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 Foucras, G.
Right arrow Articles by Guéry, J.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Foucras, G.
Right arrow Articles by Guéry, J.-C.
The Journal of Immunology, 2002, 168: 179-187.
Copyright © 2002 by The American Association of Immunologists

Chronic Soluble Antigen Sensitization Primes a Unique Memory/Effector T Cell Repertoire Associated with Th2 Phenotype Acquisition In Vivo1

Gilles Foucras2,*, Alexandra Gallard*, Christiane Coureau*, Jean-M. Kanellopoulos{dagger} and Jean-Charles Guéry3,*

* Institut National de la Santé et de la Recherche Médicale, Unité 28, Institut Fédératif de Recherche 30, Hôpital Purpan, Toulouse, France; and {dagger} Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale, Unité 277, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR V{beta} repertoire, different from the TCR V{beta} profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IAd-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 T cell subsets play a central role in the regulation of immune responses. After Ag encounter on professional APC, naive CD4 T cells differentiate into mature effector T cells that can be subdivided into two functionally distinct populations based on their cytokine secretion profiles (1). Th1 secrete IFN-{gamma}, TNF-{beta}, and IL-2; mediate inflammatory responses; activate macrophages; and stimulate cell-mediated immunity. Th2 cells, which produce IL-4, IL-5, IL-10, and IL-13, induce IgE production by B cells and eosinophil differentiation and activation. Although Th cells are important in the eradication of extracellular pathogens, they are also prominent mediators of allergic immune responses (2, 3). In vitro experiments have clearly established that the Th1 or Th2 differentiation program can be induced in the same T cell precursors depending on the presence of IL-12 or IL-4, respectively (4). Furthermore, much progress has been made in characterizing the cytokine-dependent signaling pathways and the transcription factors that control Th1 and Th2 lineage commitment (5). However, the mechanisms that regulate the activation, expansion, and differentiation of CD4 T lymphocytes into the type 2 effector subset in vivo are still poorly understood. Besides the cytokines present in the microenvironment at the time of naive CD4 T cell priming (4), it is now clear that the polarization of Th cells can be influenced by other factors, including the dose and the route of Ag administration (6, 7), the strength of TCR-ligand interactions (6, 8, 9), the genetic background (10, 11), and the hormonal status of the host (12).

Th2 responses that predominate in overwhelming infections and allergic disorders are often associated with chronicity of antigenic stimulation. Indeed, we have previously shown that continuous release of low amounts of protein Ags, including soluble extracts from pathogenic micro-organisms, induces the selective development of Th2 cells in mice susceptible to Leishmania major infection (7, 11). To understand the mechanisms that govern Th2 differentiation in this model, we analyzed the precursor origin of Ag-specific IL-4-producing CD4 T cells. Recently, we have shown that the public T cell clonotypes that expand after hen egg lysozyme (HEL)4 priming in adjuvant were less frequently expressed in soluble HEL-induced Th2 cells, suggesting that this mode of Ag administration might have selected a different pool of precursor cells (13). In this paper we have characterized the TCR repertoire of memory/effector Th2 cells induced by chronic administration of HEL. We show that soluble HEL-induced Th2 cells contain a highly frequent public clonotype that is much less frequent in primary effector T cells from adjuvant-primed mice. Furthermore, CD4 T cells expressing this Th2-associated repertoire recognize an IAd-restricted HEL determinant contained in sequence 13–105 of the protein, selectively presented by dendritic cells (DC). These data provide direct evidence that chronic soluble Ag sensitization primes a distinct memory/effector repertoire associated with Th2 phenotype acquisition in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunizations

Female BALB/c (H-2d) mice, 8 wk of age, were purchased from Centre d’Elevage R. Janvier (Le Genest St. Isle, France). B10.D2 mice were originally purchased from Harlan (Oxon, U.K.), and were bred and maintained in our animal facility. HEL was obtained from Sigma (St. Louis, MO). HEL peptides 7–31, 12–27, 23–32, 46–61, and 71–85 (purity, >85%) were purchased from Neosystem (Strasbourg, France). A set of 15-mer HEL peptides overlapping by three residues was synthesized using the pin synthesis system (Chiron Mimotopes, San Diego, CA) as described previously (14). The HEL13–105 fragment was prepared by cyanogen bromide cleavage of HEL protein as previously described (15). Soluble HEL was delivered by mini-osmotic pumps (Alzet 2001; Alza, Palo Alto, CA) implanted under the skin. After 12 days, mice were immunized s.c. into the hind footpads with HEL (3.5 nmol) emulsified in IFA or CFA containing H37Ra mycobacteria (Difco, Detroit, MI). Draining popliteal lymph nodes were removed 9 days after immunization.

Quantitation of serum IgE

IgE concentration was determined in serum by ELISA, using IgE-specific LO-ME-3 rat mAb (LO/IMEX, University of Louvain, Brussels, Belgium) and biotinylated R35–92 IgE-specific rat mAb (BD PharMingen, San Diego, CA) as second-step reagent. Bound biotin-labeled mAb were revealed using alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Avondale, PA). The IgE concentration was quantified from three titration points using a purified mouse IgE mAb, IgE-3 (BD PharMingen), to generate standard curves. For determination of Ag-specific IgE, the procedure was essentially the same, except that biotin-OVA was used as a second step reagent. Biotinylation of OVA was performed using a standard procedure.

Sample preparation

Popliteal lymph node cells (LNC) were depleted of CD8-positive cells by sequential incubation with KT1.5 mAb (16) culture SN and M-450 anti-rat IgG Dynabeads (Dynal, Oslo, Norway), followed by magnetic cell separation. CD4 T cells were isolated in the same way using a mixture of the following mAb: anti-CD8 KT1.5, anti-B220 RA3, and anti-MHC II M5/114. The purity of CD4 T cells controlled by flow cytometric analysis was >95%. In some experiments CD4 T cells were separated into V{beta}14+ and V{beta}14- cells. Briefly, CD4 T cells were sequentially labeled with anti-V{beta}14-biotin 14.2 mAb (BD PharMingen) and streptavidin-coated M-280 Dynabeads (Dynal). V{beta}14+ and V{beta}14- CD4 T cells were then selected by magnetic cell separation following the manufacturer’s recommendations.

T cell assays and cytokine production analysis

CD8-depleted LNC were cultured (5 x 105/well) in 96-well culture plates (Costar, Cambridge, MA) in synthetic HL-1 medium (HYCOR, Irvine, CA) supplemented with 2 mM L-glutamine (Life Technologies, Cergy Pontoise, France) and 50 µg/ml gentamicin (Sigma) with the indicated Ag concentrations. Cultures were incubated for 3 days in a humidified atmosphere of 6% CO2 in air, and then expanded for 3 additional days in complete medium. Complete medium was RPMI 1640 medium supplemented with 10% FCS (ATGC Biotechnologie, Noisy Le Grand, France), 1% pyruvate, 1% nonessential amino acids, 1% L-glutamine (Life Technologies), 50 µM 2-ME, and 50 µg/ml gentamicin (Sigma). Supernatants from at least triplicate cultures were collected after 72 h of culture and pooled for cytokine analysis. IFN-{gamma} and IL-4 concentrations were determined by sandwich ELISA as previously described (17). Intracytoplasmic staining for IFN-{gamma} and IL-4 was performed on purified CD4 T cells as previously described (13, 18). Data were collected on 20,000 events on an XL Coulter cytometer (Coultronics, Margency, France), and results were analyzed using CellQuest software (BD Biosciences, Mountain View, CA).

