Peripheral Blocks Clonal Expansion but Fails to Prevent the Differentiation of Th1 Cells

This information is current as Erika-Nell Malvey, Marc K. Jenkins and Daniel L. Mueller of September 23, 2021. J Immunol 1998; 161:2168-2177; ; http://www.jimmunol.org/content/161/5/2168 Downloaded from References This article cites 55 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/161/5/2168.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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Peripheral Immune Tolerance Blocks Clonal Expansion but Fails to Prevent the Differentiation of Th1 Cells1

Erika-Nell Malvey,* Marc K. Jenkins,† and Daniel L. Mueller2*

.Clonal anergy in Ag-specific CD4؉ T cells is shown in these experiments to inhibit IL-2 production and clonal expansion in vivo We also demonstrate that the defect in IL-2 gene inducibility can be achieved in both naive and Th1-like memory T cells when repeatedly exposed to aqueous peptide Ag. Nevertheless, this induction of clonal anergy did not interfere with the capacity of naive T cells to differentiate into Th1-like effector cells, nor did it prevent such helper cells from participating in T-dependent IgG2a anti- responses and delayed-type reactions. Thus, clonal anergy can contribute to the development of Ag-specific immune tolerance by limiting the size of a Th cell population, but not by disrupting its effector function. The Journal of Immunology, 1998, 161: 2168–2177. Downloaded from he has the remarkable ability to mount a naling cascades (17–19) necessary for the induction of activating highly specific response against invading while protein-1-dependent IL-2 gene transcription (20–22). Importantly, T ignoring self molecules. This specificity is determined in the clonal anergy observed in vitro has not been associated with a part by the T , which expresses a randomly generated global defect in activation events. Specifically, the secretion and unique TCR that recognizes a peptide Ag bound to an MHC of IFN-␥ by anergic CD4ϩ Th cells is not blocked after the in- molecule (1). Because MHC molecules can bind both self as well ϩ

duction of clonal anergy (23). Furthermore, anergic CD8 CTL are http://www.jimmunol.org/ as foreign peptides, the specificities of the peripheral TCR reper- capable of lysing target cells (24). Finally, anergy induction cannot toire or function of self-reactive T cells must be regulated such that effectively interfere with the delivery of helper signals for the stim- the immune system ignores the self peptides. The physical elimi- ulation of polyclonal IgG secretion in vitro (25). Consistent nation of autoreactive T cells during development is the with these functional results, anergic T cells can be shown to de- primary mechanism used by the immune system to establish such velop increases in intracellular calcium free ion concentrations that self-tolerance (2, 3). However, not all self peptides are present in result in the dephosphorylation and nuclear translocation of the the thymus. Therefore, the immune system must either ignore a nuclear factor of activated T cells (NFAT) upon stimulation (22). tissue-specific self peptide (4), or develop an active self-tolerance Thus, the clonal anergy mechanism appears to selectively interrupt that relies on the suppression (5, 6), physical elimination (7, 8), or signal transduction to the nucleus, resulting in a defective prolif- by guest on September 23, 2021 functional inactivation (9, 10) of mature autoreactive T cells. erative capacity, yet it may not eliminate the potential of a T cell Inactivation of T cells by the tolerance mechanism called clonal to participate in an effector cell response. anergy was originally described using a tissue culture system of These findings of selectivity in the inhibitory effects of clonal cloned T cells (11, 12). Clonal anergy has since been defined as a anergy raise uncertainty as to how this mechanism might contrib- reversible, induced tolerance state in which the T lymphocyte can- ute to the development of immune tolerance in vivo. Proliferative not produce its autocrine growth factor IL-2 or proliferate in re- T cell clonal anergy has previously been reported in animals made sponse to the Ag it recognizes (13). In vitro, this unresponsive state tolerant of foreign Ag by the systemic administration of aqueous is induced by stimulation of the T cell through its TCR in the peptides in the absence of infection or adjuvant (26). Clonal anergy absence of costimulatory signals, such as those occurring as a re- has also been observed after the systemic exposure of animals to sult of the interaction of molecules on the APC with CD28 such as staphylococcal enterotoxin B (SEB)3 (27). receptors on the T cell (14, 15). In the absence of such costimu- Interestingly, TCR-V␤8 transgenic mice immunized against teta- latory signals, T cells fail to proliferate, and TCR occupancy un- nus toxoid showed no evidence of tolerance within the tetanus accompanied by proliferation down-regulates the T cell’s respon- toxoid-specific Th cell population following exposure to SEB, de- siveness (16). Biochemical analyses have suggested that anergic T spite the ability of the SEB to induce proliferative unresponsive- cells lose the capacity to synthesize IL-2 because of a defect in the ness in the naive T cell population (28). Likewise, systemic expo- Ras coupling of the Ag receptor to downstream p21 -dependent sig- sure of naive mice to the lymphocytic choriomeningitis (LCMV) glycoprotein-derived peptide GP33 induced tolerance within CD8ϩCTL precursors, whereas mice that had first been † Departments of *Medicine and Microbiology, and Center for Immunology, Univer- infected with LCMV instead developed destructive spleen immu- sity of Minnesota Medical School, Minneapolis, MN 55455 nopathology in response to systemic administration of the aqueous Received for publication February 5, 1998. Accepted for publication April 30, 1998. GP33 peptide (29). These findings suggest either that memory T The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cells are insensitive to clonal anergy induction, or that the anergy with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by a grant from the National Institutes of Health (PO1 AI35296) as well as by a Graduate School Grant-In-Aid research award from the University of Minnesota. E.M. is also supported by an Immunology Pre-Doctoral 3 Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; DTH, de- Training Grant from the National Institutes of Health (AI07313). layed-type hypersensitivity; i.d., intradermal; LCMV, lymphocytic choriomeningitis 2 Address correspondence and reprint requests to Dr. Daniel L. Mueller, University of virus; OVAp, ovalbumin peptide 323–339; Tg, transgenic; IFA, incomplete Freund’s Minnesota Medical School, Box 334 FUMC, Minneapolis, MN 55455. adjuvant.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 2169

