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The Journal of Immunology, 2004, 172: 1619-1629.
Copyright © 2004 by The American Association of Immunologists

The Role of TNF-Related Activation-Induced Cytokine–Receptor Activating NF-{kappa}B Interaction in Acute Allograft Rejection and CD40L-Independent Chronic Allograft Rejection1

Carole Guillonneau2,*, Cédric Louvet2,*, Karine Renaudin{dagger}, Jean-Marie Heslan*, Michèle Heslan*, Laurent Tesson*, Caroline Vignes{ddagger}, Cécile Guillot*, Yongwon Choi§, Lawrence A. Turka, Maria-Cristina Cuturi*, Ignacio Anegon3,4,* and Régis Josien3,4,*

* Institut National de la Santé et de la Recherche Médicale Unit 437, and Institut de Transplantation et de Recherche en Transplantation, Nantes, France; {dagger} Service d’Anatomie Pathologique du Centre Hospitalier Régional Universitaire de Nantes, Nantes, France; {ddagger} Plateforme de Microscopie Confocale et Imagerie de l’IFR26, Faculté de Médecine, Nantes, France; and Departments of § Pathology and Laboratory Medicine and Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We analyzed the role of TNF-related activation-induced cytokine (TRANCE), a member of the TNF family expressed on activated T cells that shares functional properties with CD40L, and its receptor-activating NF-{kappa}B (RANK) which is mostly expressed on mature dendritic cells, during allogenic responses in vivo using a rodent heart allograft model. TRANCE mRNA was strongly up-regulated in acutely rejected allografts on days 4 and 5 posttransplantation whereas RANK was detected as early as day 1 but did not show further up-regulation during the first week. Immunofluoresence analyses of heart allografts showed that 80 and 100% of TRANCE and RANK-expressing cells were T cells and APCs, respectively. We show for the first time that short-term TRANCE blockade using a mouse RANKIg fusion molecule can significantly prolong heart allograft survival in both rat and mouse models. Similarly, rat heart allografts transduced with a RANKIg encoding recombinant adenovirus exhibited a significant prolongation of survival (14.3 vs 7.6 days, p < 0.0001). However, TRANCE blockade using RANKIg did not appear to inhibit allogeneic T and B cell priming humoral responses against RANKIg. Interestingly, TRANCE blockade induced strong up-regulation of CD40 ligand (CD40L) mRNA in allografts. Combined CD40L and TRANCE blockade resulted in significantly decreased chronic allograft rejection lesions as well as allogeneic humoral responses compared with CD40L blockade alone. We conclude that TRANCE-RANK interactions play an important role during acute allograft rejection and that CD40L-independent allogeneic immune responses can be, at least in part, dependent on the TRANCE pathway of costimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An important role for TNF family members in transplantation immunology has recently emerged. For instance, FasL and TNF-{alpha} are strongly up-regulated in allografts during acute rejection (1) and are likely to play a role in the effector mechanisms leading to graft destruction. More attention has been paid recently to the role of the CD40 pathway of costimulation during allograft rejection. It is now well established that the CD40 pathway is critical in the generation and the control of T-dependent immune responses (2). In rodent (3, 4) and primate (5, 6, 7, 8) models, anti-CD40 ligand (CD40L)5 mAbs prevent acute allograft rejection. However, whether CD40L blockade prevents chronic rejection is still a matter of debate. Despite its crucial role in most Th cell-mediated immune responses, some responses against pathogens such as the lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis viruses can develop in the absence of CD40 (2). This indicates that some pathogens can activate CD4+ T cells using a CD40L/CD40 independent pathway. Therefore, this also suggests that some acute or chronic alloimmune responses may be CD40-independent in vivo.

TNF-related activation induced cytokine (TRANCE), also known as receptor-activating NF-{kappa}B ligand (RANK) ligand, osteoprotegrin ligand, osteoclast differentiation factor, or TNFSF11, is a recently described member of the TNF superfamily of proteins that has emerged as a key regulator of the immune system and of bone development and homeostasis (9, 10). TRANCE, expressed by osteoblast and stromal cells, is necessary and sufficient to induce osteoclast differentiation and activation (11, 12). The activity of TRANCE can be inhibited by the soluble decoy receptor osteoprotegerin that is also produced by osteoblast and stromal cells (13). Moreover, osteoprotegerin can be neutralized by TRAIL, suggesting that TRANCE is part of a complex network that regulates a diverse set of functions (9). In the immune system, TRANCE is expressed by activated CD4+ and CD8+ T cells, with the highest level of expression on CD4+ T cells (14). TRANCE mediates its effects via the TRANCE receptor, also known as RANK or TNFRSF11a. Although RANK mRNA is widely expressed, the distribution of RANK protein appears restricted to dendritic cells (DC) (5), activated T and B cells, osteoclasts, and osteoclast lineage cells (14, 15, 16, 17). High levels of RANK are found on mature DC (15), while low levels of expression are found on activated T and B cells (14). In mice, RANK is expressed by myeloid DC, but is absent, or present at lower levels on lymphoid DC (our unpublished observations).

TRANCE has two major effects on mature DC: 1) it promotes their survival, probably by up-regulating the expression of anti-apoptotic molecules such as BcL-xL (15); 2) it induces the production of multiple cytokines in mature DC such as the proinflammatory cytokines IL-1 and IL-6 and also two cytokines involved in T cell activation and differentiation, IL-12 and IL-15 (14). Therefore, during a productive T cell/DC interaction, TRANCE probably provides survival and regulatory cytokine signals to the DC to amplify its T cell stimulatory capacity. Indeed, we have shown that, in vivo, TRANCE enhanced primary and secondary Ag-specific immune responses induced by S/C injection of mature DC pulsed with a soluble Ag (18). Moreover, following LCMV or vesicular stomatitis virus infection in mice and in the absence of the CD40L/CD40 pathway, the immune system can use the TRANCE/RANK pathway to activate CD4+ T cells (19). Finally, TRANCE-deficient mice lack all lymph nodes but have normal spleen structure and Peyer’s patches (20, 21) and although their DC appear normal, these mice exhibit defects in their early differentiation of T and B lymphocytes (20).

We hypothesized that the TRANCE pathway might be involved in allogeneic immune responses in vivo. We therefore studied the expression of TRANCE and RANK during acute allograft rejection and assessed the effect of TRANCE blockade on acute and chronic allograft rejection in heart allograft models.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cardiac allograft models

Rat model. Eight- to 12-wk-old male Lewis.1W (LEW.1W, haplotype RT1u) and Lewis.1A (LEW. 1A, haplotype RT1a) congeneic rats, obtained from the Centre d’Elevage Janvier (Le Genest-Saint-Isle, France), differed in their entire MHC region. Heterotopic LEW.1W heart transplantation into LEW.1A recipients was performed using Ono and Lindsey’s technique (22) and graft function was evaluated daily by palpation through the abdominal wall. All sentinel rats housed in the same colony were pathogen-specific free.

Mouse model. C57BL/6 mice received vascularized cardiac allografts from BALB/c mice. Donor specific splenocyte transfusion (DST) consisted in 5 x 106 spleen cells given i.v. on the day of transplantation. Fusion proteins/Abs were given at doses of 0.2 mg on days 0, 2, and 4.

Reagents

The RANKIg fusion molecule, made up of the extracellular part of murine RANK and the Fc portion of human IgG1 has been previously described (14, 15). RANKIg specifically binds to murine TRANCE, but not to other known members of the TNF superfamily (14). In control experiments, we found that RANKIg could bind rat TRANCE-tranfected but not untransfected 293T cells as well as activated rat CD4+ T cells (data not shown). RANKIg cDNA was subcloned into a pMT vector (Invitrogen, Carlsbad, CA) and was used to transfect S2 cells. Control human IgG1 was purchased from Sigma-Aldrich (St. Louis, MO). The rat anti-murine RANK mAb (clone 1E6) has been produced in our laboratory and described previously (21). Polyclonal goat anti-murine RANK and TRANCE Abs were purchased from R&D Systems (Minneapolis, MN). Anti-rat TCR{alpha}{beta} (clone R7/3), MHC class II monomorphic (clone OX6), and CD68 (clone ED1) producing hybridomas were obtained from the European Cell Culture Collection (Porton Down, U.K.). Abs were purified from supernatants and conjugated to FITC (Bioatlantic, Nantes, France). Streptavidin-Alexa 568 was purchased from Molecular Probes (Eugene, OR).

Quantitative RT-PCR

Total RNA was purified on a cesium chloride gradient (23) and reverse transcribed into cDNA as previously described (24). Real-time quantitative PCR was performed in an Applied Biosystems GenAmp 7700 Sequence Detection System using SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, CA). The following oligonucleotides were used in this study: rat TRANCE forward: CAGCATCGCTCTGTTCCTGTA; rat TRANCE reverse: GCAAACCTGTATTTTCACGGAG; rat CD40L forward: CAGATGATTGGGTCGGTGC; rat CD40L reverse: CACTGAAGAACAGATGCTGCATT; rat RANK forward: TCTCAGATGTCTTTTCGTCAACAG; rat RANK reverse: AGCCACCACTACCACAGAGATG; rat hypoxanthine phosphoribosyl transferase (HPRT) (5) forward: GCGAAAGTGGAAAAGCCAAGT; rat HPRT reverse: GCCACATCAACAGGACTCTTGTAG. A constant amount of cDNA corresponding to the reverse transcription of 100 ng of total RNA was amplified in 25 µl of PCR mix containing 300 nM of each primer; 200 µM dATP, dGTP, dCTP; 400 µM dUTP; 3 mM MgCl2; 0.25 U of uracyl-N-glycosylase; 0.625 U of AmpliTaq Gold DNA polymerase and 2.5 µl of 10x SYBR Green buffer. The reaction started with a step of 2 min at 55°C to allow the uracyl-N-glycosylase to eliminate putative PCR contaminants, followed by 10 min at 95°C to activate the AmpliTaq Gold DNA polymerase, and then 40 cycles consisting each of 15 s at 95°C and 1 min at 60°C. The real-time PCR data were plotted as the {Delta}Rn fluorescence signal vs the cycle number. The {Delta}Rn values were calculated by the Applied Biostystems 7700 sequence detection software using the formula: {Delta}Rn = (Rn+) -(Rn-), where Rn+ is the fluorescence signal of the product at any given time, Rn- is the mean fluorescence signal during cycles 3–13 and referred to as the baseline. The Ct value is defined as the cycle number at which the {Delta}Rn crosses a threshold. The threshold is set above the background fluorescence to intersect the exponential portion of the amplification curve of a positive reaction. The Ct is inversely proportional to the log amount of template within the PCR. HPRT was used as an endogenous control gene to normalize RNA amounts. The transcript accumulation index (TAI) is expressed as the fold change in mRNA levels between a given sample (transplanted heart) (Q) and a calibrator (a native normal heart in Fig. 1 and an acutely rejected heart allograft from an untreated recipients in Fig. 4) (CB), where the calibrator represents the 1-fold expression of each gene. The TAI is calculated as: TAI = 2-{Delta}{Delta}Ct, where {Delta}{Delta}Ct = (CtTarget-CtHPRT)Q-(CtTarget-CtHPRT)CB. A prerequisite for the use of the 2-{Delta}Ct method is that the efficiency of each gene-specific PCR is >96%, as determined by the slope of the curve Ct = f(log[target DNA]) (25).



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  		FIGURE 1. Expression of TRANCE, CD40L, and RANK mRNA in heart grafts. The expression of TRANCE (A), CD40L (B), and RANK (C) mRNA was assessed in allogeneic and syngeneic heart grafts by real-time quantitative PCR during the first week posttransplantation. Allogeneic heart grafts were rejected in 6.8 ± 1.6 days. The TAI represents the fold change in the amount of transcript in a given sample relative to a normal heart. Each time point is expressed as the mean ± SD of the TAI in three to six animals in each group. *, p < 0.05 for the allogeneic vs the syngeneic group.

 


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  		FIGURE 4. TRANCE blockade in allograft recipients does not inhibit cytokine expression in heart grafts but induces strong up-regulation of CD40L. Five days after transplantation, total RNA was extracted from cardiac allografts that had been untreated or transduced with Addl324 or AdRANKIg. Real-time quantitative RT-PCR was used to analyze transcript levels. The TAI represents the fold change in the amount of transcript in a given sample relative to allografts from untreated recipients (acute rejection) that were used as calibrator for each gene expression. Each time point is expressed as the mean ± SD of the TAI in three to four animals in each group. *, p < 0.05 as compared with untreated or Addl324-treated grafts.

 
Recombinant adenovirus coding for RANKIg, CD40Ig, and gene transfer

The cDNA sequences coding for the extracellular portion of mouse RANK fused to the coding sequences of the constant domains of human IgG1 (14, 15) was subcloned into the pTrack-CMV shuttle plasmid and a recombinant adenovirus (AdRANKIg) was generated according to standard protocols, as previously described (26). The adenoviral vector coding for the extracellular portion of mouse CD40 fused to the coding sequences of the constant domains of human IgG1 (AdCD40Ig) has been described previously (27). The CD40Ig molecule interacts with mouse and rat (27) CD40L. The noncoding adenoviral vector control adenovirus (Addl324) has been described previously (28). The quantification of infectious adenoviral particles (IP) was performed using a replication center assay as previously described (27, 29). Adenovirus stocks were tested for the absence of replication-competent adenoviruses by PCR amplification of the E1 adenoviral region (the detection limit was 1 adenoviral particle in 109 IP). For gene transfer into the heart, recombinant adenoviruses (1–5 x 1010 IP in 150 µl) were slowly injected into the apex and ventricular walls of the heart at four different points as previously described in detail (30). For gene transfer into the liver, recombinant adenoviruses (1 x 1010 IP in 150 µl) were injected into the portal vein as previously described (31).

ELISAs for the detection of RANKIg and CD40Ig

Serum RANKIg was detected using a sandwich ELISA. Plates (Nunc Maxisorb; Nunc, Roskilde, Denmark) were coated overnight at 4°C with anti-mouse RANK mAb (clone 1E6) (50 µl at 10 µg/ml). Plates were incubated (90 min at 37°C) with blocking buffer (PBS containing 0.1% Tween and 10% rat serum) and then incubated with serial dilutions of rat serum in PBS containing 0.1% Tween (2 h at 37°C). After washing, a biotin-conjugated rat IgG-absorbed donkey anti-human IgG (Jackson Laboratories, West Grove, PA) was added and incubated for 1 h at 37°C. Plates were then incubated with HRP-conjugated streptavidin (45 min at 37°C) (Vector Laboratories, Burlingame, CA), the reaction was developed using ABTS (Boehringer-Mannheim, Mannheim, Germany) and the absorbance of duplicate samples read at 405 nm. Purified RANKIg diluted in rat serum was used as a standard. The ELISA detection limit was 0.02 ng/ml.

ELISAs for the detection of Abs against RANKIg

Plates (Nunc Maxisorb) were coated overnight at 4°C with RANKIg (10 µg/ml), blocked with PBS containing 0.1% Tween and 10% rat serum and then incubated with serial dilutions of rat serum in PBS containing 0.1% Tween (1 h at 37°C). After washing, a biotin-conjugated mouse anti-rat IgG Ab (Jackson Laboratories) was added and incubated for 1 h at 37°C. Plates were then incubated with HRP-conjugated streptavidin (45 min at 37°C) (Vector Laboratories), the reaction was developed using ABTS (Boehringer-Mannheim) and the absorbance of duplicate samples read at 405 nm.

ELISAs for the detection of Abs against adenoviruses

Anti-adenovirus Abs were detected by ELISA using a previously described technique (28). Briefly, plates (Nunc Maxisorb) were coated overnight at 4°C with adenoviruses (109 particles in 50 µl PBS), fixed for 20 min with 1% formaldehyde, blocked, washed, and incubated with serial dilutions of sera (all with PBS containing 0.1% Tween and 0.1% BSA). Human IgG-absorbed biotin-conjugated donkey anti-rat IgG ({gamma}-chain specific) or rat IgM (µ-chain specific) (Jackson Laboratories) was added and incubated for 2 h at 37°C. Binding was detected as described above for the detection of CD40Ig by ELISA.

Immunofluorescence and immunohistology studies

Heart allografts were removed embedded in Optimal Cutting Temperature compound (OCT Compound; Tissue Tek, Miles Laboratories, Elkhardt, IN), immediately snap-frozen in liquid nitrogen, and then stored at -70°C until use. Five-micrometer cryostat sections were cut at -20°C, air dried, fixed in acetone for 10 min at room temperature, and stored at -20°C.

For immunofluoresence studies of TRANCE and RANK expression, sections were thawed, fixed in formol calcium for 1 min at room temperature, and incubated overnight at 4°C with anti-TRANCE or anti-RANK polyclonal Abs at 5 µg/ml, followed by biotinylated anti-goat Abs and then a streptavidin-Alexa 568 conjugate. Sections were then incubated with a FITC-conjugated mouse anti-rat leukocyte Ags mAb for 30 min at room temperature and mounted in ProLong AntiFade Kit (Molecular Probes). The fluorescence was observed by confocal microscopy (Leica TCS-SP1; Heidelberg, Germany) ({lambda}ex, 490, and 598 {lambda}em, 516 nm, and 630 nm, respectively). The green and red emissions were collected using two photomultiplier tubes. The grayscale digital images were visualized with a 24-bit imaging system including Leica’s TCS-NT software. The images generated were imported into Adobe Photoshop 6.0 (San Jose, CA), pseudo-colored, and in some cases overlapped to produce merged images. Cell counting was performed manually on images taken at a magnification of x400 by identifying TRANCE or RANK+ cells and then evaluating the coexpression by each cell of the different cell markers (MHC class II, TCR{alpha}{beta}, and TCR{gamma}{delta}). A minimum of 10 sections were analyzed for each marker.

For immunoperoxidase studies, sections were thawed and labeled using a 3-step indirect immunoperoxidase technique. Nonspecific staining was controlled by omission of the primary Ab. Positive cells in each graft were quantified using a square grid (15 fields, x40 objective) and expressed as the percentage of the area of biopsy occupied by cells (32).

Histological and morphometric analysis of cardiac grafts

The upper third of the graft was fixed in paraformaldehyde, embedded in paraffin, and coronal 5 µm sections were stained with H&E-safran. Tissues were analyzed by a pathologist (K.R.) blinded to the study. Three sections of at least 3 different biopsy levels were analyzed for each graft. Only vessels that displayed a clear internal elastica interna were scored. An average of 24 vessel sections were analyzed for each graft. The percentage of vessel occlusion by intimal thickening was determined using the following scoring system: 0 = no occlusion; 1 = <20%; 2 = 20–50%; 3 = 50–80%; and 4 = >80%. Vasculitis was quantified using the following scoring system: 0 = no leukocyte adhesion to the endothelium; 1 = leukocyte adhesion to the endothelium; 2 = leukocyte infiltration of the intima; 3 = fibrosis of the intima; and 4 = leukocyte infiltration of the media. The percentage of pathological vessels was scored taking into account the presence of vessel occlusion and/or vasculitis. Myocardial fibrosis was quantified using the following scoring system: 0 = no fibrosis; 1 = focal; 2 = diffuse-moderate; and 3 = diffuse-severe. Interstitial myocardial leukocyte infiltration was quantified using the following scoring system: 0 = no leukocyte infiltration; 1 = minimal; 2 = mild; 3 = discrete; 4 = moderate; and 5 = severe. Mast cells and eosinophils were evaluated after May-Grünwald-Giemsa staining of cryostat sections and the number of positive cells were counted (10 fields, x40 objective).

Immunization against SRBC

SRBC (109 in 800 µl sterile PBS) were injected i.v. on the day of transplantation. Serum levels of anti-SRBC Abs were determined at day 17 after immunization by incubation of serially diluted heat-inactivated serum with SRBC followed by incubation with human IgG-absorbed biotin-conjugated F(ab') 2 goat anti-rat IgG ({gamma}-chain specific) and with FITC-coupled streptavidin. Ab binding was detected by cytofluorimetry (FACSCalibur; BD Biosciences, San Jose, CA).

Alloantibody detection

Alloantibodies were measured by cytofluorimetry following incubation of serially diluted heat-inactivated serum with splenocytes that had been cultured with Con A for 3 days, followed by incubation with human IgG-absorbed biotin-conjugated F(ab ')2 goat anti-rat IgG ({gamma}-chain specific) or rat IgM (µ-chain specific) (Jackson Laboratories) and with FITC-coupled streptavidin. The results were expressed as mean channel fluorescence for each serum dilution.

Allogeneic CTL detection

Splenocytes were isolated and used directly as effector cells. 51Cr (100 µCi for 1 h at 37°C)-labeled LEW.1W, BN and LEW.1A spleen Con A (2 µg/ml, in the presence of IL-2 at 100 U/ml for 3 days) blasts were used as target cells. The effector and target cells were plated in triplicate in round-bottom 96-well microtiter plates (104 target cells/well) at E:T ratios ranging from 100:1 to 12.5:1 in the culture medium defined above. After incubation (6 h at 37°C), the plates were centrifuged, 51Cr was measured using a beta counter, and the percentage of specific lysis was calculated as 100 x (cpm experimental release - spontaneous release)/cpm (maximal release - spontaneous release). Maximal release: cells incubated with 1% SDS; spontaneous release: cells incubated with medium. Spontaneous release was always <10%.

Statistical analysis

Statistical significance was evaluated using a one-way ANOVA test (Fischer exact test) and Kaplan-Meier analysis of graft survival (log-rank test); p <= 0.05 were considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TRANCE, CD40L, and RANK mRNA in cardiac allografts during acute rejection

We first analyzed, by real-time quantitative PCR, the expression of TRANCE mRNA in acutely rejected heart allografts in LEW.1A recipient rats as compared with syngeneic grafts. Previous studies have shown that the acute allograft rejection that occurs during the first week in this model is associated with a strong up-regulation of both Th1- (IFN-{gamma}, IL-2) and Th2- (IL-4, IL-13) related cytokines, of the TNF superfamily members FasL and TNF-{alpha} and of the activated macrophage products iNOS and IL-10 (1, 24, 33). TRANCE mRNA up-regulation was detected on day 3 and peaked on day 5 in acutely rejected allografts (Fig. 1A). TRANCE expression dramatically decreased by day 7, when all grafts had been rejected in this strain combination (see Table I). In syngeneic heart grafts, TRANCE showed a very low but statistically significant up-regulation on days 3 and 4. Considerable interindividual variations in TRANCE mRNA expression were observed in both allogeneic and syngeneic grafts, accounting for the large SD observed in Fig. 1A. Because TRANCE and CD40L share functional properties (9), we also analyzed the expression of CD40L mRNA. Similar to TRANCE, CD40L mRNA expression was strongly up-regulated on days 4 and 5 during acute allograft rejection (Fig. 1B). Low levels of CD40L mRNA were detected on days 3, 4, and 7 in syngeneic grafts (Fig. 1B).


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  		Table I. Allograft survival

 
We next analyzed the expression of RANK by quantitative RT-PCR. As compared with syngeneic grafts, RANK mRNA expression was increased in rejected allografts (Fig. 1C). Moreover, on day 1, RANK expression was already increased 5-fold and did not further significantly increase until acute rejection. These kinetics of expression contrast with those of TRANCE in the same grafts which showed a progressive increase in expression from day 1 to day 5 (Fig. 1A). The levels of RANK mRNA detected in syngeneic grafts on days 3 – 7 were not statistically different from those found in normal hearts.

Expression of TRANCE and RANK proteins in allografts during acute rejection

We next analyzed the expression of TRANCE and RANK proteins in cardiac allografts during acute rejection using two-color immunofluoresence and confocal microscopy. This analysis was performed on day +5 when TRANCE and RANK mRNA expression were strongly up-regulated in acutely rejected allografts and significantly more than in syngeneic grafts (Fig. 1). Although a mAb to rat CD40L has been described recently (34), it did not stain CD40L on tissue sections, precluding any comparative expression study of TRANCE and CD40L. The expression of TRANCE was detected in this study using a polyclonal Ab against mouse TRANCE. We confirmed that this Ab could stain rat activated CD4+ T cells as well as rat-TRANCE transfected 293-T cells (data not shown). As shown in Fig. 2, on day 5 posttransplantation, TRANCE expression was detected on graft infiltrating cells during acute allograft rejection. We found that all TRANCE-expressing cells were MHC class II- (Fig. 2, G–I) and that 80% of TRANCE-expressing cells coexpressed TCR{alpha}{beta} (Fig. 2, A–F) However, roughly 20% of TRANCE expressing cells did not stain for TCR{alpha}{beta} (Fig. 2, C–E) or {gamma}{delta} (data not shown). The nature of these cells remains unknown.



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  		FIGURE 2. Expression of TRANCE and RANK proteins in allografts. Cryostat sections of heart allografts harvested on day 5 posttransplantation were stained with anti-TRANCE (A–I) or anti-RANK (J–L) goat polyclonal Abs followed by biotinylated anti-goat Abs and then streptavidin-Alexa 568 conjugate. Sections were then stained with FITC-conjugated anti-TCR{alpha}{beta} (A–F) or anti-MHC class II (G–L). Roughly 80% of TRANCE-positive cells were TCR{alpha}{beta} T cells (small arrows, B–F) and 20% stained negative for TCR (large arrows, E–F). All TRANCE positive cells were MHC class II negative (arrows, H–I). Staining intensity for RANK was lower than for TRANCE and gave a higher background signal. Virtually all RANK-positive cells coexpressed MHC class II (arrow, K–L) and were TCR negative (not shown).

 
RANK protein expression was analyzed by immunohistology using a polyclonal Ab against mouse RANK. As shown in Fig. 2, low expression of RANK could be detected on a substantial number of cells on day 5. Virtually all RANK expressing cells coexpressed MHC class II (Fig. 2, J–L) and were negative for TCR (data not shown) suggesting that RANK was expressed on APCs. Several studies have shown that, on day 5 after a heart transplantation most if not all donor MHC class II+ cells have disappeared from the graft due to migration to recipient spleen where they interact with T cells (35, 36, 37). Therefore, it is likely that RANK+ cells are APC from recipient origin.

In vivo TRANCE blockade prolongs heart allograft survival

To assess the role of TRANCE-RANK interactions during allograft rejection, we used a fusion molecule made up of the extracellular domain of murine RANK and the Fc portion of human IgG1 (14). We previously showed that RANKIg specifically bound to murine TRANCE in vitro, but not to other known members of the TNF superfamily (14). In control experiments, we found that murine RANKIg could bind rat TRANCE-transfected but not untransfected 293T cells as well as activated rat CD4+ T cells (data not shown). Allograft recipients treated with 3 i.p. injections of 0.5 mg RANKIg on days 0, 2, and 4 posttransplantation exhibited a significant prolongation of graft survival (21.5 ± 16 days, p = 0.01) as compared with control IgG-injected rats (Table I) (7 ± 0 days). A schedule of two IV injections of 1 mg of RANKIg on days 0 and 2 also led to a comparable prolonged allograft survival (26 ± 12.8 days). However, 3 of 4 animals treated on the day of transplantation with 1 mg of RANKIg rejected their allografts in 8 days, while the remaining survived until day 34. The effect of TRANCE blockade was also assessed in mice using a heart allograft model (BALB/c->B6). TRANCE blockade alone using RANKIg administration did not enhance allograft survival as compared with control mice. However, when B6 mice were given 5 x 106 BALB/c donor spleen cells i.v. on the day of transplantation and treated with either human IgG1 or RANKIg on days 0, 2, and 4 (0.2 mg per injection), a significant prolongation of allograft survival (58.1 ± 32.3, p < 0.001) was observed with 3 of 10 animals exhibiting long term suvival (>100 days) (Table I). Therefore, short-term TRANCE blockade during the first week posttransplantation delays acute heart allograft rejection in both rat and mouse models.

To assess the effect of a prolonged treatment with RANKIg in the rat model, we generated a recombinant adenovirus encoding the RANKIg molecule and transduced heart allografts. Heart grafts were injected with recombinant adenovirus or Addl324 after transplantation into allogeneic hosts. We previously showed that this gene delivery method led to prolonged local and systemic production of the transgene (27, 30). In control in vitro experiments, we detected RANKIg in the supernatant of COS cells transduced with RANKIg (0.5 to 2 µg/ml as measured by ELISA) but not in that of Addl324-transduced cells. Allogeneic grafts transduced with 5 x 1010 IP AdRANKIg (n = 11) exhibited a significant prolongation of survival as compared with grafts transduced with noncoding adenoviruses (Table I) (n = 10) (14.3 ± 3.3 vs 7.6 ± 1.6 days, p < 0.0001). Mean allograft survival was equivalent to that observed with RANKIg injection. To confirm in vivo production of RANKIg in transduced allografts, allografts were stained with a mAb against murine RANK (21). As shown in Fig. 3A, AdRANKIg transduced allografts exhibited a strong and patchy expression of RANK whereas Addl324 transduced allografts stained negative (data not shown). Moreover, we measured the levels of RANKIg fusion protein in the sera of allograft recipients. High levels of RANKIg were detected in the sera of recipients of AdRANKIg-transduced but not Addl324-transduced heart allografts and these levels decreased with time (Fig. 3B). The majority of animals that had received RANKIg transduced allografts exhibited a strong humoral response against RANKIg (Fig. 3D) and against adenoviruses (Fig. 3E). Taken together, these results indicate that the TRANCE blockade alone prolonged cardiac allograft survival but that immunization against the RANKIg fusion molecule and the adenoviral vector might have impaired its blocking action and have shortened the expression of RANKIg, respectively.



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  		FIGURE 3. Expression of RANKIg and detection of anti-RANKIg Abs and anti-adenovirus Abs in recipients of heart allografts expressing RANKIg after gene transfer with or without CD40L-CD40 interaction blockade. A, Expression of RANKIg in allografts transduced with AdRANKIg. Cryostat sections of a cardiac allograft harvested 5 days after transplantation and gene transfer were stained with anti-mouse RANK mAb (clone 1E6) followed by polyclonal anti-mouse Ab conjugated to peroxydase and VIP substrate. No staining was observed in hearts transduced with Addl324 (not shown). B, Detection of serum RANKIg after AdRANKIg-mediated gene transfer. Serum from animals transplanted and transduced with AdRANKIg alone (1010 IP, intragraft, n = 4) was harvested at the indicated time points after gene transfer and analyzed by ELISA (mean ± SD). RANKIg levels in Addl324 or nontreated animals were below the ELISA detection limits (0.02 ng/ml). C, Detection of serum RANKIg after AdRANKIg and AdCD40Ig mediated gene transfer. Serum from animals transplanted and transduced with both AdRANKIg into the graft and AdCD40Ig into the portal vein (1010 and 5 x 1010 IP, respectively, n = 5) was harvested at the indicated time points after gene transfer and analyzed by ELISA (mean ± SD). *, p < 0.05 as compared with RANKIg-treated grafts of B. D, Detection of anti-RANKIg Abs. Serum from animals transplanted and transduced with AdRANKIg or with both AdRANKIg and AdCD40Ig was harvested at day 17 and 100 after gene transfer and analyzed by ELISA (mean ± SD). Each histogram represents the maximum values observed for each animal. Positive samples for AdRANKIg: day 17 for animal 2 and 3 and day 100 for animal 4. Positive samples for AdRANKIg + AdCD40Ig: day 17 for animal 5. E, Detection of anti-adenovirus Abs. Serum from animals transplanted and transduced with Addl324 (n = 6), AdRANKIg (n = 4), AdCD40Ig (n = 6) or with both AdRANKIg and AdCD40Ig (n = 5) was harvested at day 17 after gene transfer and analyzed by ELISA (mean ± SD). *, p < 0.05 for AdRANKIg + AdCD40Ig and AdCD40Ig as compared with the Addl324 and AdRANKIg groups.

 
The effect of TRANCE blockade on allogeneic immune responses in allograft recipients

To analyze the immune mechanisms of TRANCE blockade-induced allograft enhancement in the rat model we first examined heart allografts by immunohistology. On day 5, AdRANKIg-transduced allografts exhibited a heavy leukocyte infiltrate similar to that observed in control allografts (data not shown). Moreover, we did not observe any difference in the phenotype (CD4, CD8, CD11b/c, CD45R, CD25, and MHC class II) of graft-infiltrating cells between the AdRANKIg and Addl324 groups (data not shown). We then measured the expression of several cytokines and surface markers by quantitative RT-PCR in heart allografts. As shown in Fig. 4, TRANCE blockade did not induce significant modification in the expression levels of IL-2, IFN-{gamma}, IL-10, IL-13, or TNF-{alpha}, suggesting that allogeneic T cell priming and differentiation had occurred normally in RANKIg treated animals. Interestingly, we found a 5-fold increase in CD40L mRNA expression in AdRANKIg-transduced allografts as compared with control Addl324-transduced allografts. TAI levels were lower than in Fig. 1 due to the use of untreated allogeneic grafts as calibrator vs native hearts in Fig. 1.

Chronic allograft rejection following CD40L blockade is partially TRANCE dependent

We and others have previously shown that long-term CD40L blockade can induce long-term or indefinite allograft survival (4, 27). However, we found that CD40L blockade did not inhibit chronic allograft rejection, suggesting CD40L-independent allogeneic immune responses in this settings (27). Because CD40Ig-treated animals exhibited strongly reduced immunization toward alloantigens (27), we hypothesized that CD40L blockade might also inhibit immunization toward RANKIg. Moreover, the strong overexpression of CD40L mRNA in AdRANKIg-transduced allografts suggested that TRANCE blockade might have enhanced the CD40L pathway of costimulation which in turn could have abrogated the effect of TRANCE blockade. This prompted us to analyze the effect of combined TRANCE and CD40L blockade in this model. For this purpose, we compared heart allografts transduced with AdCD40Ig or both AdCD40Ig and AdRANKIg. Comparable long-term allograft survival was obtained with combined CD40L and TRANCE blockade (100% of grafts) as compared with CD40 blockade alone (83% of grafts, p = NS). As compared with animals treated with RANKIg alone, cotreatment with CD40Ig significantly increased serum levels of RANKIg (Fig. 3C) and significantly inhibited the production of anti-RANKIg (Fig. 3D) and anti-adenovirus Abs (Fig. 3E).

Long-term heart allografts were then examined for the presence of chronic allograft rejection. As we have previously shown, CD40Ig-transduced allografts exhibited chronic allograft rejection as assessed by the presence of vasculopathy, fibrosis and leukocyte infiltration (Fig. 5A). In allografts from CD40Ig + RANKIg-treated animals (n = 11) we observed a significant decrease in fibrosis (0.86 ± 0.19), infiltration (1.91 ± 0.16), vessel occlusion (1.59 ± 0.32), and vessel lesions (1.68 ± 0.3) as compared with CD40Ig alone (1.42 ± 0.18, p = 0.04; 2.42 ± 0.15, p = 0.026; 2.46 ± 0.26, p = 0.036; 2.35 ± 0.13, p = 0.033, respectively) (Fig. 5A). The diminution of leukocyte infiltration in CD40Ig + RANKIg-transduced allografts was related to a decrease in macrophages and T cells infiltration whose numbers were decreased to the levels observed in syngeneic grafts (Fig. 5B). Interestingly, TRANCE blockade totally inhibited the infiltration by mast cells that occurred in CD40Ig-transduced allografts (Fig. 5C). Mast cell infiltration has been shown to correlate with chronic allograft rejection (38).



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  		FIGURE 5. TRANCE blockade inhibits chronic allograft rejection. The grafts were harvested at day 120 after transplantation and treatment with AdCD40Ig with or without AdRANKIg. A, Morphometric analysis of chronic rejection lesions in RANKIg-treated animals. Paraffin sections were stained with H&E-safran and scored by a pathologist in a blinded fashion. Sections from three different levels of the cardiac base were used to score between 19–50 vessels per graft. The severity of each criteria was graded using a scoring system described in Materials and Methods. Syngeneic, n = 3; CD40Ig, n = 13; CD40Ig + RANKIg, n = 11. B, Quantitative immunohistological analysis of graft-infiltrating leukocytes for the indicated leukocyte populations. Serial cryostat mid-ventricular cross-sections of transplants were analyzed morphometrically and data are expressed as a percentage of biopsy area occupied by cells ± SE. Naive, n = 3; syngeneic, n = 3; CD40Ig, n = 11; CD40Ig + RANKIg, n = 5. C, Mast cell density in allografts. Serial cryostat mid-ventricular cross-sections of transplanted hearts were stained with May-Grünwald-Giemsa. Tissues were analyzed morphometrically and the results are expressed as the number of mast cells per tissue section (mean ± SD). Naive, n = 3; syngeneic, n = 3; CD40Ig, n = 11; CD40Ig + RANKIg, n = 5. *, p < 0.05 as compared with CD40Ig-treated animals.

