CTLA-4/B7 Interaction Induction of T Cell Anergy in the Absence Of

Induction of T Cell Anergy in the Absence of CTLA-4/B7 Interaction Kenneth A. Frauwirth, Maria-Luisa Alegre and Craig B. Thompson This information is current as of October 1, 2021. J Immunol 2000; 164:2987-2993; ; doi: 10.4049/jimmunol.164.6.2987 http://www.jimmunol.org/content/164/6/2987 Downloaded from References This article cites 62 articles, 47 of which you can access for free at: http://www.jimmunol.org/content/164/6/2987.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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Induction of T Cell Anergy in the Absence of CTLA-4/B7 Interaction1

Kenneth A. Frauwirth,* Maria-Luisa Alegre,† and Craig B. Thompson2*

Immunologic tolerance in T lymphocytes is maintained through both thymic and peripheral contributions. One peripheral tolerance mechanism is the induction of T cell anergy, a form of nonresponsiveness resulting from incomplete T cell activation, such as stimulation through the TCR in the absence of costimulation. Recent reports have suggested that engagement of the inhibitory receptor CTLA-4 by its B7 ligand is critical for the initiation of anergy. We tested the importance of CTLA-4 in anergy induction in primary T cells with an in vitro anergy system. Using both CTLA-4/B7-blocking agents and CTLA-4-deﬁcient T cells, we found that T cell anergy can be established in the absence of CTLA-4 expression and/or function. Even in the absence of CTLA-4 signal transduction, T cells activated solely through TCR ligation lose the ability to proliferate as a result of autocrine IL-2 production upon subsequent receptor engagement. Thus, CTLA-4 signaling is not required for the development of T cell anergy. The Journal of Immunology, 2000, 164: 2987–2993. Downloaded from

he activation of resting T lymphocytes is a highly regu- fects in anergic cells appear to be similar to the acute effects of lated process requiring two distinct signaling events. CTLA-4 cross-linking, namely blockade of proliferation and IL-2 T Upon interaction of the TCR with its cognate peptide/ production (25, 26), reduction of mitogen-activated protein kinase MHC complex on an APC, signal 1 is initiated by the TCR-asso- activation (27, 28), and inhibition of AP-1 and NF-␬B activity

ciated CD3 complex. However, full activation does not occur un- (29). Indeed, administration of anti-CTLA-4 blocking Abs during http://www.jimmunol.org/ less a second, costimulatory signal (signal 2) is also received. An anergy induction appears to inhibit the induction of T cell hypo- important costimulatory molecule on resting T cells appears to be responsiveness in several systems (20, 21, 30–32), and these ob- CD28 (1–3), which initiates signaling after binding to members of servations are the basis for models invoking CTLA-4 signal trans- the B7 family on the APC; however, other T cell surface mole- duction in the induction of anergy. cules, such as CD2 (4), LFA-1 (5–7), and CD27 (8), have been Although the hypothesis that CTLA-4 is the anergy initiator reported to have costimulatory activity. molecule is an attractive one, many in vitro anergy systems ex- T cells that receive signal 1 in the absence of signal 2 enter a clude B7.1/2-expressing APC, and B7.1/2-blocking agents have state of nonresponsiveness to subsequent stimulation (9–11). This been used to induce anergy when B7-expressing cells are present nonresponsiveness, termed anergy, is characterized by a failure to (33–38). Because interaction with B7 is required for CTLA-4 sig- by guest on October 1, 2021 proliferate or secrete IL-2 upon Ag receptor engagement even in naling, it is difficult to reconcile the model with these data. We the presence of costimulation. Anergic T cells have been reported therefore undertook a direct examination of the role of CTLA-4 in to have defects in the Ras and mitogen-activated protein kinase the initiation of T cell anergy. Using both reagents that block the signaling pathways (12–14), reduced Ca2ϩ flux (15), and altered CTLA-4/B7 interaction and CTLA-4-deficient T cells, we found tyrosine kinase activity (15, 16). These signaling deficiencies ap- that CTLA-4 signaling is not required for anergy induction in vitro. pear to lead to reduced NF-␬B and AP-1 transcriptional activities (17–19). Anergy was originally described in CD4ϩ T cell clones, Materials and Methods but it has since been shown to occur in primary CD4ϩ and CD8ϩ Animals T cells, both in vitro and in vivo (20–24), and may be a significant Female C57BL/6 mice (4–8 wk old) were purchased from The Jackson component of peripheral tolerance. Laboratory (Bar Harbor, ME). Lck-Bcl-xL transgenic mice have been de- Several groups have recently proposed that CTLA-4, a molecule scribed previously (39). F5 TCR transgenic (40)/recombination-activating Ϫ/Ϫ 3 ϩ/Ϫ expressed on activated T cells, acts as the trigger for anergy (20, gene 1 (RAG1 ) /CTLA-4 mice were provided by Dr. Philip Ash- ton-Rickardt (University of Chicago, Chicago, IL). 2C TCR transgenic 21). CTLA-4, like CD28, binds to members of the B7 family, but (41)/RAG2Ϫ/Ϫ/CTLA-4ϩ/Ϫ mice were provided by Dr. Thomas Gajewski has an inhibitory effect on T cell activation (25, 26), making (University of Chicago). DO.11.10 TCR transgenic mice (42) were the gift CTLA-4 a logical candidate for initiating anergy. Further, the de- of Dr. Kenneth Murphy (Washington University, St. Louis, MO). All mice were maintained in the University of Chicago Animal Barrier Facilities. Abs and reagents *Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104; and †Department of Medicine, UC10-4F10-11 (4F10; hamster anti-CTLA-4) and 145-2C11 (2C11; ham- University of Chicago, Chicago, IL 60637 ster anti-CD3⑀) were purified from hybridoma supernatants by passage Received for publication October 21, 1999. Accepted for publication January 6, 2000. over protein A-Sepharose columns. FITC-labeled hamster IgG, anti- Thy1.2, anti-CD3, anti-TCR, anti-CD25, PE-labeled hamster IgG, anti- The costs of publication of this article were defrayed in part by the payment of page Thy1.2, anti-CD69, anti-CD44, and anti-CD62L were purchased from charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. PharMingen (San Diego, CA). Murine CTLA4-Ig was provided by Genet- ics Institute (Cambridge, MA). Influenza nucleoprotein 366–374 peptide 1 This work was supported by National Institutes of Health Grants P01DK49799 ASNENMDAM (H-2Db restricted, F5 TCR reactive) was provided by Dr. and 5P01AI35294. 2 Address correspondence and reprint requests to Dr. Craig B. Thompson, Abramson Family Cancer Research Institute, University of Pennsylvania, BRB II/III Room 450, 3 Abbreviations used in this paper: RAG, recombination-activating gene; Tg, 421 Curie Boulevard, Philadelphia, PA 19104-6160. transgenic.

