Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

Abstract

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.

Figure 1

A phenotypic profile and the purity of each sorted lymphocyte subset. The numbers in each profile indicate the percentages of cells within indicated quadrants.

Figure 2

CD3-stimulated IFN-γ production and antitumour cytotoxicity in the T cell subsets. (a) IFN-γ production. Each purified T-cell population was stimulated with anti-CD3 antibody for 48 hr and the IFN-γ levels of the culture supernatants were subjected to ELISA. (b) Cytotoxic activity against K562 cells. (c) Cytotoxic activity against Raji cells. Each purified cell population was stimulated with anti-CD3 antibody for 4 days and cytotoxic assays were performed. All data represented are the means± SE from eight independent experiments.

Figure 3

Cytokine-stimulated IFN-γ production and antitumour cytotoxicity in various lymphocyte subsets, and enhancement of the cytotoxicity against Raji cells by blocking NKG2A. (a) IFN-γ production. Each purified population was stimulated with a combination of IL-2, IL-12 and IL-15 for 48 hr and IFN-γ levels of the culture supernatants were subjected to ELISA. Cytotoxic activity against K562 cells (b) or Raji cells (c). Each purified cell population was stimulated with cytokines for 4 days and cytotoxic assays were performed. All data represented are the means± SE from five to eight independent experiments. (d) The augmentation of the cytotoxicity against Raji cells of cytokine-stimulated NK cells by blocking NKG2A. Purified NK cells were cultured for 4 days and incubated with NKG2A antibody (200 µg/ml) (mouse IgG2b antibody as a control) at 4° for 10 min and washed twice and then cytotoxic assays were performed. The data represented are the means± SE from four independent experiments.

Figure 4

The expression of cytoplasmic perforin and granzyme B in NK cells and both NK-type T cells after cytokine stimulation. (a) 72 hr after stimulation of PBMC with a combination of IL-2, IL-12 and IL-15, either cytoplasmic perforin or granzyme B of CD57⁺ cells, CD56⁺ cells and normal CD8⁺ cells were examined. (b) A time course analysis of the proportion of either cytoplasmic perforin- or granzyme-B-expressing cells in each lymphocyte subset. By gating each subset in the three-colour flow cytometric analysis, the percentage of perforin- or granzyme-B-positive cells in each subset was examined after cytokine stimulation at the indicated time points (n = 4).

Figure 5

CD3-stimulated proliferation and apoptosis of each T-cell subset. Each T-cell population was stimulated with immobilized anti-CD3 antibody for the indicated hours and the cells were pulsed with [³H]thymidine for 12 hr before the cells were harvested (a). Representative results are shown from repeated experiments with similar results. Each T-cell population was stimulated for 48 hr and stained with propidium iodide (PI) and FITC–annexin V and then was analysed by flow cytometry (b). Representative results were shown from repeated experiments with similar results.

Figure 6

NK-type T cells as a major source for the production of IFN-γ in CD3-stimulated PBMC. NK-type T-cell-depleted PBMC were cultured with anti-CD3 antibody for 48 hr and their IFN-γ production capacities were compared with those of whole PBMC. All data represented are the means ± SE from four independent experiments.

Figure 7

The proportions of NK cells and NK-type T cells increase proportionally with age. The data are based on the PBMC obtained from 56 individuals.

Figure 8

The IFN-γ production capacity of PBMC correlates either with the age or the proportion of CD57⁺ T cells. The data are based on the findings of several independent experiments from 32 individuals.

Figure 9

T-cell receptor β repertoire of CD56⁺ T cells, CD57⁺ T cells and normal CD8⁺ T cells.PBMC from four individual healthy volunteers were stained as described in Materials and Methods. % of Vβ T cells in CD56⁺ T cells = (% CD56⁺ Vβ T cells/% CD56⁺ αβ T cells) × 100; % of Vβ T cells in CD57⁺ T cells = (% CD56^– CD57⁺ Vβ T cells/% CD56^– CD57⁺ αβ T cells) × 100; % Vβ T cells in CD56^– CD57^– αβ T cells (normal CD8⁺ T cells) = (% CD56^– CD57^– Vβ T cells/% CD56^– CD57^– αβ T cells) × 100.