Of T Cell Proliferation Clonal Anergy Is Maintained Independently

Clonal Anergy Is Maintained Independently of T Cell Proliferation Sara Colombetti, Fabio Benigni, Veronica Basso and Anna Mondino This information is current as of October 2, 2021. J Immunol 2002; 169:6178-6186; ; doi: 10.4049/jimmunol.169.11.6178 http://www.jimmunol.org/content/169/11/6178 Downloaded from References This article cites 48 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/169/11/6178.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Clonal Anergy Is Maintained Independently of T Cell Proliferation1

Sara Colombetti, Fabio Benigni, Veronica Basso, and Anna Mondino2

Ag encounter in the absence of proliferation results in the establishment of T cell unresponsiveness, also known as T cell clonal anergy. Anergic T cells fail to proliferate upon restimulation because of the inability to produce IL-2 and to properly regulate the

G1 cell cycle checkpoint. Because optimal TCR and CD28 engagement can elicit IL-2-independent cell cycle progression, we investigated whether CD3/CD28-mediated activation of anergic T cells could overcome G1 cell cycle block, drive T cell proliferation, and thus reverse clonal anergy. We show here that although antigenic stimulation fails to elicit G1-to-S transition, anti- CD3/CD28 mAbs allow proper cell cycle progression and proliferation of anergic T cells. However, CD3/CD28-mediated cell division does not restore Ag responsiveness. Our data instead indicate that reversal of clonal anergy speciﬁcally requires an IL-2-dependent, rapamycin-sensitive signal, which is delivered independently of cell proliferation. Thus, by tracing proliferation and Ag responsiveness of individual cells, we show that whereas both TCR/CD28 and IL-2-generated signals can drive T cell Downloaded from proliferation, only IL-2/IL-2R interaction regulates Ag responsiveness, indicating that proliferation and clonal anergy can be independently regulated. The Journal of Immunology, 2002, 169: 6178Ð6186.

tight control of T cell proliferation is required for the has been defined as clonal anergy (3Ð5). The establishment of maintenance of a correct balance between immunity clonal anergy requires TCR-generated signaling and new protein A against foreign pathogens and tolerance against normal synthesis, because it can be inhibited if cyclosporin A and cyclo- http://www.jimmunol.org/ tissues. Indeed, whereas the expansion of pathogen-specific lym- heximide are provided at the time of TCR occupancy (2, 6, 7). It phocytes must be rapidly elicited to guarantee the development of was later shown that clonal anergy could also be induced in the protective immune responses, proliferation of potentially self-re- presence of optimal T cell activation by preventing IL-2-IL-2R active T cells must be limited to prevent the establishment of un- interaction (8) or IL-2-dependent T cell proliferation (9). Sloan- wanted autoimmune diseases. Proliferation of T lymphocytes re- Lancaster et al. (10) similarly reported that partial agonists, which quires the coordinate engagement of a number of cell surface signal through the TCR but do not cause proliferation despite the receptors. The first critical event is the simultaneous occupancy of presence of CD28 costimulation, failed to lead to T cell prolifer- the Ag-specific TCR and of the CD28 costimulatory receptor. This ation and instead induced clonal anergy. Similarly, inhibition of by guest on October 2, 2021 generates several intracellular signals required for IL-2 gene tran- G1-to-S transition by the addition of n-butyrate (11) or rapamycin scription and protein production and for cell surface expression of (12) at the time of stimulation impairs the ability of T cells to the IL-2R. The second critical event requires the interaction of respond to subsequent stimulation. By contrast, the addition of autocrine IL-2 with its newly expressed surface receptor. This ul- exogenous IL-2 to suboptimally stimulated T cells or to anergic T timately results in T cell proliferation. If T cells fail to progress cells drives extensive T cell proliferation and elicits reversal of through the cell cycle and to proliferate on Ag encounter, they clonal anergy (9, 13). become unresponsive to subsequent restimulation. These data support the view that TCR occupancy normally re- Understanding how proliferation and Ag responsiveness are reg- sults in the production of negative regulatory factors, which are ulated in T lymphocytes is critical for the development of proper then degraded or inhibited during IL-2-driven G1-to-S phase tran- treatments for a number of clinical conditions, such as autoimmune sition. This model predicts that in the absence of G1-to-S transition diseases, organ transplantation, and tumors; thus, these issues have or proliferation the anergic factors might accumulate inside the been extensively investigated over the past years. Jenkins et al. (1, cells and inhibit subsequent T cell responses (9, 14). 2) first showed that suboptimal T cell activation, provided by Even though the nature of the putative anergic factors remains chemically fixed Ag-bearing APC or by immobilized anti-CD3 controversial, it has been postulated that their activity could be mAb, fails to elicit T cell proliferation and instead hampers the directly linked to the regulation of G1-to-S phase transition. For ability of the cells to produce IL-2 and to proliferate on subsequent instance, a number of reports have indicated the G1 cyclin-depen- optimal restimulation. This state of lymphocyte unresponsiveness dent kinase inhibitor p27Kip as a putative anergic factor, because its deregulated expression directly correlates with T cell respon-

Cancer Immunotherapy and Gene Therapy Program, DIBIT/S. Raffaele Scientific siveness (15, 16). Indeed, suboptimal stimulation with costimula- Institute, Milan, Italy tory deficient APC, which results in T cell anergy, fails to down- Kip Received for publication August 7, 2002. Accepted for publication October 1, 2002. regulate p27 and elicits cell cycle arrest in early G1 (15). Kip The costs of publication of this article were defrayed in part by the payment of page Moreover, overexpression of p27 not only prevents G1-to-S charges. This article must therefore be hereby marked advertisement in accordance transition upon optimal stimulation (16) but also inhibits IL-2 tran- with 18 U.S.C. Section 1734 solely to indicate this fact. scription (15). Altogether, the existing information suggest that 1 This work was supported by a New Unit Start Up Grant of the Associazione Italiana proper regulation of p27Kip, and more generally of the G cell per la Ricerca sul Cancro (to A.M.). 1 cycle checkpoint, might simultaneously regulate proliferation and 2 Address correspondence and reprint requests to Dr. Anna Mondino, Cancer Immu- notherapy and Gene Therapy Program DIBIT/S. Raffaele Scientific Institute, Via clonal anergy in T cells and that forcing G1-to-S transition could Olgettina, 58, 20132 Milan, Italy. E-mail address: [email protected] be exploited to break T cell anergy and restore Ag responsiveness.