 
We next examined the humoral allogeneic response in recipients of long term allograft recipients. As we have previously shown, CD40L blockade strongly but incompletely inhibited IgG alloantibody production in allograft recipients (Fig. 6). This residual IgG allogeneic humoral response was significantly reduced in the sera of recipients of CD40Ig + RANKIg-transduced allografts at days 10 (data not shown) and 120 (Fig. 6) after transplantation. Levels of IgM alloantibodies were also significantly reduced (60%) after transplantation and RANKIg treatment at early time points vs Addl324-treated recipients.



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  		FIGURE 6. Suppression of alloantibody production in recipients grafted with RANKIg + CD40Ig-transduced hearts. Serial dilutions of sera from LEW.1A animals nongrafted (naive) (n = 2) or grafted with LEW.1W hearts transduced with the noncoding adenovirus Addl324 (n = 6) or AdRANKIg (n = 4) or AdCD40Ig (n = 6) or AdRANKIg + AdCD40Ig (n = 7) were incubated with LEW.1W Con A blasts and analyzed by cytofluorimetry for the binding of IgG alloantibodies. Sera were collected at day 120 after transplantation. Results are expressed as MCF ± SD. *, p < 0.001 as compared with AdCD40Ig, AdRANKIg, and Addl324-treated grafts.

 
At late time points after transplantation (120 days), the anti-donor CTL activity of splenocytes from recipients treated with both RANKIg and CD40Ig was comparable to that of animals treated with CD40Ig alone (9.9 ± 0.9%, n = 5 and 9.2 ± 1.4%, n = 4, respectively) and both were significantly decreased as compared with that of Addl324-treated recipients that had rejected their grafts (15.7 ± 2.3%, n = 4, p < 0.05), but increased as compared with naive nontransplanted animals (4 ± 0.3%, n = 4, p = <0.05). Finally, combined CD40L and TRANCE blockade did not inhibit systemic T cell dependent Ab responses against SRBC, arguing against the possibility of a general immunosuppression in these allograft recipients (Fig. 7).



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  		FIGURE 7. Systemic T cell-dependent Ab responses against cognate Ags in recipients of AdCD40Ig + AdRANKIg-transduced grafts. Rats grafted with hearts transduced with Addl324 (n = 2) or AdCD40Ig + AdRANKIg (n = 4) were immunized with SRBC on the day of transplantation. Levels of anti-SRBC IgG Abs were analyzed in serially diluted heat-inactivated serum by cytofluorometry at day 17 posttransplantation. Results are expressed as mean channel fluorescence (MCF ± SD).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several members of the TNF superfamily of proteins play crucial roles during immune responses. Here, we provide evidence for the role of a recently described member of this superfamily known as TRANCE in allogeneic immune responses in vivo. TRANCE and its receptor RANK are strongly up-regulated during acute allograft rejection and TRANCE blockade prolonged heart allograft survival both in rats and mice. Moreover, TRANCE blockade improved the chronic allograft rejection that occurred following long-term CD40L blockade, suggesting that CD40L-independent allogeneic immune responses involve the TRANCE pathway of costimulation.

In the immune system, TRANCE expression has been found restricted to activated T cells. We have previously shown that TRANCE is not expressed on the surface of resting murine T cells but under appropriate T cell stimulation in vitro, TRANCE can be detected on the surface of murine T cells after 4 h with a peak between 36 and 48 h, followed by a sustained expression for at least 48 h (14). This is an important difference between TRANCE and CD40L whose expression on activated CD4+ T cells is early but transient (2). This suggests that DC might firstly use CD40L and then TRANCE as a survival and activation signal provided by T cells. Our results indicate that activated T cells infiltrating allografts likely express both TRANCE and CD40L. Although 80% of TRANCE-expressing cells in acutely rejected grafts were T cells, roughly 20% of these cells were negative for both TCR and MHC class II. Importantly, TRANCE can be expressed on bone marrow stromal cells, osteoblasts (9), and endothelial cells (39) and it is possible that TRANCE expression is induced during inflammatory processes on some endothelial or mesenchymal cells in the heart.

In previous studies, we have shown that RANK is highly expressed on mature but not immature DC (15), whereas low levels of expression have also been detected on activated T and B cells (14). Graft infiltrating cells are, in our model, mainly monocytes/macrophages (>70%) and activated T cells (10–15%), whereas B cells are poorly represented (33). Because maximal leukocyte infiltration occurs at day 5 (33), it is likely that the early as well as late RANK mRNA overexpression in rejected grafts does not depend on activated T cells or macrophages but rather on the up-regulation of RANK on resident donor DC or on graft infiltration by recipient DC. This is consistent with the fact that virtually all RANK-expressing cells are MHC class II+. Previous studies have shown that resident tissue DC rapidly migrate and therefore mature following allogeneic transplantation (36). In contrast, early tissue infiltration by DC is a common feature during inflammatory responses (40, 41). An early allograft infiltration by recipient DC has recently been clearly demonstrated by Saiki et al. (37) and another study has shown that the recruitment of recipient DC in kidney grafts can be induced by transplant surgery alone (42). Host DC then recirculate to lymphoid organs where they can prime recipient T cells by the indirect pathway of allostimulation (37).

Our results indicate that TRANCE-RANK interactions play a role during allograft rejection. However, TRANCE blockade delays but does not inhibit acute allograft rejection. In the same strain combination as that used in this study, recipient rats treated with a blocking CD40L mAb throughout the first week posttransplantation exhibited a prolongation of allograft survival similar to that observed with RANKIg (43). However, long-term blockade of the CD40L pathway alone using CD40Ig (27) but not of the TRANCE pathway was sufficient to induce long-term allograft survival. Because high levels of circulating RANKIg were detected in the sera of animals grafted with a AdRANKIg-transduced allografts (>100 µg/ml) during the first month, it is unlikely that we would be able to obtained better allograft survival than those described here with TRANCE blocking reagents alone. However, it is possible that the anti-RANKIg Abs that appear in these animals might decrease the efficiency of TRANCE blockade. It is interesting to note that a short-term treatment with RANKIg induced a better allograft survival than a prolonged treatment as that obtained with RANKIg adenovirus transduction. This might be related to a deleterious effect of adenovirus injection of heart tissue or to inhibition of active regulatory mechanisms induced by TRANCE (44). Although TRANCE blockade appeared to strongly up-regulate CD40L mRNA expression in heart allografts, these results suggest that the immune system cannot fully compensate for the transient absence of the CD40L or TRANCE pathway to mount an effective T cell response and therefore that these pathways of DC activation do not have completely overlapping functions.

The CD40L pathway of costimulation plays an important role in T cell and T-dependent B cells responses. However, CD40L-deficient mice can mount effective immune responses against several pathogens such as LCMV or the influenzae virus. Bachman et al. (19) have shown that TRANCE is critical for CD40L-independent Th cell responses against LCMV. CD40L blockade has shown great promise in clinical transplantation. However, several studies have demonstrated that short-term as well as prolonged CD40L blockade inhibits acute but not chronic rejection (27, 45, 46). When CD40L blockade was combined with injection of donor spleen cells at the time of grafting, chronic heart allograft rejection was prevented in a mouse model (47) but not in a rat model (43). The key feature of chronic rejection in heart allografts is a progressive vasculopathy that includes intimal thickening, smooth muscle cell proliferation, and vessel obstruction. The mechanisms of these lesions are not fully understood although factors such as oxido-reduction, alloantibodies, mastocytes, CTLs, and IFN-{gamma} have been proposed (48). Chronic rejection lesions following CD40L blockade are diminished by association with blockade of other costimulatory pathways, such as those triggered by inducible costimulator (49) or programmed death-1 (50). Our results strongly suggest that the TRANCE pathway plays at least a partial role in the chronic allograft rejection that occurred in recipients of CD40Ig transduced heart allografts. The mechanisms of this effect need to be clarified, however, our results suggest a role for alloantibodies. We have shown that RANK is expressed on activated B cells (14) and TRANCE deficient mice exhibit a defect in their early B cell progenitors (20). Although we could not detect any significant in vitro biological effect of recombinant TRANCE on B cells (14), it is possible that in the absence of CD40L signaling, TRANCE-RANK interactions play a role in B cell function, despite the fact that anti-viral Ab responses were not affected in RANKIg-treated mice (19). In contrast, the inhibitory effect of TRANCE blockade on alloantibody production in CD40Ig-treated animals might be explained by a synergistic inhibition of Th cell activation similar to that observed by the simultaneous blockade of both pathways in mice in the LCMV model (Abs not affected by stimultaneous blockade of CD40 and RANK) (19). This effect on T cells is suggested by the fact that TRANCE blockade almost completely inhibits T cell infiltration in long-term allografts from CD40Ig-treated rats.

Finally, a recent report indicates that TRANCE could induce angiogenesis (51) in human endothelial cells, suggesting that TRANCE-RANK interactions might play a direct role in the early stage of vasculopathy. The fact that TRANCE can also be induced on some endothelial cells by TGF-{beta} (39), a cytokine that is strongly up-regulated in rejected allografts (24), suggests an autonomous model of TRANCE-dependent endothelial cell proliferation.

Mastocytes have been associated with chronic rejection (52) and eosinophils play a role in chronic rejection in the absence of CD40-CD40L interactions and CD8+ cells (46). We did not observe eosinophils in any of the experimental conditions of our chronic rejection model in rats. The fact that mastocytes were increased in hearts with chronic rejection after CD40Ig-treatment and were significantly inhibited by treatment with RANKIg suggests that these cells and some of their products, such as bFGF, may play a role in the pathogeneis of chronic rejection. These results also suggest a possible role for TRANCE in mastocyte biology that could explain previous reports of mast cell activation by surface molecules on T cells (53). Finally, CTLs and CD8+ T cells have been implicated in the pathogenesis of chronic rejection in certain models (48) and following interruption of CD40-CD40L interactions, depletion of CD8+ cells synergizes with the neutralization of IL-4 for the prevention of chronic rejection (46). CTL activity was comparable in recipients treated with RANKIg and CD40Ig vs CD40Ig alone and permanent depletion of CD8+ cells in rats treated with CD40Ig did not modify the intensity of chronic rejection lesions (our unpublished data), suggesting that CD8+ cells do not play a major role in this chronic rejection model.

In conclusion, our results indicate that the TRANCE pathway of costimulation plays a role during acute as well as chronic allograft rejection. The fact that TRANCE blockade delayed but did not inhibit acute rejection indicates, however, a partial role and that TRANCE-RANK interaction is not required for efficient allogeneic T cell priming in vivo. It is possible that, as recently shown for another member of the TNF superfamily, namely CD134 (54), the role of the TRANCE-RANK pathway is more critical for effector or memory cell activation than for naive T cells. This is also suggested by the fact that both CD4+ and CD8+ memory, but not naive T cells, constitutively express TRANCE mRNA in vivo (14). Taken together, these results indicate a high-level complexity in the regulation of these independent costimulation pathways during T cell-APC interaction.


   Acknowledgments
 
We are grateful to Claire Usal, Bernard Martinet, and Emmanuel Merieau for animal care, and to the researchers that contributed with reagents. We thank the Vector Core of the University Hospital of Nantes, (Nantes, France), supported by the Association Française contre les Myopathies, for producing the adenoviral vectors used in this study.


   Footnotes
 
1 This work was supported by a grant from the Roche Organ Transplantation Research Foundation (ROTRF, 438528894). Back

2 C.G. and C.L. contributed equally to this report. Back

3 I.A. and R.J. are both senior and corresponding authors. Back

4 Address correspondence and reprint requests to Drs. Régis Josien or Ignacio Anegon, Institut National de la Santé et de la Recherche Médicale Unité 437, Centre Hospitalier Régional Universitaire de Nantes, 30 Boulevard Jean Monnet, 44093 Nantes Cedex 1, France. E-mail addresses: rjosien{at}nantes.inserm.fr or ianegon{at}nantes.inserm.fr Back

5 Abbreviations used in this paper: CD40L, CD40 ligand; LCMV, lymphocytic choriomeningitis virus; TRANCE, TNF-related activation induced cytokine; RANK, receptor-activating NK-{kappa}B; DC, dendritic cell; HPRT, hypoxanthine phosphoribosyl transferase; TAI, transcript accumulation index; AdRANKIg, recombinant adenovirus; Addl324, control adenovirus; IP, infectious adenoviral particles; DST, donor-specific splenocyte transfusion. Back

Received for publication June 18, 2003. Accepted for publication November 19, 2003.


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