DIFFERENTIAL EFFECTS OF COSTIMULATOR SIGNALS AND... : Transplantation (original) (raw)
The ability to delete graft-specific alloreactive T cells from the recipient peripheral T cell pool offers the potential of permanent graft tolerance without lifelong pharmacologic immunosuppression. While antigen recognition by T lymphocytes has classically been associated with an activation response involving blastogenesis and expression of lymphokines and their receptors, it has recently been recognized that T cell receptor (TCR*) ligation can also induce programmed cell death (PCD). This death response is generally considered to play a major role in immune tolerance, especially in the thymus, where cell death serves to delete potentially autoreactive T cell clonotypes (1). After T cells leave the thymus, proliferation and lymphokine secretion are the predominant responses to TCR engagement. An important finding with respect to allograft tolerance was that activated peripheral T lymphocytes die by PCD upon subsequent TCR cross-linking (2, 3). However, the signal requirements to induce cell death have been shown to be varied based on the results of several studies. In experiments using T cell clones, cell death has been shown to be dependent upon interleukin (IL)-2 (4); however, in other experimental systems, IL-2 may rescue T cells from anergy or death (5) and abrogate peripheral tolerance. The activation state of the T cell may also be of importance in determining susceptibility to TCR-mediated cell death. Cell death has been shown to be dependent upon the phase of the cell cycle for mature T cells(6, 7). Cell cycle phase for mature T cells is modified by T cell activation, which includes TCR ligation, ligation of costimulatory receptors, and lymphokine effects (8). The costimulatory signals mediated by T cell surface molecules CD28 and CTLA-4(and their ligands B7-1 and B7-2) have been shown to be essential requirements for T cell activation (9-11). The absence of proper costimulation during T cell activation has been shown to result in anergy (12) and alloantigen tolerance(13, 14). However, there have been no data regarding the possible role of T cell costimulation in the inhibition of TCR-mediated cell death.
In this study we examined the effects of activation state, IL-2 availability, costimulator, and accessory cell signaling in a model of TCR-mediated cell death of mature, highly purified, CD4+ T cells. We rigorously purified the CD4+ T cells to isolate them from costimulator and accessory cell activity that can enhance endogenous IL-2 production(15) and promote T cell proliferation rather than death. We find that: (a) resting CD4+s die when stimulated by plastic-immobilized anti-TCR/CD3 monoclonal antibody (mAb) in the absence of exogenous IL-2 and accessory cells; (b) activated blasting CD4+s die when restimulated by plastic-immobilized anti-TCR/CD3 mAb in the presence of exogenous IL-2, but die upon IL-2 withdrawal with or without anti-TCR/CD3 stimulation; (c) costimulatory anti-CD28 mAb inhibited cell death of resting CD4+s stimulated by anti-TCR/CD3 mAb in the absence of IL-2, but failed to significantly inhibit cell death of blasting CD4+s induced by TCR/CD3 religation; and (d) splenic adherent cells could inhibit cell death mediated by anti-TCR/CD3 religation in blasting CD4+ T cells, which suggests that accessory cells may provide signals other than CD28 that protect against cell death.
MATERIALS AND METHODS
Antibodies. Purified hamster mAb recognizing the mouse TCR/CD3 complex (145-2C11, specific for the ε-chain of the CD3-TCR complex[16]) was obtained from Accurate Scientific (Westbury, NY). Hamster IgG (Accurate Scientific) was used as control antibody for 2C11. Purified anti-CD28 (murine) mAb was obtained from Pharmingen (San Diego, CA). Hybridoma culture supernatant was used as a source of monoclonal antibody against IL-2 (S4B6-1), CD8/Ly2 (53-6.72), mouse macrophage (F4/80), Iab antigen (M5/114.15.2), murine anti-μ (331.12), and murine anti-NK 1.1 (PK 136). Hybridomas were obtained from American Type Culture Collection(Rockville, MD).