␮ ␮ Whereas entry of resting lymphocytes into G1 is regulated by CD28 mAb (5 g/ml) or with anti-CD3 mAb (0.1 g/ml) and rIL-2 (10 IU/ml). TCR-generated intracellular signals (17), G1-to-S phase transition has been described to depend on IL-2-IL-2R-mediated signaling Western blot analysis (18Ð20). More recently, several reports indicated that IL-2-independent T lymphocyte proliferation occurs both in vivo (21Ð23) Control and stimulated T cells were harvested, washed twice with ice cold PBS, and lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 0.5% Nonidet and in vitro (24Ð26). Furthermore, optimal engagement of the P-40, 150 mM NaCl, 100 ␮g/ml PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml leu- TCR and of CD28 has been shown to drive IL-2 independent cy- peptin, 1 mM PMSF; Sigma-Aldrich, Milan, Italy) for 20 min on ice. The clin/cdks activation and p27Kip degradation (24, 27, 28). We thus soluble fraction was separated by centrifugation at 13,000 rpm for 15 min set out to investigate whether optimal engagement of the TCR and at 4¡C, and the protein content was determined by the Bradford assay of CD28 on the surface of anergic T cells could force cell cycle (Bio-Rad, Milan, Italy). Samples containing an equal amount of proteins were mixed with an equal volume of 2ϫ Laemmli buffer (33), boiled, and progression and by doing so elicit reversal of clonal anergy. separated on standard SDS-PAGE. Proteins were then transferred onto ni- The results reported here indicate that anti-CD3 and anti-CD28 trocellulose membranes (Protran; Schleicher & Schuell, Milan, Italy). Non-

mAbs are indeed able to overcome G1 cell cycle block in anergic specific binding sites were blocked by incubating the membranes in PBS T cells and drive cell proliferation but that this is not sufficient to containing 5% nonfat dry milk and 0.05% Tween 20. Immunodetection was accomplished by incubating the membranes first with primary Abs (1 restore Ag responsiveness. Instead, our results indicate that an IL- ␮g/ml) and then with HRP-conjugated secondary Abs directed against 2-dependent, rapamycin-sensitive signal, induced independently mouse IgG or rabbit IgG (1/5000 dilution; Amersham, Lifescience, Milan, from IL-2-driven cell proliferation, is specifically required for the Italy) diluted in PBS (pH 7.4), containing 1% dry nonfat milk, 1% BSA reversal of clonal anergy. (Sigma), and 0.1% Tween 20. The immunocomplexes were then detected by chemiluminescence (SuperSignal; West Dura Extended Duration Sub-

strate; Pierce, Milan, Italy). Expression and phosphorylation of retinoblas- Downloaded from Materials and Methods toma (Rb) were analyzed by 6% SDS-PAGE as described (15). The fol- T cell clones and anergy induction lowing Abs were used: cyclin D3 (C-16), p27Kip (C-19), p21Cip (F-5), and Rb (C-15) Ab (Santa Cruz Biotechnology, Milan, Italy). The A.E7 T cell clone was maintained in RPMI supplemented with 2 mM L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, 20 mg/ml gen- Propidium iodide staining tamicin, and 50 ␮M 2-ME (Invitrogen Life Technologies, Milan, Italy), and 5% heat-inactivated FBS (Euroclone, Milan, Italy) at 37¡Cina5% T cells were stimulated as indicated in the figures, washed with PBS, CO atmosphere by periodic stimulation with a peptide derived from pi- counted, and fixed overnight at 4¡C in 70% ethanol. Cells were then pel- http://www.jimmunol.org/ 2 ϫ 6 geon cytochrome c (PCC 3; Primm, Milan, Italy) and irradiated leted; resuspended (1 10 cells/ml) in DNA staining buffer containing 81Ð104 ␮ B10.BR (Harlan, Milan, Italy) splenic APC as previously described (29). 0.1% sodium citrate, 0.05% Nonidet P-40, 50 g/ml propidium iodide, and Ϫ Ϫ ␮ The chicken OVA-specific wild-type and IL-2 / DO11.10 T cell lines 50 g/ml RNase A (Sigma); and incubated for at least1hatroom tem- were generated by stimulating lymph node cells derived from DO11.10 perature. PI contents were assessed by flow cytometry (FACScan; BD Bio- (30) and DO11.10/IL-2Ϫ/Ϫ mice (22) with irradiated syngeneic BALB/c sciences, Milan, Italy) using standard CellQuest (BD Biosciences) splenic APC (Charles River, Calco, Italy) pulsed with the OVA-derived acquisition/analysis.