Philip Ashton-Rickardt (University of Chicago). The H-2Kb-restricted 2C negative controls. Cells were simultaneously stained with PE- or FITC-

TCR-reactive peptide SIYRYYGL and the OVA323–339 peptide conjugated anti-Thy1.2 mAb to assess T cell purity. After a final wash, ISQAVHAAHAEINEAGR (I-Ad restricted, DO.11.10 TCR reactive) were cells were resuspended in FACS buffer and analyzed on a FACSsort flow purchased from Multiple Peptide Systems (San Diego, CA). cytometer (Becton Dickinson, Mountain View, CA). Forward and side scatter gates were used to exclude dead cells. Data from ϳ104 Cell lines and culture live cells were analyzed using CellQuest software (Becton Dickinson). For cell cycle d 6 The OVA323–339/I-A -specific TH1 clone pGL10 was the gift of Dr. analysis, 10 cells were fixed in 25% PBS/75% ethanol for1hat4°C. The Thomas Gajewski (University of Chicago). All cells were maintained in fixed cells were resuspended in propidium iodide staining solution (3.8 mM DMEM (Life Technologies, Grand Island, NY) supplemented with L-glu- sodium citrate, 0.125 mg/ml RNase A, and 0.01 mg/ml propidium iodide), tamine, penicillin, streptomycin, HEPES buffer, MEM nonessential amino incubated on ice for 30 min, and then analyzed by flow cytometry as de- acids, 2-ME, and 10% FBS (Life Technologies), at 37°C in a 7% CO2 scribed above. atmosphere. T cell purification Results Murine T cells were isolated from spleen and mesenteric lymph nodes using the StemSep negative selection system (StemCell Technologies, Induction of anergy in primary T cells in vitro Vancouver, Canada) following the manufacturer’s instructions. Briefly, Many of the studies used to define T cell anergy have been per- single-cell suspensions were prepared by manual disruption of spleen and lymph nodes and passage of cells through nylon mesh. Erythrocytes were formed using T cell clones. To determine whether anergy could depleted by hypotonic lysis. The cells were washed and resuspended in also be established in normal mouse T cells, an anergy induction PBS supplemented with 2% FBS and 5% heat-inactivated normal rat serum system using immobilized anti-CD3 Ab (mAb 2C11) to stimulate (Jackson ImmunoResearch Laboratories, West Grove, PA). The cells were primary T cells was developed, based on the protocol of Jenkins et incubated with StemSep T cell enrichment Ab cocktail (a mixture of bio- Downloaded from tinylated anti-B cell, anti-erythrocyte, and anti-myeloid Abs) at 4°C, al. (10). Culturing purified C57BL/6 T cells on anti-CD3-mAb- washed, and resuspended in PBS plus 2% FBS. The cells were then incu- coated dishes for 2 days followed by 2 days of rest (culture in the bated with StemSep anti-biotin tetramer (a bifunctional, anti-biotin/anti- absence of Ab) resulted in a profound nonresponsiveness to re- dextran tetramer), followed by incubation with dextran-linked magnetic beads and passage over magnetized columns to deplete non-T cells. The stimulation through Ag receptor. The T cells failed to proliferate or nonretained fraction typically contained Ͼ95% Thy1ϩ cells, as determined secrete IL-2 when stimulated with splenic APC plus anti-CD3 by flow cytometry. To purify CD4ϩ or CD8ϩ T cells, the appropriate