TCR V{beta} analysis by flow cytometry

Purified CD4 T cells from 6-day cultures were incubated with appropriate amounts of biotin- or FITC-labeled anti-V{beta} Abs and PE-labeled anti-CD4 L3T4 mAb (BD PharMingen), followed by streptavidin-FITC (BD Phar- Mingen) when necessary. After incubation with propidium iodide to exclude dead cells, data were collected on 10,000 events gated on CD4 living T cells.

T cell hybridomas

The BV8S2-J1S5 2E8 T cell hybridoma specific for HEL107–116/IEd antigenic complexes was obtained as previously described (13). V{beta}14 T cell hybridomas were generated from HEL-restimulated pooled LNC from soluble-Ag treated mice (n = 4). After 4 days of culture, living cells, isolated on a Ficoll gradient (Lympholyte-M; Cedarlane), were sequentially labeled with FITC-14.2 anti-V{beta}14 mAb (BD PharMingen) and anti-FITC-coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). V{beta}14+ cells were then positively selected using MiniMACS columns and fused with thymoma BW5147 {alpha}-{beta}- as described previously (19). Hybrids were screened for their reactivity against HEL using bone marrow-derived DC (BM-DC) as APC. T cell hybridomas were cloned by limiting dilution. BM-DC were prepared from BALB/c bone marrow cells essentially as described previously (20).

TCR-{beta} repertoire analysis with Immunoscope technique

Total RNA was extracted using the TRIzol procedure (Life Technologies, Cergy Pontoise, France). cDNA was synthesized using oligo(dT)17 or random primers (Life Technologies) and Moloney murine leukemia virus reverse transcriptase (Roche, Mannheim, Germany). The CDR3 size distribution of BV14-BC-rearranged TCR was analyzed with Immunoscope as previously described (21). Semiquantitative analysis of BV segment usage was essentially performed as described previously (22). Run-off reactions were performed using the 6-carboxyfluorescein (FAM)-labeled BV14-, J2S7-, or the CDR3-specific primers. The sequence of the BV14-J2S7 CDR3 primer was 5'-TACTGTTCTCCTAGACTCCAGG-3'.

Quantitative transcript analysis

Public BV8S2-J1S5 and BV14-J2S7 rearrangement quantification was performed by real-time quantitative RT-PCR using an ABI PRISM 5700 sequence BioDetector (PE Biosystems, Foster City, CA), according to the manufacturer’s instructions. Total RNA was extracted and reverse transcribed as indicated above. Primers and probes included: BV8S2 sense, 5'-ATCCATTATTCATATGGTGCTGGC-3'; J1S5 antisense, 5'-AGTCCCCTCTCCAAAAAGCG-3'; BV8S2-J1S5 CDR3-specific probe FAM, 5'-GCGGTACAGGGAACAACCAGGCT-3'-6-carboxytetramethylrhodamine (Tamra); BV14 sense, 5'-GGACGACCAATTCATCCTAAGC-3'; BJ2S7 antisense, 5'-CTAAAACCGTGAGCCTGGTGC-3'; BV14-J2S7 CDR3-specific probe, FAM-5'-CGAAGTACTGTTCTCCAAGACTCCAGGCAC-3'-Tamra; TCR {beta}-chain sense, 5'-CACAATCCTCGCAACCACTTC-3'; TCR {beta}-chain antisense, 5'-GTGAGCCCTCTGGCCACTT-3'; and TCR {beta}-chain probe, FAM-5'-TCCTCCTGTGAAAGCCCATGGAACTG-3'-Tamra. The 2E8 and IIIE4/1A10 TCR {beta}-chains were cloned into pCR2.1 plasmid using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands) and were used as an external standard. The number of copies was estimated by OD determination. Cycling conditions were 2 min at 50°C, 10 min at 95°C, followed by 40 repeats of 95°C for 15 s and 60°C for 60 s. Data were analyzed with the sequence detection software supplied with the instrument and were expressed as the ratio of public CDR3 over TCR BC mRNA copies.

Predeveloped primer probe sets (Applied Biosystems) were used for the quantitation of IL-4 and 18S RNA. Real-time PCR analysis was conducted essentially as described in the manufacturer’s instructions.

Sequence of TCR {alpha}- and {beta}-chain determination

TCR chain sequences were determined on PCR products purified with exonuclease I (10 U) and shrimp alkaline phosphatase (0.5 U) in a final volume of 10 µl. Sequences were performed in a protocol adapted from Applied Biosystems using the Dye Terminator DNA sequencing kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TCR V{beta} repertoire analysis of Th2 cells induced by chronic Ag administration

BALB/c mice were implanted s.c. with mini-osmotic pumps delivering continuously over a 10-day period approximately 750 ng/h HEL. Mice were then immunized with HEL in adjuvant, and the polarization of the CD4 T cell response was tested in immune LNC upon in vitro culture with the Ag. In agreement with previous experiments (7, 11, 13), such immunization protocol selectively induced a strong expansion of Ag-specific IL-4-producing CD4 T cells independently of the type of adjuvant used for antigenic challenge (Fig. 1GoA). These Th2 cells were functional in vivo, since a dramatic and long-lasting up-regulation of the serum IgE level was observed in mice pretreated with soluble HEL (Fig. 1GoB). Similar results were obtained with OVA as model Ag for which we could design an assay to measure the OVA-specific IgE response. Serum IgE levels were high in soluble OVA pretreated mice for >5 wk and were associated with the production of OVA-specific IgE (data not shown).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. Continuous administration of low dose soluble Ag induces strong memory recall type 2 T and B cell response in BALB/c mice. A, Mice (n = 3) untreated or treated with 10 nmol HEL in a pump were immunized with 3.5 nmol HEL in CFA or IFA. CD8-depleted LNC from each group were stimulated with HEL (10 µM) in vitro. After 6 days of culture, negatively enriched CD4 T cells were stimulated with PMA/ionomycin and stained intracellularly for IFN-{gamma} and IL-4. Results are from one representative experiment of three performed. B, Mice, sensitized or not with soluble HEL as described above, were immunized i.p. with 50 µg/mouse of HEL in CFA. The total serum IgE level was quantified in individual mice by sandwich ELISA. Results from individual mice are shown and are from one representative experiment of two performed

