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The Journal of Immunology, 2001, 167: 5034-5041.
Copyright © 2001 by The American Association of Immunologists

Embryonic Thymic Epithelium Naturally Devoid of APCs Is Acutely Rejected in the Absence of Indirect Recognition1

Ricardo Pimenta-Araujo2,*, Laurent Mascarell{dagger}, Michèle Huesca{dagger}, Ana Cumano* and Antonio Bandeira*

* Unité du Développement des Lymphocytes and {dagger} Unité d’Immunophysiologie Moléculaire, Centre National de Recherche Médical, Unité de Recherche Associée 1961, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4+ and CD8+ T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1+ cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4+ T cells do not reveal any significant delay in rejection, even when CD8+ T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transplants of tissues or organs are naturally rejected in genetically different individuals of the same or of different species due to recognition of MHC Ags and/or minor histocompatibility differences.

Allograft rejection is triggered by passenger leukocytes that recirculate and colonize tissues. According to the report of Lafferty et al. (1, 2), tissues artificially depleted of hemopoietic-derived cells (HC)3-professional APCs are rendered less immunogenic, revealing long term allograft survival. Moreover, T cell culture with parenchymal or endothelial cells lacking costimulatory molecules induces allospecific nonresponsiveness (3, 4, 5, 6). Indeed, dendritic cells (DC) have a unique ability to prime naive CD4+ and CD8+ T cells, both in vitro and in vivo (7).

Fetal thymic lobes (TL) of E14 treated in vitro with 2-deoxyguanosine (dGuo) before transplantation into fully mismatched recipients are accepted at a frequency similar to that observed in syngeneic recipients (8). This nucleotide analogue kills dividing immature thymic cells when used at toxic concentrations, and graft acceptance was correlated with depletion of DCs. Curiously, treated explants still reveal significant hemopoietic cell contamination (9) and yet remain accepted. Moreover, allograft survival of dGuo-treated E14TL is observed although expression of class I and class II MHC molecules in the thymic epithelium (TE) is kept at normal levels (10), and T cells of the recipient have normal in vitro proliferation against donor cells (8). However, this state of tolerance is not very robust, because tolerance can be broken by injection of DC (11). In consequence, pure TE has been considered a nonimmunogenic tissue (8, 12).

Third branchial pouches from E10 embryos (E10BPs) harbor the epithelial thymic bud (13), at a stage before hemopoietic colonization. Thus, pure TE can be obtained through dissection of this embryonic structure, without the need for a depleting treatment, that being toxic for developing thymocytes (10, 14) can also affect the TE stroma (10). Furthermore, TE was shown to induce tolerance in birds (15) and in mice (16) to xeno- and allogeneic donor type tissues, respectively, using immunodeficient recipients. In the murine model, the allogeneic TE-nude chimeras reveal lifelong tolerance to skin, heart, islets (R. Pimenta-Araujo, unpublished observations) and thyroid (G. Castro, unpublished observations) of the TE donor origin. Tolerance is maintained by the production of TE-selected regulatory T cells that control donor MHC-reactive T cells (17). Given that TE lacks immunogenicity and also selects a population of regulatory cells, adult immunocompetent recipients should accept allogeneic grafts of pure TE naturally devoid of HC.

At odds with the current dogma in the field, our data show that fresh, nonmanipulated TE is indeed strongly immunogenic and capable of inducing an acute rejection response, in the absence of donor HC-derived APCs. Rejection takes place without T cell indirect recognition of Ags through the host APCs (18, 19, 20, 21, 22). Allograft rejection of embryonic tissues (TE, heart) deprived of HC occurs in the absence of direct, indirect, and cross-priming pathways. Here, we provide in vivo evidence for the existence of a novel pathway of allograft rejection involving direct recognition of parenchymal tissues.


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

BALB/c (H-2d) and C57BL/6 (B6) (H-2b) mice were purchased fromIFFA-CREDO (L’Abresle, France). C57BL/6-nu/nu (B6 nude) (H-2b) mice were purchased from Centre de Développement des Techniques Advancées pour l’experimentation animale (Orleans, France). MHC class II-/-/CD4+ were a kind gift from Dr. L. Glimcher (19) and result from a cross between class II-/- mice and the 36.5-transgenic mouse expressing I-E{alpha}b exclusively in the thymus (23). The resulting line, although selecting a normal CD4+ subset, does not express MHC class II in the periphery.