mechanism cannot regulate all effector functions by an expanded T rificed, lymph node and spleen cells were isolated, and the KJ1-26ϩCD4ϩ cell clone in the peripheral immune system. cells were enumerated by flow cytometry. In some experiments, day 30 ␮ Previously, in vivo models of T cell activation have not had the nu/nu mice were then primed s.c. with 100 g OVAp in CFA at the base of the tail and sacrificed at day 40. Alternatively, nu/nu mice were primed power to quantitate the effector cell capabilities of first with OVAp in CFA at day 15, and then tolerized at days 25, 30, and following the induction of an Ag-specific tolerance. Specifically, 35 with i.p. OVAp injections before sacrifice at day 40. T cells isolated differences in the frequencies of Ag-reactive T cells that develop as from these mice were subsequently transferred i.v. into unirradiated syn- a consequence of clonal expansion and/or activation-induced cell geneic BALB/c recipients, such that each host animal received 0.25 to 2 ϫ 106 KJ1-26ϩCD4ϩ donor cells in 0.5 ml PBS. Some of the donor cells death have complicated the analysis. Therefore, we have devel- were also used immediately for in vitro proliferation and lymphokine pro- oped an experimental system that both allows for the induction of duction assays. T cell tolerance in mice using an aqueous peptide Ag, and that permits the subsequent recovery and identification of Ag-specific Flow cytometry cells from these tolerant animals. Using this system, the functional Peripheral blood leukocytes, lymph node cells, or splenocytes were harvested, capacities of equal numbers of normal and tolerant T cells can be and 106 cells were incubated on ice with biotinylated anti-clonotypic mAb directly compared following their adoptive transfer into normal KJ1-26 (31), followed by streptavidin Cy-Chrome (PharMingen, San Diego, CA) together with anti-CD4-phycoerythrin (Caltag, Burlingame, CA). After recipient mice. In this study, we describe the results of experiments several washes, cells were either immediately analyzed or were fixed in 0.5% designed to assess the functional effects of clonal anergy induction paraformaldehyde before the acquisition of 10,000 lymphocyte-gated events on both naive and Ag-experienced Th cells. Our findings indicate using forward and side scatter. -matched irrelevant mAb dye conju- that exposure to an aqueous peptide Ag in vivo results in the in- gates served as negative controls in all experiments. Flow cytometry was per- duction of a clonal anergy that limits the expansion of T cells formed using a FACScan and CellQuest Software (Becton Dickinson, Moun- Downloaded from tain View, CA). responding to that Ag, and yet fails to interrupt the generation and delivery of Th1-like effector activities by these lymphocytes. Ex vivo intracellular IL-2 measurement For intracellular IL-2 detection, peripheral lymph node (inguinal, axillary, Materials and Methods brachial, mesenteric, and periaortic) and spleen cells were harvested 3 h Mice after OVAp injection i.v., and 5 ϫ 106 cells were immediately incubated on ␥ ice with anti-FcR mAb 2.4G2 (American Type Culture Collection, Ma- http://www.jimmunol.org/ BALB/c and BALB/c nu/nu (nu/nu) mice, 6 to 8 wk old, were purchased nassas, VA) plus 10% rat serum (Sigma, St. Louis, MO), followed by from Charles River (Wilmington, MA) through a contract with National anti-CD4-FITC (Caltag) and the biotinylated KJ1-26 mAb, and then Cancer Institute at National Institutes of Health (Frederick, MD), and streptavidin Cy-Chrome. Cells were fixed in 2% formaldehyde (Fisher, housed under specific -free conditions. The DO11.10 TCR-trans- Pittsburgh, PA) and permeabilized in 0.5% saponin (Sigma) before staining genic (TCR-Tg) mouse line has been described previously (30) and was with phycoerythrin-conjugated rat anti-murine IL-2 mAb (PharMingen), as bred to homozygosity in our animal facility in accordance with the guide- described (32). Following three washes, 10,000 lymphocyte-gated events ϩ lines of National Institutes of Health. CD4 T cells in these mice have been in addition to 1,000 KJ1-26ϩCD4ϩ-gated events were individually col- engineered to specifically recognize the chicken OVA peptide 323–339. lected. Results indicate the percentage of T cells with increased IL-2-phy- All mice used for any given experiment were sex and age matched. coerythrin staining in 1,000 cells from the KJ1-26ϩCD4ϩ subpopulation. Data are expressed as the mean Ϯ SEM for replicate animals.

Immunizations and Ags by guest on September 23, 2021 Anti-DNP Ab production Normal BALB/c mice were immunized with 100 ␮g OVA peptide 323– 339 (OVAp) (synthesized and purified within Microchemical Facilities at BALB/c mice were bled by retroorbital puncture, and sera collected both University of Minnesota, Minneapolis, MN) emulsified in incomplete before immunization (preimmune) and 10 to 12 days after primary or sec- Freund’s adjuvant (IFA) (Difco, Detroit, MI) by s.c. injection in a volume ondary Ag challenge. Sera were stored at Ϫ20°C, and Ab titers were de- of 100 ␮l at the base of the tail. DNP-specific Abs were subsequently termined by ELISA using DNP-BSA as the capture Ag. DNP-specific IgG1 elicited by secondary immunization at the same site either with 10 or 100 and IgG2a were detected using isotype-specific, horseradish peroxidase- ␮g DNP-conjugated OVA (Pierce, Rockford, IL) (DNP-OVA) emulsified labeled, goat anti-mouse IgG1 and anti-mouse IgG2a Abs, respectively in CFA (Difco), or with CFA alone as a negative control. Alternatively, (Southern Biotechnology, Birmingham, AL). The Ab titer is calculated anti-DNP Ab production was determined in some experiments after only based on that dilution of a serum that has a concentration of anti-DNP primary s.c. immunization with DNP-OVA in CFA. BALB/c mice that capable of eliciting a one-fourth maximal OD within the peroxidase assay. received an adoptive T cell transfer were immunized 1 or 2 days later s.c. Titers shown typically represent the geometric mean Ϯ SEM of replicate at the base of the tail with either 10 or 100 ␮g DNP-OVA emulsified in animals. CFA, or with CFA alone as a negative control, to induce a T cell clonal expansion and the production of anti-DNP Ab. For the measurement of ex DTH reaction vivo intracellular IL-2 levels in Ag-stimulated T cells, 250 ␮g OVAp was Pinna thickness was determined for both ears using an engineer’s micro- injected i.v. into adoptive transfer recipient BALB/c mice 1 day after cell ␮ transfer. Delayed-type hypersensitivity (DTH) was also assessed 1 day meter both before and 24 h after i.d. injection of 10 g OVA into one ear after adoptive T cell transfer into normal BALB/c mice by intradermal and PBS into the opposite ear. The DTH response was determined as the ␮ ␮ difference between the pre- and postinjection measurements. Data shown (i.d.) injection of 10 g OVA in 10 l PBS into the pinna of one ear (with Ϯ PBS alone injected into the other ear as a negative control). Finally, a are the mean SEM of replicate animals. ␮ regimen of three i.p. injections of 100 g OVAp in PBS at 5-day intervals In vivo T cell clonal expansion was utilized in some experiments to induce immune tolerance within the BALB/c mice. Five days after immunization with OVAp in CFA, animals were sacrificed, and draining lymph nodes (inguinal and periaortic) were harvested. The TCR-Tg model of in vivo T cell tolerance total viable lymph node cell number was determined by trypan blue ex- clusion microscopy. Lymph node cells were also analyzed by flow cytom- DO11.10 lymph node and spleen cells were harvested and treated with ϩ ϩ etry to detect the presence of KJ1-26 CD4 TCR-Tg T cells, as described ACK lysis buffer (Biofluids, Rockville, MD), followed by anti-CD8 mAb ϩ ϩ above. The total number of KJ1-26 CD4 T cells recovered was calcu- 3.155 plus rabbit complement (Cedarlane, Westbury, NY) to deplete RBC ϩ ϩ ϩ lated by multiplying the percentage of KJ1-26 CD4 cells times the total and CD8 T cells. Subsequently, ϳ20 ϫ 106 OVA-specific KJ1- ϩ ϩ viable lymph node cell count. Data shown represent the mean Ϯ SEM of 26 CD4 TCR-Tg T cells were injected i.p. into ␥-irradiated (200 rad) ϩ replicate animals. nu/nu mice. The number of KJ1-26 cells injected was calculated based on ϩ ϩ multiplying the percentage of KJ1-26 CD4 cells measured by flow cy- T cell proliferation assay tometry times the number of live cells purified. At days 15, 20, and 25 after T cell transfer, some nu/nu mice were injected i.p. with 100 ␮g OVAp to Harvested lymph node cells from nu/nu mice were cultured with 5 ϫ 105 induce tolerance in the TCR-Tg cells. Negative control mice received PBS irradiated (3000 rad) normal BALB/c splenocytes and 1 ␮M OVAp for injections without Ag. At day 30 after transfer, the nu/nu mice were sac- 48hin200␮l complete medium (1:1 Eagle’s Hanks’ amino acids (EHAA) 2170 PEPTIDE Ag-INDUCED TOLERANCE IN Th1 CELLS

