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HIV-Producing T Cells in Cerebrospinal Fluid
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JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 37(2) 1 October 2004 pp 1237-1244
Neuenburg, Jutta K MD*†; Sinclair, Elizabeth PhD‡; Nilsson, Annelie BA†; Kreis, Christophe PhD*; Bacchetti, Peter PhD§; Price, Richard W MD†; Grant, Robert M MD, MPH*
From the *Gladstone Institute of Virology and Immunology, San Francisco, CA; †Department of Neurology, University of California, San Francisco, CA; ‡Department of Medicine, University of California, San Francisco, CA; and §Department of Epidemiology and Biostatistics, University of California, San Francisco, CA.
SUMMARY
In HIV-1-infected subjects, the magnitude of HIV-1 viral load in cerebrospinal fluid (CSF) correlates with the CSF white cell count. To determine whether HIV-1-producing T cells appear in CSF and whether their percentage and number correlate with viral load in CSF, we developed a flow cytometric assay that detects HIV-1-producing T cells by identifying intracellular p24 HIV-1 antigen. We found that most CSF T cells were not HIV-1 producing, even when cell-free viral load in CSF was high. Most activated T cells in CSF were also not HIV-1 producing, but the activated CD38+ CD4 T-cell fraction in CSF was independently associated with the fraction of HIV-1-producing T cells in CSF. We conclude that HIV-1-producing T cells appear in CSF and that their percentage and number correlate with cell-free viral load in CSF, even though the CSF total white cell count remains the best predictor for CSF viral load. In HIV-1 infection, CSF white cell counts seem to contain a large number of uninfected cells. White cell counts and viral load in CSF may result from systemic inflammation and immune activation.
INTRODUCTION
HIV-1 infection causes significant injury to the brain, which is reflected in HIV-1-associated neurocognitive deficits and HIV-1 encephalopathy. Cerebrospinal fluid (CSF), which surrounds brain tissue, may reflect cellular events in brain tissue. The magnitude of HIV-1 viral load in cell-free CSF correlates with the number of CSF white cells, which we found to be mostly T cells. Among T cells, CD4+ cells can be productively infected with HIV-1. It is unknown whether HIV-1-producing T cells appear in CSF and, if so, whether the percentage and number of HIV-1-producing T cells in the CSF correlate with cell-free viral load. Alternatively, viral load and increased white cell counts in the CSF may result from systemic inflammation and immune activation.
Although antiretroviral therapy reduces viral load and immune activation in CSF and blood and ameliorates the clinical manifestations of HIV-1 infection, there is evidence of persistent HIV-1-specific brain pathology in the era of antiretroviral therapy. In living subjects, HIV-1 brain disease can lead to a loss of brain white matter density detectable in magnetic resonance imaging and spectroscopy. A loss of brain white matter density can also be seen in other systemic diseases in which immune activation is present, for example, multiple sclerosis, lupus erythematosus, and Behçet disease.
Even though we are not examining brain tissue in this study, a study of CSF T cells can give us clues regarding the inflammatory process of the nervous system during HIV-1 infection. The viral load in blood and the magnitude of T-cell activation predict clinical and immunologic injury during HIV-1 infection. Less information is available about the viral load and magnitude of T-cell activation as well as their consequences in the CSF and brain tissue.
Elsewhere, we describe the use of multiparameter flow cytometry to characterize T cells in CSF using cross-sectional samples of HIV-infected and -uninfected subjects. (J. K. Neuenburg et al, submitted for publication). We now describe a subgroup of these study subjects, some of whom have been followed over time. Their specimens were assessed for intracellular expression of p24 HIV-1 capsid antigen. The presence of intracellular HIV-1 p24 capsid antigen inside T cells characterizes an HIV-1-producing cell. To determine whether HIV-1-producing T cells appear in CSF and, if so, whether their percentage and number in the CSF correlate with cell-free viral load, we developed a novel flow cytometric assay that detects intracellular HIV-1 p24 capsid antigen and allows simultaneous detection of surface markers. We explored the relationships between the frequency and number of HIV-1-producing T cells in CSF and several clinically relevant parameters of HIV-1 disease, including blood CD4 T-cell count, activation levels of CD4+ T cells in CSF and blood, and cell-free CSF and blood viral load.
