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, 2000, 165: 4994-5003.
Copyright © 2000 by The American Association of Immunologists

Dendritic Cells Prime In Vivo Alloreactive CD4 T Lymphocytes Toward Type 2 Cytokine- and TGF-ß-Producing Cells in the Absence of CD8 T Cell Activation1

Gilles Foucras2, Jérôme D. Coudert2, Christiane Coureau 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms that influence the polarization of CD4 T cells specific for allogeneic MHC class II molecules in vivo are still poorly understood. We have examined the pathway of alloreactive CD4 T cell differentiation in a situation in which only CD4 T cells could be activated in vivo. In this report we show that priming of adult mice with allogeneic APC, in the absence of MHC class I-T cell interactions, induces a strong expansion of type 2 cytokine-producing allohelper T cells. These alloantigen-specific CD4 T cells directly recognize native allogeneic MHC class II molecules on APC and secrete, in addition to the prototypic Th2 cytokines IL-4, IL-5, and IL-10, large amounts of TGF-ß. The default Th2-phenotype acquisition is not genetically controlled and occurred both in BALB/c and C57BL/6 mice. CD8 T cells are the principal cell type that controls CD4 T cell differentiation in vivo. Furthermore, we demonstrate that strong Th2 priming can be induced not only with allogeneic splenocytes but also with a low number of bone marrow-derived dendritic cells. Finally, using a passive transfer system, we provide direct evidence that CD8 T cell expansion in situ promotes alloreactive Th1 cell development principally by preventing their default development to the Th2 pathway in a mechanism that is largely IFN-{gamma} independent. Therefore, this work demonstrates that type 2 cytokine production represents a dominant pathway of alloreactive CD4 T cell differentiation in adult mice, a phenomenon that was initially thought to occur only during the neonatal period.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The functional significance of Th cell subsets in the eradication of intracellular pathogens (1) and in the genesis of organ-specific autoimmune disease is now well documented (2). Conversely, the respective roles of the different types of effector Th cells in allotransplantation is still a matter of debate (3). It is widely accepted that Th1-dominated immune responses are mainly involved in graft rejection (4), probably by promoting cellular immunity and by providing help to CD8 T cells. Thus, it has been hypothesized that immune deviation by favoring the generation of alloreactive Th2-type cells or regulatory T cells could promote allograft acceptance by antagonizing Th1 cell development (5). Indeed, it is now well established that neonatal induction of lymphoid chimerism (6, 7, 8, 9, 10), as well as transplantation tolerance in adult mice injected with nondepleting anti-CD4 and anti-CD8 mAb (5), are associated with the development of IL-4-producing cells. However, the beneficial role of Th2 cells in allograft survival has been recently challenged by reports showing that deleterious responses could also be associated with Th2-type immunity (11, 12).

Although much progress has been made in our understanding of the molecular basis of allorecognition (13), the mechanisms that influence the expansion and differentiation in vivo of CD4 T cells specific for allogeneic MHC class II molecules are still poorly understood. The current hypothesis states that, due to their high precursor frequency, the alloreactive T cell repertoire may consist of experienced T cells committed to their Th1 or Th2 lineage. Therefore, direct alloantigen recognition in adult life would preferentially prime a cross-reactive Th1 memory repertoire that would subsequently skew the entire immune response to the Th1 pathway, resulting in rapid allograft rejection. Conversely, Th2 development would be confined to the neonatal period, when all host T cells are naive and most likely uncommitted to a particular Th phenotype (8, 14). However, this hypothesis is challenged by experiments showing that the frequency of IL-4-producing cells was similar between neonatally tolerized mice and adult mice repeatedly stimulated with alloantigen in MHC class II-disparate strain combinations (6). Therefore, it is possible that the Th1 development of alloreactive CD4 T cell responses observed in MHC class I and class II disparate combinations (8) was greatly influenced by CD8 T cell priming. Indeed, several reports have shown that CD8 T cells can regulate Th2 immunity in vivo in response to viral infection (15, 16) or to protein Ag challenge (17), in chronic graft-vs-host disease (18), and in systemic autoimmunity (19).

The studies described in this paper were aimed at analyzing the mechanisms involved in Th phenotype acquisition of alloreactive CD4 T cells in vivo in the absence of NK and CD8 T cell activation. The experimental model we have chosen involved in vivo priming of the parental strain B6 or BALB/c mice with semiallogeneic F1 APC. ß2-microglobulin (ß2m)4-deficient, MHC class I-negative mice were first used to study CD4 T cell priming only. Semiallogeneic (BALB/c x C57BL/6)F1 (CB6F1) APC that expressed simultaneously self and allogeneic MHC products were used instead of fully allogeneic cells to avoid NK cell activation (20). Furthermore, this strategy allowed us to analyze alloreactive CD4 T cell development in different genetic backgrounds with the same APC populations, so that alteration in Th phenotype acquisition could not be attributed to the APC origin. We show that while immunization of adult BALB/c mice with semiallogeneic splenocytes induces the differentiation of donor-specific CD4 T cells toward the Th1 phenotype, a strongly polarized Th2-type response occurs in combinations in which ß2m-dependent cells or CD8 T cells are absent. We next analyzed the roles of endogenous IL-4 production, host genotype, and dendritic cells (DC) in the generation of alloreactive Th2 responses in vivo. Finally, we provide direct evidence that the in situ recruitment of a low number of CD8 T cells can effectively control CD4 T cell differentiation in vivo.


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

BALB/c (H-2d), C57BL/6 (H-2b), and CB6F1 mice were purchased from Centre d’Elevage R. Janvier (Le Genest St Isle, France). H-2b mice with disrupted ß2m genes (21) were backcrossed to BALB/c mice as previously described (22). ß2m-deficient (ß2m°) mice on BALB/c or C57BL/6 background were used after 10 backcrosses. CD8-deficient (CD8°) (23), ß2m° (21), IAß-deficient (24), and ß2m° IAß-deficient mice in the C57BL/6 background were initially obtained from the Centre National de la Recherche Scientifique (Centre de Développement des Techniques Avancées, Orléans, France). BALB/c ß2m°, C57BL/6 ß2m°, C57BL/6 CD8°, and CB6F1 ß2m° mice were bred and maintained in our specific pathogen-free animal facility. Adult mice (8–12 wk old) were immunized s.c. in the hind footpads with 30–50 x 106 irradiated (2400 rad) spleen cells (SC) or 0.3–0.5 x 106 bone marrow (BM)-derived DC (BM-DC) from normal or ß2m° CB6F1 mice. Five to 6 days after immunization, the draining popliteal and inguinal lymph nodes were removed and further processed as described below.

In vivo mAb treatment

For CD8 cell depletion, the anti-CD8{alpha} 53-6-72 rat IgG2a mAb (TIB 105, American Type Culture Collection (ATCC), Manassas, VA) was purified from ascites fluid by protein G chromatography. For in vivo CD8 depletion, mice were injected i.p. with 500 µg 53-6-72 mAb for 3 consecutive days (at days 6, 5, and 4), the day of immunization (day 0), and 3 days later. For blocking endogenous IL-4, mice were injected with 800 µg i.p. of 11B11 (HB 188, ATCC) or isotype control LO-DNP-2 rat mAb, as previously shown (22), at day 0 and with 400 µg at days 2 and 4.

