Cerebrospinal fluid CD4+ T cell infection in humans and macaques during acute HIV-1 and SHIV infection

Abstract

HIV-1 replication within the central nervous system (CNS) impairs neurocognitive function and has the potential to establish persistent, compartmentalized viral reservoirs. The origins of HIV-1 detected in the CNS compartment are unknown, including whether cells within the cerebrospinal fluid (CSF) produce virus. We measured viral RNA+ cells in CSF from acutely infected macaques longitudinally and people living with early stages of acute HIV-1. Active viral transcription (spliced viral RNA) was present in CSF CD4+ T cells as early as four weeks post-SHIV infection, and among all acute HIV-1 specimens (N = 6; Fiebig III/IV). Replication-inactive CD4+ T cell infection, indicated by unspliced viral RNA in the absence of spliced viral RNA, was even more prevalent, present in CSF of >50% macaques and human CSF at ~10-fold higher frequency than productive infection. Infection levels were similar between CSF and peripheral blood (and lymph nodes in macaques), indicating comparable T cell infection across these compartments. In addition, surface markers of activation were increased on CSF T cells and monocytes and correlated with CSF soluble markers of inflammation. These studies provide direct evidence of HIV-1 replication in CD4+ T cells and broad immune activation in peripheral blood and the CNS during acute infection, likely contributing to early neuroinflammation and reservoir seeding. Thus, early initiation of antiretroviral therapy may not be able to prevent establishment of CNS viral reservoirs and sources of long-term inflammation, important targets for HIV-1 cure and therapeutic strategies.

Fig 1. Plasma viremia and CSF cellular composition in acutely SHIV-infected macaques.

(A) Longitudinal plasma viral RNA following SHIV-1157ipd3N4 infection in rhesus macaques (N = 18). Data for individual animals is shown in gray and the black trend line depicts the median value. Biospecimen collections are indicated by symbols at bottom. (B) The frequency of T cell subsets and monocytes in CSF determined by flow cytometry is plotted over time PI. T cell subset frequency was calculated as the percentage of all T cells; monocytes as the percentage of all viable CSF cells. (C) The CD4+/CD8+ T cell ratio in CSF (left) and PBMC (right) is shown in uninfected and SHIV-infected macaques 4 and 12 weeks PI. (D) The proportion of memory CD4+ T cell subsets in CSF (left) and PBMC (right) was assessed by CD28 and CD95 staining and flow cytometry. The frequency of central memory (CM; CD95+ and CD28+), effector memory (EM; CD95+ and CD28-negative), and naive (CD95-negative) is shown over time.

Fig 2. Cell-associated SHIV RNA during acute infection.

(A) Schematic of workflow used to isolate mononuclear cell populations and estimate infected cell frequency in CSF (top) and PBMC or LNMC (bottom) directly ex vivo from SHIV-infected macaques. (B) Viral RNA RT-qPCR positivity among FACS sorted CD4+ T cells (top) and monocytes (bottom) in CSF of uninfected and SHIV-infected macaques 4, 8, and 12 weeks PI. The number of cells analyzed for each animals is indicated on the y-axis, with stacked bars reflecting the size of each sort replicate. Bar coloring indicates positivity for unspliced only (blue) or spliced (red) SHIV RNA; viral RNA-negative replicates are colored gray. Fraction of animals with SHIV RNA+ cells is indicated for each time point. (C) SHIV gag RNA+ CD4+ T cell frequency is shown for CSF, PBMC, and LNMC 4 and 8 weeks PI. Open symbols depict the limit of detection in samples where no vRNA was detected. Significant differences between tissues was assessed using Mann-Whitney test. (D) Paired analysis comparing the infected CD4+ T cell frequency between matched CSF and either PBMC or LNMC is shown. (E) Correlation between gag vRNA+ infected CD4+ T cell frequency between CSF and PBMC (left) or LNMC (right) is shown. (F) Correction between plasma viremia and gag vRNA+ infected CD4+ T cell frequency (percent, log10) in PBMC (left), CSF (middle), and LNMC (right) is shown. Rho and p-value indicate Spearman correlation.

Fig 3. Cellular activation in CSF and PBMC during acute SHIV infection.

T cell and monocyte activation assessed by surface staining flow cytometry for CD38 and CD169, respectively, following SHIV infection in macaques. (A) CSF CD4+ and CD8+ T cell CD38 expression, reported as the median fluorescent intensity (MFI), is shown at the indicated weeks PI. (B) CSF monocyte CD169 MFI and CXCL10 gene expression (copies per 10⁶ cells) measured by RT-qPCR is shown at the indicated weeks PI. (C) PBMC CD4+ and CD8+ T cell CD38 surface protein expression and (D) PBMC monocyte CD169 expression is shown at the indicated weeks PI. Bars and whiskers indicate median and interquartile range values, respectively. Significant differences by Mann-Whitney test are indicated as follows: *, P <0.05; **, P <0.01; ***, P <0.0001.

Fig 4. HIV-1 infection of CD4+ T cells in CSF during acute infection in humans.

(A) Single-cell analysis of vRNA content in memory CD4+ T cells from PBMC of an acutely infected PLWH from the RV254 cohort. CRF01_AE HIV-1 mRNA was measured by RT-qPCR using assays specific for LTR, gag, and env in cells FACS sorted at one per well. Each symbol depicts vRNA content for one cell. Symbol color represents the combination of vRNAs detected in the cell, as indicated in the key. Surface CD4 protein expression for env vRNA+ cells (red line) was measured by flow cytometry and is indicated relative to vRNA-negative cells (gray) in inset. (B) HIV-1 vRNA RT-qPCR positivity among CD4+ T cells isolated by flow cytometry from CSF of PWOH and people living with acute HIV-1. Bars indicate the number of cells analyzed for each individual; stacked bars reflect the size of each replicate pool of CD4+ T cells. Bar coloring indicates HIV-1 vRNA positivity for unspliced (blue, gag) or spliced plus unspliced (red, env and gag) mRNA; vRNA-negative replicates are colored gray. (C) Distribution of HIV-1 gag (left) and env (right) vRNA+ CD4+ T cell frequency in CSF and PBMC is shown for individuals represented in (B). HIV-1 gag vRNA+ CD4+ T cell analysis in CSF was limited to four individuals. Bars indicate median values and whiskers span the interquartile range. (D) Paired analysis comparing the infected CD4+ T cell frequency between CSF and PBMC is shown. Significant differences between tissues was assessed using Wilcoxon matched-pairs rank test. (E) HIV-1 vRNA positivity among monocytes and CD8+ T cells (F) isolated from CSF as in (B); purple, env+ and gag-negative. Fraction of individuals with vRNA+ cells is indicated.

Fig 5. Cellular activation in CSF during acute HIV infection.

(A) CD38+ T cell frequency was measured by flow cytometric detection of surface staining on CD4+ (top) and CD8+ (bottom) T cells in CSF (left) and PB (right) of PWOH and PLWH in acute infection. (B) Monocyte activation measured by CD169 (top) and CD38 (bottom) surface staining and flow cytometry in CSF (left) and PB (right). Significant differences by Mann-Whitney test are indicated as follows: *, P <0.05; ** P <0.01; *** P <0.0001. (C) Correlation between CSF IP-10 concentration and activated CD38+HLA-DR+ CD4+ T cells (left), CD38+HLA-DR+ CD8+ T cells (middle), and CD163 surface expression by monocytes (right) in CSF of PLWH. Rho and P values indicate Spearman correlation.