METHODS
Study subjects were seen at the San Francisco General Hospital (SFGH) General Clinical Research Center (GCRC) and gave written informed consent. The study protocol was approved by the University of California, San Francisco Committee on Human Research. Subjects were recruited from clinical studies at the SFGH Neurology Clinic through hospital flyers and newspaper ads. The inclusion criteria were HIV-1 infection documented by detection of HIV-1 RNA in blood for the HIV-1 group and HIV-1 seronegativity in blood for the control group. Lumbar punctures and blood sampling were performed as described elsewhere.26 In this study, 10 to 12 mL of CSF was used for the p24 assay and 8 to 12 mL of CSF was used for surface staining of activation markers.
Viral Load Testing
Plasma and CSF viral loads were determined by the ultrasensitive and standard Roche Amplicor reverse transcriptase-polymerase chain reaction (RT-PCR; Roche, Branchberg, NJ) using a lower limit of detection of 20 and 400 copies/mL, respectively. If viral load was above the dynamic range of the assay, the specimen was diluted and retested.
DISCUSSION
The magnitude of HIV-1 viral load in cell-free CSF correlates with the number of white cells in CSF. To determine whether HIV-1-producing T cells appear in CSF and, if so, whether the percentage and number of infected cells in the CSF correlate with cell-free viral load, we developed a flow cytometric assay that detects intracellular HIV-1 p24 capsid antigen as a measure of HIV-1-producing T cells. Cell-free HIV-1 viral load in the blood has been associated with the rate of clinical progression in untreated subjects. In treated subjects, the change in plasma viral load relative to pretreatment baseline is a strong predictor of CD4 T-cell count decline. The biologic relevance of cell-free measures of viral load has been questioned, however. Indeed, HIV-1 requires host cells for replication and probably for transmission as well. Most of viral replication seems to occur in lymphoid tissues in which T cells and macrophages are closely approximated in a manner that appears to greatly enhance viral production. For these reasons, we have been interested in measuring viral expression in cells of tissues. CSF, which surrounds brain tissue, may reflect cellular events in brain tissue. The flow cytometry-based assay described here proved to be a sensitive and specific measurement of p24 HIV-1 capsid antigen expression in T cells, allowing us to identify HIV-1-producing T cells. Previous attempts to identify HIV-1-infected cells in fresh PBMC samples have failed because of abundant and highly variable background staining, which seems to be caused, in part, by nonspecific staining of monocytes and granulocytes with anti-HIV-1 p24 antibodies (data not shown). In contrast, anti-p24 staining in cultured cells has been successful, probably because of the following facts. In a stimulated culture, the percentages of HIV-1-producing cells are higher than in vivo; therefore, a high background is not as critical. Second, background staining in culture is reduced because of the adherence of monocytes to the culture dish and the relative absence of granulocytes after the Ficoll step. Anti-p24 staining has also been demonstrated in cell lines, where percentages of infected cells are also higher and the background staining is minimal because of the absence of monocytes and granulocytes. Our variation of the intracellular staining protocol, which includes removal of monocytes and granulocytes by magnetic bead separation and removing remaining monocytes from analysis by gating out of CD14+ cells, enables us to reliably detect HIV-1-producing T cells that might be present at very low percentages. The capacity to identify and quantify virus expression in specific T cells by flow cytometry may prove to be useful for research that aims to characterize target cells for HIV-1 and to evaluate viral and T-cell dynamics in tissues.
We found that HIV-1-producing T cells are uncommon in CSF. Typically, less than 1% of T cells were HIV-1 producing, with the highest value of 37 specimens being 2.39%. As reported earlier in a different set of patients, we found a strong relationship between CSF white cell count (which consists mainly of T cells) and CSF viral load. We now find that CSF viral load is weakly associated with the percentage of T cells in CSF that express viral p24 antigen (P = 0.11). CSF viral load is strongly associated with the calculated number of HIV-1-producing cells, however, expressed as cell numbers per microliter of CSF (P = 0.0006). Hence, the strong association between numbers of HIV-1-producing cells and cell-free viral load in CSF is driven by the association between HIV-1 viral load and total CSF T-cell number, and the link between viral load and inflammatory cells in the CSF involves infected as well as uninfected cells. CD4+ T cells and CD4+/CD14+ cells from the monocyte/macrophage lineage can be readily infected with HIV-1, and their turnover is increased in HIV-1 infection.42 CD8+ T cells and B cells are not readily infected with HIV-1, but the turnover of these uninfected cell types is also increased in HIV-1 infection. The abundance of activated cells that do not produce HIV-1 in CSF may reflect increased proliferation or decreased destruction of uninfected cells in CSF compared with blood.
