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The Journal of Immunology, 1999, 162: 3367-3377.
Copyright © 1999 by The American Association of Immunologists

T Cell Repertoire Alterations of Vascularized Xenografts1

Sophie Brouard*, Bernard Vanhove*, Katia Gagne*, Avidan Neumann{dagger}, Patrice Douillard{ddagger}, Anne Moreau§, Cristina Cuturi* and Jean Paul Soulillou2,*

* Institut National de la Santé et de la Recherche Médicale (INSERM)-Unité 437: "Immunointervention dans les Allo et Xénotransplantations" and Institut de Transplantation et de Recherche en Transplantation (ITERT), Centre Hospitalier Universitaire-Hotel Dieu, Nantes, France; {dagger} Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel; {ddagger} Brigham and Women’s Hospital, Boston, MA 02115; and § Service d’Anatomopathologie, Centre Hospitalier Universitaire-Hotel Dieu, Nantes France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of T cells in the rejection of vascularized xenografts has been little explored. Because of the high potential diversity of xenoantigens, it has been suggested that xenotransplantation could induce a strong cellular response that could contribute to delayed rejection. Alternatively, alterations in molecular interactions could impair the T cell response. Because the analysis of TCR repertoire in vivo indirectly reflects the nature and the magnitude of T cell xenorecognition, we took advantage of the possibility of obtaining long term survival of hamster heart xenografts in rat recipients treated with a combination of cobra venom factor and cyclosporin A (CsA), to analyze T cell infiltration and, for the first time, Vß TCR usage, at the complementarity-determining region 3 level, in accommodated and rejected xenografts, compared with allografts. After withdrawal of CsA (on day 40), the analysis of Vß family expression and corresponding complementarity-determining region 3 lengths in rejected xenografts revealed a Gaussian pattern, in contrast to a much more restricted pattern in rejected allografts (p = 0.002), suggesting that, after withdrawal of CsA, all the underrepresented T cell clones are rapidly expanded in xenografts. These results correlate with the rapid kinetics of rejection (4 ± 1 days), the high number of T cells, the rapid expression of markers of activation (IL-2 receptor {alpha}-chain and class II receptor), and the strong deposit of IgG Abs in rejected xenografts. Taken together, these results suggest that the intensity and diversity of the T cell response to xenografts could be stronger than the response to allografts in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Following allotransplantation of a foreign tissue, T cells can recognize graft MHC Ags in two ways: 1) "direct" recognition of Ags on donor cells, involved in acute allograft rejection 1 ; and 2) "indirect" recognition (or cross priming) of processed donor Ags presented on recipient APCs as peptides combined with MHC molecules 2 . More recently, it has been shown that this second "physiological" pathway also plays a role in allograft rejection 3 and could be involved in the induction of self tolerance or tolerance in some models of allotransplantation 4 .

Alterations of the TCR Vß complementarity-determining region 3 (CDR3)3 length distribution have been observed in vitro and in vivo during allorecognition, using various different techniques 4, 5, 6, 7, 8 . However, although xenorecognition seems to take place equally through direct and indirect pathways in vitro 9 and could involve an exacerbated diversity of presented peptides, there has been no available in vivo study of the TCR repertoire in the xeno situation. While there are indications that T cells play a role in the rejection of nonvascularized xenografts 10 , their role in the rejection of vascularized xenografts remains unclear, and the characteristics of this response in vivo have not yet been explored. One of the most important questions about T cell recognition of xenogeneic tissues to be clarified is whether the diversity of peptide determinants triggering the xenogeneic response makes that response much more extensive than the response to allogeneic tissues, and whether the TCR repertoire shows a more (or less) perturbed pattern in the xenogeneic than in the allogeneic response. This question is indirectly addressed by the degree to which the TCR repertoire is restricted or extended following xenorecognition in vivo, and one of the purposes of our study was therefore to analyze TCR usage at the CDR3 level in xenografts.

Peptides processed from Ags and presented by MHC molecules are specifically recognized by the TCR, a membrane bound heterodimer of three-dimensional structure, composed of {alpha}ß or {delta}{gamma} chains 11, 12 . The TCR-{alpha} and -ß genes are composed of V{alpha} J{alpha} C{alpha} or of Vß Dß Jß Cß segments, respectively, and the variability of the TCR domains is the result of rearrangements of the V, (D), and J segments and of the combinatorial association of the {alpha}- and ß-chains 13 . The CDR3, encoded by the VJ or VDJ junctions and representing much of the diversity of TCR sequences, interacts with antigenic peptides bound to the MHC molecule, while CDR1 and CDR2 interact with the {alpha} helix of the MHC binding site 13, 14 . In this respect, analysis of CDR3 lengths and sequences should prove particularly relevant regarding indirect presentation, since CDR3 interacts with peptides presented by MHC molecules.

