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The Journal of Immunology, 2006, 176: 5880-5889.
Copyright © 2006 by The American Association of Immunologists

CD25+Foxp3+ Regulatory T Cells Facilitate CD4+ T Cell Clonal Anergy Induction during the Recovery from Lymphopenia1

Tracy L. Vanasek*, Sarada L. Nandiwada*, Marc K. Jenkins{dagger} and Daniel L. Mueller2,*

* Department of Medicine and {dagger} Department of Microbiology, and Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell clonal anergy induction in lymphopenic nu/nu mice was found to be ineffective. Exposure to a tolerizing peptide Ag regimen instead induced aggressive CD4+ cell cycle progression and increased Ag responsiveness (priming). Reconstitution of T cell-deficient mice by an adoptive transfer of mature peripheral lymphocytes was accompanied by the development of a CD25+Foxp3+CTLA-4+CD4+ regulatory T cell population that acted to dampen Ag-driven cell cycle progression and facilitate the induction of clonal anergy in nearby responder CD25–CD4+ T cells. Thus, an early recovery of CD25+ regulatory T cells following a lymphopenic event can prevent exuberant Ag-stimulated CD4+ cell cycle progression and promote the development of clonal anergy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Individuals with primary immunodeficiency and reduced lymphocyte counts are at heightened risk for the development of autoimmunity (1). Likewise, lymphopenia is not uncommon in systemic inflammatory diseases such as systemic lupus erythematosus (SLE), and those affected with rheumatoid arthritis or multiple sclerosis demonstrate a primary defect in either thymic output or peripheral T cell homeostasis (2, 3). Mouse models of systemic lupus erythematosus and diabetes demonstrate similar evidence of premature thymic atrophy and/or peripheral lymphopenia (4, 5). Finally, lymphopenia is associated with resistance to the development of transplantation tolerance (6). Thus, clinical and experimental data suggest an association between T cell lymphopenia and defects in immune self-tolerance. Despite these data, immunodepleting agents such as cyclophosphamide and antithymocyte globulin remain the standard of care for the treatment of severe necrotizing vasculitis and prevention of acute allograft rejection, respectively (7, 8). Although effective to quickly suppress dangerous immunopathology, such treatments are not designed to achieve durable Ag-specific tolerance and carry substantial risk of infection.

Under nonlymphopenic conditions, naive CD4+ T cells can be induced into an unresponsive state termed clonal anergy by repeated systemic exposure to soluble Ag (9). Anergy is an inability of CD4+ T cells to produce IL-2 or to proliferate upon subsequent Ag challenge, as a consequence of multiple intracellular signaling defects (10, 11, 12). This contrasts with the aggressive priming of CD4+ T effector cells and generation of immunological memory that follows the recognition of Ag in the presence of adjuvant, tissue injury, and/or infection (9, 13, 14). Certain biochemical signals (e.g., activation of the mammalian target of rapamycin) that occur as T cells move through cell cycle during a protective immune response to Ag appear to durably increase the recall Ag responsiveness of the T cells in vivo (15).

Within the lymphopenic immune system there is a strong homeostatic drive to increase the total number of T cells, and this can lead to cell cycle progression that is independent of exogenous (foreign) Ag or infection (16, 17). Lymphopenia-induced proliferation is known to require the recognition of a self peptide/MHC complex, and may continue until a sufficiently diverse repertoire develops that can efficiently compete for every self peptide/MHC complex (18, 19, 20, 21, 22, 23). These observations raise the question of whether this homeostatic drive can also promote a more intense cell cycle progression in response to self Ag recognition that antagonizes the development of clonal anergy. If true, any attempt by the immune system to recover from lymphopenia through homeostatic proliferation carries the risk of selecting for T cells with the highest potential for clinical autoreactivity (4, 5, 24, 25). Nevertheless, CD4+ regulatory T cells can also quickly expand during the course of immune reconstitution, and such cells may act to prevent the development of overt autoimmunity in individuals recovering from lymphopenia (26, 27, 28).

We have investigated the regulation of CD4+ T cell clonal anergy induction within the setting of lymphopenia. As described below, we have found T cells to be resistant to clonal anergy induction immediately following adoptive transfer into athymic nu/nu (nude) mice as a consequence of unrestrained cell cycle progression. This system thus afforded us the opportunity to examine CD4+ regulatory T cells for their ability to influence the development of anergy. We now confirm that the adoptive transfer of mature CD4+ T cells into lymphopenic mice leads to a spontaneous expansion of a large population of CD25+Foxp3+CTLA-4+CD4+ regulatory T cells, and demonstrate that these CD25+ T cells have the capacity to dampen the Ag-induced drive to proliferate and facilitate the induction of clonal anergy after partial immune reconstitution.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

DO11.10 (DO11) TCR-transgenic (TCR-Tg)3 were bred to homozygosity and maintained in our animal facility (29). CD4+ T cells in these mice are uniformly reactive to chicken OVA peptide 323–339 (OVAp)/I-Ad complexes and express a clonotypic TCR detectable with the mAb KJ1-26 (30). Rag-2–/– DO11 TCR-Tg mice were purchased from Taconic Farms through their Emerging Models Program. The HA TCR-Tg mice (31) were obtained from A. Khoruts (University of Minnesota, Minneapolis, MN) and were maintained in our animal facility. CD4+ T cells in these mice are specific for peptide 111–119 of influenza hemagglutinin (HAp) presented by class II I-Ed MHC molecules and can be detected with the anti-clonotypic mAb 6.5 (31). Wild-type (WT) BALB/c and BALB/c nu/nu (nude) recipient mice, 5–8 wk old, were purchased from Charles River Laboratories through a contract with the National Cancer Institute at the National Institutes of Health (Frederick, MD). All mice were housed under specific pathogen-free conditions and used in accordance with National Institutes of Health guidelines and the University of Minnesota Institutional Animal Care and Use Committee. Mice were age and sex matched for all experiments.