BM-DC

Mouse BM cells were labeled with 30-H12 anti-Thy.1.2 (TIB 107, ATCC) and RA3-3A1 anti-B220 (TIB 146, ATTC) mAb. T and B cells were then depleted using anti-rat IgG M-450 Dynabeads (Dynal, Oslo, Norway). Cells were cultured as previously described (25), with some modifications. Culture medium was RPMI 1640 (Life Technologies, Cergy Pontoise, France) supplemented with 10% FCS (ATGC Biotechnologie, Noisy Le Grand, France), 1% pyruvate, 1% nonessential amino acids, 1% L-glutamine, 50 µM 2-mercaptoethanol, and 50 µg/ml gentamicin (Sigma, St. Louis, MO). After overnight culture at 106 cells/ml in a 24-well plate without cytokine, cells were replated (2.5 x 105 cells/well) in the presence of murine GM-CSF (10 ng/ml; Sigma). On day 3, half of the culture supernatant was replaced with fresh medium containing GM-CSF. Cultures were duplicated on day 5, and DC were harvested by gentle pipetting on days 7–8. After Ficoll separation, living cells were incubated sequentially with biotin-GR-1 anti-Ly6G mAb (PharMingen, San Diego, CA) and streptavidin-coated M-280 Dynabeads (Dynal). Granulocytes were then removed by magnetic cell separation. The DC preparation was >95% CD11c+ by flow cytometric analysis using N418 anti-CD11c mAb (ATCC, HB 224).

T cell assays

For cytokine production analysis, CD8-depleted lymph node cells (LNC) from mice immunized with allogeneic SC were cultured at 3 x 105 cells/well in 96-well culture plates (Costar, Cambridge, MA) in the presence of various concentrations of irradiated SC of the indicated origin. For removal of CD8 cells, LNC were incubated with KT1.5 mAb (26) culture supernatant, washed, and subsequently incubated with sheep anti-rat IgG M-450 Dynabeads (Dynal) previously adsorbed with 10% normal mouse serum. CD8-positive cells were then selectively depleted with a magnet (BioSource, Camarillo, CA). CD4 T cell enrichment was performed as above by incubating LNC with anti-B220 RA3-3A1 (ATCC, TIB 146), anti-CD8 KT1.5, and anti-class II M5/114 (ATCC, TIB 120) mAb. Cells were cultured in HL-1 synthetic medium (Hycor, Irvine, CA) supplemented with 2 mM L-glutamine and 50 µg/ml gentamicin (Sigma). Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air. Supernatants from replicate cultures, usually three to four wells, were collected after 72 h and pooled for cytokine analysis. For T cell proliferation assays, cell cultures were pulsed 8 h with 1 µCi [3H]TdR (40 Ci/nmol, Radiochemical Center, Amersham, U.K.) before harvesting on glass fiber filter. Incorporation of [3H]TdR was measured by direct counting using an automated ß-plate counter (Matrix 9600, Packard, Meriden, CT).

Cytokine assays

IFN-{gamma}, IL-4, IL-5, and IL-10 were quantified by two-site sandwich ELISA as previously described (22). For IFN-{gamma}, polyvinyl microtiter plates (Falcon 3912; Becton Dickinson, Oxnard, CA) were coated with anti-IFN-{gamma} AN-18.17.24 mAb (27), and peroxidase-conjugated XMG1.2 IFN-{gamma}-specific mAb (28) was used as detection mAb. For IL-4, IL-5, and IL-10 determination, two-site ELISA were performed with paired mAb, all purchased from PharMingen. Absorbance was read on an automated microplate ELISA reader (Emax, Molecular Devices, Sunnyvale, CA). Cytokines were quantified from two to three titration points using standard curves generated with purified recombinant mouse cytokines, and results are expressed as cytokine concentration in ng or pg/ml. Detection limits were 15 pg/ml for IFN-{gamma} and IL-4, 3 pg/ml for IL-5, and 30 pg/ml for IL-10. For TGF-ß determination, acid-activated samples were incubated overnight at 4°C on plates coated with anti-TGF-ß 2G7 mAb (29). After washing, bound TGF-ß was revealed by addition of biotin-anti-TGF-ß1 mAb (R&D Systems, Minneapolis, MN) followed by streptavidin-alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA). Human recombinant TGF-ß1 (R&D Systems) was used as standard; the sensitivity of the assay was 7 pg/ml.

Flow cytometric analysis of intracellular cytokine synthesis

LNC were cultured with allogeneic irradiated (2400 rad) SC from C57BL/6 ß2m° mice as indicated above. After 72 h of culture, cells were harvested, washed, and cultured for an additional 72 h in complete medium. After Ficoll separation, living cells were collected, resuspended at 106/ml, and stimulated with PMA (Sigma, 50 ng/ml) plus ionomycin (Sigma, 0.5 µg/ml) for 4 h. Two hours before cell harvest, brefeldin A (Sigma) was added at a concentration of 10 µg/ml. Cells were harvested, washed in the presence of brefeldin A, and stained using biotinylated anti-CD4 mAb (PharMingen) followed by streptavidin-CyChrome (PharMingen). Labeled cells were then fixed with 2% paraformaldehyde (Fluka Chemie, Buchs, Switzerland). Intracytoplasmic staining was performed as previously described (30). After washing and 10-min incubation in saponin medium, cells were incubated for 30 min at room temperature with the appropriate concentration of FITC- or PE-conjugated cytokine-specific mAb in saponin buffer. The following mAbs were used: PE-11B11 anti-IL-4 (PharMingen), FITC-JES5-16E3 anti-IL-10 (PharMingen), and FITC-labeled XMG1.2 (28). Cells were washed first in saponin buffer and then with PBS/FCS to allow membrane closure. Data were collected on 20,000 CD4+ cells on an XL Coulter cytometer (Coultronics, Margency, France) and analyzed using the CellQuest software (Becton Dickinson, Mountain View, CA).

CD8 T cell passive transfer experiments

Pooled LNC and SC from B6->CB6F1 BM chimeras and from normal or IFN-{gamma}-deficient B6 mice were incubated with the following mAb mixture: anti-class II M5/114 (TIB 120, ATCC), anti-CD11b M1/70 (TIB 128, ATCC), anti-B220 RA3-3A1 (TIB 146, ATCC), and anti-CD4 GK1.5 (TIB 207, ATCC). Cells were incubated with Low-Tox-M rabbit complement (Cedarlane, Hornby, Ontario, Canada) according to manufacturer procedure. Highly enriched CD8 T cells were obtained by negative selection using anti-rat IgG M-450 Dynabeads (Dynal) after labeling of cells with the previous mAb cocktail supplemented with anti-Ly-6G Gr-1, anti-Ly-76 TER-119, and anti-Pan-NK DX5 mAb, all purchased from PharMingen. Purity was routinely >90% as assessed by flow cytometry analysis. CD8 T cells were subsequently injected i.v. into CD8° B6 mice. For the analysis of in situ recruitment of CD8 T cells, immune LNC were stained with PE-anti-CD4 (PharMingen), PE-anti-B220 (PharMingen), FITC-anti-CD8 (26), and biotin-H57 (HB 218, ATCC) anti-TCR mAb, followed by streptavidin-CyChrome. Data were collected on 5000 B220–CD4– cells on an XL Coulter cytometer (Coultronics) and analyzed using the CellQuest software.

Generation of CB6F1 BM chimeras

After RBC lysis, BM cells from 8- to 10-wk-old B6 mice were labeled with mAb 30H12 anti-Thy 1.2 (TIB 107, ATCC) and incubated in Low-Tox-M rabbit complement (Cedarlane) to eliminate T cells. CB6F1 mice (7 wk old) received an i.p. injection of PK 136 anti-NK cells (HB 191, ATCC) (100 µg) and were lethally irradiated (900 rad). The day after, they were i.v. injected with 10 x 106 T cell-depleted B6 BM cells. These chimeras were used 6 wk after reconstitution.

