The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haribhai, D.
Right arrow Articles by Chatila, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haribhai, D.
Right arrow Articles by Chatila, T. A.
The Journal of Immunology, 2007, 178: 2961-2972.
Copyright © 2007 by The American Association of Immunologists, Inc.

Regulatory T Cells Dynamically Control the Primary Immune Response to Foreign Antigen1

Dipica Haribhai*, Wen Lin{dagger}, Lance M. Relland*, Nga Truong{dagger}, Calvin B. Williams2,* and Talal A. Chatila2,{dagger}

* Division of Rheumatology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226; and {dagger} Division of Immunology, Allergy and Rheumatology, Department of Pediatrics, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The population dynamics that enable a small number of regulatory T (TR) cells to control the immune responses to foreign Ags by the much larger conventional T cell subset were investigated. During the primary immune response, the expansion and contraction of conventional and TR cells occurred in synchrony. Importantly, the relative accumulation of TR cells at peak response significantly exceeded that of conventional T cells, reflecting extensive cell division within the TR cell pool. Transfer of a polyclonal TR cell population before immunization antagonized both polyclonal and TCR transgenic responses, whereas blocking TR cell function enhanced those responses. These results define an inverse quantitative relationship between TR and conventional T cells that controls the magnitude of the primary immune response. The high frequency of dividing TR cells suggests degenerate TCR specificity enabling activation by a broad spectrum of Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Among the several subpopulations of regulatory T (TR)3 cells identified to date, the naturally arising CD4+CD25+ TR cells have emerged as particularly critical for the maintenance of immunological tolerance (1). CD4+CD25+ TR cells arise in the thymus, represent 5–10% of CD4+ T cells in the periphery, and are distinguished by their specific and universal expression of the forkhead type transcription factor Foxp3, which serves as a master switch factor for their development and function (2, 3, 4). In vitro, TR cells are anergic, do not produce IL-2, and fail to proliferate in response to mitogenic stimulation (5, 6). They constitutively express the IL-2R{alpha}-chain (CD25) and a number of other surface markers that are not specific for TR cells (1, 7). Lack of specific markers has complicated the in vivo identification and analysis of TR cells.

The mechanisms by which the smaller subset of TR cells regulate the responses of the much larger subset of conventional T cells are of considerable interest. The essential function of TR cells in the maintenance of tolerance to self-Ags is established, with dominant, contact-dependent suppression of autoimmune responses emerging as a central feature of TR cell activity (8). Indeed, the absence of TR cells due to Foxp3 deficiency results in a fatal lymphoproliferative disease marked by autoimmunity and inflammation in multiple organs (4, 9, 10, 11, 12, 13, 14, 15).

More problematic is the role of TR cells in the control of immune responses to foreign Ags. This dilemma is due to the minimal overlap of their TCR repertoire with that of conventional T cells and their decided bias toward self-reactivity (16, 17, 18). Consistent with such regulation, TR cell depletion and/or blocking of TR cell activity by means of an anti-CD25 mAb enhances the immune response to infectious agents and to immunization (19, 20, 21, 22). This process is postulated to involve increased APC function, an enhanced expansion phase or a prolonged contraction phase of the primary immune response (23, 24). Although control of immune responses is beneficial in most settings, TR cell inhibition of effector T cell responses to infections may allow for persistence of pathogens such as parasites, viruses, and indolent bacterial species (25). Therefore, a detailed understanding of mechanisms involved in TR cell control of immune responses to foreign Ags is essential to the rational manipulations of these responses.

Given the fact that in vitro TR cells are generally viewed as anergic, the stimulatory effects of anti-CD25 mAb treatment on the immune response to foreign Ags could be interpreted as the result of overcoming a tonic, basal inhibitory effect by TR cells. However, adoptive transfer studies with TCR transgenic TR cells demonstrate that TR cells can proliferate in vivo in response to primary immunization with cognate Ag and that they suppress the expansion of TCR transgenic conventional T cells (26, 27). These data suggest that Ag-driven proliferation of TR cells occurs in vivo. However, two caveats limit a broad interpretation of these results. The first is the forced expression of the same transgenic TCR on both conventional and TR cells. This condition is not likely to occur in normal mice, because these two populations have largely distinct TCR repertoires. The second issue is the inflated frequencies of Ag-specific conventional and TR cells used in these experiments. This approach does not replicate the stoichiometry of the interaction between conventional and TR cells seen in a normal polyclonal response.

Attempts to study the more physiological in vivo polyclonal TR cell response to foreign Ags have yielded conflicting results and are subject to the same difficulties in interpretation noted above. Although some adoptive transfer studies demonstrate suppression by polyclonal TR cells of primary and memory immune responses (19), others show lack of suppression by the same population in the context of a strong stimulus (28). In the latter study, proliferation of polyclonal TR cells was not observed under either weak or strong stimuli. Even assuming equal frequencies of precursor Ag-specific cells in both populations, these findings suggest that in a primary immune response, the emergence of an effective Ag-specific TR cell response might lag behind that of effector T cells, simply due to the much smaller size of the TR cell pool (one-tenth that of effector T cells).

In this study, we investigated the mechanisms by which polyclonal TR cells regulate the immune responses to foreign Ags. To facilitate the accurate identification and tracking of Foxp3+ TR cells in vivo, we derived mice with a bicistronic Foxp3 locus (Foxp3EGFP) that coexpresses the enhanced GFP (EGFP) under control of the endogenous Foxp3 promoter/enhancer elements. These Foxp3EGFP mice were immunized with CFA to generate a strong Th1 immune response to mycobacterial Ags. Our studies reveal that TR cells proliferate vigorously in vivo after immunization. The kinetics of TR cell expansion and contraction parallel that of conventional T cells. The extent of cell division within the TR cell population is significantly greater, leading to a relative increase in TR cells in the draining lymph nodes. Manipulating the TR:conventional T cell ratio by adoptive transfer of TR cells before immunization, or blocking TR cell function alters the magnitude of the primary immune response. These data establish a dynamic and quantitative mechanism by which TR cells control the magnitude of the primary responses to foreign Ags, and they implicate degenerate TR cell Ag specificity as a central feature of this mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of Foxp3EGFP mice

Foxp3 genomic DNA was isolated from a bacterial artificial chromosome clone (Genome Systems) and subcloned into the plasmid vector pKO (Lexicon). A DNA cassette composed of an internal ribosomal entry sequence (IRES) linked to downstream EGFP and SV40 poly-A sequences (BD Clontech) was inserted by blunt-end ligation at the SSPI restriction site immediately downstream of the Foxp3 translational stop codon and upstream of the endogenous polyadenylation signal. A PGK-neo cassette was also inserted at the EcoRI site in intron 9 of Foxp3 in the same orientation as Foxp3 and was flanked (floxed) by two LoxP sites to allow excision by Cre-mediated recombination. The targeting construct also included a diphtheria toxin gene (DT) for negative selection against randomly inserted targeting constructs.

Targeting plasmids were introduced by electroporation into SCC10 embryonic stem cells and subjected to G418 selection. Resistant clones were screened by Southern blotting. Successfully targeted clones were injected into C57BL/6 blastocysts, and chimeric males were mated with wild-type (WT) BALB/c females to derive N1 females that have transmitted the bicistronic allele together with the PGK-neo cassette insert (Foxp3EGFPneo) in the germline. The PGK-neo cassette was removed by mating founder males with Cre-deleter female mice that harbor a constitutive, ubiquitously expressed Cre recombinase allele (29). Male offspring hemizygous for Foxp3EGFP allele (minus the PGK-neo cassette) developed normally and were free of disease. Foxp3EGFP+ mice were further backcrossed for up to seven generations on the BALB/c background. The mice were housed under specific pathogen-free conditions and used according to the guidelines of the institutional Animal Research Committees at the University of California in Los Angeles and at the Medical College of Wisconsin. The Foxp3EGFP mice have been deposited in the induced mutant resource repository at The Jackson Laboratory. The stock numbers and designations of the Foxp3EGFP strains are stock no. 006769 and C.Cg-Foxp3<tm1Tch>/J mice on the BALB/c background, and stock no. 006772 and B6.129X1-Foxp3<tm1Tch>/J for mice on the C57BL/6J background.

PCR screening of the mutant allele was also achieved by PCR amplification using genomic DNA and the following EGFP-specific primers: forward sense 5'-CGGCAAGCTGACCCTGAAGT-3' and reverse 5'-GGATGTTGCCGTCCTCCTTG-3'. PCR-based discrimination between WT and mutant alleles was conducted by analyzing for the presence of the residual LoxP-sequence retained in the mutant allele following Cre-mediated excision of the PGK-neo cassette. The following primers were used: sense 5'-GCGTAAGCAGGGCAATAGAGG-3' and antisense 5'-GCAT GAGGTCAAGGGTGATG-3'.