Adoptive transfer and in vivo treatments of mice

Lymph nodes (axillary, brachial, inguinal, and mesenteric) and spleens of TCR-Tg mice were harvested into complete media containing 10% FCS (Atlas Biologicals), 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 5 x 10–5 M 2-ME in RPMI 1640 (Mediatech), and prepared for adoptive transfer as described previously (9). In some experiments, cells were labeled with CFSE (Molecular Probes) before transfer, using a modification of a technique previously described (32). Briefly, lymph node and spleen cells in PBS at a concentration of 1 x 107 cells/ml were incubated in 2.5 µM CFSE for 5 min at 37°C. The labeling reaction was stopped by the addition of complete media. The CFSE-labeled T cells were washed twice with PBS before i.v. transfer of 2.5–5 x 106 cells to recipient syngeneic mice. OVAp 323–339 was produced in our microchemical facility (University of Minnesota), dissolved in PBS, and filter sterilized for use. HAp 111–119 was kindly provided by A. Khoruts (University of Minnesota). OVAp and HAp were delivered i.v. at doses of 100 or 250 µg. Rapamycin (RAPA) was obtained from S. N. Sehgal (Wyeth-Ayerst Research, Princeton, NJ). A stock solution of 1 mg/ml RAPA in 100% ethanol was prepared. RAPA was then suspended in 0.2% carboxymethylcellulose, as previously described (33), and delivered i.p. at a dose of 0.5 mg/kg/day for 4 days beginning on the day of adoptive transfer. The rat anti-mouse CD25 mAb PC61 obtained from A. Khoruts (University of Minnesota) was purified from hybridoma cell culture supernatants using standard protein G-Sepharose chromatography techniques. Anti-CD25 mAb (400 µg) was injected i.p. into mice every 3 days during the experiment starting on the day of DO11 T cell adoptive transfer and throughout the period of the CD4+ cell immune reconstitution. Control mice given the PC61 mAb starting only after the CD4+ cell reconstitution and at the time of OVAp administration demonstrated no effect of the Ab treatment on either cell cycle progression or recall IL-2 production (data not shown).

Flow cytometry

Spleen cells were washed with staining buffer (PBS containing 2% FCS and 0.2% azide), and then incubated with anti-Fc mAb 93 (eBioscience) to block FcRs. Cells were then stained with the combination of PerCP-cyanin 5.5-labeled anti-CD4 (RM4-5) mAb (BD Pharmingen) and allophycocyanin-labeled DO11.10 TCR (KJ1-26) mAb (Caltag Laboratories) as well as one or more of the following: biotin-labeled anti-CD25 (7D4), PE-labeled anti-CTLA4 (4F10), (BD Pharmingen); PE-labeled anti-IL-2 (JES6-5H4), FITC- or PE-labeled anti-CD25 (PC61), PE-labeled anti-CD69 (H1.2F3), PE-labeled streptavidin, (eBioscience). Intracellular molecules were detected essentially as previously described (34, 35). Briefly, washed and Fc-blocked spleen cells were first incubated with PerCP-cyanin 5.5-labeled anti-CD4 mAb and allophycocyanin-labeled KJ1-26 mAb. Cells were then washed one time with PBS, fixed in 2% formaldehyde (Sigma-Aldrich) for 20 min at room temperature, permeabilized in 0.5% saponin (Sigma-Aldrich), and incubated at room temperature for 20 min with PE-labeled anti-IL-2 mAb, PE-labeled anti-CTLA4 mAb, or PE-labeled irrelevant mAb. Finally, cells were washed once in 0.5% saponin and once in staining buffer. For all experiments, at least 1000 KJ1-26+CD4+ events were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo (Tree Star) software.

Measurement of cell cycle progression in vivo

Cell cycle progression was monitored using the CFSE dye as a marker of cell division (32). The average division of CFSE-labeled KJ1-26+ CD4+ T cells at various times after stimulation was calculated as previously described (15). Briefly, based on the peaks of CFSE fluorescence intensity within the population, each T cell was assigned to a particular cell division group d (with d = 0 to n cell divisions), and the number of T cell events (E) observed within each cell division group (Ed) was determined. Average division was calculated using the following equation:

Formula

Cell purification and in vitro suppression assay

The suppressive properties of CD4+ subpopulations isolated from intact DO11 mice, or from DO11-reconstituted nude mice, were tested by their addition to CD25+ cell-depleted normal DO11 lymph node and spleen cells. In some experiments, CD25+ T cells from normal DO11 mice were positively selected using CD25 mAb and MACS magnetic streptavidin microbeads (Miltenyi Biotec), according to manufacturer’s instructions. To obtain CD25+CD4+ and CD25–CD4+ subpopulations from DO11-reconstituted nude mice, CD4+ cells were first isolated using CD4 Dynabeads (Dynal Biotech) followed by CD4 Detachabead to remove the CD4 magnetic beads. The CD25+CD4+ and CD25–CD4+ subpopulations were then separated using CD25 mAb and MACS-positive selection (Miltenyi Biotec). To assay for in vitro suppression, the purified CD25+ cells from intact DO11 mice were compared with CD25+CD4+ and CD25–CD4+ subsets from DO11-reconstituted nude mice following addition to CD25-depleted DO11 lymph node and spleen cell cultures in the absence or presence of 10 µM OVAp. IL-2 secretion was measured in the 48 h supernatants by capture ELISA.