FIGURE 1. Schematic of aqueous pep- tide Ag-induced tolerance using donor T cells from DO11.10 TCR-Tg mice and T cell-deficient BALB/c nu/nu host mice. A, Induction of tolerance in naive T cells after adoptive cell transfer into nu/nu mice. B, Priming of control naive or tolerant T cells in the nu/nu recipient mice. C, Induction of tolerance in Ag-experienced Th cells within nu/nu mice. Downloaded from http://www.jimmunol.org/ medium (Biofluids) and RPMI 1640 (Celox, Hopkins, MN) containing the peripheral blood (Fig. 2, A and B). Recipient nu/nu mice were 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, penicillin, gentami- then challenged i.p. with OVAp in PBS three times at 5-day in- ϫ Ϫ5 ␮ cin, and 5 10 M 2-ME). T cells were then pulsed with 0.25 Ci of tervals beginning on day 15 after transfer (3ϫOVAp pretreatment) [3H]thymidine (Dupont-NEN, Boston, MA) for 12 to 16 h, followed by harvesting of the cells on a PHD cell harvester (Cambridge Technology, to induce tolerance within this TCR-Tg OVA-responder popula- Cambridge, MA) and liquid scintillation counting of the samples to deter- tion. Exposure to aqueous 3ϫOVAp i.p. did not significantly alter mine the level of thymidine incorporation. the percentage of KJ1-26ϩCD4ϩ T cells observed in either lymph nodes (Fig. 2C) or spleen (data not shown) on day 30 after transfer, In vitro IL-2 and IFN-␥ production ϩ ϩ

and the total number of KJ1-26 CD4 cells recovered from the by guest on September 23, 2021 IL-2 secretion into the supernatant was monitored by bioassay at 48 and 3ϫOVAp-treated mice averaged 8.5 Ϯ 1 ϫ 106 vs 8.7 Ϯ 1.1 ϫ 72 h of stimulation using CTLL-2 cells (33) that express a rBcl-x molecule 106 in 3ϫPBS-treated control mice (n ϭ 10). Thus, an overall (kindly provided by Dr. C. Thompson, University of Chicago, Chicago, IL), as previously described (34). IFN-␥ secretion during the experiment decrease in OVA-responder cell frequency as a result of peripheral was determined by ELISA using PharMingen capture and detection mAbs. deletion (36–38) did not accompany the exposure of T cells to Lymphokine production results are expressed as the amount secreted per aqueous peptide administration in the nu/nu mice. ϩ ϩ OVA-specific KJ1-26 CD4 T cell present in the culture. Recovered 3ϫOVAp-pretreated KJ1-26ϩCD4ϩ T cells did, Statistical analysis however, demonstrate decreased proliferative responsiveness to OVAp rechallenge in vitro (Fig. 3A), as well as a markedly re- Ϯ Data are expressed as the mean SEM of replicate animals. In some cases, duced capacity to secrete their autocrine growth factor IL-2 (Fig. experimental treatment groups were compared using Student’s paired or unpaired t tests. 3, B and C). In nine separate experiments, IL-2 production (cor- rected for the number of KJ1-26ϩCD4ϩ T cells present) in cultures Results from 3ϫOVAp-treated nu/nu recipient animals was inhibited an Exposure of TCR-Tg T cells to aqueous peptide Ag in nude mice average of 93 Ϯ 2% compared with control 3ϫPBS-treated ani- induces clonal anergy mals. This failure to detect the production of IL-2 by 3ϫOVAp- treated T cells was not simply the consequence of a change in the Previously, it had been observed that an immune tolerance to the kinetics of secretion, as determined by time-course studies (data chicken OVA peptide 323–339 (OVAp) develops in animals fol- not shown); furthermore, T cells recovered from nu/nu animals lowing i.v. exposure to the Ag in PBS because of the induction of immunized s.c. with OVAp in CFA retained the capacity to secrete T cell clonal anergy (26, 35). To prove this, some of these exper- ϩ IL-2 (Fig. 3C). Thus, exposure of CD4 TCR-Tg T cells in nu/nu iments have relied on an adoptive transfer of OVA-specific mice to repeated aqueous peptide-Ag infusions resulted in clonal CD4ϩTCR-Tg DO11.10 T cells into normal BALB/c mice before anergy induction. the Ag infusion, and the subsequent detection of these OVA-re- active T cells with the KJ1-26 anti-clonotypic mAb (26, 30, 31). In the experiments described in this work, adoptive transfer of Development of T cell clonal anergy in vivo does not prevent ␥ DO11.10-pooled lymph node and spleen cells into lightly irradi- differentiation to an IFN- -producing phenotype ated T cell-deficient BALB/c nu/nu mutant mice was performed to It previously has been observed that the s.c. immunization of allow for a detailed analysis of the effects of tolerance induction on DO11.10 TCR-Tg T cells in BALB/c adoptive transfer animals the effector functions of a relatively large population of Ag-spe- with OVAp in CFA results in the differentiation of these cells to an cific T cells in vivo (see illustrations, Fig. 1). Within 15 days of i.p. IFN-␥-producing Th1 phenotype (39, 40). Consistent with this, transfer, significant numbers of clonotype-positive (KJ1-26ϩ) increased IFN-␥ production was observed in cultures containing CD4ϩ T cells spontaneously left the peritoneum and appeared in KJ1-26ϩCD4ϩ T cells recovered from nu/nu mice after OVAp/ The Journal of Immunology 2171

FIGURE 2. Exposure of OVA-specific CD4ϩ T cells to repeated aqueous peptide Ag injections in nu/nu mice fails to induce their elimination. A, Flow-cytometric analysis of CD4 and TCR-clono- type KJ1-26 expression patterns in the PBL of wild- type, DO11.10 TCR-Tg, and nu/nu BALB/c mice. B, Appearance of KJ1-26ϩCD4ϩ T cells in the pe- ripheral blood of nu/nu mice at days 5, 10, and 15 after i.p. adoptive transfer of DO11.10 TCR-Tg spleen and lymph node cells. C, OVA-specific KJ1- 26ϩCD4ϩ T cells in the pooled lymph nodes of con- trol and aqueous peptide Ag-treated nu/nu mice at day 30 after adoptive cell transfer. Recipient nu/nu mice received three i.p. injections at 5-day intervals consisting of either PBS alone (3ϫPBS i.p.) or 100 ␮g OVAp in PBS (3ϫOVAp i.p.), beginning 15 days after adoptive cell transfer (as described in Fig. Downloaded from 1A). Values shown in the upper right quadrants rep- resent the percentage of KJ1-26ϩCD4ϩ T cells present in 10,000 collected events. http://www.jimmunol.org/

CFA immunization in response to OVAp rechallenge in vitro (Fig. gene inducibility and reduced proliferative responsiveness, as mea- 3C). Cells from the 3ϫOVAp i.p.-pretreated group, on the other sured by in vitro thymidine incorporation. This predicted that the hand, failed to secrete a significant quantity of IFN-␥ into the induction of clonal anergy would also result in T cells with re- culture medium during reexposure to Ag. Therefore, clonal anergy duced capacity for clonal expansion in vivo. We investigated this was induced in naive T cells in the absence of a differentiation to possibility by pretreating TCR-Tg T cells in the nu/nu mice either a Th1-like phenotype. In addition, no evidence for clonal diversion with 3ϫPBS i.p. or the aqueous 3ϫOVAp i.p. infusions, and then by guest on September 23, 2021 to a Th2-like phenotype was obtained in these experiments, since transferring these T cells into normal naive BALB/c recipients. 3ϫOVAp-treated T cells also failed to develop the capacity to These adoptively transferred BALB/c mice were subsequently im- secrete either IL-4 or IL-5 (data not shown). munized s.c. either with CFA alone (as a negative control) or with To test whether systemic exposure to aqueous peptide-Ag pre- DNP-OVA in CFA to test the capacity of the OVA-reactive T cells cludes later differentiation of an anergic T cell to a helper pheno- to undergo a clonal expansion during the next 5 days. As illustrated type, 3ϫOVAp-pretreated adoptive transfer nu/nu mice were chal- in Figure 4A, KJ1-26ϩCD4ϩ T cells exposed to the aqueous pep- lenged s.c. on day 30 with OVAp in CFA (Fig. 1B). When tide-Ag pretreatment (3ϫOVAp) did indeed demonstrate defective recovered from these animals 10 days later (on day 40), KJ1- clonal expansion and accumulation within the draining lymph 26ϩCD4ϩ cells demonstrated little improvement in their capacity nodes, with only about one-quarter the increase in cell number as to secrete IL-2 (13% of control 3ϫPBS-pretreated T cells; p ϭ that seen with normal T cells transferred from PBS-treated nu/nu 0.025). On the other hand, IFN-␥ production in these cultures in mice. response to Ag restimulation was now greatly augmented and Interestingly, draining lymph nodes in mice transferred with equal to that of a normal OVAp/CFA-primed helper cell popula- control (3ϫPBS i.p.-pretreated) T cells and then primed s.c. with tion (Fig. 3C). This result suggested that the development of clonal DNP-OVA/CFA contained more than twice as many leukocytes anergy in a T cell in vivo cannot prevent its differentiation to a (almost entirely KJ1-26Ϫ) as lymph nodes from similar animals Th1-like phenotype, and indicated that defective IL-2 production immunized with CFA alone 5 days earlier. This DNP-OVA-de- can coexist with intact IFN-␥ gene inducibility. In support of this pendent increase in total cell number, however, was not observed second hypothesis, the induction of clonal anergy with 3ϫOVAp when the transferred responder T cells came from tolerant infusions i.p. in animals previously primed s.c. with OVAp in CFA (3ϫOVAp i.p.-pretreated) nu/nu mice (Fig. 4B). Thus, the induc- (Fig. 1C) also failed to interfere with the secretion of IFN-␥ in tion of clonal anergy in vivo in response to aqueous peptide-Ag vitro by the helper cells, even though IL-2 gene inducibility in the exposure led to a stable (for at least 5 days) impairment in clonal ϩ ϩ KJ1-26 CD4 T cells was reduced 97% relative to animals expansion by Ag-specific responder T cells, as well as to a defec- treated only with OVAp in CFA s.c. ( p ϭ 0.014) (Fig. 3C). tive capacity to recruit other (Ag-nonspecific) leukocytes into the draining lymph node. Clonal anergy induction blocks T cell clonal expansion, Defective clonal expansion in this system may reflect either de- bystander lymph node cell recruitment, and IL-2 production in creased production of or responsiveness to a growth factor such as vivo IL-2, or increased death in the Ag responders. Tolerant T cells Our experiments using TCR-Tg T cells and nu/nu adoptive transfer survived well in BALB/c adoptive transfer recipients for 5 days in mice confirmed that the repeated exposure of naive CD4ϩ T cells the absence of Ag stimulation (Fig. 4A). Likewise, reexposure of to aqueous peptide Ag in vivo leads to a down-regulation of IL-2 3ϫOVAp-treated nu/nu mice to s.c. OVAp/CFA stimulation failed 2172 PEPTIDE Ag-INDUCED TOLERANCE IN Th1 CELLS

FIGURE 4. Exposure to aqueous OVAp infusion blocks subsequent T cell clonal expansion in vivo. Spleen and lymph node cells were recovered from 3ϫPBS i.p.- or 3ϫOVAp i.p.-pretreated mice (as described in Fig. 1A) and adoptively transferred i.v. into normal BALB/c recipients. Twenty- four hours later, these mice were immunized s.c. with 100 ␮g DNP-OVA in CFA (DNP-OVA/CFA s.c.; hatched bars) or CFA alone (CFA s.c.; filled Downloaded from bars). Draining lymph nodes were harvested 5 days later, lymph node cells were counted, and then KJ1-26ϩCD4ϩ cells were analyzed by flow cy- tometry in 10,000 total events. A, Total number of KJ1-26ϩCD4ϩ T cells identified in draining lymph nodes. B, Total lymph node cell number. Data in A and B represent the mean Ϯ SEM for either 4 (filled bars) or 11 (hatched bars) separate recipient animals per experimental group. http://www.jimmunol.org/

