Toll-like receptor ligands induce human T cell activation and death, a model for HIV pathogenesis

Abstract

Background: Recently, heightened systemic translocation of microbial products was found in persons with chronic HIV infection and this was linked to immune activation and CD4(+) T cell homeostasis.

Methodology: We examined here the effects of microbial Toll-like receptor (TLR) ligands on T cell activation in vitro.

Conclusions/findings: We show that exposure to TLR ligands results in activation of memory and effector CD4(+) and CD8(+) T cells. After exposure to each of 8 different ligands that activate TLRs 2, 3, 4, 5, 7, 8, and 9, CD8(+) T cells are activated and gain expression of the C type lectin CD69 that may promote their retention in lymphoid tissues. In contrast, CD4(+) T cells rarely increase CD69 expression but instead enter cell cycle. Despite activation and cell cycle entry, CD4(+) T cells divide poorly and instead, disproportionately undergo activation-induced cell death. Systemic exposure to TLR agonists may therefore increase immune activation, effector cell sequestration in lymphoid tissues and T cell turnover. These events may contribute to the pathogenesis of immune dysfunction and CD4+ T cell losses in chronic infection with the human immunodeficiency virus.

Figure 1. Stimulation of peripheral blood cells by Toll-like Receptor ligands increases expression of CD38 on CD4+ and CD8+ T lymphocytes.

PBMC were cultured overnight in medium alone, or stimulated with individual TLR ligands (Poly-I:C, LPS, Flagellin, or CpG DNA) or with plate-bound anti-CD3 antibody. Expression of CD38 and HLA-DR was monitored by flow cytometry among: (A) CD4+ T cells and (B) CD8+ T cells. Percentages of cells expressing each marker are shown. This experiment is representative of 5.

Figure 2. TLR ligands induce high level CD69 expression on CD8+ T cells and Ki- 67 expression in CD4+ T cells.

Intracellular expression of Ki-67 (A) and surface expression of CD69 (B) were monitored after 7 days of cell culture in medium or with the stimuli as indicated. Bars represent means and the lines standard errors of the mean (SEM) of 15 separate experiments using PBMC of healthy controls. Black boxes = CD4+ T cells and gray boxes = CD8+ T cells. * = nominally significantly different (p<0.05 by Wilcoxon Sign Rank test when compared to results obtained in medium alone.

Figure 3. TLR ligands preferentially activate CD4+ central memory and effector memory T cells and CD8+ effector memory T cells.

Intracellular Ki-67 expression (3A, 3C) and cell surface CD69 (3B, 3D) were analyzed after 7 days' incubation of PBMC in medium alone, or in medium supplemented with plate bound anti-CD3 antibodies, or the indicated TLR ligand. Figures 3A and B are representative results among phenotypically defined naïve (CD45RA⁺CD45RO⁻CCR7⁺), central memory (CD45RA⁻CD45RO⁺CCR7⁺), and effector memory (CD45RA⁻CD45RO⁺CCR7⁻) CD4⁺ and CD8⁺ T cells. Values represent percentages of cells staining for Ki-67 or CD69. Figures 3C and 3D reflect the mean data from 15 separate experiments. Values nominally significantly different from medium alone values (Wilcoxon Sign Rank Test) are shown with an asterisk.

Figure 4. Toll-like receptors 3 and 5 are expressed by a subset of T cells, but expression is not always sufficient for activation by TLR ligands.

(A) Whole PBMCs were isolated from healthy donors and expression of TLRs 2, 3, and 5 were evaluated by flow cytometry on gated CD3+ T cells. (B) Whole PBMCs, purified T cells (>95% CD3⁺), or purified T cells separated from whole PBMCs by a transwell, were cultured in medium alone, or in medium supplemented with plate bound anti-CD3, poly I:C or flagellin A. Expression of CD38 on memory (CD45RO⁺CD45RA⁻) CD4⁺ T cells was assessed by flow cytometry following overnight culture. Dotplots shown are representative of 8 separate experiments.

Figure 5. Activation as a result of TLR ligand exposure preferentially induces T cell death especially among CD4+ T cells.

PBMCs were labeled with CFSE and incubated for 6 days in the presence of anti-CD3 antibody, medium alone or TLR ligands alone (PGN, poly-I:C, LPS (10 or 20 ng/ml), Flagellin A, Flagellin B or imiquimod). After 6 days of incubation, the CD4⁺ and CD8⁺ T cells were examined for dilution of CFSE dye and for binding of Annexin V. Numbers in right upper corners represent percentage of Annexin V-binding cells. Numbers in the left lower corner represent percentage of cells that diluted dye without Annexin-V binding. This experiment is representative of three separate experiments.