Cellular Composition of Cerebrospinal Fluid in HIV-1 Infected and Uninfected Subjects

Abstract

In order to characterize the cellular composition of cerebrospinal fluid (CSF) in a healthy state and in the setting of chronic pleocytosis associated with HIV-1 (HIV) infection, multi-parameter flow cytometry was used to identify and quantitate cellular phenotypes in CSF derived from HIV-uninfected healthy controls and HIV-infected subjects across a spectrum of disease and treatment. CD4+ T cells were the most frequent CSF population and the CD4:CD8 ratio was significantly increased in the CSF compared to blood (p = 0.0232), suggesting preferential trafficking of CD4+ over CD8+ T cells to this compartment. In contrast, in HIV-infection, CD8+ T cells were the major cellular component of the CSF and were markedly increased compared to HIV-uninfected subjects (p<0.001). As with peripheral blood, the CSF CD4:CD8 ratio was reversed in HIV-infected subjects compared to HIV-uninfected subjects. Monocytes, B cells and NK cells were rare in the CSF in both groups, although absolute counts of CSF NK cells and B cells were significantly increased in HIV-infected subjects (p<0.05). Our studies show that T cells are the major cellular component of the CSF in HIV-infected and uninfected subjects. The CSF pleocytosis characteristic of HIV infection involves all lymphocyte subsets we measured, except for CD4+ T cells, but is comprised primarily of CD8+ T cells. The reduced proportion of CD4+ T cells in the CSF may reflect both HIV-related peripheral loss and changes in trafficking patterns in response to HIV infection in the central nervous system.

Figure 1. Flow cytometry gating strategy to define blood and CSF white blood cell (WBC) populations.

Data was compensated and gated using FlowJo to define TruCOUNT™ beads, (i) from both blood (left panel) and CSF (right panel) data. Remaining events were displayed in a CD45 vs FSC plot (ii) to gate out debris and define a total WBC population. CD14 was used to gate monocytes (iii) from the total WBC gate, and CD14− cells were displayed on a CD45 vs SSC plot (iv) to define lymphocyte and granulocyte gates. Lymphocytes were subdivided into CD3+ T cells and CD3− lymphocytes (v), CD3− lymphocytes were displayed on a CD19 vs CD56&16 (vi) plot to define B cells and NK cells respectively. CD3+ T cells were displayed on a CD4 vs CD8 plot (vii) to define CD4+ and CD8+ T cells.

Figure 2. HIV-infected and uninfected subjects have distinct blood and CSF lymphocyte proportions and counts.

Proportions of CD3+, CD4+ and CD8+ T cells; B cells; and NK cells present in the blood (A) and CSF (B) of HIV-uninfected (HIV-) and HIV-infected (HIV+) subjects are depicted as a percentage of total lymphocyte population. Absolute CSF cell counts of lymphocyte subsets for HIV-uninfected and HIV infected (C) subjects are shown. Median and interquartile range for each cell population are shown in the box plots. Whiskers are set at 10–90%. Mean values are indicated by (+).

Figure 3. ART influences the absolute lymphocyte subset counts present in the CSF of HIV-infected subjects.

Absolute number of CSF monocytes (A), CD3+ T (B), CD4+ T (C), CD8+ T (D), B (E) and NK (F) cell subsets present in CSF of the 4 patient subgroups (Off, Rx Viremic, Rx VL<500 and HIV-uninfected) are shown. Median and interquartile range for each cell population are shown in the box plots. Whiskers are set at 10–90%. Mean values are indicated by (+). ANOVA for significant overall differences between the 3 HIV-infected treatment groups was conducted, with the p value depicted at the top of each panel. Post-hoc analysis using Dunn’s test comparing differences between pairs of groups, if statistically significant, are as indicated in each panel.

Figure 4. Comparison of T cell subsets in CSF and blood of HIV-infected and uninfected subject.

(A) Relation of CD4+ T cells present in the CSF to those present in the blood amongst the 4 subject groups with regression line (solid line; slope  = 1.395, r² 0.7608, p<0.0001) and 95% confidence intervals (dotted lines). (B) Relation of CD8+ T cells present in the CSF to those present in the blood amongst the 4 subject groups with regression line (slope  = 0.9768, r² 0.5794, p<0.0001) and 95% confidence intervals. (C) Comparison of CSF and blood CD4:CD8 ratio between the 4 subject groups.