Real-time quantitative RT-PCR

CD25+CD4+ and CD25–CD4+ cell populations from intact DO11 mice and DO11-reconstituted nude mice were purified as described above. One million cells were lysed with TRIzol (Invitrogen Life Technologies) and RNA was extracted according to the manufacturer’s instructions. RNA was further purified using the RNA Easy Mini kit (Qiagen). Total RNA equivalent to the cell number from each sample was reverse transcribed using the Superscript II Platinum Two Step qRT-PCR kit (Invitrogen Life Technologies). PCR primers were synthesized in our microchemical facility (University of Minnesota) and real-time PCR was conducted using a Cepheid Smart Thermocycler by adding SybrGreen (Molecular Probes) to the reaction mixtures. Primers were designed to amplify the junction region of exons 7 and 8 of the Foxp3 mRNA. The primers contained the following sequences: Foxp3 (forward): 5'-AAA GGA GAA GCT GGG AGC TAT G-3'; Foxp3 (reverse): 5'-CCT GAG TAC TGG CTA CGA T-3'. Hprt mRNA was used as a positive control to normalize the Foxp3 data. The Hprt primers were designed to amplify the junction of exons 7 and 8 and contained the following sequences: Hprt (forward): 5'-TGA AGA GCT ACT GTA ATG ATC AGT CA-3'; Hprt (reverse): 5'-AGC AAG CTT GCA ACC TTA ACC A-3'. Data are expressed as the amount of Foxp3 mRNA present in a sample relative to Hprt.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Clonal anergy induction is defective in the setting of T cell lymphopenia

To explore clonal anergy induction in the setting of T cell lymphopenia, OVAp-reactive DO11 CD4+ T cells were adoptively transferred into athymic nu/nu (nude) BALB/c recipient mice and then immediately exposed to repeated (three times) i.v. injections of Ag (OVAp) in the absence of an adjuvant. In a parallel group of WT recipient animals, this regimen of prolonged TCR stimulation led to a state of unresponsiveness to Ag rechallenge by day 13 that resulted in a defect in the in vivo production of IL-2 upon Ag rechallenge (Fig. 1A). In contrast, the KJ1-26+CD4+ T cells exposed to the 3x OVAp regimen within the nude mice retained a significantly higher capacity to synthesize IL-2 (p = 0.001). Therefore, clonal anergy could not successfully be induced in the lymphopenic environment.


Figure 1
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  		FIGURE 1. Defective tolerance induction in the setting of lymphopenia. WT or nude mice were pretreated i.v. three times 3 days apart with 100 µg of OVAp (or PBS as a control) beginning 1 day after adoptive transfer of 4 x 106 DO11 T cells. Six days later, recipient mice were challenged i.v. with 250 µg of OVAp for 3 h. A, Intracellular IL-2 content among Ag-specific KJ1-26+CD4+ T cells, as a percentage of the 3x PBS-treated controls. B, Percentage of KJ1-26+CD4+ T cells accumulating within the spleens of partially reconstituted nude mice on day 13 of the analysis. Error bars represent the SEM for duplicate mice. The p value was determined using the Student t test. This experiment was repeated with similar results.

 
Ag-stimulated cell cycle progression is stronger during T cell lymphopenia

Further analysis of the proportion of Ag-reactive KJ1-26+ CD4+ T cells that remained within the spleens of the WT mice after the 3x OVAp infusion regimen revealed little change in their frequency as compared with 3x PBS-treated control animals, consistent with an ineffective clonal expansion response in the absence of infection or adjuvant (Fig. 1B) (9). In contrast, a significant increase in the percentage and total number of KJ1-26+ CD4+ T cells was observed within the spleens of nude recipient mice chronically exposed to Ag, as compared with nude mice exposed to PBS alone (Fig. 1B and data not shown). This enhanced clonal expansion response together with the persistent recall Ag responsiveness of the 3x OVAp-treated KJ1-26+CD4+ T cells in lymphopenic mice thus was more reminiscent of a successful T cell priming event than of an induction of immunological tolerance.

Previously, our work had indicated that aggressive in vivo cell cycle progression during the primary response to Ag antagonizes the development of clonal anergy (15). We, therefore, postulated that the resistance to anergy induction observed in the nude mice was caused by this lymphopenia-induced enhanced drive for cell cycle progression. To test this, the intensity of cell cycle progression in lymphopenic mice was characterized based on the rate of CFSE dye dilution in KJ1-26+CD4+ T cells immediately exposed to a single infusion of Ag (1x OVAp). KJ1-26+CD4+ T cells in both WT and nude recipients did demonstrate a reduced CFSE fluorescence intensity indicative of multiple rounds of cell division in response to Ag (Fig. 2A). However, the CFSE fluorescence of the KJ1-26+CD4+ T cells recovered from 1x OVAp-treated nude mice was always much lower than in the WT mice, consistent with a faster rate of cell division. A mathematical examination of the flow cytometry data confirmed that KJ1-26+CD4+ T cells in nude mice had a significantly higher average cell division rate (5.58 ± 0.35 divisions/T cell over 5 days) than T cells stimulated within the WT recipients (2.79 ± 0.05; p = 0.016) (Fig. 2B). Such an increase in the rate of cell division predicted a generation of daughter cells in the lymphopenic mice that was nearly eight times greater than in the WT case. Therefore, an enhanced cell cycle progression may have accounted at least in part for the greater clonal expansion originally observed in the 3x OVAp-treated lymphopenic animals.


