CSF provides an accessible and useful window into CNS infection and immune responses, albeit with the general caveat that brain and CSF responses are not always congruent.27
In this study, we demonstrate that flow cytometry can be used to analyze the activation state of CSF T cells, and that T-cell characterization affords novel insight into the pathogenesis and treatment of CSF HIV-1 infection. The 4 subject groups included in the study encompassed a diverse population with respect to HIV-1 infection, degree of immunosuppression, and treatment effect, allowing comparisons of blood and CSF cells across a broad range of CD8 and CD4 T-cell activation. The levels of activation of CSF CD8 cells and, to a lesser extent, CD4 cells were highly correlated with their blood counterparts across the subject groups and the range of cell activation. CSF CD8 T-cell coexpression of CD38 and HLA-DR was parallel to that in peripheral blood, though it was approximately 9% higher. This is consistent with the transmigration of these cells from blood to CSF with only minor selection or modification affecting these phenotypic characteristics, so that CSF T cells largely retain the activation profiles of those circulating in blood.
HIV-1 infection and treatment outcome had a marked effect on T-cell activation in both blood and CSF, with a stepwise decrease in the level of blood CD8 CD38/HLA-DR coexpression with partial and full viral suppression. Thus, although suppressive therapy had a greater impact, even failed treatment related to the development of drug resistance had a substantial and statistically significant effect, confirming previous reports related to blood cells19,28
and now extending this finding to CSF T cells. Comparison of activation in the 2 viremic groups in relation to plasma HIV-1 concentrations clearly showed that failed treatment lowered both blood and CSF CD8 activation compared to untreated subjects across the range of plasma viral loads () and that the reduced activation was not simply related to the (statistically insignificant) lower plasma HIV-1 RNA levels in the drug-resistant failures. The reason for this reduction in activation in relation to plasma viral load is not clear but has been suggested to relate in some way to reduced immunopathogenicity of drug-resistant virus.19
Whatever the mechanism, wild-type virus is associated with a higher level of activation than resistant virus at a given level of plasma viremia.
By contrast, a similar downward shift in activation in relation to CSF viral levels was not seen in failures compared to offs, so that the relation of blood and CSF CD8 T-cell activation to CSF viral load did not differ in the 2 viremic groups (). Thus, there was a fundamental difference in the effects of virologic failure on the relation of activation to viral RNA levels in the 2 compartments. We favor the interpretation that the level of immune activation had a modulating effect on local CSF viral replication and that both resistant and wild-type viruses replicated similarly at a given level of immune activation in this compartment. Thus, although in the blood, activation depends on whether infection is caused by wild-type or resistant virus, in the CSF this relation does not hold; rather, activation determines a similar level of replication for both types of virus. The reasons for this are not clear, but may relate to the fact that systemic virus originates in lymphoid tissue, whereas CSF virus relies on transmigration of lymphocytes and monocytes into a non-lymphoid organ. Cell activation may be an important determinant of this transmigration. Whatever the mechanism, this relation between activation and CSF HIV-1 RNA may explain some of the effects of antiretroviral treatment on CSF HIV-1 RNA concentrations, including the disproportionate effect of failed treatment reported earlier in this cohort13
and noted again for the study visits reported here, with CSF HIV-1 RNA levels more than 10-fold lower in the failures than the offs, despite nearly equal plasma HIV-1 RNA levels ().
The multivariate analysis of the possible contributions to CSF HIV-1 RNA concentrations suggested that the measured variables could be segregated into 3 groups: the plasma HIV-1 RNA concentration; the CD8 T-cell activation, with the blood CD8 activation occluding the effect of CSF CD8 activation (when blood CD activation was added to the model, CSF activation was no longer significant); and the CSF neopterin and WBC counts, with the former occluding the effect of the latter (addition of CSF neopterin to the model eliminated the WBC count as a significant contributor). These variables are clearly intimately interrelated, both statistically and biologically.
The combination of our earlier observations13
with these findings related to CD8 T-cell activation can be synthesized into a coherent set of observations and derivative pathogenic hypothesis as follows: First, systemic HIV-1 infection drives “generalized immune activation”29
that includes both CD8 and CD4 T cells and, likely, monocyte/macrophages.30
Second, CD8 T-cell activation, whether measured in blood or CSF, serves as a useful index of this generalized immune activation.21,31
Third, resistant virus is a less potent driver of systemic activation than wild-type virus. Fourth, by virtue of their origin from blood, CSF T cells retain their activated blood phenotype, which is determined by the level of systemic immune activation rather than local factors; indeed, statistically, blood CD8 activation more directly accounts for the contribution of T-cell activation than CSF CD8 activation. Fifth, HIV-1 replication of both wild-type and resistant viruses in the CSF is similarly modulated by the level of this local immune activation. Sixth, although the mechanism of this modulation is uncertain, it may involve the increased availability of target CD4 T cells due to their increased cell traffic (an idea supported by the correlation between CD8 activation and CSF pleocytosis) and to their higher capacity for supporting viral replication as a result of their activated state. Thus, activation state might contribute to CSF viral replication and local viral amplification by at least 2 mechanisms: an effect on cell migration (cell availability) and an effect on viral output from the local cells (viral production). Other major contributors to CSF HIV-1 concentration include the magnitude of systemic infection, as indicated by the plasma HIV-1 RNA concentration, and intrathecal macrophage activation, indicated by CSF neopterin.