peptide (OVAp323Ð339; Primm). The cells were restimulated weekly with OVA-pulsed APC and expanded in exogenous rIL-2 (10 IU/ml; Roche, Analysis of cell division by CFSE dilution Milan, Italy). All experiments were performed at least 10 days from the last T cells were washed twice with PBS and resuspended at a density of 2 ϫ Ag exposure. At this time, the cells appeared to be in the G -G stage of 7 0 1 10 cells per ml in PBS. An equal volume of 5 ␮M carboxyfluorescein by guest on October 2, 2021 the cell cycle. diacetate succinimidyl ester (Molecular Probes., Milan, Italy) in PBS was Where indicated, a fraction of the cells was harvested and rested in fresh added, and the cells were gently mixed for 8 min at room temperature. medium for 5 days (referred to as “control” T cells). At the same time, a Unbound carboxyfluorescein diacetate succinimidyl ester, or the deacety- ⑀ similar number of cells were cultured for 16 h on plate-bound anti-CD3 lated form, CFSE, was quenched by the addition of an equal volume of ␮ mAb (clone 145-2C11; 4 g/ml) (31), removed from the Ab, and then FBS. The labeled cells were washed twice in complete medium and stim- rested for an additional 5 days in fresh medium (referred to as “anergic” T ulated as indicated in the figures. At the time of harvest, CFSE-labeled cells cells), as previously described (8). Similarly to control T cells, anergic cells were washed twice in PBS, and the vital dye TO-PRO-3 (Molecular appeared to be in the G0-G1 phase of the cell cycle. Probes) was added to each sample (1 nM final concentration) before ac- Thereafter, viable cells were separated on a Lympholyte-M (Cedarlane, quisition to distinguish live and dead cells. Cell division analysis was per- Hornby, Ontario, Canada) density gradient and restimulated with increas- formed on a BD Biosciences FACSCalibur dual-laser cytometer using ing amounts of the PCC-derived peptide and irradiated APC, with immo- standard CellQuest acquisition/analysis. If not differently indicated, cells ␮ bilized anti-CD3 mAb (2 g/ml) and anti-CD28 mAb (clone 37.51) (5 were harvested and analyzed 5 days after CFSE labeling and 15,000 events ␮ g/ml) (32), or with rIL-2 (10 IU/ml). Proliferation of control and aner- were collected. CFSE labeling remained stable for up to 3 wk in our cell ϫ 4 gized A.E7 T cells was performed in 96-well plates (2 10 cells/well) by cultures. [3H]thymidine incorporation after 72 h of culture as previously described (8). The presence of IL-2 in culture supernatants of activated T cells was determined 24 h after stimulation by capture ELISA as previously de- Results scribed (22). Functional assay was performed in 96-well plates using 1 ϫ CD3/CD28-mediated stimulation of anergic T cells bypasses the 5 10 cells per well. The ELISA standard curve was generated using recom- G1 cell cycle block and drives cell division binant murine IL-2 (BD PharMingen, Milan, Italy), and the level of de- tection was 1 ng/ml. Because TCR- and CD28-generated signaling can mediate the G1- to-S phase transition via IL-2-independent mechanisms (24, 27, T cell activation 28), we investigated whether a combination of anti-CD3 and anti- Control and anergic A.E7 cells were stimulated with Ag (1 ␮M) and irra- CD28 mAbs would overcome the G1 cell cycle block reported for diated B10.BR splenocytes (T:APC ratio, 1:4), with immobilized anti-CD3 anergic T cells and by that cause reversal of clonal anergy. mAb (2 ␮g/ml) and anti-CD28 mAb (5 ␮g/ml), or with rIL-2 (10 IU/ml) T cell anergy was induced in cloned CD4ϩ Th lymphocytes by in the absence or in the presence of rapamycin (200 nM; Calbiochem, chronic TCR engagement in the absence of costimulation as pre- Merck, Milan, Italy). At the end of the stimulation, the cells were either analyzed by flow cytometry or removed from the Abs, rested in fresh viously described (8). Briefly, nonproliferating A.E7 T cells were medium for 10 days, and then restimulated with Ag/APC. harvested and rested in fresh medium (referred to as control T Wild-type and IL-2Ϫ/Ϫ DO11.10 T cells were stimulated for the indi- cells) or cultured for 16 h on plate-bound anti-CD3 mAb, removed cated periods of time with immobilized anti-CD3 mAb (1 ␮g/ml) and anti- from the Ab, and rested for an additional 5 days in fresh medium (referred to as anergic T cells) (Fig. 1A). At the end of the resting 3 Abbreviations used in this paper: PCC, pigeon cytochrome c; Ag/APC, Ag-pulsed time, control and anergic T cells appeared to be in the G0-G1 phase irradiated splenocytes; mTor, mammalian target of rapamycin; Rb, retinoblastoma. of the cell cycle (refer to Figs. 2 and 3). In addition, control and 6180 CELL CYCLE AND T CELL ANERGY

FIGURE 2. Regulation of G1 cell cycle proteins in control (CT) and anergic (AN) T cells on Ag/APC- or CD3/CD28 mAbs-mediated activation. T cell anergy was induced as described in Fig. 1. Control and anergic

T cells were stimulated with either Ag/APC (A) or anti-CD3/CD28 mAbs Downloaded from (B) for 24 h. Thereafter, the cells were lysed, and protein content was analyzed by SDS-PAGE and Western blot analysis with the Abs as indicated. p27Kip and p21Cip were detected on the same membrane filter by sequential hybridization. The arrows indicate the relative mobility of the proteins. http://www.jimmunol.org/ (Fig. 2). Although Ag-dependent stimulation of control cells resulted in the up-regulation of cyclin D3 and of p21Cip, in the hy- perphosphorylation of Rb, and in the down-regulation of p27Kip FIGURE 1. Chronic TCR engagement results in the establishment of T levels (Fig. 2A), it failed to induce G1-related events in anergic T cell clonal anergy.A, Schematic representation of anergy induction. Rest- cells. Thus, as also reported elsewhere (11, 16), anergic T cells are ing A.E7 cells were either left untreated (control cell) or cultured for 16 h unable to progress through the G1 cell cycle checkpoint upon Ag/ on plate-bound anti-CD3 mAb (anergic cells). Thereafter, the cells were APC encounter. rested in fresh medium for 5 days and restimulated as indicated. B, Control In contrast, stimulation with anti-CD3/CD28 mAbs elicited sim- by guest on October 2, 2021 (CT) and CD3-treated cells (anergic (AN)) were challenged with irradiated ilar up-regulation of cyclin D3 and of p21Cip, hyperphosphoryla- splenic APC and the indicated amount of the PCC-derived peptide, with tion of Rb and down-regulation of p27Kip (Fig. 2B) in both control ␮ immobilized anti-CD3/anti-CD28 mAbs (2 and 5 g/ml, respectively), or and anergic T cells. This indicates that optimal TCR/CD28 en- 3 with rIL-2 (10 IU/ml). After 48 h, [ H]thymidine was added to each well, gagement elicits proper regulation of the G cell cycle proteins and and the cells were harvested and assayed for proliferation after an addi- 1 entry of anergic cells in the S phase. tional 16 h of culture. C, IL-2 production was measured in the supernatants of CD3/CD28-stimulated T cells after 24 h of culture by capture ELISA. To determine the fraction of anergic T cells able to proceed from The SDs of triplicate cultures are reported. Values reflect one of several G1 to S-G2-M after CD3/CD28-mediated stimulation, we mea- (more than five) independent similar experiments. sured DNA contents by propidium iodide staining. This technique allows one to discriminate cells in G0-G1, recognized by a typical DNA content equal to 1N, from cells in S-G2-M which have a anergic cells expressed similar surface levels of the TCR (V␤3: DNA content Ͼ1N (2N for cells in the M phase). Preliminary mean fluorescence intensity, 41.66 Ϯ 0.34 vs 41.35 Ϯ 0.12), of experiments indicated that by 48 h of stimulation, 20Ð40% of CD3 (27.01 Ϯ 1.39 vs 23.83 Ϯ 3.66; see also Ref. 34), and of stimulated A.E7 T cells had a DNA content greater than 1N, sug- Ϯ Ϯ CD25 (23.87 0.07 vs 26.54 0.17). gesting that by this time the cells had entered the S-G2-M phase Control and CD3-treated anergic T cells were thus restimulated (data not shown and Fig. 3). with either Ag-pulsed APCs (Ag/APC) or with a combination of Control and anergic T cells were therefore stimulated with either anti-CD3 and anti-CD28 mAbs. Proliferation and IL-2 secretion Ag/APC or immobilized anti-CD3/CD28 mAbs for 48 h, fixed, were then measured by [3H]thymidine incorporation and by cap- stained with PI, and analyzed by flow cytometry. As expected, ture ELISA, respectively. On Ag rechallenge, anergic A.E7 T cells whereas control cells increased their DNA content upon Ag/APC- proliferated to much lower extents than control T cells (Fig. 1B) stimulation, anergic T cells failed to do so (Fig. 3A). In contrast, and showed defective IL-2 production even in response to optimal upon CD3/CD28 stimulation, both control and anergic cells had a TCR and CD28 engagement (Fig. 1C), as previously described (8, sizable fraction of cells with a DNA content Ͼ1N, and thus in Ϯ Ϯ 34). Interestingly, despite the failure to produce IL-2, control and S-G2-M (23.83 3.92% and 34.89 5.68%, respectively; Fig. anergic T cells produced comparable amounts of IFN-␥ (not 3B). After 48 h of stimulation, no differences were detected in the shown) and proliferated to similar extents in response to immobi- total number of live cells or in the fraction of the cells with DNA Ͻ lized anti-CD3/CD28 mAbs (Fig. 1B). content 1N (subG1, apoptotic cells), suggesting that anergic T This last observation prompted us to investigate whether the cells were not preferentially dying in our cell culture conditions

