Joaquin Navarro

DC-SIGN (CD209) Expression Is IL-4 Dependent and Is Negatively Regulated by IFN, TGF-β, and Anti-Inflammatory Agents

Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) is a monocyte-derived dendritic cell (MDDC)-specific lectin which participates in dendritic cell (DC) migration and DC-T lymphocyte interactions at the initiation of immune responses and enhances trans-infection of T cells through its HIV gp120-binding ability. The generation of a DC-SIGN-specific mAb has allowed us to determine that the acquisition of DC-SIGN expression during the monocyte-DC differentiation pathway is primarily induced by IL-4, and that GM-CSF cooperates with IL-4 to generate a high level of DC-SIGN mRNA and cell surface expression on immature MDDC. IL-4 was capable of inducing DC-SIGN expression on monocytes without affecting the expression of other MDDC differentiation markers. By contrast, IFN-α, IFN-γ, and TGF-β were identified as negative regulators of DC-SIGN expression, as they prevented the IL-4-dependent induction of DC-SIGN mRNA on monocytes, and a similar inhibitory effect was exerted by dexamethasone, an inhibitor of the monocyte-MDDC differentiation pathway. The relevance of the inhibitory action of dexamethasone, IFN, and TGF-β on DC-SIGN expression was emphasized by their ability to inhibit the DC-SIGN-dependent HIV-1 binding to differentiating MDDC. These results demonstrate that DC-SIGN, considered as a MDDC differentiation marker, is a molecule specifically expressed on IL-4-treated monocytes, and whose expression is subjected to a tight regulation by numerous cytokines and growth factors. This feature might help in the development of strategies to modulate the DC-SIGN-dependent cell surface attachment of HIV for therapeutic purposes.

Interferon beta-1a therapy enhances CD4 + regulatory T-cell function: An ex vivo and in vitro longitudinal study in relapsing−remitting multiple sclerosis

Interferon beta-1a (IFNâ-1a) has demonstrated efficacy in multiple sclerosis (MS), although its mechanism of action remains only partly understood. We evaluated the ex vivo and in vitro effects of IFNâ-1a (Rebif) on regulatory T-cell (T(Reg)) function in 22 relapsing-remitting MS patients and 16 healthy controls. T(Reg) function was significantly enhanced after 3 and 6 months of IFNbeta-1a therapy. Furthermore, there was a trend towards increasing proportions of total CD4(+)CD25(+) and CD4(+)CD25(+)GITR(+) T(Reg) after 6 months of IFNbeta-1a therapy when compared with baseline. In conclusion, IFNbeta-1a therapy enhances T(Reg) function, and this may be relevant in the inflammatory environment of MS lesions.

Increased levels of activated subsets of CD4 T cells add to the prognostic value of low CD4 T cell counts in a cohort of HIV-infected drug users.

ObjectiveTo identify subsets of CD4 T lymphocytes that can predict the development of AIDS and to assess whether increased levels of these cellular markers could provide additional independent prognostic information to the CD4 T cell count and plasma HIV-1-RNA levels. Design and methodsIn a prospective study, a cohort of 85 HIV-positive intravenous drug users [clinical categories of the CDC classification A (n = 48) and B (n = 37)] were followed for a period of 37 ± 13 months. Memory and activated CD4 and CD8 T cells were quantitated by three-colour flow cytometry at baseline and expressed as a percentage of total CD4 and CD8 lymphocytes. Clinical evaluations were performed at 6 month intervals. The relationships between these lymphocyte subsets and progression to AIDS were studied using Kaplan–Meier plots and proportional hazards regression models. ResultsAfter adjustment for the level of CD4 T cells and plasma HIV-1-RNA levels, the elevation in the subset CD4+CD38+DR+ was the marker within the functionally distinct subsets of CD4 T lymphocytes with additional prognostic value in bivariate Cox regression models. In multivariate models, increased percentages of CD4+CD38+DR+ T cells provided the strongest independent prognostic information for progression to AIDS (relative hazard, 1.07;P < 0.0001). ConclusionOur results suggest that high levels of CD4+CD38+HLA-DR+ T cells reflect the increasing degree of CD4 T cell activation during the progression of HIV infection, and could be used together with the CD4 T cell and HIV-RNA levels to evaluate more accurately the progressive cellular immune impairment associated with the risk of progression to AIDS.

