Role of interleukin-2 in superantigen-induced T-cell anergy

Abstract

T-cell anergy is a state of immunological tolerance characterized by unresponsiveness to antigenic stimulation. Previous studies have shown that anergy is induced in T cells following stimulation in the absence of adequate costimulatory signals. These cells fail to respond to stimulation via the T-cell receptor (TCR), and fail to produce normal levels of interleukin-2 (IL-2). We present results here which show that low concentrations of the superantigen staphylococcal enterotoxin A (SEA) in the absence of antigen-presenting cells induced both proliferation and anergy in the A.E7 T-cell clone. Furthermore, under these conditions, the A.E7 clone remained responsive to exogenous IL-2. Fluorescence-activated cellular cytometry analysis revealed unaltered expression of the TCR/CD3 complex in the anergized clone; however, both CD4 and CD25 expression increased after 24 hr of stimulation by SEA under these conditions. Interestingly, a low level of IL-2 production was measured during the induction of anergy. Most strikingly, stimulation of the A.E7 clone by SEA in combination with exogenous IL-2 resulted in a more pronounced state of anergy. These results suggest that the induction of anergy is a process that is essentially independent of the production of IL-2.

Figure 1

The proliferative response to SEA. The A.E7 T-cell clone was cultured with the designated concentrations of SEA for 48 hr in the presence (▴) or absence (•) of irradiated B10.A splenocytes. The results are representative of six independent experiments. The background proliferation for the A.E7 clone was 154 c.p.m. in this experiment.

Figure 2

Dose-dependent induction of anergy with SEA. The A.E7 T-cell clone was stimulated for 48 hr in culture (stage 1) with SEA in the absence of APCs. The cells were washed and re-cultured for two days in medium alone. In the second stage, the T cells were cultured for 48 hr with either medium alone (•), irradiated APCs (▪), SEA (1 ng/ml) presented by irradiated APCs (▴) (top panel), or rhIL-2 (20 U/ml) (▾) (bottom panel), and the proliferative response was determined. The open symbols represent cells that were not stimulated by SEA in the first stage of the assay. These data are representative of three independent experiments. *P < 0·05, †P < 0·01, ‡P < 0·001. The background proliferation of the A.E7 cells, together with APCs (no SEA), was 487 c.p.m. in this experiment.

Figure 3

Failure to modulate CD3 expression during induction of anergy by SEA. The A.E7 clone was stimulated with 25 ng/ml of SEA for 30, 60, 120 or 240 min, and the CD3ε expression was determined. The profile for unstained cells is shown in the shaded peak. Expression of CD3 in unstimulated resting cells is indicated by the zero time point.

Figure 4

Modulation of CD4 and CD25 expression during the induction of anergy. The A.E7 clone was stimulated with SEA (25 ng/ml) for 0, 6, 24 and 48 hr, and the expression of CD4 and CD25 was determined by FACS analysis. The percentage of positive cells and the mean channel fluorescence intensity are shown in the top left and top right corners, respectively, of each panel. The profile for unstained cells is shown in the top panel of both columns.

Figure 5

IL-2 production of A.E7 cells stimulated by SEA. The A.E7 clone was stimulated with the designated concentrations of SEA for 6 or 8 hr in the presence (solid symbols) or absence (open symbols) of irradiated B10.A splenocytes and the level of IL-2 production was determined. Results are representative of three independent experiments.

Figure 6

Failure of IL-2 to prevent anergy induction. (a) The A.E7 clone was cultured in the first stage of the assay with medium, SEA (25 ng/ml), SEA plus rhIL-2 (20 U/ml), or IL-2 alone (20 U/ml) for 48 hr. (b) A.E7 cells were cultured in the first stage with either ECDI-fixed APCs, SEA (25 ng/ml) with ECDI-fixed APCs, or SEA presented by ECDI-fixed APCs plus IL-2 (20 U/ml). (c) A.E7 cells were cultured in the first stage with immobilized anti-CD3 antibody with or without rhIL-2 (20 U/ml) present for 48 hr. The cells were washed and rested for two days before initiation of the second stage, where the proliferative response of the T cells to SEA (1 ng/ml) presented by irradiated APCs (filled bars) or to IL-2 (20 U/ml) (hatched bars) was determined. The results are representative of three independent experiments. *P < 0·05, †P < 0·01, ‡P < 0·001, when compared with cells receiving medium alone in the first stage. §P < 0·001 when compared with cells receiving only SEA or anti-CD3 (no IL-2) in the first stage. The background proliferation of the A.E7 cells together with APCs (no SEA) was 401 c.p.m. in this experiment.

Figure 7

Time course of the effect of IL-2 on anergy induction. A.E7 cells were cultured with medium alone or SEA for 48 hr. At the designated times after initiation of SEA stimulation (0, 16, 24, 40 and 44 hr), IL-2 (20 U/ml) was added to the cultures. At the end of the first stage, the cells were washed and rested for 2 days, and the proliferative response to either SEA (1 ng/ml) presented by irradiated APCs or to rhIL-2 (20 U/ml) was determined. The results are representative of four independent experiments. ‡P < 0·001 when compared with cells cultured without IL-2 in the first stage. The background proliferation of the A.E7 cells together with APCs (no SEA) was 252 c.p.m. in this experiment.

Figure 8

Effect of various concentrations of IL-2 on the induction of anergy. A.E7 cells were cultured with medium alone or SEA either alone or with 1, 10 or 20 U/ml of IL-2 in the first stage. The proliferative response of the A.E7 cells in the second stage to SEA presented by irradiated APCs (solid bars) or IL-2 (hatched bars) was determined. The results are representative of three independent experiments. ‡P < 0·001, when compared with cells receiving medium alone in the first stage. §P < 0·001, when compared with cells receiving only SEA (no IL-2) in the first stage. The background proliferation of the A.E7 cells together with APCs (no SEA) was 1249 c.p.m. in this experiment.