From www.bloodjournal.org by guest on January 12, 2015. For personal use only. Molecular Analysis of T-cell Receptor Repertoire in Bone Marrow Transplant Recipients: Evidence for Oligoclonal T-cell Expansion in Graft-Versus-Host Disease Lesions By Xiping Liu, Vera Chesnokova, Stephen J. Forman, and Don J. Diamond We have analyzed the T-cell receptor (TCR) Vp repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT)from related and unrelated donors at theCity of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR Vp families, whereas examination of peripheral blood (PB) withdrawn at thesame time did notreveal a similar phenomenon. In one such family, TCR Vp2 is predominantly expressed in 7 of 7 biopsy specimens examined. Vp2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single Vp2-bearing TCRs in GVHD leafter diagsions, and in the PB of some patients nosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used t o probe Southern blots of amplified Vp2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute t o the disease pathology. 0 1996 by The American Societyof Hematology. A as a lymphocytic infiltrate at the site of disease.”14 A possible mechanism which underlies the development of GVHD isthat antigenic differences of the BM recipient willbe subject to immunologic challenge as a result of donor Tcell recognition of recipient processed antigen as foreign.”,” This may be especially true in the case of MUD transplant donors selected through the NMDP (National Marrow Donor Program), because their only genetic similarity to the BMT recipient may be within a portion of the major histocompatability complex (MHC) class I and class I1 I O C ~ . ~There . ’ ~ is still a strong likelihood that mismatches may arise which are undetectable using conventional approaches for establishing MHC identity.IR Confounding the immunologic analysis of the disease is the paucity of knowledge concerning the putative antigens, and the specific lymphoid cells involved in inducing GVHD, although antigen-specific T cells” or natural killer cells2” are likely contributors to the pathology of GVHD. An exception to our limited knowledge about which antigens stimulate GVHD are studies involving the the minor histocompatibility antigens (MiHA). A growing bodyof biochemical studies has identified new structural information in the identification of these antigens.”.** Both in vivo clinical data and in vitro studies strongly suggest that MiHA differences play a role in the development of GVHD.’.*’ The increased reliance on unrelated or haploidentical donors for BMT recipients makes it crucial to find means to limit the pathology caused by GVHD.24An approach to limiting the severity of GVHD, especially among NMDP unrelated donors, was developed over a decade ago and it uses T-cell depletion (TCD) of the marrow graft to eliminate mature T cells.2s~Z6 This leads to a reduction in both acute and chronic GVHD,27although studies throughout the 1980s and into the early 1990s have shown multiple problems with this approach.**The problems include graft failure in up to 10% of patients, as well as an increased relapse rate, particularly in patients who have chronic myelogenous leukemia (CML).27Recently, variations of the original protocol including selective in vitro depletion of the CD8’ T-cell subset for BMT in CML have shown a benefit in terms of reducing GVHD, but graft failure still remains higher than in patients LLOGENEIC bone marrow transplantation (ABMT) has emerged as a successful therapyin the treatment of otherwise incurable malignant hematologic disorders.’ It has been most effective in the treatment of acute and chronic leukemia and many patients enjoy long-term, disease-free survival as a result of the success of this therapy.’.’ The patient who is treated with ABMT undergoes a process of immune reconstitution over a 6- to 12-month period involving all aspects of the immune ~ y s t e m The . ~ redevelopment of immunocompetence can be hampered by acute and chronic graft-versus-host-disease (GVHD).5.6Despite the use of prophylactic medications for GVHD, approximately 20% to 30% of patients receiving matched sibling donor (MSD) allografts and 75% of those patients receiving matched unrelated donor (MUD) allografts develop graft-versus-host reaction that can lead to increased morbid it^.^.' GVHD is mediated by T cells that are derived from the BM graft.’ Studies in animal models have shown that specific T-cell subsets contribute to the pathology of GVHD,’” although there is no consensus as to the predominate subset implicated in a particular organ involvement.” Although GVHD is considered as a disease of allorecognition, the pathologic profile, especially in skin, resembles a number of autoimmune diseases. Common to these syndromes is the function of mature T cells in mediating the pathology, such From the Department of Hematology and Bone Marrow Transplantation and the Division of Immunology, City of Hope Medical Center and the Beckman Research Instilute, Duarte, CA 91010. Submitted August 30, 1995; accepted November 14, 1995. Supported in part by Grants No. P o l - C A 30206 and CA 33572 from the National Cancer Institute, Department of Health and HUman Services, Bethesda, MD. Address reprint requests to Don J. Diamond, PhD, Department of Hematology and BMT, City of Hope Medical Center, 1500 E Duarte Rd, Duarte, CA 91010. The publication costsof this article were defrayedin part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8707-0007$3.00/0 3032 Blood, Vol 87,No 7 (April l ) , 1996: pp 3032-3044 From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3033 MOLECULAR ANALYSIS OF TCR REPERTOIRE IN GVHD Peripheral Blood with unmanipulated More selective T-cell depletion, with emphasis on those subsets that manifest an aggresPatients had blood drawn at the time of admission (20 mL) to sive phenotype, might address the problems associated with establish a reference TCR repertoire before total body irradiation GVHD without disturbing the beneficial effect of the graft and cytoreduction. BM donors had blood drawn on the day of BM harvest (day 0) to establish a reference donor TCR repertoire. We on preventing relapse. Our approach is to characterize the T also processed a small amount (1 to 5 mL) of BM from the donor cells that infiltrate the GVHD lesion to evaluate the potential on the day of the BM harvest for our TCR studies. Subsequent to for immunotherapeutic intervention as a means to limit tissue the transplant on days +21, 35, 49, 63, and day 100 PB (20 mL) destruction. was drawn from the BMT recipient. In addition, blood was drawn Because both human and animal studies implicate mature again at approximately day 180 for patients who were being tapered T cells in the development of GVHD, we and others have off pharmacologic immunosuppression. If the patient developed visiconducted research to better define the types of T or other ble and/or clinical signs of graft-versus-host reaction and a biopsy lymphoid cells associated with the disease ~ t a t e . ’ ~ .Our ~ ~ - ~ *was performed on the liver, gut, or skin for the diagnosis of GVHD, previous phenotypic studies have shown that the majority of a blood sample was also withdrawn for analysis lymphocytes infiltrating GVHD lesions of BMT recipients are CD3+/a/?