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. Author manuscript; available in PMC: 2010 Apr 15.
Published in final edited form as: J Immunol. 2002 Oct 1;169(7):3622–3629. doi: 10.4049/jimmunol.169.7.3622

Effector CD4 Cells Are Tolerized Upon Exposure to Parenchymal Self-Antigen1

Amy D Higgins 1, Marianne A Mihalyo 1, Adam J Adler 1,2
PMCID: PMC2855047  NIHMSID: NIHMS187897  PMID: 12244153

Abstract

It has long been established that exposure of naive T cells to specific Ag in the absence of adjuvant leads to tolerization. Nonetheless, the potential of effector CD4 cells to be tolerized has been less well characterized. To address this issue, we have used an adoptive transfer system in which naive TCR transgenic hemagglutinin (HA)-specific CD4+ T cells are initially primed to express effector function upon exposure to an immunogenic recombinant vaccinia virus expressing HA, and then exposed to forms of HA that are tolerogenic for naive CD4 cells. HA-specific effector CD4 cells residing in both the spleen as well as in two separate nonlymphoid tissues were tolerized upon exposure to high doses of exogenous soluble HA peptide. Additionally, tolerance could also be induced by bone marrow-derived APCs that cross-present parenchymally derived self-HA. Thus, effector CD4 cells are susceptible to similar tolerogenic stimuli as are naive CD4 cells.

Antigen encounter by naive T cells in the absence of adjuvant (i.e., inflammation) leads to tolerance (1, 2). Although this paradigm has been well established for naive T cells (reviewed in Refs. 3–5), it has been less clear whether the same rule applies for T cells that have previously been primed to express effector/memory function. Although numerous studies have demonstrated that autoreactive effector T cells can be tolerized upon exposure to high doses of exogenous soluble autoantigen (reviewed in Ref. 6), it has not been determined whether effector T cells can be tolerized by physiological sources of noninflammatory Ag, such as self-Ags that might be expressed at low levels. This question might be relevant to situations in which self-reactive T cells are primed by cross-reactive pathogens, in which the ability of self-Ag to induce tolerance might limit the extent of autoimmune damage.

The relative susceptibilities of naive and effector T cells to tolerogenic stimuli have not been assessed. However, given that naive and effector/memory T cells exhibit numerous functional differences, such as their TCR vs costimulatory signaling requirements for activation (7–12), the subset of cytokines they express (13–15), TCR-mediated intracellular signaling events (16, 17), migratory properties (18–20), and the necessity of TCR signaling for homeostasis (21–23), they might also be differently susceptible to tolerization.

We have previously developed a model system to study tolerization vs priming in which naive TCR transgenic CD4 cells specific for the influenza Ag hemagglutinin (HA)3 are either tolerized upon adoptive transfer into transgenic mice expressing HA as a parenchymal self-Ag, or primed upon transfer into nontransgenic (NT) mice that have been infected with a recombinant vaccinia virus expressing HA (vacc-HA) (24–26). Although functionally distinct bone marrow-derived APCs presenting self or vaccinia-derived HA appear to program tolerization vs priming soon after the initial APC-CD4 cell interactions, prolongation of the duration of viral-HA exposure had a modest effect in down-modulating the level of effector function in primed CD4 cells (26), suggesting that effector CD4 cells might be sensitive to tolerogenic stimuli. In the current study, we directly assessed the potential of effector CD4 cells to be tolerized by first priming naive HA-specific CD4 cells with vaccinia HA, and then exposing them to forms of HA that induce tolerization of naive CD4 cells. Effector CD4 cells were not only tolerized following exposure to high doses of exogenous soluble HA peptide, but also upon encountering low levels of parenchymally expressed self-HA. Additionally, tolerization of effector CD4 cells to parenchymally derived self-HA was mediated by cross-presenting bone marrow-derived APCs, which we have previously shown to mediate tolerization of naive CD4 cells (24). Thus, effector CD4 cells are susceptible to similar tolerogenic stimuli as are naive CD4 cells.

