CD28null T cells are a highly enriched subset of proinflammatory T cells in patients with autoimmune diseases that are oligoclonal and autoreactive. In this study, we analyzed the role of CD152 signaling on the longevity of human CD28null T cells. Using a sensitive staining method for CD152, we show that human CD4+CD28null and CD8+CD28null T cells rapidly express surface CD152. Serological inactivation of CD152 using specific Fab or blockade of CD152 ligands using CTLA-4Ig in CD4+CD28null and CD8+CD28null T cells enhances apoptosis in a Fas/FasL-dependent manner. CD152 cross-linking on activated CD28null cells prevents activation-induced cell death as a result of reduced caspase activity. Apoptosis protection conferred by CD152 is mediated by phosphatidylinositol 3′-kinase-dependent activation of the kinase Akt, resulting in enhanced phosphorylation and thereby inhibition of the proapoptotic molecule Bad. We show that signals triggered by CD152 act directly on activated CD28null T lymphocytes and, due to its exclusive expression as a receptor for CD80/CD86 on CD28null T cells, prevention of CD152-mediated signaling is likely a target mechanism taking place during therapy with CTLA-4Ig. Our data imply strongly that antagonistic approaches using CD152 signals for chronic immune responses might be beneficial.

The elderly and patients suffering from immunopathologies such as multiple sclerosis, Wegner’s granulomatosis, ankylosing spondylitis, and rheumatoid arthritis (RA)4 show expanded populations of CD4+ and CD8+ T cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Those clonally expanded T cells have lost the expression of CD28 molecules, indicating a shift in their dependence on CD28-mediated costimulation. CD4+CD28null T cells are infrequent in healthy individuals (0.1–3.4% of T cells) but, most interestingly, numbers can increase to up to >10% corresponding with severity of RA, especially with extra-articular manifestation and advanced joint destruction (1). The limited TCR diversity of CD4+CD28null populations in RA patients suggests that these T cells recognize a limited spectrum of Ags which have been shown to be putative autoantigens (11). Suggestive evidence shows that CD8+CD28null T cells show suppressive capacity in a mouse model of RA (12, 13). However, CD4+CD28null T cells are incapable of providing B cell help, show cytolytic activity, and produce high levels of IL-2 and IFN-γ, giving them the ability to function as proinflammatory cells (7, 14, 15). Thus, they likely contribute to and stabilize the Th1 response, which is the underlying immunological process driving immunopathologies such as of RA.

Apoptosis of Ag-experienced T cells is of particular importance for the organism to ensure that an ongoing inflammation does not continue after the clearance of the pathogen. The primary form of apoptosis of clonally expanded T cells is activation-induced cell death (AICD), which is mainly controlled by the Fas (CD95) system (16, 17). Fas-mediated AICD is considered the primary mechanism for deletion of mature CD4+ T cells in the periphery, whereas it is not involved in thymic-negative selection (18). In the absence of appropriate costimulation, TCR signaling induces Fas and Fas ligand (FasL) expression (19). Ligation of Fas initiates the recruitment of Fas-associated death domain protein and caspase 8, which then triggers the proteolytic caspase cascade, resulting in the cleavage of various proteins and finally apoptotic cell death (20, 21, 22). Several mechanisms exist to counterregulate death processes either at the receptor, mitochondrial, or caspase level. One survival mechanism is mediated by phosphatidylinositol 3′-kinase (PI3′K) which activates Akt/protein kinase B, an antiapoptotic kinase that inactivates proapoptotic molecules such as Bad and the forkhead transcription factor FKHRL1 (23, 24). CD152 costimulatory signaling has been shown to down-regulate Fas and FasL expression and to up-regulate the antiapoptotic molecule Bcl-2 during an immune response (25). This response is mediated by activation of PI3′K and reduced phosphorylation of FKHRL1 (25). Importantly, some effector T cells survive and contribute to the generation of long-term memory (26). CD28null T cells are long-lived and persist for years in RA patients and unaffected siblings (27). Their longevity is attributed to resistance against apoptosis which correlates with enhanced expression of the antiapoptotic molecule Bcl-2 (28). Their persistence might contribute to long-term memory driving the inflammatory process.

CD28 and its homolog CD152 (CTLA-4) are located in close proximity to each other on chromosome 2 (29). In contrast to CD28, CTLA-4 (CD152) is a major down-regulator of immune responses (30, 31, 32, 33). CD152, which shares its ligands with CD28, mediates inhibition of IL-2 transcription and cell cycle progression (31), even in previously activated T cells (34). CD152 has been reported to bind to PI3′K and the phosphatases PP2A and SHP-2 (35, 36, 37, 38, 39). The coupling of CD152 to signaling mediators along with the activation of PI3′K and Bcl-2 indicates that CD152 indeed initiates specific signaling pathways in addition to competing with CD28 for B7 ligands. In CD4+CD28null T cells an “initiator complex” consisting of the nuclear proteins nucleolin and hnRNP-D0A whose binding to the CD28 promoter is necessary for CD28 expression is lost (9, 40). How this phenomenon effects the expression of CD152 or the responsiveness toward CD152-mediated signals is not known.

In this study, we investigated the expression and functional role of CD152 on individual primary CD28null cells using a unique sensitive immunofluorescent labeling technique. We show for the first time that following stimulation, CD152 surface expression is indeed induced at high frequencies in CD4+CD28null as well as CD8+CD28null T cells. CD152 inhibition leads to enhanced proliferation showing that CD152 signaling is controlling homeostasis. Interestingly, inhibition of CD152 sensitized CD28null cells for apoptosis. We identified that apoptosis protection by CD152 was dependent on the suppression of the Fas/FasL system and is mediated by PI3′K-dependent activation of the kinase Akt, leading to inhibition of the proapoptotic molecule Bad. Thus, CD152 regulates cell cycle progression and AICD by targeting Akt. This novel activity of CD152 on CD28null T cells could explain their resistance to apoptosis and their persistence and accumulation in the organism and may be important for the etiology of autoimmune diseases.

The following Abs against human Ags were used: anti-CD152 (BN13), anti-CD4 (TT1), and anti-CD8 (GN11/134D7) were purified from hybridoma supernatants with protein G and controlled by HPLC and FACS analysis and used in their respective form of FITC, PE, and Cy5 conjugates. Anti-CD28 (CD28.2), anti-CD3 (UCHT1), anti-Bcl-2 (6C8), anti-Fas (G247-4), and anti-FasL (NOK-1) were purchased from BD Pharmingen, anti-phospho-Akt (Ser473; 193H12), anti-Akt (pan; C67E7), and anti-phospho-Bad (Ser112; 40A9) were purchased from New England Biolabs. Human CTLA-4Ig (Abatacept) was supplied by Bristol-Myers Squibb. Anti-CD152 Fab were prepared with the Immunopure Fab preparation kit (Pierce) and used at 50 μg/ml. The Fab were analyzed by HPLC. The anti-FasL Ab was used at a concentration of 50 μg/ml in neutralization experiments. Control Abs such as mouse IgG2a and annexin-V-PE or annexin-V-FITC conjugates were purchased from BD Pharmingen. Magnetic microbeads anti-CD4, anti-CD62L, anti-CD90, and anti-FITC multisort microbeads were purchased from Miltenyi Biotec. Sulfate polystyrene latex microspheres of 5 ± 0.1 μm in diameter were obtained from Interfacial Dynamics. The IDO inhibitor 1-methyl-dl-tryptophan was purchased from Sigma-Aldrich and used at 200 μM. The PI3′K inhibitor LY-294002 hydrochloride was purchased from Sigma-Aldrich and used at a concentration of 30 μM.