+T-cell receptor (TCR) containing T lymphoBiopsy cytes.” This is despite the fact that during the early phases When there was evidence of clinical GVHD a biopsy was taken of the hematopoietic reconstitution there are large numbers from the involved area for study. A 4-mm punch biopsy was taken of CD3’IyS’ T lymphocytes in the p e r i p h e ~ y . ~In ~ . ’this ~ from the affected skin, and divided into two pieces. One piece was report, we have extended these studies by examining at the analyzed morphologically for evidence of clinical disease by staff molecular level the productively rearranged TCR repertoire pathologists at the City of Hope, and the other portion was frozen in reconstituting peripheral blood (PB) and GVHD lesions in liquid nitrogen (LN2t) and made available for our molecular of BMT patients. We present evidence for specific T-cell analyses. In the case of gut biopsies, several needle biopsies were taken from the affected area, and were subdivided as described for expansion in GVHD lesions of patients, and in certain cases, skin. The portion to be used in this study was placed immediately within the PB at the time of development of GVHD. MATERIALS AND METHODS Patients Patients who received allogeneic BMT at the City of Hope were eligible for this study, and ranged in age between 15 and 56 years, with a median age of 33 years. Patient accrual for this study was initiated in January 1992, and at that time PB specimens were obtained from all patients undergoing ABMT at the City of Hope. As the frequency and degree of seventy of GVHD is far greater in the MUD versus the MSD group,3s we restricted the accrual to only individuals undergoing a MUD transplant in 1993 and 1994. The group of patients who have been enrolled in our study do not statistically differ in GVHD incidence from the ABMT population treated between 1992 and 1994 at the City of Hope (data not shown). All patients received their BM grafts from either an HLA-identical sibling, or an HLA-matched unrelated donor. No patient received a T-cell-manipulated marrow, and all patients received immunosuppression with cyclosporine A (CSA), methotrexate, with or without prednisone, after BMT. Patients With GVHD Biopsy of skin, gut, or liver was taken for the primary purpose of clinical diagnosis, and secondarily some material was made available for use in our study. We did not rebiopsy any patient for the purpose of this study after GVHD treatment was initiated. No biopsy specimens were taken from sites not suspected to be GVHD involved, because the review of our protocol by the Institutional Review Board (IRB) disallowed it. GVHD grade was established by microscopic evaluation of biopsy samples, performed by members of the Department of Hematopathology (City of Hope). The clinical staging’6 and pathologic grading3’ of GVHD were based on published reports. The clinical signs included skin rash, abnormal liver function tests, or gastrointestinal dysfunction, which led to a biopsy of the effected organ. In all patients studied in this report, when GVHD was diagnosed, the therapeutic dose of immunosuppressive agent(s) was increased (Table 1). The treatment profile and clinical characteristics of the eight patients we have analyzed for this report are summarized in Table 1. At the date of occurrence of GVHD, patients were still receiving oral CSA, 3 to 5 mgkg. in saline, andthen frozen in LN2 until processing for molecular studies. Patients who developed chronic GVHD also had biopsies performed, which were subdivided for use in clinical diagnosis as described above, and for analysis by molecular methods. Biopsy material from some of the patients analyzed in this report were also sectioned, and analyzed morphologically with the aid oflymphocytespecific monoclonal antibodies (MoAbs).” Molecular Methods Processing of PB and BM. Ten to 20 mL of heparinized blood was drawn from donors and patients at various times, diluted 1:1 with phosphate-buffered saline (PBS), and the peripheral blood lymphocytes (PBLs) isolated by centrifugation through Ficoll-HyPaque (Pharmacia, Piscataway, NJ). In the case of the donor, at the time ofBM harvesting, we obtained the excess BM cells in which the mononuclear cells were previously separated by Ficoll-HyPaque centrifugation. In some cases, we obtained donor BM from the NMDP. The equivalent of 1 to 5 mL of BM was generally available for RNA processing. Preparation of RNA and cDNA. The interface layer containing PBLs was isolated and washed twice with PBS. RNA was prepared using a modification of the method of Chomczynski and S a ~ c h i ~ ~ using the TRIzol (Bethesda Research Laboratories [BRL], Gaithersberg, MD) reagent. Total RNA was also isolated from biopsy specimens. Biopsy specimens (skin, gut, or stomach) were placed in isotonic saline immediately after the procedure, then quickly frozen in LN2, and stored until used at -80°C. The frozen tissue was placed in 1 mL of TRIzol (BRL) reagent in a microcentrifuge tube, and then homogenized in situ for 1 minute using a Tissue-Tearor (Biospec Products). Total RNA was then isolated using the same method as for PBLs described above, and the absorbance monitored at 260 and 280 nm. Typically, we recovered 5 to 10 pg of RNA per skin biopsy specimen, and 20 to 50 pg from gut biopsy samples. cDNA from both PBLs and tissue was generated with 2 pg RNA using 5 U of avian myeloblastosis virus (AMV) reverse transcriptase (RT) in a 50-pL reaction at 42°C for 20 minutes, that also contained as a primer oligo dT12.1x and deoxyribonucleotides at 250 pmoVL each. Fifty microliters of ddH20 was added, the sample boiled for 3 minutes, spun out, and the supernatant was used for polymerase chain From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3034 LIU ET AL Table 1. Summary of Patient Characteristics ~ Description UPN 1 + Disease 768 CML 789 AML 916 ALL 2081 CML 2085 CML 2093 CML 2094 CML 2098 CMMoL Donor Type HLA Type Preparative Regimen A:33, 814, B35. DR3. Busulfan (4 mglkg) + CTX DR4 (60 mglkg) MSD A3, A26,87,835, FTBl DR1, 1,320 c G +~ VP-16 DRwl5 (60 mglkg) MSD *A2. A26.87. Bw57. FTBl 1,320 cGy + VP-l6 DR4, DRwll (60 mglkg) MUD A l . A3, 88. Bw57. DR3, FTBl 1,320 cGy + CTX (60 DR7 mg/kg) A3, MSD M U D A3, All, B18, B35. DR7, FTBl 1,320 cGy DR17 mglkg) M U D A2, A3, 67, Bw57, DR7, FTBl 1,320cGy DR11 mglkg) M U D A2, A24, 844, Bw58, FTBl 1,320 cGy DR13 DRl1, mdkg) M U D A2, A26, Bw41, Bw62, FTBl 1,320 cGy DR4 mglkg) Date of Post-BMT GVHD Prophylaxis Transplant CsA + MTX + MPSE 9/29/92 CSA + MTX 1/12/93 CsA + MTX + MPSE 10/4/94 GVHD SkinBiopsy (d) Grade Posttransplant II 122t II 371 II - CsA + anti-Tac + MTX 6/15/94 II - + CTX (60 CsA + MTX + MPSE 7/13/94 + CTX (60 CsA + anti-Tac + MTX 9/19/94 CsA + MTX + MPSE + CTX (60 CsA + MTX 32 109 37 74 183 (C) 157 - 35 79 (St) 11/7/94 II 42 11/18/94 - 82 + MPSE + CTX (60 II ~~ Ill Donors for BMT recipients are either siblings who are matched at major HLA loci (MSD) or unrelated donors selected to be matched as closely as possible, and identically at HLA A, B, and DR loci (MUD). The pathologic grade of GVHD is noted as determined by the Department of Hematopathology using standard criteria, with (-) signifying no pathologic evidence of GVHD.