Relationship of Virologic, Immunologic, and Clinical Parameters in Infants with Vertically Acquired Human Immunodeficiency Virus Type 1 Infection

We have investigated the relationship among the HIV-1 biologic phenotype, replicative capacity of virus isolates, HIV-RNA copy number in plasma, p24 antigenemia, CD4+ T lymphocyte counts in peripheral blood, and the clinical status in a cohort of 13 HIV-infected children younger than 12 mo of age, born of HIV-1 seropositive mothers. Six out of 13 HIV-1 isolates from these patients were classified as rapid/high and seven as slow/low. We have found a significantly positive correlation between the replication rate of HIV isolates and their capacity to induce syncytia in vitro. Most of the serial HIV-1 isolates obtained from infants with AIDS had the rapid/high phenotype and induced syncytia, whereas only two out of 23 HIV-1 isolates obtained from infants without AIDS showed these properties. In sequential analysis of HIV-1 isolates from infants with AIDS, the presence of viral isolates with rapid/high and SI phenotype was associated with higher levels of HIV-1 RNA in plasma, CD4+ T cell depletion, and clinical progression. By contrast, infants whose viruses exhibited nonsyncytium-inducing phenotype throughout the follow-up showed lower levels of HIV RNA, stable CD4+ T cell counts, and mild symptomatic HIV infection. Our findings indicate that infants who carried viruses with more cytopathic biologic phenotype and who had higher viral RNA coy numbers in blood were more likely to have lower CD4+ T cell counts and more likely to have AIDS.

Circulating dendritic cells subsets and regulatory T-cells at multiple sclerosis relapse: Differential short-term changes on corticosteroids therapy

Glucocorticoids remain the treatment of choice for MS relapses. However, little is known on the effect of intravenous methylprednisolone (IVMP) on dendritic cells (DCs) and regulatory T-cells (TReg). Our main goal was to quantify circulating myeloid and plasmacytoid DCs (mDCs and pDCs), and TReg at MS relapse versus healthy controls; and to analyse the short-term changes after IVMP for MS relapse. MS patients at relapse compared to controls showed higher %CD4+CD25high+ TReg (p<0.01). After 5-days of IVMP, activated T-lymphocytes (p=0.001), pDCs (p<0.0001), and CD11c+ mDCs (p<0.0001) decreased. By contrast, CD4+CD25+ and CD4+CD25high+ TReg further increased (p<0.0001 both). Changes on these subsets may play a relevant role in the immunosuppressive activity of this drug.

Immunological synapse formation inhibits, via NF-κB and FOXO1, the apoptosis of dendritic cells

The immunological synapse (IS) is a cell–cell junction formed between CD4+ T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-κB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

Naïve and memory CD4+ T cells and T cell activation markers in HIV-1 infected children on HAART.

The objective of this study was to investigate the relationship between peripheral blood CD4+ T cell subsets and routine viro‐immunological markers in vertically HIV‐1‐infected children undergoing highly active antiretroviral therapy (HAART). CD4+ and CD8+ T cell subsets were examined by three‐colour flow cytometry. Plasma viraemia was quantified by a standardized molecular assay. A negative correlation between the %CD4+ T cells and both viral load and the %CD8+ T cells was observed. A strong positive correlation between the %CD4 T cells and naïve, CD38+ and non‐activated CD4+ T cell subsets was found, whereas the %CD4 T cells correlated negatively with the numbers of memory, activated and memory‐activated CD4+ T cell subsets. Elevated percentages of CD8 T cells were associated with increased memory and CD4+ CD62L‐T cell subsets, whereas the naïve and CD4+ HLA‐DRCD38+ subsets negatively correlated with the CD8%. Co‐expression of CD62L on memory CD4+ cells and high expression of HLA‐DR (but not of CD38) were associated with high viral load. No association between viral load and naïve CD4+ T cells was observed. Specific CD4+ T cell subsets may be more informative than routine surrogate markers in defining the evolution of HIV infection and immune reconstitution in children.

Pentoxifylline inhibits acute HIV-1 replication in human T cells by a mechanism not involving inhibition of tumour necrosis factor synthesis or nuclear factor-κB activation

Objective and design:To study the in vitro activity of pentoxifylline (PTX), which may be of benefit in AIDS, on cell proliferation, tumour necrosis factor (TNF)-α, interferon (IFN)-γ (a type 1 cytokine) and interleukin (IL)-10 (a type 2 cytokine) production, viral replication and CD4+ depletion in acutely HIV-1-infected human T cells. Methods:T cells were stimulated with anti-CD3 antibody or phytohaemagglutinin (PHA) and infected with HIV-1 in presence or absence of PTX. Cell proliferation, CD4+ cell number, nuclear factor (NF)-κB activation, p24 antigen release or lymphokine content of the supernatants were evaluated by [3H]-thymidine incorporation, cytofluorimetry, electrophoretic mobility shift assays and specific enzyme-linked immunosorbent assay, respectively. Results:In HIV-1-infected T cells, PTX inhibited cell proliferation and p24 release and prevented CD4+ depletion associated with HIV replication. Moreover, PTX reduced TNF-α, IFN-γ and IL-10 production and NF-κB activation. PTX inhibited with similar potency IFN-γ, TNF-α and cell proliferation. However, the inhibition of p24 release and specially of IL-10 production required significantly lower doses of PTX. Exogenous addition of IL-2 or TNF-α in presence of PTX restore T-cell proliferation and NF-κB activation respectively, but did not affect p24 inhibition. Conclusions:Our data suggest that the inhibitory effect of PTX on HIV replication cannot be satisfactorily explained by the inhibition of NF-κB or TNF-α. Moreover, PTX cannot be primarily considered as a TNF-α inhibitor and has several immunomodulatory and antiviral properties which could be of benefit against HIV-1 at various levels.