Isolation of CD4+CD28null and CD8+CD28null cells from peripheral blood of healthy donors was performed by preparing PBMCs by density gradient centrifugation followed by a positive selection of CD14+ APCs with anti-CD14 Microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. The CD4+CD28null and CD8+CD28null cells were then FACS sorted from the CD14 fraction by FACSAria (BD Biosciences). CD4+CD28null and CD8+CD28null were cultured with irradiated CD14+ cells as APCs in a ratio of 1:2 (T cell:APC) at a concentration of 2–4 × 106 total cells/ml in RPMI 1640 (Invitrogen) supplemented with 10% human serum and stimulated by 1 μg/ml anti-CD3. In CD152 cross-linking experiments, CD4+CD28null or CD8+CD28null cells were first stimulated by 20 μg/ml Con A or 10 μg/ml anti-CD3 for 4 days and restimulated by the addition of Ab-coupled polystyrene latex microspheres. These microspheres were coupled with either 2 μg/ml anti-CD3 and 3 μg/ml isotype control Ab or with 2 μg/ml anti-CD3 and 3 μg/ml anti-CD152 as described before (25).

Surface expression of CD152 was detected using magnetofluorescent liposomes (34). T cells were incubated with unconjugated hamster anti-CD152 Ab at a concentration of 1 μg/ml for 15 min at 4°C. Then, cells were washed twice and incubated with Cy5-liposomes, conjugated to anti-ham Fab for 30 min at 4°C. Unbound liposomes were removed by washing the cells twice with PBS-BSA. The specificity of CD152 staining was controlled by isotype control Ab conjugated with Cy5-liposomes as well as by incubation of cells with Cy5-liposomes only. Cytometric analyses were performed using a FACSCalibur (BD Biosciences) and CellQuest (BD Biosciences) or FlowJo software (Tree Star). Dead cells were excluded by forward and sideward scatter gating and propidium iodide (PI) staining.

Cell cycle progression was measured by labeling of T cells with CFSE (Molecular Probes). In brief, 1 × 107 cells/ml were washed in PBS and stained with CFSE (5 μM in PBS/0.1% BSA) for 6 min at room temperature in the dark. The reaction was stopped by resuspending the cells in RPMI 1640 medium. To measure cell death, cells were stained with annexin V-PE or annexin V-FITC (BD Pharmingen) and PI according to the manufacturer’s instructions to exclude early apoptotic and late apoptotic/dead cells, respectively. Routinely, staining was controlled by calcium chelation with 2 mM EGTA. Active caspases were stained with CaspACE FITC-VAD-FMK (Promega). Two × 106 cells/ml were incubated with 10 μM FITC-VAD-FMK for 30 min at 37°C.

In this study, we show that among CD8+CD28null as well as CD4+CD28null cells from peripheral blood of healthy donors a high percentage of cells express IFN-γ (Fig. 1,A). With 40–77% of cells expressing IFN-γ in CD4+CD28null and CD8+CD28null, respectively, the frequencies are 2- to 3-fold higher compared with CD28+ cells. With regard to the expression of the proinflammatory cytokine IL-17, there is no difference discernable between CD28null and CD28-competent cells (Fig. 1,A). To further characterize ex vivo CD28null cells, we analyzed the intracellular expression of CD28-related molecule CD152. There was no CD152 detectable in CD4+CD28null cells (Fig. 1,B, middle panel) or CD8+CD28null cells ex vivo (Fig. 1,B, lower panel). A positive control of intracellular CD152 staining in stimulated total CD4+ cells is shown (Fig. 1,B, upper panel). Next, we analyzed whether CD28null T cells are able to express surface CD152 upon stimulation, since only the surface-expressed CD152 is functional (34). We used our sensitive fluorescent liposome-based staining technique (25, 34) to detect CD152 on CD28null cells from healthy donors 48 h after stimulation with anti-CD3 (Fig. 1,C). As shown in Fig. 1,C, ∼5% of all CD4+ cells do not express CD28. Of these CD28null cells, almost 30% express CD152 on the cell surface (Fig. 1,C, upper panel). Among the CD8+ population of PBMCs, ∼40% of the cells lack the expression of CD28 at the surface. Of these CD8+CD28null cells, 20% are capable of expressing CD152 at the plasma membrane (Fig. 1,C, lower panel). The kinetics of surface CD152 expression is similar in CD4+CD28null and CD8+CD28null T cells, with the peak expression 48 h after onset of stimulation (Fig. 1 D). Surface CD152 expression is higher in CD4+CD28null cells with an average of 20% surface CD152- positive cells compared with CD8+CD28null cells where 15% are positive for surface CD152 after 48 h of activation.

FIGURE 1.

CD4+CD28null and CD8+CD28null cells express CD152 at their cell surface. A, PBMCs of healthy donors were ex vivo stimulated with PMA and ionomycin for 6 h. Expression of IL-17 and IFN-γ was analyzed by gating on CD8+CD28+, CD8+CD28null, CD4+CD28+, or CD4+CD28null cells. B, Intracellular expression of CD152 in CD28null and CD28+ cells was analyzed in ex vivo PBMCs (middle and lower panels) or in PBMCs stimulated with anti-CD3 for 48 h gated on total CD4+ cells (upper panel). The numbers indicate the percentage of CD152-positive cells. C, PBMCs of healthy donors were stimulated with anti-CD3 (1 μg/ml) for 48 h. Expression of CD4, CD8, and CD28 was analyzed by conventional staining. Surface CD152 expression was analyzed by liposome-enhanced staining and controlled by blocking with 50-fold excess of unconjugated anti-CD152 Ab. Shown is the surface CD152 staining of CD4+CD28null (upper row) and CD8+CD28null cells (lower row) by gating with the indicated gates. The numbers indicate the percentage of surface CD152-positive cells. D, Kinetics of surface CD152 expression on CD4+CD28null and CD8+CD28null cells. PBMCs of healthy donors (n = 5) were stimulated as described in A and surface CD152 expression was analyzed at the indicated time points after stimulation by liposome-enhanced staining. Depicted is the percentage of surface CD152-positive cells.

FIGURE 1.

CD4+CD28null and CD8+CD28null cells express CD152 at their cell surface. A, PBMCs of healthy donors were ex vivo stimulated with PMA and ionomycin for 6 h. Expression of IL-17 and IFN-γ was analyzed by gating on CD8+CD28+, CD8+CD28null, CD4+CD28+, or CD4+CD28null cells. B, Intracellular expression of CD152 in CD28null and CD28+ cells was analyzed in ex vivo PBMCs (middle and lower panels) or in PBMCs stimulated with anti-CD3 for 48 h gated on total CD4+ cells (upper panel). The numbers indicate the percentage of CD152-positive cells. C, PBMCs of healthy donors were stimulated with anti-CD3 (1 μg/ml) for 48 h. Expression of CD4, CD8, and CD28 was analyzed by conventional staining. Surface CD152 expression was analyzed by liposome-enhanced staining and controlled by blocking with 50-fold excess of unconjugated anti-CD152 Ab. Shown is the surface CD152 staining of CD4+CD28null (upper row) and CD8+CD28null cells (lower row) by gating with the indicated gates. The numbers indicate the percentage of surface CD152-positive cells. D, Kinetics of surface CD152 expression on CD4+CD28null and CD8+CD28null cells. PBMCs of healthy donors (n = 5) were stimulated as described in A and surface CD152 expression was analyzed at the indicated time points after stimulation by liposome-enhanced staining. Depicted is the percentage of surface CD152-positive cells.