G1-to-S transition could be differentially regulated after Ag/APC- (not shown). or CD3/CD28-mediated restimulation of anergic T cells. We ﬁrst To demonstrate that CD3/CD28-mediated stimulation could

analyzed the expression of G1 cell cycle proteins by Western blot promote cell proliferation, we labeled the cells with the ﬂuorescent The Journal of Immunology 6181

FIGURE 4. CD3/CD28-mediated stimulation drives comparable proliferation of control and anergic A.E7 cells. T cell anergy was induced as

described in Fig. 1. Control (CT) and anergic (AN) T cells were then Downloaded from labeled with the ﬂuorescent dye CFSE and left untreated (untr.) or stimulated with Ag-pulsed irradiated splenocytes (Ag/APC) or with anti-CD3/ CD28 mAbs for 5 days and then analyzed by ﬂow cytometry. A, Repre- sentative FACS histograms reporting the degree of CFSE dilution are shown. B, Percentage of control and anergic T cells that divided more than once (and thus fell in the indicated M2 gate) calculated in four independent

experiments (mean Ϯ SD). http://www.jimmunol.org/

CD3/CD28-mediated signals drive the expansion of IL-2 deﬁcient T cells

To better define whether anti-CD3/CD28 mAbs could drive G1- to-S transition and cell proliferation in the absence of IL-2, we generated a T cell line from the DO11.10 TCR-transgenic IL-2- deficient mice (22) and analyzed the ability of these cells to respond to CD3/CD28 stimulation. Wild-type and IL-2Ϫ/Ϫ T cells by guest on October 2, 2021 FIGURE 3. CD3/CD28-mediated stimulation of anergic T cells by- were stimulated with immobilized anti-CD3 and anti-CD28 mAbs passes G cell cycle block and results in G -to-S cell cycle progression. T 1 1 for 48 h. Thereafter, the cells were harvested and lysed, and protein cell anergy was induced as described in Fig. 1. Control (CT) and anergic contents were analyzed by Western blot. After stimulation, up- (AN) T cells were then stimulated with either Ag/APC (A) or with anti- Cip CD3/CD28 mAbs (B) for 48 h. Thereafter, the cells were counted, fixed, regulation of cyclin D3 and of p21 and down-regulation of Kip stained with propidium iodide, and analyzed by flow cytometry. Repre- p27 were detected in both cell types (Fig. 5A), suggesting that sentative FACS histograms of the DNA content of unstimulated (untreated optimal TCR/CD28 engagement can regulate the G1 cell cycle or stimulated with APC only), Ag/APC-stimulated, and CD3/CD28-stim- proteins in the absence of autocrine IL-2. Interestingly, cyclin D3 ulated control and anergic T cells are reported. C, Percentage of cells in the up-regulation was less pronounced in both anergic (Fig. 2B) and Ͼ Ϫ/Ϫ S-G2-M phase (DNA content 1N) in resting (untr.) and stimulated (Ag/ IL-2 (Fig. 5A) T cells than in the control and wild-type pop- APC, CD3/CD28) control (Ⅺ) and anergic (f) T cells calculated in four ulation. This could be due to the fact that activation of both anergic Ϯ Ϫ Ϫ independent experiments (mean SD). and IL-2 / T occurs in the absence of IL-2, which has been shown to elicit and sustain cyclin D3 expression (26, 35). We next asked whether anti-CD3/CD28 mAbs could drive com- dye CFSE. This dye passively diffuses into the cells, it is retained plete cell cycle progression and elicit cell division in the absence Ϫ Ϫ at the cell surface, and it segregates equally between daughter cells of IL-2. To this aim, wild-type and IL-2 / T cells were labeled upon cell division, resulting in the sequential halving of cellular with CFSE, stimulated with immobilized anti-CD3/CD28 mAbs, fluorescence intensity with each successive generation and allow- and analyzed by flow cytometry 5 days later (Fig. 5B). As control, ing one to trace the fate of single cells by flow cytometry. Thus, T cells were stimulated with rIL-2 normally used to propagate this control and anergic T cells were labeled with CFSE, stimulated clone in culture. Results show that similarly to wild-type T cells Ϫ Ϫ with Ag/APC and anti-CD3/CD28 mAbs, and analyzed 5 days (not shown), IL-2 / DO11.10 T cells completed several rounds later by flow cytometry. Whereas only control cells divided on of cell division as detected by serial CFSE dilution during CD3/ Ag/APC-mediated stimulation, both control and anergic T cells CD28- and IL-2-mediated stimulation. completed several cycles of cell division upon CD3/CD28-medi- Together, these results support the idea that CD3/CD28 can reg- ated stimulation (Fig. 4). Interestingly, a comparable fraction of ulate cell cycle-related events and induce T cell proliferation via control and anergic cells proliferated on immobilized anti-CD3/ IL-2 independent mechanisms. CD28 mAbs, despite the fact that IL-2 was undetectable in culture supernatants of anergic T cells. CD3/CD28-mediated proliferation does not restore Ag Altogether, these results suggest that optimal cross-linking of responsiveness the TCR and of the CD28-costimulatory receptor overcomes the Because IL-2-dependent G1-to-S transition was previously shown G1 cell cycle block and drives anergic T cell proliferation. to prevent and reverse clonal anergy (9, 13), we next investigated 6182 CELL CYCLE AND T CELL ANERGY Downloaded from