 
As a first step in analyzing the TCR repertoire in soluble HEL-induced Th2 cells, CD4 T cells obtained as described in Fig. 1GoA were stained with a panel of TCR V{beta}-specific mAbs. As shown in Fig. 2GoA, the TCR V{beta} profiles were similar in CD4 T cells from mice immunized with HEL in CFA or IFA. They were dominated by V{beta}2-, V{beta}4-, V{beta}6-, and V{beta}8.1.2-positive cells. Interestingly, when the same analysis was performed on Ag-stimulated CD4 T cells from mice pretreated with soluble HEL a different pattern of V{beta}-chain usage was observed (Fig. 2GoA). This Th2-associated repertoire was characterized by a reduced frequency of all TCR V{beta}-chains that dominate the T cell response from adjuvant-primed mice, and a dramatic up-regulation of TCR V{beta}14+ CD4 T cells. This TCR V{beta} repertoire skewing in soluble HEL-induced Th2 cells was observed regardless of the type of adjuvant used to immunize the mice (Fig. 2GoA). Measuring the expression of the remaining BV genes among both populations was performed by a PCR-based semiquantitative analysis (22). As shown in Fig. 2GoB, the BV14 mRNA level was selectively increased in Th2 cells from soluble HEL-treated mice, while BV8S2 mRNA was reduced. No other major skewing in the TCR repertoire could be observed for the other BV gene segments analyzed.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. TCR repertoire analysis of soluble Ag-induced Th2 cells. A, Ag-specific CD4 T cells, obtained as described in Fig. 1Go, were stained with PE-labeled anti-CD4 GK1.5 and FITC- or biotin-labeled anti-V{beta} mAbs and avidin-FITC. Identical results were obtained in three independent experiments. B, The relative abundance of seven TCR BV was determined by semiquantitative PCR and expressed as a ratio for Th2 (HEL-pump, HEL-CFA)/Th1 (HEL-CFA) CD4 T cells. C, The frequency of V{beta}8.2 and V{beta}14 T cells was determined in HEL-restimulated CD4 T cells from mice pretreated with the indicated amounts of soluble HEL in a pump. D, HEL-reactive CD4 T cells (2 x 105 cells/well) from control or HEL pump-treated mice were restimulated with plate-bound anti-V{beta}14 14.2 mAb (BD PharMingen) in the presence of 2 µg/ml anti-CD28 mAb (BD PharMingen). IL-4 secretion was assessed in 48-h culture supernatants by ELISA.

 
We have previously shown that soluble HEL administration induced anergy in CD4 T cells specific for dominant and subdominant epitopes of the HEL protein (13). Since these T cells expressed a V{beta}8.2 TCR, we next analyzed the frequency of V{beta}8.2 and V{beta}14 T cells in HEL-restimulated CD4 T cells from mice treated with various doses of HEL in pump. The data in Fig. 2GoC show that continuous administration of low dose soluble HEL (5 µg/mouse) over a 10-day period resulted in a 2-fold reduction in V{beta}8.2 frequency (Fig. 2GoC). Increasing the dose of HEL further reduced the size of the V{beta}8.2 T cells from 25 to 8%. Conversely, a dose-dependent up-regulation of V{beta}14 T cells was seen in HEL-reactive CD4 T cells from soluble HEL-treated mice (Fig. 2GoC). Thus, there was a reciprocal regulation of V{beta}8.2 and V{beta}14 T cell expansion depending on the dose of soluble Ag administered in the pump. V{beta}14 T cells were functional memory/effector Th2 cells, since they produced very high amounts of IL-4 (Fig. 2GoD), but no IFN-{gamma} (not shown), following in vitro stimulation with anti-V{beta}14 mAb. By contrast, T cells from mice primed with HEL in adjuvant did not produce either IL-4 (Fig. 2GoD) or IFN-{gamma} (not shown) when stimulated under the same conditions. Indeed, the data illustrated in Fig. 3GoA show that IL-4 transcripts were more abundant in ex vivo V{beta}14+ T cells from mice treated with soluble HEL. By contrast, isolated IL-4 expression was low in CD4 T cells from HEL-CFA-primed mice and was similar between V{beta}14+ and V{beta}14- T cells. After in vitro expansion with HEL (as in Fig. 1Go), we analyzed the TCR V{beta}-chain usage for the most frequent V{beta} (V{beta}6, V{beta}8.2, and V{beta}14) within T cells producing IL-4 determined by intracellular staining (Fig. 3GoB). The data illustrated in Fig. 3GoC show that V{beta}14+ CD4 T cells represent >30% of the IL-4-producing T cells. Taken together, these data demonstrate that V{beta}14 T cells account for a large part of IL-4 expression in CD4 T cells from soluble HEL-pretreated mice.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. Analysis of IL-4 transcripts in V{beta}14 T cells. BALB/c mice, sensitized or not with soluble HEL in a pump (10 nmol/mouse), were immunized with 3.5 nmol HEL in CFA. A, CD4 T cells were separated into V{beta}14+ and V{beta}14- cells and assayed for IL-4 and 18S RNA by real-time fluorogenic PCR. Transcript abundance for IL-4 is normalized to 18S RNA. B and C, CD8-depleted LNC from soluble HEL-treated mice were stimulated with HEL (10 µM) in vitro. After 6 days of culture, negatively enriched CD4 T cells were stimulated with PMA/ionomycin, surface labeled with anti-CD4 (FL-1, FITC), anti-V{beta} mAb (FL-4, CyChrome) and stained intracellularly for IL-4 (FL-2, PE). B, CD4/IL-4 staining. C, Cell surface expression of the indicated TCR V{beta}-chains was analyzed on IL-4+ CD4 T cells.

 
We next focused our attention on the V{beta}14 repertoire by determining CDR3 size variation using the Immunoscope procedure (21). CDR3 size distribution analysis of BV14-BC PCR products revealed a dominant peak corresponding to a size of 5 aa (Fig. 4GoA). Run-off reactions performed with nested BJ-specific primers identified a recurrent peak of the corresponding size in T cells bearing BV14-J2S7-rearranged gene segments. We estimated that 70% of the V{beta}14 TCR with a 5-aa CDR3 loop were contained in the BV14-J2S7 T cells that represented approximately 20% of all V{beta}14 T cells (Fig. 4GoB). Direct sequencing of BV14-J2S7 PCR products from HEL-treated mice gave a readable SLGEQ motif, indicating high homogeneity of sequence in the 5-aa band (Fig. 4GoA). Indeed, cloning before sequencing indicated that the SLGEQ CDR3 motif was largely predominant and represented approximately 75% of the BV14-J2S7 rearrangements (Fig. 4GoA and data not shown). Together, these data strongly suggest that the frequency of BV14-J2S7 clonotypic T cells was high in the HEL-specific CD4 T cells from soluble Ag-treated mice.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. Immunoscope analysis of rearranged BV14 TCR in soluble HEL-induced Th2 cells. A, CDR3 size analysis of Th2 cells from soluble HEL-treated mice (n = 3) was performed on BV14-BC PCR products using the Immunoscope procedure. BV14-J2S7 PCR products were directly sequenced or sequenced after bacterial cloning. B, Semiquantitative analysis of BJ segment gene usage within BV14-rearranged TCR was obtained using data from Immunoscope calculation.