Figure 2
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  		FIGURE 2. Increased recall Ag responsiveness following Ag-stimulated cell cycle progression in lymphopenic recipients. WT or nude mice adoptively transferred with 4 x 106 CFSE-labeled DO11 T cells were treated with a single i.v. infusion of 100 µg of OVAp or PBS as a control. Some animals were treated with simultaneous i.p. infusions of 0.5 mg/kg RAPA in carboxymethylcellulose, whereas other mice received the vehicle alone. Five days later, animals were rechallenged i.v. with 250 µg of OVAp for 3 h. A, CFSE dye dilution (log FL1) of KJ1-26+CD4+ T cells recovered from the spleens of WT (filled histograms) or nude (open histograms) recipient mice. B, Average division calculation for the Ag-stimulated KJ1-26+CD4+ T cell groups as shown in A. C, Plot of the relationship between cell division history and mean recall Ag-induced IL-2 production in KJ1-26+CD4+ T cells from WT (filled symbol) or nude (open symbol) recipients pretreated i.v. on day 1 with 100 µg of OVAp in the presence of RAPA (circles) or vehicle control (squares). IL-2 production is calculated as the percentage of the T cell response observed in control animals (diamond symbol) receiving a PBS pretreatment alone. *, The mode number of cell divisions observed in the KJ1-26+CD4+ T cell population as a result of the OVAp pretreatment. Error bars represent the SEM. Data shown are representative of two independent experiments.

 
A single i.v. infusion of Ag in the absence of adjuvant is a relatively poor stimulus for T cell clonal anergy induction in WT mice (15); nevertheless, it was apparent in these experiments that the T cells exposed to 1x OVAp in nude mice achieved an even greater level of recall Ag responsiveness as they underwent progressively more rounds of cell division (Fig. 2C). To determine whether the strength of cell cycle progression during primary Ag challenge directly regulated the level of recall Ag responsiveness in the nude mice, we examined the effects of the antiproliferative agent RAPA on both the lymphopenia-enhanced cell cycle progression and the eventual level of Ag responsiveness achieved. As previously described, treatment of WT mice with RAPA during a primary Ag challenge significantly inhibited cell cycle progression by the KJ1-26+CD4+ T cells (Fig. 2, A and B) (15). Similarly, treatment of nude mice with RAPA during the time of the primary i.v. OVAp administration slowed the cell cycle progression and reduced the average division rate of the KJ1-26+ CD4+ T cells by 36 ± 0.09%. Interestingly, RAPA also inhibited the lymphopenia-induced proliferation of a fraction of the transferred KJ1-26+CD4+ T cell population in the absence of Ag. KJ1-26+CD4+ T cells in both WT and nude animals demonstrated reduced recall IL-2 production in association with their blunted primary proliferative response when RAPA was present at the time of priming (Fig. 2C). Thus, these data showed that within the lymphopenic host, a homeostatic drive toward immune reconstitution promotes an overly aggressive cell cycle progression response during Ag stimulation that prevents the development of clonal anergy.

Recovery from lymphopenia reduces the homeostatic drive for excessive Ag-induced T cell proliferation

We previously demonstrated that KJ1-26+ CD4+ T cells will become anergic even in nude recipients when a 3x OVAp infusion regimen first begins at least 15 days after the T cell adoptive transfer (35). An adaptive tolerance that resembles this peptide-induced clonal anergy has also been observed to develop over extended periods of time in 5C.C7 TCR-Tg CD4+ T cells adoptively transferred into lymphopenic (CD3{epsilon}–/–) mice that express this T cell’s specific Ag (pigeon cytochrome c) as a transgene (36). Therefore, chronic TCR stimulation can induce T cell clonal anergy within immunodeficient mice, but only after a partial reconstitution of the lymphopenic immune system has taken place.

We directly compared Ag-stimulated cell cycle progression in nonreconstituted or partially reconstituted nude recipients by the adoptive transfer of a second, CFSE-labeled DO11 T cell population. OVAp challenge was found to elicit significantly fewer average cell divisions by the CFSE-labeled cohort of KJ1-26+CD4+ T cells in the partially reconstituted nude recipients (2.65 ± 0.13) as compared with nude mice that had not received an initial DO11 T cell adoptive transfer on day 0 (4.29 ± 0.31; p = 0.001) (Fig. 3A). In fact, OVAp-induced cell cycle progression in the partially reconstituted nude recipients closely resembled that observed in WT mice (data not shown). Once again, those KJ1-26+CD4+ T cells that had divided the most during the primary Ag exposure in the nonreconstituted nude mice also demonstrated a higher level of recall Ag responsiveness than naive T cells, consistent with priming (Fig. 3B). In contrast, KJ1-26+CD4+ T cells exposed to Ag after partial reconstitution of the nude mice showed only poor production of IL-2 in response to an OVAp rechallenge. Thus, the results confirmed that a partial reconstitution of the lymphopenic immune system can reduce the drive for aggressive cell cycle progression during primary Ag challenge and restore the ability to induce clonal anergy.