“ The days (d) after transplant refers to the date of the biopsy procedure. The preparative regimen is shown, and for 718 patients included FTBl (fractionated total body irradiation), and/ or busulfan, CTX (cyclophosphamide), and VP-16 (etoposide) at the indicated dosages. Every ABMT patient undergoes prophylaxis for GVHD, and receives a combination of CsA (50-250 mglkg),MTX (methotrexate), MPSE (methyl-prednisolone). Abbreviations: C, colon; St, stomach. * Patient 916 was mismatched and heterozygous (A2IA26 vA2/A2) at the HLA A locus versus the donor. t Biopsied skin was unavailable for molecular analysis from patients UPN 768 and 789. Confirmed diagnosis of GVHD led to increases in dosage of CsA and MPSE given to the patient after biopsy was completed. reaction (PCR) analysis. The quality of the RNAwas evaluated using an oligonucleotide pair which detects a 661-bp fragment of the human P-actin cDNA’~(data not shown). If a band could be detected after 35 cycles, subsequently all 26 V0 gene oligonucleotides were tested (Table 2). PCR conditions and quanritation. For Vp repertoire studies, 1 of 26 5’ VD-specific primers (Table 2), andan antisense oligonucleotide (Cpl,2) that is complementary to both Col and Co2 genes,40 were incubated (5 pmol each) with 5.0 pL cDNA in a 50-pL reaction volume. To that was added MgCI2to1.5 mmol/L, 200 pmol/L dXTPs, and 3 U ofthe Stoffel fragment4’ of Taq polymerase in standard reaction buffer for the enzyme (Perkin Elmer Cetus, Norwalk, CT) . The Vp PCR products were approximately 350 bp (Table 2). As an internal control for Vp gene segment synthesis, each reaction contained 5’ and 3‘ C a primers which amplify a 591-bp fragment (CO nucleotides 7 to 598). Theoligonucleotides were synthesized with an Applied Biosystems 391 DNA synthesizer (Applied Biosystems, Foster City, CA) and purified by solid-phase extraction on a Sep-PakTMCIRcartridge (Waters, Milford, MA). Amplification steps for cDNA were performed as follows: “hot start” at 80°C for 10 minutes, followed by 35 3-step cycles of 94°C for 25 seconds, 53°C for 25 seconds, and 75°C for 25 seconds. A final one-step cycle of 72°C for 7 minutes was used. All PCR reactions were performed using the GeneAmp 9600 (ABI-Perkin-Elmer, Norwalk, CT). For purposes of quantitation, 10pCi of (a-32P) dCTP+dATP was added to each reaction. Radiolabeled products ( l / 5th of reaction) were separated on 1.3% agarose (Seakem L E FMC Bioproducts, Rockland, ME) gels, dried under vacuum, and then exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA). Quantitation was accomplished using ImageQuant software after scanning the screen with the Phosphorimager (Molecular Dynamics). Quantitative aspects of TCR repertoire analysis using PCR. To substantiate the reliability and reproducibility of the PCR studies that are an integral part of this report, we conducted a series of titration and validation studies of TCR product made from PB of normal volunteers. Test reactions were performed to determine the maximum number of PCR cycles that would still cause the linear incorporation of 32Pnucleotide triphosphate into double-strand DNA product. Standard reaction conditions as described above were used, except that the cycle number was varied between 15 and 45repetitive cycles. Two pairs of oligonucleotides which respectively amplify the C a (591 bp) fragment and V(D)JC fragment from the rearranged Vp (360 bp) TCR (vp2,7<pl,z , Table 2) were co-incubated in our standard reaction mix. Since the incorporation of 32Pnucleotide triphosphate plateaued at 40 cycles for C a synthesis, we chose to use 35 cycles in all further PCR. Interestingly, both Vp2 and VD7 synthesis did not plateau, even at 45 cycles (data not shown). We also determined the amount of oligo-dT primed cDNA that would give an adequate signal, without saturation of the available enzyme in the PCR. cDNA was made from PBL RNA as described above. Two pairs of oligonucleotides were added, as described for the above experiment, and 35 cycles of synthesis were performed in the GeneAmp 9600, with the cycles times and temperatures as described above. Five microliters of cDNA results in a level of 32Pnucleotide triphosphate incorporation in double-stranded TCR product that is linearly dependent with the amount of input cDNA (data not shown). That volume of input cDNA has been used in PCR reactions for our subsequent repertoire studies. Molecular cloning and sequence analysis. PCR amplification products from patients and blood donors at various time points were purified from 1.3% agarose separating gels electrophoresed in TAE (Tris-Acetate-EDTA, pH 7.5) with the help of glass beads (GeneClean 11; B10 101, La Jolla, CA). The fragments which contain ragged ends (terminal 3’ dATP addition) as aresult of their synthesis using modified Taq polymerase (Stoffel Fragment) were cloned into a modifiedpGEM vector (Promega, Madison, WI) containing 3’ From www.bloodjournal.org by guest on January 12, 2015. For personal use only. MOLECULAR ANALYSIS OF TCRREPERTOIRE IN 3035 GVHD Table 2. Oligonucleotides UsedTO Detect TCR Va and Ca Gene Segments V, Gene 5’ Nucleotide Sequence 3‘ 1 GCACAACAGTTCCCTGA TCATCAACCATGCAAGCC GTCTCTAGAGAGAAGAAG ACATATGAGAGTGGATITGT ATACTTCAGTGAGACACAG TKCCTAACTATAGCTCTG AGGCCTGAGGGATCCG CCTGAATGCCCCAACAG ATITACITAACAACAACGTT CCTAAATCTCCAGACAAAG CTCCAAAAACTCATCCTGT TCAACAGTCTCCAGAATAA AAAGGAGAAGTCTCAGAT CAAGGAGAAGTCCCCAAT GGTGAGGGTACAACTGC GTCTCTCGAAAAGAGAAGA AGTGTCTCTCGACAGGC AAAGAGTCTMCAGGATGA CAGATAGTAMTGACTITCA GATGAGTCAGGAATGCCA CAATGCCCCAAGAACGC AGCTCTGAGGTGCCCCAGAA~C~C TCCAACCTGCAAGGCTTGACGACT AAGTGATCTTGCGCfGTGTCCCCA GCAGGGTCCAGGTCAGGACCCCCA CCCAGITGGAAAGCCAGTGACCC CACCTCCTTCCCATKAC AACAAGGTGTTCCCACCCGAGGTCG CCAGAACCCTGACCCTGCCGTG ATCATAAATTCGGGTAGGATCC 2 3 4 5.1 5.2 6.1 7 8 9 10 11 12 13.1 13.2 14 15 16 17 18 19 20 W21 W22 w23 w24 CP3.2 C& Ca5‘ Ca3‘ Fragment (bp) 360 360 360 370 380 350 350 355 390 355 355 350 370 370 380 360 360 380 380 360 310 360 360 360 360 360 59 1 591 PCR primers from 26 V@ families are shown. The oligonucleotides Vp1-195p and V ~ ~ Z O - W Zare ~ ~ ’modifications from published sequences. c&, c&. Cas’,and Ca3’ were designed by Liu et al.= The fragment sizes are denoted in nucleotides (bp) based on comparison with the 100-bp DNA Ladder (GIBCO-BRL). terminal thymidines at both ends (pGEM-T). Recombinants in JMI09 bacteria were identified using ,%galactosidase screening on X-gal plates with isopropyl-0-D-thiogalactoside (IPTG). The plasmids containing inserts were separately grown as 10-mL cultures, and plasmid DNA prepared from them by the alkaline lysis method with an added RNAse digestion step (1 p@mL for 60 minutes at 37°C). Sequence analysis was initially performed using complementary primers for the Sp6 or T7 promoter sites flanking the cloning sites. Further sequence information was obtained using antisense primers to CB or 17- to 20-bp primers complementary to sequences unique to individual TCRs. Sequencing was performed using denatured double-stranded plasmid DNA with a deaza-GTP containing Sequenase Version 2.0 kit (USB, Cleveland, OH) and ”S-dATP (ICN, Costa Mesa, CA). Gene segments were identified by comparison with the Genbank database using IGSUITE (Intelligenetics, Palo Alto, CA). Hybridization of oligomer probes to VB-specific PCR products. Ten microliters from the 50-pL PCR reaction was size-fractionated on a 1.3% agarose gel, and the bands localized by staining with ethidium bromide ( 1 pg/mL). After electrophoresis, the gel was denatured for 30 minutes in 1.5 m o m NaCV0.5 m o m NaOH, neutralized for 30 minutes in 3.0 mom NaCV0.5 mol& Tris-HCI, pH 7.5, and then transferred to nylon membranes in 2OX SSC (3.0 mol/ L sodium chloride, 0.3 moVL sodium citrate, pH 7.0)by capillary force for 16 hours. The DNA was fixed to the filter by UV illumina- tion (Stratalinker; Stratagene, La Jolla, CA). The membrane was prehybridized for 3 hours at 50°C in a solution containing 1% sodium dodecyl sulfate (SDS), 6x SSPE (1 X SSPE: 180 mmol/L NaCI, 10 m o Y L NaP04, pH 7.7, 1 mmovL EDTA), and 1OX Denhardt’s solution (0.1%bovine serum albumin, 0.1 % polyvinylpyrrolidone, 0.1% Ficoll), 50 p g / d denatured salmon sperm DNA, and 20 @g/ mL tRNA. Oligonucleotides were labeled at the 5’ end by phosphorylation with T4 polynucleotide kinase and { y-”Pj ATP (see