Quantitative real-time PCR analysis

Real-time PCR for Foxp3 expression was performed as described previously (15). Samples were run in triplicate, and the relative expression was determined by normalizing expression of each target to the endogenous reference, hypoxanthine phosphoribosyltransferase (Hprt) transcripts. Primers and internal fluorescent probes were as follows: Foxp3 exon7, sense 5'-CCCAGGAAAGACAGCAACCTT-3, antisense 5'-TTCTCACAACCAGGCCACTTG-3', and probe, 5'-FAM-ATCCTACCCACTGCTGGCAAATGGAGTC-TAMRA-3'; Foxp3 exon 11, sense 5'-GGA GGC AAG TCC TAC GTG TAC C-3', antisense 5'-CTA GAT AGG GAG CAG AGG CCC-3', and probe 5'-FAM-TGG AAA CCG GGC GAT GAT GTG C-TAMRA-3'.

Antibodies

PE anti-mouse Thy1.2, allophycocyanin anti-mouse Thy1.1, allophycocyanin anti-mouse CD44, allophycocyanin BrdU Flow Kit, and PE anti-mouse CD4 were obtained from BD Biosciences. Pacific Blue anti-mouse CD4, Pacific Orange anti-mouse CD8, biotin anti-DO11.10 TCR, PE-Texas Red anti-mouse CD62L, allophycocyanin Cy5.5 anti-mouse CD19, and streptavidin-PE Cy5.5 were obtained from Invitrogen Life Technologies. Pacific Blue anti-mouse Foxp3 was purchased from eBioscience and used following the manufacturer’s instructions. The clone PC61 was purchased from the American Tissue Culture Collection, and the Abs were generated and purified in the laboratory.

Flow cytometry

For cell surface markers, single-cell suspensions of lymphocytes were stained as previously described (30). Intracellular Foxp3 staining was conducted using an anti-murine Foxp3-Pacific Blue Ab (15). Samples were analyzed on an LSRII using a live cell gate, and a minimum of 1 x 106 live cell events were collected per sample.

Suppression assays

Plates were coated with anti-CD3 mAb at 2.5 µg/ml in PBS for 6 h at 37°C. CD4+ T cells were enriched from whole splenocytes using mouse CD4 columns (R&D Systems), and CD4+EGFP+ and CD4+EGFP– cells were further purified by cell sorting. Purified cell populations were suspended in culture medium containing RPMI 1640, 10% FBS, 5 x 10–5 M 2-ME, 1 mM glutamine, and 1 µg/ml anti-CD28 mAb. Cells were added to coated wells, and the number of responder cells (R) was kept constant at 5 x 104 cells per well. The number of suppressor cells (S) was titrated to achieve the R:S ratios as indicated (see Fig. 2D). Triplicate wells were set at each R:S ratio. Cultures were incubated for 48 h at 37°C with 5% CO2, pulsed with 0.4 µCi/well [3H]TdR for an additional 18 h, harvested onto fiber filtermats using a Micro96 harvester (Skatron), and counted.


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 2. Analysis of EGFP+ lymphocytes. A, Frequency of EGFP+CD4+ T cells in the peripheral blood of hemizygous males and heterozygous females. Results are means ± SEM of percentage of EGFP+CD4+ T cells in circulation (males, 5.30 ± 0.50; females, 2.82 ± 0.46; n = 5 mice each; p = 0.0064). B, Foxp3 mRNA segregates with EGFP+ cells. Peripheral lymphoid CD4+EGFP+ and CD4+EGFP– populations of Foxp3EGFP+ males were isolated by cell sorting and analyzed for Foxp3 mRNA expression by real-time PCR. The Foxp3 mRNA levels of CD4+EGFP– cells were assigned an arbitrary value of 1, those of CD4+EGFP+ cells were 74 ± 3.0 (n = 3; p = 0.0017). C, EGFP fluorescence colocalizes with Foxp3 expression in CD4+ and CD8+ T cells. Lymph node cells from Foxp3EGFP+ males were stained with an anti-murine Foxp3 mAb, and the T cells were then examined by flow cytometry for colocalization of EGFP fluorescence and Foxp3 staining. D, Suppressor function of CD4+EGFP+ cells. Cell-sorted suppressor CD4+EGFP+ (S) and responder CD4+EGFP– (R) cells were cultured in 96-well plates either alone or mixed at the indicated R:S ratios while keeping the responder cells constant at 5 x 104 cells/well. The cells were stimulated with anti-CD3 and -CD28 mAbs, and their proliferative responses were assessed by [3H]thymidine incorporation. Results shown are mean cpm ± SEM values of 12–15 replicates at each R:S ratio from five independent experiments.

 
Immunization

Foxp3EGFP mice were immunized s.c. with CFA emulsified 1:1 with PBS (100 µl per mouse). At various times after immunization, the draining lymph nodes were removed and the total number of lymphocytes and those of the respective subsets were determined by counting with a hemocytometer and by flow cytometric analysis, respectively.

Immunocytochemistry

Tissues were fixed in 4% formaldehyde in PBS for 1 h at 4°C, transferred to 20% sucrose in PBS, and incubated 1 h at 4°C, embedded in the OCT reagent, and frozen in liquid nitrogen. Sections were cut at a thickness of 7 µm, fixed with acetone for 10 min at 4°C, and blocked with 5% BSA in PBS for 30 min in a humidified chamber. Abs and secondary reagents were diluted in the blocking buffer to a final concentration of 2 µg/ml. Primary reagents were applied to the sections, and incubated for 12 h in a humidified chamber at 4°C. In the case of biotinylated primary Abs (anti-I-Ad), blocking reagents (Vector Laboratories) were each applied for 15 min at room temperature before applying the primary Ab. Following incubation with primary Abs, the slides were washed and secondary reagents were applied as appropriate for 1 h at 4°C. The slides were then mounted in Prolong Gold antifade reagent with 4',6'-diamidino-2-phenylindole (Molecular Probes). Sections were visualized by laser scanning confocal microscopy with a Leica TCS SP II microscope equipped with 488, 568, and 633 nm lasers. Images were collected using Leica acquisition software at x10, x20, x40, and a x63 objective with a x4 optical zoom for close-up pictures. The Abs and emissions collected were as follows: Alexa 488 rabbit-anti-GFP IgG fraction (Molecular Probes), collected at 500–545 nm; PE-Texas Red anti-B220, collected at 615–700 nm; biotinylated anti-I-Ad with streptavidin-Alexa 568 (Molecular Probes), collected at 615–700 nm; and allophycocyanin-anti-CD4, -anti-CD8, and -anti-CD11c, all collected at 650–740 nm.

Image analysis

Layered images were aligned in Adobe Photoshop version 8.0. The conversion ratio was determined to be 0.53 µm2/pixel. T cell zones were arbitrarily delineated in regions away from B cells and B cell follicles using the lasso function and were subsequently outlined with the Adobe Photoshop stroke command. The borders of B cell follicles were similarly delineated using the lasso function, but they were both contracted and expanded by 22 pixel lengths (16 µm) from the original selection before being outlined with the stroke command. This 32-µm area between the contracted/expanded selections was considered to be the B/T cell border, and the region within the contracted selection was considered the to be the B cell zone. The absolute pixel count of each outlined region was determined using the histogram function, and the density of cells in each zone was determined using the following equation: D = (x)(109)(n3.71)–1mm–3, where x = cell count and n = pixel count. TR cells were counted after subtracting other fluorochromes to simplify the identification of cells.

It should be noted that in calculating the number of conventional T cells, we subtracted the number of EGFP+ TR cells from the total number of CD4+ T cells. Although this partial correction still slightly overestimates the number of conventional CD4+ T cells seen in Foxp3EGFP+/– female mice, only the calculation of conventional CD4+ T cells is affected. Importantly, both the baseline values and the experimental values for TR densities would increase proportionally in hemizygous male mice, and there would be no change in the observed effect.

Isolation of EGFP+ and EGFP– T cells

CD4+ T cells from lymph nodes and spleens were purified by negative selection using mouse CD4 columns following the manufacturer’s recommendations (R&D Systems). Purified CD4+ T cells were stained with anti-CD4-PE mAb and sorted into CD4+EGFP+ and CD4+EGFP– populations using a FACSAria or a FACSVantage SE cell sorter (BD Biosciences).

