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The Journal of Immunology, 2000, 165: 3469-3483.
Copyright © 00 by The American Association of Immunologists

Coronary Arteries from Human Cardiac Allografts with Chronic Rejection Contain Oligoclonal T Cells: Persistence of Identical Clonally Expanded TCR Transcripts from the Early Post-Transplantation Period (Endomyocardial Biopsies) to Chronic Rejection (Coronary Arteries)1 ,2

C. A. Slachta*, V. Jeevanandam{dagger}, B. Goldman{ddagger}, W. L. Lin* and C. D. Platsoucas3,*

Departments of * Microbiology and Immunology, {dagger} Surgery, and {ddagger} Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic cardiac allograft rejection presents pathologically as graft arteriosclerosis (GA) characterized by recipient T cell and monocyte infiltration. To determine whether oligoclonal T cells are present in coronary arteries of cardiac allografts from patients with GA, we conducted sequencing analysis of ß-chain TCR transcripts from these explanted coronary arteries using the nonpalindromic adaptor-PCR. Substantial proportions of identical ß-chain TCR transcripts in three of five patients were observed, clearly demonstrating the presence of oligoclonal T cells. TCR transcripts from the arteries of two other patients were relative heterogeneous. High proportions of identical CDR3 ß-chain TCR motifs were found in each patient. GENEBANK/EMBL/SWISS PROT database comparison of all sequences revealed that these ß-chain TCR transcripts were novel. Using Vß-specific PCR (independent amplification), we found in patient GA03 that the TCR transcript that was clonally expanded in the left anterior descending artery after nonpalindromic adaptor-PCR was also clonally expanded in the right coronary artery of the same allograft. These results demonstrate that this TCR transcript was clonally expanded at different anatomic sides of the cardiac allograft in a systemic manner. In two patients identical ß-chain TCR transcripts that were found to be clonally expanded in the coronary arteries of their explanted cardiac allografts were also found to be clonally explanted in endomyocardial biopsies collected 17 and 21 mo earlier from each patient. The presence of oligoclonal populations of T cells in the rejected graft suggest that these T cells have undergone specific Ag-driven proliferation and clonal expansion early on within the graft and persist throughout the post-transplantation period.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic rejection of cardiac allografts is the major limitation of graft survival 1 year post-transplantation, and it is found in ~50% of the cardiac allograft recipients 5 years post-transplantation (1, 2, 3, 4, 5). Chronic rejection is associated with graft arteriosclerosis (GA)4 or transplantation-associated arteriosclerosis, which pathologically presents as diffuse, concentric, stenosing fibrointimal proliferation within the coronary arteries of allografts and eventually leads to obliterative arteriopathy and graft failure (1, 2, 3, 4, 5, 6, 7). The pathology of GA is different in several aspects from that of naturally occurring arteriosclerosis (6, 7).

Cell-mediated immune responses and perhaps injury of the arteries by an allogeneic immune response may be the primary cause of the development of GA (3, 4, 5, 6, 7, 8, 9, 10). T cells may play a major role in the pathogenesis of GA. The development of GA can be enhanced in rats with cardiac allografts by prior sensitization of recipient animals to donor splenic lymphocytes (11). In contrast, rats transplanted with cardiac allografts from syngeneic animals did not develope GA (12). These results suggest the involvement of an alloantigen-driven T cell response in the pathogenesis of chronic rejection. Although this immune response appears to be the dominant factor, Ag-independent and nonimmunological factors, such as ischemia-reperfusion, viral infection, hyperlipidemia, insulin resistance, and hypertension, may also contribute to the development of GA (reviewed in Refs. 13, 14, 15).

Mononuclear cell infiltrates are commonly associated with GA and have been observed in the intima (16) or subendothelial space (17) of cardiac allograft arteries of patients with GA. These infiltrates are comprised predominantly of T cells and monocytes (6, 16, 17, 18, 19, 20, 21, 22, 23, 24) of recipient origin (24), which are believed to represent an immune response of the donor to the graft. These T cells may play a major role in the immunopathogenesis of chronic rejection. The majority of the infiltrating T cells appear to be CD8+CD4- (17, 20, 21, 22, 23), although equal ratios of CD4+CD8- and CD4-CD8+ lymphocytes have also been reported (18). Perforin-positive CTL have been identified in coronary arteries of patients with GA and may contribute to the development of disease (25). These CD8 cells may be responsible, directly or indirectly, for endothelial cell injury, which is an important step in the development of GA (17).

More than 90% of T cells in the peripheral blood express the {alpha}ß TCR, which is comprised of two highly polymorphic disulfide-linked peptide chains, the {alpha}-chain and the ß-chain. Both are members of the Ig supergene family (26). T cells of the recipient recognize organ grafts by two different pathways of Ag presentation, the direct and the indirect (27, 28, 29, 30, 31, 32, 33, 34, 35). Antigenic specificity for the TCR is primarily associated with the sequences of the hypervariable, or CDR3, part of the ß-chain TCR transcript (36, 37, 38, 39).

To elucidate whether T lymphocytes infiltrating coronary arteries of cardiac allografts from patients with chronic rejection contain oligoclonal populations of T cells, we amplified ß-chain TCR transcripts from the coronary arteries of five explanted cardiac allografts with GA by the nonpalindromic adaptor PCR (NPA-PCR) (40, 41, 42, 43) and by Vß-specific PCR. The NPA-PCR method is specifically designed for the amplification of transcripts with unknown 5' ends, such as TCRs and Igs (40, 41, 42, 43). The amplified transcripts were cloned and sequenced. Sequence analysis revealed substantial proportions of identical ß-chain TCR transcripts, strongly suggesting the presence of oligoclonal T cells infiltrating coronary arteries with GA. In one patient, identical TCR clones were found to be clonally expanded in the left anterior descending (LAD) and the right coronary artery (RCA) of the same explanted allograft with chronic rejection. Additionally, sequence analysis of archived endomyocardial biopsy material from two patients with GA demonstrated the presence of clonally expanded ß-chain TCR transcripts identical with those found to be clonally expanded in the coronary arteries of these patients many months later.