Table 5 ) . The labeled probe was separated from unreacted ATP by passage over a NENSORB 20 Nucleic Acid Purification Cartridge (Du Pont-NEN, Boston, MA). Hybridization was performed in 1.0% SDS and 6x SSPE at the TM of the DNA-hybrid for 16 hours. The filters were washed three times for 15 minutes in 6x SSPE and 1.0% SDS at 20”C, followed by a single wash-step in 1X SSPWl.O% SDS at the TMof the hybrid for 30 minutes. The filters were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY). Quantitation was accomplished using ImageQuant software after scanning the screen which was exposed to the radioactive filter with the Phosphorimager (Molecular Dynamics). RESULTS V0 Family Usage in Eight BMT Recipients We identified 8 patients for which RNA had beensuccessfully prepared from PB at the appropriate intervals indicated in Materials and Methods, and in 6 of 8 patients at least one skin biopsy specimen was available in which RNA had also been successfully prepared. In addition, RNA was prepared from a PB sample taken on, or a few days before, a skin or gut biopsy specimen was taken on suspicion of GVHD (Tables l and 3). Clinical information for these patients is shown in Table 1. Five of eight patients had MUD donors, 3 had MSD donors, and in 1 MSD case the recipient was m i s matched at the HLA A locus with respect to the donor (A2/ A26 v A2/A2, Table 1). Inallbut three instances, microscopic evaluation of skin biopsy samples showed them to beinfiltrated with lymphocytes characteristic of GVHDi9 (and data not shown). We also prepared RNA from colon and stomach biopsy specimens from patients who had previous GVHD positive skin biopsies (Table 1). Results from the Vp repertoire studies from PB are shown in Fig 1. The nomaiized percentage of expression for each V,8 family is tabulated and presented underneath an example of a V 0 TCR profile from one analyzed patient (UPN 916, d32). Most V@ have expression levels that vary less than a few fold between patient samples, and there is little evidence for bias in expression of individual family members within a sample. The levels of expression of most Vps in the samples shown in Fig 1 compare well with published analyses and our determinations from normal individuals4*(and data not shown). The importance of these results is the contrast they provide to our observations of V@ family usage in skin biopsies. The results of the repertoire studies in biopsied skin are shown in Fig 2. A remarkable aspectof the data is the radically different profile of usagein skin versus P 8 sampledat the same time (Figs 1 and 2). in contrast to the results with PB, among 26 VP families examined,only 1 family is consistently overexpressed in all of the biopsied skin samples tested. The VD2 family shows predominant expression in biopsied skin, whether or not it is frompositivelyidentified GVHD lesions (Fig 2), although the absolute levels of Vp2 TCR RNA From www.bloodjournal.org by guest on January 12, 2015. For personal use only. LIU ET AL 3036 Sample - Table 3. Time Points ofPeripheral Blood Samples From the Eight Patients Discussed in This Report Analyzed by PCR and Southern Hybridization UPNl 768 789 916 2081 2085 2093 2094 2098 PER BM 421-34 X X X X X X 635-48 449-62 463-99 d100-dl79 X X X d180-d364 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 1Y 2Y X X Died Died Died X The numbers following the d(day) refer to days posttransplant. 1Y (lyear) and 2Y (2 year) were samples collected from a single patient (no. 768). Peripheral blood samples were taken pretransplant from the recipient (PER), and a portion of the bone marrow from the donor (BM). A I 12 l1 I O 9 eO.1 4.5 6.5 8 I Vs Family # 7 6 5.2 5.1 4 3 2 1 Flpi + d32 1 + + 6 I 15.8 120811 d157 120851 d74 + d35 + d42 eO.1 17.4 eO.1 eO.17.9 6.5 6.3 2.8 5.8 12.4 0.4 eO.1 3.7 6.0 2.3 2.3 1.4 1.7 2.1 16.2 eO.1 eO.1 2.7 3.1 m m I 4.1 5.39.6 5.0 0.65.66.1 5.5 3.4 eO.1 I cO.1 4.8 4.0 6.6 1.7 4.3 2.3 3.5 5.0 3.3 1.8 p.0 cO.1 eO.1 3.1 2.3 4.2 4.3 3.4 1.6 2.6 - d82 12098116.6 ~ 0 . 1 7.9 9.0 I I I I 9.4 eO.1 8.7 eO.1 eO.1 11.7 3.8 5.2 8.7 3.6 1.7 1.7 18.0 14.7 B + d74 12081]10.1 0.9 2.2 1.1 3.7 1.7 4.8 1.6 4.0 1.5 + dlS7 m 2.0 2.9 4.0 18.2 2.2 + 11.6 1.5 5.3 4.9 3.1 0.3 0.8 4.4 I 0.9 7.2 17.3 11.8 d35 3.5 0.4 4.5 1.3 0.3 5.8 5.1 22.1 6.1 4.9 3.1 12.0 + d42 12.3 - d82 12098j10.5 2.2 1.5 0.8 2.0 0.3 0.2 1.926.1 6.5 0.3 0.4 1.8 0.5 3.2 3.3 7.6 CO.l 0.9 0.3 0.7 2.3 14.5 are lower in biopsies uninvolved with GVHD (data notshown). The Vp2 gene segment is representedin PB atthe level of 7.0% to 14.0%in normal individuals, although in skin there is no evidence of its preferential usage in a previous study."?Vpl 1 Fig 1. Southern hybridization analysis of PCR products from PB. (A) Results using Vpl-12. PB was analyzed for expression of Vp TCR familiesonthe same date as the biopsy sample was takenfromtheindicated patients. The presence of GVHD wasconfirmedbythe Department of Pathology, and is indicated by a plus or minus symbol and the number of days postBMT of occurrence (Table 1). Techniques of PCR using simultaneous dual amplificationof Vp and C a fragments are described in Materials and Methods. Fragments were separated on 1.30/0 agarose gels and transferred by capillary action t o nylon as described in Materials and Methods. To detect the expression of the Vp-Cp fragments, 32Plabeled oligonucleotide CpM (Table 2) was hybridized to the membrane-bound doublestranded DNA PCR product. The normalized percentages for each Vp family are indicated, and for patient 916, the gel bands are shown. Data analysis was done as described in Materials and Methods. (B) Results using Vp13.1-24 same as(AI, except the Vp families examined are 13.1-24. Vp2 as a reference fragment wasgenerated at thesame time as Vp13.1-24. is the only other family that showed consistently moderately high expression in skin biopsies, although its expression is far less than Vp2. As expected, other Vp families are also expressed in skin, although there is no consistency inthe level From www.bloodjournal.org by guest on January 12, 2015. For personal use only. MOLECULAR ANALYSIS OF TCR REPERTOIRE IN GVHD 3037 A 1 9 13.1 101112 Fig 2. Southern hybridization analysis of PCR products from skin biopsy specimens. (AI Shown are the results for testing the expression of Vpl-12 from skin biopsy R N A using PCR analysis as described in Fig 1. GVHDpositive biopsy samples are distinguished by a plus symbol l+) and the two GVHD-negative biopsy samples are indicated by a minus symbol (-1, next to the U P N number and the days postBMT of occurrence. Methods for preparation of RNA from biopsy specimens are described in Materials and Methods. Hybridization to Southern blots waswith CPM (Table 2). and the visualization and quantitation of radioactive bands was done as described in Materials and Methods and Fig 1. R N A from each biopsy specimen was analyzed for expression of actin mRNA. t o normalize expression of Vp TCR families(data not shown). (B1 Analysis of Vp13.1-24: same as (AI, except that the Vp oligonucleotides used are V/513.1-24. As a comparison for relative band intensities, the Vp2 family was amplified at thesame time (data not shown). + d32 m + d74 120811 + dl57 + d35 120851 120931 + d42 m 1 1.0 CO.1 I cO.1 0.3 cO.1 m m I -dl09 -d82 1 cu.1 0.5 cu.1 CO.1 CO.1 cO.1 C0.1 cO.1 1.9 CO.l CO.1 CO.1 1.0 I ~ 0 . 1 cO.1 23 66.3 6.5 CO.1 CO.1 ~ 0 . 1 15.4 CO.1 1 6 2 5.2 3 45.1 7 8 cu.1 CO.1 [cO.1 cO.1 cO.1 4.0 CO.1 I Vfi Family # 11.3 CO.l 1.1 3.6 0.8 CO.1 CO.1 cO.1 1.1 1.5 CO.l CO.l 72.3 14.7 2.5 ~ 0 . 1 50.8 9.8 I 1.1 I 57.8 4.0 I ~0.1 cO.1 ~ 0 . 