Cerebrospinal fluid (CSF) CD8+ T-cells that express interferon-gamma contribute to HIV associated neurocognitive disorders (HAND)

Abstract

Background: HIV associated neurocognitive disorders (HAND) continue to affect cognition and everyday functioning despite anti-retroviral treatment (ART). Previous studies focused on mechanisms related to monocyte/macrophage mediated inflammation. However, in the ART era, there is increasing evidence for the involvement of CD8+ T-cells in CNS pathogenesis.

Methods: To investigate the relationship between T-cell responses and neurocognitive impairment (NCI), cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T-cell intracellular cytokine (IFNγ, IL-2, TNFα) and lytic marker (CD107a) expression were assessed in HIV infected subjects who underwent comprehensive neurocognitive (NC) evaluation and either initiated or changed ART.

Results: Data were collected from 31 participants at 70 visits. The frequency of cytokine expressing T-cells in CSF was significantly higher than in peripheral blood for CD4+T-cells: TNFα, IL-2, IFNγ and CD8+T-cells: IL-2 and IFNγ. Analysis of T-cell activity and NCI as a function of CSF HIV RNA levels suggested a general association between NCI, high CSF CD8+ (but not CD4+T-cell) cytokine expression and CSF HIV RNA <103 copies/ml (p<0.0001). Specifically, CSF CD8+ T-cell IFNγ expression correlated with severity of NCI (r = 0.57, p = 0.004). Multivariable analyses indicated that CSF CD8+T-cell IFNγ and myeloid activation (CD163) contributed equally and independently to cognitive status and a composite variable produced the strongest correlation with NCI (r = 0.83, p = 0.0001). In contrast, CD8+ cytolytic activity (CD107a expression) was negatively correlated with NCI (p = 0.05) but was dependent on CD4 levels >400/μl and low CSF HIV RNA levels (<103 copies/ml). In our longitudinal analysis of 16 subjects, higher CSF CD8+IFNγ expression at baseline predicted NC decline at follow-up (p = 0.02). Severity of NCI at follow-up correlated with level of residual HIV RNA in CSF.

Conclusions: Presence of IFNγ expressing CD8+ T-cells, absence of cytolytic CD8+ T-cells, high myeloid activation, and failure of ART to suppress HIV replication in CSF contribute to increased risk of HAND.

Fig 1. The frequency of HIV specific CD4 T-cells expressing IFNγ in the CSF was higher than in peripheral blood.

PBMC (1a) and CSF (1b) cells from the same HIV+ donor were cultured overnight with HIV p24 antigen and stained for intracellular IFNγ expression.

Fig 2. CD8+ T-cells from NC impaired subjects expressed increased levels of constitutive and p24 induced IFNγ, IL-2, and TNFα at higher CSF HIV RNA levels (>400 copies/ml).

Constitutive and HIV p24 antigen induced CSF CD8+ IFNγ, IL-2, and TNFα expression by normal (triangles) and neurocognitive impaired (circles) subjects were compared at 3 CSF HIV RNA levels. Expression levels were square root transformed. Differences between impaired and unimpaired subjects were significant at higher HIV RNA levels (>400 copies/ml) in CSF (WRS = Wilcoxon Rank Sum p<0.001)

Fig 3. When HIV RNA was detectable in CSF, constitutive CD8+IFNγ and IL-2 expression correlated positively with NCI (Fig. 3a) and CD4+ and CD8+CD107a expression correlated negatively with NCI (3b) Percent cytokine (3a) or lytic marker (3b) expression were plotted as a function of neurocognitive global deficit score (GDS).

Expression levels and GDS were square root transformed. a: When HIV RNA was detectable in CSF, there was a positive correlation between constitutive CSF CD8+IFNγ (solid line, diamonds r = 0.57, p = 0.004) and IL-2 expression (dotted line, circles r = .49, p = 0.01) and NCI. b: In contrast, CSF CD4+CD107a+ (solid line, triangles r = 0.49, p = 0.001) and CD8+CD107a (dotted line, circles r = .44, p = 0.004) expression were negatively associated with NCI.

Fig 4. Constitutive and HIV p24 induced CD8+CD107a expression (lytic activity) in CSF and peripheral blood were only detected when HIV RNA levels were <1000 copies/ml and CD4 levels were >400/μl.

a: The percentage of HIV p24 induced CD4+ (solid diamond) and CD8 (open circle) and constitutive (cCD107a) CD4+ (open triangle) and CD8+ (x) expression in CSF were plotted as a function of log HIV RNA in CSF. CD107a expression >1% was only detected when HIV RNA was <1000 copies/ml. b: The percentage of constitutive CD8+CD107a+ in CSF was plotted as a function of plasma CD4 levels. CD8+CD107a expression >1% was detected when CD4 levels were >400/μl. Cytokine expression and CD4 levels were square root transformed.

Fig 5. CD8+IFNγ + expression and soluble CD163 in CSF represent two significant and independent correlates of NCI (5a). The composite association is stronger than either alone (5b).

a: The percentage of constitutive CSF CD8+IFNγ expression (solid circles, dotted line, r = 0.59 p = 0.004) and soluble CSF CD163 (open diamonds, solid line, r = 0.42, p = 0.004) were plotted as a function of neurocognitive global deficit score (GDS). b: The percentage of constitutive CSF CD8+IFNγ expression and pg of soluble CSF CD163 were summed for visits at which both values were available and the composite score plotted as a function of neurocognitive GDS (solid triangles, r = 0.83, p<0.0001). Expression levels and GDS were square root transformed.

Fig 6. When HIV RNA is suppressed, the percent of CD8+ T-cells in CSF (6a) and the percent of HIV specific CD8+ IFNγ+ T-cells in CSF (6b) both correlate with severity of NCI (GDS).

a, b: When HIV RNA was suppressed in CSF, CD8+ T-cells (6a: r = 0.66, p = 0.002) and HIV p24 induced CD8+ T-cell IFNg expression (6b: r = .53, p = 0.02) correlated positively with NCI (GDS). Cell numbers, expression levels and GDS were square root transformed.

Fig 7. Neurocognitive outcome (GDS) can be viewed as a function of levels of chemoattractant (CXCL10) in CSF combined with the phenotype of CD8+ T-cells (CD107a or IFNγ+) available for recruitment into the CNS.

Using all patient visits, best fit lines/curves for constitutive CSF CD8+CD107a (r = 0.28 p = 0.05), CD8+IFNγ expression (r = 0.47 p = 0.001) (both left axis), and CSF CXCL10 (r = 0.51 p = 0.004) (right axis) were plotted as a function of neurocognitive GDS. The dotted line indicates the conventional cut-off for clinically significant NCI, crossing near the inflection point for CXCL10 and the intersection of falling CD107a levels and rising CD8+IFNγ expression. CD8+T-cell expression levels, CXCL10, and GDS were square root transformed.