 
BV14-J2S7 CD4 T cells recognize an IAd-restricted HEL epitope selectively presented by DC

To determine the fine specificity of T cells expressing the public BV14-J2S7 rearrangement, we generated lysozyme-specific T cell hybridomas from soluble HEL-induced V{beta}14+ CD4 T cells. We next tested the ability of the LPS-induced B cell blasts or BM-DC to present the HEL epitope to six V{beta}14 T cell hybridomas. As a comparison, V{beta}8.2 2E8 T hybridoma cells against the dominant HEL107–116/IEd antigenic complex were also included in the assay. The 2E8 T cells bear the BV8S2-J1S5 public rearrangement found in HEL-reactive CD4 T cells from HEL-adjuvant-immunized BALB/c mice (23). The HEL107–116 epitope can be presented by both APC populations, with BM-DC being a more potent stimulator than LPS-induced B cell blasts (Fig. 5Go, A and C) or A20 B lymphoma cells (data not shown). Similar results were obtained with V{beta}8.2 T cells specific for the subdominant HEL12–25/IAd determinant (not shown). In marked contrast, V{beta}14 T cell hybridomas could be activated by HEL presented by BM-DC (Fig. 5GoB), but not by B cells (Fig. 5GoD). Although both T cell hybridomas were better activated with the highest concentration of BM-DC (Fig. 5Go, A and B), V{beta}14 T cells were more sensitive to the density of APC compared with 2E8 T cells. Likewise, optimal activation of IIIH12 T cells required 100 times more Ag to achieve 50% maximal T cell activation compared with V{beta}8.2 2E8 T cells. Similar results were obtained with the six V{beta}14 T hybridomas listed in Fig. 6Go. They were all IAd-restricted as shown by blocking experiments with class II-specific mAb (Fig. 5GoE). Using HEL fragments prepared by cyanogen bromide cleavage to map recognition sites (15), we found that the determinant recognized by V{beta}14 T cells was contained in the sequence 13–105 of the protein (Fig. 5GoF). However, using a set of 15-mer synthetic peptides, overlapping by three residues, encompassing this region (not shown) as well as various known epitopes of HEL in the H-2d haplotype (24, 25), we were unable to identify a shorter determinant (Fig. 5GoF).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 5. APC requirement, restriction, and specificity of BV14-J2S7 CD4 T cells. A–D, We compared the capacity of BM-DC and LPS-induced B cell blasts to activate the 2E8 T cell hybridoma specific for the IEd-restricted, dominant HEL107–116 epitope and the V{beta}14 T cell hybridoma IIIH12/2G5. T cell hybridomas (0.5 x 105/well) were cultured with the titrated amounts of BM-DC (A and B) or LPS-induced B cell blasts (C and D) obtained from BALB/c mice in the presence of various concentrations of HEL. Data are expressed as the IL-2 concentration determined by ELISA in 24-h culture supernatants. E, For MHC class II restriction analysis, the indicated V{beta}14 Th2 hybridomas were cultured with 3 x 104 BM-DC and 3 µM HEL in the presence of anti-IAd MKD6 or anti-IEd 14.4.4S mAb (10 µg/ml). IL-2 was measured as indicated above, and data are expressed as a percentage of the response obtained in the absence of mAb. F, A representative V{beta}14 T cell hybridoma (IIH12/2G5) tested for its reactivity to HEL, HEL13–105 fragment prepared by cyanogen bromide cleavage (CnBr HEL13–105), and the indicated HEL peptides. BALB/c BM-DC were used as APC. Data are expressed as IL-2 production in 24-h culture supernatants.

 


View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 6. TCR chain sequence determination reveals a highly conserved AV3-J17 rearrangement. After RNA extraction on HEL-reactive T cell hybridoma and cDNA synthesis, PCR were performed with BV14-BC and a set of AV-AC-specific couple of primers and analyzed on 2.5% agarose gel. PCR products were eventually sequenced using an AC or BC internal primer and the Dye Terminator sequencing kit.

 
CD4 T cells bearing a clonotypic {alpha} and {beta} TCR preferentially expand after chronic Ag stimulation

We then sequenced TCR {alpha} and {beta} transcripts from the six V{beta}14 T cell hybridomas obtained. All of them expressed a 5-aa-long CDR3 loop, and five used the BJ2S7 gene segment. Four of the six T cell clones tested exhibited the SLGEQ CDR3 motif identified in the whole HEL-reactive Th2 cell population (Fig. 6Go). By sequencing the rearranged TCR AV genes we found that V{beta}14 T cell hybridomas were highly restricted to the AV3-J17 gene segment usage. Furthermore, the CDR3 region (10 aa long) was perfectly conserved at the amino acid level (Fig. 6Go) and characterized by the sequence SPASSGSWQL. Together, these results demonstrate that V{beta}14 Th2 cells contained a public clonotype characterized by a preferred CDR3 motif at both chains of the TCR. Although amino acid residues in {alpha} and {beta} CDR3 loops were highly conserved, variations in codon usage were found in N diversity regions, indicating different clonal origins of these T cells (Fig. 6Go). Thus, a strong Ag-driven selection must have occurred during the immune response to soluble HEL in vivo.

We next determined whether this Th2-associated clonotype could be detected in HEL-reactive CD4 T cells from mice primed with protein Ag in adjuvant. Analysis of the CDR3 size distributions of TCR bearing a rearranged BV14 gene segment showed that there was a modest expansion of the 5-aa-long BV14-J2S7 rearrangement in HEL-reactive CD4 T cells from mice primed with Ag in adjuvant compared with soluble Ag-sensitized mice (Fig. 7Go, A and B). As shown in Fig. 7GoB, although HEL-IFA immunization induces some degree of Th2 responsiveness (Fig. 1Go), this was not associated with an increased expansion of the BV14-J2S7 clonotype compared with HEL-CFA-primed mice. Analysis of the AV3-C and AV3-J17 run-off products in the same T cell populations revealed that the peak corresponding to a CDR3 of 10 aa was strongly increased in soluble HEL-treated mice (Fig. 7GoC). We directly sequenced the AV3-J17 PCR products, and we found the expected SPASSGSWQL CDR3 motif (Fig. 7GoD). In marked contrast, the 10-aa band was absent in the AV3-J17 profile from HEL-reactive CD4 T cells from adjuvant-primed mice (Fig. 7GoC).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. Immunoscope analysis of the TCR {alpha}- and {beta}-chains. CD8-depleted popliteal LNCs from soluble HEL-treated or control mice, as described in Fig. 1Go, were stimulated in vitro with HEL (10 µM). CDR3 size distribution of rearranged BV14 (A and B) and AV3 TCR (C) chains was performed by Immunoscope on cDNA prepared from CD4 T cells. Fluorescent primers used for run-off reactions are indicated. B, The relative frequency of 5 aa peaks within BV14-J2S7 rearrangements is presented after normalization to BC fluorescence. D, Single-peak AV3-J17 rearrangement was directly sequenced with the AV3-specific primer.