FIGURE 5. CD3/CD28 engagement regulates the G1 cell cycle proteins and elicits proliferation of IL-2 Ϫ/Ϫ T cells.A, Wild-type (WT) and IL- 2Ϫ/Ϫ DO11.10 T cells were either left untreated (Ϫ) or stimulated with immobilized anti-CD3 and anti-CD28 mAbs (0.1 and 5 ␮g/ml, respectively) for the indicated times. Western blot analysis of total cell lysates http://www.jimmunol.org/ were then performed with the Abs as indicated. p27Kip and p21Cip were detected on the same membrane filter by sequential hybridization. B, IL- 2Ϫ/Ϫ DO11.10 T cells were labeled with the fluorescent dye CFSE and either left untreated (thin line) or stimulated with anti-CD3/CD28 mAbs (CD3/CD28) or suboptimal anti-CD3 mAb and rIL-2 (IL-2) for 5 days and then analyzed by flow cytometry. Representative FACS histograms report- FIGURE 6. Proliferation of anergic T cells induced by optimal CD3/ ing the degree of CFSE dilution are shown. CD28 engagement does not restore Ag responsiveness. A, Schematic representation of the experimental design. T cell anergy was induced as described in Fig. 1. Control (CT) and anergic (AN) T cells were then allowed by guest on October 2, 2021 to proliferate in response to immobilized anti-CD3/CD28 mAbs for 5 days, whether proliferation induced by anti-CD3/CD28 mAbs could re- as described in Fig. 4, and rested for the next 10 days. In parallel, the cells store Ag responsiveness in anergic T cells. were also stimulated with rIL-2. Thereafter, control and anergic T cells Control and anergic T cells were first allowed to proliferate in were labeled with CFSE and either left untreated or stimulated with Ag/ response to immobilized anti-CD3 and anti-CD28 mAbs or to APC or with rIL-2 (IL-2) and analyzed by flow cytometry 5 days later. B, rIL-2 (first stimulation; refer to Fig. 6A for a schematic represen- Representative FACS histograms reporting the degree of CFSE dilution in tation of the experiment). Proliferation during this time was mon- the absence (thin line) and in the presence (thick line) of stimulation are itored by CFSE dilution in separate samples (not shown). There- depicted. C, Percentage (mean Ϯ SD of three independent experiments) of after, the cells were rested for 10 days, labeled with CFSE, and control and anergic cells pretreated as indicated (first stimulation) dividing restimulated with Ag/APC or rIL-2. CFSE dilution in response to to secondary rechallenge with Ag/APC. Ag/APC rechallenge was then used to compare Ag responsiveness in untreated, CD3/CD28-treated, and IL-2-treated cells. As expected, only untreated control cells, and not untreated anergic T IL-2-driven proliferation restored the ability of anergic T cells cells, proliferated in response to Ag/APC and completed up to four not only to proliferate in response to an Ag rechallenge but also to rounds of cell division (Fig. 6B, left panels). Surprisingly, similar produce IL-2 (data not shown). Thus, whereas the IL-2 produced results were obtained with cells allowed to proliferate in response by CD3/CD28-stimulated anergic cells represented only 2.5 Ϯ 0% to immobilized anti-CD3/CD28 mAbs (Fig. 6B, middle panels). of the IL-2 produced by control cells, the amount of IL-2 produced Thus, only CD3/CD28-stimulated control T cells, and not CD3/ by anergic T cells allowed to proliferate in response to rIL-2 was CD28-stimulated anergic cells, divided in response to Ag/APC 87.5 Ϯ 22.5% of the amount produced by control T cells. By (Fig. 6, B, middle panels, and C). This indicates that despite com- contrast, IL-2 was never detected on restimulation of anergic T pleting several rounds of cell division, anergic T cells still fail to cells allowed to proliferate on immobilized anti-CD3/CD28 mAbs respond to antigenic rechallenge. (not shown). In contrast to anergic T cells that had divided in response to Finally, all the cells proliferated to similar extents in response to immobilized mAbs, anergic T cells allowed to proliferate in re- rIL-2 (Fig. 6B, dotted lines), indicating that untreated and CD3/ sponse to rIL-2 regained, at least in part, the ability to respond to CD28-stimulated anergic cells were viable in our cultures but were Ag/APC (Fig. 6, B, right panels, and C). The IL-2-dependent an- specifically unable to respond to Ag/APC. ergy reversal was detected, although to variable extents, in several Despite several rounds of cell division, the total number of vi- independent experiments (the average proliferative response to able cells recovered from the cultures of CD3/CD28-stimulated Ag/APC rechallenge of rIL-2-treated anergic T cells was 77.81 Ϯ control and anergic T cells was only 100Ð200% of the one recov- 36.42% of control cells). ered from untreated population. To exclude the possibility that The Journal of Immunology 6183