Statistical analysis

Results are expressed as mean ± SD, and overall differences between variables were evaluated by the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo priming of alloreactive CD4 T cells in the absence of ß2m-dependent T cells results in a strong Th2-dominated response

We have analyzed whether the presence of ß2m-dependent T cells could influence the differentiation of alloreactive CD4 T cells following in vivo priming with semiallogeneic SC. Immune LNC from adult BALB/c mice immunized with irradiated CB6F1 splenocytes were stimulated in vitro with syngeneic or allogeneic B6 APC, and the polarization of the CD4 T cell response was determined by measuring cytokine production in 72-h culture supernatants. Before culture, LNC were selectively depleted of CD8 T cells to avoid the interference of this T cell population during the in vitro assay. As shown in Fig. 1GoA, immunization of BALB/c mice with semiallogeneic splenocytes induced the differentiation of donor-specific CD4 T cells toward the Th1 phenotype. Although IL-4, IL-10 (Fig. 1GoA), and IL-5 (not shown) could not be detected, a moderate production of TGF-ß was observed in response to allogeneic B6 APC. In striking contrast, immunization of ß2m° BALB/c mice with semiallogeneic ß2m° splenocytes resulted in the generation of B6-specific T cells able to secrete in addition to IFN-{gamma} dramatic amounts of IL-4, IL-10, TGF-ß (Fig. 1GoB), and IL-5 (not shown). Similar cytokine profiles were obtained using CB6F1 APC for in vitro restimulation (not shown). The proliferative responses were similar in both combinations, and identical results were obtained using ß2m° APC (not shown). The phenotype of H-2b-specific CD4 T cells was further analyzed in secondary cultures after one round of stimulation with B6 ß2m° APC. As shown in Fig. 2GoA, T cells primed in the wild-type (WT) combination developed into secondary T cells secreting high levels of IFN-{gamma}. Production of IL-4, IL-10, and TGF-ß was low or undetectable in the supernatants from these cultures. In sharp contrast, T cells primed in vivo in the absence of ß2m secreted low levels of IFN-{gamma} and impressive amounts of IL-4, IL-10, and TGF-ß following secondary in vitro stimulation. No cytokine production was observed when syngeneic ß2m° BALB/c or ß2m° IAß-deficient B6 splenocytes were used as APC (not shown). To analyze the heterogeneity of these secondary T cell populations, we next determined the intracellular cytokine production of IFN-{gamma}, IL-4, and IL-10 in CD4 T cells restimulated with PMA plus ionomycin (Fig. 2GoB). As expected, CD4 T cells primed in the WT combination expressed primarily IFN-{gamma} (40%), whereas the opposite cytokine profile was observed in secondary CD4 T cells from ß2m° mice. Analysis of IL-4 and IL-10 synthesis at the single-cell level revealed different populations of cells that produced either IL-4 or IL-10, or both cytokines simultaneously (20%). Approximately 3% of cells produced both IFN-{gamma} and IL-4 (Th0 type) or IFN-{gamma} alone (Th1 type). Thus, in vivo priming of alloreactive CD4 T cells in the absence of ß2m-dependent T cells resulted in a Th2-dominated immune response associated with the up-regulation of TGF-ß-producing cells.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Cytokine production by allohelper T cells from BALB/c mice immunized with CB6F1 SC in the absence of MHC class I-T cell interactions. WT (A) or ß2m° (B) BALB/c mice (n = 4) were immunized in the hind footpads with 50 x 106 semiallogeneic SC from WT or ß2m° mice. Six days later, CD8-depleted immune LNC (3 x 105 cells/well) were cultured in triplicate with the indicated amount of syngeneic ({circ}) or fully allogeneic C57BL/6 (•) irradiated (2400 rad) SC in HL-1 synthetic medium. Cytokine production in 72-h culture supernatants was measured by ELISA. Results (mean ± SD) are from one representative experiment of five performed.

 


View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2. Alloantigen-specific and intracellular cytokine synthesis in secondary CD4 T cells. A, CD8-depleted LNC from ß2m° BALB/c mice, immunized with ß2m°CB6F1 APC, were cultured for 3 days in the presence of allogeneic APC as described in Fig. 1Go. Cells were then expanded in complete medium containing 5% FCS. After 6 days of culture, highly enriched (>95%) CD4 T cells (105 cells/well) were restimulated with irradiated SC from ß2mo allogeneic C57BL/6 mice. Cytokine production was analyzed after 48 h of culture by ELISA, and results are expressed as mean ± SD of three to four mice/group. B, Intracellular cytokine synthesis was examined in secondary CD4 T cells after PMA and ionomycin stimulation as described in Materials and Methods. Data from three to four mice per group are expressed as mean ± SD and are representative of three experiments performed.

 
Blocking endogenous IL-4 prevents Th2 cell expansion

To address whether IL-4 was required in vivo for the expansion of B6-specific Th2 cells, we examined the polarization of the T cell response in ß2m° BALB/c mice immunized with ß2m° CB6F1 SC in the presence of 11B11 mAb. Results in Fig. 3Go show that B6-reactive CD4 T cells from untreated BALB/c ß2m° mice or mice injected with a control rat mAb secreted high amounts of IL-4, IL-10, and TGF-ß upon in vitro stimulation with I-Ab-expressing B6 ß2m° APC. Administration of anti-IL-4 11B11 mAb at the time of allogeneic priming resulted in a significant inhibition of IL-4 and IL-10 production by B6-specific T cells (Fig. 3Go, A and B) and was not associated with the up-regulation of IFN-{gamma} secretion (not shown). On the contrary, the generation of TGF-ß-producing cells was moderately affected by blocking IL-4 in vivo (Fig. 3GoC). Thus, the development of IL-4-/IL-10-producing CD4 T cells during in vivo allogeneic priming in the absence of ß2m was dependent on the endogenous production of IL-4.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. Blocking endogenous IL-4 inhibits the expansion in vivo of type 2 cytokine- but not TGF-ß-producing allohelper T cells. BALB/c mice ß2m° were immunized with 50 x 106 CB6F1 ß2m° SC into the hind footpads. Animals were injected i.p. with PBS (•), anti-IL-4 11B11 mAb ({triangleup}), or control LO-DNP-2 mAb ({circ}) at days 0, 2, and 4 of the experiment. The polarization of the T cell response was tested 6 days after immunization, as in Fig. 1Go. Data are from one representative experiment of three performed. *, p < 0.05.