 
High frequency of BV14-J2S7 public TCR correlates with soluble Ag priming

Quantitative analysis of the clonal size of the BV14-J2S7 public rearrangement shows that this repertoire was frequently represented among HEL-reactive CD4 T cells that expand in vitro from soluble HEL-treated mice (Fig. 8Go, A and B). Conversely, the BV8S2-J1S5 public rearrangement (Fig. 8GoA) that normally proliferated in both HEL-IFA and HEL-CFA-immunized mice was tolerized in mice that received systemic soluble Ag, in agreement with our previous observation (13). Thus, the BV14-J2S7 CDR3 motif was strongly up-regulated in Ag-specific CD4 T cells from soluble Ag-treated mice and represented around 1–2% of CD4 T cells, a result compatible with the estimate frequency of this rearrangement calculated from the Immunoscope analysis (Fig. 4Go and data not shown). This increased frequency of BV14-J2S7 T cells was dependent on the dose of soluble Ag administered in vivo and was correlated with the expansion of IL-4-producing T cells (Fig. 8GoB). Since in vitro stimulation might have introduced a bias in terms of clonotype expansion that may not reflect the development of memory T cells in vivo, we analyzed the BV14-J2S7 TCR {beta}-chain frequency in CD4 T cells from immune lymph nodes ex vivo. Data in Fig. 8GoC show that its frequency was increased by 5-fold in T cells from mice that received HEL in pump compared with untreated BALB/c mice. These CD4 T cells subsequently expand following in vitro stimulation with HEL (Fig. 8GoC) and develop along the Th2 pathway.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 8. High frequency of BV14-J2S7 public rearrangement in soluble Ag-induced Th2 cells. BALB/c mice, sensitized or not with soluble HEL in a pump (10 nmol/mouse), were immunized with 3.5 nmol HEL in CFA or IFA as indicated. A, CD8-depleted draining LNC (5 x 105 cells/well) from six mice per group were stimulated in vitro with HEL (10 µM) in HL-1 synthetic medium. IFN-{gamma} and IL-4 production was determined in 72-h culture supernatants. cDNA were prepared from purified CD4 T cells obtained as indicated in Fig. 1Go. Quantitative measurement of TCR {beta}-chain bearing the BV8S2-J1S5 or BV14-J2S7 public rearrangements was performed by real time quantitative PCR and normalized after BC mRNA level determination. Data are expressed as the mean ± SD of eight mice per group. B, CD8-depleted LNC from mice (n = 3) untreated or treated with the indicated amounts of HEL in a pump were restimulated for 6 days as described in Fig. 1Go. Purified CD4 T cells were stimulated with PMA/ionomycin and stained intracellularly for IL-4. Clonotypic BV14-J2S7 CDR3 mRNA copies were quantified by real-time quantitative PCR and normalized on BC mRNA copies. C, The BV14-J2S7 clonotype expansion was quantified in sequential sampling during in vitro restimulation of CD8-depleted LNCs from control or soluble HEL-pretreated mice.

 
Non-MHC-linked genetic polymorphism determines BV14-J2S7 Th2 clonotype expansion

We have previously shown that continuous protein Ag sensitization induces strong Ag-specific Th2 cell development in mice with the BALB background (11, 17). Whereas Th1 cell unresponsiveness was observed in all strains tested after soluble Ag delivery, Th2 development was shown to depend on a non-MHC-linked genetic polymorphism and was predictive of disease outcome following L. major infection (11). We have compared the effect of chronic soluble Ag sensitization on Th2 repertoire development between BALB/c and B10.D2 mice. In agreement with our previous experiments (11, 17), we show that chronic soluble HEL administration does not induce Th2 cell development in B10.D2 mice, unlike in BALB/c mice (see Fig. 9Go, A and B). Furthermore, this lack of Th2 priming in B10.D2 mice is associated with a lack of expansion of T cells expressing the public BV14-J2S7 rearrangement (Fig. 9Go, C and D). On the contrary, continuous administration of soluble HEL resulted in a strong reduction in the clonal size of HEL-specific T cells expressing the public BV8S2-J1S5 rearrangement (Fig. 9Go, C and D), which could be correlated with the down-regulation of IFN-{gamma} production in both BALB/c and B10.D2 mice (Fig. 9Go, A and B). Thus, priming of the Th2-associated BV14-J2S7 clonotype by chronic Ag sensitization is controlled by a non-MHC-linked genetic polymorphism. By contrast, the induction of T cell unresponsiveness in HEL107–116/IEd-specific BV8S2-J1S5 T cells does not appear to be genetically controlled.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 9. Effect of genetic background on BV14-J2S7 Th2 cell development induced by soluble HEL administration. BALB/c (A) or B10.D2 (B) mice, sensitized or not with soluble HEL in a pump (10 nmol/mouse), were immunized with 3.5 nmol HEL in CFA. CD8-depleted draining LNC (5 x 105 cells/well) were stimulated in vitro with HEL (10 µM) in HL-1 synthetic medium. IFN-{gamma} and IL-4 production was determined in 72-h culture supernatants. cDNA were prepared from ex vivo-purified CD4 T cells from BALB/c (C) or B10.D2 (D) immunized as indicated above. The frequency of TCR {beta}-chain transcripts bearing the HEL107–116/IEd-specific BV8S2-J1S5 rearrangement or the Th2-associated BV14-J2S7 CDR3 motif was quantified by fluorogenic PCR as described in Fig. 8Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chronic exposure to soluble Ag in adults (13) or neonatal administration of Ag in IFA (26) has been shown to lead to inactivation of the public clonotypes directed against dominant HEL epitopes, indicating that the Th2 cell development observed in these models was not due to clonal immune deviation. Indeed, we now provide direct evidence that systemic Ag administration concomitantly induces the recruitment of a distinct repertoire of T cell precursors primed toward IL-4 production. This Th2-associated repertoire is focused on a particular TCR chain usage in which we could identify a highly frequent AV3/BV14 clonotype characterized by conserved sequences in the CDR3 loops. Interestingly, the AV3/BV14 clonotype was not recruited in HEL-reactive CD4 T cells generated under adjuvant-guided priming conditions. Although we could identify the BV14-J2S7 CDR3 motif in HEL-reactive T cells from HEL-CFA- or IFA-immunized mice, its frequency was much lower compared with that in soluble Ag-treated animals. Moreover, V{beta}14+ T cells from HEL/CFA-primed mice did not exhibit detectable effector function, since they were unable to secrete either IFN-{gamma} or IL-4, unlike V{beta}14+ T cells from soluble HEL-treated mice that produced large amounts of IL-4. Likewise, we could not detect significant expansion in the AV3-J17 rearrangement corresponding to a 10-aa long CDR3 loop. Thus, T cell clones expressing both public AV3-J17 and BV14-J2S7 chains were present at very low frequency in mice primed with protein Ag in adjuvant, including IFA that induces some degree of Th2 cell development (see Fig. 1Go) (13). Taken together, our data demonstrate that the expansion of the AV3/BV14 repertoire was strongly dependent on the mode (chronic low dose) and the form (soluble) of protein Ag administration and was associated with the development of memory/effector Th2 cells. Indeed, both Th2 responsiveness and BV14-J2S7 clonotype expansion were dependent on the dose of soluble Ag administered in pump. The frequency of BV14-J2S7 T cells in immune lymph node before in vitro stimulation was increased by at least 5-fold, indicative of the induction of a memory response in soluble-Ag sensitized mice (see Figs. 8GoC and 9C). Accordingly, up-regulation of IL-4 transcripts was prominent in ex vivo-isolated V{beta}14+ CD4 T cells. Another important point to stress is that similar Th2 cell expansion occurred after antigenic challenge in IFA or CFA (see Fig. 1Go). Since CFA is a strong Th1-polarizing agent, the demonstration that a Th2 response could still develop under this condition of antigenic challenge indicates that HEL-reactive Th cells were already committed to the Th2 phenotype. This is in agreement with previous work showing that administration of IL-12 at the time of immunization in adjuvant had only a modest effect on Th2 responses (27), probably as a consequence of the loss of IL-12 responsiveness by Th2 cells (27, 28). Lastly, upon secondary antigenic challenge, strong IgE production was observed in soluble Ag-sensitized mice, indicatory of a type 2 T and B cell memory responses.