mAb (Fig. 1), but were hyper-responsive to exogenously added http://www.jimmunol.org/ StemSep Ab cocktails (containing the components of the T cell enrichment IL-2 (data not shown), indicating that the lack of response was not Ab cocktail plus either anti-CD8 or anti-CD4 Abs, respectively) were used in place of StemSep T cell enrichment Ab cocktail. due to T cell death during the restimulation. This was confirmed by manual cell count (data not shown). In vitro anergy induction A major fate of purified T cells was to undergo apoptotic death Purified murine T cells were cultured on immobilized anti-CD3 mAb during the initial 4-day culture, however, limiting the yields of ␮ (2C11) for 2 days in the presence or absence of either 50 g/ml soluble viable anergic cells. This difficulty in maintaining cell survival was CTLA4-Ig or 25 ␮g/ml soluble anti-CTLA-4 mAb (4F10). Cells were har- vested, resuspended in fresh medium, and cultured for 2 additional days in not limited to the anergic cell population, as freshly isolated T cells the absence of anti-CD3 mAb (“rest”). T cells were collected, counted, and cultured in the absence of stimulation also displayed low levels of tested for anergy by measuring cytokine secretion and proliferation upon viability after 4 days (data not shown). To avoid these obstacles, T by guest on October 1, 2021 restimulation with irradiated C57BL/6 splenocytes and titrated doses of cells isolated from animals bearing a transgene for the anti- anti-CD3 mAb. For proliferation assays, 5 ϫ 104 T cells plus 2 ϫ 105 splenocytes were cocultured in a final volume of 200 ␮l. For cytokine apoptotic protein Bcl-xL under the control of the Lck proximal ϫ 5 ϫ 6 assays, 2.5 10 T cells plus 1 10 splenocytes were cocultured in a promoter (39) were used. The Bcl-xL transgenic (Tg) T cells main- final volume of 1 ml. In some experiments with TCR transgenic T cells, the tained high viability in culture with or without anti-CD3 mAb specific peptide was used instead of anti-CD3 mAb in the restimulation cultures. stimulation (39) and, like their nontransgenic counterparts, became anergic when treated with immobilized anti-CD3 mAb (Fig. 1, b Cytokine and proliferation assays and c). Bcl-xL Tg T cells left untreated in culture for 4 days gen- IL-2, IL-4, and IFN-␥ levels in 1-day restimulation culture supernatants erally had proliferative and cytokine responses comparable to were measured by sandwich ELISA. Primary and biotinylated secondary those of freshly isolated T cells, indicating that the culture condi- anti-cytokine Abs were purchased from PharMingen and used at the con- tions themselves did not contribute significantly to nonresponsive- centrations recommended by the manufacturer. Alkaline phosphatase-conjugated avidin was purchased from Jackson ImmunoResearch Laboratories ness (data not shown). and used at a 1/3000 dilution. Colorimetric alkaline phosphatase substrate During anergy induction, the T cells were induced to express (Sigma, St. Louis, MO) was used at 1 mg/ml in 10% diethanolamine buffer, several activation markers, including CD25 (Fig. 2a) and CD69 and quantitation was performed on a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA). Data were analyzed using SoftMax (data not shown), both of which dropped back to near initial levels Pro software (Molecular Devices) by comparison with a standard curve during the rest period. Culture with anti-CD3 mAb caused a generated using recombinant cytokines at known concentrations. Prolifer- marked reduction in surface CD3 levels, but CD3 was re-expressed 3 ation after 3 days of restimulation was determined by [ H]thymidine in- to nearly starting levels by the end of the rest period (Fig. 2b). T corporation. Cells were pulsed for 7–8 h with 1 ␮Ci of [3H]thymidine (ICN, Costa Mesa, CA)/well, transferred to glass-fiber filters with a 96-well cells also went through blastogenesis during anergy induction (Fig. 3). cell harvester (Tomtec, Hamden, CT), and analyzed by liquid scintillation Several reports have linked anergy induction with G1 arrest (43– using a 1205 Betaplate scintillation counter (Wallac, Turku, Finland). Data 45). The DNA content of T cells was therefore analyzed over the points for all analyses are presented as the mean of triplicate wells. course of anergy induction. Treatment with immobilized anti-CD3

Flow cytometry mAb induced progression through S and G2/M phases (Fig. 3), and Levels of CD3, TCR, CD25, CD69, CD44, and CD62L were determined cells returned to G1 phase during the rest period. Upon restimula- on freshly purified, anti-CD3-treated, and rested T cells by two-color flow tion, however, the anergized T cells were unable to exit the G1 cytometry. Following harvest, cells were washed once in FACS buffer (1% phase of the cell cycle, unlike T cells that had been left untreated BSA and 0.01% sodium azide in PBS, pH 7.4) and incubated for 30 min in culture for 4 days (Fig. 3). These findings are consistent with on ice in 100 ␮l of FACS buffer containing FITC-conjugated (anti-CD3, anti-TCR, anti-CD25 mAbs) or PE-conjugated (anti-CD69, anti-CD44, anti- results from other models, in which anergy is preceded by an initial CD62L mAbs) mAb; FITC- and PE-conjugated hamster IgG were used as proliferative response (21, 46–48). The Journal of Immunology 2989 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. CD25 and CD3 levels on T cells during anergy induction. T cells were stained with FITC-conjugated anti-CD25 mAb (a) or anti-CD3 mAb (b) after purification (untreated), culture on immobilized mAb 2C11 FIGURE 1. Immobilized anti-CD3 mAb anergizes primary T cells. (2 days Ab), and rest culture (2 days rest), and CD25 and CD3 levels were measured by flow cytometry. For CD3, staining with FITC-conjugated Splenic T cells were purified from normal C57BL/6 (a and c) or Bcl-xL transgenic (b and c) mice, cultured for 2 days in dishes coated with 10 hamster IgG (isotype control) is also shown (b). Fluorescence intensity ␮g/ml of anti-CD3 mAb 2C11, and then rested for 2 days in fresh medium (arbitrary units) is shown on the horizontal axis, and cell number is given in uncoated dishes. To assay proliferation (a and b), cells were restimulated on the vertical axis. for 3 days with irradiated C57BL/6 splenocytes plus titrated soluble mAb 2C11, and incorporation of [3H]thymidine was measured as described in mAb/Bcl-x Tg T cell system to investigate the role of CTLA- Materials and Methods. IL-2 secretion (c) was determined by ELISA of L culture supernatants after 21 h of restimulation with irradiated splenocytes 4/B7 interactions in anergy induction. Freshly isolated T cells Ͼ ϩ plus 10 ␮g/ml of soluble mAb 2C11. Proliferation and IL-2 production ( 95% Thy1 after purification) did not express detectable B7.1 were compared with those of freshly isolated (untreated) T cells (open or B7.2, but up-regulated both B7.2 (transiently) and B7.1 during symbols/hatched bars). Data are representative of two independent exper- anti-CD3 mAb treatment (data not shown), raising the possibility iments for wild-type C57BL/6 cells and Ͼ10 independent experiments for that B7 molecules on the T cells themselves could act as the li-