This reasoning, which underlies the simple model of CSF HIV-1 infection diagrammed in , suggests that the bulk of the virus detected in CSF derives from amplified infection that is modulated by the level of immune activation. The simple schematic in the figure depicts the principal compartments participating in the origin of CSF infection—systemic (blood), leptomeninges (CSF), and brain—and the hypothesized role of immune activation in transitory, autonomous, and amplified CSF infection. In the systemic compartment, HIV-1 replication leads to blood viremia and stimulates generalized immunoactivation, including systemic T-cell activation, which can be measured using CD8 CD38 and HLA-DR surface coexpression as an index. Although this activation modulates HIV-1 replication, systemic HIV-1 replication is the prime mover in this interaction. In the leptomeninges compartment, systemic viremia (especially CD4 T-cell–associated infection) is responsible for initial seeding. This direct hematogenous seeding is responsible for transitory CSF infection that predominates during primary HIV-1 infection. In the brain compartment, chronic brain infection sustained within longer-lived perivascular and parenchymal macrophages and microglial cells (together encompassed by the abbreviation MΦ), termed autonomous infection, can also spill over into the CSF. Macrophages presumably also participate in the generalized immune activation caused by HIV-1, as suggested by the elevated CSF neopterin and its correlation with CD8 T-cell activation in CSF and blood. The multivariate analysis suggests that macrophage activation contributes to CSF pleocytosis as well. Activated CSF T cells originate in the blood and importantly contribute to local intrathecal (and perhaps perivascular) amplified infection by supplying susceptible CD4 T cells that sustain local replication within the CSF space. This amplified infection may be continuously reseeded from the blood or from the brain sources (ie, from transitory or autonomous mechanisms) and modified by local viral evolution and selection. The level of CD4 T-cell activation is a major factor in sustaining the level of this local amplification. Though measurements of CD4 T cells do not correlate as well with CSF HIV-1 levels as CD8 activation does, this may be explained by direct infection of the former and their preferential destruction.
FIGURE 5 Model of CSF HIV-1 infection and its relation to immune activation and treatment responses. Transitory CSF infection (1) predominates during primary HIV-1 infection. Autonomous infection (2), a chronic brain infection sustained within longer-lived perivascular (more ...)
Responses of CSF infection to treatment may depend on both direct and indirect effects. The chief direct target may be systemic infection, reflected in the plasma HIV-1 response, whereas direct effects may be weaker behind the BCB and BBB because of reduced drug penetration. However, targeting systemic infection will secondarily reduce CSF infection by several mechanisms, including reduction of new transitory seeding as both the influx of hematogenously circulating infected cells and their virus output are reduced. Reduction of systemic infection will also reduce systemic T-cell activation. Because these cells are the source of activated CSF T cells, they will also be reduced in the CSF and in turn will lead to lower amplification. Effects on macrophage activation may also reduce autonomous infection and even amplified infection through modulation of T-cell chemotaxis.
This model is also consistent with observations of genetically compartmentalized infection32,33
and with suggestions that CSF HIV-1 derives principally from short-lived cells, likely lymphocytes.32
When applied to understanding treatment effects, this model suggests that reducing systemic immune activation with ART may contribute to suppressing CSF infection, not only in the failures but also in the successes. Because of the BBB and BCB, most antiviral drugs do not achieve concentrations in brain or CSF comparable to those in plasma or systemic organs and therefore might be expected to be less effective in these compartments.34
However, as in this group of subjects, most studies of treatment on CSF HIV-1 have shown effective viral suppression.5,14–16
Although in some patients CSF HIV-1 RNA decays with treatment more slowly than plasma RNA does, in many CSF virus falls just as rapidly through the first phase of decay, and good overall therapeutic responses seem to be the rule with occasional notable exceptions.5,35–37
How is this possible, with CSF’s lower drug access? Reduction of immune activation, leading to both reduced influx of infected cells and down-regulation of local amplification, may contribute importantly. Autonomous infection within meningeal and perivascular macrophages and microglial cells38
likely is also modulated as part of the generalized immunoactivation, as suggested by the correlation of CD8 activation with CSF neopterin (a marker of local macrophage activation and intrathecal immunoactivation25,39
) and the contribution of CSF neopterin to the multivariate model of CSF HIV-1 RNA concentrations. Indeed, local macrophage activation, as indicated by neopterin concentrations, likely also contributes to CSF pleocytosis, perhaps through chemotactic signals or other mechanisms.
CNS HIV-1 infection is an important component of systemic infection and a critical target of therapy. These studies suggest that the level of systemic immune activation is an important modulator of this infection and that its downregulation by ART may contribute to controlling HIV-1 in this compartment and explain the better-than-predicted responses of CSF HIV-1 to ART and both the preventative40
effects of treatment on ADC, although importantly the current study was confined to subjects without neurological impairment.