How to explain the selective development of the AV3-J17/BV14-J2S7 clonotype to the Th2 pathway? These T cells may originate from a memory/effector cross-reactive T cell population primed against environmental Ags, as it has been recently shown for Leishmania homolog of receptors for activated C kinase-specific T cells in L. major model in BALB/c mice (29). We consider this possibility as improbable, since we have found that the frequency of the BV14-J2S7 clonotype in naive splenic CD4 T cells was approximately 10-6 (data not shown), a frequency similar to that measured for the dominant epitope-specific T cell clones (13). Most likely, they may originate from naive T cell precursors that under this condition of chronic Ag exposure would have been preferentially selected in vivo to develop along the Th2 pathway due to their TCR specificity. This is supported by the strong bias in the Th2 cell repertoire that we have observed, indicating that Ag-driven selection had occurred in vivo. Thus, ligand-TCR interactions might be a critical factor in the initiation of the Th2 differentiation program, in agreement with the hypothesis that conventional CD4 T cells are the original source of IL-4 during Th2 development (30). Indeed, there are several observations suggesting that the strength of MHC-peptide-TCR interactions can influence CD4 T cell differentiation (6, 9). The HEL determinant recognized by BV14-J2S7 Th2 cells is probably generated less efficiently than the immunodominant epitope HEL107–116/IEd (see Fig. 5Go) and is preferentially presented by DC. Thus, the combination of both events, i.e., selective Ag presentation by professional APC and low density of ligand available for T cells, could favor Th2 cell differentiation (31, 32). In addition, DC expressing the Th2 epitope could interact with T cells bearing a low affinity TCR, resulting in partial signaling and subsequent Th2 commitment. Indeed, lowering MHC-peptide-TCR interactions using altered peptide ligands that contain amino acid substitution at key TCR contact residues (32, 33, 34) or mutating the TCR at a single position that contacts MHC class II-bound peptide have been shown to result in Th2 cell differentiation (8). Finally, the type of professional APC involved in Th2 cell activation could also be at play. Accumulating evidence indicates that there are several subsets of DC that differ in phenotype and function (35). Chronic Ag exposure might target Ag presentation to a particular DC subset, such as DC2, thereby promoting Th2 cell development (35).

What are the mechanisms that lead to the emergence of different repertoires in vivo depending on the mode of protein Ag administration? We hypothesize that the following sequence of events may occur in vivo under chronic soluble Ag delivery. Presentation of dominant HEL determinants by nonprofessional APC, such as B cells (36, 37), would result in the induction of anergy in T cells bearing the public clonotypes (e.g., BV8S2-J1S5) specific for dominant HEL107–116/IEd determinant. This mechanism might be dependent on the engagement of CTLA-4 on T cells and/or from the lack of proinflammatory cytokines, such as IL-12, in the microenvironment (38, 39). On the contrary, the selective capacity of DC to process the IAd-restricted cryptic epitope, contained in the HEL sequence 13–105, might restrain Ag presentation to this professional APC population, resulting in AV3/BV14 T cell activation and expansion as discussed above. Despite the different approaches used to identify the epitope recognized by the AV3/BV14 clonotype, we were unable to restrict a core sequence shorter than the HEL13–105 fragment. The difficulty for its characterization could be due to the fact that this antigenic determinant is unstable or only presented after processing of large peptidic fragments. Another appealing possibility is that Th2 cells might recognize post-translationally altered forms of the HEL sequence, similarly to what has been shown for the MHC class I-restricted H-Y Ag (40, 41) and more recently for the HEL determinant 48–62 presented by IAk molecules (42). Alternatively, soluble Ag administration could prime a T cell repertoire directed to a cryptic determinant of HEL, selectively presented by DC under a Th2 prone microenvironment. Indeed, it has been reported that DC exposed to IL-6 and native protein Ag in vitro could efficiently prime T cells against cryptic determinants in vivo (25). Whether such a IL-6-dependent mechanism occurs in our model remains to be investigated.

Thus, quantitative analysis of clonal T cell populations reveals a selective distribution of TCR clonotypes among effector Th cells depending on the mode of protein administration. Immunization in CFA or IFA primes identical T cell repertoires directed to immunodominant epitopes of the Ag, with selectivity in Th phenotype acquisition being determined by the presence of mycobacteria (13). In contrast, chronic Ag sensitization induces the selective expansion of a unique T cell repertoire that differentiates into type 2 memory/effector CD4 T cells. This Th2-associated clonotype recognizes a distinct antigenic determinant selectively processed by DC. In animal models of allergic asthma, immunotherapy with a dominant epitope of the protein failed to down-modulate Th2 responsiveness to the whole protein and eventually aggravated the disease (43, 44). Furthermore, it has been recently shown in humans that immediate and delayed-type hypersensitivities to the dermatophyte fungus Trychophyton Ags were associated with CD4 T cell responses to distinct epitopes of the Tri r2 protein (45). Our study identifies a potential mechanism that might explain such observations and support the hypothesis that TCR-dependent pathways might be the initial driving forces in CD4 T cell differentiation in vivo.


   Acknowledgments
 
We are grateful to W. E Paul for critical reading of the manuscript. We thank D. Voeglé and P. A. Cazenave for providing us with anti-TCR mAbs.