Materials and Methods

Mice

Adoptive transfer recipients were on the B10.D2 (H-2d), Thy-1.2+ background (except for bone marrow chimeras; see below). C3-HAlow and C3-HAhigh transgenic mice both express the influenza HA gene (A/PR/8/34 Mount Sinai strain) under the control of the rat C3 (1) promoter, which directs HA expression to a variety of nonlymphoid organs. Although both transgenic founder lines express HA in the same subset of tissues, HA protein expression in the C3-HAhigh mice appears to be at least 1000-fold higher than in the C3-HAlow mice (24, 25). The 6.5 TCR transgenic mice express a clonotypic TCR that recognizes an I-Ed-restricted HA epitope (110SFERFEIFPKE120) (27), and were backcrossed to a B10.D2, Thy-1.1 + congenic background.

Bone marrow chimeras

Bone marrow chimeras were generated as previously described (26). In short, C3-HAhigh hosts backcrossed to a B6 (H-2b, Thy-1.2+) background were depleted of NK cells by i.p. injection of 15 µl of rabbit antiasialo GM1 gammaglobulin (Wako Chemicals, Richmond, VA) 1 day before receiving 1000 rad ionizing radiation, followed by 4 × 106 T cell-depleted bone marrow cells prepared from NT B10.D2, Thy-1.2+ donors. Chimeras were allowed a minimum of 2 mo recovery before experimentation.

Adoptive transfers

Adoptive transfers of 2.5 × 106 naive Thy-1.1+ CFSE-labeled 6.5 clonotypic CD4 cells into Thy-1.2+ recipients were performed as previously described (26). As indicated, some recipients were infected i.p. with 1 × 106 PFU vacc-HA 1 day before adoptive transfer. In vivo peptide treatments were performed via injection into the retro-orbital sinus of 280 µg of soluble peptide corresponding to the HA I-Ed-restricted epitope. Clonotypic CD4 cells were recovered for analysis at the indicated times from either the spleen (26), or from the liver and lung following perfusion using previously established protocols (28). For retransfer experiments, day 6 spleen preparations from multiple vacc-HA-infected NT primary recipients were pooled and relabeled with CFSE, and preparations containing 2 × 106 clonotypic effector CD4 cells (roughly corresponding to a single primary recipient) were adoptively transferred into the indicated secondary recipients.

Flow cytometry

FACS analysis was performed as previously described. In short, clonotypic CD4 cells were either identified as Thy-1.1+(using PerCP-conjugated anti-Thy-1.1; BD PharMingen, San Diego, CA) and CFSEdim (26), or as CD4+ (using CyChrome-conjugated anti-CD4; BD PharMingen) and 6.5+(24, 25). For intracellular cytokine staining, 1 × 107 splenocytes were cultured for 5 h with 100 µg/ml synthetic HA peptide and 5 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) before surface staining with anti-Thy-1.1 PerCP, fixation, and permeabilization, and finally staining with PE-conjugated anti-cytokine mAbs (BD PharMingen) (26). Intracellular cytokine staining of liver and lung resident clonotypic CD4 cells was performed similarly except that 2.5 × 106 liver- or lung-extracted lymphocytes were cultured with 7.5 × 106 splenocytes prepared from naive NT B10.D2 mice. For both surface CD25 staining (using PE-conjugated anti-CD25; BD PharMingen) as well as intracellular cytokine staining, background staining levels were determined using a PE-conjugated IgG isotype control (BD PharMingen). All quantitative FACS data are expressed as the mean ± SEM. To allow direct comparison of data collected from separate experiments, all samples were analyzed on the same flow cytometer (FACScan; BD Biosciences, San Jose, CA) using identical settings.

Results

Simultaneous exposure of naive CD4 cells to virally derived and self cognate Ag results in impaired virally induced effector function