CD4+ cell purification. Spleens were harvested from 4- to 6-week-old C57BL/10 mice (H2b). Single-cell suspensions of spleen cells were depleted of adherent cells during a 90-min incubation at 37 °C on plastic dishes. Nonadherent cells were adjusted to a concentration of 107/ml in RPMI 1640 (Gibco, Grand Island, NY) and incubated with a mixture of mAb supernatant consisting of: 25% anti-CD8 (53-6.72), 25% antimacrophage (F4/80); 25% anti-Ia (M5/114.15.2); and 25% anti-μ (331.12). One milliliter of antibody supernatant was mixed with 107 cells and incubated for 40 min at 4 °C. Cells were washed and then mixed with Dynal magnetic beads (Lake Success, NY) precoated with sheep anti-rat IgG at a bead to cell ratio of 40:1. The cell:bead mixture was subjected to a magnetic field and nonmagnetic binding cells were recovered. A second treatment with magnetic beads was performed at a bead to cell ratio of 20:1, and nonmagnetic binding cells were recovered. Nonbinding cells (CD4+) were further depleted of NK cells by incubation with mAb supernatant PK 136 (1 ml/107 cells) for 40 min at 4 °C. Cells were washed and treated with a 1:10 dilution of rabbit complement (Cedarlane, Hornby, Ontario, Canada) for 40 min at 37°C. CD4+ cells were >95% pure by two-color flow cytometry. CD4+ cells were negative for MAC-1, Ia, and NK1.1 by flow cytometry.
In vitro activation and culture. Culture wells were precoated overnight with 20 μg/ml of anti-TCR/CD3 complex mAb or control hamster IgG in 0.1 M bicarbonate buffer as indicated. For titration of immobilized 2C11, mixtures of mAb and control Ab (20 μg/ml each) were used. A total of 50μl of mAb mixture were plated per well (i.e., 40 μl of 2C11 + 10 μl of HIgG: 80% anti-TCR/CD3 mAb). This approach allows a linear titration of surface density of immobilized anti-TCR/CD3 mAb while maintaining total protein concentration. Anti-CD28 mAb was used at 20 μg/ml and was co-immobilized with anti-TCR/CD3 mAb (2C11) in 0.1 M bicarbonate buffer at a 50/50 v/v ratio. Cells were cultured at 2×105 cells/well in 96-well culture plates in 200 μl of RPMI 1640 supplemented with 10% FCS(Hyclone, Logan, UT), penicillin/streptomycin, and glutamine in 5% CO2. Unless otherwise specified, 25 Cetus U/ml recombinant human IL-2 (Genzyme, Cambridge, MA) were added to all medium. When used, anti-IL-2 mAb supernatant(S4B6-1) was added 25% v/v to culture medium. Primary activation of splenic CD4+s was achieved by 3 days of culture on immobilized anti-TCR mAb, followed by harvesting, washing, and reculturing in wells without antibody for 18 to 24 hr in IL-2-containing medium. Cells were then harvested, washed, and recultured at 2×105 cells/well for 18 to 24 hr on immobilized anti-TCR mAb or control Ig (restimulation).
In some experiments, splenic adherent cells were prepared by culturing splenocytes without IL-2 for 24 hr, removing nonadherent cells, and culturing the remaining adherent cells for 2 to 3 days, with daily removal of nonadherent cells and medium replacement (17). In experiments where such splenic adherent cells were used, polystyrene beads 6μm in diameter (Polysciences, Warrenton, PA) were coated with antibodies(2C11 and HIgG) using a borate buffer protocol provided by Polysciences. The bead to cell ratio used for these experiments was 5:1. Polystyrene beads were observed to be phagocytosed by the splenic adherent cells, but beads were present in sufficient excess to permit abundant TCR/CD3 cross-linking, as evidenced by bead/cell rosetting.