1 cO.1 cO.1 0.8 3.4 0.3 77.5 13.9 CO.1 CO.1 CO.1 CO.1 CO.1 CO.1 CO.1 CO.l I 1.8 0.4 95.5 2.0 B 21 22 23 24 + d32 m + dl57 120811 120851 + d35 120931 + d42 L204 + d74 I 1 co.1 CO.1 0.2 cO.1 c0.1 cO.1 cO.1 1co.1 CU:I co.1 co.1 co.1 W : I co.1 co.1 co.1 crrld 19 17 18 Riopsy The striking overcxpression of the V02 gene segment in the biopsy samples that we examined led us to determine whether the mixedpopulationofVD2-bearing TCRs had individual members that werespecificallyamplified in the GVHD lesion.Combinatorialmultiplicationfollowingthe I CO.1 CO.1 0.8 0.2 co.1 co.1 co.1 co.1 co.1 co.1 co.1 co.1 co.11 CO.1 ~ 0 . 1CO.1 CO.1 ~ 0 . 1 c0.1 29.7 ~ 0 . 1~ 0 . 1 CO.1 ~ 0 . 1 CO.1 co.1 co.1 co.1 co.1 co.1 cu.1 cu.1 qu.1 co.1 16 13.2 13.1 14 15 C0.1 0.9 cO.1 1.2 ~ 0 . 1cO.1 3.9 2.3 of expression of the other VD family members in our cohort of patient skin biopsy samples. VD4 has significant expression in some cases. a s do V07 and VD I8 in isolated cases (Fig 2A and B). Since in none o f the C;ISCS that we have tested was there a VD limlily that superseded V02 expression in biopsied skin, we pursued a more detailed look at the individual TCR composition of the V02 Family in individual patient tissue and PR samples by sequence analysis. Seqrrcwcv Atwlysis of' VD2 E.vprcwion iu P R Sp"'if,lc~rl.s CO.l co.1 co.1 co.1 I cO.1 cO.1 I -dl09 0.3 20 U.Z co.1 co.1 co.1 0.J CU.l co.1 co.1 CU.1 CU.1 co.1 co.1 co.1 CO.1 0.6 CO.1 co.1 co.1 0.3 CO.1 I I I I co.1 co.1 co.1 rules of association of V, D. and J genesegments and allowing for the generation of diversity by N nucleotides at -IO1: the V-D and D-J joins results in thecalculationof potentially different VD TCRS.'~"Therefore. examination of a small number of VD TCR chains by sequence analysis in PB or the lymphocytic infiltrate of a GVHD lesion without preferential expansion of a specific T-cell clone(s) should result in a random assortment of joining sequences without duplication of any one sequence. We tested that proposition by subcloning the VP2-containing fragments that were generated by PCR methods from our series of biopsiedskin lesions. of which tive were positive and two were negative for GVHD (Fig 2). In addition, WC cloned VD2 PCR fragments from several PB samples from patients 768 and 789 of' whom we did not have a biopsy specimen (Table 3). The fragments were cloned withoutmoditication into the "T" From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3038 LIU ET AL vector (Promega), individual bacterial clones were picked, and inserts were partially sequenced so as to identify the Vp family as Vp2, and to characterize the joining region. A Sequence Analysis of Vp2 TCR Joining Segments From Skin Biopsy Specimens B Approximately 1 0 0 bp located upstream of the CO junction with the J segment was sequenced using the dideoxy technique. This stretch of sequence includes the V(D)J joining segment and enough of the V gene to unambiguously identify it as V02 (Fig 3 for an illustration and Fig 4). Sequences were generated from 6 individuals, 5 of whom developed clinically definable GVHD. In 5 of 7 biopsy samples it was possible to determine a TCR sequence represented multiple times from independent plasmid clones (Fig 4A and Tables 4 and 5 ) . Further sequence analysis of each of these amplified TCRs showed them to be examples of productively rearranged TCRs with in-frame protein coding sequences (data not shown). In the two cases where we were unable to detect multiple copies of a single TCR sequence, there was a negative diagnosis for GVHD [916 (-) and 2098, Fig 4A and Table 41. In those cases, every independent Vp2 TCR plasmid clone that was sequenced had a unique primary structure (Table 4). We have recently sequenced multiple V02 TCRs from three additional biopsy specimens that were negative for GVHD, and we have not found amplified TCR sequences (data not shown). Similarly, in those patient samples in which we derived a predominant TCR sequence, all of the other TCR sequences in the sample were also unique (Table 4). Despite the presence of multiple TCRs containing the identical JP in a sample, the joining segment was unique at multiple nucleotides, precluding the derivation of closely related TCRs from the same cell. The multiple differences also made it improbable that errors caused by infidelity of either the Stoffel fragment of Taq polymerase or the Sequenase I1 version of the "7 polymerase were responsible for generating the diversity. Vp2 Sequence Diversity in PB Before and After BMT Previously, we had conducted PCR analysis of the TCR VD repertoires from the PB samples indicated in Table 3. We found little or no difference in the expression levels of Vp2 as well as the other V@ family members in samples withdrawn before and after transplant, or simultaneously with GVHD diagnosis (data not shown). To address whether clonal diversity or oligoclonality existed in those samples, an approximately equal number of Vp2 TCRs were sequenced from the BMT donor, the recipient pretransplant and the day of GVHD diagnosis, as well as on a date postGVHD treatment (day 188 for UPN 789, Fig 4 9 and 2Y for unique patient number [UPN] 768, see below). All of the TCRs were of unique sequence for patient 768, except for a single class of TCRs, which are highlighted in Fig 4B. An equivalent situation was found with patient 789 on day 35, although by day 188 in which the GVHD had resolved, we found no sequences that matched the unique TCR found on day 35 (Fig 4B). The significance of these results for the development of GVHD is that they suggest that a small number, possibly one clone, of T cells became amplified L V p l L Vp2 LVg3 LVpn Dpl Jp1.1-6 n u o n n o o n mmmemne 1 Cp2 /. I (N regiondiversification) RNA Transcrlptlon and Processing IPrimers Probe Jp2.1-7 1mmmmmn 0m / / L L e n t 4 m C Dp2 Cpl x? 4 ISObpl ~~ \VD2 NDpN I l JB2.3 l I ~ CP2 Fig 3. Diversification of TCRs by combinatorial association of V, D, and J segments. (A) The chromosomal locus of the TCR p gene is shown at the top of the figure which depicts the arrangement of gene segments as they are aligned on the chromosome?'The arrows originate fromsegments that werejuxtaposed t o create the TCR that was found t o be highly expressed during GVHD in patient 768. The selection of segments could apply t o any of the Vp2 TCRs found in patient blood or skinlesions described in this report (see Fig 4). (B) The gene segments which form the mature TCR become juxtaposed in chromosomal DNA from individualT cells. Between the ends of the V (3'), (5') D (3'), and (5') J segments are inserted nonchromosomal nucleotides (NI that markedly increase the diversity of TCRs. The ends of the gene segments that receive N nucleotide additionsalso have deletions of chromosomally encoded DNA. The rearranged DNA is transcribed into nuclear RNA which isprocessed to form the cytoplasmic mRNA that encodes the productivelyrearranged TCR p protein (see ref 44 for more details). (C) The expanded structure (not t o scale) of the mature RNA depicted in (B) is shown with references t o the gene segments and positions of N nucleotide insertions which form thecomplete sequence. Cloned PCR products whosesequences are shown in Fig 4 were generated using the primers shown in (Cl. The CDR3 sequences (Table 5) derived from data presented in Fig 4 are complementary t o the region of the TCR underneath the "probe" designation. PCR fragments from PB and skin (Fig 5A) were made using the primers depicted in (C). within the GVHD lesion, and in some cases the progeny of this clone as represented by the identical TCR could be detected in the PB. However, sequencing a small number of TCR mRNAs is not a sensitive enough technique to preclude that a given clonotypic TCR is not expressed. That possibility could be approached by sequencing a greater poolof plasmids, or switching to a different method of analysis that isuniquely suited to the polymorphism of TCR gene sequences? Detection of Specijc Vp2 TCR Using Probe CI CDR3 Region Our sequencing studies served an additional purpose by providing the primary structure of the conserved TCR sequence in GVHD skin lesions. These data assisted in the design of a probe that was potentially specific for a single rearranged TCR clonotypic sequence. We hypothesized that using sequence information from the most variable portion of the TCR between the V segment and the J segment would result in a unique sequence TCR probe.