FIGURE 3. Repeated exposure to aqueous peptide Ag leads to a T cell duced IL-2 production by anergic T cells may actually protect defect in proliferation and IL-2 secretion, but not IFN-␥ production. Be- them from apoptosis. To address these issues, we examined intra- ginning 15 days after adoptive transfer of TCR-Tg cells into nu/nu mice, ϩ ϩ animals received either PBS alone (3ϫPBS i.p.) or OVAp (3ϫOVAp i.p.) cytoplasmic IL-2 levels by flow cytometry in KJ1-26 CD4 T injections three times i.p. over 15 days, as described in Figure 1A. Animals cells ex vivo following i.v. infusion of OVAp. No differences in were sacrificed at day 30, and pooled spleen and lymph node CD4ϩ T cells cell survival after Ag stimulation were noted between the 3ϫPBS were isolated. KJ1-26ϩCD4ϩ cells were enumerated by flow cytometry, as and 3ϫOVAp pretreatment groups (data not shown). Instead, it well as cultured in the presence of 1 ␮g/ml OVAp Ag and irradiated syn- was observed that pretreatment of the TCR-Tg T cells in nu/nu geneic APC to induce their proliferation (A) and production (B) of IL-2. mice with 3ϫOVAp i.p. virtually eliminated their subsequent ca- by guest on September 23, 2021 Ϯ Proliferation data are expressed as the mean cpm SEM for T cells from pacity to synthesize IL-2 upon i.v. OVAp rechallenge in the three nu/nu mice per group, with the data shown representing one of nine BALB/c adoptive transfer recipient mice (Fig. 5, A and B). There- separate experiments. IL-2 production data are expressed as the average ϩ ϩ fore, poor clonal expansion by anergic T cells in vivo correlated IL-2 secreted per KJ1-26 CD4 T cell for each experimental group. Each pair of points and connecting line in the graph represents one individual best with defective IL-2 production. experiment. The geometric mean IL-2 production for each group in nine As predicted by the in vitro IL-2 production results, tolerant experiments is represented by short horizontal lines. C, All nu/nu mice 3ϫOVAp-pretreated T cells subsequently challenged s.c. with began pretreatments with peptide Ag beginning at least 15 days after adop- OVAp in CFA within the nu/nu mice also failed to regain the tive cell transfer. In some experiments, animals were treated first with capacity to accumulate intracytoplasmic IL-2 after adoptive trans- 3ϫOVAp i.p., then injected s.c. with OVAp in CFA on day 30, and sub- fer into BALB/c recipients (Fig. 5, C and D). Likewise, T cells sequently sacrificed and assayed for lymphokine production on day 40 initially primed s.c. with OVAp/CFA and subsequently treated ϫ 3 (3 OVAp i.p. OVAp/CFA s.c.) (see Fig. 1B). In other experiments, an- with 3ϫOVAp infusions i.p. to induce tolerance within the nu/nu imals were first primed with OVAp in CFA s.c., and then treated 10 days mice, failed to accumulate intracytoplasmic IL-2 when adoptively later with the 3ϫOVAp injections i.p., followed by sacrifice and lympho- transferred to normal BALB/c mice and rechallenged with OVAp kine production assay on day 40 (OVAp/CFA s.c.33ϫOVAp i.p.) (see Fig. 1C). Results are also shown for animals treated only with i.p. OVAp (data not shown). Taken together with the observed deficiency in injections (3ϫOVAp i.p.), and for animals immunized only with s.c. clonal expansion, these IL-2 data suggested that peptide-induced OVAp in CFA (OVAp/CFA s.c.). Negative control animals (3ϫPBS i.p.) clonal anergy leaves both naive and effector T cells defective for received only i.p. PBS and s.c. CFA injections in combination. In vitro IL-2 Ag-induced autocrine growth in vivo. Furthermore, they indicated (stipled bars) and IFN-␥ (filled bars) production per KJ1-26ϩCD4ϩ T cell that exposure of tolerant T cells to peptide Ag under immunogenic are shown as the geometric mean Ϯ SEM and arithmetic mean Ϯ SEM, conditions neither reverses the anergy nor allows for a significant respectively, of results obtained from pooled T cells (3–5 nu/nu mice per outgrowth of normal cells that may have escaped tolerance. group) in three separate experiments. Clonal anergy induction in vivo fails to regulate the delivery of helper signals for IgG2a production to result in a significant reduction in the number of anergic KJ1- In the next series of experiments, this nu/nu mouse adoptive trans- 26ϩCD4ϩ T cells recovered (data not shown). IL-2 secretion is fer system was used to investigate the effects of clonal anergy likely to contribute to the development of the autocrine growth induction on the capacity of a population of TCR-Tg T cells to response, and may also act as a cell-survival factor by regulating perform Th1-like helper functions in vivo. Normal BALB/c mice the death-repressor proteins Bcl-2 and Bcl-xL (34, 41). On the were again used as final adoptive transfer recipents of TCR-Tg other hand, IL-2 production during an Ag response also appears to cells initially parked in nu/nu mice, and these animals were then prime T cells for activation-induced cell death (37); therefore, re- tested for Th cell activity in the production of DNP-specific The Journal of Immunology 2173

BALB/c mice and subsequent s.c. immunization with DNP-OVA in CFA (Fig. 6A). The ability of these naive T cells to help in the production of anti-DNP IgG2a in vivo apparently reflected their capacity to differentiate into potent Th1-like effector cells during the 10- to 12-day course of the DNP-OVA/CFA priming, since this population of cells displayed an undifferentiated lymphokine pro- duction profile in vitro and demonstrated no capacity to act as helpers in a 48-h DTH reaction in vivo (Fig. 3C, and data not shown). Remarkably, KJ1-26ϩCD4ϩ T cells recovered from tol- erant nu/nu mice (3ϫOVAp i.p. pretreated) were at least as potent as the control naive cells in providing help for IgG2a anti-DNP Ab (Fig. 6, A and B). In fact, immunization of BALB/c adoptive trans- fer recipients with a low (10 ␮g) dose of DNP-OVA in CFA re- vealed that the tolerant 3ϫOVAp-pretreated T cells could be a more potent helper population than either 3ϫPBS-pretreated naive T cells, or Ag-experienced TCR-Tg T cells that had initially been primed in the nu/nu mice with OVAp in CFA (Fig. 6C). Therefore, under controlled conditions in which T cells were shown to be anergic at the level of the IL-2 gene, this peptide-induced tolerance Downloaded from mechanism failed to eliminate the delivery of Th signals for the production of IgG2a in vivo. Induction of clonal anergy in naive T cells does not prevent later Ag-induced differentiation to a Th1 phenotype capable of