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Since the main function of CD152 on activated CD4 T cells is traditionally seen to be the control of cell cycle progression (31, 41), we next analyzed whether CD152 controls cell cycle progression of activated CD28null T cells. Experiments were conducted with FACS-enriched CD28null T cells. CD4+CD28null cells which constitute ∼1% of all PBMCs and 3–5% of CD4+ cells, as well as CD8+CD28null cells which correspond to ∼8% of all PBMCs and 30–40% of CD8+ T cells, were isolated by FACS enrichment to a purity of >95% (Fig. 2,A). CD28null T cells were activated with anti-CD3 and APCs in the presence or absence of Ab blockade of CD152 with specific anti-CD152 Fab. The usage of monovalent anti-CD152 Fab instead of whole Abs excludes the possibility of unintentional cross-linking of CD152. Inhibition of CD152 on CD28null T cells had no observable effect on their proliferation up to 48 h after stimulation, neither for CD4+CD28null nor CD8+CD28null T cells (Fig. 2,B). Dramatic increases in frequencies of proliferating cells were seen in CD4+CD28null cells 120 h after beginning the stimulation, which increased from 41 to 61%. The effect was even more pronounced for CD8+CD28null cells where the number of cycling cells increased from 31 to 54% when CD152 was inactivated. In both cases, the 20–30% enhanced frequencies of proliferating cells in the presence of CD152 blockade correlates well with the frequency of surface CD152-expressing CD28null cells (Fig. 1). These results indicate that signals originating from CD152 are responsible for cell cycle arrest in CD28null T cells (31, 41). This also indicates that CD152 tightly controls the proliferation of these quiescent CD4+CD28null and CD8+CD28null T cells.

FIGURE 2.

Serological blockade of CD152 enhances proliferation. A, Purity of CD28null FACS-sorted populations. Shown are the frequencies of CD4+CD28null cells (upper panel) and CD8+CD28null cells (lower panel) after FACS sort. B, CD4+CD28null (upper panel) and CD8+CD28null (lower panel) cells were FACS sorted, labeled with CFSE, and stimulated with 1 μg/ml anti-CD3 in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. Proliferation was measured 48 and 120 h after stimulation.

FIGURE 2.

Serological blockade of CD152 enhances proliferation. A, Purity of CD28null FACS-sorted populations. Shown are the frequencies of CD4+CD28null cells (upper panel) and CD8+CD28null cells (lower panel) after FACS sort. B, CD4+CD28null (upper panel) and CD8+CD28null (lower panel) cells were FACS sorted, labeled with CFSE, and stimulated with 1 μg/ml anti-CD3 in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. Proliferation was measured 48 and 120 h after stimulation.

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A hallmark of CD28null T cells is their longevity, which is due to resistance to apoptosis (42, 43). In this study, we examined the role of CD152 for the induction of AICD, the major form of apoptosis for CD4 T cells, in CD4+CD28null and CD8+CD28null T cells (Fig. 3). CD152-mediated signaling was blocked by the addition of anti-CD152 Fab to CD28null cells stimulated with anti-CD3 and APCs. The frequency of non- and apoptotic cells was determined by double staining with PI and annexin V. PI was used to assess plasma membrane integrity in dead and late apoptotic cells. Annexin V binds phosphatidylserine (PS), which is exposed at the outer leaflet of the plasma membrane at early stages of apoptosis. The binding of annexin V to PS is dependent on Ca2+ so that the specificity of the interaction can be controlled by using a Ca2+-chelating EGTA-containing buffer (Fig. 3). As shown in Fig. 3,A, there is an increase by 25% of apoptotic CD8+CD28null cells at day 4 after stimulation when CD152 was neutralized. An increase in apoptotic cells was also visible on day 7. A similar effect was observed in CD4+CD28null cells where the frequency of apoptotic cells rose by 20% from 11.7% to 30.1% only 2 days after the onset of stimulation when CD152 was blocked (Fig. 3,B). This enhanced frequency of apoptotic T cells was similar on day 4 after stimulation when the frequency of annexin V-positive cells doubled from ∼20 to 40% (Fig. 3 B). Thus, in the presence of functional CD152 less apoptosis occurred, supporting the concept that CD152 could be involved in their resistance to apoptosis.

FIGURE 3.

CD152 blockade enhances AICD in both CD4+CD28null and CD8+CD28null T cell subsets. A, CD8+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On days 4 and 7, AICD was analyzed by staining the cells with annexin V. B, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On days 2 and 4, apoptosis was analyzed by staining the cells with annexin V.

FIGURE 3.

CD152 blockade enhances AICD in both CD4+CD28null and CD8+CD28null T cell subsets. A, CD8+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On days 4 and 7, AICD was analyzed by staining the cells with annexin V. B, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On days 2 and 4, apoptosis was analyzed by staining the cells with annexin V.

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A prerequisite for apoptosis is the activation of caspases. Activated caspases were detected by staining of cells with the fluorescence-labeled inhibitor of caspases VAD-FMK and subsequent cytometric analysis. As shown in Fig. 4,A, the serological inactivation of CD152 by anti-CD152 Fab during stimulation leads to a marked increase in cells expressing active caspases as compared with CD28null cells being stimulated in the presence of control Fab. The frequencies of activated CD8+CD28null cells expressing activated caspases increase at day 7 after stimulation from 45 to 54% when CD152 is inactivated (Fig. 4,A, upper panel). Also, in CD4+CD28null cells, a 4- to 5-fold increase in cells expressing active caspases from 7.5 to 33.8% is observable (Fig. 4,A, lower panel). Similar results were obtained on day 4 after onset of stimulation (data not shown). This result indeed corroborates our data obtained with annexin V binding (Fig. 3,B) and illustrates that CD152 is responsible for the resistance against apoptosis in CD28null T cells. Interestingly, enhanced proliferation induced by CD152 blockade was not the cause of enhanced apoptosis, as we show in Fig. 4 B that serological blockade of CD152 enhanced frequencies of annexin V-binding cells dramatically in proliferating as well as in nonproliferating cells.

FIGURE 4.

CD152 engagement in CD28null T cells inhibits apoptosis independent of proliferation. A, CD4+CD28null and CD8+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On day 7, apoptosis was analyzed unambiguously by staining active caspases with the FITC-labeled caspase inhibitor VAD-FMK. B, PMBCs were stained with CFSE and stimulated with anti-CD3 (1 μg/ml) for 5 days. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On day 5, proapoptotic CD4+CD28null T cells were identified by cytometric detection of annexin V binding. Top panels show annexin V binding cells of all CD4+CD28null cells. Middle panels show CFSE analysis (filled curve, nonstimulated cells; nonfilled curve, stimulated cells). Percentage of proliferating cells is indicated. Lower panels show proapoptotic cells (annexin V+) of proliferating and nonproliferating CD4+CD28null T cells. C, CD4+CD28null cells were FACS sorted and stimulated with Con A (10 μg/ml) for 60 h. Cells were restimulated either with anti-CD3 + isotype or anti-CD3 + anti-CD152 coupled to microspheres (2 μg/ml anti-CD3 and 3 μg/ml isotype or anti-CD152) for 4 days. Apoptosis was analyzed by staining with the FITC-labeled caspase inhibitor VAD-FMK.