Indeed, we found that the relationship between viral load, HIV-1-producing cells, and CD4+ T-cell activation were different in CSF compared with blood. Viral load, HIV-1-producing cells and the level of CD4+ T-cell activation were associated with each other in CSF, in contrast to blood, where an association was not evident. Interactions between viral replication and T cells may be different in the 2 tissue compartments. Extensive cell-to-cell contact in blood and lymphoid tissues may increase viral replication while concurrently enhancing loss of activated CD4+ T cells because of increased apoptosis, sequestration, or effector-cell killing. In contrast to blood, cell concentrations are much lower in CSF (1-5 cells per microliter of fluid), which may decrease cell-to-cell contact and allow activated CD4+ T cells to circulate, remain uninfected, or stay infected for longer periods of time. In this way, the linkage between viral replication and T-cell destruction that occurs in hematologic tissues may not occur in the CSF compartment. Therefore, in CSF, virus and target cells (activated CD4) might be high at the same time, whereas in blood, when virus is high, target cells might be low, and vice versa, because of the consumption of activated CD4 target cells in blood and not in CSF. Unlinking of viral replication and T-cell destruction in CSF may also contribute to the higher levels of T-cell activation that we observed in CSF (compared with blood). The mechanisms that allow activated CD4+ T cells in CSF to remain uninfected or stay infected for longer periods of time despite cocirculation with HIV-1-producing T cells and functional CD8 effector cells are not known.
Low blood CD4 T-cell count is a strong predictor of the percentage of HIV-1-producing T cells in CSF. This relationship remained after controlling for (1) CSF cell count, (2) antiretroviral therapy, (3) blood viral load, or (4) CSF viral load. Low CD4+ T-cell counts have been associated with high plasma viral load and high T-cell activation in blood, linking late-stage HIV-1 disease and inflammation. The increased percentage of HIV-1-producing T cells in CSF during late-stage HIV-1 disease may reflect increased systemic levels of viral replication or increased trafficking of infected cells into tissues. Cell trafficking from blood to CSF is consistent with our finding that all T-cell subsets (CD4 and CD8, activated and nonactivated) were correlated between blood and CSF. The fact that percentages of HIV-1-producing T cells in blood and CSF (P = 0.12) are somewhat linked is consistent with trafficking between blood and CSF or systemic effects that affect both fluids. Systemic determinants might regulate T-cell biology in blood and CSF, or upregulation of signals in the choroid plexus might lead to trafficking of certain cells types to the CSF.
HIV-1-producing T cells in the CSF may contribute to HIV-1-specific brain pathology, which also occurs in later disease stages and continues to be an autopsy finding even in the era of highly active antiretroviral treatment (HAART).15-18 Interestingly, the relationship between low CD4+ T-cell count and higher frequency of HIV-1-producing T cells was stronger in treated subjects (see Fig. 1C), who were mostly suffering virological drug failure, than in untreated patients. Although clinical dementia is markedly improved during antiretroviral therapy, the persistence of HIV-1-specific pathologic changes during the HAART era raises the concern that clinical progression may accumulate over long periods of time among people surviving with HIV-1. Long-term clinical evaluation is required to address this concern.
We conclude that HIV-1-producing T cells appear in CSF and that their percentage and number correlate with cell-free viral load in CSF, even though the CSF total white cell count remains the best predictor for CSF viral load. Most CSF T cells were not HIV-1 producing, even when cell-free viral load in CSF was high. Most activated T cells in CSF were also not HIV-1 producing, but the activated CD38+ CD4 T-cell fraction in CSF was independently associated with the fraction of HIV-1-producing T cells in CSF. In HIV-1 infection, CSF white cell counts seem to contain a large number of uninfected cells. White cell counts and viral load in CSF may both result from systemic inflammation and immune activation.
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