   Footnotes
 
1 This work was supported by the Institut National de la Santé et de la Recherche Médicale and grants from Association Française contre les Myopathies, Université Paul Sabatier, and Ministère de l’Education Nationale de la Recherche et de la Technologie (PRFMMIP 1998, 2000). Back

2 On a leave of absence from Ecole Nationale Vétérinaire de Toulouse (Toulouse, France). Current address: Laboratory of Immunology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892. Back

3 Address correspondence and reprint requests to Dr. Jean-Charles Guéry, Institut National de la Santé et de la Recherche Médicale, Unité 28, Hôpital Purpan, place du Dr. Baylac, 31059 Toulouse, France. E-mail address: jean-charles.guery{at}toulouse.inserm.fr Back

4 Abbreviations used in this paper: HEL, hen egg white lysozyme; BM-DC, bone marrow-derived dendritic cells; DCs, dendritic cells; FAM, 6-carboxyfluorescein; LNC, lymph node cells; Tamra, 6-carboxytetramethylrhodamine. Back

Received for publication April 3, 2001. Accepted for publication October 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, R. L. Coffman. 1986. Two types of murine helper T cell clone. I. Definition according to profile of lymphokine activities and secreted proteins. J. Immunol. 136:2348.[Abstract]
  2. Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[Medline]
  3. Cookson, W.. 1999. The alliance of genes and environment in asthma and allergy. Nature 402:B5.[Medline]
  4. O’Garra, A.. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275.[Medline]
  5. Glimcher, L. H., K. M. Murphy. 2000. Lineage commitment in the immune system: the T helper lymphocyte grows up. Genes Dev. 14:1693.[Free Full Text]
  6. Constant, S. L., K. Bottomly. 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15:297.[Medline]
  7. Guéry, J-C., F. Galbiati, S. Smiroldo, L. Adorini. 1996. Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and {beta}2-microglobulin-deficient mice. J. Exp. Med. 183:485.[Abstract/Free Full Text]
  8. Blander, J. M., D. B. Sant’Angelo, K. Bottomly, Jr C. A. Janeway. 2000. Alteration at a single amino acid residue in the T cell receptor {alpha} chain complementarity determining region 2 changes the differentiation of naive CD4 T cells in response to antigen from T helper cell type 1 (Th1) to Th2. J. Exp. Med. 191:2065.[Abstract/Free Full Text]
  9. Malherbe, L., C. Filippi, V. Julia, G. Foucras, M. Moro, H. Appel, K. Wucherpfennig, J.-C. Guéry, N. Glaichenhaus. 2000. Selective activation and expansion of high affinity CD4+ T cells in resistant mice upon infection with Leishmania major. Immunity 13:771.[Medline]
  10. Reiner, S. L., R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.[Medline]
  11. Guéry, J.-C., F. Galbiati, S. Smiroldo, L. Adorini. 1997. Non-MHC-linked Th2 cell development induced by soluble protein administration predicts susceptibility to Leishmania major infection. J. Immunol. 159:2147.[Abstract/Free Full Text]
  12. Krishnan, L., L. J. Guilbert, A. S. Russel, T. G. Wegmann, T. R. Mosmann, M. Belosevic. 1996. Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific IFN-{gamma} responses and increased production of T helper 2 cytokines. J. Immunol. 156:644.[Abstract]
  13. Foucras, G., L. Gapin, C. Coureau, J. M. Kanellopoulos, J. C. Guéry. 2000. IL-4 producing CD4 T cells arise from different precursors depending on the conditions of antigen exposure in vivo. J. Exp. Med. 191:683.[Abstract/Free Full Text]
  14. Gapin, L., J. P. Cabaniols, R. Cibotti, D. M. Ojcius, P. Kourilsky, J. M. Kanellopoulos. 1997. Determinant selection for T-cell tolerance in HEL-transgenic mice: dissociation between immunogenicity and tolerogenicity. Cell. Immunol. 177:77.[Medline]
  15. Katz, M. E., R. M. Maizels, L. Wicker, A. Miller, E. E. Sercarz. 1982. Immunological focusing by the mouse major histocompatibility complex: mouse strains confronted with distantly related lysozymes confine their attention to very few epitopes. Eur. J. Immunol. 12:535.[Medline]
  16. Tomonari, K., S. Spencer. 1990. Epitope specific binding of CD8 regulates activation of T cells and induction of cytotoxicity. Int. Immunol. 2:1189.[Abstract/Free Full Text]
  17. Guéry, J.-C., F. Galbiati, S. Smiroldo, L. Adorini. 1996. Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and {beta}2-microglobulin-deficient BALB/c mice. J. Exp. Med. 183:485.
  18. Openshaw, P., E. E. Murphy, N. A. Hosken, V. Maino, K. Davis, K. Murphy, A. O’Garra. 1995. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182:1357.[Abstract/Free Full Text]
  19. Adorini, L., E. Appella, G. Doria, Z. A. Nagy. 1988. Mechanisms influencing the immunodominance of T cell determinants. J. Exp. Med. 168:2091.[Abstract/Free Full Text]
  20. Lutz, M. B., N. Kukutsch, A. L. J. Ogilvie, S. Rö{beta}ner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77.[Medline]
  21. Pannetier, C., M. Cocher, S. Darche, A. Casrouge, M. Zöller, P. Kourilsky. 1993. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments. Proc. Natl. Acad. Sci. USA 90:4319.[Abstract/Free Full Text]
  22. Azuara, V., J. P. Levraud, M. P. Lembezat, P. Pereira. 1997. A novel subset of adult gamma delta thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire. Eur. J. Immunol. 27:544.[Medline]
  23. Cibotti, R., J.-P. Cabianols, C. Pannetier, C. Delarbre, I. Vergnon, J. M. Kanellopoulos, P. Kourilsky. 1994. Public and private V{beta} T cell repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice. J. Exp. Med. 180:861.[Abstract/Free Full Text]
  24. Gammon, G., H. M. Geysen, R. J. Apple, E. Pickett, M. Palmer, A. Ametani, E. E. Sercarz. 1991. T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes. J. Exp. Med. 173:609.[Abstract/Free Full Text]
  25. Drakesmith, H., D. O’Neil, S. C. Schneider, M. Binks, P. Medd, E. Sercarz, P. Beverley, B. Chain. 1998. In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen. Proc. Natl. Acad. Sci. USA 95:14903.[Abstract/Free Full Text]
  26. Maverakis, E., J. T. Beech, S. S. Wilson, A. Quinn, B. Pedersen, E. E. Sercarz. 2000. T cell receptor complementary determining region 3 length analysis reveals the absence of a characteristic public T cell repertoire in neonatal tolerance: the response in the "tolerant" mouse within the residual repertoire is quantitatively similar but qualitatively different. J. Exp. Med. 191:695.[Abstract/Free Full Text]