directing a DTH reaction http://www.jimmunol.org/ To determine whether this failure of in vivo tolerance induction to regulate T cell participation in the production of the anti- DNP IgG2a represents a general resistance of Th1 effector func- FIGURE 5. T cells exposed to aqueous OVAp develop reduced induc- tions to the effects of clonal anergy, T cells were also examined ibility of intracytoplasmic IL-2 ex vivo. In A and B, spleen and lymph node for their ability to direct a DTH response after anergy induction. cells were recovered from 3ϫPBS i.p.- or 3ϫOVAp i.p.-pretreated ani- ϩ ϩ ϫ As expected, naive KJ1-26 CD4 T cells recovered from mals, whereas in C and D, cells were harvested from 3 PBS i.p.- or ϫ 3ϫOVAp i.p.-pretreated nu/nu mice subsequently primed with OVAp/ 3 PBS i.p.-pretreated control nu/nu mice and transferred into CFA s.c. (as described in Fig. 1, A and B). All cell populations were then normal BALB/c recipients were incapable of inducing DTH in by guest on September 23, 2021 adoptively transferred i.v. into normal BALB/c recipients. The next day, response to i.d. ear injection with OVA (Fig. 7). Exposure of the recipient animals were challenged i.v. with 250 ␮g OVAp in PBS (OVAp T cells to 3ϫOVAp i.p. administration in the nu/nu mice to i.v.; hatched bars) or PBS alone (PBS i.v.; filled bars). Mice were sacrificed induce tolerance also failed to result in the development of a 3 h later and immediately analyzed by flow cytometry for the presence of capacity to mediate DTH. This was consistent with the finding ϩ ϩ ϩ IL-2 cells within the KJ1-26 CD4 T cell populations of spleen (A, C) that these cells also demonstrated little ability to secrete IFN-␥ and lymph node (B, D). Data are expressed as the mean Ϯ SEM response Ϫ ϩ (Fig. 3C). In contrast, s.c. immunization with OVAp in CFA of recipient animals (five mice per group). KJ1-26 CD4 T cells in test induced the generation of a potent Th1 effector cell population animals showed no increase in the percentage of IL-2ϩ cells after exposure in normal untransferred BALB/c mice that was capable of elic- to OVAp (data not shown). iting a DTH reaction to OVA (data not shown). These results indicated that the stimulation of KJ1-26ϩCD4ϩ anti-hapten IgG2a Abs. An anti-DNP response was utilized be- T cells with OVAp under anergy-inducing conditions either was cause DNP-specific B cells are present in naive BALB/c animals at insufficient to induce differentiation to a DTH-effector pheno- relatively high frequency; therefore, no priming of the B cell rep- type, or resulted in a block of any newly acquired DTH-effector ertoire was necessary to elicit anti-hapten IgG2a responses when activity. To begin to distinguish between these two possibilities, adequate T cell help was available (42). The s.c. challenge of nor- TCR-Tg T cells in nu/nu animals were first made anergic with mal BALB/c animals with the hapten-carrier conjugate DNP-OVA 3ϫOVAp injections i.p., or were injected i.p. with 3ϫPBS in CFA was observed to elicit significant serum titers of anti-DNP alone as a control. The nu/nu mice were then immunized s.c. on IgG2a only when these mice had first been primed s.c. with OVAp day 30 with OVAp in CFA to induce the differentiation of in IFA to increase the frequency of OVA-reactive T cells (Fig. 6A). OVA-specific DTH Th cells (Fig. 1B). As expected, positive Thus, anti-DNP IgG2a production in these BALB/c mice demon- control 3ϫPBS-pretreated KJ1-26ϩCD4ϩ T cells recovered on strated the expected requirement for a primed and clonally ex- day 40 from these OVAp/CFA s.c. immunized nu/nu mice were panded Th cell population, presumably of a Th1-like phenotype found to be fully capable of performing a DTH response within (43, 44). 24 h after adoptive transfer into naive BALB/c mice and sub- Using the nu/nu mouse T cell tolerance model and secondary sequent ear challenge with OVA (Fig. 7). T cells pretreated with adoptive transfer of TCR-Tg T cells into normal BALB/c mice, we 3ϫOVAp i.p. also demonstrated equally strong dose-dependent next assessed the capacity of clonal anergy induction to block the DTH reactivity if primed s.c. with OVAp/CFA before adoptive development or delivery of effector cell activities by CD4ϩ Th transfer into the BALB/c mice (Fig. 7), consistent with their cells. Naive KJ1-26ϩCD4ϩ T cells recovered from control 3ϫPBS acquired ability to secrete IFN-␥ (Fig. 3C), and despite a per- i.p.-pretreated nu/nu mice were found to be competent to provide sistent inability to produce IL-2 (Fig. 5, C and D). These same helper activity for the production of anti-DNP IgG2a in a dose- T cells were also potent Th cells for the production of anti-DNP dependent fashion following their adoptive transfer into the normal IgG2a (Fig. 6C). The results indicate that exposure of naive T 2174 PEPTIDE Ag-INDUCED TOLERANCE IN Th1 CELLS Downloaded from http://www.jimmunol.org/

FIGURE 6. Exposure of T cells to systemic administration of aqueous peptide Ag fails to inhibit their capacity to provide help for the production of anti-hapten IgG2a in vivo. A, Spleen and lymph node cells were recovered from 3ϫPBS i.p. (open squares)- or 3ϫOVAp i.p. (filled squares)-pretreated mice, and various numbers of OVA-specific T cells were adoptively transferred i.v. into normal BALB/c recipients, as indicated. Positive control mice were primed 10 days previously with 100 ␮g OVAp in IFA, and received no adoptive cell transfer (filled circle). All mice were subsequently immunized s.c. with 100 ␮g DNP-OVA in CFA. Sera were collected 12 days later for anti-DNP IgG2a determination by ELISA. Each point represents a single animal, and the data shown are representative of two additional independent experiments. B, Anti-DNP IgG2a production by recipient mice in response to ␮ ϫ 6 ϩ ϩ

immunization s.c. with 100 g DNP-OVA in CFA. Animals either received no adoptive cell transfer (No T cells), or received 2 10 KJ1-26 CD4 T by guest on September 23, 2021 cells from 3ϫPBS i.p.- or 3ϫOVAp i.p.-pretreated nu/nu mice, or they received cells from nu/nu mice that had been primed 10 days earlier with OVAp/CFA s.c. C, Anti-DNP IgG2a production by adoptive transfer recipient mice in response to immunization s.c. with 10 ␮g DNP-OVA in CFA. The T cells used for adoptive cell transfer (2 ϫ 106) are identical to those shown in Figure 3C, while “No T cells” indicates animals that received no cell transfer. Results shown in B and C represent the geometric mean Ϯ SEM of eight mice per group. cells to aqueous peptide Ag in the absence of infection or ad- The induction of clonal anergy in Th1-like effector cells fails to juvant is not sufficient to cause their differentiation into DTH regulate their capacity to mediate a DTH reaction effectors, and instead induces clonal anergy at the level of the The finding that differentiation to an IgG2a- and DTH-helper phe- IL-2 gene. Furthermore, these experiments suggest that the de- notype could occur even in tolerant T cells implies that clonal velopment of peptide-induced tolerance in vivo cannot interfere anergy induction in vivo does not regulate Th1 effector function with the capacity of anergic T cells to subsequently develop a per se. To formally test this, KJ1-26ϩCD4ϩ T cells were first Th1-like effector phenotype if exposed to Ag in adjuvant, even primed s.c. in nu/nu mice with OVAp in CFA to induce the dif- though they never regain the ability to secrete IL-2. ferentiation of Th1 effector cells. Animals were then either treated

FIGURE 7. Aqueous peptide Ag-induced T cell clonal anergy does not inhibit the generation and delivery of help for DTH. T cells were first exposed to peptide Ag in nu/nu mice, as de- scribed in Figure 3C, and then were transferred (either 1 or 2 ϫ 106 cells per recipient) into naive BALB/c mice. Negative control mice received no adoptive cell transfer (No T cells). The next day, mice were challenged with PBS alone in one ear (filled bars) and 10 ␮g OVA in PBS in the other (hatched bars). Ear swelling was measured 24 h later. Data are shown as the mean change in ear size Ϯ SEM for n ϭ 5 animals per treatment group (except n ϭ 3 for 2 ϫ 106 3ϫOVAp i.p.3OVAp/CFA s.c. and n ϭ 4 for 1 ϫ 106 OVAp/CFA s.c.33ϫOVAp i.p.). The Journal of Immunology 2175

FIGURE 8. Model of peptide Ag-induced tolerance in vivo. Primary T cell activation with peptide Ag in the absence of infection or adjuvant fails to expand the clone, cannot in- duce differentiation of cells to a Th1 pheno- type, and instead induces a clonal anergy that leaves the Ag responder with impaired prolif- erative capacity. In contrast, immunogenic stimulation in the presence of adjuvant activ- ity induces both a and expan- sion into a differentiated Th1 population with retained proliferative capacity. Secondary ex- posure of differentiated helper cells to the Ag in the absence of adjuvant does not directly impair their functional effector activity, but it does reduce their capacity to secrete IL-2 and proliferate. Likewise, the anergic T cell can be induced to differentiate into a functional

Th1 cell in response to secondary challenge Downloaded from with immunogenic Ag. Nevertheless, this clone will remain at low frequency as a result of its reduced proliferative capacity and, therefore, cannot mediate an effective Th response. http://www.jimmunol.org/

beginning on day 25 with 3ϫPBS injections alone, or given tolerance as a result of the elimination of the majority of Ag re- 3ϫOVAp injections i.p. to induce T cell tolerance (Fig. 1C). In- sponders and anergy induction within the survivors (36). Similarly, deed, the capacity of OVAp/CFA-primed KJ1-26ϩCD4ϩ T cells CD4ϩOVA-reactive T cells underwent an abortive clonal expan- to promote the development of OVA-dependent DTH in BALB/c sion following i.p. infusion of peptide Ag, and surviving cells were recipients was not found to be adversely affected by three infusions shown to have an IL-2 production defect in vitro, consistent with of OVAp i.p. before the final adoptive transfer (Fig. 7). Likewise, the induction of clonal anergy (26). This combination of activa- by guest on September 23, 2021 these same primed/tolerant T cells remained fully competent to tion-induced cell death and clonal anergy induction would be ex- mediate an anti-DNP IgG2a response (Fig. 6C), despite the fact pected to hinder T cell clonal expansion in response to a later that they had lost the capacity to accumulate intracytoplasmic IL-2 exposure to the Ag, and both most likely contribute to peripheral (data not shown). immune tolerance. We speculate that naive T cells responding to aqueous peptide Discussion Ag undergo an abortive differentiation as a result of TCR stimu- The development of an efficient peripheral immune system de- lation in the presence of a relatively low level of B7 molecule pends on the establishment of tolerance to tissue-specific self Ag, expression. While insufficient to induce T cell proliferation or the while retaining reactivity to foreign pathogens, a process that can- development of DTH-helper activity, perhaps because of the not rely solely on the of autoreactive T cell pre- counter-regulatory influences of simultaneous CTLA-4 ligation, cursors within the thymus. It follows, then, that the onset of an this activation event does appear to leave the T cell susceptible to such as or rheumatoid ar- clonal anergy (39, 50). Upon further TCR occupancy in the ab- thritis in an individual may represent either a specific breakdown sence of infection or adjuvant, the capacity of the T cells to pro- of such self-tolerance to a particular Ag, or may result from a more duce IL-2 is progressively lost (Fig. 8). global defect in the development and/or maintenance of self-tol- We have examined the ability of repeated i.p. peptide-Ag ad- ϩ erance in the periphery. The experiments shown in this study ministration to influence the capacity of CD4 T cells to partici- aimed to establish the mechanism of T cell tolerance induced in the pate in the production of anti-hapten IgG2a Ab, and to mediate periphery of adult mice following aqueous peptide-Ag infusion, in DTH, by utilizing a modification of the TCR-Tg adoptive transfer an attempt to define those factors likely to be important in restoring system originally pioneered by Kearney and Jenkins (26). In this self-tolerance to tissue-specific Ag in patients with . new model, TCR-Tg T cells were first adoptively transferred into Numerous reports have indicated previously that a systemic ex- T cell-deficient nu/nu mice, and then were exposed to peptide Ag, posure of the mature peripheral immune system to deaggregated either under immunogenic conditions or in a fashion capable of forms of a protein Ag or simple peptides, in the absence of adju- inducing T cell tolerance. This system of adoptive cell transfer vant or infection, fails to productively engage the immune system afforded us the opportunity to recover peptide-affected T cells at and instead reduces the individual’s responsiveness to that Ag (45– high number for use in various functional assays. Using an anti- 49). Recently, several studies have taken advantage of TCR-Tg clonotypic mAb to enumerate Ag-specific TCR-Tg T cells after the technology to probe the nature of this Ag unresponsiveness under peptide treatments, the study confirmed that systemic peptide Ag conditions in which Ag responders can be carefully monitored. In administration in the absence of adjuvant fails to induce either a one of these experiments, i.v. exposure of CD8ϩLCMV glycop- clonal expansion of the Ag-reactive T cells or their deletion. Closer rotein-specific T cells to a peptide derived from that Ag led to a examination of the T cells recovered from the peptide-treated 2176 PEPTIDE Ag-INDUCED TOLERANCE IN Th1 CELLS nu/nu mice also identified the development of a profound IL-2 within the nu/nu mice. Neither naive cells nor T cells exposed to production defect in surviving Ag responders, despite continued the aqueous peptide Ag pretreatment alone had such a helper ca- high level expression of both CD4 and the Ag-specific TCR. This pacity. The failure of 3ϫOVAp i.p.-pretreated T cells to directly loss of IL-2 inducibility was not accompanied by a diversion to the help in the development of a DTH response is consistent with their Th2 phenotype (51), since these peptide-treated T cells never inability to secrete IFN-␥, and may reflect insufficient IL-12 pro- gained the ability to produce IL-4 or IL-5 (E. Malvey, unpublished duction by APC or the effects of CTLA-4 ligation during the observation). Therefore, our results suggest that clonal anergy is course of the clonal anergy induction (40). Regardless, our data induced in T cells after the series of i.p. injections with peptide Ag provide no support for the notion that clonal anergy induction di- in PBS. verts differentiation away from the Th1 phenotype, as anergic T Peptide-induced anergy, if similar to in vitro models of Ag un- cells responded to Ag stimulation in the presence of adjuvant- responsiveness, might be expected to inhibit the clonal expansion induced costimulatory signals with the development of Th1 helper of Ag-reactive cells in vivo because of a block in signal transduc- activity. tion between the TCR and the IL-2 gene. Peptide-pretreated KJ1- Finally, these experiments demonstrate that clonal anergy at the 26ϩCD4ϩ T cells did, in fact, demonstrate only poor clonal ex- level of the IL-2 gene can coexist in vivo with intact Th1 effector pansion in vivo in response to Ag rechallenge, following adoptive function. Specifically, we assessed the capacity of primed Th1-like transfer into normal BALB/c recipients. Additionally, these toler- memory cells to be made tolerant to Ag upon repeated i.p. expo- ant T cells were incapable of recruiting other Ag-nonspecific leu- sure to the OVAp. It was shown that these Th1 cells lost the ca- kocytes into the draining lymph node during the course of the Ag ϫ pacity to synthesize IL-2 following the 3 OVAp i.p. injections, Downloaded from response. Consistent with these data, the in vivo induction of IL-2 synthesis within the tolerant T cells was found to be markedly consistent with the development of clonal anergy. Nevertheless, defective. These results argue conclusively against a role for either such tolerant T cells retained their capacity to immediately induce peripheral elimination or suppression in this TCR-Tg immune tol- a DTH reaction, as well as participate in the production of anti- erance model. While it is currently uncertain which factors are hapten IgG2a. Thus, primed effector cells are indeed susceptible to most responsible for the development of a strong clonal expansion anergy induction at the level of the IL-2 gene, but this clonal an- response or recruitment of bystander cells in vivo, the results of ergy tolerance mechanism is incapable of interfering with the ac- http://www.jimmunol.org/ these experiments suggest that tolerance develops because the tivation of T cells for the delivery of Th1-dependent activities elaboration of at least one critical factor by the responding T cells important to the development of DTH and IgG2a Ab responses. is blocked as a consequence of the induction of clonal anergy. These findings suggest that the most important role for clonal Does the induction of clonal anergy in vivo result in a complete anergy in the development of peripheral T cell tolerance may be functional inactivation of the T cell? The evidence suggests oth- the regulation of autocrine growth-factor production and clonal erwise. First, peptide-pretreated T cells appeared fully competent expansion, with the resultant maintenance of potentially deleteri- to participate in the production of anti-hapten IgG2a after chal- ous T cells at low frequency. In pathologic states in which auto- lenge with the hapten-carrier protein in adjuvant. Cognate help for reactive T cells presumably reach high frequency, this mechanism IgG2a isotype switching is thought to rely both on the delivery of may no longer have the inherent capacity to control their actions. by guest on September 23, 2021 the CD40 ligand as well as on the production of IFN-␥ by Th cells Similarly, the design of future therapies for autoimmune disease responding to carrier-derived peptide Ag presented on the surface based on this tolerance mechanism should anticipate that clonal of the hapten-specific B cells (52, 53). Therefore, the production of anergy may not inhibit the delivery of all potentially dangerous high titer anti-DNP IgG2a in animals transferred with high num- Th1 activities. bers of peptide Ag-pretreated TCR-Tg cells implies that these T It should be noted that Gilbert et al. (54) previously determined cells could be induced to differentiate to a Th1-like phenotype that the delivery of help for IgG production in vitro is sensitive to upon exposure to DNP-OVA in adjuvant, and provide help to the induction of T cell clonal anergy. Likewise Finck et al. (55) DNP-specific B cells during the course of the response. This helper demonstrated a capacity of the B7/CD28 antagonist CTLA-4Ig to activity was not simply the result of an outgrowth of T cells that interrupt the production of pathogenic and improve had escaped tolerance induction, nor did it result from a reversal of survival in (NZB/NZW)F lupus-prone mice, a therapy perhaps the anergy: after transfer into the BALB/c recipients, 3ϫOVAp 1 predicted to promote clonal anergy induction in the self Ag-spe- i.p.-pretreated T cells maintained an anergic phenotype based on cific Th cells. Both experiments, however, lacked the power to their weak ability to undergo a clonal expansion. Furthermore, establish whether the development of tolerance stemmed from a aqueous peptide Ag-pretreated T cells intentionally primed s.c. reduced expansion of critical Ag-specific helper cells or from an with OVAp in CFA within the nu/nu mice never regained a normal ability of the tolerance mechanism to interrupt the delivery of Th ability to produce IL-2 in vivo. activities. Our own in vitro studies have also recently addressed this issue and determined that under conditions in which T cell numbers were held Finally, Marusic and Tonegawa (56) recently showed that a sin- constant, the delivery of Th signals for the induction of B cell poly- gle i.p. injection of peptide Ag in IFA induced clonal anergy clonal Ab production was only partially sensitive to clonal anergy within a large population of TCR-Tg T cells specific for myelin induction (25). The secretion of IL-4 and IL-5 by anergic T cells basic protein. In their experiments, anergy was associated with a ␥ responding to Ag on B cells was found to be moderately depressed in defect in IFN- production, and was in fact sufficient to protect the these long-term tissue culture experiments. Nevertheless, CD40 li- mice from experimental autoimmune encephalomyelitis. This ap- gand was found to be inducible and fully functional after activation of parent inconsistency with the findings of our system may stem anergic T cells, and IFN-␥ could still be secreted in a normal fashion, from their use of IFA in the single peptide Ag infusion, since consistent with these in vivo results. systemic Ag persists considerably longer after i.p. injection with Additional evidence that anergic T cells remain functionally re- IFA than with PBS (M.K.J., unpublished observation). The con- sponsive to Ag stimulation includes the finding that aqueous pep- tinuous presence of nonimmunogenic peptide most likely desen- tide-Ag-pretreated TCR-Tg T cells also developed the capacity to sitizes T cells to subsequent Ag stimulation, and may promote the direct a DTH response following priming s.c. with Ag in adjuvant appearance of a more profound functional anergy. The Journal of Immunology 2177

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