Bcl-xL transgenic cells. Error bars represent 1 SD from the mean of trip- gands for CTLA-4. We therefore added either CTLA4-Ig, which licate wells. binds B7 molecules with high affinity, or ␣-CTLA-4 mAb (mAb 4F10) to prevent any potential CTLA-4/B7 interactions. Addition of CTLA4-Ig to cultures was completely ineffective at preventing Blocking CTLA-4/B7 does not prevent anergy induction nonresponsiveness (Fig. 4, a and b), even at levels that totally The T cell inhibitory receptor CTLA-4 is thought to be important abrogated CD28 costimulation in parallel cultures (Fig. 4c). Like- for the induction and/or maintenance of peripheral tolerance (20, wise, coculture with soluble ␣-CTLA-4 mAb consistently had no 21, 30–32, 49–51). We therefore used the immobilized anti-CD3 appreciable effect on anergy induction (Fig. 5). 2990 CTLA-4 AND ANERGY Downloaded from FIGURE 3. Anergized cells enter cell cycle, but arrest in G1 phase upon restimulation. Purified Bcl-xL transgenic T cells were cultured in medium alone for 4 days (upper panels) or were subjected to anergy induction culture of 2 days on immobilized mAb 2C11 plus 2 days of rest (lower panels) and then stimulated for 3 days with irradiated C57BL/6 splenocytes plus 10 ␮g/ml of soluble mAb 2C11. Dead T cells and splenocytes were removed by centrifugation over Ficoll, and recovered cells (Ͼ95% Th1.2ϩ; http://www.jimmunol.org/ not shown) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry after 2 days of primary culture (left panels) and 3 days of restimulation (right panels). Fluorescence intensity is shown on the vertical axis, and forward scatter is shown on the horizontal axis.

Analysis of CD4ϩ/CD8ϩ subsets after anergy induction in some experiments revealed a skewing toward CD8ϩ cells, although the by guest on October 1, 2021 reasons for this are unknown. The reports linking CTLA-4 to anergy either evaluated only CD4ϩ cells (20) or concluded that ␣-CTLA-4 treatment most dramatically affected CD4ϩ cells (21), raising the concern that our failure to prevent anergy by CTLA-4 blockade might be due at least in part to this CD8ϩ skewing. We therefore compared anergy induction in purified CD4ϩ and CD8ϩ T cells. Culture on immobilized anti-CD3 mAb inhibited subsequent proliferative and IL-2 responses of both subsets, and treatment with ␣-CTLA-4 mAb was unable to prevent anergy in either CD4ϩ (Fig. 6)orCD8ϩ (data not shown) cells. Likewise, CTLA4-Ig did not block induction of anergy in T cells purified from DO.11.10 TCR (OVA/I-Ad-specific) transgenic mice, and neither CTLA4-Ig nor anti-CTLA-4 mAb inhibited anergy induc- FIGURE 4. Blockade of CTLA-4/B7 interactions with CTLA4-Ig does d ϩ tion in the OVA/I-A -specific CD4 clone pGL10 (data not not prevent anergy induction. Purified Bcl-xL transgenic T cells were cul- shown). tured on immobilized mAb 2C11 in the absence (anti-CD3) or the presence (anti-CD3/CTLA4-Ig) of 50 ␮g/ml of soluble CTLA4-Ig, rested, and re- CTLA-4-deficient T cells can be anergized stimulated as described. Proliferation (a) and IL-2 secretion (b) were mea- The inability of either CTLA4-Ig or ␣-CTLA-4 mAb to prevent T sured as in Fig. 1 and compared with responses of freshly isolated (untreated) cells. Data are representative of three independent experiments. cell nonresponsiveness in this system strongly suggested that Error bars represent 1 SD from the mean of triplicate wells. c, CTLA4-Ig CTLA-4 is not required for anergy induction; however, it was pos- activity was confirmed by inhibition of CD28-dependent proliferation. Freshly sible that neither reagent completely blocked CTLA-4/B7 interac- purified T cells were stimulated with irradiated splenocytes plus soluble mAb tions, allowing a small, but sufficient, amount of CTLA-4 signaling 2C11 in the absence (untreated) or the presence of 50 ␮/ml of soluble CTLA4- to occur. We therefore made use of CTLA-4-deficient T cells to Ig, and proliferation was measured as described in Fig. 1. address this concern. CTLA-4 knockout mice develop a fatal lymphoproliferative disease within 3–4 wk of birth (49, 50). However, when the CTLA-4 deficiency was bred onto TCR transgenic, To study anergy in the complete absence of CTLA-4, we puri- RAGϪ/Ϫ mice, the exclusive expression of either the 2C (Ld-al- fied 2C TCR Tg T cells from CTLA-4ϩ and CTLA-4Ϫ age- loreactive (41)) or F5 (Db/influenza nucleoprotein-reactive (40)) matched mice and cultured them with immobilized anti-CD3 mAb receptor led to T cell populations that avoided the lymphoprolif- as described above. As shown in Fig. 7, both proliferation and IL-2 erative disease (M. Alegre, unpublished observations). secretion were greatly reduced upon restimulation in CTLA-4ϩ The Journal of Immunology 2991 Downloaded from http://www.jimmunol.org/