In vivo suppression

A total of 2 x 105 CD4+EGFP+ T cells was adoptively transferred into BALB/c mice. Mice were immunized the following day with CFA. At 10 days after immunization, the draining lymph nodes were analyzed by cell count and FACS.

For suppression of Ag-specific T cells, 2.5 x 105 CD4+Rag–/– DO11.10+ T cells were adoptively transferred i.v. into BALB/c mice. Two to 3 h later, 5 x 105 CD4+EGFP+ T cells were also adoptively transferred i.v. into the same mice. Mice were immunized s.c. with 2 nmol of OVA (323–339) peptide emulsified in CFA. At the peak of the immune response, draining lymph nodes were analyzed by cell count and by FACS.

Antagonizing TR cell function with a blocking anti-CD25 mAb

Mice were given 250 µg of PC61 mAb i.p. at day –4 and day –1. Mice were immunized with CFA s.c. on day 0. Lymphocytes from draining lymph nodes were analyzed by cell count and by FACS.

BrdU treatment and detection

Mice were given a single i.p. injection of 1 mg BrdU, followed by continuous administration of BrdU (0.8 mg/ml) in the drinking water. On the same day as the i.p. injection, mice were immunized s.c. with CFA. The water containing BrdU was changed on a daily basis. At various times after immunization, the draining lymph nodes were analyzed by cell count and by FACS. Unimmunized mice were used as controls. Single-cell suspensions from the draining lymph nodes were subject to cell surface staining for CD4, CD8, CD62L, and CD44 followed by intranuclear staining for BrdU incorporation using a BrdU Flow Kit (BD Pharmingen) following the manufacturer’s protocol. Analysis was based on sequential gating using live cells followed by a CD4+ gate.

Conversion of polyclonal and monoclonal T cells

A total of 5 x 106 CD4+EGFP– T cells were adoptively transferred into BALB/c Thy1.1 mice. Transfer recipients were immunized s.c. the following day with CFA. Ten days after immunization, the draining lymph nodes were analyzed by cell count and flow cytometry. In similar experiments, 5 x 105 CD4+DO11.10+ T cells from DO11.10 RAG–/– mice were adoptively transferred into BALB/c recipients. In some experiments, cells were labeled with CFSE before adoptive transfer as described previously (31). Mice receiving DO11.10 T cells were immunized the following day with 2 nmol OVA (323–339) peptide emulsified in CFA. Five days after immunization, the draining lymph nodes were examined by cell count and flow cytometry.

Statistical analysis

Comparisons involving different T cell populations derived from the same mice were performed by the Student’s two-tailed paired t test. For other studies comparing populations between mice the Student’s two-tailed unpaired t test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Derivation and characterization of Foxp3EGFP mice

A cassette encoding an IRES followed the EGFP, and the polyadenylation signal from SV40 (SV40 poly-A) was inserted into the 3' untranslated region of Foxp3 to generate a bicistronic locus encoding both Foxp3 and EGFP under the control of the Foxp3 promoter (Foxp3EGFP) (Fig. 1). Both male and female mice harboring the Foxp3EGFP allele were phenotypically indistinguishable from their WT littermates, were fertile, and exhibited normal T and B cell development. EGFP expression was found restricted to the T cell lineage, primarily to the CD4+ T cell population (see below). The number of EGFP+CD4+ T cells found in the peripheral blood of heterozygous females was on average roughly half that of hemizygote males, consistent with random X-inactivation in females (Fig. 2A). Real-time PCR analysis of Foxp3 mRNA expression from EGFP+ and EGFP–CD4+ T cells that were purified by cell sorting revealed that Foxp3 mRNA localized (>70-fold enrichment) to EGFP+CD4+ T cells (Fig. 2B). Analysis of Foxp3 expression by intracellular staining with anti-murine Foxp3 revealed that ≥97.5% of Foxp3+ T cells detected were EGFP+, with the remainder being EGFP dim (Fig. 2C). A rare population of Foxp3+EGFP+CD8+ T cells was also detected (Fig. 2C). The percentage and total number of EGFP+CD4+ and CD8+ T cells in the thymus, inguinal lymph nodes, and spleen is shown in Table I. Finally, CD4+EGFP+ cells suppressed the proliferation CD4+EGFP– effector T cells in response to stimulation with anti-CD3 and anti-CD28 mAb (Fig. 2D). These results confirmed that EGFP accurately and specifically identifies Foxp3+ TR cells, consistent with other mice expressing fluorescent proteins under the control of the Foxp3 promoter (32, 33).


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 1. Derivation of mice with a bicistronic Foxp3EGFP locus. A, Targeting strategy. An IRES-EGFP-SV40 poly-A cassette was inserted into an SSPI restriction site in the Foxp3 3'-untranslated sequence in exon 11 immediately downstream of the Foxp3 stop codon and upstream of the poly-A adenylation signal. Neo, Floxed PGK-neo cassette; DT, diphtheria toxin gene. The PGK-neo cassette is later removed by the Cre recombinase. B, Southern blot analysis of KpnI-digested DNA derived targeted embryonic stem (ES) cells (left panel) and mouse tail biopsies (right panel). The introduction of a novel KpnI site carried within the EGFP cassette reduces the size of a KpnI genomic fragment detected with a distal 3' probe from 9.5 kb in the WT allele to 6.5 kb in the mutant (MUT) allele. C, PCR genotyping of Foxp3EGFP mice using EGFP-specific primers (upper panel) or primers spanning the residual 34-bp LoxP site in intron 9 following Cre-mediated excision of the floxed PGK-neo cassette.

 

View this table:
[in this window]
[in a new window]

  		Table I. The percentage and total number of EGFP+CD4+ and CD8+ T cellsa

 
Foxp3+ cells accumulate in the draining lymph nodes during a primary immune response

To study the role of Foxp3+ TR cells in the immune responses to foreign Ags, Foxp3EGFP+/– female mice were immunized with CFA. The draining lymph nodes were examined at the times indicated by cell count and analytical flow cytometry. The results were compared with those of unimmunized control mice.

Expansion of the total lymphocyte compartment as well as individual CD4+ and CD8+ conventional T cell populations peaked at day 10, as expected, before contracting again back to baseline levels by day 20 (Fig. 3, A–C). Individual CD4+EGFP+ and CD8+EGFP+ TR cell populations followed a similar time course of expansion and contraction. In the early stages of the primary response, the EGFP– and EGFP+ T cell populations accumulated in the draining lymph nodes at a similar rate. For example, on day 5, a 1.2-fold expansion is seen in both the CD4+EGFP– (conventional) and CD4+EGFP+ (regulatory) T cell populations. However, by day 10, the conventional CD4+ T cell population expanded by an average of 2.3-fold, whereas the CD4+EGFP+ TR cells expanded by an average of 3.3-fold (Fig. 3D). Thus, the ratio of CD4+EGFP+ T cells:CD4+EGFP– T cells increased by 43%, demonstrating a significant and preferential accumulation of TR cells at the peak of the primary immune response (p = 0.005, two-tailed paired t test) (Fig. 3D). Similarly, by day 10, the comparatively much smaller CD8+EGFP+ T cell population had expanded ~3.5-fold compared with a 2-fold increase in the CD8+EGFP– T cell population (p = 0.006, two-tailed paired t test) (Fig. 3D). The differences in fold expansion between the CD4+EGFP+ and CD8+EGFP+ TR cell populations and their respective conventional counterparts narrowed by day 15 postimmunization, and both populations approached their baseline status by day 20 postimmunization. These data suggested that the ratio of TR:conventional T cells in the course of a primary immune response is tightly controlled, implicating TR cells in the control of these responses.


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 3. Flow cytometry-based analysis of TR kinetics in the draining lymph nodes of Foxp3EGFP+/– mice after immunization with CFA. In each panel, the dashed line represents the average steady-state number of lymphocytes per lymph node in unimmunized Foxp3EGFP+/– female controls. All plots show raw data that has not been corrected for the 50% of TR that do not express EGFP. A, Total number of lymphocytes per lymph node. B, Number of CD4+EGFP– (right panel) and CD4+EGFP+ (left panel) T cells per lymph node. C, Number of CD8+EGFP– (right panel) and CD8+EGFP+ (left panel) T cells per lymph node. D, Fold expansion calculated from the baseline. The number of mice (m) and lymph nodes (ln) analyzed at each time point are as follows: day 2 m = 3, ln = 9; day 5 m = 10, ln = 35; day 10 m = 15, ln = 55; day 15 m = 11, ln = 38; day 20 m = 4, ln = 12; unimmunized controls m = 47, ln = 258. All data represent mean values ± SEM. *, p < 0.05 and **, p ≤ 0.005.