FIGURE 4.

CD152 engagement in CD28null T cells inhibits apoptosis independent of proliferation. A, CD4+CD28null and CD8+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On day 7, apoptosis was analyzed unambiguously by staining active caspases with the FITC-labeled caspase inhibitor VAD-FMK. B, PMBCs were stained with CFSE and stimulated with anti-CD3 (1 μg/ml) for 5 days. CD152 blocking anti-CD152 Fab or isotype Fab were used at a concentration of 50 μg/ml. On day 5, proapoptotic CD4+CD28null T cells were identified by cytometric detection of annexin V binding. Top panels show annexin V binding cells of all CD4+CD28null cells. Middle panels show CFSE analysis (filled curve, nonstimulated cells; nonfilled curve, stimulated cells). Percentage of proliferating cells is indicated. Lower panels show proapoptotic cells (annexin V+) of proliferating and nonproliferating CD4+CD28null T cells. C, CD4+CD28null cells were FACS sorted and stimulated with Con A (10 μg/ml) for 60 h. Cells were restimulated either with anti-CD3 + isotype or anti-CD3 + anti-CD152 coupled to microspheres (2 μg/ml anti-CD3 and 3 μg/ml isotype or anti-CD152) for 4 days. Apoptosis was analyzed by staining with the FITC-labeled caspase inhibitor VAD-FMK.

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In contrast to blockade of CD152 by anti-CD152 Fab, which leads to an increase in apoptosis, signals derived from CD152 then in turn should lead to a reduced number of cells undergoing apoptosis. Following stimulation of CD4+CD28null T cells with Con A for 3 days to ensure activation of all T cells, the CD4+CD28null cells received either a CD152 signal or no signal. This was approached by stimulating CD4+CD28null cells with microspheres that have been coupled with either anti-CD3 and isotype-matched control Ab (IgG2a) or anti-CD3 and anti-CD152. This ensures that in the first case the cells receive no signal via CD152 or in the second case the cells are triggered by CD152 while T cells of both arms of the study receive similar triggering of the TCR-CD3 complex. When measuring the frequencies of apoptotic cells according to the expression of activated caspases at day 4 after stimulation with Abs coupled to microspheres, there is a drastic reduction in apoptotic cells when CD4+CD28null cells were stimulated by CD152 cross-linking concordantly with CD3 compared with CD3 stimulation alone (Fig. 4 C). The frequency of viable CD4+CD28null T cells increased dramatically when cells received a CD152 signal compared with cells that did not receive a signal. Thus, CD152-mediated signaling induces resistance against apoptosis in CD28null T cells.

CTLA-4Ig (Abatacept) consists of the extracellular ligand-binding domain of human CD152 fused to a modified constant region of IgG (44, 45). In conventional CD28-expressing cells, the efficacy of the treatment is explained by inhibition of activation due to the lack of CD28-mediated costimulation (45, 46). In CD28null cells, the stimulation in the presence of CTLA-4Ig leads to an increase of cells expressing active caspases. Although there was no alteration detectable at early and late time points, on day 7 after stimulation the blockade of B7 by CTLA4Ig led to an increase of ∼10% of T cells expressing active caspases as shown for CD8+CD28null T cells and CD4+CD28null T cells (Fig. 5,A). These results obtained by cytometric analysis of active caspases were corroborated by analyzing the frequency of CD28null T cells binding annexin V (Fig. 5,B). Whereas earlier time points showed no change in apoptotic cells, on day 9 after stimulation in CD8+CD28null cells, the inhibition of B7-1 and B7-2 using CTLA-4Ig led to an increase of 15% in apoptotic cells, whereas CTLA-4Ig treatment of CD4+CD28null T cells during their stimulation resulted in drastically enhanced frequencies of annexin V-positive cells from 18 to >40%. These results show that the engagement of B7-1 and B7-2 on APCs led to increased apoptosis in CD28null T cells, most likely due to the lack of CD152-mediated signal transduction. It has been described that engagement of CD80/CD86 on APCs by CTLA-4Ig can lead to the induction of IDO, which will result in the generation of tryptophan metabolites (kynurenines) known to induce apoptosis in T cells (47, 48). We excluded that the increased apoptosis observed in CD28null T cells after blockade of CD152 by CTLA-4Ig due to CD80/CD86 engagement is caused by the CTLA-4Ig-mediated induction of IDO in APCs using the IDO inhibitor 1-methyl-tryptophan (Fig. 5 C). The inhibition of IDO by 1-methyl-tryptophan did not reverse the induction of apoptosis in CD28null cells, indicating that the apoptosis-enhancing effect of CTLA-4Ig in CD28null cells is cell autonomous and not mediated via IDO induction in APCs.

FIGURE 5.

Enhanced apoptosis in CD28null cells is mediated by CTLA-4Ig (Abatacept). A, CD4+CD28null and CD8+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. B7-1/B7-2 blocking CTLA-4Ig and isotype-Ig were used at a concentration of 50 μg/ml. Apoptosis was analyzed at the indicated time points by staining with the FITC-labeled caspase inhibitor VAD-FMK. B, As in A, at the indicated time points, apoptosis was analyzed by staining the cells with annexin V. C, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. B7-1/B7-2 blocking CTLA-4Ig and isotype-Ig were used at a concentration of 50 μg/ml; the IDO inhibitor 1-methyl-tryptophan was used at a concentration of 200 μM. Apoptosis was analyzed on day 7 by staining with FITC-labeled VAD-FMK.

FIGURE 5.

Enhanced apoptosis in CD28null cells is mediated by CTLA-4Ig (Abatacept). A, CD4+CD28null and CD8+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. B7-1/B7-2 blocking CTLA-4Ig and isotype-Ig were used at a concentration of 50 μg/ml. Apoptosis was analyzed at the indicated time points by staining with the FITC-labeled caspase inhibitor VAD-FMK. B, As in A, at the indicated time points, apoptosis was analyzed by staining the cells with annexin V. C, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. B7-1/B7-2 blocking CTLA-4Ig and isotype-Ig were used at a concentration of 50 μg/ml; the IDO inhibitor 1-methyl-tryptophan was used at a concentration of 200 μM. Apoptosis was analyzed on day 7 by staining with FITC-labeled VAD-FMK.

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The major apoptosis-inducing receptor in AICD is Fas (CD95). First, we analyzed the effect of CD152 neutralization on the expression of CD95 in CD28null cells (Fig. 6,A). In this study, we show that the inhibition of CD152-mediated signaling by anti-CD152 Fab has no significant effect on the surface expression of CD95 at all time points analyzed. Additionally, we examined the expression of the antiapoptotic protein Bcl-2, which was virtually unchanged in cells with or without blockade of CD152 signal transduction (Fig. 6,B). To test whether CD152 is involved in the processing of Fas-regulated induction of AICD, we analyzed the induction of apoptosis in CD4+CD28null cells after CD152 blockade by anti-CD152 Fab in the presence or absence of additional blockade of FasL. As shown in Fig. 6,C, the inhibition of CD152 signaling results in a marked decrease of surviving cells by 40–50% compared with cells treated with isotype control. The additional blockade of FasL in the presence of anti-CD152-Fab almost completely reversed the apoptosis-sensitizing effect induced by CD152 blockade (Fig. 6 C). Thus, the CD152-mediated effect is dependent on the Fas/FasL pathway in CD28null T cells and CD152-mediated signals are able to inhibit Fas/FasL-induced AICD.

FIGURE 6.