CD3/CD28-activated anergic T cells could die shortly after acti- there was no preferential loss of proliferating cells over nonre- vation and that an unresponsive population could be selected by sponding cells. this stimulation, cell survival was monitored by flow cytometry In a second set of experiments, control and anergic T cells were over time in two independent sets of experiments. labeled with CFSE, rested in fresh medium, or allowed to prolif- In the first set of experiments, control and anergic T cells were erate on immobilized anti-CD3/CD28 mAbs and then restimulated labeled with CFSE, stimulated with anti-CD3/CD28 mAbs or with either Ag/APC or rIL-2. Further dilution of the CFSE content rIL-2, and then stained with the fluorescent dye TO-PRO-3 at the during the second stimulation allows measuring Ag responsiveness time of the flow cytometry analysis (Fig. 7A). This dye has been of the cells that proliferated during the first stimulation. As shown previously used to identify viable and dead cells in each pool of above (Fig. 6), whereas control cells proliferated to Ag/APC and to divided cells (36). Although a higher fraction of TO-PRO-3ϩ cells rIL-2, anergic T cells failed to respond to Ag/APC and instead was detected in CD3/CD28-stimulated cells when compared with proliferated to rIL-2 (Fig. 7B, left panels). As also shown above, IL-2-stimulated cells (ϳ30% and ϳ2%, respectively), the fraction whereas both control and anergic T cells proliferated in response to of TO-PRO-3ϩ cells was similar in CD3/CD28-stimulated control CD3/CD28 during the first stimulation (Fig. 7B, right panels, thin and anergic cells (30.52% and 20.24%, respectively). This indi- lines), only control cells, and not anergic cells, further diluted their cates that the majority of the cells that proliferated in response to CFSE contents on Ag/APC restimulation (Fig. 7B, right panels, immobilized mAbs were viable at the end of the culture and that thick lines). By contrast, all the cells further diluted their CFSE content in response to rIL-2, supporting the idea that anergic T cells, which proliferated to CD3/CD28, remain viable and able to respond to rIL-2 but incapable of responding to Ag/APC. Together, these results indicate that Ag unresponsiveness can be Downloaded from maintained despite several rounds of cell division and that instead an IL-2-generated signal is specifically required to obtain reversal of clonal anergy. A rapamycin-sensitive signal independent of IL-2-driven

proliferation is specifically required for the reversal of clonal http://www.jimmunol.org/ anergy Previous findings and our own data indicate that IL-2-generated signals are important in preventing the establishment of T cell anergy (9, 13, 37), and in restoring Ag responsiveness to already anergized T cells (Fig. 6). Because the ability of IL-2 to prevent the establishment of T cell anergy was completely prevented by the addition of the immunosuppressive agent rapamycin (12), we investigated whether rapamycin would also prevent the ability of by guest on October 2, 2021 IL-2 to reverse clonal anergy. Control and anergic T cells were thus labeled with CFSE and either left untreated or stimulated with IL-2 in the absence or in the presence of rapamycin (200 nM) for 15 days (refer to Fig. 8A for the experimental plan). Thereafter, the cells were collected, washed, and either left untreated or restimulated for 5 days with Ag/APCs. We first analyzed the ability of rapamycin to block IL-2-driven proliferation. Although rapamycin partially blocked control T cell division in the first 5 days of culture (Fig. 8B, left panels), no differences between IL-2- and IL-2 plus rapamycin-treated cells could be observed after 20 days of culture (Fig. 8B, right panels). This occurred despite the fact that rapamycin was active throughout the duration of the culture (our unpublished observation). The same result was obtained with an- FIGURE 7. Survival of control and anergic T cells undergoing CD3/ ergic T cells (Fig. 8B). Even though the inefficacy of rapamycin CD28-induced stimulation. T cell anergy was induced as described in Fig. was initially surprising, this result is consistent with the possibility 1. Thereafter, the cells were labeled with CFSE and analyzed after 5 days of stimulation (A) or rested for an additional 10 days and rechallenged as that rapamycin does not completely prevent cell cycle progression indicated (B) (refer to Fig. 6A for a schematic representation of the exper- but merely delays entry into the S phase. We then compared the imental design). A, CFSE-labeled control (CT) and anergic (AN) T cells ability of untreated, IL-2-treated, and IL-2 plus rapamycin-treated were either left untreated or allowed to proliferate in response to immo- cells to respond to Ag rechallenge. As also shown in Fig. 6, bilized anti-CD3/CD28 mAbs (CD3/CD28) or rIL-2 (IL-2) for 5 days. Just whereas the majority of untreated control T cells proliferated in before the analysis, TO-PRO-3 was added to each sample. Representative response to Ag/APC, only a fraction of the anergic T cells re- dot plots reporting the degree of CFSE dilution (proliferation) and TO- sponded to the antigenic stimulation (Fig. 8C, left panels). As also PRO-3 staining (cell death) are depicted. B, Control and anergic T cells shown above (Fig. 6), Ag unresponsiveness was lost upon IL-2- were labeled with CFSE and cultured for 5 days in fresh medium (left driven cell proliferation. Indeed, both control and anergic T cells panels) or on anti-CD3/CD28 mAbs-coated plates (CD3/CD28, right pan- stimulated for 20 days in IL-2 responded to a similar extent to els). Thereafter the cells were rested for 10 days and either left untreated (thin lines) or restimulated with Ag/APC (thick lines) or rIL-2 (dotted Ag/APC (Fig. 8C, middle panels). By contrast, anergic T cells lines). Representative FACS histograms of one of two similar experiments stimulated with IL-2 in the presence of rapamycin failed to further (performed with duplicates) are shown. The overlays demonstrate that the dilute their CFSE content on Ag/APC rechallenge (Fig. 8C, right cells, which proliferated during the first stimulation, are still able to divide panels). This indicates that although rapamycin does not block T on rechallenge, as indicated by further dilution of their CFSE content. cell proliferation, it completely prevents IL-2-mediated reversal of 6184 CELL CYCLE AND T CELL ANERGY

to elicit G1-to-S transition via IL-2-independent mechanisms. We thus investigated the ability of anergic T cells to respond to plate- bound anti-CD3/CD28 mAbs. The results reported here provide a number of new important ﬁndings: 1) although Ag/APC fail to