Cell death determination and apoptosis by DNA content analysis. Cell death was determined using trypan blue exclusing and light microscopy. Flow cytometry using an EPICS Profile II (Coulter, Miami, FL) was used to assess cell death by propidium iodide (Sigma, St. Louis, MO) staining of semipermeabilized cells. DNA content < G1(2N DNA) of the cell cycle has been shown to correlate with the oligonucleotide fragments characteristic of apoptosis (18). For DNA content measurements, cells were treated with 0.1% sodium citrate and 0.1% Triton X-100, pH 7.2, and stained with 50 μl/ml propidium iodide. Data are expressed as the mean ± SEM of at least 3 separate experiments.
XTT assay for lymphocyte growth and proliferation. The use of the tetrazolium salt XTT to assay cell proliferation and growth has been described(19). Briefly, 1×105 cells/well are plated in 96-well microtiter plates in 150 μl of medium. XTT/PMS (Sigma) solution(37.5 μl) was added to each well and incubated for 8 hr at 37 °C and 5% CO2. XTT metabolism was determined by reading OD 450 nm on an ELIZA multiscan spectrophotometer. Data are expressed as mean ± SEM of at least 3 experiments.
RESULTS
Highly purified, resting splenic CD4+ T cells die when stimulated by anti-TCR/CD3 mAb in the absence of IL-2, but proliferate if IL-2 is present. As shown in Figure 1, splenic CD4+ T cells were stimulated for 3 days on plastic-immobilized anti-TCR/CD3 mAb(2C11) in the presence or absence of exogenous IL-2. Figure 1A shows cell death as measured by trypan blue exclusion, revealing up to 80% cell death by day 3 of culture when exogenous IL-2 was absent. Conversely, cell death was shown to be below control values when CD4+ cells were stimulated by 2C11 in the presence of IL-2. Figure 1B shows cell growth as measured by XTT metabolism in a 3-day assay of resting splenic CD4+s stimulated by 2C11 in the presence or absence of exogenous IL-2. Over the 3-day period, cell growth was enhanced by 2C11 in the presence of IL-2 but depressed in the absence of exogenous IL-2 compared with controls.
Death of resting splenic CD4+ cells in the absence of IL-2 is apoptotic. Figure 2 shows the results of a 3-day assay where CD4+s were stimulated by anti-TCR/CD3 mAb in the presence or absence of exogenous IL-2, and DNA content analysis was performed by propidium iodide staining and flow cytometry. Apoptotic cell death has been shown to correlate with DNA content less than G1 (2N) when measured by propidium iodide staining and flow cytometric cell cycle analysis (18). As shown in Figure 2A, CD4+s stimulated by 2C11 in the presence of IL-2 show minimal apoptosis (13%) on day 3; cell division is denoted by developing S and G2 phases. In contrast, CD4+s stimulated by anti-TCR/CD3 mAb in the absence of exogenous IL-2 (Fig. 2B) showed 58% apoptotic cell death on day 3, with no evidence of cell division.
Blasting splenic CD4+s die upon restimulation with anti-TCR mAb in the presence of IL-2, while IL-2 withdrawal results in cell death of blasting CD4+s. Figure 3A shows cell death measured by trypan blue exclusion for blasting CD4+s restimulated by 2C11 in the presence and absence of exogenous IL-2. When IL-2 was omitted from culture, anti-IL-2 mAb (S4B6-1) was added to bind endogenously produced IL-2. In the presence of IL-2, cell death occurred in a dose-dependent response to increasing concentrations of anti-TCR/CD3 mAb. However, in the absence of IL-2, cell death was observed throughout the anti-TCR mAb dosage, including in the absence of anti-TCR/CD3 mAb. These data correlate with the growth inhibition seen in Figure 3B using XTT metabolism as the readout. In the presence of IL-2, there was progressive growth inhibition with increasing dose of anti-TCR/CD3 mAb, whereas in the absence of IL-2, growth inhibition was seen throughout, including in the absence of anti-TCR/CD3 mAb. It can also be seen that growth inhibition was significantly greater throughout the dose-response curve in the absence of IL-2. In data not shown, cell death of blasting CD4+s induced by anti-TCR stimulation was shown to be apoptotic by flow cytometric DNA content analysis.