 
Induction of type 2 cytokine-producing cells does not depend on the genetic background of the host

Induction of Th2 responses in vivo has been found to be highly dependent on a non-MHC-linked genetic polymorphism (1, 31). To test the effect of genetic background in our model, we have compared the polarization of alloreactive CD4 responses between ß2m° BALB/c and B6 mice. The same APC population was used to immunize both strains. Data in Fig. 4GoA show that in vivo priming of alloreactive CD4 T cells in the absence of ß2m-dependent T cells induced a strongly Th2 polarized immune response both in BALB/c and B6 mice. Both CD4 T cell populations secreted high amounts of IL-4, IL-5, IL-10, and TGF-ß in response to their respective allogeneic stimulators, although B6 CD4 T cells produced less IL-4 than did BALB/c cells (see Fig. 7Go, below). However, these results should be interpreted with caution, because these mice differ not only by genetic background but also by MHC haplotype. Thus, the lower IL-4 production observed in B6 mice may also reflect difference in the allorepertoire involved between the two combinations and/or differences at the level of APC used for restimulation. Secondary T cells were then analyzed for their intracellular expression of cytokines. As shown in Fig. 4GoB, the frequency of IL-4-single-positive Th2 as well as IL-4-/IFN-{gamma}-double-positive Th0 cells was strongly up-regulated in both backgrounds. However, IL-4-producing cells were less frequent in B6 as compared with BALB/c (48% vs 58%, respectively). Analysis of the coexpression of IL-4 and IL-10 showed that the decreased frequency in CD4 IL-4+ in B6 mice mainly affected cells coexpressing both IL-4 and IL-10. Finally, the overall frequency of IFN-{gamma}-producing CD4 T cells was higher in B6 than in BALB/c mice (30% vs 11%, respectively). In conclusion, although some quantitative differences may exist between the two strains, the strong Th2 priming that occurred in the absence of ß2m-dependent T cells was not dependent on a genetic polymorphism.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 4. Th2 polarization of alloreactive CD4 T cells is not dependent on the genetic background of the host. A, Pooled LNC (3 x 105 cells/well) from C57BL/6 ß2m° or BALB/c ß2m° mice (n = 2) immunized 6 days before with irradiated SC from CB6F1 ß2m° mice were cultured in the presence of APC (3 x 105 irradiated SC/well) as indicated. Th2-type cytokines (IL-4, IL-5, and IL-10) and TGF-ß1 concentrations (mean ± SD) were measured by ELISA in 72-h culture supernatants. B, After 6 days of culture in the presence of allogeneic APC, pooled LNC were stimulated with PMA/ionomycin and then surface stained for CD4 and intracellularly for the respective cytokines IFN-{gamma}/IL-4 and IL-10/IL-4, as in Fig. 2Go. Data from individual mice are shown and are from one representative experiment of four performed.

 


View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 7. DC prime alloreactive CD4 T cells toward type 2 cytokine and TGF-ß-producing cells in vivo in the absence of MHC class I-T cell interactions. CB6F1 BM-DC (0.5 x 106/mouse) from WT or ß2m° mice were used to immunize normal BALB/c or ß2m° BALB/c and B6 mice, respectively. Highly enriched (>95%) CD4 T cells (2 x 105 cells/well) were cultured for 3 days in the presence of irradiated SC from B6 or BALB/c mice. Cytokine production was analyzed in 72-h culture supernatants by ELISA, and results are expressed as mean ± SD of three mice per group. Data are from one representative experiment of three performed. *, p < 0.05.

 
The ß2m-dependent T cells that control alloreactive Th2 cell development in vivo are CD8 cells

The allogeneic interaction in the WT donor/recipient combination involves both MHC class I and class II molecules; therefore, it is likely that CD8 T cell activation might be an important factor controlling the alloreactive Th2 response. To address this point, we tested whether CD8° B6 mice immunized with WT CB6F1 SC would behave like ß2m° B6 mice injected with ß2m°CB6F1 cells. As shown in Fig. 5GoA, although variations occurred among individual mice, no significant difference could be observed between both combinations regarding the production of IL-4 and IL-10 (Fig. 5GoA). This was confirmed by analyzing the intracellular cytokine synthesis of secondary CD4 T cells (Fig. 5GoB). The frequency of IL-4- and IL-10-producing cells was almost identical between the two strains. To test whether the Th2 polarization was due to the developmental absence of CD8 molecule or CD8 T cells, experiments were performed in BALB/c mice injected with depleting anti-CD8 mAb before and after priming with irradiated CB6F1 SC. As shown in Fig. 6GoA, the effective depletion of CD8 cells in vivo at the time of alloantigenic priming resulted in the down-regulation of IFN-{gamma} synthesis and the appearance of IL-4-producing cells in primary T cell cultures. The reversal of the polarization of B6-specific CD4 T cells from a Th1- to a Th2-dominated profile was even more impressive in the secondary stimulation (Fig. 6GoB).



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 5. The ß2m-dependent T cells that control alloreactive Th2 cell development in vivo are CD8 cells. A, CD8-depleted LNC (3 x 105 cells/well) from C57BL/6 WT, CD8°, or ß2m° mice immunized 6 days before with irradiated SC from WT or ß2m° CB6F1 mice, as indicated, were cultured in the presence of irradiated BALB/c SC (3 x 105/well). IL-4 and IL-10 concentrations were measured in 72-h culture supernatants by ELISA. Data from individual mice are shown, and bars represent the mean of five to six mice per group from two experiments of four performed with similar results. B, After 6 days of culture, pooled LNC (two mice per group) were stimulated with PMA/ionomycin and then surface stained for CD4 and intracellularly for the respective cytokines IFN-{gamma}/IL-4 and IL-10/IL-4 as in Fig. 2Go. Data are from one representative experiment of two performed.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 6. Depletion of CD8 T cells in vivo unmasks Th2 cell priming. A, BALB/c mice were immunized with irradiated CB6F1 SC into the hind footpads. Animals were injected i.p. with 53-6-72 anti-CD8 mAb (500 µg/mouse) for 3 consecutive days (days –6, –5, and –4) and then at days 0 and 3 of the experiment. The polarization of the T cell response was tested 5 days after immunization as in Fig. 1Go. B, For secondary stimulation, CD8-depleted immune LNC cultured in the presence of allogeneic APC were harvested at day 6 of culture. Highly enriched (>95%) CD4 T cells (105 cells/well) were restimulated with irradiated C57BL/6 splenocytes (3 x 105 cells/well). Cytokine production (IFN-{gamma} and IL-4) was analyzed in 48-h culture supernatants by ELISA. Data are shown for individual mice, and bars represent the mean of three to four mice per group. Data are from one representative experiment of three performed.

 
DC can prime alloreactive CD4 T cells for type 2 cytokine production in vivo

Donor DC by interacting directly with recipient T cells have been shown to be important in causing graft rejection (32, 33). We next tested whether this professional APC population could substitute splenocytes in this system. DC were obtained from CB6F1 BM progenitors cultured in the presence of GM-CSF for 7–8 days. Living BM-DC, depleted of granulocytes, were used to immunize parental strains. As shown in Fig. 7Go, while CB6F1 DC primed Th1 development in the BALB/c mice, a strong Th2-dominated response, accompanied with TGF-ß synthesis, was induced in both ß2m° combinations. Again, IL-4 production was significantly lower in the B6 as compared with the BALB/c background, confirming our previous observation (Fig. 4GoA). Similar response patterns were observed when WT CB6F1 DC were injected into CD8° BALB/c (not shown) or B6 mice (Fig. 8GoB). Only 0.3–0.5 x 106 DC were necessary to generate such responses, which is 100-fold less than the amount of splenocytes used so far. The magnitude of the response was also consistently higher with DC as compared with total SC (not shown). Purified recipient CD4 T cells were used in the MLR, indicating that alloreactive CD4 T cells directly recognize the native I-Ab molecules displayed on the surface of B6 APC. Indeed, no cytokine production was observed using APC from MHC- or IA-ß-deficient B6 mice (not shown). Therefore, these results demonstrate that CD4 T cell priming by DC in vivo in the absence of ß2m-dependent CD8 T cells resulted in a strong expansion of CD4 T cells characterized by their capacity to secrete type 2 cytokines and TGF-ß.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 8. CD8 T cell requirement for alloreactive Th1 cell development in vivo. Negatively selected lymph node CD8 cells (>95% CD8+) from B6 mice were transferred by i.v. injection (2–3 x 106/mouse) into B6 CD8° mice 1 day before immunization with CB6F1 BM-DC (0.5 x 106/mouse). A, Analysis of CD8 T cell recruitment was performed on LNC 6 days after immunization. B, CD4 T cells (2 x 105 cells/well) were cultured for 3 days in the presence of irradiated SC from BALB/c mice. Cytokine production was analyzed in 72-h culture supernatants by ELISA. Results are the mean ± SD from three mice per group and are from one of four representative experiments. *, p < 0.05.