FIGURE 5. Blockade of CTLA-4/B7 interactions with anti-CTLA-4 ϩ mAb does not prevent anergy induction. Purified Bcl-x transgenic T cells FIGURE 6. CD4 T cells can be anergized in the presence of anti- L ϩ were cultured on immobilized mAb 2C11 in the absence (anti-CD3) or the CTLA-4 mAb. CD4 T cells were purified from Bcl-xL transgenic mice presence (anti-CD3/anti-CTLA-4) of 25 ␮g/ml of soluble mAb 4F10, and treated as described in Fig. 6, except that neglected cells (T cells by guest on October 1, 2021 rested, and restimulated as described. Proliferation (a) and IL-2 secretion cultured in medium alone for 4 days) were used instead of freshly isolated (b) were measured as described in Fig. 1. Data are representative of four cells for comparison. a, Proliferation. b, IL-2 secretion. Data are represen- independent experiments. Error bars represent 1 SD from the mean of tative of two independent experiments. Error bars represent 1 SD from the triplicate wells. mean of triplicate wells. and CTLA-4Ϫ T cells after culture on plate-bound anti-CD3 mAb. Ϫ Although the CTLA-4 cells consistently gave higher overall re- anergy induction. One explanation for this discrepancy may be our sponses, the degree of inhibition by the anergizing treatment was use of an in vitro system. Perez et al. analyzed anergy induced in ϩ comparable (IL-2 secretion: ϳ84% inhibition with CTLA-4 vivo by soluble Ag, and Walunas et al. studied induction of anergy Ϫ cells, ϳ79% with CTLA-4 cells; proliferation: ϳ94% inhibition by staphylococcal enterotoxin B, also in vivo. Although it is pos- ϩ Ϫ with CTLA-4 cells, ϳ89% with CTLA-4 cells; averaged over sible that the requirements for anergy in vivo differ from those in the top three peptide concentrations). Treatment with immobilized vitro, one must also interpret in vivo results carefully due to the anti-CD3 mAb also inhibited subsequent proliferative responses ϩ Ϫ increased complexity of in vivo systems. Injection of anti-CTLA-4 equally in CTLA-4 and CTLA-4 F5 TCR Tg T cells (Fig. 7c). Ab may alter the basal responses of T cells other than those being assayed. Unimmunized CTLA-4-deficient mice develop a severe Discussion lymphoproliferative disease (49, 50), indicating that CTLA-4 reg- To study anergy in primary T cells, we established an in vitro ulates immune responses to environmental and self-Ags. An in- system using immobilized anti-CD3 mAb as the anergizing agent. crease in IL-2 levels due to an enhancement of general T cell We found that induction of anergy follows an activation phase, activity could prevent or reduce anergy induction (52, 53), inde- which includes expression of CD25 and CD69, modulation of pendent of any cell-autonomous role for CTLA-4. Consistent with CD3, blastogenesis, and progression through the cell cycle. We our in vitro results, a recent study found that blockade of CTLA-4 further demonstrated that anergy can be induced in primary T cells did not have any effect on anergy induction in vivo in a nasal in the absence of any CTLA-4/B7 interaction. Neither CTLA4-Ig tolerance model (48). nor ␣-CTLA-4 mAb was able to prevent anergy induced by im- Earlier models of anergy have generally proposed that TCR/ mobilized anti-CD3 mAb, and CTLA-4-deficient T cells were as CD3 ligation leads to both positive and negative signals in T cells. susceptible as CTLA-4-expressing cells to anergy induction. Thus, The decision between activation and anergy would depend on the CTLA-4 is not required for initiating T cell anergy in this system. balance of those signals, with costimulatory pathways tipping the These results conflict with reports from the Abbas (20) and balance from anergy to activation. Kaufman et al. (54) have for- Bluestone (21) groups, which identified CTLA-4 as critical for mulated such a model in mathematical terms, predicting anergy vs 2992 CTLA-4 AND ANERGY

tor and IL-2 signaling are required. The model predicts that anergy will follow an activation phase if dissociation of the activating ligand is slow, resulting in a prolonged signal, and this is consistent with our results using anti-CD3 mAb. An important consideration in trying to build models to under- stand anergy induction is that the phenomena being studied as anergy are, in fact, a collection of responses to tolerizing stimuli, with different requirements and mechanisms. For example, the ability of anergized cells to differentiate into effector cells or to produce cytokines other than IL-2 (particularly IL-4, IL-10, and IFN-␥) depends on the anergy induction protocol used, as does the ability of CD28 costimulation and exogenous IL-2 to prevent anergy. Thus, CTLA-4 may be important for some pathways to anergy, but is not a universal trigger. Analysis of anergy induction is further complicated by the observation that T cells exposed to various tolerizing conditions can develop into regulatory cells able to suppress the responses of nontolerized cells (55–59). One mechanism of suppression, observed after injection of bacterial superantigen, involves the production of TGF-␤ (59). Induction of TGF-␤ secretion is also a result of CTLA-4 signaling (60), sug- Downloaded from gesting that some apparent effects of CTLA-4 on anergy may, in fact, represent contributions to suppressive mechanisms. There are considerable data to support a role for CTLA-4 in the down-regulation of immune responses, and a number of strategies to restore or enhance in vivo immune responses by inhibiting CTLA-4 signal transduction have been proposed (61, 62). How- http://www.jimmunol.org/ ever, our findings indicate that T cell anergy can be established in the absence of CTLA-4 function, and that studies to establish or reverse Ag-specific anergy should not focus exclusively on CTLA-4. Continued characterization of alternate mechanisms for anergy induction using either the TCR or other T cell-specific receptors is warranted.