Anti-CD28 mAb inhibits cell death of resting CD4+s stimulated by anti-TCR/CD3 mAb. Figure 4A shows that when resting CD4+s are stimulated by co-immobilized 2C11 and anti-CD28 mAb(50% v/v) in the absence of exogenous IL-2, cell death was significantly inhibited (50%). Addition of anti-IL-2 mAb (S4B6-1, 25% v/v) incompletely inhibited the effect of anti-CD28 by increasing cell death. However, in blasting CD4+s, cell death induced by 2C11 religation, in the presence of IL-2, was not significantly inhibited by co-immobilized anti-CD28 mAb(Fig. 4B).
Cell death induced by TCR/CD3 religation in blasting CD4+ T cells could be inhibited by splenic adherent cells. Figure 5 shows the results of co-culturing splenic adherent cells with CD4+ blasts during restimulation with 2C11 in the presence of exogenous IL-2. Cell death was assessed by trypan blue exclusion as well as flow cytometric analysis of DNA content less than G1. Splenic adherent cells reduced trypan blue-measured cell death by 50% and DNA content less than G1 was similarly reduced compared with controls.
DISCUSSION
Programmed cell death of lymphocytes resulting in negative selection of potentially autoreactive T cell receptors is recognized as integral to the ontogeny of the mammalian immune system (20). However, recent evidence now suggests that mature, postthymic T lymphocytes may still be susceptible to PCD (2, 6, 21, 22). This susceptibility to PCD, particularly via the T cell antigen receptor, suggests that the postthymic immunoreactive potential of the host may be modified. Hypothetically, an avenue may now exist to induce permanent immune tolerance toward an allograft by PCD of postthymic, graft-specific T cells. In this series of experiments, we characterize the conditions under which highly purified mature splenic CD4+ T cells undergo PCD.
T cell activation, in addition to ligation of the TCR itself, requires accessory signals such as CD4, CD8, ICAM-1, CD45, and CD28(23-25). CD4+ cells were highly purified from splenic single-cell suspensions so that TCR-mediated cell death in the absence of accessory or costimulator signals could be studied. InFigure 1, resting CD4+ cells are stimulated by immobilized anti-TCR mAb in the presence and absence of IL-2. When IL-2 is present, cell death is minimal over the 3-day time course(Fig. 1A) and, in fact, cell growth occurs(Fig. 1B). However, in the absence of exogenous IL-2 and accessory signals, cell death (Fig. 1A) and growth inhibition (Fig. 1B) are seen. This is interpreted to suggest that in the resting, unstimulated state, strong TCR signaling in the absence of accessory, costimulator, or IL-2 signals results in PCD. These data are consonant with findings reported by Groux et al.(26) in human medullary thymocytes. InFigure 2 we show by flow cytometry that the cell death seen in Figure 1 exhibits characteristics of apoptosis. These findings support the conclusion that TCR ligation translates a death signal for resting CD4+ T cells in the absence of accessory, costimulator, or IL-2 signals, and that IL-2 can prevent cell death.
Once T cells are activated, religation of the TCR can result in PCD(2, 3). In Figure 3, activated, blasting CD4+ T cells are restimulated with anti-TCR mAb over a range of mAb concentrations and in the presence or absence of IL-2. In the absence of exogenous IL-2, anti-IL-2 mAb (S4B6-1) was added to block the effect of endogenously secreted IL-2. IL-2 withdrawal results in significant cell death and growth inhibition across the mAb doses and in the absence of mAb stimulation. However, when exogenous IL-2 is present, cell death and growth inhibition are dose dependent upon increasing mAb concentrations. In the absence of mAb, minimal cell death is observed with corresponding growth enhancement, as evidenced by increased XTT metabolism. In data not shown, cell death was found to exhibit DNA content less than G1 in cell cycle analysis, consistent with apoptosis (18). In comparison with conditions of IL-2 withdrawal, IL-2 is protective against cell death and growth inhibition at all concentrations of anti-TCR/CD3 mAb, and only at the highest concentration of anti-TCR/CD3 mAb do cell death and growth inhibition approach the levels observed in the absence of IL-2. The finding that IL-2, vis-a-vis its absence, protects activated CD4+ T cells against TCR-mediated cell death is consonant with the findings described above for resting cells. This protective effect of IL-2 in both activated and resting T cells is in contrast to findings obtained in T cell clones where IL-2 was found to promote cell death (4). Furthermore, the finding that resting cells undergo TCR-mediated PCD in the absence of IL-2 suggests that cell cycling is not a requirement for accessing the death pathway(7). Although IL-2 is protective against cell death when compared with conditions of IL-2 withdrawal, it is significant that in the presence of IL-2, anti-TCR/CD3 religation induces cell death and growth inhibition, which suggests that this cell death pathway is not entirely IL-2 dependent.
