Sonia Fernandez

CD4+ T-Cell Deficiency in HIV Patients Responding to Antiretroviral Therapy Is Associated With Increased Expression of Interferon-Stimulated Genes in CD4+ T Cells

Most patients with human immunodeficiency virus (HIV) who remain CD4(+) T-cell deficient on antiretroviral therapy (ART) exhibit marked immune activation. As CD4(+) T-cell activation may be mediated by microbial translocation or interferon-alpha (IFN-α), we examined these factors in HIV patients with good or poor CD4(+) T-cell recovery on long-term ART. Messenger RNA levels for 3 interferon-stimulated genes were increased in CD4(+) T cells of patients with poor CD4(+) T-cell recovery, whereas levels in patients with good recovery did not differ from those in healthy controls. Poor CD4(+) T-cell recovery was also associated with CD4(+) T-cell expression of markers of activation, senescence, and apoptosis, and with increased serum levels of the lipopolysaccharide receptor and soluble CD14, but these were not significantly correlated with expression of the interferon-stimulated genes. Therefore, CD4(+) T-cell recovery may be adversely affected by the effects of IFN-α, which may be amenable to therapeutic intervention.

Interferon-alpha, immune activation and immune dysfunction in treated HIV infection

Type I interferons (IFNs) exert anti‐viral effects through the induction of numerous IFN‐stimulated genes and an immunomodulatory effect on innate and adaptive immune responses. This is beneficial in controlling virus infections but prolonged IFN‐α activity in persistent virus infections, such as HIV infection, may contribute to immune activation and have a detrimental effect on the function of monocytes and T and B lymphocytes. Activation of monocytes, associated with increased IFN‐α activity, contributes to atherosclerotic vascular disease, brain disease and other ‘age‐related diseases’ in HIV patients treated with long‐term antiretroviral therapy (ART). In HIV patients receiving ART, the anti‐viral effects of IFN‐α therapy have the potential to contribute to eradication of HIV infection while IFN‐α inhibitor therapy is under investigation for the treatment of immune activation. The management of HIV patients receiving ART will be improved by understanding more about the opposing effects of IFN‐α on HIV infection and disease and by developing methods to assess IFN‐α activity in clinical practice.

Vaccine-induced IgG2 anti-HIV p24 is associated with control of HIV in patients with a 'high-affinity' FcγRIIa genotype

Objectives:We have previously shown that vaccination with a recombinant fowlpox virus carrying the genes for HIV Gag-Pol and interferon-gamma (IFN-γ) was associated with partial control of HIV replication after antiretroviral therapy (ART) was ceased but not with increased anti-HIV T-cell responses. Because IFN-γ enhances IgG2 production, and IgG2 antibodies to HIV antigens and the ‘high-affinity’ polymorphism of FcγRIIa (the major Fc receptor for IgG2) have been associated with a favourable outcome of HIV infection, we examined the association of IgG2 antibodies to HIV p24 and ‘high-affinity’ polymorphisms of FcγRIIa with control of HIV replication in these patients. Methods:Plasma from weeks 0 (cessation of ART 1 week after the last vaccination), 9 and 20 was available from patients who had received the full construct vaccine, a partial construct (without IFN-γ) or placebo. IgG2 and IgG1 anti-p24 and anti-gp41 were assayed and all patients were genotyped for the FcγRIIa 131 R/H polymorphism that affects IgG2 binding. Results:At week 0, IgG2 anti-p24 was present in five of nine full construct patients but none of 14 partial construct or placebo patients and was associated with a smaller increase in plasma HIV RNA over 20 weeks. Patients with IgG2 anti-p24 and the ‘high-affinity’ polymorphism of FcγRIIa exhibited lower HIV replication than other patients at week 20. Conclusion:The role of IgG2 anti-HIV antibodies and FcγRIIa in the control of HIV replication should be investigated further. Inclusion of an IFN-γ gene in DNA vaccine constructs might be a means of enhancing IgG2 antibody production.

