P-glycoprotein in blood CD4 cells of HIV-1-infected patients treated with protease inhibitors

Objective

Many factors are involved in the virological failure of antiretroviral treatments such as low pharmacological plasma levels of drugs, poor adherence to therapy and emergence of viral resistance. P‐glycoprotein (P‐gp) has been demonstrated to play a role in multidrug resistance in the therapy of solid tumours, haematological malignancies and Plasmodium falciparum infection. HIV‐1 protease inhibitors (PIs) have been described to be substrates of P‐gp. In vitro and in vivo studies performed in mice have demonstrated that P‐gp may affect the oral bioavailability and intracellular accumulation of PIs. P‐gps have been detected on peripheral CD4 blood cells in HIV‐1‐infected, but antiretroviral‐naive patients.

Method

We quantified P‐gp expression and performed functional tests of P‐gp activity in the CD4 cells in HIV‐1‐infected patients, with and without virological failure, treated with PIs, and in healthy patients (control group).

Result

Out of the 18 HIV‐infected patients studied, P‐gp expression and function were found in the CD4 cells of six patients (four of 10 without, and two of eight with virological failure). Out of the 43 healthy patients studied, P‐gp expression and function were found in the CD4 cells of 11 patients (26%). We found P‐gp in peripheral CD4 cells of patients treated with PIs, with and without virological failure, within the same frequency than in antiretroviral naive patients or than in non HIV‐infected patients.

Conclusions

P‐gp expression in peripheral CD4 blood cells does not seem to be enhanced by PI treatment and does not seem to be linked particularly to virological failures. These facts do not preclude of the role of P‐gp on PI absorption or efficacy in other compartments of the body such as gut, lymph nodes or brain in HIV‐1 PI‐treated patients.

     

IL-2 therapy and thymic production of naive CD4 T cells in HIV-infected patients with severe CD4 lymphopenia

OBJECTIVES: IL-2 therapy increases memory and naive CD4 T cells in HIV-infected patients, but its effect on thymopoiesis is unknown. To investigate this effect, we quantified T-cell receptor rearrangement excision circles (TREC) in CD4 T cells from lymphopenic AIDS patients treated with highly active antiretroviral therapy and IL-2. METHODS: CD4 cell subsets were evaluated by flow cytometry using anti-CD45RO/RA, CD62L, Ki67 and CD95 monoclonal antibodies. The proportion of recent thymic emigrant had been quantified by a real-time polymerase chain reaction assay for signal joint TREC in peripheral blood mononuclear and purified CD4 T cells. RESULTS: At initiation of IL-2, TREC copies/microl of blood were correlated with naive T cell numbers and age. Both naive and TREC numbers/microl significantly increased over time in all patients, with a wide range of TREC increases. Higher percentages of CD4+CD45RO-negative cells positive for the Ki67 cell-cycle marker were found in patients with a low TREC increase, but remained stable under IL-2. TREC and naive cell recovery were correlated; they also correlated with the numbers of TREC and naive cells at the start of IL-2, and with age, suggesting a thymic origin for naive T-cell recovery. A mathematical model showing the linear recovery of naive cells and TREC under IL-2 also strongly suggested that a naive T-cell increase reflects thymic export and involves little net death and proliferation. CONCLUSION: Although we cannot rule out a mechanism of altered proliferation or death rate, the thymus plays an important role in the long-term recovery of naive T cells under IL-2 therapy.     

CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

Viral phenotype affects the thymic production of new T cells in HIV-1-infected children

OBJECTIVE: To determine whether viral phenotype has any effect on thymic production of new T cells in HIV-1-infected children. DESIGN: Differences in CD4+ T-cell counts and a marker of thymic output [T-cell antigen receptor (TCR) rearrangement excision circles (TRECs)], between HIV-1-infected children with non-syncytium-inducing (NSI) and syncytium-inducing (SI) viral strains were determined. PATIENTS AND METHODS: A cross-sectional study in 90 samples from vertically HIV-1-infected-children (median age 4.9 years) treated with combination therapy, and a longitudinal study in three children that underwent a change from NSI to SI phenotype were carried out. Viral load, viral phenotype, CD4+ T-cell counts, and quantification of TRECs values were determined. RESULTS: Children with SI virus showed significant lower levels of CD4+ T cells and a lower thymic production of new T cells than children with NSI. These reductions were independent of the treatment and the age of the children. However, there were no differences in viral load with the phenotype between those groups. In children with both NSI and SI viral phenotype, there was a significant correlation between CD4+ T-cell counts and TRECs values. CONCLUSION: The decrease of CD4+ T cells in presence of T-tropic viruses would be mainly due to a lower production of new CD4+ T cells as consequence of the inhibitory effect of these T-tropic strains on thymic function. This effect is not due either to the amount of circulating virus or to the replication kinetics of those strains, but rather depends on the ability of T-tropic viruses to infect T-cell precursors using CXCR4 receptors, which are highly expressed in immature thymocytes.     

Selective expansion of polyfunctional pathogen-specific CD4(+) T cells in HIV-1-infected patients with immune reconstitution inflammatory syndrome

Since the introduction of highly active antiretroviral therapies (ART), the prognosis for HIV-1 patients has improved immensely. However, approximately 25% of patients can experience a variety of inflammatory symptoms that are collectively known as immune reconstitution inflammatory syndrome (IRIS). Studying the etiology and immunopathology of IRIS has been hampered by the fact that the symptoms and associated opportunistic infections are highly varied. We hypothesized that there is a common mechanism underlying IRIS pathogenesis and investigated a patient group with IRIS related to different pathogens. Functional and phenotypic characterization of PBMC samples was performed by polychromatic flow cytometry after in vitro stimulation with relevant antigenic preparations. In most patients, IRIS events were characterized by the robust increase of preexisting polyfunctional, highly differentiated effector CD4(+) T-cell responses that specifically targeted the antigens of the underlying co-infection. T-cell responses to HIV-1 or other underlying infections were not affected and did not differ between IRIS and non-IRIS patients. These data suggest that patients with IRIS do not have a generalized T-cell dysfunction; instead, IRIS represents a dysregulated CD4(+) T-cell response against residual opportunistic infection antigen. These studies were registered at www.clinical-trials.gov as NCT00557570 and NCT00286767.     

Antibodies to soluble CD4 in HIV-1-infected individuals

A direct enzyme immunoassay (EIA) using the recombinant soluble form of CD4 (sCD4) produced in rodent cells as antigen was applied to detect antibodies to CD4 in sera from HIV-1- and HIV-2-infected patients. High titers of antibodies to sCD4 were found in sera from 12.6% of the HIV-1-infected persons included in this study, but not in 120 normal human sera. The reactivity of these antibodies with sCD4 was confirmed by a Western blot analysis. A possible anti-idiotypic origin of those antibodies was thought to be unlikely in view of the lack of inhibition of the binding of the biotin-labeled monoclonal antibody (mAb) anti-Leu3a by sCD4 positive sera. Attempts to correlate the evolution of the disease with the presence or absence of antibodies to sCD4 in a panel of well documented HIV-1-seropositive cases did not reveal any clear correlation. Sera from HIV-2-infected people (nine sera analysed), sera from HIV-1-infected chimpanzees (10 sera analysed) and sera from humans immunized with a recombinant vaccinia virus expressing gp160 (10 sera analysed) scored negative for antibodies to sCD4. The possible origin and biological significance of the observed antibodies to sCD4 are discussed.     

Interleukin 7 reduces the levels of spontaneous apoptosis in CD4+ and CD8+ T cells from HIV-1-infected individuals

Apoptosis has been suggested as one of the major mechanisms of CD4+ T cell depletion during the course of HIV type 1 (HIV-1) infection. Here, we show that interleukin 7 (IL-7), a nonredundant cytokine that plays essential roles in the generation and homeostasis of the T cell compartment of the immune system, exerts strong antiapoptotic effects ex vivo on both CD4+ and CD8+ T cells derived from HIV-1-infected subjects. The level of IL-7-mediated reduction of apoptosis was inversely correlated with the number of circulating CD4+ T cells, indicating a higher sensitivity to IL-7 effects in patients with more advanced disease. The antiapoptotic effect of IL-7 was uncoupled from the induction of cellular proliferation or endogenous HIV-1 replication. These results provide a further rationale for consideration of IL-7 as an agent of immune reconstitution in HIV-1 infection.     

Persistence of distinct HIV-1 populations in blood monocytes and naive and memory CD4 T cells during prolonged suppressive HAART

OBJECTIVE: Reservoirs of HIV-1 are a major obstacle to virus eradication. There is therefore a need to clearly understand the molecular nature of the virus populations that persist in patients with sustained suppression of plasma viraemia on highly active antiretroviral therapy (HAART). DESIGN: We performed a detailed analysis of the genotypes of HIV-1 quasispecies isolated from highly purified blood cell types taken from three selected patients with sustained undetectable viral loads on HAART for 7 years. METHODS: We used polychromatic flow cytometry to sort naive and memory CD4 T cells, CD14 monocytes, and CD56+CD3- natural killer (NK) cells from the total peripheral blood mononuclear cells after 7 years of HAART. Clonal analysis was used to determine coreceptor use and drug-resistance genotypes of HIV-1 quasispecies in the sorted blood cell types. RESULTS: We detected HIV-1 DNA in memory and naive CD4 T cells and in CD14 monocytes, but not in the CD56+CD3- NK cells. Phylogenetic analysis demonstrated that the various blood cells types of two of the three patients harboured genetically distinct HIV-1 quasispecies. Drug-resistance mutations were also distributed differently from one cell type to another. This compartmentalization suggests a minimal virus trafficking between blood cell types during suppressive HAART. CONCLUSIONS: We observed a cell-specific compartmentalization of the residual virus populations during prolonged suppressive HAART. The coexistence of numerous HIV-1 quasispecies with different resistance genotypes and coreceptor use in cellular reservoirs may be relevant for future antiretroviral treatment strategies.     

Depletion of naive CD4 T cells by CXCR4-using HIV-1 variants occurs mainly through increased T-cell death and activation

OBJECTIVE: Using SCID-Hu mice models and in vitro culture systems, it has been shown that syncytium inducing/CXCR4 using (X4) HIV-1 variants affect thymic function through infection and killing of CXCR4 thymocytes. The effect of X4-emergence on naive, memory and effector T-cell subset kinetics in vivo is, however, not known. DESIGN: Prospective cohort study. METHODS: Analysis of changes in naive, memory and effector CD4 and CD8 T-cell numbers and cell division before and after the emergence of X4 variants. RESULTS: Significantly lower numbers of CD4 T cells in patients with X4 variants (n = 18) compared to patients with non-syncytium inducing/CCR5 using variants (n = 74) were due to increased loss of naive and CD27 memory CD4 T cells. In addition, emergence of X4 variants was associated with a small but significant decline in naive CD8 T-cell numbers and increased proportions of dividing CD4 and CD8 naive, memory and effector T cells. CONCLUSION: Loss of naive T cells may suggest thymic dysfunction, however, such an effect would explain only part of the accelerated naive CD4 T-cell decline because of the longevity of naive T cells. Our data suggest that the accelerated naive CD4 T-cell decline induced by X4 variants is caused mainly by increased death and recruitment to the memory compartment of these cells.     

Dendritic cells with TGF-beta1 differentiate naive CD4+CD25- T cells into islet-protective Foxp3+ regulatory T cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that beta-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4(+)CD25(+)Foxp3(+) T cells from na?ve islet-specific CD4(+)CD25(-) T cells in the presence of TGF-beta1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-beta1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4(+)CD25(+)Foxp3(+) T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.     

HIV-1-specific IFN-gamma/IL-2-secreting CD8 T cells support CD4-independent proliferation of HIV-1-specific CD8 T cells

Functional and phenotypic characterization of virus-specific CD8 T cells against cytomegalovirus, Epstein-Barr virus, influenza (flu), and HIV-1 were performed on the basis of the ability of CD8 T cells to secrete IFN-gamma and IL-2, to proliferate, and to express CD45RA and CCR7. Two functional distinct populations of CD8 T cells were identified: (i) dual IFN-gamma/IL-2-secreting cells and (ii) single IFN-gamma-secreting cells. Virus-specific IFN-gamma/IL-2-secreting CD8 T cells were CD45RA-CCR7-, whereas single IFN-gamma CD8 T cells were either CD45RA-CCR7- or CD45RA+CCR7-. The proportion of virus-specific IFN-gamma/IL-2-secreting CD8 T cells correlated with that of proliferating CD8 T cells, and the loss of HIV-1-specific IL-2-secreting CD8 T cells was associated with that of HIV-1-specific CD8 T cell proliferation. Substantial proliferation of virus-specific CD8 T cells (including HIV-1-specific CD8 T cells) was also observed in CD4 T cell-depleted populations or after stimulation with MHC class I tetramer-peptide complexes. IL-2 was the factor responsible for the CD4-independent CD8 T cell proliferation. These results indicate that IFN-gamma/IL-2-secreting CD8 T cells may promote antigen-specific proliferation of CD8 T cells even in the absence of helper CD4 T cells.     

Increased frequency of CD27- (naive) B cells and their phenotypic alteration in HIV type 1-infected patients

To investigate HIV-1-related B cell disorders, the quantity of peripheral CD27 negative (CD27-) B cells, their CD38, CD95, and bcl-2 intensities, and their apoptosis susceptibility were examined by flow cytometry analysis in 16 drug-naive patients, 27 HAART-treated patients, and 20 uninfected controls. CD27- B cells have been recognized as naive B cells. The mean percentage of CD27- B cells was significantly higher in drugnaive patients (88.1%) and in HAART-treated patients (83.9%) than in controls (68.6%) (p < 0.01). The intensities of CD38 and CD95 on CD27- B cells were significantly higher in drug-naive patients than in controls (p < 0.01). The intensity of CD95 on CD27- B cells in HAART-treated patients was lower than that of drug-naive patients, but significantly higher than that of controls (p < 0.01). The intensity of bcl-2 on CD27- B cells in drug-naive patients was lower than that of controls. In drug-naive patients, CD27-B cells with high CD38 expression represented low bcl-2 expression. The CD27- B cells of drug-naive patients showed an increased susceptibility to apoptosis, characterized by diminished cell size and a high frequency of annexin-V binding, compared with controls and HAART-treated patients. These findings suggested that HIV-1 infection affects peripheral CD27- (naive) B cells as well as CD27+ (memory) B cells and that CD27- B cells might be activated and rendered highly susceptible to apoptosis by HIV-1 infection. Some phenotypic alterations in CD27- B cells may continue after the reduction of HIV-1 loads by effective antiviral therapy.     

Increased circulating CD3+/CD31+ T cells in patients with acute coronary syndrome

The number of circulating endothelial progenitor cells (EPCs) is considered to be a surrogate marker for coronary artery disease (CAD). Recent studies have identified a novel T-cell subset labeled with CD3⁺/CD31⁺, which is necessary for EPC colony formation and constitutes the central cluster. However, the clinical relevance of the CD3⁺/CD31⁺ T cells in CAD remains unclear. We sought to clarify whether circulating CD3⁺/CD31⁺ T cells are increased in patients with acute coronary syndrome (ACS). Circulating CD3⁺/CD31⁺ T cells were determined in 16 ACS patients undergoing emergency percutaneous coronary intervention (PCI) and in 16 control subjects with angiographically normal coronary arteries. Although no differences between the groups were found in baseline patient characteristics, the ratio of circulating CD3⁺/CD31⁺ T cells before PCI was higher in ACS patients as compared with that in control subjects (51.8 % ± 7.8 % vs 31.8 % ± 9.6 %, respectively; P < 0.001). The increased ratio of CD3⁺/CD31⁺ T cells in ACS patients was not altered 24 h after PCI, but became comparable with that in control subjects within 6 months after PCI. These results suggest that mobilization of CD3⁺/CD31⁺ T cells occurs in ACS, but is no longer detectable at 6 months after PCI.     

Naive CD4(+) T cells and recent thymic emigrant levels in treated individuals with HIV: clinical relevance

An increase in the levels of naive T cells after the administration of HAART is an indicator of the quality of immune reconstitution. We investigated whether levels of naive CD4 T cells (CD4(+)/CD45RA(+)) and of recent thymic emigrants (RTEs; CD4(+)/CD45RA(+)/CD31(+)) achieved in chronically treated HIV-infected patients could predict the length of time patients could interrupt antiretroviral treatment before their CD4 counts reached values < or =350 cells/mm(3) or HIV-1 RNA levels increased to > or =100,000 copies/ml (Tibet cohort). Serial measurements revealed that the level of naive CD4 T cells among patients was extremely variable (5-95%), but the values for each patient remained stable throughout the study. We then focused on those patients who showed percentages of naive CD4 T cells above 60% or below 30%. The levels of naive T cells, RTEs, mononuclear cells containing T cell receptor excision circles (TRECs), and the strength of CD4 helper responses to HIV p24 antigen during treatment failed to predict the duration of treatment interruptions. In contrast, the median survival time of patients with a CD4 nadir > or =350 cells/mm(3) was 2-fold higher than that of patients with a CD4 nadir <350. However, the probability of restarting therapy in these two groups of patients was independent of the levels of naive T cells, RTEs, or TRECs.