Decreased CXCR3+ CD8 T cells in advanced human immunodeficiency virus infection suggest that a homing defect contributes to cytotoxic T-lymphocyte dysfunction

Abstract

To exert their cytotoxic function, cytotoxic T-lymphocytes (CTL) must be recruited into infected lymphoid tissue where the majority of human immunodeficiency virus (HIV) replication occurs. Normally, effector T cells exit lymph nodes (LNs) and home to peripheral sites of infection. How HIV-specific CTL migrate into lymphoid tissue from which they are normally excluded is unknown. We investigated which chemokines and receptors mediate this reverse homing and whether impairment of this homing could contribute to CTL dysfunction as HIV infection progresses. Analysis of CTL chemokine receptor expression in the blood and LNs of untreated HIV-infected individuals with stable, chronic infection or advanced disease demonstrated that LNs were enriched for CXCR3(+) CD8 T cells in all subjects, suggesting a key role for this receptor in CTL homing to infected lymphoid tissue. Compared to subjects with chronic infection, however, subjects with advanced disease had fewer CXCR3(+) CD8 T cells in blood and LNs. CXCR3 expression on bulk and HIV-specific CD8 T cells correlated positively with CD4 count and negatively with viral load. In advanced infection, there was an accumulation of HIV-specific CD8 T cells at the effector memory stage; however, decreased numbers of CXCR3(+) CD8 T cells were seen across all maturation subsets. Plasma CXCL9 and CXCL10 were elevated in both infected groups in comparison to the levels in uninfected controls, whereas lower mRNA levels of CXCR3 ligands and CD8 in LNs were seen in advanced infection. These data suggest that both CXCR3(+) CD8 T cells and LN CXCR3 ligands decrease as HIV infection progresses, resulting in reduced homing of CTL into LNs and contributing to immune dysfunction.

FIG. 1.

Chemokine receptor expression on bulk and HIV-specific CD8 T cells in chronic and advanced HIV infection. (A) Contour plots demonstrating gates used for lymphocytes, CD8 T cells and tetramer⁺ CD8 T cells, and chemokine receptors. SSC, side scatter; FSC, forward scatter. (B) Representative examples of cell-surface expression of chemokine receptors in study subjects with either chronic (top row) or advanced (bottom row) HIV infection. SSC, side scatter. (C and D) The levels of chemokine receptor expression on bulk CD8 T cells (C) and HIV-specific CD8 T cells (D) were compared between HIV-positive study subjects with chronic and advanced infection. (E) Levels of CXCR3 expression on bulk and EBV-specific CD8 T cells from five HIV-negative subjects.

FIG. 2.

CD8 T-cell CXCR3 expression correlates positively with peripheral CD4⁺ T-cell count and negatively with HIV viral load. The levels of CXCR3 expression on bulk CD8 T cells or HIV-specific CD8 T cells as a function of peripheral CD4 T cells (A) or HIV viral load (B) are shown.

FIG. 3.

Maturation phenotype and CXCR3 expression on bulk and HIV-specific CD8 T cells. (A) Representative contour plot demonstrating gates used for definition of four maturation subsets: naïve (CD27⁺ CD45RA⁺), CM (CD27⁺ CD45RA⁻), EM (CD27⁻ CD45RA⁻), and EMRA (CD27⁻ CD45RA⁺) CD8 T cells. (B and C) Distribution of bulk CD8 T cells (B) and HIV-specific CD8 T cells (C) according to maturation phenotype. (D and E) Lower levels of CXCR3 were seen across all maturation subsets of bulk (D) and HIV-specific (E) CD8 T cells from subjects with advanced versus chronic infection, suggesting that the maturation defect seen in subjects with advanced infection did not account for the decreased CXCR3 expression on CD8 T cells.

FIG. 4.

Plasma levels of CXCR3 ligands in healthy controls (n = 5) and subjects with chronic HIV infection (n = 8) and advanced HIV infection (n = 10) as measured by enzyme-linked immunosorbent assay or cytometric bead assay.

FIG. 5.

Comparison of results from lymph node samples obtained by either excisional biopsy or FNA. (A) Representative dot plots demonstrating scatter properties and mononuclear staining for samples obtained by excisional biopsy and FNA of the same lymph node are shown. SSC, side scatter; FSC, forward scatter. (B) Cumulative flow cytometry data on the distribution of CD3, CD4, and CD8 T cells in FNA or excisional biopsy (EB) samples from three subjects. (C) Homeostatic chemokine levels as measured by qPCR in lymph node samples obtained by FNA or excisional biopsy from three subjects.

FIG. 6.

CXCR3 levels on bulk CD8 T cells from paired blood and lymph node samples in subjects with chronic or advanced HIV infection. (A) Contour plots of lymphocyte gating (left panels), CD8 T-cell staining (middle panels) of the lymphocyte gated population, and CXCR3 expression of gated CD8 T cells (right panels) in the blood and lymph nodes of representative study subjects with chronic or advanced HIV infection. SSC, side scatter; FSC, forward scatter. (B) Percentages of CD8 T cells expressing CXCR3 in paired blood and lymph node samples of individuals with chronic (n = 6) and advanced (n = 6) HIV infection.

FIG. 7.

Expression levels of CD8 and chemokine mRNA in lymph node tissue from subjects with chronic or advanced HIV infection. Lymph node CD8 (A) and inflammatory chemokine (B) mRNA copy numbers normalized to copy numbers of glyceraldehyde-3-phosphate dehydrogenase mRNA from study subjects with chronic (n = 6) or advanced (n = 6) HIV infection.