Figure 3
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  		FIGURE 3. Effect of partial immune reconstitution on Ag-induced cell cycle progression and clonal anergy induction. CFSE-labeled DO11 T cells were adoptively transferred into nude mice that were either nonreconstituted or previously reconstituted with unlabeled Rag-2+/+ DO11 T cells for 20 days. One day after transfer of the CFSE-labeled marker population, recipients were injected i.v. with OVAp (100 µg) or with PBS as a control. Three days later, a second infusion of OVAp (250 µg) was given, and the splenic KJ1-26+CD4+ T cells were analyzed 3 h later. A, OVAp-induced CFSE dye dilution (log FL1) of splenic marker KJ1-26+CD4+ T cells. Dotted lines to the right of the original Rag-2+/+ DO11-reconstituting population (shaded histograms) indicate the highest log FL1 autofluorescence of unlabeled T cells. Hatched lines indicate the cell division history of the CFSE-labeled marker T cell population (filled histograms). Plots are representative of four individual mice within two independent experiments. B, The relationship between cell division history and mean recall Ag-induced IL-2 accumulation within the marker KJ1-26+CD4+ T cells from nonreconstituted ({circ}) or Rag-2+/+ DO11 T cell-reconstituted (•) nude recipients was plotted. IL-2 production is calculated as the percentage of the T cell response observed in control animals that were initially infused with PBS alone ({square}). Only those marker cells with CFSE fluorescence greater than the autofluorescence of the reconstituting population were included in this analysis. *, The mode number of cell divisions observed in a marker T cell population as a result of the OVAp pretreatment. Error bars represent the SEM. Data shown in B are the average of two independent experiments. C, Splenic KJ1-26+CD4+ T cells were examined for surface CD25 expression as a function of CFSE dye content. Both unlabeled reconstituting Rag-2+/+DO11 T cells (left quadrants) and the CFSE-labeled marker KJ1-26+CD4+ T cell populations (right quadrants) are shown. Horizontal lines in each plot indicate the maximum log FL2 fluorescence of isotype control Ab-stained cells. Plots are representative of duplicate animals. This experiment was repeated more than three times with similar results. 

 
Recovery from lymphopenia is associated with the expansion of a large CD25+Foxp3+CTLA-4+CD4+ regulatory T cell subpopulation

Knoechel et al. (28) recently reported that following an adoptive transfer of Rag–/–DO11 T cells into Rag-deficient and lymphopenic mice that constitutively expressed a soluble form of OVA as a transgene, the T cells caused an early wasting disease that resulted in the death of about half of the recipients. Nevertheless, beyond 14 days after the T cell adoptive transfer (in surviving animals) a tolerance to OVA developed and these partially reconstituted lymphopenic animals regained their health. In their studies, this late immune tolerance was associated with a self Ag (OVA)-dependent generation of a subpopulation of CD25+Foxp3+KJ1-26+CD4+ regulatory T cells. Both Rag-sufficient TCR-Tg and polyclonal CD25+CD4+ regulatory T cells have been previously shown to undergo an MHC class II-dependent clonal expansion following their adoptive transfer into Rag-deficient lymphopenic hosts, and still retain their suppressive activity (27). Therefore, self Ag-specific CD25+CD4+ regulatory T cells might also be expected to arise over time following a partial reconstitution of lymphopenic nude mice with DO11 T cells to promote the establishment of immunological tolerance.

Based on this information, we sought evidence that our nude mice are resistant to clonal anergy induction because they lack regulatory T cells. Freshly isolated Rag-sufficient DO11 T cells were found to contain a small CD25+CD4+ subpopulation (data not shown), but these putative regulatory T cells did not become enriched in response to an OVAp primary Ag challenge performed immediately after T cell adoptive transfer (Fig. 3C). In contrast, 24 days after their adoptive transfer into nude recipients in the absence of OVAp, a sizable proportion of the reconstituting Rag-sufficient KJ1-26+CD4+ T cells appeared to have undergone multiple rounds of cell division (data not shown) and expressed a high level of CD25 (Fig. 3C).

Consistent with a regulatory T cell phenotype, the CD25+KJ1-26+CD4+ T cells that reconstituted nude mice demonstrated a high level of intracellular CTLA-4 and reduced expression of CD45RB (Fig. 4A and data not shown). Furthermore, in response to the infusion of OVAp this CD25+ subpopulation demonstrated little capacity to accumulate intracellular IL-2. Nevertheless, a partial induction of CD69 expression was consistently observed following stimulation, suggesting that the CD25+KJ1-26+CD4+ T cells still retained some Ag reactivity (Fig. 4A and data not shown). Foxp3 expression has been shown to be a very good marker for the development of regulatory T cell function (37). CD25+KJ1-26+CD4+ T cells purified from nude mice after partial immune reconstitution expressed high levels of Foxp3 mRNA, relative to CD25–CD4+ T cells found in either WT DO11 mice or the same partially reconstituted nude mice (Fig. 4B). These same CD25+KJ1-26+CD4+ T cells also demonstrated a capacity to inhibit IL-2 production by activated CD25–KJ1-26+CD4+ T cells in an in vitro assay system (Fig. 4C). Therefore, the CD25+ T cells that arose during a partial reconstitution of lymphopenic mice had a similar phenotype as the well-characterized natural CD25+CD4+ regulatory T cells (38, 39, 40).


