Linda A. Ehler

HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors

It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.

Maintenance of Large Numbers of Virus-Specific CD8+ T Cells in HIV-Infected Progressors and Long-Term Nonprogressors

The virus-specific CD8+ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8+ T cell responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis. The frequencies of CD8+ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular IFN-γ in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8–18.0%) of circulating CD8+ T cells were found to be HIV specific. High frequencies of HIV-specific CD8+ T cells were not limited to long-tern nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8+ T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8+ T cells exist even in patients with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8+ T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive disease.

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21high- and CD21low-expressing B cells, the CD21low fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21high fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21low B cell population but not in the CD21high fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro.

Macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell expressed and secreted), which are the natural ligands of the CC-chemokine receptor CCR5, inhibit replication of MT-2- negative strains of HIV-1 by interfering with the ability of these strains to utilize CCR5 as a coreceptor for entry in CD4(+) cells. The present study investigates the capacity of natural killer (NK) cells isolated from HIV-infected individuals to produce CC-chemokines and to suppress HIV replication in autologous, endogenously infected cells as well as to block entry of MT-2-negative HIV into the CD4(+) T cell line PM-1. NK cells freshly isolated from HIV-infected individuals had a high number of mRNA copies for MIP-1alpha and RANTES. NK cells produced significant amounts of RANTES, MIP-1alpha, and MIP-1beta constitutively, in response to stimulation with IL-2 alone and when they were performing their characteristic lytic activity (K562 killing). After CD16 cross-linking and stimulation with IL-2 or IL-15 NK cells produced CC-chemokines to levels comparable to those produced by anti-CD3-stimulated CD8(+) T cells. Furthermore, CD16 cross-linked NK cells suppressed (49-97%) viral replication in cocultures of autologous CD8/NK-depleted PBMC to a degree similar to that of PHA or anti-CD3-stimulated CD8(+) T cells. In 50% of patients tested, NK-mediated HIV suppression could be abrogated by neutralizing antibodies to MIP-1alpha, MIP-1beta and RANTES; in contrast, CD8(+) T cell-mediated suppression was not significantly overcome upon neutralization of CC-chemokines. Supernatants derived from cultures of CD16 cross-linked NK cells stimulated with IL-2 or IL-15 dramatically inhibited entry of a MT-2-negative strain of HIV, BaL, in the CD4(+)CCR5(+) PM-1 T cell line. These data suggest that activated NK cells may be an important source of CC-chemokines in vivo and may suppress HIV replication by CC-chemokine-mediated mechanisms in addition to classic NK-mediated lytic mechanisms.

Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals

The presence of HIV-1 in latently infected, resting CD4+ T cells has been clearly demonstrated in infected individuals; however, the extent of viral expression and the underlying mechanisms of the persistence of HIV-1 in this viral reservoir have not been fully delineated. Here, we show that resting CD4+ T cells from the majority of viremic patients are capable of producing cell-free HIV-1 spontaneously ex vivo. The levels of HIV-1 released by resting CD4+ T cells were not significantly reduced in the presence of inhibitors of cellular proliferation and viral replication. However, resting CD4+ T cells from the majority of aviremic patients failed to produce virions, despite levels of HIV-1 proviral DNA and cell-associated HIV-1 RNA comparable to viremic patients. The DNA microarray analysis demonstrated that a number of genes involving transcription regulation, RNA processing and modification, and protein trafficking and vesicle transport were significantly upregulated in resting CD4+ T cells of viremic patients compared to those of aviremic patients. These results suggest that active viral replication has a significant impact on the physiologic state of resting CD4+ T cells in infected viremic patients and, in turn, allows release of HIV-1 without exogenous activation stimuli. In addition, given that no quantifiable virions were produced by the latent viral reservoir in the majority of aviremic patients despite the presence of cell-associated HIV-1 RNA, evidence for transcription of HIV-1 RNA in resting CD4+ T cells of aviremic patients should not necessarily be taken as direct evidence for ongoing viral replication during effective therapy.

The Differential Ability of HLA B*5701+ Long-Term Nonprogressors and Progressors To Restrict Human Immunodeficiency Virus Replication Is Not Caused by Loss of Recognition of Autologous Viral gag Sequences

ABSTRACT Although the HLA B*5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57+ progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restricted gag epitopes by CD8+ T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B*57+ patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions of gag. Furthermore, CD8+ T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4+ T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8+ T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restricted gag epitopes and are not the primary mechanism of virus evasion from immune control in B*5701+ HIV-infected patients. Qualitative features of the virus-specific CD8+ T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B*5701+ LTNPs and progressors to restrict virus replication.