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

Explanted cardiac allografts from five adult patients (Table IGo; mean age, 45.8) who had undergone primary, single-organ, orthotopic heart transplantation and developed chronic rejection were used in this study. Post-transplantation care in all cases was routine. These studies have been approved by the Temple University Hospital Institutional Review Board.


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  		Table I. Demographic and clinical characteristics of patients with chronic cardiac allograft rejection

 
Cardiac explants

Coronary arteries from cardiectomy tissue obtained immediately before each patient’s retransplantation were used. Each patient was diagnosed with moderate (n = 1) to severe (n = 4) GA upon pathological examination, based on the observation of circumferential fibrointimal thickening, lumenal narrowing, and stenosis (20–90%; mean, 56%) in the main coronary arteries as well as the presence of stenosis in multiple small epicardial coronary branches. Samples of coronary arteries were taken from the proximal, middle, and distal thirds of each of two major epicardial arteries (RCA and LAD) and were chosen to represent the most abnormal portion of each segment. Tissue were snap-frozen within 1–2 h of procurement. The endomyocardium of the explants from patients GA05 and GA09 demonstrated focal moderate acute cellular rejection consistent with International Society of Heart and Lung Transplantation (ISHLT) rejection grade 3A/4. The remaining three explants demonstrated either no acute cellular rejection (GA02, GA03), or grade 1A (minimal) acute rejection (GA06). Evidence of a Quilty effect (endocardial lymphoid infiltrate) (44) was present in all five explants studied.

Endomyocardial biopsies (EMBX)

EMBXs were obtained from patients GA05 and GA09 as part of routine post-transplantation care and monitoring. EMBX were snap-frozen and stored in liquid nitrogen. EMBXs used in this study were collected 13 and 16 mo post-transplantation (and 17 and 28 mo before retransplantation with a second cardiac allograft) from patients GA05 and GA09, respectively. These EMBXs were chosen on the basis of the pathology observed in each specimen. Rejection grade in each EMBX was diagnosed in accordance with the guidelines established by the ISHLT (45). EMBX from patient GA09 demonstrated grade 1A rejection (focal perivascular or interstitial infiltrate with no evidence of necrosis) as well as evidence of atherosclerosis. EMBX from patient GA05 exhibited grade 3A rejection (necrosis as well as evidence of diffuse inflammatory processes) and mild atherosclerosis.

Controls

Human PBMC were prepared as previously described (40) and were used as methodological controls. Control epicardial arteries, designated control arteries 1 and 2, respectively, were obtained from a nontransplanted normal adult male heart and from autopsy material of an adult female who died due to unrelated disease (diabetes mellitus).

Histology and immunohistochemical staining

Cardiectomy sections were embedded in paraffin. Serial 6-µm sections were stained using hematoxylin and eosin and were evaluated for the presence of GA by routine pathological examination. Immunohistochemistry was performed (46, 47) using the anti-CD3 mAb, clone NCL-CD3-PS1 (Novocastra, Newcastle upon Tyne, U.K.) and an isotype-matched nonspecific mouse IgG as a negative control.

RNA isolation

RNA from sections of LAD or RCA, EMBX, and from PBMC were prepared using a guanidinium thiocyanate solution (Stratagene, La Jolla, CA) as recommended by the manufacturer.

cDNA synthesis

Double-stranded cDNA was synthesized from oligo(dT)-NotI (Promega, Madison, WI)-primed total RNA by use of the Superscript II (Life Technologies, Grand Island, NY) cDNA synthesis kit. Double-stranded cDNA was blunt ended (for efficient adaptor ligation) using T4 DNA polymerase.

Nonpalindromic adaptor-PCR (NPA-PCR)

NPA-PCR was conducted as previously described with minor modifications (40, 41, 42, 43). Briefly, double-stranded blunt-ended cDNA was ligated with an equivalent molar concentration of nonpalindromic adaptor, which consisted of two complimentary oligonucleotides (Table IIGo), EcoRI-XmnI and XmnIG, that were preannealed to each other. cDNA and adaptor were incubated at 16°C overnight with T4 DNA ligase (1.2 U) and purified on a G-50 column (5 Prime 3 Prime, Boulder CO). NotI restriction endonuclease (20 U) treatment for 2 h at 37°C was then used to remove ligated adaptor from the 3' end of the cDNA. The resultant cDNA was repurified on a G-50 column, and 0.5 vol of eluate was amplified using two rounds of PCR in a 100-µl reaction. In the first amplification 5' amplification primer was the adaptor primer, EcoRI-XmnI, and a human HCß3 primer was used as the 3' amplification primer (100 pmol each; Table IIGo). In the second amplification, the adaptor primer was again used as the 5' amplification primer, and the HCß2 primer, located 5' to the HCß3 primer (nested design), served as the 3' amplification primer. In addition to the primers and cDNA, each PCR contained the following: 2.5 mM MgCl2, 5 U Taq DNA polymerase with appropriate buffer (Promega), and 0.25 mM dNTPs (Pharmacia, Piscataway, NJ). PCR for the first amplification (30 cycles) included denaturation (94°C, 45 s), annealing (45°C, 45 s), and elongation (72°C, 45 s), followed by final incubation at 72°C for 10 min. PCR for the second amplification (35 cycles) included denaturation (94°C, 45 s), annealing (50°C, 45 s), elongation (72°C, 45 s), and a final incubation at 72°C for 10 min.