Figure 4
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  		FIGURE 4. CD25+CD4+ DO11 T cells reconstituting nude mice express high levels of intracellular CTLA-4 and Foxp3, and are hyporesponsive to OVAp stimulation. DO11 T cells were transferred into either WT or nude mice. A, Twenty days after adoptive transfer, recipient mice received an i.v. infusion of OVAp (250 µg). Later (2.5 h later), splenic KJ1-26– and KJ1-26+CD4+ T cells from WT recipients and KJ1-26+CD4+ T cells from nude recipients were identified by flow cytometry and analyzed for surface CD25 expression and either surface CD69, intracellular CTLA-4, or intracellular IL-2, as indicated. Quadrant gates indicate the log FL1 and log FL2 fluorescence of isotype control Ab-stained cells. B, CD25+CD4+ and CD25–CD4+ T cells were purified from intact Rag-2+/+ DO11 Tg animals (WT), or from Rag-2+/+ DO11 T cell-reconstituted nude (NU) mice as described in Materials and Methods. Foxp3 mRNA levels in equivalent numbers of T cells were then determined using real-time quantitative RT-PCR. Foxp3 levels are expressed as arbitrary units, normalized to Hprt mRNA levels present in each sample. C, CD25+CD4+ and CD25–CD4+ T cells were purified from Rag-2+/+ DO11-reconstituted nude (NU) mice, and examined for in vitro suppressive activity as compared with CD25+ T cells purified from intact Rag-2+/+ DO11 TCR-Tg (WT) animals. Suppressor T cell and CD25–CD4+ responder T cell populations were mixed in the ratios shown and examined for the production of IL-2 (at 48 h) in response to stimulation with 10 µM OVAp using a capture ELISA. Error bars indicate SEM. Data shown are representative of two independent experiments.

 
Endogenous TCR gene recombination is necessary for optimal CD25+CD4+ regulatory T cell generation during partial reconstitution of lymphopenic mice to fully dampen Ag-induced cell cycle progression

An examination of TCR transgene and CD25 expression following partial immune reconstitution of nude mice with Rag-sufficient DO11 T cells did reveal a dimming of the clonotypic TCR staining within the large CD25+KJ1-26+CD4+ regulatory T cell population, perhaps consistent with endogenous Tcra gene rearrangement and the expression of a second TCR having self Ag specificity (Fig. 5A). Such recognition of particular self peptide/MHC specificities appears in general to be important, because Rag–/–TCR-Tg mice lacking in TCR diversity are often deficient in CD25+CD4+ regulatory T cells (41, 42). Thus, we reasoned that a partial reconstitution of nude mice with Rag-deficient DO11 donor T cells having limited TCR diversity would fail to give rise to the CD25+KJ1-26+CD4+ subset and would test whether the control Ag-induced cell cycle progression depended on the presence of these regulatory T cells. The generation of CD25+KJ1-26+CD4+ regulatory T cells (in the absence of OVAp) was observed to be reduced following reconstitution of the nude mice with Rag–/– DO11 donor T cells (Fig. 5A). Furthermore, reconstitution with Rag–/– DO11 T cells had a decreased capacity to suppress the proliferation of CFSE-labeled OVAp-stimulated KJ1-26+CD4+ T cells as compared with Rag-sufficient donor cells (Fig. 5B). Thus, it appeared that a TCR-diverse CD25+CD4+ regulatory T cell subset that developed early on during immune reconstitution acted to reduce the intensity of Ag-induced cell cycle progression.


Figure 5
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  		FIGURE 5. Reconstitution with Rag-deficient TCR-Tg T cells fails to counterregulate Ag-stimulated cell cycle progression. Nude mice were partially reconstituted with either Rag-2+/+ or Rag-2–/– DO11 T cells for 20 days. Following that time period, a CFSE-labeled marker Rag-2–/– DO11 T cell population was adoptively transferred, and 1 day later, 100 µg of OVAp (or PBS as a control) was infused i.v. to stimulate cell cycle progression. A, CFSE– reconstituting Rag-2+/+ (left) and Rag-2–/– (right) DO11 CD4+ T cell populations, stained with anti-CD25 (log FL2) and anti-clonotypic TCR KJ1-26 (log FL4). Gates for CD25 expression are based on the log FL2 fluorescence of isotype-stained cells. B, OVAp-induced CFSE dye dilution (log FL1) of splenic marker KJ1-26+CD4+ T cells in the Rag-2+/+ (left) and Rag-2–/– (right) DO11 T cell-reconstituted nude mice. Dotted lines separate the reconstituting (gray tracings) and marker (black histograms) T cell populations. The experiments shown were repeated twice with similar results.

 
Suppression of OVAp-dependent cell cycle progression in lymphopenic mice can occur following reconstitution with CD4+ T cells having no reactivity to OVAp

These findings indicated that a diverse TCR repertoire and broad self peptide/MHC specificity within the population of reconstituting CD4+ T cells was more important than a high level of clonotypic TCR expression to the counterregulation of OVAp-induced proliferation within nude mice. This predicted that suppression of the proliferation of CFSE-labeled KJ1-26+CD4+ T cells by the original DO11-reconstituting T cell population was not the result of intraclonal competition for OVAp/I-Ad complexes (43, 44). Consistent with this, a partial reconstitution of the nude mice with Rag+/+ HAp-reactive HA TCR-Tg CD4+ T cells proved equally effective in dampening the OVAp-induced KJ1-26+CD4+ T cell proliferation, regardless of whether these T cells were stimulated with HAp (Fig. 6). Note that suppression was only found to occur when the DO11- or HA-reconstituting T cells expressed Rag proteins and were capable of endogenous Tcra gene rearrangements (Fig. 5 and data not shown).