 
T and B cell zone-specific changes in the density of CD4+EGFP+ T cells in draining lymph nodes

TR cells are hypothesized to regulate both T and B cell responses, implying that they will be found within both T and B cell zones. Therefore, we determined the location and density of EGFP+ cells within the draining lymph nodes during the primary response by immunocytochemistry. Analysis using hemizygous male mice revealed large clusters of EGFP+ cells in their draining lymph nodes that made it difficult to accurately count the number of cells in a given area. Heterozygous female mice were therefore selected for this analysis, because only half of their TR cell population expresses EGFP due to random X chromosome inactivation (Fig. 2 and data not shown). Restricting analysis to heterozygous females minimized the TR cell clustering effect.

One draining lymph node from each heterozygous female mouse used in Fig. 3 was sectioned and stained with conjugated Abs specific for CD4, CD8, B220, CD11c, and EGFP. Lymphocytes were visualized by scanning confocal laser microscopy, and the images were aligned to create a composite of each lymph node. Anti-EGFP Ab was used to enhance image quality and to accurately detect these cells. Representative data from each time point at x20 and x40 magnification are shown in Fig. 4A. At early time points (day 2 and day 5), most EGFP+ cells are localized in T cell areas. By day 10, EGFP+ cells are found within B cell zones, but relatively few cells are seen within the germinal centers from day 15 and day 20 lymph nodes.


Figure 4
View larger version (87K):
[in this window]
[in a new window]

  		FIGURE 4. Localization and density of TR cells in the draining lymph nodes during the course of the primary immune response. A, Immunocytochemistry of draining lymph nodes after immunization. Representative sections demonstrating that TR cells (green) were found largely in T cell (blue) areas or at the B/T borders 2, 5, 10, 15, and 20 days after primary immunization. TR cells also migrated into B cell (red) follicles as the immune response developed. Top panels, x20 and bottom panels, x40 magnification of the same section. B, The density of TR cells and conventional T cells increases in draining lymph nodes following primary immunization. The data is plotted for T cell areas, B cell areas, and B/T borders as defined in Materials and Methods. Shaded areas are the observed density range ± SEM in unimmunized mice. The number of lymph nodes analyzed at each time point are as follows: day 2 = 3; day 5 = 4; day 10 = 5; day 15 = 4; day 20 = 4; unimmunized controls = 6. Each lymph node was from a separate mouse. C, TR cells show cell-cell contact with conventional T cells, B cells, APCs, and other regulatory T cells. C1–2, Both CD4+ and CD8+ T cells show expression of EGFP. C3–4, TR cells come into contact with B cells (long arrows) and conventional T cells (short arrows). C5–6, TR cells come into contact with other TR cells and are able to form TR cell aggregates (large arrowheads). C7–8, TR cells may also contact APCs (small arrowheads) while simultaneously contacting other cell types. *, p < 0.05 and **, p < 0.005.

 
Visual inspection of the images also suggested that the relative density of EGFP+ cells in the T cell zones increased by day 10. To further examine this possibility, the density of EGFP+ cells was determined by counting these cells in multiple areas from multiple lymph nodes at each time point. The T and B cell zones and the B/T borders were delineated as described in Materials and Methods. These quantitative measurements demonstrated that, in female Foxp3EGFP+/– mice, the density of EGFP+ cells within the B cell zones increased by day 10 (Fig. 4B, top left panel), from a mean density of 46 x 103 cells/mm3 in unimmunized controls to 59 x 103 cells/mm3 in immunized mice (p = 0.046). This result was followed by a decrease into the normal range for EGFP+ cells found in the B cell zones on day 15, and a secondary increase in the density of these cells on day 20 (66 x 103 cells/mm3; p = 0.034). A similar biphasic response was also observed for TR cells at the B/T borders. The decrease in the TR cell density on day 15 likely reflects a relative expansion of the B cell zones. The density of conventional CD4+ T cells found in the B cell zones is increased on day 10 and day 20, but plateaus on day 15, consistent with the TR cell data (Fig. 4B).

At baseline, the density of EGFP+ cells in the T cell zones was 254 x 103 cells/mm3. The TR cell density increased on the 10th day after immunization to 354 x 103 cells/mm3 (p = 0.038), and remained near this level at day 15 and day 20 (Fig. 4B). Examination of EGFP+ cells found at the B/T borders under high-power magnification revealed direct contact between EGFP+ cells and CD4+ T cells, CD8+ T cells, B cells, and CD11c+ APCs (Fig. 4C). Furthermore, clusters of EGFP+ cells are seen, suggesting the possibility of TR cell-cell interaction. Together, the data in Figs. 3 and 4 demonstrate that both the number and relative concentration of TR cells increase in the draining lymph nodes following immunization, consistent with a role in regulating both B and T cell primary responses.

TR cells control the magnitude of the primary response

To investigate the influence of TR cells on primary responses, we antagonized TR cell function by pretreating Foxp3EGFP+/– female mice with an anti-CD25 mAb. This approach, which takes advantage of the high level of expression of CD25 on TR cells and the critical function of IL-2 in TR cell function, blocks TR cell activity while sparing the expansion of conventional T and B cells (34). Accordingly, mice were treated with anti-CD25 mAb (clone PC61) on days –4 and –1 before their immunization with CFA. Ten days after immunization, draining lymph nodes were harvested and analyzed for lymphocyte cell numbers and phenotype (Fig. 5A). The total number of lymphocytes per draining lymph node was increased after treatment with anti-CD25 mAb (13.54 vs 8.45 x 106; n = 14 and 15 respectively; p = 0.001), primarily due to the expansion of CD4+ T cell and CD19+ B cell populations (Fig. 5B). Analysis of EGFP expression in the CD4+ T cell compartment showed a decrease in the percentage of CD4+EGFP+ T cells in anti-CD25 mAb-treated mice as compared with sham-treated mice (4.4 vs 6.8%; p = 0.007). Simultaneous staining for CD25 also showed a similar decrease in the CD25+EGFP+ population (Fig. 5C). This reflected the expansion of CD4+EGFP– cell population in the anti-CD25 mAb treated mice, because the total number of CD4+EGFP– cells was increased after treatment with anti-CD25 mAb as compared with the sham treatment (3.77 vs 2.72 x 106; p = 0.02). The total number of CD4+EGFP+ T cells was not significantly changed due to the 55% increase in lymph node size after PC61 treatment (Fig. 5D).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Pretreatment with an anti-CD25 mAb results in an increase in the magnitude of the T and B cell responses. A, Experimental timeline. B, The total number of lymphocytes from the draining lymph nodes with and without pretreatment. C, Dot plot analysis showing the percentage of CD4+EGFP+ and EGFP– T cells, with and without PC61 treatment. The CD4+ populations were also analyzed for CD25 expression as a function of EGFP expression. (PC61 pretreatment, n = 15; controls, n = 14.) D, The total number of CD4+EGFP+ and EGFP– T cells per lymph node, with and without PC61 pretreatment is shown. All data represent mean values ± SEM. *, p < 0.05 and **, p < 0.005.

 
Next, we determined whether tipping the balance in favor of TR cells would suppress the magnitude of the T and B cell expansion following immunization with CFA. CD4+ T cells were isolated from Foxp3EGFP mice by negative selection, the EGFP+ and EGFP– populations were purified by cell sorting, and 2 x 105 EGFP+ TR cells or 5 x 106 EGFP– (control) T cells were adoptively transferred into BALB/c recipients. The following day, recipient mice and untransferred mice were immunized with CFA. The draining lymph nodes were analyzed for cell number and phenotype 10 days after immunization. The adoptive transfer of TR cells resulted in a 22% decrease in lymph node cellularity, and a 39% decrease in the number of CD4+ T cells relative to the controls (Fig. 6A; p = 0.047 and p = 0.002, respectively). B cell numbers were unchanged. The adoptive transfer of EGFP+ TR cells increased the proportion of TR cells in the draining lymph nodes from 11% to 14% of the total CD4+ population (p = 0.008; data not shown). This change in the proportion of TR to conventional CD4+ T cells is due to the decreased numbers of the latter population, because only a small number of transferred TR cells (~500 cells/lymph node) were identified in the lymph nodes of transfer recipients at the peak of the response.