CD152 regulates resistance against apoptosis by activation of Akt. A, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab and isotype Fab were used at a concentration of 50 μg/ml. The surface expression of CD95 (Fas) was analyzed at the indicated time points. B, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab and isotype Fab were used at a concentration of 50 μg/ml. The intracellular expression of Bcl-2 was analyzed at the indicated time points. Numbers indicate the percentage of Bcl-2 high cells (24 h) or the geometric mean of fluorescence intensity (48 h). C, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab, isotype Fab, and anti-FasL Ab (NOK-1) were used at a concentration of 50 μg/ml. Apoptosis was analyzed at day 7 after stimulation. Viable cells were defined as annexin V/PI or caspase/PI and the numbers of living cells in isotype-treated cells set to 100%. The graph represents the mean ± SEM of seven independent experiments. D, CD4+CD28null cells were FACS sorted, stimulated with Con A (10 μg/ml) for 60 h, and restimulated with Ab-coupled polystyrene latex microspheres coupled with anti-CD3 + isotype control Ab (light gray) or anti-CD3 + anti-CD152 (black). Total amount of Akt (left panel) and phosphorylated Akt (right panel) was analyzed after 30 min of restimulation by flow cytometry using pan-Akt or phospho-Akt (Ser473)-specific Abs. Isotype control staining is shown as blank. The numbers indicate the geometric mean of fluorescence intensity. E.1, As in D, activation of Akt was analyzed by flow cytometry after stimulation with anti-CD3 + anti-CD152 (black) or anti-CD3 + anti-CD152 in the presence of the PI3′K inhibitor LY-294002 at a concentration of 30 μM (gray). Isotype control staining is shown as nonfilled curve. The numbers indicate the geometric mean of fluorescence intensity. E.2, As in D, cells were restimulated with anti-CD3 + isotype control Ab (black) or with anti-CD3 + isotype control Ab in the presence of the PI3′K inhibitor (gray). E.3, As in D, cells were restimulated with anti-CD3 + isotype control Ab (gray) or anti-CD3 + anti-CD152 (black) in the presence of the PI3′K inhibitor. F, CD4+CD28null cells were FACS sorted and stimulated with Con A (10 μg/ml) for 60 h. Activation of Bad was analyzed by flow cytometry after stimulation with anti-CD3 + isotype control Ab (light gray) or anti-CD3 + anti-CD152 (black) using a phospho-Bad (Ser112)-specific Ab. The numbers indicate the percentage of phospho-Badhigh cells.

FIGURE 6.

CD152 regulates resistance against apoptosis by activation of Akt. A, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab and isotype Fab were used at a concentration of 50 μg/ml. The surface expression of CD95 (Fas) was analyzed at the indicated time points. B, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab and isotype Fab were used at a concentration of 50 μg/ml. The intracellular expression of Bcl-2 was analyzed at the indicated time points. Numbers indicate the percentage of Bcl-2 high cells (24 h) or the geometric mean of fluorescence intensity (48 h). C, CD4+CD28null cells were FACS sorted and stimulated with anti-CD3 (1 μg/ml) in the presence of CD14+ cells as APCs. CD152 blocking anti-CD152 Fab, isotype Fab, and anti-FasL Ab (NOK-1) were used at a concentration of 50 μg/ml. Apoptosis was analyzed at day 7 after stimulation. Viable cells were defined as annexin V/PI or caspase/PI and the numbers of living cells in isotype-treated cells set to 100%. The graph represents the mean ± SEM of seven independent experiments. D, CD4+CD28null cells were FACS sorted, stimulated with Con A (10 μg/ml) for 60 h, and restimulated with Ab-coupled polystyrene latex microspheres coupled with anti-CD3 + isotype control Ab (light gray) or anti-CD3 + anti-CD152 (black). Total amount of Akt (left panel) and phosphorylated Akt (right panel) was analyzed after 30 min of restimulation by flow cytometry using pan-Akt or phospho-Akt (Ser473)-specific Abs. Isotype control staining is shown as blank. The numbers indicate the geometric mean of fluorescence intensity. E.1, As in D, activation of Akt was analyzed by flow cytometry after stimulation with anti-CD3 + anti-CD152 (black) or anti-CD3 + anti-CD152 in the presence of the PI3′K inhibitor LY-294002 at a concentration of 30 μM (gray). Isotype control staining is shown as nonfilled curve. The numbers indicate the geometric mean of fluorescence intensity. E.2, As in D, cells were restimulated with anti-CD3 + isotype control Ab (black) or with anti-CD3 + isotype control Ab in the presence of the PI3′K inhibitor (gray). E.3, As in D, cells were restimulated with anti-CD3 + isotype control Ab (gray) or anti-CD3 + anti-CD152 (black) in the presence of the PI3′K inhibitor. F, CD4+CD28null cells were FACS sorted and stimulated with Con A (10 μg/ml) for 60 h. Activation of Bad was analyzed by flow cytometry after stimulation with anti-CD3 + isotype control Ab (light gray) or anti-CD3 + anti-CD152 (black) using a phospho-Bad (Ser112)-specific Ab. The numbers indicate the percentage of phospho-Badhigh cells.

Close modal

We have shown earlier that CD152 cross-linking activates PI3′K which is responsible for resistance against AICD in murine Th cells (25). To test whether CD152 mediates antiapoptotic effects via Akt, we stimulated CD4+CD28null cells with immobilized anti-CD3 for 60 h followed by a stimulation with Ab-coated microspheres in which the cells either received a CD152 signal (anti-CD3 plus anti-CD152) or only a CD3 signal (anti-CD3 plus isotype), which results in reduced apoptosis as measured by reduced numbers of cells expressing activated caspases (Fig. 4,C). The activation of Akt was examined by flow cytometry using an Ab recognizing the phosphorylated (Ser473) and therefore activated form of Akt. Fig. 6,D (right panel) shows that the signal transduction by CD152 leads to a marked increase in cells expressing the active form of Akt. This is illustrated by the increase of the geometric mean of fluorescence intensity rising from 128 in cells without a CD152 signal compared with 157 in cells receiving a CD152 signal. This effect is not due to a different amount of total Akt protein since the detection with a pan-Akt Ab displays similar amounts irrespective of a CD152 signal (Fig. 6,D, left panel). This induction of Akt activity is mediated by PI3′K as the inhibition of this kinase completely abolishes the CD152-mediated positive effect on Akt induction as measured by the geometric means of fluorescence intensity (Fig. 6,E.1). That the PI3′K inhibition is mainly acting on CD152 and not CD3-induced Akt phosphorylation is illustrated in Fig. 6, E.2 and E.3. The inhibition of PI3′K by Ly-294002 reduces the geometric mean of fluorescence from 157 to 117 when CD4+CD28null cells were stimulated with anti-CD3 plus anti-CD152 for 30 min. In contrast, the inhibition of PI3′K in cells stimulated with anti-CD3 alone has only a minor inhibitory effect as indicated by the drop in the geometric mean of fluorescence from 128 to 110. The overlay of the phosphorylated Akt stainings of anti-CD3 plus anti-CD152 and anti-CD3 plus isotype-stimulated cells in the presence of Ly-294002 reveals no significant difference in fluorescence intensity. These results demonstrate that the observable reduction of phosphorylated Akt in cells stimulated with anti-CD3 plus anti-CD152 in the presence of the PI3′K inhibitor is for the most part caused by the inhibition of enhanced CD152-induced activation of Akt. This result shows that signals derived from CD152 lead indeed to the PI3′K-dependent activation of Akt. A target molecule of activated Akt is the proapoptotic molecule Bad. Akt is able to mediate its antiapoptotic function by phosphorylating and thereby inhibiting Bad. To test whether CD152 signaling induces inactivation of Bad, we analyzed phosphorylated Bad of preactivated CD28null T cells stimulated with either anti-CD3 plus anti-CD152 or anti-CD3 plus isotype control by using a phospho-Bad-specific Ab (Ser112). Fig. 6 F shows that CD152-mediated signal transduction leads to a frequency of 25% of cells highly expressing phosphorylated Bad. This result clearly illustrates that CD152 signals via PI3′K and Akt which inactivates Bad.

In the present study, we have identified CD152 as a major regulator of CD28null T cell quiescence and longevity. By using an enhanced staining technique, we showed that individual CD4+CD28null and CD8+CD28null cells rapidly express CD152 at their cell surface. This surface expression of CD152 serves as an important regulator of cell cycling in CD28null T cells, as it is responsible for keeping them in a proliferative quiescent state. Furthermore, we showed that the resistance against apoptosis typical for CD28null cells is due to CD152-mediated signal transduction. Signal transduction mediated by CD152 was interfering with the Fas/FasL pathway of apoptosis.

We show here that CD28null T cells are indeed able to activate the expression of CD152 because we could clearly show accumulation of CD152 protein intracellularly plus induced surface CD152 expression upon T cell activation. This finding was unexpected because CD28 and CD152 are homologous genes, very likely generated by gene duplication, and due to the chromosomal location of CD152 that is in close proximity to the CD28 gene whose transcription is prevented in CD28null T cells (40, 49). Our data also show that since CD152 molecules accumulate in activated CD28null T cells a control mechanism of its trafficking similar to CD28-competent T cells is also present in CD28null T cells (50). This also shows that access to the gene locus of CD152 plus intracellular storage of CD152 protein is independent of CD28 expression and signaling.

In this work, we were also able to unequivocally detect expression of CD152 at the cell surface of ∼20–30% of either CD4+CD28null or CD8+CD28null T cells 48 h after stimulation in vitro. The frequency of surface CD152-expressing cells as well as the kinetics of expression upon activation resembles the frequency and kinetics detected in conventional human CD28+ T cells as well as in murine Th cell and CTLs (25, 34, 50). Surprisingly, no CD152 was stored intracellularly in ex vivo-monitored CD28null T cells which could be expected in Ag-experienced cells.

CD28null T cells are fairly unresponsive with respect to low/no proliferation upon stimulation compared with conventional CD28-expressing cells (51). This is also attributed to the Ag experience of CD28null cells, which is accompanied by several rounds of replication. As we show in our study, this phenomenon of cell cycle arrest is closely regulated by CD152. The blockade of CD152 releases an inhibitory signal, leading to enhanced numbers of cells entering the cell cycle. This also means that surface CD152 in CD28null T cells is indeed responsible for keeping these Ag-experienced cells in check and preventing their expansion which could be deleterious to the whole organism since these cells have been shown to be autoreactive (52). Failure of CD152 to control their expansion could lead to induction or enhanced disease progression, e.g., RA. The finding that enhanced disease progression with respect to extra-articular manifestation and joint destruction of RA patients correlates significantly with enhanced appearance of CD28null T cells supports this concept (1). Potential target molecules repressed by CD152 could be G1 kinases of the cell cycle machinery as described before in the murine system (31). Taken together, the data suggest that CD152 “locks” the responsiveness of CD28null T cells.

Most prominent is the effect of CD152 signaling in CD28null cells for the induction of resistance against apoptosis. Reduced CD152-mediated signal transduction leads to a marked increase in cells undergoing AICD after stimulation. This could be observed in both CD4+ as well as CD8+CD28null cells. The increase in apoptotic cells between 20 and 30% strikingly resembles the number of surface CD152-expressing CD28null cells, indicating that the surface CD152-expressing cells are especially affected by the CD152 blockade. Conversely, the cross-linking of CD152 on CD4+CD28null cells leads to a reduction of AICD by 20–30%. Given that competition of CD152 with CD28 is excluded in CD28null cells, our results demonstrate that indeed CD152-mediated signaling pathways are responsible for the effects on apoptosis. Because CD152 has a much higher avidity for the ligands CD80 and CD86 compared with CD28, inhibitory actions mediated by CD152 are often attributed to the mere competition for ligand-binding CD28 with CD80 and CD86 (53). Incidentally, CD152 expression in CD28null T cells and its function stresses the point that competition of CD152 and CD28 for its ligands is negligible. This in turn implies that unique CD152-mediated signals must be at work, at least in CD28null T cells. Most interestingly, the reciprocal blockade of the CD152 ligands CD80 and CD86 by CTLA-4Ig has also an apoptosis-enhancing effect on CD28null T cells. This result also stresses the point that it is the lack of CD152-mediated signals that leads to increased apoptosis in CD28null T cells.

CD152 induces resistance against AICD by repressing the induction of Fas/FasL-mediated apoptosis. The mechanism of CD152-mediated resistance against apoptosis is mediated via the antiapoptotic kinase Akt (protein kinase B) acting in a PI3′K-dependent pathway. In contrast to our recent study using murine Th2 cells (25), our results show that in human CD28null cells the expression of Bcl-2 is unaffected by CD152 signaling. This result points likely to differences in the CD152-mediated signals due to different differentiation states of murine Th 2 cells and CD28null cells which are Th1-like (Fig. 1). However, Bcl-2 in turn has been described to be already expressed in higher levels in CD28null T cells (28). We show that Fas expression on CD28null T cells is not regulated by CD152. Because CD28null T cells are Th1-like (Fig. 1), FasL is very likely also not regulated by CD152 as shown for murine Th1 cells (25). Here we can clearly show that Bad, a downstream target of Akt, is enhanced phosphorylated after signaling of CD152. The CD152-induced increase of Bad phosphorylation is very likely dependent on the activation of PI3′K since the inhibition of PI3′K in CD28-expressing cells almost completely reversed the effect of CD152 on Bad phosphorylation (data not shown).

The mechanism whereby CTLA-4Ig reduces inflammation in diseases driven by memory cells is only now becoming elucidated (54, 55, 56), but still not fully understood. We show here that B7 engagement by CTLA-4Ig on APCs during activation of CD28null T cells leads to enhanced apoptosis of CD28null T cells. Prevention of CD152 signaling in this T cell population appears to “unlock” resistance against apoptosis. Although inhibition of CD152 signaling in CD28null T cells might induce initially proliferation, these cells would eventually die by apoptosis. Thus, the net effect of CTLA-4Ig treatment of activated Th1-like CD28null T cells would be anti-inflammatory. In chronic autoimmune diseases such as RA, where most of the T cells of the inflamed joints are effector and memory cells, many of which are CD28null, preventing CD152 signal transduction with CTLA-4Ig could well explain one of the actions of CTLA-4Ig in chronic T cell responses.

Thus, CD152 mediates resistance against AICD in CD28null cells by inactivation of proapoptotic molecules. Antagonistic approaches for CD152 should be considered to eliminate inflammatory T lymphocytes.

We thank Alexander Scheffold and Bernhard Fleischer for providing valuable reagents.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Bristol-Myers Squibb and Deutsche Forschungsgemeinschaft Br1680/3-4.