For this purpose, we used a hamster to rat concordant model of heart xenotransplantation, in which recipients did not hyperacutely reject their grafts because they were treated with cobra venom factor (CVF) and cyclosporin A (CsA). This protocol produced 60% long term survivors (LTS), the remaining 40% of recipients rejecting their graft, despite treatment, on day 8.2 ± 2.3 15 . However, if CsA is withdrawn on day 40, a time at which any putative T cell-mediated rejection would be likely to take place via "indirect" presentation, XLTS (xenografted LTS) fail to attain tolerance and reject their grafts in 4 ± 1 days. We studied for the first time the degree to which the Vß T cell repertoire at the CDR3 is perturbed in response to xenografts, as well as to allografts, in similarly treated recipients of the same strain, before and after withdrawal of CsA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and surgical procedure

Inbred adult rats (200–250 g) from the LEW.1A (RT1a), LEW.1W (RT1u) congeneic strains and outbred adult Syrian hamsters (100–150 g) were purchased from Janvier (Savigny/Orge, France). All animals were males, 5 to 7 wk of age, maintained in our animal facility under standard conditions according to institutional guidelines. Heterotopic cardiac transplantations were performed aseptically as described by Ono and Lindsey 16 . Ether was used as anesthetic in all procedures. Rejection was indicated when the graft ceased to beat and was confirmed by direct observation of the graft after anesthesia and histological examination.

Immunosuppressive protocol and experimental groups

Two models of cardiac transplantation were studied: a concordant xenograft model using Syrian hamsters as donor and LEW.1A rats as recipients, and an allograft model in which LEW.1W rats were donors and LEW.1A rats, once more, the recipients. Recipients of both types of graft were similarly treated. Purified CVF, kindly provided by Dr. David White (Cambridge, U.K.), was administered to the recipients i.m. at a dose of 0.25 mg/kg on the day before transplantation and at a dose of 0.125 mg/kg every 2 days for 10 days. CsA (Sandimmun, Novartis, Basel, Switzerland) was administered orally at a dose of 10 mg/kg once daily. Several experimental groups of xenografts were studied: a first group was composed of recipients of accommodated xenografts harvested on days 15 and 40 (n = 17). These particular days were chosen for harvesting on the basis that a graft would have a 100% chance of LTS if it beat for longer than the mean + 2 SD (>13.2 days) of treated rejected hearts (8.2 ± 2.3 days) 15 . A second group was composed of the nonaccommodated recipients rejecting their xenografts on 8.2 ± 2.3 days despite treatment (n = 10). A third group was composed of accommodated long term survivors (XLTS) (>40 days) in which the administration of CsA was stopped on day 40 and that rejected their graft in the following 4 ± 1 days (n = 6).

The LEW.1A recipients of LEW.1W allograft hearts were divided into several groups. A first group was composed of recipients harvested on day 15 (n = 5) and day 40 after transplantation (n = 3), when the allograft was still beating and the recipients still being treated with CsA. A second group was composed of allografted LTS (ALTS) in which CsA was withdrawn on day 40 and that rejected their grafts in the following 18 ± 4 days (n = 4). In addition, allografts from recipients receiving the same treatment were harvested at day 4 after withdrawal of CsA. All these recipients were studied at these particular dates to allow optimal comparison of the allo- with the xenograft settings.

Histology and immunohistology

Immunohistology was performed as previously described 15 . Graft and spleen samples were snap-frozen, embedded in Tissue Tek (OCT compound, Sakura Tinetek, Torrance, CA), cut into 5-µm sections, and fixed in acetone. Sections were then labeled using a three-step indirect immunoperoxidase technique with, as primary Abs, the following mouse anti-rat mAbs: OX1 and OX30 directed at the common leukocyte Ag CD45, present on all leukocytes; R7-3 recognizing a constant determinant on rat TCR{alpha}ß; Ox 33, recognizing B cells; ED 2 recognizing macrophages and some dendritic cells; 3.2.3 recognizing the NKR-P1 receptor of NK cells (Serotec, Oxford, England); OX6 recognizing the Class II receptor; and OX39 recognizing the IL2 receptor {alpha}-chain. All these Abs, except the latter, were obtained from the European Collection of Animal Cell Culture (Salisbury, U.K.), purified in our laboratory, and pretested on splenocytes from healthy rats to assess their optimal dilution. Nonspecific staining was controlled for by the omission of the first Ab. For Ig labeling, sections were labeled using a three-step indirect immunoperoxidase technique with, as primary Abs, serial dilutions (1/500, 1/1,000, 1/2,000, 1/4,000, 1/8,000, 1/16,000, 1/32,000) of mouse mAbs to rat IgM and IgG (kindly provided by Dr. H. Bazin, University of Louvain, Louvain, Belgium). Results are expressed as the highest dilution titer of mouse anti-rat IgM and IgG showing positive labeling (end-titer dilution). Graft samples for histology were fixed in 10% formalin solution, paraffin embedded, sectioned, and stained with hematoxylin and eosin.