CD28 has been shown to be an important costimulatory signal for T cell activation. CD28 ligation can block T cell anergy (12), induce tumor immunity (27), and enhance IL-2 production by T cells (15). Blocking of the B7 ligand(s) on accessory cells can prevent graft rejection (28), presumably by preventing costimulation through CD28 or CTLA4 pathways. As shown in Figure 4, the effect of co-immobilized anti-CD28 mAb was tested in the TCR-mediated cell death of resting(Fig. 4A) and blasting (Fig. 4B) CD4+ T cells. In Figure 4A, resting CD4+s were stimulated by 2C11 in the absence of exogenous IL-2, conditions that result in cell death. However, in the presence of co-immobilized anti-CD28 mAb, cell death was significantly reduced to control levels at day 3. When anti-IL-2 mAb was added, the effect of anti-CD28 was incompletely blocked and cell death increased. In contrast, anti-CD28 mAb did not significantly reduce cell death when tested on blasting CD4+s restimulated with anti-TCR/CD3 in the presence of IL-2 (Fig. 4B).
Clearly the role of costimulator signals in TCR-mediated cell death is complex. The data obtained with anti-CD28 mAb in resting CD4+ T cells in the absence of IL-2 (Fig. 4A) suggest that enhanced T cell IL-2 production mediated by CD28 (10, 15, 29) may prevent cell death; however, addition of anti-IL-2 mAb only partially restored cell death, which suggests that (a) CD28 binding may generate signals apart from IL-2 that inhibit cell death or (b) anti-IL-2 may not completely block autocrine IL-2 signals generated by CD28 stimulation. The possible role of non-IL-2 signals blocking TCR-mediated cell death is further supported by (a) the failure of anti-CD28 mAb to significantly prevent cell death of blasting CD4+ T cells in the presence of IL-2, and (b) the ability of splenic adherent cells to inhibit cell death of blasting CD4+s.
IL-12 may be an important costimulator signal influencing TCR-mediated cell death. IL-12 was shown to inhibit Th1 apoptosis in a model of gp 120/CD4-mediated cell death (30) and in human HIV disease (31). IL-12 has been shown to be secreted by macrophages (32). IL-12 has been shown to co-operate with B7 in the induction of T cell proliferation and γ-interferon production (33). When splenic adherent cells were added to cultures of blasting CD4+s undergoing TCR religation in the presence of IL-2 (Fig. 5), splenic adherent cells were shown to inhibit cell death by over 50% and reduce DNA content < G1 (apoptosis) by 25-30%. These results suggest that splenic adherent cells may protect against PCD by a mechanism distinct from IL-2. In addition to cytokines such as IL-12, a possible mechanism may involve blocking cell death mediated by the Fas antigen and its ligand FasL, which have been shown to induce cell death in activated T cells (34, 35).