4

Abbreviations used in this paper: RA, rheumatoid arthritis; AICD, activation-induced cell death; FasL, Fas ligand; PI3′K, phosphatidylinositol 3′-kinase; PI, propidium iodide; PS, phosphatidylserine.

1
Pawlik, A., L. Ostanek, I. Brzosko, M. Brzosko, M. Masiuk, B. Machalinski, B. Gawronska-Szklarz.
2003
. The expansion of CD4+CD28 T cells in patients with rheumatoid arthritis.
Arthritis Res. Ther.
5
:
R210
-R213.
2
Lamprecht, P., F. Moosig, E. Csernok, U. Seitzer, A. Schnabel, A. Mueller, W. L. Gross.
2001
. CD28 negative T cells are enriched in granulomatous lesions of the respiratory tract in Wegener’s granulomatosis.
Thorax
56
:
751
-757.
3
Giscombe, R., S. Nityanand, N. Lewin, J. Grunewald, A. K. Lefvert.
1998
. Expanded T cell populations in patients with Wegener’s granulomatosis: characteristics and correlates with disease activity.
J. Clin. Immunol.
18
:
404
-413.
4
Markovic-Plese, S., I. Cortese, K. P. Wandinger, H. F. McFarland, R. Martin.
2001
. CD4+CD28 costimulation-independent T cells in multiple sclerosis.
J. Clin. Invest.
108
:
1185
-1194.
5
Schirmer, M., C. Goldberger, R. Wurzner, C. Duftner, K. P. Pfeiffer, J. Clausen, G. Neumayr, A. Falkenbach.
2002
. Circulating cytotoxic CD8+CD28 T cells in ankylosing spondylitis.
Arthritis Res.
4
:
71
-76.
6
Duftner, C., C. Goldberger, A. Falkenbach, R. Wurzner, B. Falkensammer, K. P. Pfeiffer, E. Maerker-Hermann, M. Schirmer.
2003
. Prevalence, clinical relevance and characterization of circulating cytotoxic CD4+CD28 T cells in ankylosing spondylitis.
Arthritis Res. Ther.
5
:
R292
-R300.
7
Namekawa, T., U. G. Wagner, J. J. Goronzy, C. M. Weyand.
1998
. Functional subsets of CD4 T cells in rheumatoid synovitis.
Arthritis Rheum.
41
:
2108
-2116.
8
Posnett, D. N., R. Sinha, S. Kabak, C. Russo.
1994
. Clonal populations of T cells in normal elderly humans: the T cell equivalent to “benign monoclonal gammapathy.”.
J. Exp. Med.
179
:
609
-618.
9
Vallejo, A. N., A. R. Nestel, M. Schirmer, C. M. Weyand, J. J. Goronzy.
1998
. Aging-related deficiency of CD28 expression in CD4+ T cells is associated with the loss of gene-specific nuclear factor binding activity.
J. Biol. Chem.
273
:
8119
-8129.
10
Colombatti, A., R. Doliana, M. Schiappacassi, C. Argentini, E. Tonutti, C. Feruglio, P. Sala.
1998
. Age-related persistent clonal expansions of CD28 cells: phenotypic and molecular TCR analysis reveals both CD4+ and CD4+CD8+ cells with identical CDR3 sequences.
Clin. Immunol. Immunopathol.
89
:
61
-70.
11
Schmidt, D., P. B. Martens, C. M. Weyand, J. J. Goronzy.
1996
. The repertoire of CD4+CD28 T cells in rheumatoid arthritis.
Mol. Med.
2
:
608
-618.
12
Freedman, M. S., T. C. Ruijs, M. Blain, J. P. Antel.
1991
. Phenotypic and functional characteristics of activated CD8+ cells: a CD11bCD28 subset mediates noncytolytic functional suppression.
Clin. Immunol. Immunopathol.
60
:
254
-267.
13
Cortesini, R., J. LeMaoult, R. Ciubotariu, N. S. Cortesini.
2001
. CD8+CD28 T suppressor cells and the induction of antigen-specific, antigen-presenting cell-mediated suppression of Th reactivity.
Immunol. Rev.
182
:
201
-206.
14
Park, W., C. M. Weyand, D. Schmidt, J. J. Goronzy.
1997
. Co-stimulatory pathways controlling activation and peripheral tolerance of human CD4+CD28 T cells.
Eur. J. Immunol.
27
:
1082
-1090.
15
Weyand, C. M., J. C. Brandes, D. Schmidt, J. W. Fulbright, J. J. Goronzy.
1998
. Functional properties of CD4+CD28 T cells in the aging immune system.
Mech. Ageing Dev.
102
:
131
-147.
16
Watanabe, T., Y. Sakai, A. Hanai, S. Masaki, K. Ohno, S. Miyawaki, A. Matsuzawa.
1993
. A new allele at the lpr gene on mouse chromosome 19 expresses properties different from the original recessive mutation.
Mamm. Genome.
4
:
346
-347.
17
Lynch, D. H., M. L. Watson, M. R. Alderson, P. R. Baum, R. E. Miller, T. Tough, M. Gibson, T. vis-Smith, C. A. Smith, K. Hunter.
1994
. The mouse Fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster.
Immunity
1
:
131
-136.
18
Green, D. R., N. Droin, M. Pinkoski.
2003
. Activation-induced cell death in T cells.
Immunol. Rev.
193
:
70
-81.
19
Lenardo, M., K. M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, L. Zheng.
1999
. Mature T lymphocyte apoptosis: immune regulation in a dynamic and unpredictable antigenic environment.
Annu. Rev. Immunol.
17
:
221
-253.
20
Pinkoski, M. J., D. R. Green.
2002
. Lymphocyte apoptosis: refining the paths to perdition.
Curr. Opin. Hematol.
9
:
43
-49.
21
Datta, S. R., A. Brunet, M. E. Greenberg.
1999
. Cellular survival: a play in three Akts.
Genes Dev.
13
:
2905
-2927.
22
Vaux, D. L., R. A. Flavell.
2000
. Apoptosis genes and autoimmunity.
Curr. Opin. Immunol.
12
:
719
-724.
23
Van, P. L., D. A. Peterson, A. K. Abbas.
1998
. The Fas/Fas ligand pathway and Bcl-2 regulate T cell responses to model self and foreign antigens.
Immunity
8
:
265
-274.
24
Van, P. L., A. Biuckians, A. K. Abbas.
1998
. Functional roles of Fas and Bcl-2-regulated apoptosis of T lymphocytes.
J. Immunol.
160
:
2065
-2071.
25
Pandiyan, P., D. Gartner, O. Soezeri, A. Radbruch, K. Schulze-Osthoff, M. C. Brunner-Weinzierl.
2004
. CD152 (CTLA-4) determines the unequal resistance of Th1 and Th2 cells against activation-induced cell death by a mechanism requiring PI3 kinase function.
J. Exp. Med.
199
:
831
-842.
26
Chambers, C. A., M. F. Krummel, B. Boitel, A. Hurwitz, T. J. Sullivan, S. Fournier, D. Cassell, M. Brunner, J. P. Allison.
1996
. The role of CTLA-4 in the regulation and initiation of T-cell responses.
Immunol. Rev.
153
:
27
-46.
27
Waase, I., C. Kayser, P. J. Carlson, J. J. Goronzy, C. M. Weyand.
1996
. Oligoclonal T cell proliferation in patients with rheumatoid arthritis and their unaffected siblings.
Arthritis Rheum.
39
:
904
-913.
28
Schirmer, M., A. N. Vallejo, C. M. Weyand, J. J. Goronzy.
1998
. Resistance to apoptosis and elevated expression of Bcl-2 in clonally expanded CD4+CD28 T cells from rheumatoid arthritis patients.