  27. Galbiati, F., L. Rogge, J.-C. Guéry, S. Smiroldo, L. Adorini. 1998. Regulation of the IL-12R {beta}2 subunit by soluble antigen and IL-12 in vivo is associated with the control of Th1 and Th2 cell development. Eur. J. Immunol. 28:209.[Medline]
  28. Szabo, S. J., N. G. Jacobson, A. S. Dighe, U. Gubler, K. M. Murphy. 1995. Developmental commitment to the Th2 lineage by extinction of IL-12 signaling. Immunity 2:665.[Medline]
  29. Julia, V., S. S. McSorley, L. Malherbe, J. P. Breittmayer, F. Girard-Pipau, A. Beck, N. Glaichenhaus. 2000. Priming by microbial antigens from the intestinal flora determines the ability of CD4+ T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major. J. Immunol. 165:5637.[Abstract/Free Full Text]
  30. Noben-Trauth, N., J. Hu-Li, W. E. Paul. 2000. Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation. J. Immunol. 165:3620.[Abstract/Free Full Text]
  31. Constant, S., C. Pfeiffer, A. Woodard, T. Pasqualini, K. Bottomly. 1995. Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells. J. Exp. Med. 182:1591.[Abstract/Free Full Text]
  32. Tao, X., S. Constant, P. Jorritsma, K. Bottomly. 1997. Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell differentiation. J. Immunol. 159:5956.[Abstract]
  33. Evavold, B. D., P. M. Allen. 1991. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science 252:1308.[Abstract/Free Full Text]
  34. Pfeiffer, C., J. Stein, S. Southwood, H. Ketelaar, A. Sette, K. Bottomly. 1995. Altered peptide ligands can control CD4 T lymphocyte differentiation in vivo. J. Exp. Med. 181:1569.[Abstract/Free Full Text]
  35. Moser, M., K. M. Murphy. 2000. Dendritic cell regulation of Th1-Th2 cell development. Nat. Immunol. 1:199.[Medline]
  36. Fuchs, E. F., P. Matzinger. 1992. B cells turn off virgin but not memory T cells. Science 258:1156.[Abstract/Free Full Text]
  37. Eynon, E. E., D. C. Parker. 1992. Small B cells as antigen-presenting cells in the induction of tolerance to soluble protein antigens. J. Exp. Med. 175:131.[Abstract/Free Full Text]
  38. Perez, V. L., L. V. Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411.[Medline]
  39. Van Parijs, L., V. L. Perez, A. Biuckians, R. G. Maki, C. A. London, A. K. Abbas. 1997. Role of interleukin 12 and costimulators in T cell anergy in vivo. J. Exp. Med. 1997:1119.
  40. Meadows, L., W. Wang, J. M. M. den Haan, E. Blokland, C. Reinhardus, J. W. Drijfhout, J. Shabanowitz, R. Pierce, A. I. Agulnik, C. E. Bishop, et al 1997. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity 6:273.[Medline]
  41. Pierce, R. A., E. D. Field, J. M. M. den Haan, J. A. Caldwell, F. M. White, J. A. Marto, W. Wang, L. M. Frost, E. Blokland, C. Reinhardus, et al 1999. The HLA-A*0101-restricted HY minor histocompatibility antigen originates from DFFRY and contains a cysteinylated cysteine residue as identified by a novel mass spectrometric technique. J. Immunol. 163:6360.[Abstract/Free Full Text]
  42. McAdam, S. N., B. Fleckenstein, I. B. Rasmussen, D. G. Schmid, I. Sandlie, B. Bogen, N. J. Viner, L. M. Sollid. 2001. T cell recognition of the dominant I-A(k)-restricted hen egg lysozyme epitope: critical role for asparagine deamidation. J. Exp. Med. 193:1239.[Abstract/Free Full Text]
  43. Janssen, E. M., M. H. Wauben, E. H. Jonker, G. Hofman, W. Van Eden, F. P. Nijkamp, A. J. Van Oosterhout. 1999. Opposite effects of immunotherapy with ovalbumin and the immunodominant T-cell epitope on airway eosinophilia and hyperresponsiveness in a murine model of allergic asthma. Am. J. Respir. Cell Mol. Biol. 21:21.[Abstract/Free Full Text]
  44. Janssen, E. M., A. J. van Oosterhout, A. J. van Rensen, W. van Eden, F. P. Nijkamp, M. H. Wauben. 2000. Modulation of Th2 responses by peptide analogues in a murine model of allergic asthma: amelioration or deterioration of the disease process depends on the Th1 or Th2 skewing characteristics of the therapeutic peptide. J. Immunol. 164:580.[Abstract/Free Full Text]
  45. Woodfolk, J. A., S. S. Sung, D. C. Benjamin, J. K. Lee, T. A. Platts-Mills. 2000. Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the trichophyton antigen, Tri r 2. J. Immunol. 165:4379.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
X. Tang, I. Maricic, and V. Kumar
Anti-TCR Antibody Treatment Activates a Novel Population of Nonintestinal CD8{alpha}{alpha}+TCR{alpha}beta+ Regulatory T Cells and Prevents Experimental Autoimmune Encephalomyelitis
J. Immunol., May 15, 2007; 178(10): 6043 - 6050.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
C. Nicolo, G. Di Sante, M. Orsini, S. Rolla, S. Columba-Cabezas, V. R. Spica, G. Ricciardi, B. M. C. Chan, and F. Ria
Mycobacterium tuberculosis in the adjuvant modulates the balance of Th immune response to self-antigen of the CNS without influencing a "core" repertoire of specific T cells
Int. Immunol., February 1, 2006; 18(2): 363 - 374.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Mandic, F. Castelli, B. Janjic, C. Almunia, P. Andrade, D. Gillet, V. Brusic, J. M. Kirkwood, B. Maillere, and H. M. Zarour
One NY-ESO-1-Derived Epitope That Promiscuously Binds to Multiple HLA-DR and HLA-DP4 Molecules and Stimulates Autologous CD4+ T Cells from Patients with NY-ESO-1-Expressing Melanoma
J. Immunol., February 1, 2005; 174(3): 1751 - 1759.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Garidou, S. Laffont, V. Douin-Echinard, C. Coureau, A. Krust, P. Chambon, and J.-C. Guery
Estrogen Receptor {alpha} Signaling in Inflammatory Leukocytes Is Dispensable for 17{beta}-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis
J. Immunol., August 15, 2004; 173(4): 2435 - 2442.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Ria, A. Gallard, C. R. Gabaglia, J.-C. Guery, E. E. Sercarz, and L. Adorini
Selection of Similar Naive T Cell Repertoires but Induction of Distinct T Cell Responses by Native and Modified Antigen
J. Immunol., March 15, 2004; 172(6): 3447 - 3453.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
L. Overbergh, B. Decallonne, D. D. Branisteanu, D. Valckx, A. Kasran, R. Bouillon, and C. Mathieu
Acute Shock Induced by Antigen Vaccination in NOD Mice
Diabetes, February 1, 2003; 52(2): 335 - 341.
[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 Foucras, G.
Right arrow Articles by Guéry, J.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Foucras, G.
Right arrow Articles by Guéry, J.-C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS