Signaling through CD28 and CTLA-4 controls two distinct forms of T cell anergy

Abstract

Primary T cell proliferative responses to TCR ligation plus CD28 costimulation are surprisingly heterogeneous. Many cells that enter G1 fail to progress further through the cell cycle, and some of these cells subsequently fail to divide upon restimulation, even in the presence of IL-2. Such IL-2-refractory anergy is distinct from the IL-2-reversible anergy induced by TCR occupancy in the absence of CD28 costimulation. Here, we focus on the contributions of cell cycle progression and costimulatory (CD28/CTLA-4) signals in the regulation of anergy. We show that CD28 costimulation is not sufficient for anergy avoidance and that activated T cells must progress through the cell cycle in order to escape anergy. Induction of this "division-arrest" form of anergy requires CTLA-4 signaling during the primary response. Also, cell division per se is not sufficient for anergy avoidance: the few T cells that undergo multiple rounds of cell division during overt CD28 costimulatory blockade do not escape the ultimate induction of clonal anergy. Anergy avoidance by primary T cells is thus a multistep process: in order to participate in a productive immune response, an individual T cell activated through its antigen receptor must receive CD28 costimulation and progress through the cell cycle. Anergy may be induced either through a combination of CTLA-4 signaling and the failure of cell cycle progression, or through a proliferation-independent mechanism in which TCR ligation occurs in the absence of CD28.

Figure 1

Regulation of T cell IL-2 production by both B7-mediated costimulation and cell division. (a) Pooled BALB/c lymph node and spleen cells were labeled with CFSE, and T cells were stimulated in the presence of endogenous B7-mediated costimulation by the addition of soluble anti-CD3 antibody (1 μg/ml). (b) Alternatively, B7-mediated costimulation was blocked during primary activation by the addition of CTLA4Ig (15 μg/ml). After 4 days, cultures were washed, plated in fresh medium, and rested for an additional 48 hours. T cells were then restimulated for 5 hours by the addition of polystyrene beads (5 μm) coated with anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) antibodies, and the frequency of CD4⁺ cells producing IL-2 was assessed by flow cytometry. Plots show IL-2 production as a function of cell division (CFSE fluorescence) in the CD4⁺ T cell subset. Vertical lines delineate the divided from the undivided cells, and horizontal lines denote the maximal fluorescence of cells stained with isotype control antibody. Values in each corner represent the proportion of the CD4⁺ events that fall in each quadrant. Cells cultured in medium with no stimulus and stained with specific antibody were less than 0.1% positive for IL-2. These data are representative of three independent experiments. The frequency of IL-2 producers among CD3/CD28-primed T cells varied from experiment to experiment (range: ∼10–35%); however, the frequency of IL-2 producers in cultures primed in the presence of CTLA4Ig was consistently reduced two- to fourfold.

Figure 2

B7-mediated costimulation and cell division differentially regulate secondary T cell proliferation. (a–o) CFSE-labeled spleen and lymph node cells were stimulated with anti-CD3 in the presence of human IgG (a–g; first and second sets of columns in o) or CTLA4Ig (h–n; third and fourth sets of columns in o). Cultures were rested for 48 hours, and Thy1.2⁺ cells that had divided twice (b, d, f, i, k, and m; shaded bars in o) or had remained undivided (c, e, g, j, l, and n; open bars in o) following primary stimulation were purified by FACS. The sorted T cells were cultured with irradiated APCs and restimulated with anti-CD3 in the presence or absence of exogenous IL-2; proliferation was assessed 4 days later by flow cytometry. One representative experiment for each condition (a–g and h–n) is depicted graphically. The mean secondary mitotic events of separate experiments (n = 2 [first and second sets of columns] or n = 4 [third and fourth sets of columns]) are plotted in o. Statistically significant differences were assessed by paired t test and are denoted by brackets: *P < 0.05; ***P < 0.001. (p) Lymph node and spleen cells were cultured with anti-CD3 in combination with anti-CD28 antibody (first and second lanes) or CTLA4Ig (third and fourth lanes). The cultures were rested for 24 hours, and the T cells were restimulated with anti-CD3–coated beads for 48 hours in the presence (second and fourth lanes) or absence (first and third lanes) of IL-2. Live cells were harvested after the primary stimulus (top panels) and after the secondary stimulus (bottom panel) by isolation over Ficoll, and lysates were subjected to immunoblot analysis using antibodies against p27^kip1 (top and bottom panels) or actin (data not shown). The results shown are representative of two independent experiments. (q) Primary, CFSE-labeled T cells were primed with anti-CD3 as in p and rested (top panel), and a portion of the cells were restimulated with either 50 U/ml IL-2 for 48 hours (middle panel) or PMA/ionomycin (PMA/Iono) for 24 hours (bottom panel). The live, CD4⁺ cells were then sorted into fractions that had divided two or more times (right lane, “D”), or had remained undivided during the culture period (left lane, “U”). The cells were lysed, and equal cell equivalents were assessed for p27^kip1 content by immunoblot analysis. The data shown are representative of two independent experiments.

Figure 3

Regulation of primary T cell division by B7–CTLA-4 interactions. (a) Pooled BALB/c lymph node and spleen cells were labeled with CFSE, and T cells were subjected to co-crosslinking of TCR and CD28 by the addition of polystyrene beads coated with anti-CD3 (1 μg/ml), anti-CD28 (1 μg/ml), and control hamster IgG (1 μg/ml). (b) In separate cultures, T cells were subjected to co-crosslinking of TCR, CD28, and CTLA-4 by the addition of polystyrene beads coated with anti-CD3 (1 μg/ml), anti-CD28 (1 μg/ml), and anti–CTLA-4 (1 μg/ml). Proliferation of the CD4⁺ T cell subset was assessed by flow cytometry 3 days later. The frequency of precursor T cells that divided in response to stimulus (R_F), and the number of daughter T cells generated by the average responding precursor T cell (C_P), were calculated as described previously (2, 3). The data are representative of two separate experiments.

Figure 4

Regulation of secondary T cell proliferation by CTLA-4–mediated signal transduction and cell division during the primary response. (a–h) CFSE-labeled splenocytes were stimulated with anti-CD3 in the presence of hamster IgG (first and second sets of columns in h), or either anti–CTLA-4 antibody (10 μg/ml; n = 2) or anti–CTLA-4 Fab (20 μg/ml; n = 2; a–g). Experiments using whole and Fab fragments of anti–CTLA-4 antibody gave similar results and are shown combined as n = 4 experiments (h, third and fourth sets of columns). Cultures were rested for 48 hours, and Thy1.2⁺ cells that had divided twice (b, d, and f; shaded bars in h) or had remained undivided (c, e, and g; open bars in h) following primary stimulation were purified by FACS. The sorted T cells were cultured with irradiated APCs and restimulated with anti-CD3 in the presence or absence of exogenous IL-2, and proliferation was assessed 4 days later by flow cytometry. One representative experiment is depicted graphically in a–g. The mean secondary mitotic events of separate experiments (n = 4, as described above) are plotted in h. The data from the control cultures (first and second sets of columns in h) are the same as depicted in Figure 1. Statistically significant differences were assessed by paired t test and are denoted by brackets: *P < 0.05; **P < 0.01; ***P < 0.001. (i) Lymph node and spleen cells were cultured with anti-CD3 in combination with anti-CD28 (first lane) or anti–CTLA-4 (second lane). The cultures were then rested for 24 hours (first panel), and a portion of the T cells were restimulated with anti-CD3–coated beads (second panel) for 48 hours. In a separate experiment, lymph node and spleen cells were stimulated as above, rested (third panel), and stimulated with IL-2 (50 U/ml; fourth panel) for 48 hours. Live cells were isolated over Ficoll, and lysates were subjected to immunoblot analysis using antibodies against p27^kip1 or actin (fifth panel). The results shown are representative of two independent experiments.