Phenotypic and functional characterization of lymphocytes derived from normal and HIV-1-infected human lymph nodes

Abstract

Lymph nodes are the major site of cell-to-cell transmission and replication of HIV-1. Trafficking of CD4+ T lymphocytes into lymph nodes provides a continual supply of susceptible target lymphocytes, and conversely, recruitment of CD8+ T lymphocytes may be critical for the host response that attempts to control HIV-1 replication. The present study was undertaken as no detailed assessment of lymphocyte subpopulations in HIV-1-infected lymph nodes has previously been reported. Peripheral blood and single-cell suspensions prepared from lymph nodes of patients with HIV-1 and control subjects were analysed using three-colour flow cytometry. Approximately 80% of the lymphocytes in control lymph nodes were CD3+ T lymphocytes, of which over 65% were CD4+. The majority of the CD4+ and CD8+ T lymphocytes obtained from both lymph nodes and blood of control subjects were immunologically naive (CD45RA+). By contrast, in HIV-1-infected patients there was a significant reduction in the proportion of CD4+ T lymphocytes and an expansion of the CD8+ T lymphocyte subset in both lymph nodes and peripheral blood. Furthermore, a high proportion of these T lymphocytes displayed a marker for immunological memory (CD45RO+). T lymphocytes derived from HIV-1-infected lymph nodes also showed altered expression of the adhesion molecules, L-selectin and very late antigen-4 (VLA-4), but not leucocyte function-associated antigen-1 (LFA-1). In an in vitro adhesion assay, lymphocytes from HIV-1-infected nodes were significantly more adhesive than control lymphocytes on fibronectin, as well as recombinant human intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) substrates. This combination of altered lymphocyte subpopulations in the HIV-1-infected lymph nodes, as well as enhanced adhesion phenotype and function, suggests that T lymphocyte traffic to lymph nodes in HIV disease may be an important determinant of pathogenesis.

Fig. 1

Representative dot plots illustrating the FACS analysis of lymph node cells derived from a control subject (a–f) and an HIV-1-infected individual (g–l) on the lymphocyte gate as defined by the Leucogate. The numerical values indicated on each quadrant represent the proportions of cells that are single-positive (upper left and lower right quadrants), double-positive (upper right quadrant) and double-negative (lower left quadrant) for the phenotypic markers designated on the abscissa and ordinate of each plot. The cursors for the quadrants were set based on negative control antibodies IgG1–FITC, IgG2a–PE and IgG2b–PerCp conjugated antibodies.

Fig. 2

The proportion of T lymphocyte subsets expressing CD45RA (□) or CD45RO (▪) in blood and lymph node of control subjects (n = 9) and patients (n = 11) with HIV (mean ± s.e.m.).

Fig. 3

The mean ± s.e.m. proportion of T lymphocytes expressing leucocyte adhesion molecules in lymph nodes and peripheral blood obtained from patients infected with HIV-1 (n = 5) and control subjects (n = 10). VLA-4, Very late antigen-4; ICAM-1, intercellular adhesion molecule-1; LFA-1, leucocyte function-associated antigen-1.

Fig. 4

Scatter diagram showing the level of adhesion to fibronectin, rh intercellular adhesion molecule-1 (ICAM-1) or rh vascular cell adhesion molecule-1 (VCAM-1) by lymph node-derived lymphocytes (LNL) and peripheral blood lymphocytes (PBL) obtained from individual HIV-1-infected patients and control subjects (n = 4). Each point represents the mean of triplicate samples and the horizontal bars represent the mean values for each group. The differences among the triplicate wells of each sample were < ± 5%.