To assess the susceptibility of effector CD4 cells to tolerization, we asked whether clonotypic HA-specific CD4 cells primed with vacc-HA retain effector function in the presence of self-HA. CFSE-labeled naive clonotypic CD4 cells were adoptively transferred into either C3-HAlow transgenic recipients, vacc-HA-infected NT recipients, or vacc-HA-infected C3-HAlow recipients. C3-HAlow transgenic mice express HA as a parenchymally derived self-Ag that is cross-presented by bone marrow-derived APCs in a manner that induces tolerization of naive clonotypic CD4 cells (24). Upon analysis of splenocytes 6 days posttransfer, the clonotypic CD4 cells were found to have undergone extensive proliferation (seven or more divisions; Fig. 1A) and accumulation (Fig. 1B) in the vacc-HA-infected NT recipient groups. Proliferation was also extensive in the noninfected C3-HAlow recipients, although consistent with our previous results (25), the clonotypic CD4 cells proliferated less robustly (Fig. 1A) and accumulated at a lower frequency (Fig. 1B) than in the vacc-HA-primed animals. Also consistent with our previous results (26), clonotypic CD4 cells primed with vacc-HA in NT recipients expressed high levels of intracellular IFN-γ upon in vitro restimulation with Ag, while counterparts tolerized in C3-HA recipients were virtually incapable of expressing this effector cytokine (Fig. 1, A and C). Interestingly, clonotypic CD4 cells recovered from vacc-HA-infected C3-HAlow recipients proliferated (Fig. 1A), accumulated (Fig. 1B), and expressed intracellular IFN-γ only slightly better than the noninfected C3-HAlow recipients, and markedly lower than the vacc-HA-infected NT recipients. Thus, self-HA limits clonotypic CD4 cell effector function elicited by vacc-HA.

FIGURE 1.

FIGURE 1

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Simultaneous exposure of naive CD4 cells to virally derived and self cognate Ag leads to impaired virally induced effector function. CFSE-labeled naive clonotypic CD4 cells were adoptively transferred into the following recipients: NT + vacc-HA (vacc-HA-infected NT mice), C3-HA (noninfected C3-HAlow transgenic mice), and C3-HA + vacc-HA (vacc-HA-infected C3-HAlow transgenic mice). Six days later, the clonotypic CD4 cells were recovered from spleens to determine their proliferative responses and abilities to express intracellular IFN-γ following in vitro restimulation. A, Representative FACS histogram plots of CFSE dilution (left panels; with a dotted reference line placed immediately to the left of the undivided cells) and intracellular IFN-γ expression (right panels; the percentage of cytokine-positive cells and the level of cytokine expression (expressed as the mean fluorescence (MF)) are shown above and below the reference bar, respectively). B, The frequencies of clonotypic CD4 cells. C, Total intracellular IFN-γ expression was calculated as the product of the percentage of IFN-γ-expressing clonotypic CD4 cells and the level of IFN-γ expression (MF) on these positive cells, and is expressed in arbitrary units, as previously described (26). n = 3–5 for each group, and the data shown are representative of two separate experiments.