activate anergic T cells and drive G1-to-S transition, optimal oc-

cupancy of the TCR and of CD28 overcomes G1 cell cycle block and drives anergic cell proliferation; 2) CD3/CD28-dependent proliferation can occur in the absence of IL-2; 3) expression of IL-2

and G1 cell cycle proteins can be independently regulated; 4) G1-

to-S-G2-M transition is not sufﬁcient to restore Ag responsiveness; and 5) IL-2-induced signaling independently regulates cell proliferation and lymphocyte responsiveness. As previously reported elsewhere (16), we also found that antigenic stimulation of anergic T cells was unable to elicit the ex-

pression of the G1-associated proteins required for the G1-to-S

transition. However, we showed that the G1 cell cycle block could be overcome by optimal engagement of the TCR and of CD28 with plate-bound anti-CD3/CD28 mAbs. Indeed, comparable levels of Cip Kip cyclins D2, D3, and E; of the inhibitors p21 and p27 ; and of Downloaded from hyperphosphorylated Rb were induced in CD3/CD28-stimulated control and anergic T cells. Furthermore, comparable numbers of control and anergic T cells entered the S phase (as detected by DNA content) and completed several rounds of cell division (as detected by CFSE dilution) upon stimulation with plate-bound anti-

CD3 and anti-CD28 mAbs. http://www.jimmunol.org/ Several possibilities could explain why a different outcome is induced upon Ag- or Ab-mediated stimulation. It is possible that the establishment of T cell anergy lowers the sensitivity of the TCR or of CD28 and thus that more potent receptor engagement is required to elicit proper intracellular signals. In agreement with this hypothesis is the ﬁnding that soluble or latex bead-bound anti- CD3/CD28 mAbs (which provide weaker receptor cross-linking

FIGURE 8. Rapamycin prevents IL-2-mediated reversal of clonal an- when compared with plate-bound mAbs) mediate proliferation of by guest on October 2, 2021 ergy, but not IL-2-driven proliferation. T cell anergy was induced as de- control, but not of anergic cells (our unpublished result). Thus, it scribed in Fig. 1. A, Schematic representation of the experimental design. is conceivable that whereas receptor occupancy by the Ag/MHC II B, CFSE-labeled control (CT) and anergic (AN) T cells were either left complexes and the B7 family members expressed on the surface of untreated (thin lines) or stimulated with rIL-2 in the absence (IL-2, thick splenic APCs only provides suboptimal receptor engagement that lines) or presence (dotted line) of rapamycin (200 nM, IL-2 plus rapamycin. Cell proliferation was determined by flow cytometry after 5 (left pan- is insufficient to drive anergic cell activation, plate-bound anti- els) and 20 days (right panels). Representative histograms are shown. C, TCR (anti-CD3) and anti-CD28 mAbs optimally activate anergic T CFSE-labeled untreated (medium), and IL-2 or IL-2 ϩ rapamycin-treated cells. It is also possible that only anti-CD28 mAb and not the B7 cells were left unstimulated (untreated; thin line) or stimulated with Ag/ molecules can activate the intracellular pathways leading to IL-2- APC (thick line) or rIL-2 (dotted line) for 5 days. Representative FACS independent p27Kip degradation, recently described in T cell histograms reporting CFSE content are shown (top, middle panel, the clones (16) and here in IL-2 deficient T cell lines (Fig. 5). Even CFSE profile obtained with rIL-2 is underneath the profile elicited by Ag/ though this possibility requires further investigation, it seems un- APC). The overlays demonstrate further dilution of CFSE content on secondary stimulation. A representative experiment (performed with dupli- likely because evidences for IL-2-independent but CD28-depen- cates) of three independent experiments is shown. dent T cell proliferation has been already described in the context of Ag recognition in vivo (21, 22). Finally, it is possible that stimulation of anergic T cells with plate-bound anti-CD3/CD28 mAbs, clonal anergy. Thus, altogether, our results indicate that an IL-2- but not with Ag-bearing APC, elicits minute amounts of IL-2, induced signal, which is delivered independently of IL-2-driven which could be sufficient to sustain clonal expansion. Even though proliferation is absolutely required to regulate clonal anergy and this remains a possibility, it is interesting that the IL-2 possibly lymphocyte responsiveness. produced does not elicit reversal of clonal anergy (discussed in more detail below). Discussion The fact that plate-bound anti-CD3/CD28 mAbs drive prolifer- By tracing proliferation and Ag responsiveness of single cells, we ation of anergic T cells and of IL-2-deficient DO11.10 T cells, show here that proliferation and clonal anergy can be indepen- which are respectively functionally and genetically incapable of dently regulated, and although both TCR/CD28- and IL-2-gener- producing IL-2, suggests that CD3/CD28-dependent but IL-2 in- ated signals elicit T cell proliferation, only IL-2/IL-2R interaction dependent cell cycle progression can occur in optimally activated has the ability to regulate Ag responsiveness. T lymphocytes. Appleman et al. (27) recently found that ligation of This study originated from the observation that whereas anergic CD28 results in p27Kip ubiquitination and degradation and subse- T cells fail to produce IL-2 and to properly regulate the expression quent activation of cyclin D2-cdk4/cdk6 and of cyclin E-cdk2. In Kip of G1-related proteins, optimal engagement of TCR/CD28 is able our hands CD28 ligation augmented p27 degradation and CFSE The Journal of Immunology 6185