Figure 6
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  		FIGURE 6. Inhibition of proliferation is not Ag specific and does not require acute activation. CFSE-labeled marker DO11 T cells were transferred into nude mice that were reconstituted for 40 days with Rag-2+/+ DO11 or HA TCR-Tg cells, as indicated. One day after transfer of the DO11 marker cells (day 41), recipient mice were infused i.v. with 100 µg of OVAp alone and/or 100 µg of HAp. Three days following the peptide infusion (day 44), splenic KJ1-26+CD4+ T cell were recovered and examined for CFSE dye dilution as in Fig. 3. Plots shown are representative of duplicate mice. This experiment was repeated with similar results.

 
CD25+CD4+ T cells facilitate clonal anergy induction

Taken together, the data suggested that the development of a CD25+Foxp3+CTLA-4+CD4+ regulatory T cell population early during the course of immune reconstitution of lymphopenic mice was necessary to inhibit Ag-induced cell cycle progression in the absence of adjuvant or infection, and this then led to an induction of clonal anergy. To firmly establish that CD25+CD4+ regulatory T cells were responsible for suppressing the Ag-induced cell cycle progression of CD25–CD4+ responder T cells during recovery from lymphopenia, the proliferation of CFSE-labeled KJ1-26+CD4+ T cells was examined following adoptive transfer into nude mice reconstituted in the presence of an anti-CD25 mAb capable of inhibiting the development of this CD25+ population. Treatment of nude mice with anti-CD25 mAb PC61 throughout the period of DO11 T cell immune reconstitution reduced the percentage of KJ1-26+CD4+ T cells expressing CD25 (as detected using the 7D4 anti-CD25 mAb) from 31 ± 2% to 12 ± 4% (Fig. 7A). Although this Ab treatment never resulted in a complete elimination of the CD25+CD4+ subpopulation, OVAp-stimulated cell division by marker KJ1-26+CD4+ T cells was nevertheless significantly enhanced (p = 0.008; Fig. 7, B and C). Remarkably, this increased cell cycle progression was also associated with a resistance to clonal anergy induction and increased recall Ag responsiveness, despite the continued development of a large CD25–CD4+ DO11-reconstituting T cell population (Fig. 7D). Thus, the rapid expansion of a CD25+Foxp3+CD4+ regulatory T cell population during partial immune reconstitution facilitated clonal anergy induction in nearby CD25–CD4+ T cells that recognized the presence of Ag.


Figure 7
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  		FIGURE 7. Depletion of CD25+CD4+ T cells during immune reconstitution leads to enhanced Ag-induced proliferation and resistance to clonal anergy induction. Nude mice were partially reconstituted with Rag-2+/+ DO11 T cells either in the absence or presence of the anti-CD25 mAb PC61. Infusions of mAb (400 µg) were initiated on day 0 (the day of T cell transfer) and repeated every 3 days during the course of the experiment. One group of mice was analyzed on day 8 for the presence of CD25+KJ1-26+CD4+ T cells within the spleens following reconstitution in the absence or presence of anti-CD25 mAb, as indicated (A). Other recipient mice were transferred with a CFSE-labeled marker DO11 T cell population on the same day (day 8) and 1 day later were infused with OVAp i.v. (100 µg). Three days into the OVAp response (day 12), recipient mice received a second i.v. infusion of OVAp (250 µg) and spleens were harvested 3 h later. OVAp-induced CFSE dye dilution (B), average division (C), and IL-2 production (D) were measured within the marker KJ1-26+CD4+ T cell population recovered from recipients reconstituted in the absence or presence of anti-CD25 mAb, as indicated. Dotted and hatched lines in B are as indicated in Fig. 3. The percentage of IL-2+KJ1–26+CD4+ T cells during recall Ag challenge was determined based on staining with an isotype control Ab (D). Data plotted in C and D are the mean ± SEM of duplicate mice. The p value was determined using the Student t test. Results shown are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD25+Foxp3+CTLA-4+CD4+ regulatory T cells were shown for the first time to facilitate the induction of clonal anergy in vivo in CD25–CD4+ T cells that recognize Ag in the absence of an infection or adjuvant. CD25+CD4+ T cells accumulated in lymphopenic mice early during the course of immune reconstitution and functioned to counterregulate the homeostatic drive for excessive cell cycle progression during Ag encounter. Their inhibition of Ag-stimulated cell cycle progression enabled them to promote clonal anergy induction. Without such a partial T cell reconstitution of the nude mice, and in the absence of CD25+CD4+ regulatory T cells, Ag-stimulated cell cycle progression was exuberant and was invariably associated with the maintenance of a high level of Ag responsiveness.

It is plausible that a resistance to T cell clonal anergy induction in the absence of CD25+CD4+ regulatory T cells also accounts for the failure of transplantation tolerance-inducing regimens during lymphopenia (6). Whether CD25+Foxp3+CD4+ regulatory T cells play a similar role in facilitating clonal anergy induction in nonlymphopenic hosts remains uncertain. Ag-specific CD25+Foxp3+CD4+ regulatory T cells generated in vivo in the presence of low doses of Ag can be shown to interfere with the priming of a second cohort of naive Ag-reactive CD25–CD4+ T cells and lead to their reduced capacity to produce IL-2 upon in vitro rechallenge (45). Nonlymphopenic CD28–/– animals also demonstrate reduced numbers of CD25+CD4+ regulatory T cells, and anergy induction by infusion of soluble peptide Ag can be ineffective in these mice (46).