Acknowledgments by guest on October 1, 2021 We thank Thomas Gajewski for providing mice and reagents, Pat Fields and Francesca Fallarino for assistance with anergy induction protocols, Jeffrey Rathmell for assistance with T cell purification, and members of the Thompson laboratory for helpful discussions and critical reading of this manuscript. References 1. Thompson, C. B., T. Lindsten, J. A. Ledbetter, S. L. Kunkel, H. A. Young, S. G. Emerson, J. M. Leiden, and C. H. June. 1989. CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines. Proc. Natl. Acad. Sci. USA 86:1333. 2. Linsley, P. S., W. Brady, L. Grosmaire, A. Aruffo, N. K. Damle, and J. A. Ledbetter. 1991. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173:721. FIGURE 7. CTLA-4-deficient TCR Tg T cells can be anergized in vitro. 3. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, and J. P. Allison. 1992. Ϫ ϩ/ϩ CD28-mediated signalling co-stimulates murine T cells and prevents induction of a and b, T cells were purified from 2C TCR Tg/RAG2 /CTLA-4 (filled anergy in T-cell clones. Nature 356:607. Ϫ/Ϫ symbols) or CTLA-4 (open symbols) mice and stimulated with anti- 4. Bierer, B. E., J. Barbosa, S. Herrmann, and S. J. Burakoff. 1988. Interaction of genic peptide and irradiated C57BL/6 splenocytes without (untreated) or CD2 with its ligand, LFA-3, in human T cell proliferation. J. Immunol. 140:3358. after (anti-CD3) culture on immobilized mAb 2C11. Proliferation (a) and 5. Altmann, D. M., N. Hogg, J. Trowsdale, and D. Wilkinson. 1989. Cotransfection of ICAM-1 and HLA-DR reconstitutes human antigen-presenting cell function in IL-2 levels (b) were measured as described in Fig. 1. c, T cells were pu- mouse L cells. Nature 338:512. Ϫ ϩ/Ϫ rified from F5 TCR Tg/RAG1 /CTLA-4 (filled symbols) or CTLA- 6. Kuhlman, P., V. T. Moy, B. A. Lollo, and A. A. Brian. 1991. The accessory 4Ϫ/Ϫ (open symbols) mice and stimulated with or without prior culture on function of murine intercellular adhesion molecule-1 in T lymphocyte activation: immobilized anti-CD3 mAb as described in a, except that soluble mAb contributions of adhesion and co-activation. J. Immunol. 146:1773. 7. Van Seventer, G. A., Y. Shimizu, K. J. Horgan, and S. Shaw. 1990. The LFA-1 2C11 was used in place of antigenic peptide for the stimulation. Data are ligand ICAM-1 provides an important costimulatory signal for T cell receptor- representative of three independent experiments for 2C T cells and two mediated activation of resting T cells. J. Immunol. 144:4579. independent experiments for F5 T cells. Error bars represent 1 SD from the 8. Kobata, T., K. Agematsu, J. Kameoka, S. F. Schlossman, and C. Morimoto. 1994. ϩ mean of triplicate wells. CD27 is a signal-transducing molecule involved in CD45RA naive T cell costimulation. J. Immunol. 153:5422. 9. Jenkins, M. K., and R. H. Schwartz. 1987. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro activation based on the kinetics of ligand binding and signal trans- and in vivo. J. Exp. Med. 165:302. 10. Jenkins, M. K., C. A. Chen, G. Jung, D. L. Mueller, and R. H. Schwartz. 1990. duction. Although this model does not exclude the possible con- Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stim- tributions of inhibitory receptors such as CTLA-4, only Ag recep- ulation with immobilized anti-CD3 monoclonal antibody. J. Immunol. 144:16. The Journal of Immunology 2993