CD31 (PECAM-1) is a marker of recent thymic emigrants among CD4 + T-cells, but not CD8 + T-cells or γδ T-cells, in HIV patients responding to ART

Some severely immunodeficient HIV patients experience poor recovery of CD4+ T‐cell counts on antiretroviral therapy (ART). Evaluation of the function of thymopoiesis in T‐cell production in individual patients requires a simple marker of T‐cells that have recently emigrated from the thymus. Here, we address whether expression of CD31 on CD4+ T‐cells, CD8+ T‐cells, regulatory T‐cells and γδ T‐cells correlates with other indicators of thymus function. Adult HIV‐1 patients (n=27) with nadir CD4+ T‐cell counts <100 per μl and a sustained virological response to ART and healthy controls (n=23) were studied. CD31 expression was assessed by flow cytometry, T‐cell receptor excision circles content by real‐time PCR and thymic volume by spiral computed tomography. Proportions of CD4+ T‐cells expressing CD45RA and CD31 declined with age in HIV patients (P=0.03) and healthy controls (P<0.0001), and correlated directly with other markers of thymus function. In controls, proportions of CD8+ T‐cells expressing CD45RA and CD31 declined with age (P=0.003) and correlated directly with some markers of thymus function, but this was not seen in HIV patients. Proportions of CD45RA+ CD31+ γδ T‐cells were higher in patients than controls (P=0.007) and did not correlate with thymus volume. In controls, proportion of γδ T‐cells co‐expressing CD45RA and CD31 increased with age (P=0.002). These data support the use of CD31 as a marker of recent thymic origin in CD4+ T‐cells, but not CD8+ T‐cells in HIV patients receiving ART. In such patients, CD31 expression is unlikely to indicate thymic origin in γδ T‐cells.

Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-alpha and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4(+) and CD8(+) T-cell sub-populations (naïve: CD45RA(+)CD57(-); central memory: T(CM) CD45RA(-)CD57(-); effector memory: T(EM) CD45RA(-)CD57(+) and terminally differentiated effector memory: T(EMRA) CD45RA(+)CD57(+)) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4(+) T-cells and RIG-I expression on CD8(+) T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.

Isotype-switched immunoglobulin G antibodies to HIV Gag proteins may provide alternative or additional immune responses to 'protective' human leukocyte antigen-B alleles in HIV controllers

Background:Natural control of HIV infection is associated with CD8+ T-cell responses to Gag-encoded antigens of the HIV core and carriage of ‘protective’ human leukocyte antigen (HLA)-B alleles, but some HIV controllers do not possess these attributes. As slower HIV disease progression is associated with high levels of antibodies to HIV Gag proteins, we have examined antibodies to HIV proteins in controllers with and without ‘protective’ HLA-B alleles. Methods:Plasma from 32 HIV controllers and 21 noncontrollers was examined for immunoglobulin G1 (IgG1) and IgG2 antibodies to HIV proteins in virus lysates by western blot assay and to recombinant (r) p55 and gp140 by ELISA. Natural killer (NK) cell-activating antibodies and Fc&ggr;RIIa-binding immune complexes were also assessed. Results:Plasma levels of IgG1 antibodies to HIV Gag (p18, p24, rp55) and Pol-encoded (p32, p51, p66) proteins were higher in HIV controllers. In contrast, IgG1 antibodies to Env proteins were less discriminatory, with only antigp120 levels being higher in controllers. High-level IgG2 antibodies to any Gag protein were most common in HIV controllers not carrying a ‘protective’ HLA-B allele, particularly HLA-B*57 (P = 0.016). HIV controllers without ‘protective’ HLA-B alleles also had higher plasma levels of IgG1 antip32 (P = 0.04). NK cell-activating antibodies to gp140 Env protein were higher in elite controllers but did not differentiate HIV controllers with or without ‘protective’ HLA-B alleles. IgG1 was increased in Fc&ggr;RIIa-binding immune complexes from noncontrollers. Conclusion:We hypothesize that isotype-switched (IgG2+) antibodies to HIV Gag proteins and possibly IgG1 antip32 may provide alternative or additional immune control mechanisms to HLA-restricted CD8+ T-cell responses in HIV controllers.

Recovery of CD4+ T Cells in HIV Patients With a Stable Virologic Response to Antiretroviral Therapy Is Associated With Polymorphisms of Interleukin-6 and Central Major Histocompatibility Complex Genes