Effector CD4 cells are tolerized by soluble peptide

Although the preceding experiment is consistent with the possibility that vacc-HA-primed clonotypic effector CD4 cells are tolerized by self-HA, it might also have been possible that this result represented a mixed response in which some of the clonotypic CD4 cells had initially encountered vaccinia-derived HA presented by immunogenic APCs, while others had initially encountered tolerogenic APCs cross-presenting parenchymal self-HA (24, 26). To clarify this issue, naive clonotypic CD4 cells were first primed in vacc-HA-infected NT recipients, and subsequently exposed to forms of HA Ag that induce tolerance in naive clonotypic CD4 cells. In the first set of experiments, vacc-HA-infected NT recipients received daily injections of soluble HA peptide starting at day 6, and clonotypic CD4 cell function was analyzed on day 14 by measuring both intracellular cytokine expression following in vitro restimulation, as well as blastogenesis following in vivo restimulation. Six days following vacc-HA-induced priming, the clonotypic CD4 cells exhibit a resting effector phenotype; they are no longer blasting (i.e., low forward scatter), but will express high levels of IL-2 and IFN-γ following restimulation (26). Daily peptide treatment resulted in dramatic reductions in the expression levels of both IFN-γ and IL-2 relative to nontreated vacc-HA-primed controls (Fig. 2A). To assess whether this effect was influenced by the duration of peptide exposure, peptide was given once on day 6, and analysis was performed on day 7. This regimen induced a reduction in IFN-γ expression, albeit the magnitude of this reduction was less than that observed when peptide was administered for 8 consecutive days. Most of the peptide delivered in a single bolus injection appears to be cleared within 4 days, as only a small percentage of naive clonotypic CD4 cells undergoes proliferation (i.e., CFSE dilution) when adoptively transferred into NT hosts 4 days following a single bolus of peptide (Fig. 2D). In light of this observation, it did not appear that vacc-HA-primed clonotypic CD4 cells exposed to a single peptide bolus on day 6 regain function following a short recovery period, as their ability to express IFN-γ was no better on day 14 than on day 7 (Fig. 2A). In contrast, a single peptide bolus given on day 6 did not impair the ability of the primed clonotypic CD4 cells to express IL-2 (Fig. 2A), suggesting that the potential to express IFN-γ is more sensitive to weakly tolerogenic stimuli than is the potential to express IL-2. Intermittent peptide treatment (days 6, 9, and 13) induced reductions in IFN-γ and IL-2 expression on day 14 that were equivalent to the daily peptide treatment group. In addition to the loss of IFN-γ and IL-2 expression potential, the peptide-treated clonotypic CD4 effectors also appeared to be defective in their ability to undergo blastogenesis following in vivo Ag restimulation. When day 6 clonotypic effectors were exposed in vivo to a single bolus of HA peptide, ~80% of them become blasts (i.e., acquired a high forward scatter) 20 h later, consistent with their having initiated cell cycle progression. As the duration of in vivo peptide treatment was extended, there was a progressive decrease in the percentage of vacc-HA-primed clonotypic CD4 cells that had undergone blastogenesis 20 h following the final peptide bolus; intermittent and daily peptide treatments from days 6 through 13 induced 20 and 8% blasting clonotypic CD4 cells, respectively (Fig. 2C). Interestingly, it appeared that the ability of the primed clonotypic CD4 cells to undergo blastogenesis was the parameter that was the least sensitive to tolerization. Thus, the intermittent peptide treatment impaired IFN-γ and IL-2 expression to an equal extent as did the daily peptide treatment; however, the former did not impair blastogenesis as effectively as did the latter. Although there were subtle differences in the frequencies of clonotypic CD4 cells in the spleens of the different treatment groups (Fig. 2B), these differences did not correlate with functional capacity. For example, the intermittent peptide-treated and nontreated vacc-HA-primed groups exhibited equivalent clonotypic frequencies, but displayed distinct functional responses.

FIGURE 2.

FIGURE 2

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In vivo treatment of effector CD4 cells with soluble peptide leads to an impaired ability to produce IFN-γ and IL-2 as well as to undergo blastogenesis following Ag restimulation. Vacc-HA-infected and non-infected NT adoptive transfer recipients of naive CFSE-labeled clonotypic CD4 cells were treated with 280 µg of soluble HA peptide as indicated, and the clonotypic CD4 cells were recovered from spleens on either day 7 or 14 for analysis (refer to legend). A, Total intracellular cytokine expression following in vitro restimulation. B, The frequencies of clonotypic CD4 cells corresponding to A and C. C, Blastogenesis of clonotypic CD4 cells. Representative FACS histogram plots of forward scatter (FSC) on gated clonotypic CD4 cells are shown on the left, with the percentage of blasting cells indicated above the reference bar. The graph on the right shows the quantitative analysis, with the experimental groups that received a peptide bolus 20 h before recovery represented with hatched bars. For A–C, n = 3–6 for each group, and the data were pooled from three separate experiments that each contained multiple treatment groups to verify consistency. D, CFSE-dilution histogram plots for naive clonotypic CD4 cells adoptively transferred into NT recipients that had been treated with 280 µg soluble HA peptide at the indicated times before transfer. Analysis was performed on spleens 3 days posttransfer. The percentage of clonotypic CD4 cells (Thy-1.1+ and 6.5+) in the initial population that did not divide (shown in the upper right corner) was calculated as previously described (52).

To assess whether the soluble peptide treatment of effector CD4 cells was inducing tolerization, or rather altering the type of effector function expressed, clonotypic effectors that had been treated with soluble peptide on days 6 and 10 were analyzed on day 14 for their ability to express the Th2 cytokines IL-4 and IL-10 following in vitro restimulation (Fig. 3A), as well as for their expression of CD25 (a marker for regulatory cells (29, 30)) (Fig. 3B). Although in the absence of peptide treatment the vaccinia-primed clonotypic CD4 cells exhibited a clear Th1 phenotype marked by high IL-2 and IFN-γ expression and low or absent IL-4 and IL-10 expression, peptide treatment greatly impaired IL-2 and IFN-γ expression without inducing a reciprocal increase in IL-4 and IL-10 expression. As a positive control for IL-4 and IL-10 staining, MiCK-2 cytokine-positive control cells (BD PharMingen) stained brightly under both cytokine-staining conditions (data not shown). Thus, the peptide-treated vacc-HA-primed CD4 cells had not been converted into Th2 effectors. Furthermore, peptide treatment did not induce CD25 expression, suggesting that the primed CD4 cells had not been converted into prototypical regulatory cells. Thus, it appears that in vivo exposure of clonotypic effector CD4 cells to soluble peptide leads to functional unresponsiveness (i.e., tolerization).

FIGURE 3.

FIGURE 3

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In vivo treatment of Th1 effector CD4 cells with soluble peptide does not induce conversion into Th2 effector or regulatory CD4 cells. Vacc-HA-infected NT adoptive transfer recipients of CFSE-labeled naive clonotypic CD4 cells were treated with or without 280 µg of soluble HA peptide on days 6 and 10, and analysis of spleens was performed on day 14. A, Total intracellular cytokine expression following in vitro re-stimulation (n = 3 for both groups). B, Representative FACS histogram plots of CD25 expression on primed clonotypic CD4 cells (vacc-HA) and peptide-treated counterparts (vacc-HA + P), with the percentage of positively expressing cells shown above the reference bar. CD25 expression on naive and blasting (i.e., activated) clonotypic CD4 cells is shown for comparison. Note that the background staining on naive clonotypic CD4 cells is slightly elevated due to a compensation effect resulting from high CFSE fluorescence.

Effector CD4 cells residing in nonlymphoid organs are tolerizable

As recent studies have demonstrated that a subset of effector/memory T cells migrates into nonlymphoid organs (19, 28), we asked whether clonotypic effector CD4 cells residing in the liver and lung of vacc-HA-primed NT recipients are similarly tolerized following soluble peptide treatment as are counterparts in the spleen (Fig. 4A). Interestingly, 14 days posttransfer, the level of inducible cytokine expression in the absence of in vivo peptide treatment varied between the different tissues. For example, IFN-γ and IL-2 expression were lower in the liver than in the spleen or lung, suggesting that tissue-specific factors might influence the level of effector function. Nonetheless, following in vivo peptide treatments on days 6 and 10, there were decreases in the expression levels of both cytokines in all three tissues following in vitro restimulation on day 14 (albeit there were variations in the degrees to which cytokine expression was reduced). As observed earlier in the spleen (Fig. 2B), the frequency of clonotypic CD4 cells in the liver and lung did not correlate with functional capacity (Fig. 4B).

FIGURE 4.

FIGURE 4

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Effector CD4 cells residing in non-lymphoid tissues are tolerizable. Vacc-HA-infected NT adoptive transfer recipients of CFSE-labeled naive clonotypic CD4 cells were treated with or without 280 µg of soluble HA peptide on days 6 and 10. On day 14, animals were perfused, and lymphocytes were prepared from the spleen, liver, and lung for analysis. A, Total intracellular cytokine expression following in vitro restimulation. n = 3 for all groups, except for the liver + peptide group, in which n = 2 because there was an insufficient number of clonotypic CD4 cells extracted from one of the samples for analysis. B, The frequencies of clonotypic CD4 cells corresponding to A.

Effector CD4 cells are tolerized upon encountering parenchymally derived self-Ag

To determine whether effector CD4 cells undergo a loss of function upon encountering a physiological source of tolerizing Ag, clonotypic effectors were harvested from spleens of vacc-HA-infected NT primary recipients at day 6, relabeled with CFSE, re-transferred into C3-HA transgenic secondary recipients, and then analyzed 8 days later from spleens. As a control, clonotypic effector CD4 cells were retransferred into vacc-HA-infected NT secondary recipients. Upon re-encountering vaccinia-derived HA, clonotypic effector CD4 cells underwent multiple rounds of division diluting CFSE fluorescence levels nearly to background (Fig. 5A), and following in vitro restimulation 8 days later were able to produce high levels of both IFN-γ and IL-2 that were similar to the levels that they exhibited before the secondary transfer (Fig. 5C). When retransferred into C3-HA transgenics that express high levels of parenchymal HA (C3-HAhigh), the clonotypic effectors also underwent a vigorous proliferative response. However, they ultimately developed a tolerant phenotype; 8 days postretransfer, they exhibited reductions in their potential to express both IFN-γ and IL-2 relative to the control group (total IFN-γ and IL-2 expression levels were reduced 5.5- and 4-fold, respectively). Furthermore, despite their initial proliferative response that was indicated by CFSE dilution, and the continual expression of HA in these transgenic secondary recipients, 8 days following retransfer, the clonotypic CD4 cells were not undergoing blastogenesis (Fig. 5D), indicating that they had also lost the ability to undergo cell cycle progression. The observation that clonotypic effector CD4 cells initially undergo a proliferative phase in response to tolerizing Ag before developing a tolerant phenotype is consistent with the response of naive counterparts that we have previously described (25, 26). Although we have previously demonstrated in C3-HA transgenic mice that naive clonotypic CD4 cells can only recognize parenchymally derived HA on cross-tolerizing APCs (24, 26), it might have been possible that tolerization of clonotypic effector CD4 cells (which are capable of trafficking into parenchymal tissues (Fig. 4) (19)) is dependent upon direct HA presentation by parenchymal cells. To explore this possibility, NT (H-2d)→C3-HAhigh (H-2b) bone marrow chimeras in which only APCs are genetically capable of presenting the HA epitope (26) were used as secondary recipients. Reductions in IFN-γ and IL-2 expression (Fig. 5C) as well as in blastogenesis (Fig. 5D) were observed in the chimeric C3-HAhigh secondary recipients that were comparable with the native counterparts, indicating that cross-presenting APCs are sufficient, and HA-presenting parenchyma are not necessary, for tolerization of effector CD4 cells. When clonotypic effector CD4 cells were retransferred into C3-HAlow transgenics that express parenchymal HA at levels that are at least 1000-fold lower than in the C3-HAhigh transgenics (25), they underwent a strong, albeit reduced, proliferative response relative to the C3-HAhigh secondary recipients (Fig. 5A). Nonetheless, the reductions in their potential to express IFN-γ and IL-2 as well as to undergo blastogenesis that ultimately developed were equivalent, if not slightly greater than that observed in C3-HAhigh secondary recipients (Fig. 5, C and D). Additionally, in neither the C3-HAlow nor C3-HAhigh secondary recipient groups did the clonotypic CD4 cells gain the potential to express IL-4 and IL-10 (data not shown). Comparison of the frequencies of clonotypic CD4 cells present in the spleens of the different secondary transfer recipients (Fig. 5B) showed no apparent correlation between frequency and function (Fig. 5C). For example, the vacc-HA and C3-HAhigh recipients exhibited similar clonotypic frequencies, yet showed marked differences in their abilities to express cytokines following in vitro restimulation. The clonotypic CD4 cell frequency in the C3-HAlow secondary recipients was lower than in the other groups, consistent with their less robust proliferative response, as measured by CFSE dilution (Fig. 5A). Thus, 8 days postretransfer, the clonotypic CD4 cell frequency roughly correlated with proliferation immediately following retransfer, but not with the functional capacity that ultimately developed.

FIGURE 5.

FIGURE 5

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Effector CD4 cells undergo tolerization upon encountering parenchymally derived self-Ag. Day 6 clonotypic effector CD4 cells recovered from vacc-HA-primed primary adoptive transfer recipients were relabeled with CFSE and retransferred into the indicated secondary recipients. Eight days later, they were recovered from spleens for analysis. A, Representative FACS histogram plots showing CFSE-dilution profiles with a dashed reference line placed immediately to the left of the undivided cells. B, Frequencies of clonotypic CD4 cells corresponding to A, C, and D. C, Intracellular cytokine expression of clonotypic CD4 cells following in vitro restimulation. Representative histogram plots of intracellular IFN-γ expression in the different secondary recipient groups, as well as in the primary effectors immediately before the retransfer (1°) are shown on the left. Total intracellular IFN-γ and IL-2 expression is shown on the right. D, Blastogenesis of clonotypic CD4 cells following recovery from secondary recipients. Clonotypic effectors given a single peptide bolus on day 6 and analyzed on day 7 (data taken from Fig. 2C) are shown as a reference (C). n = 5–8 for each group, and the data were pooled from three separate experiments that each contained multiple treatment groups to verify consistency.

Taken together, the preceding experiments indicate that effector CD4 cells are tolerized upon exposure to cognate Ag either when supplied exogenously in a soluble form, or when expressed as a parenchymally derived self-Ag.

Discussion

It has long been established that exposure of naive T cells to Ag in the absence of adjuvant (i.e., inflammation) leads to tolerization (1, 2); however, the potential for primed (i.e., effector/memory) T cells to be tolerized has been less well studied. Our current study indicates that following exposure to two different forms of noninflammatory Ag that are known to induce tolerization of naive T cells (i.e., soluble peptide (31, 32) and parenchymally derived self-Ag (33)), Th1 effector CD4 cells undergo tolerization as assessed by their diminished capacity to express IFN-γ and IL-2 as well as to undergo blastogenesis following restimulation with Ag. A number of previous studies have shown that ongoing autoimmune responses can be ameliorated by high dose exogenous soluble autoantigen administration (reviewed in Ref. 6). In some cases, disease amelioration was found to be mediated by the deletion of autoreactive effectors (34), or by the conversion of Th1 into Th2 autoreactive effectors (35, 36). A more recent study has demonstrated that high dose exogenous soluble Ag can also induce tolerization of memory Th1 CD4 cells, through a combination of deletion and unresponsiveness (37). Our current results appear to differ from these previous models in that our observed tolerization involves the induction of unresponsiveness rather than deletion or the conversion of a Th1 into a Th2 response. Nonetheless, we cannot rule out the possibility that tolerized effector CD4 cells might eventually undergo deletion at time points later than what we have currently examined. The most significant result of our current study is that tolerization of effector CD4 cells is not only mediated by high dose exogenous soluble Ag treatment, but also by a physiological source of tolerizing Ag (i.e., low levels of parenchymally derived self-Ag).

Our observation that clonotypic effector CD4 cells are efficiently tolerized following multiple exposures to soluble peptide (the ability to express IFN-γ was reduced by up to 10-fold) differs from a previous study in which a polyclonal effector/memory CD4 cell population was inefficiently tolerized by exogenous soluble Ag; the capacity to facilitate Ig class switching was not affected, and the ability to mediate a delayed-type hypersensitivity response was only reduced ~2-fold (38). One possible explanation for this difference is that our current study made use of a primed clonotypic CD4 cell population expressing a high affinity TCR for the tolerizing peptide (39), while the primed polyclonal CD4 cell population in the earlier study (38) most likely contained high as well as low affinity receptors. Perhaps effector/memory T cells expressing high affinity receptors are more susceptible to tolerization than low affinity clones, as is the case for naive T cells (40). Thus, in a polyclonal effector/memory CD4 cell population, low affinity clones that escape peptide-induced tolerization might be sufficient to provide help to B cells and to mediate a moderate delayed-type hypersensitivity response.

The duration of Ag exposure appears to play a role in determining the extent of tolerization, as effectors exposed to multiple boluses of peptide over 8 days become less functional than those exposed to a single bolus, suggesting that the level of cumulative Ag exposure might determine the degree of tolerization. Thus, it was surprising that retransfer of effectors into secondary hosts expressing low levels of parenchymal HA was tolerized as efficiently, if not slightly better, than counterparts retransferred into secondary hosts expressing much higher levels of parenchymal HA, suggesting that the level of cumulative Ag exposure might not directly determine the efficacy of tolerization. Perhaps the length of time that effectors are exposed to tolerizing Ag is more critical in this regard. Additionally, although proliferation clearly precedes the development of tolerance in both naive (25, 26) as well as in effector clonotypic CD4 cells (Fig. 5), the magnitude of the effector proliferative response induced by tolerizing Ag does not appear to directly correlate with the efficiency of tolerization; effectors exposed to low levels of parenchymal HA underwent less vigorous proliferation, but were tolerized as well, if not slightly better, than those exposed to much higher levels of parenchymal HA.