11. Quill, H., and R. H. Schwartz. 1987. Stimulation of normal inducer T cell clones 38. McIntosh, K. R., P. S. Linsley, and D. B. Drachman. 1995. Immunosuppression with antigen presented by purified Ia molecules in planar lipid membranes: spe- and induction of anergy by CTLA4Ig in vitro: effects on cellular and antibody cific induction of a long-lived state of proliferative nonresponsiveness. J. Immu- responses of lymphocytes from rats with experimental autoimmune myasthenia nol. 138:3704. gravis. Cell. Immunol. 166:103. 12. DeSilva, D. R., W. S. Feeser, E. J. Tancula, and P. A. Scherle. 1996. Anergic T 39. Chao, D. T., G. P. Linette, L. H. Boise, L. S. White, C. B. Thompson, and cells are defective in both Jun NH2-terminal kinase and mitogen-activated protein S. J. Korsmeyer. 1995. Bcl-xL and Bcl-2 repress a common pathway of cell death. kinase signaling pathways. J. Exp. Med. 183:2017. J. Exp. Med. 182:821. 13. Li, W., C. D. Whaley, A. Mondino, and D. L. Mueller. 1996. Blocked signal 40. Mamalaki, C., J. Elliott, T. Norton, N. Yannoutsos, A. R. Townsend, P. Chandler, ϩ transduction to the ERK and JNK protein kinases in anergic CD4 T cells. E. Simpson, and D. Kioussis. 1993. Positive and negative selection in transgenic Science 271:1272. mice expressing a T-cell receptor specific for influenza nucleoprotein and endog- 14. Fields, P. E., T. F. Gajewski, and F. W. Fitch. 1996. Blocked Ras activation in enous superantigen. Dev. Immunol. 3:159. ϩ anergic CD4 T cells. Science 271:1276. 41. Sha, W. C., C. A. Nelson, R. D. Newberry, D. M. Kranz, J. H. Russell, and 15. Gajewski, T. F., D. Qian, P. Fields, and F. W. Fitch. 1994. Anergic T-lymphocyte D. Y. Loh. 1988. Selective expression of an antigen receptor on CD8-bearing T clones have altered inositol phosphate, calcium, and tyrosine kinase signaling lymphocytes in transgenic mice. Nature 335:271. pathways. Proc. Natl. Acad. Sci. USA 91:38. 42. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen of 16. Boussiotis, V. A., G. J. Freeman, A. Berezovskaya, D. L. Barber, and intrathymic apoptosis of CD4ϩ CD8ϩ TCRlo thymocytes in vivo. Science 250: L. M. Nadler. 1997. Maintenance of human T cell anergy: blocking of IL-2 gene 1720. transcription by activated Rap1. Science 278:124. 43. Gilbert, K. M., D. N. Ernst, M. V. Hobbs, and W. O. Weigle. 1992. Effects of 17. Kang, S. M., B. Beverly, A. C. Tran, K. Brorson, R. H. Schwartz, and tolerance induction on early cell cycle progression by Th1 clones. Cell. Immunol. M. J. Lenardo. 1992. Transactivation by AP-1 is a molecular target of T cell 141:362. clonal anergy. Science 257:1134. ϩ 44. Gilbert, K. M., and W. O. Weigle. 1993. Th1 cell anergy and blockade in G 18. Sundstedt, A., and M. Dohlsten. 1998. In vivo anergized CD4 T cells have 1a phase of the cell cycle. J. Immunol. 151:1245. defective expression and function of the activating protein-1 transcription factor. J. Immunol. 161:5930. 45. Powell, J. D., C. G. Lerner, and R. H. Schwartz. 1999. Inhibition of cell cycle 19. Mondino, A., C. D. Whaley, D. R. DeSilva, W. Li, M. K. Jenkins, and progression by rapamycin induces T cell clonal anergy even in the presence of D. L. Mueller. 1996. Defective transcription of the IL-2 gene is associated with costimulation. J. Immunol. 162:2775. 46. Lanoue, A., C. Bona, H. von Boehmer, and A. Sarukhan. 1997. Conditions that Downloaded from impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells. J. Im- ϩ munol. 157:2048. induce tolerance in mature CD4 T cells. J. Exp. Med. 185:405. 20. Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, and 47. Sun, J., B. Dirden-Kramer, K. Ito, P. B. Ernst, and N. Van Houten. 1999. Anti- A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires gen-specific T cell activation and proliferation during oral tolerance induction. CTLA-4 engagement. Immunity 6:411. J. Immunol. 162:5868. 21. Walunas, T. L., and J. A. Bluestone. 1998. CTLA-4 regulates tolerance induction 48. Tsitoura, D., R. DeKruyff, J. Lamb, and D. Umetsu. 1999. Intranasal exposure to and T cell differentiation in vivo. J. Immunol. 160:3855. protein antigen induces immunological tolerance mediated by functionally dis- ϩ 22. Chai, J. G., and R. I. Lechler. 1997. Immobilized anti-CD3 mAb induces anergy abled CD4 T cells. J. Immunol. 163:2592. ϩ

in murine naive and memory CD4 T cells in vitro. Int. Immunol. 9:935. 49. Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, and http://www.jimmunol.org/ 23. Dubois, P. M., M. Pihlgren, M. Tomkowiak, M. Van Mechelen, and J. Marvel. A. H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and 1998. Tolerant CD8 T cells induced by multiple injections of peptide antigen fatal multiorgan tissue destruction, revealing a critical negative regulatory role of show impaired TCR signaling and altered proliferative responses in vitro and in CTLA-4. Immunity 3:541. vivo. J. Immunol. 161:5260. 50. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakeham, A. Shahinian, 24. Hayashi, R. J., D. Y. Loh, O. Kanagawa, and F. Wang. 1998. Differences between K. P. Lee, C. B. Thompson, H. Griesser, and T. W. Mak. 1995. Lymphoprolif- responses of naive and activated T cells to anergy induction. J. Immunol. 160:33. erative disorders with early lethality in mice deﬁcient in Ctla-4. Science 270:985. 25. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, 51. Lin, H., J. C. Rathmell, G. S. Gray, C. B. Thompson, J. M. Leiden, and J. M. Green, C. B. Thompson, and J. A. Bluestone. 1994. CTLA-4 can function M. L. Alegre. 1998. Cytotoxic T lymphocyte antigen 4 (CTLA4) blockade ac- as a negative regulator of T cell activation. Immunity 1:405. celerates the acute rejection of cardiac allografts in CD28-deﬁcient mice: CTLA4 26. Krummel, M. F., and J. P. Allison. 1995. CD28 and CTLA-4 have opposing can function independently of CD28. J. Exp. Med. 188:199. effects on the response of T cells to stimulation. J. Exp. Med. 182:459. 52. DeSilva, D. R., K. B. Urdahl, and M. K. Jenkins. 1991. Clonal anergy is induced