Association of CD8+ T lymphocyte subsets with the most commonly used markers to monitor HIV type 1 infection in children treated with highly active antiretroviral therapy

In contrast to adults, there is no information about children concerning the effects of the new antiretroviral therapy on the chronic activation and expansion of CD8+ T cells. We have investigated the relationship between blood CD8(+) T cell subsets, with percent CD4+ cells (%CD4), percent CD8+ cells (%CD8), and plasma viral load (VL), in 39 vertically HIV-1-infected children receiving highly active antiretroviral therapy (HAART) (mean age, 7.6 years; range, 2-15.6 years). CD8+ subsets were examined by three-color multiparametric flow cytometry, and VL was quantified by standard assays. There was a strong positive correlation between activated CD8+ T cells and VL. An increase in memory and memory-activated CD8+ T cells correlated with increased VL, whereas nonactivated memory cells and CD28+ CD8+ T cells correlated negatively with VL. Naive and effector cells did not correlate with VL, although the CD8+ CD45RA -CD62L- subset correlated with increased VL. Activated CD8(+) T cells did not correlate with %CD4, but an increase in memory-activated and effector CD8+ T cells was associated with lower %CD4. Increased naive CD8+ and CD28 +CD8+ T cells showed a positive correlation with %CD4 and a negative correlation with %CD8. In conclusion, in HIV-1-infected children receiving HAART, the activation of CD8+ T cells is associated with high VL, whereas CD8 +CD28+ and nonactivated CD8+ memory cells are associated with lower viral load. Naive CD8+ and CD28 +CD8+ T cells are associated with an improved immunological status.

Quantitative Abnormalities of Peripheral Blood Distinct T, B, and Natural Killer Cell Subsets and Clinical Findings in Obstetric Antiphospholipid Syndrome

Objetive. Few studies have assessed immunophenotypic abnormalities on lymphocyte subsets in patients with antiphospholipid syndrome (APS). We performed an extended immunological study to define alterations of distinct T, B, and natural killer (NK) cell subsets in obstetric patients with APS and their relationship with APS–associated complications. Methods. Patients and controls: 36 women with APS [Sydney criteria, Group A1 without thrombosis (n = 26), Group A2 with thrombosis (n = 10)]; and 36 age matched women with recurrent abortion without antiphospholipid antibodies (disease controls; Group B), 36 healthy parous women (healthy controls; Group C), and 36 healthy nonparous women (healthy controls; Group D). Thrombotic events occurred after history of abortions in all A2 women. Three-color whole-blood flow cytometry was used to characterize the distinct immunophenotypes. Results. A1 patients had significantly higher percentages of CD4+CD45RA–CCR7+ central memory cells (A1 vs D), higher percentages of activated CD4+CD25+ T cells (A1 vs D), and lower percentages and absolute counts of CD4+CD45RA–CCR7– effector memory cells (A1 vs D). GroupA2 patients had higher percentages and absolute numbers of CD19+CD27–IgD+ naive B cells (A2 vs A1 vs all controls), lower percentages and absolute numbers of CD3–CD56+CD16+ NK cells (A2 vs all controls), and higher percentages of activated CD4+DR+ (A2 vs all controls), CD8+DR+ (A2 vs A1 vs C vs D), CD4+CD38+DR+ (A2 vs D), and CD4+CD25+DR+ T cells (A2 vs all controls). Increased percentages of CD8+DR+ T cells [relative risk (RR) 2.43, 95% CI 1.09–5.44, p = 0.02] and of naive B cells (RR 3.05, 95% CI 1.30–7.11, p = 0.009) were associated with development of thrombosis. Conclusion. In obstetric patients with APS we documented significant changes in T, B, and NK cell homeostasis. Increased levels of CD8+DR+ and CD19+CD27–IgD+ cells might identify obstetric patients with APS at risk of having thrombosis.