The patients on therapy were largely chosen because they had suffered virologic drug failure during at least 1 time point. Viremic subjects were selected because they would have higher levels of CSF white cell counts, which facilitates the flow cytometric characterization of cells. Eight of 13 subjects who supplied 37 paired specimens (CSF and blood) were taking antiretroviral therapy during at least 1 time point. Only 1 subject was treatment naive. Of the 37 pairs of specimens, 19 (51%) were collected during combination antiretroviral therapy, including at least 3 drugs involving at least 2 classes (eg, nucleoside analogue reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, protease inhibitors). Because of this preselection, therapy did not completely suppress viral load at most time points: 17 (84.2%) of the 19 blood specimens and 16 (89.5%) of the 19 CSF specimens taken during therapy had viral loads >50 copies/mL. The high viral loads in these subjects likely reflect drug resistance as a result of exposure to prior therapy or nonadherence. Accordingly, antiretroviral therapy was only weakly associated with lower viral load in this group. None of the study subjects suffered from opportunistic central nervous system (CNS) diseases at the time their CSF and blood samples were taken.
We developed an intracellular flow cytometric assay to detect HIV-1–producing T cells by detecting intracellular p24 HIV-1 capsid antigen. We removed monocytes and remaining granulocytes in blood after Ficoll testing using CD11b magnetic beads. We identified T cells in CSF and blood with CD3 (T-cell marker) and the absence of CD14 (monocyte marker). The p24+CD3+CD14− T-cell population was considered to represent HIV-1–producing T cells. Analysis of CSF and blood from uninfected controls showed that the median false-positive rate of p24+CD3+CD14− cells was 0.20% for blood and 0.25% for CSF. p24 staining above background was detected in 22 (59.5%) of 37 blood specimens and in 35 (94.6%) of 37 CSF specimens. We therefore concentrated on the p24 percentage data for CSF in this report, also because the p24 staining intensity for CSF cells was higher.
HIV-1–producing T cells were found in a median of 0.28% of T cells in blood and 0.66% of T cells in CSF. The percentage of T cells that were HIV-1 producing was higher in CSF than in blood (median difference = 0.27%; 95% CI: 0.20%–0.52%; N = 37; P < 0.0001), as indicated by the majority of CSF data points seen above the identity line in Figure 1B. There was a trend toward correlation of HIV-1–producing T cells in both compartments (P = 0.12), suggesting trafficking of cells between compartments or systemic regulation of cells for both compartments. This trend linking CSF and blood HIV-1–producing T-cell percentages was unchanged after controlling for CSF white cell count and therapy.
The concentration of HIV-1–producing T cells in CSF was estimated by multiplying the CSF white cell count per microliter and the CSF p24 percentage. This estimated cell concentration correlated strongly with viral load in CSF (P = 0.0006), but the percentage of HIV-1–producing T cells in CSF alone (p24 percentage) correlated only weakly (P = 0.11) with cell-free viral load. In regression analysis, with each doubling of the HIV-1–producing T-cell count, CSF viral load was estimated to be 0.26 log10 copy/mL higher. For example, if the HIV-1–producing T-cell count increased from 15 to 30 cells/μL, viral load would increase from 1840 to 3322 copies/μL. An analysis of CSF p24 percentage, HIV-1–producing cell count, and CSF white count variables (all controlled for therapy) indicated that CSF white count was the most statistically significant independent correlate of cell-free viral load in the CSF (P < 0.0001). Therefore, unexpectedly, the analysis of p24 percentage left the association almost unchanged and did not add additional predictive value to the CSF white count.
The CD4 T-cell count in blood is a marker of HIV-1 disease stage and predicts the risk of clinical disease progression.30 A low CD4 T-cell count in this study was associated with a high CSF p24 percentage (estimate = −0.38, 95% CI: −0.75 to −0.01; P = 0.046), even after controlling for CSF white count and therapy (estimate = −0.39, 95% CI: −0.78–0.00; P = 0.049) and for CSF viral load and blood viral load (estimate= −0.43, 95% CI: −0.88–0.02; P = 0.059). For each doubling of the CD4 T-cell count in blood, the percentage of HIV-1–producing T cells was estimated to decrease 0.39% (controlled for therapy and white cell count). Antiretroviral therapy was associated with a decrease in the percentage of HIV-1–producing T cells. Unexpectedly, however, the relationship between CD4 and CSF p24 percentage was stronger in treated subjects (see Fig. 1C).
HIV-1–infected subjects with higher CSF white cell counts tended to have lower percentages of CSF monocytes, indicating that the observed increase in white cell counts predominantly involved T cells. Granulocytosis in CSF was not observed.
The magnitude of HIV-1 viral load in cell-free CSF correlates with the number of white cells in CSF.5–8 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.30,31 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.32 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.33–36 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,37 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,38,39 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,40,41 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,5,26 we found a strong relationship between CSF white cell count (which consists mainly of T cells) and CSF viral load (see Fig. 1A). 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.42–47 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 (see Fig. 1D). 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.24,48 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.25 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.49,50
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.
The authors thank all study participants. The authors also thank the Gladstone Virology Core Laboratory for performing the viral load assays and Sarah Mays for computer assistance.
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