dilution in CD3-stimulated T cells (our unpublished data). Fur- not in G0,G1b,orS-G2 phase (11, 12). Our results expand these thermore, CD3/CD28 stimulation elicited comparable down-regu- original observations and further indicate that neither G1-to-S- Kip lation of p27 in IL-2-sufficient (control A.E7 cells, wild-type G2-M phase transition nor cell division elicited by either CD3/ DO11.10 T cells) and IL-2-deficient (anergic A.E7 and DO11.10 CD28 or by IL-2 is sufficient to obtain anergy reversal. Indeed, IL-2Ϫ/Ϫ) T cells. Down-regulation of p27Kip in CD3/CD28-acti- even in the case of IL-2, we were able to dissociate T cell prolif- vated DO11.10 IL-2Ϫ/Ϫ T cells also correlated with cell prolifer- eration from anergy reversal because the addition of rapamycin ation. This was previously reported not to be the case in a recent failed to prevent IL-2-driven cell proliferation and instead com- publication by Powell et al. (38). This difference can, however, be pletely prevented the ability of IL-2 to restore Ag responsiveness. attributed to the fact that these authors used soluble and not plate- A similar result was recently obtained by Vanasek et al. (39), who bound anti-CD3/CD28 mAbs, and only looked at p27Kip expres- showed that rapamycin induced T cell tolerance in vivo despite sion early after stimulation. It is possible that either stronger re- residual T cell expansion. ceptor cross-linking or longer stimulation must be provided to The inability of rapamycin to inhibit proliferation in our cultures elicit more complete p27Kip degradation and cell division. Thus, could be explained by rapamycin merely delaying and not inhib- even though the possibility that optimal CD28 stimulation elicits iting cell cycle progression (compare day 5 with day 20 of Fig. 8, production of IL-2 or of a related cytokine responsible for subse- and refer to Ref. 40) and by the existence of IL-2-dependent but quent cell division remains to be elucidated, our results support the rapamycin-insensitive proliferative pathways (41). Indeed, there is possibility that CD28-generated signals might directly control cell now evidence that IL-2-dependent proliferation is driven not only cycle progression, via an IL-2-independent mechanism. by activation of the phosphatidylinositol 3-kinase-protein kinase It is interesting that although maximal TCR and CD28 ligation B/AKt-mammalian target of rapamycin (mTor) pathway but also ␤ ␥ Downloaded from was able to bypass the G1 cell cycle block and to elicit similar by IL-2R - and -chain-generated signals involving Bcl-2, c- proliferation of control and anergic T cells, it failed to elicit de- Myc, and Janus kinase 3-STAT5-dependent transcription (42). It is tectable IL-2 production by anergized T cells. This suggests that interesting that the limited efficacy of rapamycin in blocking T cell the signals involved in IL-2 gene transcription and in the regula- expansion was previously underestimated by measuring prolifer- 3 tion of the G1 checkpoints might be of a different nature in our ation by conventional [ H]thymidine incorporation (12) and was anergized Th clone. Boussiotis et al. (15) previously suggested that instead revealed by following division of individual CFSE-labeled deregulated expression of the cyclin-dependent kinase inhibitor T cells over time (S. Colombetti and A. Mondino, manuscript in http://www.jimmunol.org/ p27Kip, which they found overexpressed in anergic human T cell preparation). clones, correlated with defective IL-2 transcription. These authors The observation that rapamycin prevents anergy reversal sug- also showed that overexpression of p27Kip inhibited IL-2 gene gests that an IL-2-generated, rapamycin-sensitive signal is specif- transcription by sequestering JAB-1 and thus inhibiting AP-1- ically required to restore Ag responsiveness. This signal would dependent transcription. Our results do not support a role for drive clonal anergy reversal not by allowing the G1-to-S transition p27Kip in defective IL-2 production in anergic A.E7 T cells. In- but rather by directly eliciting degradation or inhibition of the neg- deed, although IL-2 was barely detectable in culture supernatants, ative anergic factors putatively expressed on TCR engagement. others (38) and we (Fig. 2) found p27Kip efficiently down-regu- At this time, it is unclear how rapamycin exerts its function. by guest on October 2, 2021 lated on CD3 and CD28 engagement. Furthermore, we found Rapamycin is known to bind the immunophilin FK506BP12, and JAB-1 highly represented in both the nuclear and the cytosolic this complex regulates the activity of mTor (43Ð45). mTor then fraction of control and anergic T cells (our unpublished observa- leads to the activation of the p70S6 kinase (46), and to the phos- tion), suggesting that JAB-1 is not a limiting factor for IL-2 gene phorylation of 4E-binding protein 1 (47, 48). Ongoing experiments transcription in anergic A.E7 T cells. Thus G1-to-S transition and are investigating which is the mTor-dependent signaling pathway transcription of the IL-2 gene seem to be independently regulated linked to the reversal of clonal anergy. by CD3/CD28-generated signaling, because cell cycle progression In conclusion, this study clearly dissociates cell cycle progres- occurs despite defective IL-2 synthesis. sion from the regulation of clonal anergy and reveals that the im- Regardless of the final mechanism that dictated CD3/CD28-de- munosuppressive effect of rapamycin is unrelated to its antiprolif- pendent anergic T cell proliferation, this was not sufficient to elicit erative effect; rather, it is directly linked to the control of reversal of clonal anergy. This could be possibly explained if lymphocyte responsiveness. In light of these results, caution chronic TCR engagement in the absence of IL-2 could result in the should be used in interpreting the immunosuppressive effect of continuous production and accumulation of the putative anergic rapamycin in vivo. factor despite cell proliferation. However, our finding that even IL-2-driven proliferation was unable to elicit reversal of clonal Acknowledgments anergy, when occurring in the presence of rapamycin (see below), We thank Dr. R. Pardi (DIBIT, Milan, Italy), Dr. Jon Pines (Wellcome/ supports the idea that proliferation per se is not sufficient to restore Cancer Research U.K. Institute), and Dr. Dan Mueller (University of Min- Ag responsiveness. nesota) for critical reading of the manuscript. The idea that proper cell cycle progression and proliferation upon activation was required to maintain lymphocyte responsive- References 1. Jenkins, M. K., and R. H. Schwartz. 1987. Antigen presentation by chemically ness was originally proposed by Jenkins and Schwartz (1). These modified splenocytes induces antigen-specific T cell unresponsiveness in vitro authors showed that TCR engagement in the absence of costimu- and in vivo. J. Exp. Med. 165:302. lation elicited T cell activation but not proliferation and resulted in 2. Jenkins, M. K., C. A. Chen, G. Jung, D. L. Mueller, and R. H. Schwartz. 1990. Inhibition of antigen-specific proliferation of type 1 murine T cell clones after the establishment of clonal anergy. In this model CD28 costimu- stimulation with immobilized anti-CD3 monoclonal antibody. J. Immunol. 144: lation was proposed to promote escape from anergy by eliciting 16. IL-2 production and cell division. It was, however, later shown that 3. Mueller, D. L., M. K. Jenkins, and R. H. Schwartz. 1989. 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