected patients. CD127 down-regulation is associated with T cell activation and with CD4(+) cell restoration after HAART.

Differences in Cellular Activation and Apoptosis in HIV-Infected Patients Receiving Protease Inhibitors or Nonnucleoside Reverse Transcriptase Inhibitors

The mechanism of CD4(+) T cell depletion seen in HIV infection is largely mediated by increased apoptosis of these cells. The benefit of protease inhibitor (PI)-based antiretroviral therapy to CD4(+) T cell recovery seems to involve more than its antiviral activity, and a direct antiapoptotic effect of PIs has been proposed to explain it. To test this hypothesis we have analyzed directly, ex vivo, the effects of two different highly active antiretroviral therapy (HAART) regimens on the levels of activation and apoptosis of T lymphocytes. A total of 126 subjects (43 receiving PIs, 35 receiving NNRTIs, 27 untreated HIV carriers, and 21 uninfected control subjects) was included in the study. Apoptosis was measured in blood lymphocytes by flow cytometry, using annexin V labeling. A broad panel of monoclonal antibodies was used to characterize the different CD4(+) and CD8(+) lymphocyte subsets. Apoptosis was significantly increased in HIV-untreated subjects, whereas apoptosis levels did not differ when comparing HIV-positive subjects undergoing HAART and uninfected control subjects. Likewise, markers of activation were elevated in HIV-positive untreated patients, and declined in subjects receiving treatment. However, activated-memory CD8(+) T cells remained significantly higher in treated patients with respect to uninfected control subjects. No differences in the level of apoptosis or in immune activation markers were recognized when comparing subjects receiving PIs and those receiving NNRTIs. Antiretroviral therapy reduces apoptosis of CD4(+) and CD8(+) lymphocytes to normal levels without differences when comparing subjects receiving PI and NNRTI triple combinations. Despite complete suppression of viral replication, activated memory CD8(+) T cells remain significantly elevated in subjects receiving HAART, suggesting the persistence of residual HIV replication. If PIs provide a positive effect on CD4(+) counts beyond an antiviral effect, mechanisms other than apoptosis should be involved.

Low-Level Exposure to HIV Induces Virus-Specific T Cell Responses and Immune Activation in Exposed HIV-Seronegative Individuals

HIV-specific T cells response and T cell activation are frequently seen in exposed seronegative individuals (ESN). In this study, we report HIV-specific response and level of T cell activation in ESN partners of HIV-infected patients presenting low or undetectable levels of HIV-RNA. We evaluated 24 HIV-serodiscordant couples. ESN were classified into three categories of exposure to HIV (very low, low, and moderate-high), considering levels of HIV-RNA in their infected partner and frequency of sexual high-risk practices within the last 12 mo. HIV-specific T cell responses and activation levels in T cell subsets were evaluated by flow cytometry. We reported that 54% of ESN had detectable HIV-specific T cells response, being the highest prevalence seen in the low exposure group (64%). Several T cell subsets were significantly increased in ESN when compared with controls: CD4+CD38+ (p = 0.006), CD4+HLA-DR−CD38+ (p = 0.02), CD4+CD45RA+CD27+HLA-DR−CD38+ (p = 0.002), CD8+CD45RA+CD27+CD38−HLA-DR+ (p = 0.02), and CD8+CD45RA+CD27−CD38+HLA-DR+ (p = 0.03). Activation of CD8+ T cells was increased in ESN with detectable HIV T cell responses compared with ESN lacking these responses (p = 0.04). Taken together, these results suggest that persistent but low sexual HIV exposure is able to induce virus-specific T cells response and immune activation in a high proportion of ESN, suggesting that virus exposure may occur even in conditions of maximal viral suppression in the HIV-infected partner.

CD4+ T Cell Recovery beyond the First Year of Complete Suppression of Viral Replication during Highly Active Antiretroviral Therapy Is Not Influenced by CD8+ T Cell Activation

CD38 expression on CD8(+) T cells was longitudinally assessed in 31 human immunodeficiency virus (HIV)-infected persons with undetectable plasma viremia who had undergone highly active antiretroviral therapy (HAART) for 12 months and were followed for a mean of 30 months thereafter. Overall, CD4(+)T cell counts increased during follow-up, whereas CD38 expression remained stable. However, a subset of patients showed declines in CD38 expression, and, conversely, another subset showed increases in CD38 expression. No association could be found between long-term gains in CD4(+) T cells and evolution of CD38 expression. Thus, activation of CD8(+) T cells does not seem to be associated with the extent of CD4(+) T cell recovery beyond the first year of successful HAART.

Phenotype and functional characteristics of HIV-specific cytotoxic CD8+ T cells in chronically infected patients: dual effects of highly active antiretroviral therapy.

Background: Cytotoxic CD8+ T cells (CTLs) are critical for the control of viral infections. Although these cells can be recognized in most HIV‐infected individuals, they fail to successfully control HIV replication. Distinct functional defects seem to limit their efficacy in HIV infection, although they have been not fully elucidated. Patients and methods: Blood lymphocytes collected from 61 HLA‐A0201+, untreated, chronically HIV‐infected individuals were examined for the presence of CTLs against epitopes from HIV Gag and Pol proteins, using tetrameric complexes. Several functional aspects of these cells were further analyzed (immunophenotype; ability to produce interferon‐&ggr; (IFN‐&ggr;) in response to the specific peptide; proliferative capacity; and cytolytic activity). Lymphoproliferative responses of these cells confronting different stimulus were also evaluated. A longitudinal analysis was carried out in a subgroup who underwent antiretroviral therapy and were followed for 6 months. Results: CD8+ T cells staining with the tetramer complexes (Tet+) were detected in 44% of patients, with TetGag+ cells being more frequently detected and at higher levels than TetPol+ cells. Most Tet+ cells expressed a memory phenotype, showed an impaired ability to produce IFN‐&ggr; when stimulated with the cognate peptide, and showed a very low expansion when cultured in the presence of the peptide. There was a negative correlation between the proportion of Tet+ cells producing IFN‐&ggr; and plasma HIV‐RNA. Although Tet+ cells diminished in most individuals after beginning antiretroviral therapy, some patients showed de novo appearance of Tet+ cells. Conclusions: Most Tet+ cells in chronic HIV‐infected individuals express a memory phenotype and show an impaired production of IFN‐&ggr; and a lower proliferative response to specific HIV antigens. Interestingly, some individuals under successful antiretroviral therapy may show de novo appearance of specific CTLs. The implications of these findings are relevant for a better understanding of virus‐host interactions.

No Major Differences in the Functional Profile of HIV Gag and Nef-Specific CD8+ Reponses between Long-Term Nonprogressors and Typical Progressors

The mechanism explaining the failure of HIV-specific CD8(+) T cell responses to successfully control HIV replication remains elusive. A total of 83 drug-naive HIV-infected individuals, 27 of whom were long-term nonprogressors (LTNP), was examined. The ability of CD8(+) T lymphocytes to produce three different cytokines (MIP-1beta, TNF-alpha, IL-2) in response to HIV Gag and Nef peptides and to polyclonal stimuli and the ability of HIV-specific CD8(+) T cells to expand in vitro were evaluated by multiparameter flow cytometry. In response to polyclonal stimulation, LTNP presented significantly higher levels of several CD8(+) T cell subsets than progressors. While most patients presented detectable Gag and Nef-specific CD8(+) responses, no significant differences in any of the CD8(+) functional T cell subsets were recognized when comparing LTNP and progressors. HIV responses were dominated by cells producing only MIP-1beta or TNF-alpha, being similar in LTNP and progressors. However, expansion of HIV-specific CD8(+) T cells was more frequent in LTNP than progressors, especially for cells producing MIP-1beta. LTNP show higher levels of CD8(+) responses against polyclonal stimuli than progressors. However, HIV-specific CD8(+) responses do not differ between them except for a more preserved ability of cells from LTNP to expand in vitro.

Enhanced HIV-specific immune responses in chronically HIV-infected patients receiving didanosine plus hydroxyurea.

Background: The role of hydroxyurea (HU) in the treatment of HIV infection remains controversial. HU potentiates didanosine (ddI) antiviral activity and might exert immunomodulatory effects. Patients and methods: Immunologic parameters were examined in HIV-infected patients enrolled in a simplification trial in which ddI-HU was provided to subjects who had been on complete virus suppression under highly active antiretroviral therapy (HAART) for longer than 6 months. A total of 84 of these patients showed plasma viraemia repeatedly below 5000 HIV-RNA copies/ml, and were the main study population. A group of 22 patients who continued on HAART and another of 22 drug-naive HIV-positive individuals were taken as controls. Results: At 12 months, the levels of naive and memory T-cell subsets were similar in patients on ddI-HU and under HAART, whereas immune activation tended to be lower in the former group. The frequency of HIV-specific CD8+ T cells (CTL) directed against 125 peptides derived from Gag, Pol, Env, Nef and HIV regulatory proteins was similar among patients on ddI-HU and untreated controls, and significantly higher than in patients under HAART. This higher CTL response in patients on ddI-HU was seen even when considering only subjects with undetectable viral load. HIV-specific CD4+ T-cell responses were absent in almost all patients on HAART, whereas they were present in up to 19% of patients on ddI-HU. Conclusion: Treatment with ddI-HU provides higher levels of HIV-specific CD8+ and CD4+ T-cell responses than standard triple drug regimens. Thus, hydroxyurea might exert a beneficial immunomodulatory effect in HIV infection.

Hydroxyurea exerts an anti-proliferative effect on T cells but has no direct impact on cellular activation

Hydroxyurea (HU) is a cytostatic drug which has been used as an anti‐HIV agent due mainly to its synergistic activity when combined with certain anti‐retrovirals. In addition, HU might have a beneficial effect on parameters involved in the pathogenesis of HIV infection, such as immune activation. To test this hypothesis, the effect of HU on T cell proliferation and T cell activation, as well as the potential association between these two phenomena, were examined in an in vitro model. HU exerted a dose‐dependent anti‐proliferative effect on T cells, and modulated the expression of different activation markers. In cells exposed to HU, expression of CD25 and CD38 diminished in a dose‐dependent manner, whereas expression of CD69 increased. However, when the expression of these markers was examined separately on proliferating and non‐proliferating lymphocytes, HU did not exert any significant effect. Thus, the effect of HU on T cell activation is not direct and seems to be mediated through its effect on T cell proliferation.