Quantitative analysis of cellular infiltrate

The area of each immunoperoxidase-labeled tissue section infiltrated by cells was determined by quantitative morphometric analysis. Positively stained cells on each slide were counted by morphometric analysis using point counting analysis 17 with a 121-intersection squared grid in the eyepiece of the microscope. Briefly, the percentage of the area of each graft section occupied by cells of a particular antigenic specificity (area infiltrate) was calculated as follows: [number of positive cells under grid intersection/(total number of grid intersections = 121)] x 100. The graft sections were examined at a magnification of x400. The accuracy of the technique is proportional to the number of points counted. Thus, to maintain a SE of <10%, 15 fields were counted for each labeled section of high density and 40 fields were counted for each labeled section with a low density (<15%). Point counting was performed by two observers. Results are expressed as the percentage of the area of the tissue section infiltrated by leukocytes (±SD) (determined with OX1, OX30 labeling) and the phenotypic composition of the infiltrate and subpopulations which are related to the percentage of total leukocytes and are expressed as the percentage of leukocytes (±SD).

Proliferation assays

In vitro, purified LEW.1A responding T cells (105cells) were stimulated with hamster splenocytes enriched with dendritic cells (105 cells). Cultures were performed in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml of penicillin/streptavidin, 1 mM sodium pyruvate and 5 x 10-5 M ß-mercaptoethanol on days 0, 3, 5 and 7 of culture. Aliquots (minimum of 106 cells) were removed from the culture and RNA was extracted from cells by Immunoscope/Reperturb analysis as described below. Cell proliferation was assessed on day 3 (n= 3), 5 (n = 3), and 7 (n = 3) by measuring [3H]TdR incorporation.

CDR3 length analysis of TCR repertoire

RNA extraction and cDNA synthesis. Total RNA from organs (hearts and spleens) were isolated by the guanidium isothiocyanate procedure and purified on a cesium chloride gradient 18 . Total RNA from cell suspensions were extracted by the method of Chomczynski and Sacchi 19 and 10 µg were reverse-transcribed, using the Boehringer cDNA (Boehringer Mannheim, Indianapolis, IN), and diluted to a final volume of 100 µl.

PCR amplification and elongation reactions for analysis of the CDR3 length. The experimental approach has been described in detail elsewere 20 . Briefly, RNA was reverse-transcribed into cDNA, which was amplified by PCR 21 using a Cß primer and one of the Vß-specific primers 4 . cDNA corresponding to 50 ng of total RNA was amplified for 40 cycles and PCR amplifications were conducted in a 9600 Perkin-Elmer Automate (Perkin-Elmer, Foster City, CA). Amplifications were started with a denaturation step of 20 s at 94°C, followed by 40 cycles, each consisting of 20 s at 94°C, 45 s at 60°C, 45 s at 72°C, ending with a step of 3 min at 72°C 22 . Each product was then used for elongation reaction with each 12 dye-labeled (*) J and Cß specific primers (0.1 µl) 4 . Water replaced cDNA in each Vß subfamily negative control.

Electrophoresis. The runoff reaction products were heat-denatured. 2 µl of the resulting mixture were loaded onto a 6% acrylamide, 8 M urea gel and electrophoresed for 5 h using an Applied Biosystems (Foster City, CA) 373A DNA sequencer.

Sequencing of PCR products. When common public dominant VßJß PCR products were found, PCR products were cloned in a proper vector to be sequenced. VßCß PCR products were subjected to a second amplification for 35 cycles at 60°C with VßJß primers. PCR products were cloned into the PCR Topo Vector (1 µl) of the Topo Ta Cloning Kit (Invitrogen, Carlsbad, CA), in accordance with the manufacturer’s instructions, and transferred into TopF' cells. Plasmid DNA was then recovered by the alkaline lysis method and digested with EcoRI to confirm insertion of clones. Colonies containing individual clones were selected and harvested for each animal presenting an expansion of the TCR profile. 40 µl of plasmid DNA were purified and 5 µg of the preparation were lyophilized and sequenced (Costom DNA sequencing service, Genset, Strasbourg, France).