All mice, including B6 x BALB/c F1 were bred under specific pathogen-free conditions in the animal facilities of the Institut Pasteur (Paris, France). The day of vaginal plug observation was considered as day 0 postcoitum (E0). Embryos were derived either from BALB/c or B6 pregnant females, washed and kept in cold balanced salt solution or HBSS (Life Technologies, Paisley, U.K.) from dissection to transplant. Unless stated otherwise, recipients were 7- to 12-wk-old male mice.

Embryonic tissues

BALB/c and B6 embryos were used as tissue donors. Embryos were staged by somite counting: E10 embryos had 24–32 somites; whereas E8 embryos ranged between 0 and 5 somites. The E8 heart primordium was dissected as seen in (24).

In vitro myeloid colony assay and quantification of precursors

Cell suspensions were obtained from 1–3 explants after treatment with trypsin-EDTA (Life Technologies) for 10 min at 37°C. Ten percent FCS was added to stop treatment. Complete dissociation was obtained by passage through a 1-ml syringe. Cell suspensions were washed twice and resuspended in complete medium; 250 µl cells were placed in 1250 µl complete medium supplemented with 10% FCS, 5 x 10-5 M 2-ME, and 1% IL-3, 1% c-kit ligand, and 4 ng/ml GM-CSF, obtained from supernatant of cDNA-transfected cell lines (a kind gift from Dr. F. Melchers, Basel, Switzerland), all 2-fold concentrated. Finally, 1 ml methylcellulose was added, and this mixture was placed in culture in six-well plates at 37°C. Hemopoietic colonies were counted at day 7 of culture and individually picked for May-Grünwald-Giemsa staining and microscopic observation. Alternatively, liquid cultures were made in the presence of S17 stromal cells and the same cytokine mixture mentioned above plus 1% IL-4 (also a kind gift from Dr. F. Melchers). Plating efficiency of the liquid culture assay was estimated in limiting dilution of Mac-1+- and Mac-1--sorted cells from E10 yolk sacs, in two independent experiments.

Transient mAb therapy

Anti-LFA-1 Ab was obtained from the KBA hybridoma cell line (a kind gift from Dr. R. G. Gill, Denver, CO). Anti-LFA-1 was administered i.p. daily (100 µg/dose) during 7 days, starting on day 0 (day of the transplant) (25). Anti-NK1.1 (PK136) was purchased from Pasteur’s cytofluorometry service, administered at day -3, and then injected weekly (200 µg/dose), as described (26). Depletion efficiency was evaluated by the presence of Ly49D+ cells in the spleen (<=0.03% Ly49D+). The YTS 169.4 Ab (a kind gift from Prof. H. Waldmann, Oxford, U.K.) was used for CD8+ cell depletion. Injections (1 mg/injection) were administered i.p. on day -1 and day 0, as described (27) and confirmed with another anti-CD8 Ab (53-6.7). The frequency of splenic CD8+ T cells was <=0.43%.

Grafts and thymectomies

Mice were opened under anesthesia by ketamine (140 mg/kg) and xylazine (7 mg/kg), both from Sigma (Munich, Germany). E10BP, E10H, and E8H were grafted underneath the kidney capsule.

Thymectomy of BALB/c and class II-/-/CD4+ male recipients was performed at the age of 4 and 6 wk, respectively, under anesthesia, using vacuum pressure after middle sternum incision. Surgical incisions were always mechanically sutured. Graft observation took place at indicated times under anesthesia, before sacrifice by cervical dislocation, or after sacrifice.

Flow cytometry

Grafts were detached from the kidney, and cell suspensions were prepared. Cells were stained with the following mAbs: anti-CD4-FITC or -APC (L3T4); anti-CD8{alpha}-PE, TriColor (CT-CD8a) or APC (53-6.7); anti-CD44-PE (IM7.8.1); anti-NK1.1-PE (PK136); anti-Ly49D-FITC (4E5); anti-TCR{alpha}{beta}-APC (H57-purified in our laboratory); and anti-CD3{epsilon}-biotin (145-2C11). Streptavidin-PerCP was used for biotin-labeled stainings. All Abs were purchased from PharMingen (San Diego, CA) or Caltag (Burlingame, CA), except the anti-TCR{alpha}{beta}.

Peripheral blood was obtained from puncture of the retro-orbital plexus and collected in 25-µl heparin-containing Eppendorf tubes. Samples were incubated with Abs 2-fold concentrated in 96-well plates. RBC lysis and leukocyte fixation with a FACS lysing solution (BD Biosciences, San Jose, CA) were accomplished at room temperature for 10 min. Cells were washed twice in balanced salt solution, and FACS medium without PI was finally added.

Stained cells were analyzed on a FACScan or on a Calibur for four-color staining (both from BD Biosciences), and data were stored and analyzed with CellQuest 3.1 software (BD Biosciences immunocytochemistry systems).

Histology

Kidneys grafted with E10BPs, E10H, and E8H were fixed in Bouin’s solution and embedded in paraffin. Serial sections were stained with H&E.

RT-PCR and semiquantitative analysis

At day 10 post-transplant, 104 cells obtained from individual graft infiltrates were washed and lysed in TRIzol reagent (Life Technologies, Gaithersburg, MD). RNA preparation and RT-PCR were performed as previously described (28). The following primers (50 pmol/reaction) were used: {beta}-actin: 3'-CAC GAT TTC CCT CTC AGC, 5'-GCA CCA CAC CTT CTA CAA; IL2: 3'-TGC TGA CTC ATC ATC GA, 5'-AGG ATG GAG AAT TAC AG; IFN-{gamma}: 3'-CGA CTC CTT TTC CGC TTC CTG AG, 5'-TGA ACG CTA CAC ACT GCA TCT TGG; IL4: 3'-CAT GGT GGC TCA GTA CTA, 5'-GTC TCT CGT CAC TGA CGG C; IL-10: 3'-CTG TCT AGG TCC TGG AGT CCA GCA GAC TCA A, 5'-TCA AAC AAA GGA CCA GCT GGA CAA CAT ACT G; TNF-{alpha}: 3'-CGC ACG TGG AAC TGG CAG AAG, 5'-GGT ACA ACC CAT CGG CTG GCA; TGF-{beta}: 3'-AGG AGC GCA CAA TCA TGT TG, 5'-CGG CAG CTG TAC ATT GAC TT. The conditions were as follows: 30 s denaturation at 94°C, 30 s annealing at 55°C, and 30 sec extension at 72°C. Thirty cycles were used for each PCR, and amplifications were performed in a PCR system 9700. Semiquantitative analyses were performed as previously described (28) by hybridization of PCR products with 32P-labeled specific oligonucleotide probes. Autoradiography was performed and radioactivity was scored using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The values are shown as OD in arbitrary units.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Allografts of E10BP are rejected by immunocompetent recipients

TE is thought to be poorly immunogenic and has the capacity to generate tolerance to other tissues. To induce tolerance to allogeneic tissues in normal mice, we grafted TE isolated before colonization by hemopoietic cells. BALB/c embryos obtained at E10 of gestation (24–32 somites) were used as donors of the third brachial arch region (E10BP). Two to six E10 BPs were grafted underneath the kidney capsule of allogeneic (B6) and syngeneic (BALB/c) adult mice (Fig. 1Go). To exclude competition for thymic precursors between the host thymus and the newly grafted thymic rudiments, we transplanted both euthymic (n = 9 syngeneic and n = 11 allogeneic) or thymectomized recipients (n = 8 syngeneic and n = 21 allogeneic). Mice were sacrificed between 3 and 10 wk post-transplant. There was no significant difference between the two groups. All syngeneic transplants had a large thymic tissue mass, with abundant vascularization (Fig. 1GoA). In contrast, B6 allogeneic recipients consistently rejected the graft showing reduced tissue remaining, (mainly cartilage), with poor vascularization (Fig. 1GoB). Histological analysis confirmed these observations (data not shown). Flow cytometry analysis revealed the typical pattern of a functional thymus in syngeneic grafts, demonstrated by the presence of double-positive (DP) CD4+CD8+ (Fig. 1GoC), suggesting normal T cell development, whereas in allografts only single-positive (SP) cells were found (Fig. 1GoD). Absolute cell numbers of total, DP, and SP populations of the euthymic (data not shown) and thymectomized group confirm a clear difference in the lymphoid content present in allogeneic (~105 cells) compared with syngeneic control grafts (~106–108; Fig. 1GoE).



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  		FIGURE 1. Rejection of E10BP allografts by adult immunocompetent recipients. BALB/c (SYNG) and B6 (ALLO) mice were grafted with two to six E10BPs and sacrificed 4–10 wk later. Macroscopic observation of syngeneic (A) and allogeneic (B) grafts. FACS profiles for the indicated markers from cell suspensions of syngeneic (C) and allogeneic (D) transplants. E, Cell number of the indicated subpopulations in syngeneic (n = 6) and allogeneic (n = 16) thymectomized recipients. Each dot corresponds to one animal. Similar results were obtained in euthymic hosts.

 
Thus, contrary to previous suggestions (8), a fully MHC-mismatched TE is a target for rejection.

TE from BALB/c, transiently parked in B6 nudes, is also rejected in secondary B6 immunocompetent recipients

In comparison with E14 thymic lobes (E14TL), E10BPs harbor a nonfunctional TE, not yet colonized by thymocyte precursors (29). To test the influence of the embryonic stage of the TE used on the rejection phenomenon described above, B6 nude mice were transiently grafted with E10BPs of BALB/c, to allow maturation of the TE and colonization by B6 hemopoietic cells. TE function was evaluated by the presence of circulating CD4+ and CD8+ cells in the blood (data not shown). At 5 wk post-transplant, grafts were dissected out from the enveloping kidney capsule. These now chimeric mature thymi, with TE-BALB/c and bone marrow-derived cells of B6 origin, were then grafted under the kidney capsule of B6 Ly5.1 mice and (B6 x BALB/c)F1 controls. One month later, the grafts were analyzed for cell numbers and FACS staining profiles (Table IGo). Secondary B6 Ly5.1 recipients, incompatible only to the BP of BALB/c, rejected the chimeric transplants, whereas chimeric thymi transplanted in syngeneic F1 controls were accepted as shown by the presence of DP cells (data not shown). Thus, functional TE allografts colonized by syngeneic hemopoietic cells are still rejected.


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  		Table I. Mature TE is rejected in adult allogeneic recipients1

 
Kinetic analysis of lymphoid cells infiltrating E10BP grafts

Next, we characterized the kinetic of cell infiltration during rejection of E10BP allografts, using B6 or BALB/c E10 embryos as donors to B6 adult recipients. All mice were grafted with six pairs of E10BPs underneath the kidney capsule. Recipient mice were sacrificed at days 8, 10, and 12 post-transplant (Fig. 2Go). Allografts show a 3-fold higher number of infiltrating cells than syngeneic grafts, and absolute numbers of CD4+ and CD8+ cells reach a maximum by day 10 (Fig. 2Go, A and B). Thereafter, these values dropped to baseline levels (day 12 onward), indicating a decrease in local inflammatory activity. Allografts showed 2-fold more CD8+ than CD4+ T cells.



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  		FIGURE 2. Kinetic analysis of infiltrating lymphoid cells in syngeneic and allogeneic grafts. E10BP from B6 (SYNG) and BALB/c (ALLO) embryos were grafted in B6 adult recipients, and analyses were performed at days (D) 8, 10, and 12 post-transplant. Absolute cell numbers of CD4+ (A), CD8+ (B), and NK1.1+ (C) are shown. D, Percentages of Ly49D+ cells (gated on the NK1.1+ cells) at day 8 from syngeneic and allografts. E, E10BP grafts performed in B6 nudes and analyzed also at day 8 post-transplant. Each dot represents one animal.

 
Because embryonic tissues may express low levels of MHC class I (30), thus being potential targets of NK activity, we investigated the role of NK1.1+ cells in mediating allograft rejection of TE. Strikingly, we found a major infiltrate of this population (Fig. 2GoC), which at day 8 even exceeded the numbers of CD8+ cells. Characterization of the expression of the Ly49D receptor, which identifies NK cells capable of killing BALB/c (H-2d) targets in vivo (31), showed no enrichment for NK1.1+Ly49D+ cells in allogeneic infiltrates compared with controls (Fig. 2GoD). Furthermore, B6 nude mice that have normal NK cell activity (32), and nevertheless accept solid allografts, showed equal numbers of NK cells in both graft types at day 8 post-transplant (Fig. 2GoE). To formerly exclude an essential role of NK1.1 cells as mediators of rejection, we grafted B6 mice with syngeneic and allogeneic E10BPs and treated them with a depleting anti-NK1.1 Ab, following a protocol that allows NK cell depletion for 8 wk (26). NK1.1-depleted B6 mice also rejected allografts (data not shown), suggesting that the high numbers of NK cells present in allograft infiltrates in normal mice are not required for graft rejection.

LFA-1 Ab treatment has been described to efficiently induce long term tolerance to heart (25) and islet allografts (33) given its role as a costimulatory molecule in TCR-mediated recognition and in cell migration. To ascertain the role of anti-LFA-1 treatment in TE immunogenicity, in comparison with the tissues formerly studied, we used the same protocol to evaluate E10BP allograft acceptance. We observed that allogeneic TE is capable of developing in a normal adult recipient under transient immune suppression (data not shown).

In conclusion, allogeneic E10BPs are acutely rejected by a T cell-mediated immune response.

Infiltrating cells of E10BP allografts display a distinct pattern of cytokine expression

Potential changes in the patterns of cytokine expression of infiltrating T cells could determine syngeneic graft acceptance and allograft rejection. We therefore measured the mRNA accumulation of IL-2, IL-4, IL-10, IFN-{gamma}, TNF-{alpha}, and TGF-{beta} by RT-PCR.

Total infiltrating cells of E10BP syngeneic and allogeneic grafts were obtained, at the peak of infiltration day 10 post-transplant. An adult thymus was used as negative control where all tested cytokines except TGF-{beta} are barely detectable (Fig. 3Go). Expression of IL-2, IFN-{gamma}, IL-10, and TNF-{alpha} is significantly increased in allogeneic grafts. No differences were observed in the patterns of IL-4 and TGF-{beta} expression between the two groups. The increase in IL-2 and IFN-{gamma} expression with no changes in IL-4 is suggestive of a Th1 polarization in these infiltrates. However, IL-10 is also markedly increased in this situation.



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  		FIGURE 3. Cytokine expression in infiltrates of syngeneic (SYNG) and allogeneic (ALLO) E10BP grafts. IL-2, IFN-{gamma}, IL-4, IL-10, TNF-{alpha}, and TGF-{beta} mRNA in total cell suspensions obtained from the grafts at day 10 post-transplant were determined by RT-PCR followed by hybridization of the amplified material with the respective 32P-labeled probes (left). Measurement of {beta}-actin mRNA by RT-PCR in individual samples is also shown. Right, Quantification by PhosphorImager of the hybridized material as OD (arbitrary U x 103). Data are representative of three independent experiments. Exposure time, 1 h.

 
Allogeneic E8 heart transplants are rejected in the absence of APCs

The onset of hemopoietic development takes place at 7.5 days postcoitum (dpc) in the yolk sac. Thus, before the establishment of circulation (starting at the eight-somite stage), tissues in the embryonic body are not yet colonized by hemopoietic cells (34).

To test the natural immunogenicity of other parenchymal tissues, we dissected the E8 heart primordium of embryos at zero- to five-somite stage (BALB/c) and grafted them under the kidney capsule of B6 (n = 6) and syngeneic controls (n = 8). Contrary to functional E10 syngeneic hearts, acceptance of these very immature tissues is given only by the size and histological analysis of the grafts, because there is no contractile activity detected in this case. One month post-transplant, syngeneic grafts were accepted and generated a large vesicle-shaped organ with mature muscle cells, a lining endothelium recovering the interior walls, and no detectable infiltrate (Fig. 4GoA), whereas allogeneic E8 heart grafts were always rejected. In the latter group, no cardiac tissue is present, and besides a dense mononuclear infiltrate, the remaining tissue is of cartilaginous origin (Fig. 4GoB). As summarized in Table IIGo, the results showed that 88% of the syngeneic grafts were accepted whereas all E8 heart primordia were rejected in allogeneic recipients. Essentially the same results were obtained with E10 heart grafts. We thus conclude that embryonic tissues devoid of HCs are immunogenic.



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  		FIGURE 4. E8H allografts analyzed at 4 wk post-transplant. Histological cross-sections stained with H&E at the indicated magnification of syngeneic (A) and allogeneic (B) transplants.

 

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  		Table II. Rejection of allogeneic E8H and E10H transplants1

 
E10BPs and E8 hearts are devoid of myeloid precursor contamination

To confirm that the transplanted tissues are devoid of hemopoietic cell contaminants, we quantified HC precursors in a variety of embryonic tissues using an in vitro assay that allows the generation of myeloid cells, including DCs. E10 brain and E10 limb buds were used as positive and negative controls, respectively, because the brain is colonized very early and the latter provides a sample for blood-borne myeloid precursors. The E8 heart primordium (zero to five somites) is the tissue that allowed us to test the complete lack of hemopoietic cells, because it is dissected at a stage before the beginning of intraembryonic hemopoietic development and still before circulation is established between the embryo and the yolk sac (34). E14TL and adult bone marrow cells were also used as positive controls (Table IIIGo). Colonies were counted for all tested tissues. Myeloid colonies were found at the expected ratios in bone marrow, brain, E14 TL, and E10 hearts. E8 hearts and E10BPs showed fewer colonies than our control for blood-borne myeloid precursors (E10 limb bud). May-Grünwald-Giemsa-stained cells from individually picked colonies systematically revealed myeloid cells with veiled morphology of the plasma membrane, typical of dendritic cells. Cells isolated from E10 BP were also tested in a liquid culture assay that allowed the generation of myeloid cells in 1:1.2 yolk sac cells. Again myeloid colonies were virtually undetectable. These results indicate that E10 BP and E8 hearts are naturally depleted of hemopoietic cells.


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  		Table III. Quantitative in vitro analysis of myeloid precursors present in embryonic tissues1

 
Indirect pathway-deficient mice (class II-/-/CD4+) reject E10BP allografts in <15 days.

The results described above rule out the direct pathway as the mechanism involved in E10BP allograft rejection. Two other possibilities are still available to explain our data. Either CD4+ T cells can be primed through indirect APC presentation of donor-derived peptides (indirect pathway; Refs. 22 and 35) or CD8+ T cells are activated via the recognition of donor peptides bound to MHC class I in the APCs of the recipient (cross-priming; Ref. 36). Class II-/- mice expressing E{alpha} exclusively in the thymus (class II-/-/CD4+ mice; H-2b), have normal numbers of naive CD4+ cells that cannot be primed in the periphery to class-II restricted Ags. These mice were used as recipients to test the role of the indirect pathway in mediating acute allograft rejection. Class II-/-/CD4+ mice were double-grafted with single pairs of E10BPs from BALB/c and B6 embryos, on the lower and upper pole of the left kidney, respectively (Fig. 5Go). Fifteen days later, the graft-infiltrating cells were counted and analyzed by FACS as previously. All allogeneic grafts systematically revealed fewer infiltrating cells (~20-fold) compared with syngeneic controls, taken as a sign of rejection. At this time point, only one-half of the syngeneic grafts showed DP cells (Fig. 5GoA). Thus, allograft rejection still takes place in the first 15 days. Another group of mice (n = 7) was sacrificed at 4 wk post-transplant, and this time all allografts were rejected whereas all syngeneic grafts were functional (Fig. 5GoB).



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  		FIGURE 5. Recipients lacking indirect recognition acutely reject E10BP allografts. Class II-/-CD4+ adult recipients were double-transplanted with 2 E10BPs of B6 (SYNG) and BALB/c (ALLO), on the upper and lower kidney pole, respectively, and sacrificed at day 15 (A) or at 1 mo (B) post-transplant. Thymectomized and CD8+ T cell-depleted class II-/-CD4+ adult mice also received syngeneic and allogeneic E10BP grafts and were studied at day 15 post-transplant (C). CD4+ cell numbers in CD8 cell-depleted recipients are compared with the four mice of A that did not show DP cells in the syngeneic grafts (data not shown). Absolute cell numbers are shown regarding each population.

 
To evaluate the importance of cross-priming in the rejection of embryonic tissues naturally deprived of professional APCs and in the absence of the indirect pathway, class II-/-/CD4+ mice were thymectomized. Two weeks later, these mice were treated with an anti-CD8+-depleting Ab and double-grafted with syngeneic and allogeneic E10BP grafts, as before. At day 15 post-transplant, the infiltrating cells were counted and analyzed (Fig. 5GoC). This approach, although not allowing observation of functional syngeneic grafts because DP cells are also depleted on Ab therapy (data not shown), permitted us to score allograft rejection by comparing total and CD4+ SP infiltrating cell numbers between allogeneic and syngeneic grafts. At day 15 post-transplant, allografts show a 4-fold decrease in total cell numbers and a 6-fold decrease in CD4+ compared with syngeneic graft numbers (Fig. 5GoC). In allografts, CD4+ SP infiltrating cells represent 14 and 17% of that found in infiltrating syngeneic grafts, in the absence of an indirect pathway alone (without CD8+ depletion) and of both indirect and cross-priming pathways (with CD8+ depletion), respectively (Fig. 5GoC). Given this similarity, we assume that the allogeneic E10BPs are rejected.

Thus, we do not observe any significant alteration in the rejection kinetics in the absence of all previously described rejection pathways. In addition, CD8+ T cells do not play an essential role in acute rejection of this tissue.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that transplants of fully allogeneic embryonic tissues, obtained at stages before hemopoietic colonization, are systematically rejected in immunocompetent adult recipients.

It is clearly established that the first hemopoietic cells appear in the yolk sac around 7.5 dpc and that at this stage they belong mostly to the primitive erythroid lineage, generating the first wave of nucleated erythrocytes (37). The emergence of hemopoietic precursors from the embryo occurs only between 9.5 and 12.5 dpc (38), and migration to the BPs starts only at 10.5–11 dpc (29). Most importantly, there is no circulation established between the yolk sac and the embryo before the eight-somite stage (34). On the basis of this knowledge, we used donor tissues that, being devoid of hemopoietic cells, should be accepted indefinitely and eventually induce tolerance (e.g., thymic epithelium) or be rejected much later due to lack of DC (e.g., E8 hearts).

Both the E10BP, which hosts the thymic epithelium anlagen, and the E8 heart primordium are highly immunogenic despite lacking HCs. Moreover, we showed that E10BPs are acutely rejected in <12 days. These results are unexpected because mature DCs are required for the initiation of adaptive immune responses in general, and in particular those involved in allograft rejection (7). In contrast, immature DCs induced unresponsiveness of allogeneic naive T cells, leading to prolonged allograft survival (39). Moreover, it has been shown in vitro that parenchymal tissues per se are not capable of inducing T cell effector activity (3, 4, 5, 6). Altogether, these lines of research led to the contention that embryonic tissues either fully deprived of DC or colonized by immature hemopoietic cells should be accepted or survive longer in allogeneic adult recipients.

Our results are well in line with recent work by Munn et al. (40). In pregnant mice treated with an inhibitor of indoleamine 2,3-dioxygenase, a tryptophan-catabolizing enzyme that keeps the systemic concentration of tryptophan at low levels during pregnancy, semiallogeneic concepti are rejected. This process starts between 7.5 and 8.5 dpc, i.e., 3–4 days after implantation of the embryo. At this stage, hemopoietic precursor activity with myeloid potential is barely detectable in the yolk sac, and therefore mature APCs are virtually absent. These data clearly contradict the idea that embryonic tissues are antigenically immature and fully support our content of an acute allograft rejection of embryonic tissues, in the absence of donor professional Ag presentation.

Our data also imply a reassessment of the danger model (41), especially concerning the importance of professional APCs as regulators of danger signals (42). Both syngeneic and allogeneic donor tissues appear to generate the same inflammatory process but reveal two different graft fates. Thus, acceptance and rejection must depend on other mechanisms of self-nonself discrimination.

Acute allograft rejection is the strongest response the immune system is capable of mounting, with precursor frequencies of alloreactive T cells involved in recognition of peptides bound to allogeneic MHC molecules of 1/20 (43), which contrasts with available precursors reactive against the presentation of a foreign peptide by self APCs of 1/104 (44). Thus far, three main pathways of activation of naive T cells have been described to induce rejection. The direct pathway relies on the recognition of donor DC. The indirect pathway and the cross-priming allow activation of naive CD4+ and CD8+ T cells, respectively, through the presentation of donor-derived peptides expressed on the surface of host APCs. The frequency of naive T cells involved in both the direct and the indirect pathways, although difficult to estimate precisely, is expected to be significantly different, in particular in the case of full MHC differences between the graft and the recipient. Consequently, rejection based on indirect recognition of alloantigens is not acute in normal, unprimed animals. Therefore, the observation that E10BPs are acutely rejected seems incompatible with a significant involvement of the indirect pathways in this process (Fig. 5Go).

During the last years, the role of the indirect pathways in allograft rejection has been extensively studied, although their precise function remains unclear, because they are supposed to be involved both in naive T cell priming and tolerance induction (45). In vitro culture systems that use physical or chemical agents to deplete tissues or organs of passenger leukocytes before transplant were suggested to interfere only with the direct pathway (1, 2). Given the fact that long term allograft acceptance is the common finding in this type of experiments, the relevance of indirect Ag recognition in these transplantation studies becomes largely questioned. In addition, time-dependent acquisition of a state of resistance against rejection, tested by donor spleen cell injections, may favor instead a role for the indirect pathway in tolerance induction (46).

Although in the present study acute allograft rejection was observed, we nevertheless addressed the potential role of the indirect recognition in the rejection of these embryonic tissues.

In MHC class II-/-/CD4+ mice, naive CD4+ T cells cannot be activated on recognition of MHC class II-bound peptides on their own DC. Rejection of E10BP and E8 heart allografts in these mice was not delayed as compared with normal wild-type hosts. These results allow us to conclude that acute rejection of these tissues occurred in the absence of the indirect pathway. We also attempted to evaluate the cross-priming pathway, via CD8 depletion in MHC class II-/-/CD4+ mice. No significant difference in the number of CD4+ T cells that infiltrated E10BP was observed as compared with untreated MHC class II-/-/CD4+ recipients that rejected the allograft. In this experimental situation, the anti-CD8 Ab treatment also depleted CD4+CD8+ cells present in the grafts, our readout for graft acceptance. We could thus not provide functional evidence showing that syngeneic E10BPs have been accepted. Consequently, we could not rule out a role of the cross-priming pathway in this experimental system. However, given the similarity of CD4+ T cell infiltration, with or without CD8+ cells, we find very unlikely that the cross-priming pathway plays a role in the acute rejection of allogeneic E10BPs. Also, CD8+ T cells are not essential in rejection of this tissue, as demonstrated for other tissues (47).

The observation that TE is rejected raises an apparent contradiction with the 2-dGuo-treated E14TL allograft acceptance (8) as well as with other in vitro culture systems for other tissues. Our results imply that besides DC depletion, these treatments must also generally affect TE and other parenchymal tissues.

Passenger leukocytes certainly increase tissue immunogenicity. The link between DC density and immunogenicity of skin grafts explains the faster rejection of thoracic skin compared with skin from other regions (48) and also correlates with strong stimulatory activity of splenocytes in proliferation in vitro assays (49). Increased islet allograft survival correlates with DC elimination, using a depleting Ab treatment in vitro, and DC injection triggers rejection in these tolerant mice (50).

In our experimental system, we could obtain tissues without passenger leukocytes in the absence of in vitro culture treatment; nevertheless, parenchymal tissue immunogenicity was apparent. In fact, culture of thymic lobes or endocrine glands, sometimes up to 4 wk (51), is an artificial protocol that may well have other effects besides simple APC depletion. Furthermore, it is always difficult to formally demonstrate histologically that there are no APCs left. For instance, UV B radiation treatment induces systemic immune suppression (52) and prevents skin hypersensitivity and heart and cornea allograft rejection (53), although no correlation was found between the number and morphology of Langerhans cells and the state of tolerance (54). This state of tolerance is dependent on urocanic acid production by keratinocytes, mimicking a physiological mechanism with potent anti-inflammatory properties, which naturally occurs only on exposure to sunlight (55). Thus, parenchymal cell derived factors can prevent alloreactive T cell responses, consequently conferring active allograft protection, even in the presence of donor APCs. Furthermore, {gamma}-radiation is not as effective as any of the above described procedures on long term allograft tolerance induction (51), although it is still commonly used to deplete hemopoietic cells. Altogether these observations demand a re-evaluation of the role of parenchymal cells in solid allograft acceptance.

Thus far, the evaluation of cytokine expression has given no clear insight on factors associated with acceptance or rejection of grafts (56). Although Th1 cytokines are preferentially increased in our allografts, the concomitant increase in IL-10 illustrates the complexity of the process and is consistent with the failure to induce tolerance via inactivation of cytokines, such as IL-2 (57) and IFN-{gamma} (58).

Acute allograft rejection in the absence of all known pathways of allograft rejection strongly suggesting that a novel pathway operating in vivo is unveiled with this experimental approach. Two major possibilities can be envisaged: 1) naive T cells are capable of being directly activated by cell types that do not belong to the hemopoietic lineage but still behave as APCs (59); 2) rejection may be driven by activated/memory cells (60) that do not require the presence of DCs for effector functions (61). Given the importance of DC in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.


   Acknowledgments
 
We thank Dr. Josselyne Salaün for demonstrating dissection of the third branchial pouches and Patricia Belo for histological preparations, Dr. Laurie Glimcher (Boston, MA) for providing us the MHC class II-/-CD4+ mice, Dr. Ronald G. Gill (Denver, CO) for the KBA hybridoma cell line, Prof. Herman Waldmann (Oxford, U.K.) for the YTS 169.4 Ab, Dr. Isabelle Godin for helpful embryological discussions, and Dr. James Di Santo for revising the manuscript.


   Footnotes
 
1 This work was supported by grants from the Association pour la Recherche sur le Cancer (to A.C.) and by a grant from the Agence Nationale de Recherche sur le SIDA. R.P.-A. is a Gulbenkian Foundation student from the Programa Gulbenkian de Doutoramento em Biologia e Medicina and is supported by Fundação para a Ciéncia e a Tecnologia-Praxis XXI Fellowship BD/9804/96 (Portugal). L.M. is supported by the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (France). Back

2 Address correspondence and reprint requests to Dr. Ricardo Pimenta-Araujo, Unité du Développement des Lymphocytes, Institut Pasteur, 25, Rue du Docteur Roux, 75724-Paris Cedex 15, France. E-mail address: raraujo{at}pasteur.fr Back

3 Abbreviations used in this paper: HC, hemopoietic cells; DC, dendritic cells; TL, thymic lobes; dGuo, 2-deoxyguanosine; E10BP, third branchial pouches from E10 embryos E10H, E10 heart; E8H, E8 heart primordium; dpc, days postcoitum; TE, thymic epithelium; DP, double-positive; SP, single-positive. Back

Received for publication June 4, 2001. Accepted for publication August 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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