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 6. Adoptively transferred polyclonal TR cells suppress both polyclonal and monoclonal primary immune responses. A, Adoptive transfer recipients of EGFP+ TR cells ({blacksquare}) show significant reductions in both the total size of the draining lymph nodes and of the CD4+ T cell compartment after immunization relative to the controls ({square}, no transfer; Figure 6, EGFP– control cell transfer). B, Adoptive transfer recipients of DO11.10 T cells (2.5 x 105 cells) and EGFP+ TR cells (5.0 x 105) show significant decreases in the percentage and number of Ag-specific DO11.10 T cells after immunization. *, p < 0.05 and **, p < 0.005.

 
The effects of adoptively transferred TR cells on lymphocyte expansion are likely to reflect inhibition of conventional T cell Ag-specific responses. However, the precursor frequency of the responding cells and the magnitude of their expansion could not be directly determined from the previous experiments. In addition, the Ag specificity of the TR cell response is an important and unresolved question. We addressed these issues by adoptively transferring 2.5 x 105 TCR transgenic DO11.10+, Rag–/–Thy1.2+ conventional T cells, and 5.0 x 105 polyclonal EGFP+Thy1.2+ TR cells into Thy1.1+ BALB/c recipients. Mice were immunized with OVA (323–339) peptide in CFA, and the number of D011.10 T cells in the draining lymph nodes was quantified by FACS at the peak of the immune response. Control mice received only the DO11.10 T cells and were immunized following the same protocol. Results showed that increasing the number of TR cells with a normally distributed polyclonal TCR repertoire before immunization resulted in 46% reduction in the frequency and a 41% reduction in the total number of the OVA-specific DO11.10 T cells (Fig. 6B; p = 0.008 and p = 0.02, respectively). As in the prior experiments, a small number of transferred TR cells were recovered from the draining lymph nodes at the peak of the response (1,900 TR cells/lymph node). These data demonstrated that increasing the number of TR cells before immunization reduces the magnitude of the primary response. The frequency of OVA-specific TR cells in the adoptively transferred polyclonal TR cell population is likely to be immeasurably low compared with the transgenic DO11.10 T cells. Therefore, these results indicated that TR cell TCR specificity need not be directed against the foreign Ags that mediate the conventional T cell responses.

The increase in TR cells after immunization is associated with heightened cell division

Several mechanisms were possible for the relative expansion of EGFP+ TR cells in the draining lymph nodes following immunization. First, the increase in TR cells might be attributed to a nondividing TR cell population that accumulated due to increased trafficking and/or preferential retention. Second, the observed increase might largely involve the local expansion of TR cells by means of cell division. Finally, TR cells could be generated by in situ conversion of conventional T cells.

To determine the proliferative activity of TR cells in the draining lymph nodes, the DNA precursor BrdU was administered to immunized mice and unimmunized controls. BrdU incorporation into DNA and the cell surface phenotype were determined by FACS after 5 and 10 days of continuous BrdU labeling. In these experiments, CD4+ T cells were divided into four main groups (Fig. 7A): TR cells that have divided (EGFP+BrdU+); TR cells without recent cell division (EGFP+BrdU–); conventional T cells that have divided (EGFP–BrdU+); and conventional T cells without recent cell division (EGFP–BrdU–). As expected, a small percentage (1.9%) of the resident lymph node conventional T cells in unimmunized mice have undergone cell division within the time frame of the experiment (Fig. 7B). A majority of these cells were CD44highCD62Lhigh/low, consistent with the homeostatic proliferation of memory T cells (35) (Fig. 7D). In contrast, 6.6% of the TR cell population divided, representing a 3-fold increase in the baseline cell division (n = 10; p = 0.0054, two-tailed paired t test). Nearly all of the dividing TR cells also had an activated phenotype (CD44highCD62Lhigh/low), in agreement with previous reports (Fig. 7D) (36). After immunization, the percentage of TR cells that divided was increased 3-fold compared with TR cells in unimmunized mice, suggesting extensive cell division (20.1 vs 6.5%; n = 10 per group; p = 0.007) (Fig. 7B, left panel). These values were used to calculate the total number of TR cells per lymph node that had divided, and the results were compared with unimmunized controls (0.46 x 105 vs 0.068 x 105 BrdU+ cells per lymph node on day 10; p = 0.0001) (Fig. 7C, left panel), confirming heightened cell division within the TR cell pool. After immunization, virtually all TR cells that had divided exhibited an activated phenotype (CD44highCD62Lhigh/low) (Fig. 7D).


Figure 7
View larger version (64K):
[in this window]
[in a new window]

  		FIGURE 7. Increased cell division in the TR cell population at steady state and after immunization. A, Dot plot analysis showing BrdU incorporation in CD4+ T cells with and without immunization (day 5, n = 5; day 10, n = 10). B, The percentage of TR cells and conventional T cells that incorporate BrdU with and without immunization. C, The total number of dividing cells in the EGFP+ and EGFP– populations. D, Phenotypic analysis of dividing and nondividing TR and conventional T cells based on CD44 and CD62L expression. *, p < 0.05 and **, p < 0.005.

 
The percentage of conventional T cells that had divided also increased ~3-fold 5 and 10 days after immunization, compared with unimmunized controls (day 5, p = 0.013 and day 10, p = 0.012) (Fig. 7B, right panel). Consistent with this observation, the number of dividing conventional T cells was also significantly increased at these time points (day 5, p = 0.022 and day 10, p < 0.0001) (Fig. 7C, right panel). Similar to their TR cell counterparts, dividing conventional T cells had an activated phenotype (CD44highCD62Lhigh/low) (Fig. 7D). Importantly, whereas the number of dividing conventional T cells was greater than the number of dividing TR cells, the percentage of dividing TR cells was nearly 3-fold greater than that seen within the conventional CD4+EGFP– population (20.1 vs 7.3%; p = 0.0006). These data demonstrated that at the peak of the primary immune response, ~20% of the TR cell pool in the draining lymph nodes is dividing, a much higher frequency of cell division than in the conventional T cell population. However, they also show the same 3-fold expansion in both the conventional and TR cell populations, implying that the rates of cell division in the two types of cells are equivalent. Thus, the preferential accumulation of TR cells described in the preceding section involves both cell division and other mechanisms. Possibilities include selective retention/survival of TR cells, efflux of conventional CD4+ effector T cells, or both.

In situ TR cell conversion contributes marginally to TR cell expansion

Conventional CD4+Foxp3– T cells can be converted to express Foxp3 after activation in the presence of TGF-beta (37). The extent of conversion in vivo and the role of converted cells in regulating immune responses is controversial. Repertoire studies comparing TCR CDR3 regions have identified limited overlap between the conventional and TR cell pools, suggesting that in vivo conversion has a minimal role in generating long-lived TR cells. To determine the contribution of conventional T cell conversion to the observed expansion of TR cells after immunization, a polyclonal population of Thy1.2+CD4+EGFP– T cells was adoptively transferred into Thy1.1+BALB/c recipients. The Thy1.2+CD4+EGFP– population was isolated by cell sorting to >99% purity, hence EGFP expression marked only those transferred cells that up-regulated Foxp3. Recipient mice were immunized the following day with CFA. The composition of the draining lymph nodes was analyzed at the peak of the T cell response on day 10. Live cells were sequentially gated on CD4 and Thy1.2 to identify the transferred population, and CD4+Thy1.2+ cells were analyzed for EGFP fluorescence (Fig. 8A). Thy1.2+ cells represented 0.9% of the CD4+ compartment found in the draining lymph nodes. Within this transferred population, 1.5% of the CD4+ T cells were also EGFP+. When corrected for lymph node size, these percentages result in ~270 CD4+Thy1.2+EGFP+ and 20,270 CD4+Thy1.2+EGFP– cells per draining lymph node. These data suggest that in vivo conversion is a very limited process that cannot explain the large number of proliferating TR cells shown in Fig. 7.


Figure 8
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 8. Conversion of conventional T cells does not account for the expansion of TR cells during the primary immune response. A, Conversion of polyclonal T cells contributes marginally to TR cell expansion after immunization. Dot plots show the sequential gating strategy. The bar graph displays the calculated number of transferred cells per lymph node that are either EGFP+ or EGFP– (n = 4). B, Conversion of a monoclonal population after immunization with the cognant Ag is also limited. Dot plot analysis shows the percentage of adoptively transferred DO11.10 T cells that express Foxp3 with and without immunization. The total number of DO11.10 Foxp3+ or DO11.10 Foxp3– T cells is also calculated (n = 6). C, Foxp3 expression after immunization in DO11.10 T cells labeled with CFSE before adoptive transfer. The dot plots illustrate the sequential gating strategy and demonstrate that lack of conversion is not due to insufficient DO11.10 T cell activation.