The CD25+CD4+ regulatory T cell population observed here during the recovery from lymphopenia is phenotypically and functionally similar to naturally occurring CD25+CD4+ regulatory T cells. CD25+CD4+ T cells have previously been shown to develop from purified CD25–CD4+ T cells that have undergone extensive homeostatic proliferation upon transfer into lymphopenic recipients (27). Purified CD25+ T cells can also give rise, through extensive lymphopenia-induced proliferation, to even greater numbers of CD25+CD4+ T cells that retain a capacity to suppress in vitro proliferation (27, 47). Therefore, it cannot be determined whether the CD25+CD4+ T cells generated during the course of immune reconstitution in these experiments arose from pre-existing natural regulatory T cells, or developed from naive T cells responding to self Ag in the lymphopenic environment. We did observe that the formation of this CD25+CD4+ subset was significantly reduced when Rag–/– DO11 cells were used as the reconstituting population, suggesting that endogenous TCR {alpha}-chain-dependent recognition of self peptide/MHC regulates their development during the immune reconstitution. Perhaps a broadened TCR diversity allowed for a large expansion of the CD25+CD4+ subpopulation without too many cells competing with each other for a single self peptide/MHC niche (43, 44). Nevertheless, the capacity of HA-reconstituting CD4+ T cells to facilitate the induction of anergy in DO11 T cells does not suggest that competition for a single self peptide/MHC niche is their mechanism of immunoregulation (43, 44).

The observation that a reduction in homeostatic drive for intense Ag-induced proliferation required reconstitution with Rag-sufficient TCR-Tg T cells was perhaps surprising, because one might have expected that an expanded population of DO11 Rag-2–/–CD4+ T cells would be fully competent to compete with newly transferred DO11 CD4+ responder T cells for peptide/MHC complexes and cause an inhibition of their proliferation (43). In fact, the sharing of Ag specificity between the reconstituting population and the responder CD4+ T cells was not required to inhibit Ag-stimulated cell cycle progression (Fig. 6). On the surface, this result appears to be at odds with that of Moses et al. (43) who showed that only TCR-Tg Rag–/– CD4+ T cells that compete for the same self peptide/MHC complex are capable of inhibiting the spontaneous proliferation of a particular TCR-Tg CD4+ T cell. It is important to note that in our experiments, the proliferative response to administered exogenous Ag given at high dose was examined rather than self peptide-dependent lymphopenia-induced proliferation.

In vitro data have also indicated that suppression by CD25+CD4+ T cells can be Ag nonspecific (48, 49). Nevertheless, it has been reported that immune regulation in vivo can appear Ag specific (50). In those experiments, HA-specific regulatory T cells were not capable of inhibiting the proliferative response of pigeon cytochrome c-specific CD4+ T cells responding to peptide-loaded dendritic cells (pulsed with both peptides), whereas the proliferative response of HA-specific responder T cells to the same limited stimulus was significantly reduced when HA-specific T regulatory cells were cotransferred. Perhaps in our system, the recognition of numerous self peptide/MHC complexes by the reconstituting Rag-sufficient CD25+CD4+ regulatory T cell population under lymphopenic conditions leads to a durable activation of this subset. This could then allow them to directly suppress either APCs or the effector CD4+ T cells themselves in an Ag-nonspecific manner, thus leading to an abortive cell cycle progression and the induction of clonal anergy in response to an Ag-recognition event.

The molecular mechanism of in vivo suppression of this Ag-induced cell cycle progression by these CD25+Foxp3 CD4+ T regulatory cells in the lymphopenic mice remains unknown. Both in vitro and in vivo investigations have indicated a capacity of CD25+CD4+ regulatory T cells to inhibit the production of IL-2 in nearby CD25–CD4+ T cells (Fig. 4C) (49, 51). In our study, the CD25+ regulatory cells themselves did appear anergic at the level of the Il2 gene, but they did not suppress the production of IL-2 by nearby Ag-stimulated CD25– T cells in vivo (Fig. 4A). Similarly, a coexistence of anergic CD25+CD4+ regulatory T cells and IL-2-producing CD25–CD4+ effector T cells has been demonstrated in the lymph nodes of OVA-expressing lymphopenic mice that had been reconstituted 30 days earlier by an adoptive transfer of Rag–/–DO11 T cells (28). Finally, it is unclear whether IL-2 plays any role in the cell division response observed in the setting of lymphopenia (34). In our hands, the anti-CD25 mAb has demonstrated no direct inhibitory effect on OVAp-induced cell cycle progression in the nude mice (Fig. 7B and data not shown). Therefore, the mechanism of inhibition of cell cycle progression by these CD25+CD4+ regulatory T cells is likely independent of any effects on Il2 gene expression. Regardless of the molecular mechanisms involved in this suppression, during the recovery from lymphopenia CD25+CD4+ regulatory T cells act to dampen Ag-stimulated cell cycle progression and facilitate instead an induction of clonal anergy.


   Acknowledgments
 
We thank Dr. Alex Khoruts for valuable discussions related to T regulatory cell development and function, and for the careful review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by National Institutes of Health Grants P01 AI35296, R01 GM54706, and P01 AI050162 (to D.L.M.). T.L.V. was also a recipient of a T32 Immunology Training Grant Predoctoral Fellowship. Back

2 Address correspondence and reprint requests to Dr. Daniel L. Mueller, Center for Immunology, Mayo Mail Code 334, 6-120 Nils Hasselmo Hall, 312 Church Street S.E., Minneapolis, MN 55455. E-mail address: muell002{at}umn.edu Back

3 Abbreviations used in this paper: TCR-Tg, TCR-transgenic; OVAp, OVA peptide; HA, hemagglutinin; HAp, HA peptide; WT, wild type; RAPA, rapamycin. Back

Received for publication December 8, 2005. Accepted for publication February 22, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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