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  		Table II. Adaptor and TCR ß-chain primer DNA sequences used in this study

 
Vß-chain-specific amplification

Vß-chain specific amplifications of TCR transcripts were used to examine, in more detail, a single Vß family or subfamily or for confirmation purposes if there was a reason to believe that ß-chain TCR clonal expansion(s) was present in a particular Vß family. On the basis of this criteria, the following Vß families were amplified: Vß2.1, Vß3.1, Vß4.1, Vß5.1, Vß9.1, and Vß12.1 (Table IIGo). Different Vß families were chosen for amplification in each patient based on the NPA-PCR results. 5' amplification primers (Vß) and the 3' amplification primer (hCß2) are shown in Table IIGo. Template cDNA was synthesized from RCA total RNA isolated from the same cardiectomies used for analysis by NPA-PCR or from archived EMBX. PCR for each amplification (35 cycles), included denaturation (94°C, 30 s), annealing (56°C, 45 s), elongation (72°C, 1 min), and final elongation (72°C, 10 min).

CD3-{delta} specific amplification

The presence of T cells in coronary arteries with GA was determined by performing PCR analysis for the presence of CD3{delta} (48). Transcripts were amplified using 36 PCR cycles (46, 47). Amplification primers are shown in Table IIGo.

Cloning and sequencing of PCR products

PCR products from either NPA-PCR or Vß-specific PCR were cloned into the pCR2.1 plasmid vector (Invitrogen, San Diego, CA), transformed into DH5{alpha}-competent cells (Life Technologies) according to the manufacturer’s instructions, and subjected to blue-white screening. White colonies were screened for TCR cDNA by hybridization with 32P-labeled HCß1 (Table IIGo). Plasmids were purified from randomly isolated hybridization-positive colonies using the Wizard Miniprep DNA Purification System (Promega) according to the manufacturer’s instructions. Plasmids were sequenced on a 6% polyacrylamide DNA sequencing gel using the ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA).

The maximum theoretical number of potentially unique ß-chain TCR transcripts is ~1012 (49). Theoretically, the probability of randomly finding two identical copies of a single ß-chain TCR transcript within a given independent sample population is negligible. During transformation of DH5{alpha}-competent cells, the plasmid/cell mix was subjected to heat shock (at 42 C for 45 s) followed by incubation on ice for 2 min and growth for 1 h in SOC medium at 37 C before plating. Under ideal conditions (log phase), Escherichia coli has a doubling time of 20 min, which would result in two doublings after 60 min (50). After heat shock, however, the cells do not immediately enter log phase, but the unlikely possibility for a few of the transformed cells to double before plating does exist. Therefore, identical TCR sequences from two different colonies (a doublet) may indicate a clonal expansion or could be the result of a singly transfected E. coli cell that doubled before plating. In the studies presented here we have sequenced 150 ß-chain TCR transcripts from normal PBMC after either NPA-PCR or Vß-specific PCR amplification (see Results). All TCR transcripts were unique compared with each other, with the exception of one transcript (Vß12.1 Dß1.1 Jß1.6), which appeared in duplicate, demonstrating that possible doubling of a singly transfected E. coli cell before plating is a rather rare event. In contrast, sequence analysis of TCR transcripts from the coronary arteries of cardiac allografts with chronic rejection yielded several examples of two identical copies of a single ß-chain TCR transcript. These transcripts may represent clonal expansions. The odds for a triplet (three identical transcripts) to be due to this heat shock artifact are negligible.

Computer analysis and comparison of sequences

Vß-chain TCR transcripts encoding the V (variable), N-D-N (diversity), J (junctional), and C (constant) regions found in each specimen and controls were obtained and compared with those in GenBank and EMBL databases using the BLAST sequence alignment program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diagnosis of GA

All explanted cardiac allografts in this study were diagnosed with chronic rejection by pathologic examination. The mean graft survival was 4.6 years, and the range was 2.5–9 years (Table IGo). All of these cardiac allografts were removed upon retransplantation of these patients. Pathological examination of the explanted hearts yielded appreciable arteriopathy with diffuse fibrointimal thickening of proximal, distal, and small branch artery segments of both LAD and RCA. Appreciable arterial stenosis was detected in both LAD and RCA of patients GA03, GA05, and GA09 (Table IGo).

CD3+ T cell infiltrates in coronary arteries with chronic rejection

Representative pathology and immunohistochemical staining for the presence of CD3+ T cells is shown in Fig. 1Go. In study patients hematoxylin and eosin staining revealed GA with chronic arteritis, fibrointimal proliferation, thickening, stenosis, and evidence of mononuclear infiltration within the intima and external area of the media (Fig. 1GoA), consistent with chronic rejection of cardiac allografts. Immunohistochemical staining of 6-µm epicardial artery tissue sections for the presence of CD3+ cells demonstrated that substantial proportions of CD3+ cells were present within the intima and external layer of the media as well as in the adventitia surrounding the chronically rejected artery (Fig. 1GoB). The intramural and epicardial lymphoid nodules observed in arteries from two of five patients contained large proportions of CD3+ cells (Fig. 1Go, A and B). Fig. 1Go, C and D, demonstrates representative high powered fields (magnification, x400) from Fig. 1Go, A and B, respectively. In a nontransplanted epicardial coronary artery (Fig. 1Go, E and F), no significant inflammation of the medium was seen; however, slight fibrointimal hyperplasia commonly associated with aging was seen. Rare CD3+ cells were observed in the nontransplanted artery (Fig. 1GoF).