We investigated whether polymorphisms in genes associated with HIV disease progression and/or immune activation affect CD4+ T-cell recovery in HIV patients who began combination antiretroviral therapy (ART) with advanced immunodeficiency and achieved stable control of plasma viremia. Patients with CD4+ T-cell counts <300 cells/μL (n = 33) and >400 cells/μL (n = 37) on ART were compared. A multiple case-control logistic regression associated carriage of BAT1(1,2) or interleukin (IL)6-174(2,2) with low CD4+ T-cell counts (P = 0.012). BAT1*2 uniquely marks the central major histocompatibility complex region of a conserved haplotype (HLA-A1,B8,BAT1*2,TNFA-308*2,DR3,DQ2). There was no association between alleles carried at CCR5Δ32, CCR5 59029, CCR5 59353, CCR2+190 (V64I), SDF1 3′UTR, IL1A+4845, IL1B+3953, IL4-589, IL10-592, IL10-R1+536, IL10-R1+1112, IL12B 3′UTR, TNFA-308, or TNFA-1031 and CD4+ T-cell counts. We suggest that immune activation and/or CD4+ T-cell apoptosis in HIV patients on effective ART is influenced by genetic factors.

Successful treatment of macrophage activation syndrome complicating adult Still disease with anakinra

A previously healthy 20‐year‐old man presented with adult Still disease (ASD). He developed life‐threatening macrophage activation syndrome (MAS), which was refractory to standard immunosuppression but responded dramatically to the IL‐1 receptor antagonist anakinra. Subsequent immunological investigations included assessment of the perforin expression of natural killer (NK) cells and CD8+ T cells, which confirmed MAS.

Immunosenescent CD57 + CD4 + T-cells accumulate and contribute to interferon-γ responses in HIV patients responding stably to ART

HIV-infected individuals responding to antiretroviral therapy (ART) after severe CD4+ T-cell depletion may retain low responses to recall antigens [eg: cytomegalovirus (CMV)] and altered expression of T-cell co-stimulatory molecules consistent with immunosenescence. We investigated the capacity of phenotypically senescent cells to generate cytokines in HIV patients receiving long-term ART (n = 18) and in healthy controls (n = 10). Memory T-cells were assessed by interferon (IFN)-γ ELISpot assay and flow cytometrically via IFN-γ or IL-2. Proportions of CD57brightCD28null CD4+ T-cells correlated with IFN-γ responses to CMV (p = 0.009) and anti-CD3 (p = 0.002) in HIV patients only. Proportions of CD57brightCD28null CD8+ T-cells and CD8+ T-cell IFN-γ responses to CMV peptides correlated in controls but not HIV patients. IL-2 was predominantly produced by CD28+T-cells from all donors, whereas IFN-γ was mostly produced by CD57+ T-cells. The findings provide evidence of an accumulation of immunosenescent T-cells able to make IFN-γ. This may influence the pathogenesis of secondary viral infections in HIV patients receiving ART.

Elevated plasma soluble CD14 and skewed CD16+ monocyte distribution persist despite normalisation of soluble CD163 and CXCL10 by effective HIV therapy: A changing paradigm for routine HIV laboratory monitoring?

Objective We investigated plasma and flow cytometric biomarkers of monocyte status that have been associated with prognostic utility in HIV infection and other chronic inflammatory diseases, comparing 81 HIV+ individuals with a range of treatment outcomes to a group of 21 healthy control blood donors. Our aim is to develop and optimise monocyte assays that combine biological relevance, clinical utility, and ease of adoption into routine HIV laboratory practice. Design Cross-sectional evaluation of concurrent plasma and whole blood samples. Methods A flow cytometry protocol was developed comprising single-tube CD45, CD14, CD16, CD64, CD163, CD143 analysis with appropriately matched isotype controls. Plasma levels of soluble CD14 (sCD14), soluble CD163 (sCD163) and CXCL10 were measured by ELISA. Results HIV status was associated with significantly increased expression of CD64, CD143 and CD163 on CD16+ monocytes, irrespective of the virological response to HIV therapy. Plasma levels of sCD14, sCD163 and CXCL10 were also significantly elevated in association with viremic HIV infection. Plasma sCD163 and CXCL10 levels were restored to healthy control levels by effective antiretroviral therapy while sCD14 levels remained elevated despite virological suppression (p<0.001). Conclusions Flow cytometric and plasma biomarkers of monocyte activation indicate an ongoing systemic inflammatory response to HIV infection, characterised by persistent alterations of CD16+ monocyte expression profiles and elevated sCD14 levels, that are not corrected by antiretroviral therapy and likely to be prognostically significant. In contrast, sCD163 and CXCL10 levels declined on antiretroviral therapy, suggesting multiple activation pathways revealed by these biomarkers. Incorporation of these assays into routine clinical care is feasible and warrants further consideration, particularly in light of emerging therapeutic strategies that specifically target innate immune activation in HIV infection.