The Role of CD4+ T Cell Help and CD40 Ligand in the In Vitro Expansion of HIV-1-Specific Memory Cytotoxic CD8+ T Cell Responses

CD4+ T cells have been shown to play a critical role in the maintenance of an effective anti-viral CD8+ CTL response in murine models. Recent studies have demonstrated that CD4+ T cells provide help to CTLs through ligation of the CD40 receptor on dendritic cells. The role of CD4+ T cell help in the expansion of virus-specific CD8+ memory T cell responses was examined in normal volunteers recently vaccinated to influenza and in HIV-1 infected individuals. In recently vaccinated normal volunteers, CD4+ T cell help was required for optimal in vitro expansion of influenza-specific CTL responses. Also, CD40 ligand trimer (CD40LT) enhanced CTL responses and was able to completely substitute for CD4+ T cell help in PBMCs from normal volunteers. In HIV-1 infection, CD4+ T cell help was required for optimal expansion of HIV-1-specific memory CTL in vitro in 9 of 10 patients. CD40LT could enhance CTL in the absence of CD4+ T cell help in the majority of patients; however, the degree of enhancement of CTL responses was variable such that, in some patients, CD40LT could not completely substitute for CD4+ T cell help. In those HIV-1-infected patients who demonstrated poor responses to CD40LT, a dysfunction in circulating CD8+ memory T cells was demonstrated, which was reversed by the addition of cytokines including IL-2. Finally, it was demonstrated that IL-15 produced by CD40LT-stimulated dendritic cells may be an additional mechanism by which CD40LT induces the expansion of memory CTL in CD4+ T cell-depleted conditions, where IL-2 is lacking.

Deleterious Effect of HIV-1 Plasma Viremia on B Cell Costimulatory Function

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4+ T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4+ T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4+ T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4+ T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4+ T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4+ T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4+ T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4+ T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.

Perturbations in B cell responsiveness to CD4+ T cell help in HIV-infected individuals

HIV infection induces a wide array of B cell dysfunctions. We have characterized the effect of plasma viremia on the responsiveness of B cells to CD4+ T cell help in HIV-infected patients. In HIV-negative donors, B cell proliferation correlated with CD154 expression on activated CD4+ T cells and with the availability of IL-2, whereas in HIV-infected viremic patients, reduced B cell proliferation was observed despite normal CD154 expression on activated CD4+ T cells. Reduced triggering of B cells by activated CD4+ T cells was clearly observed in HIV-infected viremic patients compared with aviremic patients with comparable CD4+ T cell counts, and a dramatic improvement in B cell function was observed in patients whose plasma viremia was controlled by effective antiretroviral therapy. The degree of B cell dysfunction in viremic patients correlated strongly with the inability of B cells to express CD25 in response to activated CD4+ T cells, resulting in an inability to mount a normal proliferative response to IL-2. Similar defects in responsiveness to IL-2 were observed in the B cells of HIV-infected viremic patients in the context of B cell receptor stimulation. These data provide new insight into the mechanisms associated with ineffective humoral responses in HIV disease.

Relationship between the size of the human immunodeficiency virus type 1 (HIV-1) reservoir in peripheral blood CD4+ T cells and CD4+:CD8+ T cell ratios in aviremic HIV-1-infected individuals receiving long-term highly active antiretroviral therapy.

It has been demonstrated that human immunodeficiency virus type 1 (HIV-1) replication persists in most infected individuals receiving highly active antiretroviral therapy (HAART). However, studies addressing the relationship between low levels of ongoing viral replication and immunologic parameters, such as the CD4+:CD8+ T cell ratio, in such individuals have been lacking. Here, a statistically significant inverse correlation is shown between the frequency of CD4+ T cells carrying HIV-1 proviral DNA and the CD4+:CD8+ T cell ratio in infected individuals receiving HAART and in whom plasma viremia had been suppressed below the limit of detection for prolonged periods of time. No correlation was found between the frequency of HIV-1-specific cytotoxic CD8+ T lymphocytes (CTLs) and the CD4+:CD8+ T cell ratios in those individuals. These data suggest that persistent, low-level, ongoing viral replication, although not sufficient to maintain HIV-1-specific CTL responses, may explain, in part, why normalization of the CD4+:CD8+ T cell ratio is not achieved in some infected individuals successfully treated with HAART.