 
In theory, only those T cells that are activated through their TCR are available for conversion. Thus, the maximum rate of conversion might be underestimated using the polyclonal population of T cells described above, where the frequency of cells specific for the Ags in CFA is unknown. To maximize the potential conversion process and directly examine Foxp3 expression, we transferred Ag-specific T cells isolated from DO11.10 x RAG–/– mice into BALB/c recipients. DO11.10 x RAG–/– mice do not normally develop Foxp3+ TR cells. Foxp3+ TR cells can be generated from DO11.10 x RAG–/– T cells by ex vivo conversion with TCR cross-linking in the presence of TGF-beta (data not shown). Recipient mice received 0.5 x 106 CD4+DO11.10+ T cells and were immunized with OVA (323–339) peptide emulsified in CFA. Control mice received the adoptively transferred T cells but were not immunized. After immunization, 3% of the total CD4+DO11.10+ T cells express Foxp3 (Fig. 8B). DO11.10+Foxp3+ T cells were not seen in unimmunized mice.

Although all adoptively transferred DO11.10 T cells were potentially available to participate in the immune response, it was possible that a large number of these cells were not activated. This situation would result in underestimating the contribution of conversion to TR cell expansion. To determine the extent and the rate of cell division for both conventional and converted DO11.10 T cells, the cells were labeled with CFSE before adoptive transfer. At the peak of DO11.10 expansion, lymphocytes from the draining lymph nodes were stained for CD4, the DO11.10 TCR, and Foxp3. Results confirm that 99% of DO11.10 T cells have undergone cell division, and that the number of converted TR cells remains at ~2% (Fig. 8C). A small but measurable population of DO11.10 T cells expresses Foxp3 before cell division. These results demonstrated that only a small percentage of conventional T cells undergoing Ag-driven proliferation converted into TR cells and, consequently, that conversion did not contribute substantially to the expanding TR cell pool.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
By engineering mice with a bicistronic Foxp3 allele that links the expression of Foxp3 with that of a fluorescent reporter protein (EGFP), the expansion and function of Foxp3+ TR cells during the course of a primary immune response could be accurately analyzed in vivo. Previous studies have described an increase in TR cells in vivo in response to antigenic stimulation (26, 27). In this study, we establish that the kinetics of TR cell expansion parallels that of conventional T cells, and that the vast majority of TR cells are found within T cell zones. More notably, our data demonstrate a relative accumulation of TR cells within the draining lymph nodes in excess of that seen for conventional T cells. Consistent with this observation, we also find a significant increase in the density of TR cells within T and B cell areas as the primary immune response progresses. Continuous BrdU labeling identified extensive TR cell proliferation as one mechanism driving the buildup of TR cells. This result suggests that TR cell activation and expansion is integral to the regulation of primary immunity. The biological relevance of this finding was confirmed in adoptive transfer experiments that demonstrated a modest increase in the ratio of TR:conventional T cells before immunization diminished the primary immune response. These results indicate that there is an inverse quantitative relationship between the number of responding TR and conventional cells that regulates the magnitude of the primary immune response to foreign Ags.

Adoptive transfer experiments may not faithfully reproduce the normal kinetics of Ag-specific responses (38, 39). Nevertheless, they illustrate that the dynamic relationship between TR cells and conventional CD4+ T is particularly sensitive to perturbations early in the response. Experiments using intravital microscopy to track transferred TR cells have demonstrated reduced CD4-dendritic cell interaction times and a reduction in the half-life of affected dendritic cells (40). Thus, one way that TR cells may work is by decreasing dendritic cell maturation and function. This mechanism certainly does not preclude TR cell regulation at later time points in the course of the immune response. For example, stimulated TR cells can kill activated targets, including T cells, B cells, and dendritic cells (41, 42, 43). Importantly, the adoptive transfer experiments also show that matched Ag specificity between conventional T cells and TR cells is not a prerequisite for effective regulation of the primary response.

Previous studies tracking TR cells in vivo have frequently identified TR cells using CD25, a strategy that fails to recognize up to 30% of TR cells and does not distinguish between TR cells and activated conventional T cells (33). In contrast, our data demonstrate that >20% of the TR cell population is dividing in the draining lymph nodes at the peak of the primary response, clearly indicating activation of TR cells after immunization. Activation of TR cells requires TCR engagement by Ag/MHC complexes and is essential for TR cell function (5, 6). Once activated, TR suppression of conventional T and B cell responses is thought to be independent of TR cell Ag specificity (41, 42, 43, 44). Our data is consistent with the interpretation that TR cell regulation of primary immunity depends upon activation of a sizable portion of the TR cell pool. Several mechanisms could contribute to this extensive activation, including the propensity of TR cells for self-reactivity, increased TR cell repertoire diversity, and reduced TR cell TCR specificity.

The BrdU data also provides comparative information on TR cells and conventional T cells at steady state in the absence of stimulation by foreign Ag. We found that ~7% of TR cells had divided during the 10 days of continuous BrdU labeling, while only 2% of conventional T cells had incorporated BrdU during the same time period. Cell division of conventional CD4+ T cells at steady state depends upon lower affinity interactions between the TCR and self-peptide MHC complexes than those required for T cell activation (45, 46). In the absence of foreign Ag, broadened TCR specificity and a TR cell bias toward self-recognition may also account for the relatively higher rates of TR cell division. Blocking steady-state TR cell proliferation with an anti-IL-2 Ab results in accelerated and multiorgan autoimmune disease in NOD mice, consistent with the activation of TR cells on self-peptide/MHC complexes (47). Previous BrdU studies show that most conventional CD4+ T cells that divide at steady state are CD44high, and homeostasis in lymphocyte-replete animals has therefore been postulated to largely involve the memory pool (35). A rapidly dividing CD44+ TR cell pool was observed in another study, and our data demonstrated a similar population (36). Importantly, we find that ~25% of the cells that divided in the draining lymph nodes were TR cells. These cells were exclusively CD44high, suggesting an activated/memory TR cell phenotype. No cells in the dividing TR cell pool have a naive phenotype (CD62LhighCD44low), although such cells can be seen in the conventional EGFP–BrdU+ population. The extensive contribution of TR cells to steady-state cell division within the CD4+ T cell compartment implicates continual TR cell activation as an important mechanism in controlling spontaneous autoimmune responses generated by conventional T cells (47).

In the thymus, agonist-mediated selection of TR cells has been observed in a TCR transgenic model (48). Although this point has been debated, most experiments examining TR cell development demonstrate that TR cells are relatively resistant to negative selection (48, 49, 50). In the absence of efficient negative selection, positive selection of TR cells might result in a TCR repertoire that is highly peptide degenerate and biased toward MHC recognition, analogous to the model that has been developed for the selection of conventional T cells (51, 52, 53). Our data demonstrating a significant increase in TR cell division at steady state and after immunization are arguably most consistent with a TR cell pool characterized by degenerate TCR recognition and activation largely on self-Ags. We propose a model whereby TR cells are progressively enlisted to regulate immune responses based on the level of conventional T cell activation and the local amount of IL-2 produced. Constitutive expression of the high-affinity IL-2R (CD25) and the dependence on exogenous IL-2 enforce TR cell participation commensurate with the conventional T cell response. The postulated degenerate nature of TR cell TCR recognition then allows for recruitment of a sufficient number of TR cells to control the magnitude of the response.

In contrast, in situ generation of TR cells did not appear to play a role in the accumulation of TR cells during the course of the primary immune response to CFA immunization. Previous studies have demonstrated that TR cells may arise from activation of conventional T cells, either by stimulation with low doses of Ag in the absence of professional APC stimulation (54), or by TCR cross-linking in the presence of TGF-beta (37). This conversion results in the expression of Foxp3 and the acquisition of a regulatory phenotype (32, 37, 55, 56). Converted TR cells exhibit suppressor function and are capable of down-regulating Ag-specific immune responses in a manner similar to that of thymus-derived or "natural" TR cells. Recent studies have documented the production of converted TR cells in the context of an adjuvant (aluminum hydroxide)-driven immune response (57). We also find that conversion can occur in vivo. However, our adoptive transfer experiments revealed that in situ TR conversion contributed little to the increase in TR cells in draining lymph nodes of CFA-immunized mice, making a measurable regulatory role for these cells less likely. These data do not rule out more extensive conversion at later time points, as has been seen in other systems (58).

