Immune activation and inflammation in HIV-1 infection: causes and consequences.

Thorough research on HIV is progressively enabling us to understand the intricate mechanisms that link HIV-1 infection to the onset of immunodeficiency. The infection and depletion of CD4(+) T cells represent the most fundamental events in HIV-1 infection. However, in recent years, the role played by chronic immune activation and inflammation in HIV pathogenesis has become increasingly apparent: quite paradoxically, immune activation levels are directly associated with HIV-1 disease progression. In addition, HIV-1-infected patients present intriguing similarities with individuals of old age: their immune systems are characterized by a loss of regenerative capacity and an accumulation of ageing T cells. In this review, we discuss the potential reasons for the establishment of sustained immune activation and inflammation from the early stages of HIV-1 infection, as well as the long-term consequences of this process on the host immune system and health. A simplified model of HIV pathogenesis is proposed, which links together the three major facets of HIV-1 infection: the massive depletion of CD4(+) T cells, the paradoxical immune activation and the exhaustion of regenerative capacity.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Comparison between HIV- and CMV-specific T cell responses in long-term HIV infected donors.

The mechanisms underlying non-progression in HIV-1 infection are not well understood; however, this state has been associated previously with strong HIV-1-specific CD8+ T cell responses and the preservation of proliferative CD4+ T cell responses to HIV-1 antigens. Using a combination of interferon-gamma (IFN-gamma) ELISpot assays and tetramer staining, the HIV-1-specific CD8+ T cell populations were quantified and characterized in untreated long-term HIV-1-infected non-progressors and individuals with slowly progressive disease, both in relation to CD4+ T cell responses, and in comparison with responses to cytomegalovirus (CMV) antigens. High levels of CD8+ T cell responses specific for HIV-1 or CMV were observed, but neither their frequency nor their phenotype seemed to differ between the two patient groups. Moreover, while CMV-specific CD4+ T cell responses were preserved in these donors, IFN-gamma release by HIV-1-specific CD4+ T cells was generally low. These data raise questions with regard to the role played by CD8+ T cells in the establishment and maintenance of long-term non-progression.

How important is the 'quality' of the cytotoxic T lymphocyte (CTL) response in protection against HIV infection?

Cytotoxic T lymphocyte (CTL) responses have been associated with protection from HIV-1 infection in people with a high degree of exposure to HIV and who show no serological evidence of HIV infection (HEPS, highly exposed persistently seronegative). However, it remains unclear how protective CTL responses could apparently develop in a minority of people, whilst the great majority of HIV-infected people make strong CTL responses yet progress to AIDS and death. In this paper we review the data which supports the hypothesis that the quality of the T-cell response, rather than its magnitude, may be an important factor that merits further investigation.

A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex.

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

RANTES: a versatile and controversial chemokine.

The activity of the chemokine RANTES is not restricted merely to chemotaxis. It is a powerful leukocyte activator, a feature potentially relevant in a range of inflammatory disorders. RANTES has attracted attention because it can potently suppress and, in some circumstances, enhance HIV replication. These characteristics are critically dependent on its ability to self-aggregate and bind to glycosaminoglycans.

Skewed maturation of memory HIV-specific CD8 T lymphocytes.

Understanding the lineage differentiation of memory T cells is a central question in immunology. We investigated this issue by analysing the expression of the chemokine receptor CCR7, which defines distinct subsets of naive and memory T lymphocytes with different homing and effector capacities and antiviral immune responses to HIV and cytomegalovirus. Ex vivo analysis of the expression of CD45RA and CCR7 antigens, together with in vitro analysis of the cell-division capacity of different memory CD8+ T-cell populations, identified four subsets of HIV- and CMV-specific CD8+ T lymphocytes, and indicated the following lineage differentiation pattern: CD45RA+ CCR7+ --> CD45RA- CCR7+ --> CD45RA- CCR7- --> CD45RA+ CCR7-. Here we demonstrate through analysis of cell division (predominantly restricted to the CCR7+ CD8+ T-cell subsets) that the differentiation of antigen-specific CD8+ T cells is a two-step process characterized initially by a phase of proliferation largely restricted to the CCR7+ CD8+ cell subsets, followed by a phase of functional maturation encompassing the CCR7- CD8+ cell subsets. The distribution of these populations in HIV- and CMV-specific CD8+ T cells showed that the HIV-specific cell pool was predominantly (70%) composed of pre-terminally differentiated CD45RA- CCR7- cells, whereas the CMV-specific cell pool consisted mainly (50%) of the terminally differentiated CD45RA+ CCR7- cells. These results demonstrate a skewed maturation of HIV-specific memory CD8+ T cells during HIV infection.

Functional heterogeneity and high frequencies of cytomegalovirus-specific CD8(+) T lymphocytes in healthy seropositive donors.

Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8(+) T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8(+) T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in "fresh" killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

RANTES activates antigen-specific cytotoxic T lymphocytes in a mitogen-like manner through cell surface aggregation.

RANTES (regulated upon activation, normal T cell expressed and secreted) is released by cytotoxic T lymphocytes (CTL), and is a potent chemoattractant factor for monocytes and T cells, also known for its ability to suppress HIV infection. At micromolar concentration, RANTES is able to activate leukocytes, and, paradoxically, to enhance HIV infection in vitro. These latter properties are dependent on its ability to self-aggregate. In order to understand further the mechanism of RANTES-induced activation, the effects of both aggregated and disaggregated RANTES on antigen-specific CD8(+) clones were studied in comparison with the effects of specific antigens and in the presence of specific inhibitors of RANTES-mediated activation. We observed large amounts of RANTES aggregated on the cell surface, which led to cell activation, including up-regulation of cell surface markers, and secretion of IFN-gamma and macrophage inflammatory protein (MIP)-1beta. Specific inhibitors of RANTES-induced activation, such as soluble glycosaminoglycans, MIP-1alpha and MIP-1beta, acted by preventing the binding of RANTES on the cell surface. These studies suggest that RANTES acted more like a mitogen than an antigen-independent activator.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Memory CD8+ T cells in HIV infection.

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent HIV infection in humans. The kinetics and general features of the CTL response are similar to those found during other persisting virus infections in humans. During chronic infection there are commonly between 0.1 and 1.0% of all CD8+ T cells in the blood that are specific for immunodominant virus epitopes, as measured by HLA class I peptide tetramers. These figures are greatly in excess of the numbers found by limiting dilution assays; the discrepancy may arise because in the latter assay, CTLs have to divide many times to be detected and many of the HIV-specific CD8+ T cells circulating in infected persons may be incapable of further division. Many tetramer-positive T cells make interferon-gamma, beta-chemokines and perforin, so are probably functional. It is not known how fast these T cells turn over, but in the absence of antigen they decay in number. Impairment of CTL replacement, because CD4+ T helper cells are depleted by HIV infection, may play a major role in the development of AIDS.

The dynamics of the cellular immune response to HIV infection: implications for vaccination.

Recent advances in measuring T-cell responses to viruses have led to new insights into how these T cells respond. In the acute infection there are massive CD8+ T-cell responses to both Epstein-Barr virus (EBV) and to human immunodeficiency virus (HIV). Many of these T cells are effector cells and only a minority appear to be capable of maintaining immunological memory. In persistent virus infections, high levels of antigen-specific effector cells persist. If virus does not persist, the effectors fade in number but memory is maintained and is primed to react rapidly to a new challenge. A vaccine that stimulates only T-cell responses may protect when these memory cells respond rapidly enough to generate high numbers of effectors before the infecting virus becomes established.

Aggregation of RANTES is responsible for its inflammatory properties. Characterization of nonaggregating, noninflammatory RANTES mutants.

The biology of RANTES (regulated on activation normal T cell expressed) aggregation has been investigated using RANTES and disaggregated variants, enabling comparison of aggregated, tetrameric, and dimeric RANTES forms. Disaggregated variants retain their G(i)-type G protein-coupled receptor-mediated biological activities. A correlation between RANTES aggregation and cellular activation has been demonstrated. Aggregated RANTES, but not disaggregated RANTES, activates human T cells, monocytes, and neutrophils. Dimeric RANTES has lost its cellular activating activity, rendering it noninflammatory. Macrophage inflammatory protein 1alpha, macrophage inflammatory protein-1beta, and erythrocytes are potent natural antagonists of aggregated RANTES-induced cellular activation. There is a clear difference in the signaling properties of aggregated and disaggregated RANTES forms, separating the dual signaling pathways of RANTES and the enhancing and suppressive effects of RANTES on human immunodeficiency virus infection. Disaggregated RANTES will be a valuable tool to explore the biology of RANTES action in human immunodeficiency virus infection and in inflammatory disease.

Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES. Characterization of active disaggregated chemokine variants.

Human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1alpha variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1alpha residues Asp26 and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1beta and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1alpha has enabled comparison of these residues in hMIP-1beta and RANTES. Aggregated and disaggregated forms of hMIP-1alpha, hMIP-1beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1alpha, hMIP-1beta, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.