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  		FIGURE 1. Histopathology and immunohistochemical staining of the left anterior descending (LAD) coronary artery of patient GA02 (A, B, C, and D) and a nontransplanted heart (E and F). A, Diffuse, concentric fibrointimal thickening with substantial mononuclear cell infiltrate within the intima, media, as well as the adventitia. Note damage to media near the large focus of infiltrating cells. Hematoxylin-eosin staining was used. Original magnification, x40. B, Immunohistochemical staining for presence of T cells (anti-CD3 mAb) in serial section of tissue described in A. Staining patterns were consistent with the mononuclear cell infiltrate observed in hematoxylin-eosin-stained sections, indicating that a substantial proportion of these infiltrating cells are T lymphocytes. Original magnification, x40. C, Mononuclear cell infiltrate within the intima along the lumenal surface. Hematoxylin-eosin staining was used. Original magnification, x400. D, Immunohistochemical staining for presence of T cells (anti-CD3 mAb) in serial section of tissue described in C. Staining patterns were consistent with mononuclear cell infiltrate observed in hematoxylin-eosin-stained sections, indicating that a substantial proportion of these infiltrating cells are T lymphocytes. Original magnification, x400. E, LAD of nontransplanted heart. Slight thickening of the intima consistent with normal aging is evident. Rare mononuclear cells noted near the lumenal surface. Hematoxylin-eosin staining was used. Original magnification, x80. F, Immunohistochemical staining for presence of T cells (anti-CD3 mAb) in serial section of tissues described in C. Rare CD3+ cells, designated by arrows, were observed. Original magnification, x80. Control sections were stained with an irrelevant mAb of the same isotype and were all negative (not shown).

 
Oligoclonal T cell populations are infiltrating coronary arteries of human cardiac allografts with GA: identical ß-chain TCR transcripts are clonally expanded in the LAD and RCA of cardiac allografts with chronic rejection

To determine whether fresh (not expanded in culture) mononuclear cells infiltrating coronary arteries of cardiac allografts with GA contain oligoclonal T cells, we amplified, cloned, and sequenced ß-chain TCR transcripts from these coronary arteries. Sequence analysis revealed substantial proportions of identical ß-chain TCR transcripts in the coronary arteries of three of five patients, demonstrating the presence of oligoclonal populations of T cells infiltrating the coronary arteries of patients with GA. ß-chain TCR transcripts from a fourth patient exhibited some degree of oligoclonality, whereas these transcripts from the fifth patient were relatively heterogeneous. Additionally, high proportions of identical CDR3 ß-chain TCR motifs were found in the coronary arteries of all five patients.

In patient GA03, sequence analysis of ß-chain TCR transcripts from the LAD, after NPA-PCR amplification and cloning, revealed substantial proportions of identical ß-chain TCR transcripts (Table IIIGo) comprised of the following: Vß5.1 Dß1.1 Jß1.1 (clone ga0314; CDR3: FDDLN), Vß6.7 Dß1.1 Jß1.1 (clone ga03116; CDR3: AGTGQAGG). Vß3.1 Dß1.1 Jß1.5 (clone ga0339; CDR3: FSMAWD), and Vß23 Dß1.1 Jß1.5 (clone ga03113; CDR3: VWTGEG). The first of these transcripts accounted for 20% (4 of 20) and the remaining 15% (3 of 20), each, of all ß-chain TCR transcripts sequenced. Remaining TCR transcripts were unique compared with each other (Table IIIGo). The Vß5.1 Dß1.1 Jß1.1 clonal expansion in the LAD of patient GA03 was confirmed by an independent amplification method (two-sided Vß5.1-specific PCR), using RNA from the RCA of this patient as starting material (Table IIIGo). The TCR transcript (clone ga0314), initially found to be clonally expanded in LAD by NPA-PCR, was also found to be clonally expanded in RCA by Vß5.1-specific PCR. Of 35 Vß5.1+ TCR transcripts sequenced from the RCA, 15 (42.8%) were identical with clone ga0314 (Vß5.1 Dß1.1 Jß1.1; Table IIIGo). The remaining TCR clones were unique compared with each other, with the exception of clone ga03543, which appeared in duplicate. Seven of these clones are shown in Table IIIGo; the remaining 12 have been reported to GenBank. Amplification, cloning, and sequencing experiments using RNA from the LAD and RNA from the RCA were conducted 11 mo apart, making contamination highly unlikely. ß-chain TCR sequence analysis of RCA from the same patient (GA03) following NPA-PCR amplification revealed the presence of two TCR transcripts identical with clone ga0314 (2 of 28) and of one transcript identical with clone ga03116 (1 of 28), which were observed in the LAD. These results demonstrate that identical ß-chain TCR transcripts are clonally expanded in the LAD and the RCA of patient GA03. Analysis of Vß3.1+ TCR transcripts in the RCA of patient GA03 after Vß3.1-specific amplification (Table IIIGo) demonstrated substantial proportions of identical TCR transcripts: clone ga03310, Vß3.1 Dß2.1 Jß2.1, 10 of 38 (26%); clone ga03306, Vß3.1 Dß1.1 Jß2.2, 7 of 38 (18%); and clone ga03321, Vß3.1Dß2.1Jß2.1, 5 of 38 (13%).


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  		Table III. ß-chain TCR transcripts (CDR3 region) expressed in coronary arteries of patient GA03

 
In addition to the presence of clonally expanded TCR transcripts in patient GA03, several conserved amino acid motifs were observed within the CDR3 of the transcripts sequenced. These include the Leu-Asn (LN) motif, which was observed in the clonally expanded Vß5.1 Dß1.1 Jß1.1 transcript (clone ga0314, 23 of 83 (28%) transcripts) and was also seen in 15 of 38 (39%) TCR transcripts sequenced after Vß3.1-specific amplification (Table IIIGo). The LN motif was found in the CDR3 of 38 of 121 (31%) ß-chain TCR transcripts sequenced from patient GA03. PBMC from normal donors were negative for the FDDLN motif, and the LN motif was observed only in 3 of 150 (2%) TCR transcripts sequenced. The TG, RG, GG, AG, and PG CDR3 motifs were observed, respectively, in 18 of 121 (15%), 14 of 121 (12%), 18 of 121 (15%), 10 of 121 (8%), and 8 of 121 (7%) of the ß-chain TCR transcripts sequenced from patient GA03 (Table IIIGo).

Six of 33 (18%) ß-chain TCR transcripts amplified by NPA-PCR from the LAD of patient GA06 were identical (clone 06npa12, Vß12.1Dß1.1Jß1.4; Table IVGo). The remaining 27 sequences were unique compared with each other. Sequence analysis of ß-chain TCR transcripts from the RCA of patient GA06 after Vß12.1-specific PCR amplification revealed 3 of 23 (13%) identical copies of the clone 6Vß1208, Vß12.1 Dß2.1 Jß2.2 (Table IVGo). Four other transcripts were each present in duplicate (Table IVGo). The CDR3 motif SG appeared in 9 of 66 (14%), the QSG motif in 5 of 66 (8%), the GTG motif in 7 of 66 (11%), the RG motif in 6 of 66 (10%), and the AG motif in 5 of 66 (8%) ß-chain TCR transcripts sequenced from patient GA06.


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  		Table IV. ß-chain TCR transcripts (CDR3 region) expressed in coronary arteries of patient GA06

 
Sequence analysis of ß-chain TCR transcripts from the LAD of patient GA09 after NPA-PCR amplification and cloning revealed unique sequences compared with each other, with the exception of clones 09npa03 and 09npa64, which each appeared in duplicate (Table VGo). However, sequence analysis of RCA ß-chain TCR transcripts from patient GA09 after Vß9.1-specific PCR amplification revealed a very strong clonal expansion. Twenty-four of 38 (63.2%) of these transcripts were identical with clone 09vb913, Vß9.1Dß1.1Jß2.7, CDR3:QNPGTGH (Table VGo). These results demonstrate that oligoclonal T cells infiltrate the coronary arteries of patient GA09 with chronic rejection. Furthermore, 30 of 68 (44%) ß-chain TCR transcripts used the CDR3 motif PG, 33 of 68 (49%) used the CDR3 motif TG, and 6 of 68 (9%) used the motif RG. In four TCR clones the TG motif was followed by a hydrophobic amino acid (L, I, T, or V; Table VGo).


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  		Table V. ß-chain TCR transcripts (CDR3 region) expressed in coronary arteries of patient GA09

 
In patient GA02 sequence analysis of ß-chain TCR transcripts from the LAD after NPA-PCR and cloning revealed mostly unique transcripts compared with each other, with the exception of three clones (ga0203aa, ga0221b, and ga0237aa) that appeared in duplicate (Table VIGo). Sequence analysis of TCR transcripts from the LAD of patient GA02 after Vß4.1-specific amplification and cloning revealed 3 of 33 identical copies (9%) of clone ga02vb413, Vß4.1 Dß1.1 Jß2.1, CDR3:VESDRGP, suggesting that this clone has been clonally expanded (Table VIGo). Three other clones (ga02vb417, ga02vb426, and ga02vb414) each appeared in duplicate. A more heterogeneous population of TCR transcripts was found in the coronary arteries of this patient compared with those discussed previously.


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  		Table VI. ß-chain TCR transcripts (CDR3 region) expressed in coronary arteries from patient GA02

 
Sequence analysis of ß-chain TCR transcripts from the LAD of patient GA05 after NPA-PCR and cloning, revealed unique transcripts compared with each other, with the exception of one clone (ga0519) that appeared in duplicate (Table VIIGo). However, 13 of 25 (52%) ß-chain TCR transcripts amplified by NPA-PCR used the GG motif in the CDR3 (Table VIIGo). Sequence analysis of Vß5.1 TCR transcripts from the RCA of patient GA05 after Vß5.1-specific amplification and cloning revealed unique transcripts compared with each other, with the exception of one clone (05vß503) that appeared in duplicate (Table VIIGo). However, sequence analysis of ß-chain TCR transcripts from the RCA of patient GA05 after Vß2.1-specific amplification and cloning revealed five TCR clones (05vß217, 05vß201, 05vß212, 05vß203, and 05vß208) that each appeared in duplicate (Table VIIGo). The GG CDR3 motif was used by a total of 24 of 88 (27%) transcripts sequenced in patient GA05 (Table VIIGo). The AG CDR3 motif was used by 16 of 88 (18%) TCR transcripts sequenced (Table VIIGo). The PG motif was used by 10 of 88 (11%) TCR transcripts sequenced. The TSG motif was used by 6 of 64 (9%) of the Vß5.1 and Vß2.1 TCR transcripts found in the RCA of patient GA05 (Table VIIGo) as well as in 8 of 35 (23%) of Vß5.1 clones obtained from EMBX of the same patient (see below). The TSG motif was observed in only 2% of the PBMC from normal donors (data not shown).


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  		Table VII. ß-chain TCR transcripts (CDR3 region) expressed in coronary arteries of patient GA05

 
Normal donor PBMC were used as methodological control and were subjected to either NPA-PCR or Vß-specific (Vß2.1, Vß3.1, Vß4.1, Vß5.1, Vß9.1, and Vß12.1) PCR amplification, followed by cloning and sequencing. Sequences of TCR transcripts in PBMC following amplification by NPA-PCR or Vß-specific PCR and cloning (Table VIIIGo) were all unique compared with each other, in agreement with previous results (40, 41, 42, 43), as anticipated for polyclonal populations of T cells, with the exception of one transcript (Vß12.1 Dß1.1 Jß1.6) that was present in duplicate (of 15) in the Vß12.1-specific PCR control. This sequence was not detected in any of the other specimens. These sequences are not shown here because of space limitations, but have been reported to the GenBank/EMBL database.


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  		Table VIII. ß-chain TCR transcripts (CDR3 region) expressed in normal donor PBMC

 
Control epicardial arteries from nontransplanted hearts were also subjected to analysis. As shown in Fig. 2Go, control artery 1 (nontransplanted heart from a normal adult male organ donor) did not exhibit CD3-{delta} transcripts compared with study arteries, indicating a lack of T cells in the sample. Analysis of control artery 2 (autopsy material from an adult female patient who died of end-stage diabetes mellitus; nonheart-related) for CD3-{delta} transcripts yielded a substantially diminished band compared with diseased arteries, demonstrating the presence of minimal T cell infiltrates, which have been associated with older diabetics (20). Neither artery expressed any of the Vß gene segments (Vß5, Vß9) that were expressed in the study arteries (Fig. 2Go); thus, no TCR sequences were obtained.



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  		FIGURE 2. A, Vß5.1 TCR transcript presence in RCA and EMBX tissue obtained from patient GA05. B, Vß9.1 transcripts present in RCA and EMBX tissue obtained from patient GA09. CD3-{delta} transcripts are shown in each specimen. GAPDH served as a positive cDNA control. Normal donor, nonstimulated PBMC-derived cDNA is shown as a positive transcript control. Control arteries 1 and 2 (described in Materials and Methods) were negative for both Vß5.1 and Vß9.1 transcripts. Control artery 1 (nontransplanted heart from a normal adult male organ donor) was negative for CD3-{delta}, whereas control artery 2 (autopsy material from an adult female who died of diabetes mellitus, a nonheart-related illness) demonstrated a slight CD3-{delta} band subsequent to 35 cycles of PCR, revealing the presence of either minimal T cell infiltrates, which may be associated with older diabetics (20 ), or PBMC contamination of the vessel. The Vß bands shown were cloned and sequenced. Neither of the control arteries expressed any of the Vß genes (Vß5, Vß9) that were expressed in the study arteries. The marker is a 100-bp DNA ladder (1.5 µg); 1.2% agarose gel was used.

 
Comparison of the nucleic acid and the deduced amino acid sequences with those in the GenBank/EMBL/SWISS PROT databases revealed that all ß-chain TCR transcripts obtained as part of this study were novel and had not been previously reported. There were no incidences of identical TCR transcripts appearing in different patients. However, high proportions of identical CDR3 motifs were found in the coronary arteries of all five patients, and some of them are shown in Table IXGo. The CDR3 motif TG was particularly prominent in patients GA03, GA06, and GA09; the LN motif and the RG motif were prominent in patients GA03 and GA02, respectively. The CDR3 motif GG was increased in patient GA05, and the PG motif was increased in patients GA09, GA02, and GA05 (Table IXGo).


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  		Table IX. CDR3 epitopes conserved in the coronary arteries of patients with chronic rejection

 
Preferential usage of the Jß1.1 gene segment was observed in the following patients: GA02, LAD, and RCA: 20 and 18% of TCR transcripts, respectively; GA03, LAD, and RCA: 40 and 43% of TCR transcripts, respectively; and GA05, RCA, and archived EMBX: 23 and 32%, respectively. These percentages are substantially higher than those for Jß1.1 segment usage in our control TCR populations, where 18 of 150 (12%) of the TCR transcripts analyzed used the Jß1.1 segment. The LAD of patients GA02 and GA03 contained a high percentage of TCR transcripts (17 and 35%, respectively) using the Jß1.5 gene segment compared with the relative frequency of Jß1.5 use (6.6%; 10 of 150) in TCR transcripts in PBMC from normal donors. Increased use of the Jß2.7 gene segment (28.5%) was found in TCR transcripts from the RCA of patient GA03. Similarly, increased use (42%) of the Jß2.1 gene segment was found in Vß3.1 TCR transcripts from the RCA of the same patient (Table IIIGo). The frequencies of Jß2.7 and Jß2.1 in PBMC from normal donors were 17.9% (27 of 150) and 17.2% (26 of 150), respectively.

Identical clonally expanded ß-chain TCR transcripts are present in both previously archived EMBX and coronary arteries with GA from the same patient

Two patients, GA09 and GA05, received their primary cardiac allografts at Temple University Hospital, and for this reason, archived (snap-frozen) EMBX were collected throughout the post-transplantation period and were available for analysis. One appropriately selected (on the basis of pathological examination) EMBX from each of these two patients was subjected to TCR transcript amplification, cloning, and sequencing. EMBX material from patient GA09 was collected 28 mo before retransplantation, and it was representative of grade 1A rejection (focal perivascular or interstitial infiltrates with no evidence of necrosis), in accordance with the ISHLT (45) guidelines.

EMBX from patient GA09 were analyzed for the presence of Vß9.1 TCR transcripts, because the Vß9.1 Dß1.1 Jß2.7 transcript, clone 09Vß913, was clonally expanded in the RCA of this patient’s chronically rejected heart (24 of 38 transcripts were identical; 63.2%; Table VGo). The same Vß9.1 Dß1.1 Jß2.7 transcript, clone 09Vß913 (100% sequence identity), was found to be clonally expanded in these EMBX, which were collected 28 mo before retransplantation (21 of 26 transcripts were identical; 80%; Table XGo).


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  		Table X. TCR transcripts (CDR3 region) expressed in archived EMBX of patients with cardiac allografts subsequently diagnosed with chronic rejection

 
EMBX from patient GA05 was obtained 17 mo before retransplantation, and it was representative of grade 3A rejection (necrosis as well as evidence of diffuse inflammatory processes), in accordance with the ISHLT (45) guidelines, and also contained evidence of mild atherosclerosis. The Vß5.1 Dß1.1 Jß1.1 transcript, clone 05Vß503, was clonally expanded in the EMBX of patient GA05 (7 of 35 transcripts were identical; 20%; Table XGo). The same transcript Vß5.1 Dß1.1 Jß1.1, clone 05Vß503, was present in duplicate in the RCA of patient GA05 with chronic rejection (2 of 30 transcripts were identical; 6.6%; Table VIIGo). Another transcript Vß5.1 Dß2.1 Jß2.3, clone 05Vß520, was also present in both the EMBX (Table XGo) and the RCA (Table VIIGo) of this patient; however, in both it was present as a single copy. These results demonstrate that T cells in cardiac allografts are clonally expanded in response to Ag relatively early in the post-transplantation period in the myocardium, and that these clonal expansions persist in coronary arteries well after the development of chronic rejection. It is of interest that the pathology of the EMBXs from both patients demonstrated the onset of GA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have determined that the coronary arteries of cardiac allografts with chronic rejection contain oligoclonal populations of T cells. ß-chain TCR transcripts from these coronary arteries contained substantial proportions of identical TCR transcripts. These results were very prominent in three of five patients with chronic rejection. T cells infiltrating coronary arteries from a fourth patient exhibited some degree of oligoclonality, and those from the fifth patient were relatively heterogeneous. High proportions of identical CDR3 ß-chain TCR motifs were found in the coronary arteries of all five patients. The ß-chain TCR CDR3 motif TG was particularly prominent in patients GA03, GA06, and GA09; the LN motif was prominent in patient GA03; the GG motif was prominent in patient GA05; and the PG motif was prominent in patients GA09, GA02, and GA05 (Table IXGo). The CDR3 region of the TCR ß-chain is presumed to be responsible for providing contact points for the binding of TCR to peptide/MHC complex (36, 37, 38, 39). The amino acid sequence of the CDR3 provides information regarding potential amino acid residues on the TCR that recognize peptide/MHC. Clonal expansions of ß-chain TCR transcripts and/or conservation of amino acid motifs within the hypervariable CDR3 regions provides strong evidence for Ag-driven (MHC/peptide) T cell clonal expansions. In this study we identified 1) strong highly selective clonal expansions of ß-chain TCR transcripts in the coronary arteries of cardiac allografts from patients with chronic rejection; and 2) conserved amino acid motifs within the CDR3 region of the ß-chain TCR within individual patients and between different patients.

Identical ß-chain TCR transcripts were clonally expanded in different anatomical sites (LAD, RCA, and EMBX) and at different times, demonstrating systemic clonal expansions of T cell clones. These T cells were very likely clonally expanded in situ in the allograft in response to as yet unidentified antigenic epitopes (peptide/MHC), very likely containing alloantigen, which appear to be constitutively expressed in these allografts. These clonally expanded T cells in the coronary arteries may play a significant role in the pathogenesis of GA. These results provide compelling evidence that chronic rejection is an Ag-driven T cell disease.

Only limited studies have been conducted on the TCR repertoire used by T cells infiltrating organ grafts with chronic rejection, and these have been focused on V region gene segment usage rather than on TCR transcript sequence analysis. Anti-HLA-DR3-reactive T cell clones, developed from renal allografts that used diverse TCR V{alpha} and Vß genes, possessed conserved CDR3 motifs (53). A restricted Vß repertoire throughout episodes of acute and on-going chronic rejection has been reported for 9 of 12 renal allograft recipients (54); however, it was not determined whether these T cells were clonally expanded. Others have reported heterogeneous populations of intragraft T cells in renal allografts undergoing rejection (55) and in some heart allografts (56) and have attributed their presence to nonspecific lymphocytes recruited to the site by locally produced cytokines. In studies performed using the LEW-ACI rat model of chronic cardiac graft rejection, limited heterogeneity of Vß gene usage in association with a wide variety of CDR3 motifs and Jß genes was observed, suggesting preferential use of particular Vß genes (57). Oligoclonal expansions of T cells were found in the peripheral blood of patients with obliterative bronchiolitis (chronic rejection), compared with lung recipients who showed no evidence of rejection (58). DeBruyne et al. (59) reported restricted Vß-gene segment use by bronchoalveolar lavage-derived T cell lines from patients with human lung allografts before and during rejection episodes.

Two different mechanisms of Ag presentation, the direct and the indirect, are responsible for the recognition of organ drafts by T cells of the recipient (27, 28, 29, 30, 31, 32, 33, 34, 35). The direct recognition pathway involves recognition of allogeneic MHC/peptide complexes expressed on dendritic cells (passenger leukocytes) of the donor by T cells of the recipient. MHC molecules of the donor may directly present peptides to the T cells of the recipient (31). It has been proposed that molecular mimicry is the basis of this recognition (29, 52). Molecular mimicry will explain the considerable extent of the allogeneic immune response. There is a large number of different class I or II MHC/peptide epitopes of the donor that may be recognized by different T cell clones of the recipient by molecular mimicry (29, 52). However, stimulation of T cells of the recipient by donor APCs that may lack costimulatory molecules will lead to anergy, reducing the significance of this response after the early post-transplantation period (60, 61, 62, 63, 64). The indirect recognition pathway involves presentation by APC of the recipient to T cells of the recipient of epitopes comprised of MHC molecules of the recipient and antigenic peptides derived from MHC Ags of the donor (27, 28, 29, 30, 31, 32, 33, 34, 35). The later should be available in high quantities in the graft, because of cell destruction at the site of the graft and possible shedding of MHC molecules. We report here that identical ß-chain TCR transcripts are clonally expanded in archived EMBX specimens and in coronary arteries of cardiac allografts (explanted 17 or 28 mo later) from the same patients with chronic rejection. It is not known whether these clonally expanded TCR recognize MHC/peptide epitopes presented in a direct or an indirect manner. Because these EMBX were removed 13 and 16 mo post-transplantation, it could be either. However, for these T cell clones to remain clonally expanded, the Ag(s) (peptide/MHC epitopes) must be expressed in both the EMBX and the coronary arteries; therefore, they are very likely alloantigens. It is of interest that only a limited number of T cell clones were clonally expanded in coronary arteries of cardiac allografts from patients with chronic rejection. These results are in agreement with those of others that T cells responsible for autoimmune responses use highly restricted TCR V region gene segments (65, 66), suggesting that their TCR can be targeted by virtue of this selected TCR expression (66). The presence of TCR transcripts clonally expanded in EMBX that persist in the graft many months later and have also been found to be clonally expanded in coronary arteries with GA from the same patient may have certain clinical implications. It is likely that these TCR clones are responsible at least in part for the pathogenesis of the disease. Because these TCR clonotypes persist for so long, the corresponding anti-Vß mAbs or other appropriate anti-TCR mAbs may be of use as in vivo therapeutics for treating or preventing chronic rejection.

The direct pathway of allorecognition is dominant during the early post-transplantation period, and the frequency of recipient T cells directly responding to donor APCs is at least 100-fold higher than that of recipient T cells recognizing donor allopeptides presented by APC (67) of the recipient. It has been suggested that the indirect recognition pathway may play a role in the pathogenesis of chronic rejection (31, 68). Although this is far from proven, several reports provide suggestive evidence that this may be the case (68, 69, 70, 71).

A strong correlation has been reported (27, 71) between donor-specific HLA-peptide alloreactivity of PBMC and the incidences of acute (27) and chronic rejection (71). The incidence of chronic rejection is much higher in patients with PBMC that continued to respond to donor HLA-DR peptides late after transplantation than in those without persistent PBMC alloreactivity (71).

Intermolecular and intramolecular epitope spreading has been demonstrated in the proliferative responses of PBMC to synthetic peptides of the hypervariable region of 32 HLA-DR alleles (71, 72). Epitope spreading has been originally described in autoimmune demyelinating diseases of the CNS (73, 74, 75, 76, 77), and it is defined as the generation of de novo immune responses to epitopes different from and noncross-reacting to those that initially induced the immune response. This definition (73, 74, 75, 76, 77) is extended now to the immunity against grafts (71, 72) and to the immunity against tumors (78). Progression to chronicity in demyelinating autoimmune disorders and in graft rejection is associated with broadening the T cell repertoire with time (79). The acquired neoreactivity, that is, the epitope spreading, appears to be the result of endogenous priming to new self-determinants (79) during the chronic inflammation conditions that are associated with CNS demyelinating disease or graft rejection. These chronic inflammation conditions may clearly result in breaking tolerance to self-Ags and may be responsible for epitope spreading. Another possible explanation, particularly in the case of CNS demyelinating disease, is de novo priming of self-reactive T cells to sequestered (auto)Ag epitopes released as a result of the primary immune response. However, there is a significant difference between epitope spreading in chronic rejection (71, 72) and that in demyelinating diseases (73, 74, 75, 76, 77). To date, all epitope spreading responses in chronic rejection are recognizing allogeneic HLA-DR epitopes, dominant or cryptic, but do not recognize self-determinants (71, 72). At present we do not know whether the clonally expanded T cells that we observed in the coronary arteries of patients with chronic rejection are recognizing primary HLA-DR epitopes or represent immune responses to secondary epitopes due to epitope spreading. However, the fact that these clonal expansions have been observed in both EMBX and coronary arteries (expanded 28 and 17 mo later) supports the view that they may represent primary immune responses. It should be noted that all the immune responses attributed to epitope spreading have been found in PBMC from patients with chronic rejection. The properties and the clonality of lymphocytes infiltrating coronary arteries from patients with chronic rejection have not been investigated. A statistically significant association has been described between intermolecular epitope spreading and the development of GA (p < 0.02), suggesting that the recruitment and response of T cells recognizing additional alloepitopes may be critical for the progression of GA (71).

Although primary and secondary responses of the recipient’s T cells in chronic rejection are directed against alloantigenic determinants, there is no information on whether other T cells infiltrating coronary arteries of cardiac allografts from patients with GA recognize self-Ags, at least in the late stages of the disease. Because of the extensive chronic inflammation in coronary arteries with GA, tolerance may be broken, and self-Ags may be recognized in an autoimmune fashion. T cell clones derived from classical atherosclerotic plaques responded by proliferation and cytokine production to oxidized low density lipoprotein (LDL) (80). This response was dependent on autologous APCs, and it was HLA-DR restricted (80). Local application of oxidized LDL to arteries promotes intimal thickening (81). Oxidized LDL is immunogenic and induces an Ab response (82). However, differences have been noted in the pathology of GA and atherosclerotic occlusive disease (6, 7). It is not known whether responses to oxidized LDL play a role in the late phase of the pathogenesis of chronic rejection.

Our observations demonstrate that the pathogenesis of chronic cardiac allograft rejection may involve clonal proliferation of T cells in response to persistent recognition of graft-derived antigenic epitopes. The fact that oligoclonal populations of T cells identified early in the post-transplantation period are also present in coronary arteries of allografts diagnosed with GA suggests a possible role for these T cells as targets of specific immunotherapy approaches as a means to prevent the development of GA.


   Footnotes
 
1 This work was supported in part by Grants PO1AI40160 and T32AI07101 from the National Institute of Allergy and Infectious Diseases and by a predoctoral fellowship from the American Heart Association. Back

2 The ß-chain TCR sequences reported in this manuscript have been submitted to GenBank under accession nos. AY006091 through AY006333. Back

3 Address correspondence and reprint requests to Dr. Chris D. Platsoucas, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. Back

4 Abbreviations used in this paper: GA, graft arteriosclerosis; NPA-PCR, nonpalindromic adaptor-PCR; LAD, left anterior descending; RCA, right coronary artery; ISHLT, International Society of Heart and Lung Transplantation; EMBX, endomyocardial biopsy; LN, Leu-Asn; LDL, low density lipoprotein. Back

Received for publication April 27, 2000. Accepted for publication July 5, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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