27. Calvo, C. R., D. Amsen, and A. M. Kruisbeek. 1997. Cytotoxic T lymphocyte in vitro by T cell receptor occupancy in the absence of proliferation. J. Immunol. by guest on October 1, 2021 antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) 147:3261. and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphoryla- 53. Madrenas, J., R. H. Schwartz, and R. N. Germain. 1996. Interleukin 2 production, tion of T cell receptor ␨ and ZAP70. J. Exp. Med. 186:1645. not the pattern of early T-cell antigen receptor-dependent tyrosine phosphoryla- 28. Lee, K. M., E. Chuang, M. Griffin, R. Khattri, D. K. Hong, W. Zhang, D. Straus, tion, controls anergy induction by both agonists and partial agonists. Proc. Natl. L. E. Samelson, C. B. Thompson, and J. A. Bluestone. 1998. Molecular basis of Acad. Sci. USA 93:9736. T cell inactivation by CTLA-4. Science 282:2263. 54. Kaufman, M., F. Andris, and O. Leo. 1999. A logical analysis of T cell activation 29. Fraser, J. H., M. Rincon, K. D. McCoy, and G. Le Gros. 1999. CTLA4 ligation and anergy. Proc. Natl. Acad. Sci. USA 96:3894. ␬ attenuates AP-1, NFAT and NF- B activity in activated T cells. Eur. J. Immunol. 55. Lombardi, G., S. Sidhu, R. Batchelor, and R. Lechler. 1994. Anergic T cells as 29:838. suppressor cells in vitro. Science 264:1587. 30. Samoilova, E. B., J. L. Horton, H. Zhang, S. J. Khoury, H. L. Weiner, and 56. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, Y. Chen. 1998. CTLA-4 is required for the induction of high dose oral tolerance. J. Shimizu, and S. Sakaguchi. 1998. Immunologic self-tolerance maintained by Int. Immunol. 10:491. ϩ ϩ CD25 CD4 naturally anergic and suppressive T cells: induction of autoim- 31. Issazadeh, S., M. Zhang, M. H. Sayegh, and S. J. Khoury. 1999. Acquired thymic mune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969. tolerance: role of CTLA4 in the initiation and maintenance of tolerance in a ϩ ϩ clinically relevant autoimmune disease model. J. Immunol. 162:761. 57. Thornton, A. M., and E. M. Shevach. 1998. CD4 CD25 immunoregulatory T 32. Judge, T. A., Z. Wu, X. G. Zheng, A. H. Sharpe, M. H. Sayegh, and L. A. Turka. cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the production. J. Exp. Med. 188:287. induction of long-term allograft survival. J. Immunol. 162:1947. 58. Taams, L. S., A. J. van Rensen, M. C. Poelen, C. A. van Els, A. C. Besseling, 33. Chen, C., and N. Nabavi. 1994. In vitro induction of T cell anergy by blocking J. P. Wagenaar, W. van Eden, and M. H. Wauben. 1998. Anergic T cells actively B7 and early T cell costimulatory molecule ETC-1/B7-2. Immunity 1:147. suppress T cell responses via the antigen- presenting cell. Eur. J. Immunol. 28: 34. Yi-qun, Z., K. Lorre, M. de Boer, and J. L. Ceuppens. 1997. B7-blocking agents, 2902. alone or in combination with cyclosporin A, induce antigen-specific anergy of 59. Miller, C., J. A. Ragheb, and R. H. Schwartz. 1999. Anergy and cytokine-me- human memory T cells. J. Immunol. 158:4734. diated suppression as distinct superantigen-induced tolerance mechanisms in 35. Van Gool, S. W., M. de Boer, and J. L. Ceuppens. 1994. The combination of vivo. J. Exp. Med. 190:53. anti-B7 monoclonal antibody and cyclosporin A induces alloantigen-specific an- 60. Chen, W., W. Jin, and S. M. Wahl. 1998. Engagement of cytotoxic T lympho- ergy during a primary mixed lymphocyte reaction. J. Exp. Med. 179:715. cyte-associated antigen 4 (CTLA-4) induces transforming growth factor ␤ ϩ 36. Tan, P., C. Anasetti, J. A. Hansen, J. Melrose, M. Brunvand, J. Bradshaw, (TGF-␤) production by murine CD4 T cells. J. Exp. Med. 188:1849. J. A. Ledbetter, and P. S. Linsley. 1993. Induction of alloantigen-specific hypo- 61. Leach, D. R., M. F. Krummel, and J. P. Allison. 1996. Enhancement of antitumor responsiveness in human T lymphocytes by blocking interaction of CD28 with its immunity by CTLA-4 blockade. Science 271:1734. natural ligand B7/BB1. J. Exp. Med. 177:165. 62. Hurwitz, A. A., T. F. Y. Yu, D. R. Leach, and J. P. Allison. 1998. CTLA-4 37. Chai, J. G., I. Bartok, D. Scott, J. Dyson, and R. Lechler. 1998. T:T antigen blockade synergizes with tumor-derived granulocyte-macrophage colony-stimu- presentation by activated murine CD8ϩ T cells induces anergy and apoptosis. lating factor for treatment of an experimental mammary carcinoma. Proc. Natl. J. Immunol. 160:3655. Acad. Sci. USA 95:10067.