 
CD8 T cell requirement for alloreactive Th1 cell development

Our data indicate that CD8 T cells directed to allogeneic MHC molecules effectively control Th phenotype acquisition in vivo. To directly visualize this phenomenon, we designed an adoptive transfer system in which a fixed number of CD8 T cells was injected into CD8° B6 mice before immunization with CB6F1 BM-DC. After 6 days the number of CD8 T cells that expanded in draining lymph nodes was quantified by flow cytometry and represented ~1% of total LNC, as shown in Fig. 8GoA. Because ~20 x 106 cells were recovered in both popliteal and inguinal lymph nodes, ~2 x 104 H-2d-reactive CD8 T cells were recruited in situ in response to semiallogeneic DC priming (Fig. 9GoB). Interestingly, analysis of alloreactive CD4 T cell polarization in primary MLR in vitro showed that transfer of CD8 T cells from B6 mice was associated with the selective down-regulation of type 2 cytokine secretion by alloreactive CD4 T cells (Fig. 8GoB). As control, we have generated B6 -> CB6F1, BM chimeras. In these mice, the CD8 T cells found in the periphery were of B6 origin. They expressed H-2b class I molecules (>98%) and were negative for H-2d molecules (data not shown). We have used these chimeras as a source of tolerant CD8 T cells. Indeed, passive transfer of these cells into CD8° B6 mice before immunization with CB6F1 BM-DC did not affect the in vivo priming of Th2 cells, which was similar between control uninjected mice and mice injected with this tolerant CD8 T cell population (data not shown and Fig. 9Go). Thus, the down-regulation of Th2 response by CD8 T cells was associated with the adoptive transfer of nontolerant CD8 T cells.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 9. IFN-{gamma}-production by CD8 T cells is partially involved in the inhibition of Th2 cell development in vivo. Negatively enriched CD8 T cells (>90% CD8+) from B6 -> CB6F1 BM chimeras, normal or IFN-{gamma}-deficient B6 mice were transferred by i.v. injection (1.5 x 106/mouse) into B6 CD8° mice 1 day before immunization with CB6F1 BM-DC (0.5 x 106/mouse). A, Analysis of CD8 T cell recruitment was performed on LNC 6 days after immunization as described in Materials and Methods. B, Absolute number of TCR {alpha}ß CD8 T cells recruited in the draining lymph nodes from three to four individual mice per group. Bars represent the mean of individual mice. C, CD4 T cells (2 x 105 cells/well) were cultured for 3 days in the presence of irradiated SC from B6 or BALB/c mice. Cytokine production was analyzed in 72-h culture supernatants by ELISA. Data are expressed as mean ± SD from three to four individual mice per group. Data are from one representative experiment of two performed. *, p < 0.05.

 
To further study the mechanisms by which CD8 T cells control alloreactive Th2 cell development in this passive transfer system, IFN-{gamma}-deficient B6 mice were used as a source of CD8 T cells. Data in Fig. 9Go show that the priming of type 2 cytokine-producing cells was up-regulated in the absence of IFN-{gamma} secretion by CD8 T cells. However, the transfer of CD8 T cells with a disrupted IFN-{gamma} gene still resulted in a significant inhibition of Th2 responsiveness as compared with control uninjected mice (data not shown) or mice injected with tolerant CD8 T cells (Fig. 9GoC). These data indicate that other effector functions of CD8 T cells might play a major role in the control of CD4 T cell activation and differentiation in vivo. Taken together, our results provide direct evidence that CD8 T cell responses in situ prevent the default development of alloreactive CD4 T cells to the Th2 phenotype, by skewing the response to the Th1 pathway in a mechanism that is largely IFN-{gamma} independent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results in this paper demonstrate that alloreactive CD4 T cell development in adult mice, in the absence of CD8 T cell activation, had several unexpected features. First, immunization of adult parental strains with semiallogeneic APC in the absence of ß2m-dependent CD8 T cells induces a rapid and vigorous expansion of CD4 T cells secreting type 2 cytokines in response to allogeneic MHC class II products. In addition to their capacity to secrete a massive amount of type 2 cytokines, these T cell populations secrete TGF-ß, a cytokine with multifunctional effects on immune cells (34). Analysis of cytokine production at the single-cell level demonstrates that they were composed of a majority of cells exhibiting a Th2 phenotype. Such Th cells were otherwise absent in the WT combinations due to CD8 T cell activation. Second, we show that DC are extremely potent APC in inducing such allohelper type 2 responses. Finally, in vivo priming of type 2 cytokine- and TGF-ß-secreting CD4 T cells is not dependent on a genetic polymorphism, because it occurs both in BALB and B6 backgrounds. Therefore, this work demonstrates that type 2 cytokine production represents the dominant default pathway of alloreactive CD4 T cell development in adult mice in vivo, a phenomenon that was initially thought to occur only during the neonatal period (8, 14).

The early differentiation of Th2-type cells in our model is dependent on the endogenous production of IL-4, because administration of 11B11 mAb strongly inhibits Th2 cell expansion. In agreement with previous works (22, 35, 36, 37), this early source of IL-4 does not appear to be the NK1.1 T cell subset (38). Indeed, strong Th2-type responses were observed in ß2m° mice that have a reduced number of this T cell population (38). Thus, the IL-4 required for Th2 priming in vivo is likely produced by CD4 T cells that directly recognize allogeneic MHC products on donor-derived APC. The capacity of allospecific T cells to develop either to the type 1 or type 2 cytokine-producing cells, depending on CD8 T cell activation, indicates that they originate from a majority of naive precursor cells not precommitted to a particular Th lineage. This is quite surprising, considering their high precursor frequency (13). What are the mechanisms that drive such polarization? Aside from the cytokine milieu at the time of initial T cell activation, it is clear that other factors such as the dose of Ag can modulate Th1/Th2 differentiation (39, 40, 41). Increasing the density of MHC-peptide ligands at the APC surface has been shown to selectively induce the differentiation of naive T cells into IL-4-producing cells in vitro (39), supporting the notion that initial induction of IL-4-secreting cells is dependent on higher levels of signaling (41). Several lines of evidence indicate that such a requirement could also be achieved in allorecognition. There is now strong evidence that alloreactive T cells recognize the MHC molecule and its associated peptide ligand (13). Among the several thousand different peptides that can be eluted from class II molecules, some of them are represented in sufficient quantity to allow their characterization (42, 43, 44). For instance, I-Ab-E{alpha}52–68 self-peptide complexes are expressed in large amounts on the surface of APC, where they can represent 10–15% of all MHC class II molecules (43). Indeed, it has been recently shown that a significant proportion of alloreactive CD4 or CD8 T cells do react to such highly expressed determinants (45, 46). It seems likely that direct recognition of MHC class II transplantation Ags is the principal pathway involved in the generation of alloreactive CD4 Th2 cells in our models. Therefore, we postulate that the recognition of allogeneic MHC class II ligands expressed at high density on APC may result in early IL-4 production in vivo, thereby directing the entire alloresponse to a Th2-dominated pathway.

An interesting feature of this study is that in addition to Th0/Th2-type cells, alloreactive lymphocytes able to secrete TGF-ß have been activated. Such cells have been initially described following oral administration of protein Ag and were associated with the establishment of Th2-like immunity. These cells, called Th3, were distinguished from Th2 cells by their ability to secrete TGF-ß and were able to suppress Th1 effector functions (47). However, the factors involved in their differentiation in vivo are still not clear. Recently, a subset of regulatory T cells (Tr1) has been defined by its ability to selectively secrete IL-10 alone or in conjunction with TGF-ß (48). These cells were induced to develop in vitro in the presence of IL-10 but not IL-4. The IL-4 independence of regulatory T cells has been illustrated in vivo, where TGF-ß-producing CD45RBlow T cells were shown to develop in IL-4-deficient mice (49). This conclusion is also supported by our experiments showing that TGF-ß production was not significantly affected by blocking endogenous IL-4, whereas this treatment had a profound effect on the expansion of type 2 cytokine-secreting cells. A significant proportion of alloreactive CD4 T cells that develop in our model secrete IL-10 without IL-4; whether these cells represent true Tr1 cells remains to be investigated. In this respect, the model system described in this report could be instrumental in designing immunization regimens able to selectively induce such regulatory cells to study their effector function in allograft rejection in vivo.

Another important conclusion from our work is that the default Th2 pathway of alloreactive CD4 T cell development occurs independently of the genetic background of the host. The importance of MHC polymorphism in the polarization of Th cells has been demonstrated in vivo in response to Leishmania major infection (1) or chronic soluble Ag administration (31), in which strong Th2-type responses were observed in the BALB but not in the B6 background. Such biased Th2 development was also observed in vitro in BALB/c T cells (50) and was associated with the loss of IL-12 responsiveness during the early phase of T cell activation (51). In contrast to the above-mentioned models, the strong priming of type 2 cytokine-producing cells uncovered in the absence of CD8 T cell immunity was similar between the two mouse strains. However, some quantitative differences were seen between BALB/c and B6 alloreactive CD4 T cells. IFN-{gamma}-producing cells still persisted at higher frequency in B6 mice and were composed of a mixture of Th1- and Th0-like cells. Another difference we could observe concerned the production of IL-4 that was consistently lower in B6 Th2 cells and was associated with a diminished frequency of IL-4 and IL-10 double-producing cells as compared with BALB/c CD4 T cells. Finally, BALB/c T cells exhibited an almost unipolar Th2 profile after one round of in vitro stimulation, whereas secondary B6 T cells were still capable of secreting a substantial amount of IFN-{gamma} in response to allogeneic APC (not shown). It is not clear whether the Th0 subset represents a terminally differentiated population or an intermediate stage in Th1 or Th2 development (52, 53). Thus, we could hypothesize that in the BALB background these Th0-type cells would preferentially develop along a Th2-dominated pathway following in vitro restimulation. Conversely, in the B6 genotype they may represent a fully differentiated subpopulation of cells such as Th1- or Th2-type lymphocytes. Whatever the quantitative differences that may exist regarding the heterogeneity of Th subsets between both strains of mice, it seems quite clear that the genotype of the host has little influence on the induction of type 2 cytokine-producing cells in this model.

In contrast to the genetic status of the host, CD8 T cells appear to be one of the principal regulators of the default Th2/Th0-phenotype acquisition in response to allogeneic APC in vivo. Indeed, while donor MHC-reactive CD4 T cells failed to produce Th2-type cytokines in the (A x B)F1->A WT combination, a similar profile of allospecific Th2-dominated responses was observed in both the (A x B)F1 ß2m°->A ß2m° and (A x B)F1 WT->A CD8° combinations, indicating that MHC class I-CD8 T cell interactions were involved. This was not due to the developmental absence of CD8 or MHC class I molecules, because administration of depleting anti-CD8 mAb in adult WT mice was sufficient to unmask Th2 cell priming. Finally, direct evidence for a role of CD8 T cells comes from passive transfer experiments of CD8 lymphocytes into B6 mice with a disrupted CD8 gene. Transfer of CD8 lymphocytes from B6 origin was able to skew the H-2d-specific CD4 response from a Th2/Th0-dominated phenotype to an almost unipolar Th1 response. This effect was correlated to the in situ recruitment of nontolerant CD8 T cells that were likely to be specific for H-2d MHC class I products, because CD8 T cells from B6->CB6 BM chimeras were ineffective in redirecting the CD4 differentiation pathway. CD8 T cells could control alloreactive Th cell development in this model through different mechanisms: 1) by direct killing of the donor APC thereby preventing chronic Ag presentation and/or 2) by the production of cytokines following their activation by allogeneic MHC class I molecules. We have directly addressed the role of IFN-{gamma} production by CD8 T cells in the regulation of DC-induced Th2 cell priming in vivo. In this report we show that the control of alloreactive Th2 responsiveness by CD8 T cells is largely IFN-{gamma} independent. This finding is rather unexpected, because several reports have put forward a major role for this cytokine in the regulation of Th2 cell development in vitro (54, 55) and in vivo in response to viral infection (16). Thus, our data support the challenging hypothesis that in alloimmune response Th2 cell activation/differentiation might be inhibited as a result of APC death, due to perforin and Fas killing by CD8 T cells, and the consequent absence of sustained stimulation rather than a result of IFN-{gamma}-mediated regulation of Th cell differentiation.

Another important question concerns the relevance of our work regarding the mechanisms of T cell priming during allograft rejection. Although it is clear that Th1-dominated responses are involved in acute allograft rejection in MHC class I and II disparate combinations in which both CD4 and CD8 T cells can be involved (4, 8), little is known about the phenotype of alloreactive effector CD4 T cells that develop in situations in which CD8 lymphocytes are absent or not activated. The hypothesis stating that allograft rejection or acceptance is respectively promoted by Th1 or Th2 cytokines has been recently challenged by experiments showing that allograft rejection could occur in the context of type 2 immune responses. For instance, elimination of CD8 T cells does not prevent allorejection, but it results in intragraft Th2-type cytokine production, leading to eosinophil infiltrates (11). Direct evidence for a role of type 2 effector function in allograft rejection comes from recent studies showing that IL-4, IL-5, and eosinophils are critically involved not only in chronic (12) but also in acute (56) rejection of MHC-class II disparate skin grafts. Although these experiments were performed by grafting B6 mice with B6bm12 skin that only differ at 3-aa residues in the IAß class II chain (57), it is likely that this phenomenon could be generalized to other models of MHC class II disparity. Indeed, we have recently shown that induction of neonatal lymphoid chimerism in mice in the absence of MHC class I-T cell interactions results in lethal host-vs-graft disease associated with a chronic Th2 response and hypereosinophilia (10). Strong Th2 priming of the host allorepertoire should be the hallmark of allogeneic interactions that occur across the MHC class II barrier only, while differences at both classes of transplantation Ag should always lead to Th1 development due to CD8 T cell activation.

In conclusion, we have demonstrated that polarization to type 2-producing cells is the default dominant pathway of alloreactive CD4 T cell development in adult mice. Indeed, strong expansion of type 2 cytokine-producing CD4 T cells can readily be obtained in mice primed with DC as soon as MHC class I-restricted CD8 T cells are not activated. Interestingly, this strong priming of type 2 cytokine-secreting cells is associated with the up-regulation of TGF-ß-production. Such a default pathway of alloreactive Th cell differentiation is not dramatically affected by the genetic status of the host and, therefore, might be relevant to various donor/recipient strain combinations. Our study may provide a framework to define the rules that govern allohelper T cell development in vivo and could be instrumental in designing new strategies for immune deviation or selective induction of IL-10- and/or TGF-ß-producing cells that have been shown to regulate both Th1- and Th2-mediated pathological immune responses (47, 48, 58).


   Acknowledgments
 
We thank M. Calise and S. Pilipenko for their skillful technical assistance. We thank Drs. P. Druet and L. Pelletier for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale and by grants from Conseil Général de Région Midi-Pyrénées, Etablissement Français des Greffes, Association pour la Recherche contre le Cancer, and Université Paul Sabatier. G.F. is on a leave of absence from Ecole Nationale Vétérinaire de Toulouse, France. J.D.C. is supported by the Ministère de l’Education Nationale de la Recherche et de la Technologie. Back

2 G.F. and J.D.C. contributed equally to this work. 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, 31 059 Toulouse, France. Back

4 Abbreviations used in this paper: ß2m, ß2-microglobulin; DC, dendritic cells; SC, spleen cells; BM, bone marrow; BM-DC, BM-derived DC; ß2m°, ß2m-deficient; CD8°, CD8-deficient; LNC, lymph node cells; CB6F1, (BALB/c x C57BL/6)F1; WT, wild type. Back

Received for publication December 6, 2000. Accepted for publication August 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reiner, S. L., R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.[Medline]
  2. O’Garra, A., L. Steinman, K. Gijbels. 1997. CD4+ T-cell subsets in autoimmunity. Curr. Opin. Immunol. 9:872.[Medline]
  3. Piccotti, J. R., S. Y. Chan, A. M. VanBurskirk, E. J. Eichwald, K. Bishop. 1997. Are Th2 helper T lymphocytes beneficial, deleterious, or irrelevant in promoting allograft survival?. Transplantation 63:619.[Medline]
  4. Dallman, M. J., C. P. Larsen, P. J. Morris. 1991. Cytokine gene transcription in vascularised organ grafts: analysis using semiquantitative polymerase chain reaction. J. Exp. Med. 174:493.[Abstract/Free Full Text]
  5. Cobbold, S., H. Waldmann. 1998. Infectious tolerance. Curr. Opin. Immunol. 10:518.[Medline]
  6. Powell, T. J., J. W. Streilein. 1990. Neonatal tolerance induction by class II alloantigens activates IL-4-secreting, tolerogen-responsive T cells. J. Immunol. 144:854.[Abstract]
  7. Abramowicz, D., J.-M. Doutrelepont, P. Lambert, P. Van der Vorst, C. Bruyns, M. Goldman. 1990. Increased expression of Ia antigens on B cells after neonatal induction of lymphoid chimerism in mice: role of interleukin 4. Eur. J. Immunol. 20:469.[Medline]
  8. Chen, N., E. H. Field. 1995. Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance. Transplantation 59:933.[Medline]
  9. Donckier, V., M. Wissing, C. Bruyns, D. Abramowicz, M. Lybin, M.-L. Vanderhaeghen, M. Goldman. 1995. Critical role of interleukin 4 in the induction of neonatal transplantation tolerance. Transplantation 59:1571.[Medline]
  10. Coudert, J., G. Foucras, C. Demur, C. Coureau, C. Mazerolles, G. Delsol, P. Druet, J.-C. Guéry. 2000. Lethal host-versus-graft disease and hypereosinophilia syndrome in mice in the absence of major histocompatibility class I-T cell interactions. J. Clin. Invest. 105:1125.[Medline]
  11. Chan, S. Y., L. A. DeBruyne, R. E. Goodman, E. J. Eichwald, D. K. Bishop. 1995. In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection. Transplantation 59:1155.[Medline]
  12. Le Moine, A., V. Flamand, F.-X. Demoor, J.-C. Noël, M. Surquin, R. Kiss, M.-A. Nahori, M. Pretolani, M. Goldman, D. Abramowicz. 1999. Critical roles for IL-4, IL-5, and eosinophils in chronic skin allograft rejection. J. Clin. Invest. 103:1659.[Medline]
  13. Sherman, L. A., S. Chattopadhyay. 1993. The molecular basis of allorecognition. Annu. Rev. Immunol. 11:385.[Medline]
  14. Forsthuber, T., H. C. Yip, P. V. Lehmann. 1996. Induction of Th1 and Th2 immunity in neonatal mice. Science 271:1728.[Abstract]
  15. Srikiatkhachorn, A., T. J. Braciale. 1997. Virus-specific CD8+ T lymphocytes downregulate T helper cell type 2 cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection. J. Exp. Med. 186:421.[Abstract/Free Full Text]
  16. Hussell, T., C. J. Baldwin, A. O’Garra, P. J. M. Openshaw. 1997. CD8+ T cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection. Eur. J. Immunol. 27:3341.[Medline]
  17. Holmes, B. J., P. A. MacAry, A. Noble, D. M. Kemeny. 1997. Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells. Eur. J. Immunol. 27:2657.[Medline]
  18. Rus, V., A. Svetic, P. Nguyen, W. C. Gause, C. S. Via. 1995. Kinetics of Th1 and Th2 cytokine production during the early course of acute and chronic murine graft-versus-host disease. J. Immunol. 155:2396.[Abstract]
  19. Saoudi, A., M. Castedo, D. Nochy, C. Mandet, R. Pasquier, P. Druet, L. Pelletier. 1995. Self-reactive anti-class Th2 cell lines derived from gold salt-injected rats trigger B cell polyclonal activation and transfer autoimmunity in CD8-depleted normal syngeneic recipients. Eur. J. Immunol. 25:1972.[Medline]
  20. Lanier, L. L.. 1997. Natural killer cell receptors and MHC class I interactions. Curr. Opin. Immunol. 9:126.[Medline]
  21. Koller, B. H., P. Marrack, J. W. Kappler, O. Smithies. 1990. Normal development of mice deficient in ß2M, MHC class I proteins, and CD8+ T cells. Science 248:1227.[Abstract/Free Full Text]
  22. 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 ß2-microglobulin-deficient mice. J. Exp. Med. 183:485.[Abstract/Free Full Text]
  23. Fung-Leung, W. P., M. W. Schilham, A. Rahemtulla, T. M. Kundig, M. Vollenweider, J. Potter, W. van Ewijk, T. W. Mak. 1991. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65:443.[Medline]
  24. Gosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[Medline]
  25. Mayordomo, J. I., T. Zorina, W. J. Storkus, L. Zitvogel, C. Celluzzi, L. D. Falo, C. J. Melief, S. T. Ildstad, W. M. Kast, A. B. Deleo, M. T. Lotze. 1995. Bone marrow-derived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic antitumor immunity. Nat. Med. 1:1297.[Medline]
  26. 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]
  27. Prat, M., G. Gribaudo, P. M. Comoglio, G. Cavallo, S. Landolfo. 1984. Monoclonal antibodies against murine {gamma} interferon. Proc. Natl. Acad. Sci. USA 81:4515.[Abstract/Free Full Text]
  28. Cherwinski, H., J. Shumacher, K. Brown, T. Mosmann. 1987. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166:1229.[Abstract/Free Full Text]
  29. Lucas, C., L. N. Bald, B. M. Fendly, M. Mora-Worms, I. S. Figari, E. J. Patzer, M. A. Palladino. 1990. The autocrine production of transforming growth factor-ß1 during lymphocyte activation: a study with a monoclonal antibody-based ELISA. J. Immunol. 145:1415.[Abstract]
  30. 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]
  31. 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]
  32. Lechler, R. I., J. R. Batchelor. 1982. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J. Exp. Med. 155:31.[Abstract/Free Full Text]
  33. Larsen, C. P., P. J. Morris, J. M. Austyn. 1990. Migration of dendritic leukocytes from cardiac allografts into host spleens: a novel pathway for initiation of rejection. J. Exp. Med. 171:307.[Abstract/Free Full Text]
  34. Letterio, J. J., A. B. Roberts. 1998. Regulation of immune responses by TGF-ß. Annu. Rev. Immunol. 16:137.[Medline]
  35. Brown, D. R., D. J. Fowell, D. B. Corry, T. A. Wynn, N. H. Moskowitz, A. W. Cheever, R. M. Locksley, S. L. Reiner. 1996. ß2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses. J. Exp. Med. 184:1295.[Abstract/Free Full Text]
  36. Zhang, Y., K. H. Rogers, D. B. Lewis. 1996. ß2-microglobulin-dependent T cells are dispensable for allergen-induced T helper 2 responses. J. Exp. Med. 184:1507.[Abstract/Free Full Text]
  37. Foucras, G., C. Coureau, Druet P Beijleveld L, A. Saoudi, J.-C. Guéry. 1998. ß2-microglobulin-dependent T cells are not necessary for alloantigen-induced Th2 responses after neonatal induction of lymphoid chimerism in mice. J. Immunol. 161:1751.[Abstract/Free Full Text]
  38. Bendelac, A., M. N. Rivera, S.-H. Park, J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535.[Medline]
  39. Hosken, N. A., K. Shibuya, A. W. Heath, K. M. Murphy, A. O’Garra. 1995. The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-{alpha}ß-transgenic model. J. Exp. Med. 182:1579.[Abstract/Free Full Text]
  40. 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]
  41. Rogers, P. R., M. Croft. 1999. Peptide dose, affinity, and time of differentiation can contribute to the Th1/Th2 cytokine balance. J. Immunol. 163:1205.[Abstract/Free Full Text]
  42. Rudensky, A. Y., P. Preston-Hurlburt, S.-C. Hong, A. Barlow, C. A. Janeway. 1991. Sequence analysis of peptides bound to MHC class II molecules. Nature 353:622.[Medline]
  43. Rudensky, A. Y., S. Rath, P. Preston-Hurlburt, D. B. Murphy, C. A. Janeway. 1991. On the complexity of self. Nature 353:660.[Medline]
  44. Hunt, D. F., H. Michel, T. A. Dickinson, J. Shabanowitz, A. L. Cox, K. Sakaguchi, E. Appella, H. W. Grey, A. Sette. 1992. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad. Science 256:1817.[Abstract/Free Full Text]
  45. Weber, D. A., N. K. Terrell, Y. Zhang, G. Strindberg, J. Martin, A. Rudensky, N. S. Braustein. 1995. Requirement for peptide in alloreactive CD4+ T cell recognition of class II MHC molecules. J. Immunol. 154:5153.[Abstract]
  46. Henderson, R. A., A. L. Cox, K. Sakaguchi, E. Appella, J. Shabanowitz, D. F. Hunt, V. H. Engelhard. 1993. Direct identification of an endogenous peptide recognized by multiple HLA-A2.1-specific cytotoxic T cells. Proc. Natl. Acad. Sci. USA 90:10275.[Abstract/Free Full Text]
  47. Weiner, H. L., A. Friedman, A. Miller, S. J. Khoury, A. Al-Sabbagh, L. Santos, M. Sayegh, R. B. Nussenblatt, D. E. Trentham, D. A. Hafler. 1994. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12:809.[Medline]
  48. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  49. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. Acritical role for transforming growth factor-ß but not interleukin-4 in the sup pression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.[Abstract/Free Full Text]
  50. Hsieh, C. S., S. E. Macatonia, A. O’Garra, K. M. Murphy. 1995. T cell genetic background determines default T helper phenotype development in vitro. J. Exp. Med. 181:713.[Abstract/Free Full Text]
  51. 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]
  52. Kamogawa, Y., L. E. Minasi, S. R. Carding, K. Bottomly, R. A. Flavell. 1993. The relationship of IL-4- and IFN{gamma}-producing T cells studied by lineage ablation of IL-4-producing cells. Cell 75:985.[Medline]
  53. Miner, K. T., M. Croft. 1998. Generation, persistence, and modulation of Th0 effector cells: role of autocrine IL-4 and IFN-{gamma}. J. Immunol. 160:5280.[Abstract/Free Full Text]
  54. Gajewski, T. F., J. Joyce, F. W. Fitch. 1989. Antiproliferative effect of IFN-{gamma} in immune regulation. III. Differential selection of TH1 and TH2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-{gamma}. J. Immunol. 143:15.[Abstract]
  55. Szabo, S., A. S. Dighe, U. Gubler, K. M. Murphy. 1997. Regulation of the interleukin (IL)-12Rß2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817.[Abstract/Free Full Text]
  56. Le Moine, A., M. Surquin, F. X. Demoor, J. C. Noël, M.-A. Nahori, M. Pretolani, V. Flamand, M. Y. Braun, M. Goldman, D. Abramowicz. 1999. IL-5 mediates eosinophilic rejection of major histocompatibility complex class II-disparate skin allografts in mice. J. Immunol. 163:3778.[Abstract/Free Full Text]
  57. McIntyre, K. R., J. G. Seidman. 1984. Nucleotide sequence of mutant I-A ß bm12 gene is evidence for genetic exchange between mouse immune response genes. Nature 308:551.[Medline]
  58. Bridoux, F., A. Badou, A. Saoudi, I. Bernard, E. Druet, R. Pasquier, P. Druet, L. Pelletier. 1997. Transforming growth factor ß (TGF-ß)-dependent inhibition of T helper cell 2 (Th2)-induced autoimmunity by self MHC class II-specific, regulatory CD4+ T cell lines. J. Exp. Med. 185:1769.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
S. Laffont, C. Seillet, J. Ortaldo, J. D. Coudert, and J.-C. Guery
Natural killer cells recruited into lymph nodes inhibit alloreactive T-cell activation through perforin-mediated killing of donor allogeneic dendritic cells
Blood, August 1, 2008; 112(3): 661 - 671.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. F. LaRosa, A. H. Rahman, and L. A. Turka
The Innate Immune System in Allograft Rejection and Tolerance
J. Immunol., June 15, 2007; 178(12): 7503 - 7509.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Laffont, J. D. Coudert, L. Garidou, L. Delpy, A. Wiedemann, C. Demur, C. Coureau, and J.-C. Guery
CD8+ T-cell-mediated killing of donor dendritic cells prevents alloreactive T helper type-2 responses in vivo
Blood, October 1, 2006; 108(7): 2257 - 2264.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Delpy, V. Douin-Echinard, L. Garidou, C. Bruand, A. Saoudi, and J.-C. Guery
Estrogen Enhances Susceptibility to Experimental Autoimmune Myasthenia Gravis by Promoting Type 1-Polarized Immune Responses
J. Immunol., October 15, 2005; 175(8): 5050 - 5057.
[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
B. Adams, N. Nagy, F. Paulart, M.-L. Vanderhaeghen, M. Goldman, and V. Flamand
CD8+ T Lymphocytes Regulating Th2 Pathology Escape Neonatal Tolerization
J. Immunol., November 15, 2003; 171(10): 5071 - 5076.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Buonocore, F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, and V. Flamand
Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo
Blood, February 15, 2003; 101(4): 1469 - 1476.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. D. Coudert, C. Coureau, and J.-C. Guery
Preventing NK Cell Activation by Donor Dendritic Cells Enhances Allospecific CD4 T Cell Priming and Promotes Th Type 2 Responses to Transplantation Antigens
J. Immunol., September 15, 2002; 169(6): 2979 - 2987.
[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