It has recently been shown that following priming in secondary lymphoid organs, a significant fraction of effector/memory T cells migrates into nonlymphoid tissues, where they express higher levels of effector function than do their counterparts remaining in the lymphoid organs; CD4 cells have greater potential to express IFN-γ (19), while CD8 cells express constitutive rather than inducible lytic activity (28). Given this apparent difference in the functionality of lymphoid vs peripheral effector/memory T cells, it was interesting that soluble peptide treatment tolerized effector CD4 cells residing in both the spleen as well as in the liver and lung (Fig. 4). An observation that might be relevant to this issue is that bone marrow-derived APCs cross-presenting parenchymal self-HA, which are both necessary and sufficient to induce tolerization of naive clonotypic CD4 cells (24, 26), are also sufficient to induce tolerization of clonotypic effector CD4 cells (Fig. 5). Given that the APC-naive CD4 cell interaction appears to occur in secondary lymphoid organs (26), one possibility is that tolerization of effector CD4 cells is also induced in secondary lymphoid organs, but subsequently becomes manifested in nonlymphoid tissues due to recirculation. Alternatively, nonlymphoid tissue-resident APCs might also induce tolerization of effector CD4 cells.

The observation that effector CD4 cells are sensitive to physiological tolerogenic stimuli has interesting implications. In the case of pathogen-derived epitopes that are cross-reactive with self (e.g., 41), subsequent to priming by immunogenic APCs presenting pathogen-derived epitopes, effector T cells might also encounter the cross-reactive self epitope presented in a tolerogenic manner (4), thus potentially limiting their ability to mediate autoimmune damage. This mechanism might be more effective when the relevant self-Ags are widely expressed and presented tolerogenically (e.g., in our C3-HA system) than when they are confined to discrete anatomical locations (e.g., the pancreatic islets (42, 43)), where tolerogenic presentation is more limited (44, 45). Along similar lines, effector CD4 cells might lose their ability to express IFN-γ more readily than IL-2 following a weak tolerizing stimulus because IFN-γ is a more critical mediator of autoimmunity. The potential of effector T cells to become tolerized is also relevant to tumor immunology. Adoptive T cell therapy strategies make use of tumor-reactive effector T cells that are expanded ex vivo before infusion into cancer patients (46). Because tumor-specific Ags can be cross-presented by tolerogenic APCs (47), and our current study indicates that cross-presenting APCs can tolerize effector T cells, these cross-tolerizing APCs might limit the duration that adoptively transferred tumor-reactive effector T cells are able to maintain their effector function. Nonetheless, it has been shown that this potential effect might be prevented by the administration of exogenous IL-2 (48). Along similar lines, when tumor vaccines are successful in priming tumor-reactive T cells that might have avoided tolerization initially (possibly due to ignorance (49)), there might be a second opportunity for tolerization, once again potentially limiting tumor immunity. A similar mechanism might help to explain the intriguing observation that tumor-specific T cells can undergo massive clonal expansion in the absence of vaccination, yet develop an unresponsive phenotype (50). Thus, under certain conditions, tumor Ags might initially be presented by immunogenic APCs, enabling clonal expansion of cognate T cells (51). Subsequently, these Ags might also be presented by tolerogenic APCs, resulting in unresponsiveness.

Acknowledgments

We are grateful to Anthony Vella for many helpful discussions and to Clinton Mathias for assistance with the liver and lung lymphocyte preparations. We also thank Leonardo Aguila, Leo Lefrancois, Claire Jacquin, and Clyde Mendonca for critical reading of the manuscript.

Footnotes

1

This work was supported by National Institutes of Health Grant AI49813 (to A.J.A.).

3

Abbreviations used in this paper: HA, hemagglutinin; NT, nontransgenic; vacc-HA, recombinant vaccinia virus expressing HA.

Note added in proof. Subsequent to the submission of our manuscript, Kreuwel et al. (53) have reported that, similar to our findings with effector CD4 cells, memory CD8 cells are also susceptible to tolerization upon encountering peripheral self-Ag.

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