Analysis of alterations of the TCR repertoire

Immunoscope software 23, 24 was used to obtain a semiquantitative analysis of the TCR repertoire at the CDR3. It provides distribution profiles of CDR3 lengths in amino acids with, typically between 7 and 11 peaks each separated by 3 nucleotides. Each peak corresponds to TCR transcripts with a given CDR3 length but may contain multiple different sequences. The CDR3 expansions arising within a repertoire profile determine the restrictions. Quantitative analysis of these restrictions determines the alterations of the repertoire. When only one sequence is found for the same CDR3 length, the peak signals a monoclonal expansion. If this monoclonal expansion is reproducibly found in several animals, it indicates a public expansion. The number of TCR transcripts with a specific CDR3 length is proportional to the area under each peak. An elevated peak indicates, in general, the existence of an oligoclonal or monoclonal expansion in the polyclonal T cell background, which shows a Gaussian profile. Reperturb software 25 was used to further identify and quantify the alterations in CDR3 distributions of each Vß family and in general in each sample. Briefly, each CDR3 profile, obtained from Immunoscope software, was translated into a probability distribution as described in detail elsewhere 25 . The CDR3 length distributions in PBL of five naive LEW.1A rats were analyzed to establish nondisturbed control profiles and were found to be Gaussian, as previously shown 4 . The average of these distributions, for each Vß family separately, was then used as a control distribution for analysis of the other samples. All perturbations, including those of the controls, were assessed by comparison with the average control profile for each individual Vß family individually. The alteration for a certain CDR3 length in a certain Vß family was then represented by the difference between its frequency in the studied distribution and the control distribution. The alteration per Vß family is defined as the sum of the absolute values of difference for all CDR3 lengths in that profile (see details elsewhere 25). No alteration gives a difference of 0%. A difference of 50% represents a very significant alteration, since it means that any correlation between the repertoire of the studied sample and the control was lost. No signal and very low signal (no clearly individualized peaks) for a certain Vß family are considered as "not available" in calculation and thus did not contribute to the Reperturb assessment of the CDR3 repertoire perturbation. The average of the alterations in all Vß families studied in a sample gives the alteration in TCR repertoire in that sample. Differences in the alteration between different groups of samples were statistically assessed with the Wilcoxon test, significance being established at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Extensive cellular infiltrate and Ig deposits in xenografts after withdrawal of CsA

The nature and magnitude of graft infiltrates in rejected and accommodated xenografts have been described in detail elsewhere 15 . In this model, on day 40 after transplantation, LTS grafts were found to have little leukocyte infiltration (11 ± 1% of total area) represented by 13 ± 1% T cells, 12 ± 1% B cells, and 4% NK cells. Macrophages dominated the graft infiltrate (58 ± 1% of leukocyte infiltrate), and polynuclear cells represented ~5% of the total infiltrate. Late accommodated and rejected hearts after withdrawal of CsA (on day 40) were further analyzed as follows. Xenografts following withdrawal of CsA on day 40 and studied at the time of rejection (on day 44 ± 1) exhibited a rapidly appearing and extensive T cell infiltrate (42 ± 10%) (p = 0.05) (Figs. 1Go and 2A). Analysis of Ig deposits showed consistently a stronger labeling of IgG and IgM in rejected xenografts than in accommodated ones (end points, 1/32,000 vs 1/16,000 and >1/32,000 vs 1/16,000, respectively, for IgG and IgM) (Fig. 2GoB). Interestingly, at rejection time, the infiltration of leukocytes (~30% of the total surface) and T cells (42 ± 10% vs 39 ± 5% of leukocyte infiltrate) was similar in allografts and xenografts (see above) after withdrawal of CsA. In contrast, no significant changes in cellular infiltrate were observed on day 4 after withdrawal of CsA in allografts. In addition, at this time, only few allograft-infiltrating cells exhibited class II and IL-2 receptor {alpha}-chain activation markers. Fig. 3Go shows that at rejection time, mononuclear infiltrated cells exhibited equal expression of activated markers, class II, and the {alpha}-chain of IL-2R. Finally, analysis by histology showed no major vascular damage in either situation.



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  		FIGURE 1. Kinetics and phenotypic analysis of the infiltrate in accommodated xenografts on day 40 after transplantation and in rejected xenografts after withdrawal of CsA. ED2 (macrophages and dendritic cells), 3.2.3 (NKR-P1), R7.3 (TCR{alpha}ß), OX6 (class II), and OX39 (IL-2R) Abs were used. Positively stained cells on each slide were counted by morphometric analysis using point counting analysis. To maintain a SE of <10%, 15 fields were counted for each labeled section of high density, and 40 fields were counted for each labeled section with a low density (<15%). Results are expressed as the percentage of the area of the tissue section infiltrated by leukocytes (±SD) (determined with OX1 OX30 labeling) and the phenotypic composition of the infiltrate subpopulations are related to the percentage of total leukocytes and are expressed as the percentage of leukocytes (±SD). Bars represent results from for three to five grafts analyzed at each time point.

 


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  		FIGURE 2. Cellular infiltrate and Ig deposit in xenografts. A, Cryostat sections of xenograft tissue labeled in rejected grafts (44 ± 1 day) after withdrawal of CsA with OX1 + OX30 (CD45) (x200) (a), R7.3 (TCR{alpha}ß) (x200) (b), or ED1 (anti-macrophages and dendritic cells) (x200) (c). B, End-titer dilutions, using dilutions of anti-rat IgG mAbs, in accommodated (1/16,000) (a) and in rejected xenograft (1/32,000) (b) after withdrawal of CsA.

 


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  		FIGURE 3. Kinetics and phenotypic analysis of the infiltrate in xenografts and allografts. Analysis were performed on day 40 after transplantation in XLTS and in rejected xenografts after withdrawal of CsA. Allografts were analyzed on day 40, on day 4 after withdrawal of CsA, and in rejected allografts 18 ± 4 days after withdrawal of CsA. R7.3 (anti-TCR-{alpha}ß), OX6 (class II), and OX39 (IL-2R) Abs were used. The phenotypic compositions of the infiltrate subpopulations are related to the percentage of total leukocytes and are expressed as the percentage of leukocytes (±SD). Results are expressed as means ± SD of three grafts, examined at each time point.

 
TCR repertoire analysis

The length of the CDR3 region in each Vß family was studied using specific primers for the Cß and 20 Vß of rat TCR in xeno- and allografted recipients 4 under CsA treatment and after CsA withdrawal. The degree of alteration of the T cell repertoire was studied using Reperturb Software. Secondly, some Vß families with altered profiles (restriction, see Materials and Methods) were analyzed using the 12 specific Jß primers 22 . When a single peak was observed, the specific CDR3 region was sequenced. Analysis of the TCR repertoire in spleen cells and PBL from naive LEW.1A rats reproducibly showed the usual Gaussian distribution of CDR3 lengths, already described in the rat in previous studies from our laboratory 4 , which is similar to the profile observed in normal mice or humans 23, 26 . Each TCR Vß family in the LEW.1A rat typically shows between 7 and 11 peaks, spaced by 3 nucleotides (Fig. 4Go). Alteration of each family of the TCR repertoire in naive LEW.1A spleen cells (n = 5) analyzed using Reperturb software ranged from 4.2 to 4.5% (Fig. 5Go), as compared with the average control profile.



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  		FIGURE 4. Vß repertoire in PBL from naive LEW.1A rats and rejected allografts and xenografts after withdrawal of CsA. A higher restricted TCR Vß repertoire is induced in allografts than in xenografts in vivo during the rejection after CsA was withdrawn. Analysis was performed for each Vß family on a naive rat (PBL) (top), an allograft at the time of rejection after withdrawal of CsA, and a xenograft at the time of rejection after withdrawal of CsA (bottom). Each profile represented the Vß repertoire of one animal which was representative of the group.

 


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  		FIGURE 5. Analysis of alterations to the total TCR repertoire in xenografts and allografts. Percentages of alterations in all Vß family patterns were calculated using Reperturb software. The CDR3 length profiles are translated into a function of distribution of the area under the curve for each CDR3 length. The alteration of each CDR3 length is calculated in terms of the differences in area between the distribution of each sample and the distribution of the naive rat control. Details of the method are given elsewhere (see Materials and Methods). Each dot represents the average of the alterations for all the 20 Vß families for each graft. Note the significant difference between the alterations in total repertoire in recipients of xenografts vs allografts at the rejection time following withdrawal of CsA. Analysis was performed on different samples for each point; 5 and 4 xenografts on days 15 and 40 after grafting; 4 and 3 allografts on days 15 and 40 after grafting; 3 xenografts rejected on day 8.2 ± 2.3; 4 xenografts and 4 allografts rejected after withdrawal of CsA. This represents the perturbations in the repertoire of TCR from each of the samples studied, harvested on day 15 after transplantation (A), on day 40 (B), and at rejection (C).

 
TCR repertoire in accommodated xenografts. Since all recipients were from the same LEW.1A rat strain, we speculated that each recipient may mount a response of its own (private response) but would also respond together to xenoantigens in a similar way (public response). We studied the Vß family profiles on days 15 and 40 (n = 17). Alterations of the repertoire analyzed using Reperturb software, which allows comparison of the degrees of alterations of TCR repertoire (average for the 20 Vß families for each animal), ranged from 10 to 21.5% at day 15 (p < 0.01 vs controls) and from 13.9 to 32.6% at day 40 (n = 17) (p < 0.01 vs controls) (Fig. 5Go). There was no statistically significant difference between days 15 and 40.

Analysis of the Immunoscope profile showed a restricted repertoire in accommodated xenografts (Fig. 6Go), most Vß families presenting one or two dominant expansions compared with the naive rat profile. However, such expansions were found in single animals only. No public expansion could be found in accommodated xenografts. One Vß5Jß1.6 oligoclonal expansion in four of five recipients, a corresponding CDR3 length of 9 amino acids, was found and sequenced, but it showed heterogenic sequences without specific selection of junctional motives (data not shown).



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  		FIGURE 6. Vß repertoire in PBL from naive LEW.1A rats, in allografts and accommodated xenografts on day 15 during treatment with CsA. Total RNA was extracted, reverse transcribed, and amplified with the 20 Vß- and Cß-specific primers. A runoff reaction on the 20 PCR products was performed using the Cß fluorescent primer. Runoff products were analyzed in a DNA sequencer (373A, Applied Biosystems). The areas of each peak were obtained with Immunoscope software. Graphs represent the intensity of fluorescence in arbitrary units as a function of runoff product size in nucleotides. Note the Gaussian pattern of naive LEW.1A repertoire and restricted repertoires in allografts and accommodated xenografts on day 15 after transplantation during treatment with CsA. Analysis was performed for each Vß family on a naive rat (PBL) (top), an allograft on day 15 after transplantation, and a xenograft on day 15 after transplantation (bottom). Each profile represented the Vß repertoire of one animal that was representative of the group.

 
TCR repertoire in rejected xenografts. Vß repertoires in early rejecting recipients under CsA therapy (day 8.2 ± 2.3, n = 10) and from LTS rejecting their grafts after withdrawal of CsA (day 44 ± 1, n = 6) were studied.

Overall, total alteration analyzed using Reperturb Software ranged from 11.6 to 21.3% in grafts rejected on day 8.2 ± 2.3 and from 14.4 to 18.1% in those rejected on day 44 ± 1, compared with PBL of naive LEW.1A rats (p < 0.01) (Fig. 5Go). Nevertheless, in xenografts rejected on days 8.2 ± 2.3, Vß families showed low signal patterns, despite usual levels of hypoxanthine phosphoribosyltransferase mRNA, probably because of the low number of infiltrating T cells in this early stage of rejection after transplantation 15 . The TCR repertoire showed a restricted pattern with no public dominant expansion reproducibly found between animals. By contrast, in XLTS grafts rejected 4 ± 1 days after withdrawal of CsA on day 40, all Vß profiles were represented, with a high signal pattern. TCR profiles were also qualitatively different in grafts rejected after withdrawal of CsA in that they were much less restricted than under CsA therapy, resulting in almost total recovery of the Gaussian pattern of the TCR repertoire after withdrawal of CsA (Fig. 4Go). Some dominant Vß13 Jß1.2, Jß1.5 and Jß2.4 rearrangements were observed in hearts from five of six recipients after withdrawal of CsA, with a dominant peak of 162 nucleotides and a CDR3 length of 8 amino acids. Spleens from five of these six rejecting recipients also presented Vß13-dominant rearrangements. Nevertheless, Vß13Jß peaks revealed heterogeneous CDR3 sequences with no selection of junctional motives (data not shown).

TCR repertoire in xeno-MLR. Since rejected hamster hearts harvested from LEW.1A recipients after the withdrawal of CsA could have recruited a larger quantity of uncommitted T lymphocytes, we then conducted an analysis using Immunoscope/Reperturb processing of the amplified CDR3 segment lengths, which results in a pattern similar to that found in vivo. TCR profiles were analyzed after 3, 5, and 7 days of culture. This analysis revealed a Gaussian pattern for the TCR repertoire on each day. The percentage of alteration (Fig. 7Go) was similar to those in vivo (Fig. 8Go). Fig. 7Go shows the Immunoscope profile as well as the tridimensional landscape Reperturb representation.



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  		FIGURE 7. Vß repertoire in PBL from naive LEW.1A rats and in MLR after 5 days of culture. Analysis was performed for each Vß family on a naive rat (PBL) and MLR after 7 days of culture. Each profile represented the Vß repertoire of one animal that was representative of the group. The alteration for each CDR3 length is represented in tridimensional view, by comparing the area between the distribution for each sample and the distribution for the naive rat control. Note the Gaussian pattern for the naive LEW.1A repertoire and MLR on day 5 after transplantation.

 


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  		FIGURE 8. Comparison of repertoires of animals with xenografts and allografts on days 15 and 40 after transplantation and at rejection time, after withdrawal of CsA. Each individual presents alterations of TCR profile. The alteration for each CDR3 length (y-axis, on the top of the figure) is calculated by comparing the area between the distribution of each sample and the distribution of the naive rat control. These alterations are represented in a bi- and tridimensional view of landscape surfaces in which smooth landscapes represent the nondisturbed repertoires, while mountains and valleys denote over- and underamplified CDR3 lengths. Each line crossing the landscape gives the alterations for a specific CDR3 length. Note a significantly higher altered TCR Vß repertoire in allografts after CsA was withdrawn. Analysis was performed for each Vß family on a naive rat (PBL) and an allograft and a xenograft at different time points after transplantation. Each profile represented the Vß repertoire of one animal that was representative of the group.

 
TCR repertoire in allografts under CsA therapy. To compare the repertoire in the responses to allo- and xenografts in a model using the same recipient, LEW.1A rats receiving a MHC-mismatched, congenic LEW.1W heart 27 were treated in the same way as those receiving a xenograft. Heart allografts were then harvested, beating on days 15 and 40 after transplantation, and at the time of rejection, which occurred 18 ± 4 days after the withdrawal of CsA on day 40. In the graft-infiltrating cells of grafts still beating on days 15 and 40, the calculated mean of alterations in the total repertoire ranged from 12.8 to 21.8% on day 15 and from 16.1 to 25.1% on day 40, in comparison to naive rat controls (Fig. 5Go), and there were no significant qualitative and quantitative differences between the alterations of the patterns observed in XLTS xenografts on days 15 and 40 after transplantation under CsA therapy (10–32.6%) (Fig. 5Go). One common dominant Vß13 rearrangement could be found in recipients of allografts on days 15 (n = 4) and 40 (n = 3) after transplantation. However, several Jß rearrangements (Jß1.2, Jß1.4, Jß2.4, and Jß2.5) and heterogeneous sequences were found and were also different from those found in xenografts.

TCR repertoire in rejected allografts. After withdrawal of CsA on day 40 (n = 4), recipients rejected their allografts in 18 ± 4 days, a time significantly longer (p < 0.001) than for xenografts (4 ± 1 days), and presented 22.9 to 32.5% of alterations in the total repertoire (Fig. 1Go). Fig. 8Go shows a general representation of the alterations for each CDR3 length in allo- and xenografts, calculated by comparing the area between the distribution of each sample and that of the naive rat control, which revealed a significantly more restricted repertoire in allografts than in xenografts after withdrawal of CsA (p = 0.002). These alterations are represented by bi- and tridimensional views of landscape surfaces in which smooth landscapes represent the nondisturbed repertoires, while mountains and valleys denote over- and underamplified CDR3 lengths.

These results show that in the same recipient strain, a significantly higher restricted TCR Vß repertoire is induced in allografts than in xenografts in vivo during the rejection process, appearing after CsA was withdrawn.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We studied the TCR repertoire, under CsA therapy and after withdrawal of CsA, in a concordant hamster to rat cardiac xenograft model in which prolongation of graft survival can be achieved by combined treatment with CVF and CsA 28, 29 . It proved possible to obtain 60% XLTS (>40 days), while the remaining 40%, despite treatment, rejected their grafts on day 8.2 ± 2.3. In a previous study, we have shown that the graft leukocyte infiltrate in the xenograft model consisted predominantly of macrophages, with only a few T and NK cells appearing later and representing ~10% on day 40 after transplantation 15 . In the same study, we showed that these numbers are not statistically different from an allograft, in the same strain, when a similar immune treatment was applied.

T cells can recognize xenoantigens in vitro via vigorous "direct" and "indirect" pathways 30 . However, while there are indications that T cells could play a role in the rejection of nonvascularized xenografts 31 , the characteristics of this response in vascularized xenografts remain unclear. One important question, therefore, is whether T cell recognition of xenogeneic tissues, possibly driven by a high diversity of peptides, is more marked than that of allogeneic tissues. In this paper, we have indirectly approached this question in vivo, by analyzing the degree to which the TCR repertoire at the CDR3 level is perturbed in response to xenografts, as well as to allografts, in similarly treated recipients of the same strain, at similar time points before and after withdrawal of CsA. Restriction of the TCR repertoire was defined as the presence of dominant CDR3 lengths within a specific Vß family.

The TCR repertoire of recipients with xenografts under CsA therapy and after CsA withdrawal showed restricted profiles of TCR repertoire but no public T cell response since no common dominant CDR3 length expansion was found. Indeed, whether two dominant Vß5 and Vß13 expansions with common length could be detected, these restrictions were not reproducibly found in all grafts, presented heterogeneous sequences of CDR3, and were not found in blood and spleen cells, suggesting a limited in situ T cell expansion, likely reflecting committed recruitment of T cells, as demonstrated in allografts 22 .

In xenografts rejected on day 8.2 ± 2.3, all Vß family PCR showed low signal patterns. Caution must be taken in the interpretation of altered patterns in these grafts which were rejected early and which were still under the influence of CsA. Indeed, these grafts were not rejected by a T cell dependant process since they only harbored a few T cells (3%) 15 , a situation that can result in an artifactual overestimation of TCR alterations.

After CsA withdrawal, the TCR Vß profile in xenografts quickly recovered a Gaussian profile with the rapid expansion of a large T cell number (Fig. 8Go). In contrast, allografts harbored T cells that showed a very significantly restricted TCR repertoire (p < 0.001) and were less vigorously rejected (18 ± 4 days) (p < 0.001) (Fig. 5Go). It is likely that the recovery of a polyclonal repertoire profile after withdrawal of CsA indicates that the T cell response might be due to the rapid expansion and high recruitment of numerous underrepresented clones that had been silenced under CsA therapy and that expanded after its withdrawal. This hypothesis, favoring the idea of a vigorous T cell response to xenoantigens in the late phase of xenorejection, fits with the large number of T cells and expression of class II and IL-2R activation markers, detected at the time of rejection, similar to that observed in the allogeneic situation, and the absence of vascular histological lesions at the time of rejection. Moreover, the different kinetics of cell infiltrate is exemplified by the study of allografts at day 4, when the infiltrate pattern was not significantly different from ALTS under CsA therapy and was less pronounced than in xenografts at the same time. The fact that rejection occurred much more rapidly than in allografts suggests, however, a contributing role for the high level of T-dependent IgG found in rejected grafts. The strong effect of CsA on the activation of endothelial cells,4 the fact that its withdrawal may further sensitize the graft vasculature to induce IgM and IgG, and the pronounced decrease activation markers during CsA therapy, with almost absent expression of class II or IL-2R in graft infiltrate (Fig. 3Go) 32 show nevertheless that this drug can control most of the recipient immune response against a xenograft in this model. Interestingly, efficient inhibition (~100% of CH50) of complement by CVF treatment both before and after the withdrawal of CsA in XLTS did not significantly modify rejection time (7.5 ± 0.7 vs 7.75 ± 0.5 days), indicating that complement-dependent vascular lesions were not likely to be involved, which fits with the intact vessels observed during histological examination. Finally, no definitive statement of the respective importance of T cell- or IgG-mediated lesions in xenograft rejection can be made at present, and the possibility that other cells (such as NK and macrophages, increasing on the day of rejection) may play a role in the xenorejection process by Ab-dependent cellular cytotoxicity cannot be excluded. Indeed, in athymic rats, rejection of hamster cardiac grafts can occur in the absence of both Abs and T cells 33 .

The fact that recipients that quickly rejected their xenografts (day 8.2 ± 2.3) while still under CsA therapy showed a potential restricted repertoire in contrast to later rejected grafts could be related to the fact that CsA controls clone expansion very well. The number of T cells in the infiltrate is probably too low under CsA to allow any meaningful analysis of TCR diversity (<1% of T cells), and, in any case, strongly suggests that this early rejection is not a T cell-mediated phenomenon but is rather due to the vigorous induction of IgM xenoantibodies 34 .

In vitro studies have shown that an xenogeneic response could be less strong than an allogeneic one 35 . Beside a theoretically increased peptide diversity, maintained or decreased coreceptor functions and costimulator pathways, which have been documented in xenogeneic recognition and which vary greatly according to the combination of species used 36, 37 , are essential in the xenorecognition process. Indeed, in vitro studies have shown that the human anti-porcine T cell response might be similar in strength to the allogeneic response 9 . Preliminary results from our laboratory showed also vigorous direct presentation when rat T cells react to hamster splenocytes enriched for dendritic cells in culture (S. Brouard et al., unpublished observations). However, these interactions varied with the model used. For instance, human CD4 interacts poorly with mice MHC class II 36 . Moreover, the cellular response to a xenograft might well involve more numerous and more complex interactions in vivo than in vitro. For instance, cooperation between adhesion molecules and the normal functions of chemokines in regulating the traffic into the graft of activated cells not primarily involved in the MLR would be likely to interfere in vivo. It seems likely that the evolutionarily close, concordant, hamster to rat combination allows functional cell cooperation during the rejection of a vascularized xenograft, as suggested by the dense T cell infiltrate and activation markers in hamster hearts.

We have shown that during acute interstitial cellular rejection of an allograft, anti-donor-committed cells are in the minority which has been estimated to be at best of 1:10 infiltrating cells 38 . Despite there being no indication that the committed-bystander ratio can be fundamentally decreased in xenografts, one explanation for the difference in TCR alterations observed in allografts and xenografts could be related to the nonspecific accumulation of uncommitted cells in xenografts only. However, the analysis of TCR bias in LEW.1A cultured in vitro against hamster stimulating cells and presenting a good proliferation confirmed the profile observed in vivo. We are currently undertaking experiments aimed at studying quantitative and qualitative interactions that are suggested by this observation.

In summary, our results support the idea that, beside Abs 39 , T cells play a significant role in delayed rejection of vascularized xenografts. They also suggest that the strong response observed might result from a high diversity of presented xenoantigens, as compared with the allogeneic situation. If confirmed in the pig to primate model, these observations suggest that controlling the T cell-mediated immune response would be essential to avoid delayed rejection of xenografts and that CsA could also be an important molecule in maintenance of xenografts.


   Acknowledgments
 
We thank Helga Smit, Claire Usal, and Valia Proust for their help in xenograft procedures; Philippe Loth for his help in animal laboratory maintenance; and G. Gorochov, M. Waer, and M. Bonneville for critical review of the data.


   Footnotes
 
1 This study was in part supported by the "Fondation Transvie." Back

2 Address correspondence and reprint requests to Dr. J. P. Soulillou, INSERM U437, CHU-Hotel Dieu, 30 Boulevarde Jean Monnet, 44093 Nantes Cedex 01, France. E-mail address: Back

3 Abbreviations used in this paper: CDR3, complementarity-determining region 3; CsA, cyclosporin A; CVF, cobra venom factor; LEW, Lewis; LTS, long term survivor; XLTS, xenografted LTS; ALTS, allografted LTS. Back

4 B. Charreau, S. Coupel, F. Goret, C. Pourcel, and J.-P. Soulillou. Association of glucocorticoids and cyclosporin A or rapamycin prevents E-selectin and IL-8 expression during LPS and TNF{alpha}-mediated endothelial cell activation in vitro and in vivo. Submitted for publication. Back

Received for publication August 17, 1998. Accepted for publication December 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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