In summary, these studies describe the results of IL-2, costimulator, and accessory cell signal manipulation on TCR-mediated cell death in two distinct activation states of highly purified CD4+ splenic T cells. Resting T cells, activated through the antigen receptor in the absence of costimulator signals or IL-2 undergo PCD. In vivo, this PCD mechanism may represent a form of postthymic clonal deletion for T cells inappropriately stimulated by tissue self-MHC, a context that would not provide lymphokine or costimulator signals to protect against cell death. Activated T cells may undergo PCD by IL-2 withdrawal and by TCR religation, probably through the Fas-FasL pathway as part of the physiologic down-regulation of the immune response. This T cell PCD is not significantly reversed by the costimulator signal CD28, but is reversed by splenic adherent cells, which suggests that an additional signal(s) may be required in addition to the CD28 pathway for inhibition of cell death. These results have important implications for approaching the problem of transplant tolerance induction by means of antigen-specific PCD. Resting alloreactive cells may be induced to undergo PCD by TCR stimulation only in the absence of accessory signals that prevent IL-2 generation. Once activated, however, alloreactive T cells may require strong TCR religation signals to induce an “overdrive” form of PCD.
Resting splenic CD4+s were stimulated by immobilized anti-TCR/CD3 mAb in the presence or absence of exogenous IL-2 for 3 days. (A) Cell death was measured by trypan blue exclusion. CD4+ cells were assessed at 24, 48, and 72 hr of culture. (B) XTT metabolism (OD 450 nm) was measured for CD4+ cells at the same time points as in panel A. IL-2, when added, was used at 25 Cetus U/ml (2C11+1L-2 = 20 μg/ml immobilized 2C11 + 25 U/ml IL-2; HIg+IL-2 = 20 μg/ml hamster IgG + IL-2; HIg-IL-2 = hamster IgG - IL-2). Results shown are means ± SEM.
DNA content analysis of resting CD4+ spleen cells stimulated by anti-TCR/CD3 mAb. CD4+ spleen cells were stimulated by 2C11 (20 μg/ml) for 3 days in the presence (A) or absence (B) of IL-2 (25 U/ml). DNA content analysis was assessed at 24, 48, and 72 hr. Percentages shown represent DNA content < G1, which was determined by flow cytometry.
Cell death of CD4+ blasts by TCR/CD3 religation. CD4+ blasts were exposed to graded surface concentrations of immobilized anti-TCR/CD3 (20 μg/ml 2C11) in the presence (□) or absence(•) of exogenous IL-2 (25 U/ml). Anti-IL-2 mAb culture supernatant (S4B6-1) was added 25% v/v to cell cultures when IL-2 was omitted. After 20 hr of culture, cell death was measured by trypan blue exclusion (A) and cell growth was measured by XTT mitochondrial metabolism (B). Results are expressed as means ± SEM.
Resting and blasting CD4+s were co-cultured with co-immobilized anti-TCR/CD3 and anti-CD28 mAbs. (A) Resting CD4+s were co-cultured on immobilized anti-TCR/CD3 (20 μg/ml 2C11) and anti-CD28 (20μg/ml), 50% v/v, in the absence of exogenous IL-2. Anti-IL-2 mAb SN(S4B6-1) was added to cultures 25% v/v when present. Cell death, measured by trypan blue exclusion, was assessed at 24, 48, and 72 hr. (B) CD4+ blasts were cultured on co-immobilized 2C11 (20 μg/ml) and anti-CD28 (20μg/ml), 50% v/v, for 20 hr in the presence of 25 U/ml IL-2. Cell death was measured by trypan blue exclusion and percentage of DNA content was measured by < G1 by flow cytometry. HIg, hamster Ig.
Inhibition of apoptotic cell death in CD4+ blasts by splenic adherent cells. Splenic adherent cells were prepared as described in_Materials and Methods_. CD4+ blasts were cultured for 24 hr on immobilized 2C11 (2C11-plastic) or immobilized HamIgG (HIg-plastic) as positive and negative controls for cell death, respectively. TCR cross-linking was accomplished by 2C11-coated polystyrene beads (2C11-beads), which were added to co-cultures of CD4+ blasts and splenic adherent cells (SAC). Hamster IgG was similarly coated on polystyrene as a negative control(HIg-beads). Cell death was measured by trypan blue exclusion and DNA content< G1 as an index of apoptosis. Unpaired t test for means of 2C11-beads+SAC versus 2C11-beads was significant with_P_<0.0001.
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