Whereas the TR cell expansion in the course of a primary immune response may take advantage of degenerate TCR recognition and a more diverse TCR repertoire, chronic antigenic stimulation, such as that seen with parasitic infections, can be associated with a more focused, Ag-specific TR cell response. This is demonstrated in the recent reports examining mouse models of Leishmania infection where Leishmania Ag-responsive TR cells were prominently represented at the infection site and suppressed the in situ action of effector T cells, allowing for parasite persistence (59). In both acute and chronic responses to foreign Ags, TR cells regulate the development of conventional T cell effector function. However, the differential dependence on foreign Ag specificity displayed by TR cells in the course of chronic vs acute responses may be the consequence of the manipulation of the local environment by pathogens leading to Ag persistence. Such divergence in TR cell response may explain some of the detrimental effects associated with fixing TR cell Ag specificity during chronic antigenic stimulation, which may allow the persistence of pathogens such as parasites in the face of an ongoing immune response (25).

In addition to CD4+EGFP+ TR cells, we also observed a small population of CD8+EGFP+ cells that follow the same kinetics as the CD4+EGFP+ population. Expansion and contraction of this Foxp3+ population during the primary response suggests a regulatory role, although this is not confirmed by our studies. The small number of these cells makes their potential effects more difficult to dissect. We have used the neonatal transfer of purified CD4+ T cells to rescue Foxp3– mice, suggesting that CD8+Foxp3+ cells are not essential for preventing autoimmunity (D. Haribhai, unpublished results). However, CD8+EGFP+ cells arise in the thymus and form a distinct lineage, making it likely that they play some role in regulating certain cell populations or immune responses. Assuming that CD8+Foxp3+ TR cells require activation via their MHC class I-restricted TCR for their function, targets might include cells of the innate immune system, APCs, and CD4+ TR cells.

In summary, this study provides new data about TR cell proliferation, localization, and Ag independence during the primary immune response. This study also confirms related observations in the literature using suboptimal markers. Collectively, the data raise many questions about the specific factors that drive a large fraction of the TR cell pool to divide in naive and immunized animals. Why is the baseline frequency of TR cell division so high? Are there specific APC populations or cytokines that modulate TR cell division? What is the role of TCR signal strength and of TLR signaling? How degenerate is TR cell TCR specificity, and might this impact TR cell function? The Foxp3EGFP mice provide a useful tool designed to answer these and other aspects of the biology of TR cells.


   Acknowledgments
 
We thank James Booth, Jennifer Ziegelbauer, and Brandon Edwards for animal care. We also thank Derrick Siebert for technical assistance, and John Routes, William Grossman, and James Verbsky for critical reading of the 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 2R01AI065617 (to T.A.C.) and R01 AI47154 (to C.B.W.), and by the Nickolett Family Foundation and the D.B. and Marjorie Reinhart Family Foundation (to C.B.W.). Back

2 Address correspondence and reprint requests to Dr. Talal A. Chatila, Division of Immunology, Allergy and Rheumatology, Department of Pediatrics, The David Geffen School of Medicine at the University of California at Los Angeles, MDCC 12-430, 10833 Le Conte Avenue, Los Angeles, CA 90095; E-mail address: tchatila{at}mednet.ucla.edu or Dr. Calvin B. Williams, Division of Rheumatology, Department of Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226; E-mail address: cwilliam{at}mcw.edu Back

3 Abbreviations used in this paper: TR, regulatory T; EGFP, enhanced GFP; IRES, internal ribosomal entry sequence; WT, wild type; R, responder cell; S, suppressor cell. Back