& Therefore, we synthesized a series of 20- to 23-bp probes that werecomplementary to the unique joining regionofthepredominant TCR as determined by sequencing studies from 7 of our From www.bloodjournal.org by guest on January 12, 2015. For personal use only. ANALYSIS MOLECULAR REPERTOIRE OF TCR IN GVHD 3039 r UPN # & Skin Biopsy Date 916(-) dl09 2081 d74 2085 dl57 2093 d35 2094 642 2098 d82 0 0 0 0 0 0 0 0 2 0 3 0 1 3 0 0 0 0 0 0 0 916(+) d32 W* 0 P 2 0 0 0 8 Y 3 l:?.l" 3 0 0 0 2 0 2 0 1 0 O T O Z 0 0 l 0 0 0 2 1 3 1 1 2 3 0 0 1 2 0 1 1 0 0 1 0 0 l 0 B' 0 1 2 0 1 0 0 1 1 e 3 2 S 1 3 0 1 0 l l 0 0 0 O b 2 0 2 1 3 1 3 2 0 5 4 7 4 4 2 5 1 s Fig 4. TCR Vp2 nucleotide sequences from GVHD skin lesions. (A) Fifty- to 100-ng aliquots of cDNA were selectively amplified using the Vp2 pair of oligonucleotideprimers into double-strandedfragments. As fully described in Materials and Methods, the 360-bp fragments (Table 2) were subcloned into the pGEM "T" vector, and individual bacterial colonies sequenced. The nucleotide sequence was determined between the 5' end of Cp through the last 50 bp of the 3' end of the Vp2 gene segment. Gene segments were identified by comparison with publishedsequences,"and those deposited in the GENBANK database accessed through IGSUITE (Intelligenetics Inc). Patient data are described in Table 1. Patient 916 had two biopsies (see Fig 2 and Table 11. Highlighted and starred numbers represent TCR molecules which were identical by sequence analysis from independent bacterial clones. All other sequenced TCR molecules had unique primary structures determined by sequence analysis. (B) Methods for PCR generation of fragments, subcloning, and sequence analysis are the same as in (A). Samples are indicated at the top of the table, and correspond to those shown in Table 3. PCR analyzed patients (Fig SA and Table S). To conserve RNA, we turned to the PCR approach and combined it with Southern hybridization analysis. Samples of RNA from PB and biopsy tissue were converted into Vp2 fragments using a single primer pair as shown in Table 2 (VD2 and C@,,,) as previously demonstrated in our repertoire studies (Figs 2 and 3). The PCR generated fragments were electrophoresed onto agarose gels. and the DNA transferred to nylon, followed by UV treatment to covalently attach the DNA.Oligonucleotide probes (Table S) were kinased with (y-?'P} ATP, andthen hybridized totheboundDNAfrom a series of samples of PB and skin biopsies for each of 7 patients who were diagnosed with GVHD (Fig SA). The hybridization profiles from patients for which we had biopsy samples are relevant to our central hypothesis of the role of TCR amplification and GVHD pathology. The hybridization profiles of the S patients withbiopsy data shown in Fig SA are consistent with the findings from our sequence studies indicating overexpression of a unique TCR sequence. In S of S patients whose skin biopsy specimens were shown to have a unique predominant TCR sequence, there was strong hybridization to the lane containing RNA from GVHD skin lesions (Fig SA). It is also of interest that CDR3 sequences determined fromamplified PBL T cells were also selective in their hybridization to PBL RNA taken only from timepoints when the patient had GVHD (Fig 5A). UPN 2081 illustrates the selectivity of the CDR3 probes. This patient had multiple biopsies, although a defined amplified rearranged TCR was only found in the GVHD positive skin biopsy sample. The skin biopsy specimen on day 37 as well as the colon biopsy sample taken on day 183 were both determined to be negative for GVHD by pathologic study, which isconsistent with the absence of a hybridization signal from the probe derived from skin on day 73. It is also noteworthy that nodetectable signal from the probe derived from the day 73 skin biopsy can be found in PB from the donor, or the recipient beforeand after transplantation (Fig SA). One possible conclusion from this exclusivity in expression is that the T-cell population bearing this TCR expanded at the site of injury and not in the general peripheralcirculation. The other 4 patients exhibit patterns that are similar in specificity as the one shown for patient UPN 208 I , yet each have unique aspects. Patients UPN 916, 2093,and2094 have strong expression of a unique TCR in the GVHD skin lesion, and in PB from the recipient withdrawn at the same time or subsequent to the diagnosis of GVHD (Fig SA). A stomach biopsy sample takenfrom UPN 2093.which showed no GVHD pathology upon microscopic analysis, also showed no hybridization totheday 35 skinprobe,which further illustrates the specificity of the CDR3 probes. Weperformed additional sequence studies of samples from patient 2093, which are presented in Table 4,and are informative in comparison with data shown in Fig SA. TCR sequences from V02 atd3S were all uniquewithoutany concordance withtheTCRpopulation in theskinbiopsy specimen. Sequences determined from the PB at d63 in two examples are identical to the TCRs whose sequence we determined in the GVHD lesion at day 35. The hybridization profile for this patient shows a strong signal in the day 35 skin lesion, butnot concurrently in the PBat day 35. In agreement with our limited sequence data from day 63 is the presence of a strong hybridization signal from PB initiating at day 63 of the Southern blot profile. This concordance in the hybridization profileand our limited sequence data frompatient 2093 suggests that strong hybridizationfrom skin or blood samples with a unique sequence probe may be predictive for the occurrence or continuation of the pathology of GVHD. There were no pre-BMT donor or recipient PB samples to compare hybridization signals for UPN 916; however, the intense signal on day 32 in skin and blood disappears from further PB specimens taken after resolution of GVHD. In From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3040 LIU ET AL Table 4. Summary of the Sequence AnalysisIndicating Semples That Have TCR Sequences Repeated Two or More Times UPN and Sample Description No. Sequences Determined No. Identical Sequence Repeats 768-PER 768-PED 768-dl25 789-PER 789-PED 789-d35 789-dl88 916 (-) Skin 916 (+) Skin 2081 Skin 2085 Skin 2093-d35 2093-d63 2093 Skin 2094 Skin 2098 Skin 17 17 17 14 15 14 14 11 12 7 15 11 14 14 27 14 0 0 6 0 0 5 0 0 11 2 and 3 5 0 2 14 17 0 ~~~ ____ Nomenclature for peripheral blood samples is from Table 3. TCR sequences from skin samples areso noted, next to thepatient's UPN (see Fig 4 and Table 1). The criteria for an identical sequence repeat is that the approximate 100 bp between the 3' end of the germline V02 gene segment and the 5' of the C0 gene does not vary by even onenucleotide in sequenceidentity. The column that lists unique sequences is acompilationofalldetermined TCR nucleotide sequences i n a sample which vary by one or more bps. In most cases, unless the identical TCR is repeated, the unique sequences tend t o vary in length and nucleotide identity by5 or more bp in the joining region (data not shown). the case of UPN 2094, the PB samples withdrawn pre-BMT exhibit little or no expression of the unique TCR determined in skin on day 42 (Fig SA). Patient UPN 2085 has an expression pattern resembling UPN 208 1, in thatlittle or no expression of a unique GVHD skin lesion TCR can be detected in the PB at any timepoint. Although a small amount of expression can be detected in the BM before transplant, the level of expression diminishes untilthe development of skin GVHD, and there is no further expression in the PB at any timepoint after transplantation that was available for study (day 180, UPN 2085, Fig 5A). Taken together, data from the skin biopsy specimens with GVHD as opposed to GVHDnegative biopsy samples show a consistent pattern of expression of oligoclonal TCRs that cannot be satisfactorily explained byPB or BM contamination. The likely, although still unproven, explanation is that the expression pattern of these unique TCRs is connected with the pathology of GVHD in these patients. Vp2 Expression Is Equivalent in Blood Samples and Biopsy Samples From GVHD Patients To rule out a trivial explanation of the highly significant results reported inFig 5A, all of the Southern blots were rehybridized with a probe that detects the repertoire of TCRs that are members of the V02 family (Table S). As expected, there is very little or no detectable variation in V02 TCR expression that would alternatively explain the results shown in Fig SA using unique TCR sequence probes (Fig SB). The signal from Vp2 TCR expression from samples of PB and skin or gutis remarkably homogeneous. This provides a suitable control to demonstrate that the variation in expression of individual Vp2 TCRs shown in Fig 5A is connected with a specific amplification process, as opposed to differentialRNA degradation or the absence of expression of all Vp2 family members in the blood or tissue samples. DISCUSSION The principal goal of this research has been to establish whether T-cell amplification occurs in the development of GVHD in BMT recipients. Our first approach to the problem was to examine the repertoire of TCRs from unmanipulated blood samples as a marker for the putative expansion of Tcell clones (Fig I). Based solely on the results from blood specimens, we were unable to demonstrate that the expression of the majority of TCR Vp families varied significantly between different individuals we examined in the midst of an ongoing immune response, namely GVHD (Fig l). The lack of sensitivity of displaying the PCR generatedTCR fragments on agarose gels as opposed to sequencing gels obscures possible variation within a family that isqualitative. and not q~antitative.~' Therefore, we later turned tosequenc- Table 5. The CDR3 Sequence Probes Usedin Southern Hybridization of Peripheral Blood and GVHD Lesions From Patients Designated by UPNs Discussed in Table l UPN 768 789 916 2081 2081 2085 2093 2094 All Probe Name J02.3D1 J01.4 JP1.5D1 [email protected] J02.1D1 J02.3D2 JP2.1D2 J02.1D2 VP2P Sequence of Probe Length Detection of Expression 20 Yes Yes Yes No Yes Yes Yes Yes Yes 23 20 21 20 20 20 20 25 TCR sequence whose joining region was unique in primary structure. The most polymorphic portion Each GVHD skin sample had a duplicated of the joining region was made into an oligonucleotide probe referred to as a "CDR3" sequence for the purposes of Southern hybridization studies of all available specimens. In one case, it was found that the identical sequence was conserved for two different patients (2093 and 2094). Patients 768 and 789 had CDR3 sequence probes derived from duplicated TCRs from peripheral blood samples withdrawn on the same date as the biopsy. In thosecases the biopsy sample was not available for molecular analysis. From www.bloodjournal.org by guest on January 12, 2015. For personal use only. MOLECULAR ANALYSIS OF TCR REPERTOIRE IN GVHD 3041 A Fig 5. (AI CDR3 probe analysis ofchronologic PB samples and skin biopsy specimens. PCR fragments of Vp2 containing TCRs were generated as described in Fig 2, and electrophoresed on 1.3% agarose gels, transferred t o nylon membranes. and then hybridized with 32P-labeledprobes as shown t o the right of the autoradiogram for each patient. A description of the probes used can be foundin Table 5. PB sample nomenclature designations can be found in Table 3. Biopsy samples are identified with the following letters: S, skin; C, colon; St, stomach. The date of the biopsy inis dicated as a superscript above the letter designation. CPM calculated using ImageOuant from phosphor screen exposures are shown underneath each lane ( ~ 1 0 ‘ ) .(B) Reprobe of membranes shownin (AI with the Vp2P probe (Table 51. Blots were stripped of probes shown in (A), and rehybridized with “P-labeled Vp2P probe. F[ 109 S’09 100 63 49 44 32 vp 2P 916 vp 2P 2081 l80 2085 157 - -”. St’9 S” 96 96 56 61 S”’ 63 49 -I 35 2835 -- 35 21 PBR PED BM v p 2P 21 PER PBD BM vp 2P 2093 S 42 2094 ing random TCR clones generated from PB at different time points for a more precise picture of the sequence diversity (see below). A significantly different resultwas observed when we repeated the repertoire analysis, using the GVHD tissue lesion as the source of mRNA (Fig 2). We observed a highly skewed repertoire that favored the expression of V02 family containing TCRs in the small sample of patients we ana- 61 59 42 36 PBR 21 PBR BM PED vp 2P lyzed. This result led us to determine nucleotide sequences of individual expressed TCR genes from the group of patients described here (Table 1 andFig 4). Two important points are addressed by this data: ( I ) random selection of rearranged TCR gene segments and expression could not account for the observed usage profile of productively rearranged TCR V02 genes in skin lesions, and (2) biased usage of amplified TCRs in GVHD lesions was a disease-specific phenomena From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3042 because the equivalent sequence analysis in patients whose biopsy specimens were negative for GVHD did not show a comparable amplified TCR usage profile. It remains to be explained why in every skin biopsy sample we examined, whether or not it is disease-free, has a bias in the usage of the VD2 family (Fig 2). Since all of the patients involved in this study have been treated post-BMT with agents that modify T-cell function regardless of GVHD status, we can hypothesize that the bias in the TCR repertoire of lymphocytes that infiltrate the skin is related to the unique circumstances of these patients. Ongoing studies are examining the extent of this TCR repertoire bias. Alignment of the sequences from each of the identified predominant TCRs showed that they arose from in-frame protein-encoding genes that encode productively rearranged Vg TCR chains (data not shown). In 6 of 7 cases, the 5‘ ends of rearranged D and J segments were flanked by N nucleotide insertions and deletions by comparison with nucleotide sequences of the corresponding germline gene segments (Table 5 and data not shown).&Usage of JP segments was random in skin biopsy or PB samples, although not all JP segments were selected for a given patient, nor were some JP segments used in any of the patients (Fig 4). However, apparent bias of usage of particular JP segments was found as a consequence of the amplification of a predominant TCR in a skin or PB sample. Results from a larger sample size of BMT patients may reveal association of JP usage with the development of GVHD (Liu et al, in preparation), as has been reported for an example of an autoimmune disease.47 The uniqueness of the TCR joining regions allowed us to predict probes that would discriminate among a family of rearranged TCR genes to detect the expression of the TCR that we determined by sequence analysis (Fig 4 and Table S). We proved this in one case, when we sequenced rearranged TCR genes that were the product of a Vp2-Jp2.3 rearrangement from a series of samples that were positive using the unique CDR3 sequence fingerprinting probe (Fig SA). The identical junctional sequence was found in the Vp2-Jp2.3 TCR pool, from a sample that was taken 2Y after the original PB sample on the day of GVHD diagnosis from UPN 768 (Table 3). The pattern of hybridization from the Southern blot (Fig 5A) together with the sequencing result at 2Y (see Results) are in agreement, and also serve to reinforce the correlation between GVHD and the expression of a unique amplified TCR (Fig 4). Nonetheless, the methods used in this report are unable to distinguish high expression of TCR mRNA from a small group of cells, as opposed to moderate expression of the same RNA by a larger number of cells. Therefore, use of in situ hybridization methods on paraffin-embedded GVHD lesions with probes generated from CDR3 sequences will be more suitable to test whether greater numbers of an oligoclonal T-cell subset or just limited numbers of TCR over-expressing T cells have infiltrated the lesion. The restricted expression of TCRs that are complementary to the derived CDR3 sequence probe gives support for their possible association with the development and/or pathology of GVHD in BMT recipients (Fig 5A). The unique primary structure of predominant TCRs in individual patients is an LIU ET AL argument in favor of their expansion as a result of antigenic differences between the donor and the recipient. In this small sample, we only found one instance where a probe from one patient hybridized to RNA from another patient in a predictable way (Fig 5A and Table S). The HLA type of these patients show a single class I and class I1 MHC allele in common (Table l). We can only speculate whether similarities or differences in HLA alleles will lead to predictable changes in the T-cell repertoire. A recent report examining the influence of class I1 HLA alleles on the JP repertoire suggested that frequencies of usage of particular JP gene segments could be shaped by the expression of alternative HLA alleles.47A study of TCR repertoire in GVHD from a donor and recipient mismatched in the class I1 MHC DR gene also showed preferential usage of a JP segment.” These studies establish a precedent that allelic members ofHLA class I1 genes can cause the altered usage of components of the TCR repertoire. Similar approaches as have been described in this report have been used in several disease states and with BMT pat i e n t ~ .A ’ ~similar approach of examination of the TCR repertoire of BMT patients using different methodologies was conducted in the Kourilsky laboratory.’” Their recent methodology involving an automated sequencer andImmunoscope (Institute Pasteur, Paris, France) software allows a precise measurement of the CDR3 rcgion, and the ability to distinguish different TCR cl ono type^.'^.^^ Yet, there aresome differences in the expression of V p families in skin samples reported by Dietrich et al” and in this report. They found a broader representation of overexpressed Vp families, although the sequencing of consecutive bacterial TCR clones from a single Vp family was done differently than this study. The discrepancy from our results is not easily explained, especially because their one reported case of skin GVHD arose from an MUD transplant patient whose conditioning regimen, transplant procedures, and post-GVHD prophylaxis were similar to what was performed on several individuals from our group of MUD patients.’” One important difference in methodologies is that only through actual sequencing of the V(D)J joins is it possible to establish whether they are in frame, and either the Immunoscope”~4x or similarly described techniques do not directly address whether TCR molecules are productively reamangedA5 TCR clonotype expansion within a GVHD lesion as studied in this report is reflective of a classic immune response stimulated by neo-expression of foreign antigen. The donors of BMT recipients wereall adults inwhichwe presume their existent immune system has been thymically educated. Variation of the HLA allelic complement between the donor and recipient may either cause presentation of neo-antigenic epitopes or allorecognition of MHC, events that would be capable of eliciting an immune response, such as whatis described in GVHD. An example of a restricted immune response that was implicated in the development of GVHD was the cross-reactivity of allorecognition of MHC and an Epstein-Barr virus e p i t ~ p e .Analogous ~~ to this report, a dominant cytotoxic T-lymphocyte (CTL) response was demonstrated in which the repertoire of antigen-specific TCR was highly restricted. Antigenic exposure can shape the Tcell repertoire, andis applicable toBMT, especially with From www.bloodjournal.org by guest on January 12, 2015. For personal use only. MOLECULAR ANALYSIS OF TCR REPERTOIRE IN GVHD respect to MUD transplants.Refinements of theapproach initiated here, which include useof in situ methods of detection of the CDR3 sequence in lesions, growth of T cells in vitrofrom PB or lesions, and MoAbblockingstudies of those cells, mayresultin effective therapeuticalternatives for BMT patients with acute or chronic GVHD. ACKNOWLEDGMENT We thank the dedicated physicians and nurses of the Hematology and BMT program for their tireless efforts in securing the patient samples usedinthis study. Nurse coordinators Pilar Fornbuena, Barbara Littrell, and Rodrigo Nunez are sincerely thanked for their skilled organization of accrual of all patient peripheral blood samples. Drs Donald David and Richard Merkreebs are acknowledged for their help in procuring the gastroenterologic biopsy specimens. The technical help of Zuzana Valo, Phyllis Hao, and Paul Bienvenue in the early stages of this study is gratefully acknowledged. The help of Drs Auayporn Nademanee and Joyce Niland inthe interpretation of clinical parameters and patient characteristics is gratefully acknowledged. The authors thank DrsPeter Parham and Joshua Ellenhorn for a critical reading of the manuscript, and for providing important suggestions for its improvement. REFERENCES 1. Forman SJ, Blume KG: Allogeneic bone marrow transplantation for acute leukemia. Hematol Oncol Clin North Am 4517, 1990 2. Thomas ED, Buckner CD, Clift RA, Fefer A, Johnson FL, Neiman PE, Sale GE, Sanders JE, Singer JW, Shulman H, Storb R, Weiden PL: Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 301597, 1979 3. 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Forman SJ, Diamond DJ: Methods lor determining expression and oligoclonality of TCRs in CVHD lesions from allogeneic BMT recipients, in Oksenberg JR (ed): The Human Antigen TCellReceptor:SelectedProtocols and Applications. Georgetown, TX. Landes (in press) From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 1996 87: 3032-3044 Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions X Liu, V Chesnokova, SJ Forman and DJ Diamond Updated information and services can be found at: http://www.bloodjournal.org/content/87/7/3032.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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