J. Immunol.
161
:
1018
-1025.
29
Lafage-Pochitaloff, M., R. Costello, D. Couez, J. Simonetti, P. Mannoni, C. Mawas, D. Olive.
1990
. Human CD28 and CTLA-4 Ig superfamily genes are located on chromosome 2 at bands q33–q34.
Immunogenetics
31
:
198
-201.
30
Egen, J. G., M. S. Kuhns, J. P. Allison.
2002
. CTLA-4: new insights into its biological function and use in tumor immunotherapy.
Nat. Immunol.
3
:
611
-618.
31
Brunner, M. C., C. A. Chambers, F. K. Chan, J. Hanke, A. Winoto, J. P. Allison.
1999
. CTLA-4-Mediated inhibition of early events of T cell proliferation.
J. Immunol.
162
:
5813
-5820.
32
Blair, P. J., J. L. Riley, B. L. Levine, K. P. Lee, N. Craighead, T. Francomano, S. J. Perfetto, G. S. Gray, B. M. Carreno, C. H. June.
1998
. CTLA-4 ligation delivers a unique signal to resting human CD4 T cells that inhibits interleukin-2 secretion but allows Bcl-xL induction.
J. Immunol.
160
:
12
-15.
33
Fife, B. T., M. D. Griffin, A. K. Abbas, R. M. Locksley, J. A. Bluestone.
2006
. Inhibition of T cell activation and autoimmune diabetes using a B cell surface-linked CTLA-4 agonist.
J. Clin. Invest.
116
:
2252
-2261.
34
Maszyna, F., H. Hoff, D. Kunkel, A. Radbruch, M. C. Brunner-Weinzierl.
2003
. Diversity of clonal T cell proliferation is mediated by differential expression of CD152 (CTLA-4) on the cell surface of activated individual T lymphocytes.
J. Immunol.
171
:
3459
-3466.
35
Chuang, E., T. S. Fisher, R. W. Morgan, M. D. Robbins, J. M. Duerr, M. G. Vander Heiden, J. P. Gardner, J. E. Hambor, M. J. Neveu, C. B. Thompson.
2000
. The CD28 and CTLA-4 receptors associate with the serine/threonine phosphatase PP2A.
Immunity
13
:
313
-322.
36
Baroja, M. L., L. Vijayakrishnan, E. Bettelli, P. J. Darlington, T. A. Chau, V. Ling, M. Collins, B. M. Carreno, J. Madrenas, V. K. Kuchroo.
2002
. Inhibition of CTLA-4 function by the regulatory subunit of serine/threonine phosphatase 2A.
J. Immunol.
168
:
5070
-5078.
37
Schneider, H., K. V. Prasad, S. E. Shoelson, C. E. Rudd.
1995
. CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.
J. Exp. Med.
181
:
351
-355.
38
Marengere, L. E., P. Waterhouse, G. S. Duncan, H. W. Mittrucker, G. S. Feng, T. W. Mak.
1996
. Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4.
Science
272
:
1170
-1173.
39
Lee, K. M., E. Chuang, M. Griffin, R. Khattri, D. K. Hong, W. Zhang, D. Straus, L. E. Samelson, C. B. Thompson, J. A. Bluestone.
1998
. Molecular basis of T cell inactivation by CTLA-4.
Science
282
:
2263
-2266.
40
Vallejo, A. N., C. M. Weyand, J. J. Goronzy.
2001
. Functional disruption of the CD28 gene transcriptional initiator in senescent T cells.
J. Biol. Chem.
276
:
2565
-2570.
41
Krummel, M. F., J. P. Allison.
1996
. CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells.
J. Exp. Med.
183
:
2533
-2540.
42
Vallejo, A. N., M. Schirmer, C. M. Weyand, J. J. Goronzy.
2000
. Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways.
J. Immunol.
165
:
6301
-6307.
43
Spaulding, C., W. Guo, R. B. Effros.
1999
. Resistance to apoptosis in human CD8+ T cells that reach replicative senescence after multiple rounds of antigen-specific proliferation.
Exp. Gerontol.
34
:
633
-644.
44
Linsley, P. S., S. G. Nadler, J. Bajorath, R. Peach, H. T. Leung, J. Rogers, J. Bradshaw, M. Stebbins, G. Leytze, W. Brady.
1995
. Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4 (CTLA-4): a disulfide-linked homodimer binds two CD86 molecules.
J. Biol. Chem.
270
:
15417
-15424.
45
Linsley, P. S., P. M. Wallace, J. Johnson, M. G. Gibson, J. L. Greene, J. A. Ledbetter, C. Singh, M. A. Tepper.
1992
. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule.
Science
257
:
792
-795.
46
Lenschow, D. J., Y. Zeng, J. R. Thistlethwaite, A. Montag, W. Brady, M. G. Gibson, P. S. Linsley, J. A. Bluestone.
1992
. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg.
Science
257
:
789
-792.
47
Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni, P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, P. Puccetti.
2002
. CTLA-4-Ig regulates tryptophan catabolism in vivo.
Nat. Immunol.
3
:
1097
-1101.
48
Fallarino, F., U. Grohmann, C. Vacca, R. Bianchi, C. Orabona, A. Spreca, M. C. Fioretti, P. Puccetti.
2002
. T cell apoptosis by tryptophan catabolism.
Cell Death Differ.
9
:
1069
-1077.
49
Vallejo, A. N., E. Bryl, K. Klarskov, S. Naylor, C. M. Weyand, J. J. Goronzy.
2002
. Molecular basis for the loss of CD28 expression in senescent T cells.
J. Biol. Chem.
277
:
46940
-46949.
50
Linsley, P. S., J. Bradshaw, J. Greene, R. Peach, K. L. Bennett, R. S. Mittler.
1996
. Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement.
Immunity
4
:
535
-543.
51
Scheuring, U. J., H. Sabzevari, A. N. Theofilopoulos.
2002
. Proliferative arrest and cell cycle regulation in CD8+CD28 versus CD8+CD28+ T cells.
Hum. Immunol.
63
:
1000
-1009.
52
Schmidt, D., J. J. Goronzy, C. M. Weyand.
1996
. CD4+CD7CD28 T cells are expanded in rheumatoid arthritis and are characterized by autoreactivity.
J. Clin. Invest.
97
:
2027
-2037.
53
Hoff, H., G. R. Burmester, M. C. Brunner-Weinzierl.
2006
. Competition and cooperation: Signal transduction by CD28 and CTLA-4.
Signal Transduction
6
:
260
-267.
54
Ndejembi, M. P., J. R. Teijaro, D. S. Patke, A. W. Bingaman, M. R. Chandok, A. Azimzadeh, S. G. Nadler, D. L. Farber.
2006
. Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway.
J. Immunol.
177
:
7698
-7706.
55
Bluestone, J. A., E. W. St Clair, L. A. Turka.
2006
. CTLA4Ig: bridging the basic immunology with clinical application.
Immunity
24
:
233
-238.
56
Kremer, J. M., R. Westhovens, M. Leon, G. E. Di, R. Alten, S. Steinfeld, A. Russell, M. Dougados, P. Emery, I. F. Nuamah, et al
2003
. Treatment of rheumatoid arthritis by selective inhibition of T-cell activation with fusion protein CTLA4Ig.
N. Engl. J. Med.
349
:
1907
-1915.