Received for publication October 25, 2006. Accepted for publication December 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562. [Medline]
  2. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  3. Fontenot, J. D., A. Y. Rudensky. 2005. A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6: 331-337. [Medline]
  4. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  5. Thornton, A. M., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188: 287-296. [Abstract/Free Full Text]
  6. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, S. Sakaguchi. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10: 1969-1980. [Abstract/Free Full Text]
  7. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  8. von Boehmer, H.. 2005. Mechanisms of suppression by suppressor T cells. Nat. Immunol. 6: 338-344. [Medline]
  9. Chatila, T. A., F. Blaeser, N. Ho, H. M. Lederman, C. Voulgaropoulos, C. Helms, A. M. Bowcock. 2000. JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome. J. Clin. Invest. 106: R75-R81. [Medline]
  10. Wildin, R. S., F. Ramsdell, J. Peake, F. Faravelli, J. L. Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, L. Perroni, et al 2001. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27: 18-20. [Medline]
  11. Bennett, C. L., J. Christie, F. Ramsdell, M. E. Brunkow, P. J. Ferguson, L. Whitesell, T. E. Kelly, F. T. Saulsbury, P. F. Chance, H. D. Ochs. 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27: 20-21. [Medline]
  12. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, F. Ramsdell. 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27: 68-73. [Medline]
  13. Godfrey, V. L., J. E. Wilkinson, L. B. Russell. 1991. X-linked lymphoreticular disease in the scurfy (sf) mutant mouse. Am. J. Pathol. 138: 1379-1387. [Abstract]
  14. Lyon, M. F., J. Peters, P. H. Glenister, S. Ball, E. Wright. 1990. The scurfy mouse mutant has previously unrecognized hematological abnormalities and resembles Wiskott-Aldrich syndrome. Proc. Natl. Acad. Sci. USA 87: 2433-2437. [Abstract/Free Full Text]
  15. Lin, W., N. Truong, W. J. Grossman, D. Haribhai, C. B. Williams, J. Wang, M. G. Martin, T. A. Chatila. 2005. Allergic dysregulation and hyperimmunoglobulinemia E in Foxp3 mutant mice. J. Allergy Clin. Immunol. 116: 1106-1115. [Medline]
  16. Hsieh, C. S., Y. Liang, A. J. Tyznik, S. G. Self, D. Liggitt, A. Y. Rudensky. 2004. Recognition of the peripheral self by naturally arising CD25+CD4+ T cell receptors. Immunity 21: 267-277. [Medline]
  17. Hsieh, C. S., Y. Zheng, Y. Liang, J. D. Fontenot, A. Y. Rudensky. 2006. An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat. Immunol. 7: 401-410. [Medline]
  18. Pacholczyk, R., H. Ignatowicz, P. Kraj, L. Ignatowicz. 2006. Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells. Immunity 25: 249-259. [Medline]
  19. Suvas, S., U. Kumaraguru, C. D. Pack, S. Lee, B. T. Rouse. 2003. CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses. J. Exp. Med. 198: 889-901. [Abstract/Free Full Text]
  20. Toka, F. N., S. Suvas, B. T. Rouse. 2004. CD4+CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1. J. Virol. 78: 13082-13089. [Abstract/Free Full Text]
  21. Moore, A. C., A. Gallimore, S. J. Draper, K. R. Watkins, S. C. Gilbert, A. V. Hill. 2005. Anti-CD25 antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance. J. Immunol. 175: 7264-7273. [Abstract/Free Full Text]
  22. Oldenhove, G., M. de Heusch, G. Urbain-Vansanten, J. Urbain, C. Maliszewski, O. Leo, M. Moser. 2003. CD4+CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J. Exp. Med. 198: 259-266. [Abstract/Free Full Text]
  23. Belkaid, Y., B. T. Rouse. 2005. Natural regulatory T cells in infectious disease. Nat. Immunol. 6: 353-360. [Medline]
  24. Suvas, S., B. T. Rouse. 2006. Treg control of antimicrobial T cell responses. Curr. Opin. Immunol. 18: 344-348. [Medline]
  25. Belkaid, Y., R. B. Blank, I. Suffia. 2006. Natural regulatory T cells and parasites: a common quest for host homeostasis. Immunol. Rev. 212: 287-300. [Medline]
  26. Klein, L., K. Khazaie, H. von Boehmer. 2003. In vivo dynamics of antigen-specific regulatory T cells not predicted from behavior in vitro. Proc. Natl. Acad. Sci. USA 100: 8886-8891. [Abstract/Free Full Text]
  27. Walker, L. S., A. Chodos, M. Eggena, H. Dooms, A. K. Abbas. 2003. Antigen-dependent proliferation of CD4+CD25+ regulatory T cells in vivo. J. Exp. Med. 198: 249-258. [Abstract/Free Full Text]
  28. Billiard, F., E. Litvinova, D. Saadoun, F. Djelti, D. Klatzmann, J. L. Cohen, G. Marodon, B. L. Salomon. 2006. Regulatory and effector T cell activation levels are prime determinants of in vivo immune regulation. J. Immunol. 177: 2167-2174. [Abstract/Free Full Text]
  29. Lakso, M., J. G. Pichel, J. R. Gorman, B. Sauer, Y. Okamoto, E. Lee, F. W. Alt, H. Westphal. 1996. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93: 5860-5865. [Abstract/Free Full Text]
  30. Williams, C. B., D. L. Engle, G. J. Kersh, J. M. White, P. M. Allen. 1999. A kinetic threshold between negative and positive selection based on the longevity of the T cell receptor-ligand complex. J. Exp. Med. 189: 1531-1544. [Abstract/Free Full Text]
  31. Haribhai, D., B. Edwards, M. L. Williams, C. B. Williams. 2004. Functional reprogramming of the primary immune response by T cell receptor antagonism. J. Exp. Med. 200: 1371-1382. [Abstract/Free Full Text]
  32. Wan, Y. Y., R. A. Flavell. 2005. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc. Natl. Acad. Sci. USA 102: 5126-5131. [Abstract/Free Full Text]
  33. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity 22: 329-341. [Medline]
  34. Kohm, A. P., J. S. McMahon, J. R. Podojil, W. S. Begolka, M. DeGutes, D. J. Kasprowicz, S. F. Ziegler, S. D. Miller. 2006. Cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells. J. Immunol. 176: 3301-3305. [Abstract/Free Full Text]
  35. Tough, D. F., J. Sprent. 1994. Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179: 1127-1135. [Abstract/Free Full Text]
  36. Fisson, S., G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, B. L. Salomon. 2003. Continuous activation of autoreactive CD4+CD25+ regulatory T cells in the steady state. J. Exp. Med. 198: 737-746. [Abstract/Free Full Text]
  37. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, S. M. Wahl. 2003. Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med. 198: 1875-1886. [Abstract/Free Full Text]
  38. Hataye, J., J. J. Moon, A. Khoruts, C. Reilly, M. K. Jenkins. 2006. Naive and memory CD4+ T cell survival controlled by clonal abundance. Science 312: 114-116. [Abstract/Free Full Text]
  39. Marzo, A. L., K. D. Klonowski, A. Le Bon, P. Borrow, D. F. Tough, L. Lefrancois. 2005. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6: 793-799. [Medline]
  40. Tadokoro, C. E., G. Shakhar, S. Shen, Y. Ding, A. C. Lino, A. Maraver, J. J. Lafaille, M. L. Dustin. 2006. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J. Exp. Med. 203: 505-511. [Abstract/Free Full Text]
  41. Gondek, D. C., L. F. Lu, S. A. Quezada, S. Sakaguchi, R. J. Noelle. 2005. Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J. Immunol. 174: 1783-1786. [Abstract/Free Full Text]
  42. Grossman, W. J., J. W. Verbsky, W. Barchet, M. Colonna, J. P. Atkinson, T. J. Ley. 2004. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity 21: 589-601. [Medline]
  43. Zhao, D. M., A. M. Thornton, R. J. DiPaolo, E. M. Shevach. 2006. Activated CD4+CD25+ T cells selectively kill B lymphocytes. Blood 107: 3925-3932. [Abstract/Free Full Text]
  44. Adeegbe, D., A. L. Bayer, R. B. Levy, T. R. Malek. 2006. Cutting edge: allogeneic CD4+CD25+Foxp3+ T regulatory cells suppress autoimmunity while establishing transplantation tolerance. J. Immunol. 176: 7149-7153. [Abstract/Free Full Text]
  45. Kirberg, J., A. Berns, H. von Boehmer. 1997. Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules. J. Exp. Med. 186: 1269-1275. [Abstract/Free Full Text]
  46. Viret, C., F. S. Wong, C. A. Janeway, Jr. 1999. Designing and maintaining the mature TCR repertoire: the continuum of self-peptide:self-MHC complex recognition. Immunity 10: 559-568. [Medline]
  47. Setoguchi, R., S. Hori, T. Takahashi, S. Sakaguchi. 2005. Homeostatic maintenance of natural Foxp3+CD25+CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 201: 723-735. [Abstract/Free Full Text]
  48. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2: 301-306. [Medline]
  49. Lerman, M. A., J. Larkin, III, C. Cozzo, M. S. Jordan, A. J. Caton. 2004. CD4+CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen. J. Immunol. 173: 236-244. [Abstract/Free Full Text]
  50. van Santen, H. M., C. Benoist, D. Mathis. 2004. Number of T reg cells that differentiate does not increase upon encounter of agonist ligand on thymic epithelial cells. J. Exp. Med. 200: 1221-1230. [Abstract/Free Full Text]
  51. Huseby, E. S., F. Crawford, J. White, J. Kappler, P. Marrack. 2003. Negative selection imparts peptide specificity to the mature T cell repertoire. Proc. Natl. Acad. Sci. USA 100: 11565-11570. [Abstract/Free Full Text]
  52. Huseby, E. S., J. White, F. Crawford, T. Vass, D. Becker, C. Pinilla, P. Marrack, J. W. Kappler. 2005. How the T cell repertoire becomes peptide and MHC specific. Cell 122: 247-260. [Medline]
  53. Ignatowicz, L., J. Kappler, P. Marrack. 1996. The repertoire of T cells shaped by a single MHC/peptide ligand. Cell 84: 521-529. [Medline]
  54. Apostolou, I., H. von Boehmer. 2004. In vivo instruction of suppressor commitment in naive T cells. J. Exp. Med. 199: 1401-1408. [Abstract/Free Full Text]
  55. Zheng, S. G., J. H. Wang, J. D. Gray, H. Soucier, D. A. Horwitz. 2004. Natural and induced CD4+CD25+ cells educate CD4+CD25– cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J. Immunol. 172: 5213-5221. [Abstract/Free Full Text]
  56. Fantini, M. C., C. Becker, G. Monteleone, F. Pallone, P. R. Galle, M. F. Neurath. 2004. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25– T cells through Foxp3 induction and down-regulation of Smad7. J. Immunol. 172: 5149-5153. [Abstract/Free Full Text]
  57. Mucida, D., N. Kutchukhidze, A. Erazo, M. Russo, J. J. Lafaille, M. A. Curotto de Lafaille. 2005. Oral tolerance in the absence of naturally occurring Tregs. J. Clin. Invest. 115: 1923-1933. [Medline]
  58. Knoechel, B., J. Lohr, E. Kahn, J. A. Bluestone, A. K. Abbas. 2005. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J. Exp. Med. 202: 1375-1386. [Abstract/Free Full Text]
  59. Suffia, I. J., S. K. Reckling, C. A. Piccirillo, R. S. Goldszmid, Y. Belkaid. 2006. Infected site-restricted Foxp3+ natural regulatory T cells are specific for microbial antigens. J. Exp. Med. 203: 777-788. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
R. W. van Olffen, N. Koning, K. P. J. M. van Gisbergen, F. M. Wensveen, R. M. Hoek, L. Boon, J. Hamann, R. A. W. van Lier, and M. A. Nolte
GITR Triggering Induces Expansion of Both Effector and Regulatory CD4+ T Cells In Vivo
J. Immunol., June 15, 2009; 182(12): 7490 - 7500.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Ghoreishi, P. Bach, J. Obst, M. Komba, J. C. Fleet, and J. P. Dutz
Expansion of Antigen-Specific Regulatory T Cells with the Topical Vitamin D Analog Calcipotriol
J. Immunol., May 15, 2009; 182(10): 6071 - 6078.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Haribhai, W. Lin, B. Edwards, J. Ziegelbauer, N. H. Salzman, M. R. Carlson, S.-H. Li, P. M. Simpson, T. A. Chatila, and C. B. Williams
A Central Role for Induced Regulatory T Cells in Tolerance Induction in Experimental Colitis
J. Immunol., March 15, 2009; 182(6): 3461 - 3468.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Bayry, E. Z. Tchilian, M. N. Davies, E. K. Forbes, S. J. Draper, S. V. Kaveri, A. V. S. Hill, M. D. Kazatchkine, P. C. L. Beverley, D. R. Flower, et al.
In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination
PNAS, July 22, 2008; 105(29): 10221 - 10226.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. W. T. Lau, S. Biester, R. J. Cornall, and J. V. Forrester
Lipopolysaccharide-Activated IL-10-Secreting Dendritic Cells Suppress Experimental Autoimmune Uveoretinitis by MHCII-Dependent Activation of CD62L-Expressing Regulatory T Cells
J. Immunol., March 15, 2008; 180(6): 3889 - 3899.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
E. N. M. Nolte-'t Hoen, E. P. J. Boot, J. P. A. Wagenaar-Hilbers, J. H. M. van Bilsen, G. J. A. Arkesteijn, G. Storm, L. A. Everse, W. van Eden, and M. H. M. Wauben
Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134
J. Leukoc. Biol., January 1, 2008; 83(1): 112 - 121.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haribhai, D.
Right arrow Articles by Chatila, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haribhai, D.
Right arrow Articles by Chatila, T. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS