specific t cell receptor gene rearrangements at the …...specific t cell receptor gene...

of February 23, 2010 This information is current as

1994;153;4798-4805 J. Immunol. Powell, HM Blau and L Steinman E Gussoni, GK Pavlath, RG Miller, MA Panzara, M

dystrophydegeneration in Duchenne muscularrearrangements at the site of muscle Specific T cell receptor gene

References

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Specific T Cell Receptor Gene Rearrangements at the Site of Muscle Degeneration in Duchenne Muscular Dystrophy’

Emanuela Gussoni,** Grace K. P a ~ l a t h , ~ ~ Robert C. Miller,* Michael A. Panzara,* Marianne Powell,§ Helen M. Blau,t and Lawrence Steinman4*

*Departments of Neurology and Neurological Sciences and ‘Molecular Pharmacology, Stanford University School of

Medicine, Stanford, CA 94305; and *Neuromuscular Research Division, California Pacific Medical Center, San Francisco, CA 941 18; and sArizona Cancer Center, Tucson, AZ 85724

Mononuclear cells infiltrate degenerating muscles of Duchenne muscular dystrophy (DMD) patients. Using a quantitative PCR, we first characterized the T cells infiltrating muscle biopsies from six DMD patients. High levels of TCR Vp2 transcripts were observed in DMD muscle tissue. TCR Vp2 transcripts from seven DMD patients and five controls were sequenced, and the VDJ junctional region analyzed in 166 clones. One specific amino acid motif, RVSG, was found in the third complementary determining region (CDR3) of TCR Vp2 chains in samples from five DMD patients, but not in controls. A specific immune reaction at the site of tissue degeneration may play an important role in the pathogenesis of DMD. The journal of Immunology, 1994, 153: 4798.

I n Duchenne muscular dystrophy (DMD),’ a fatal X chromosome-linked recessive disease, skeletal muscle inexorably degenerates (1). Typically, boys with

this disease lose the ability to walk, and become wheel- chair-bound by the age of 8 to 12 yr. Patients usually die in their mid-twenties of respiratory failure caused by skeletal muscle weakness and an accompanying cardiomyop- athy. The primary cause of the disease is the absence of the protein dystrophin (2-4). Dystrophin associates with an integral membrane glycoprotein complex, which is con- nected to extracellular matrix proteins, such as laminin (5-8). Dystrophin plays an important role in the stabili- zation of the plasma membrane during muscle contraction (9, 10).

Received for publication June 8, 1994. Accepted for publication August 11, 1994.

The costs of publication of this article were defrayed in part by the payment of

accordance with 18 U.S.C. Section 1734 solely to indicate this fact. page charges. This article must therefore be hereby marked advertisement in

ation. C.K.P. is a postdoctoral research associate supported by the Muscular ’ E.G. is a postdoctoral fellow supported by the Muscular Dystrophy Associ-

Dystrophy Association. This work was supported by grants to H.M.B. and L.S. from the Muscular Dystrophy Association, SmithKline Beecham, and the Na- tional Institutes of Health.

’ Current address: Dr. Emanuela Cussoni, Division of Genetics, Childrens’ Hospital, Enders, Room 570, 320 Longwood Ave., Boston, MA 021 15.

’ Current address: Dr. Grace K. Pavlath, Department of Pharmacology, Room 5001, Emory University, Atlanta, GA 30322.

Address correspondence and reprint requests to Dr. Lawrence Steinman, Stanford University, Beckman Center Room 6002, Stanford, CA 94305-5429.

‘ Abbreviations used in this paper: DMD, Duchenne muscular dystrophy; CDR3, third complementarity-determining region; ALS, amyotrophic lateral sclerosis.

Copyright 0 1994 by The American Association of Immunologists

As the disease progresses, secondary events occur both within the dystrophin-deficient muscle fibers, in which free calcium increases, and in the area surrounding the degenerating fibers, in which mononuclear cells, including both CD4+ and CD8+ T cells, infiltrate from the blood vessels into the parenchyma (11-16). Several studies have shown that corticosteroids and other forms of immune suppression, such as cyclosporin A, improve muscle strength and actually prolong ambulation by 2 to 3 yr (17- 21). Corticosteroid treatment of boys with DMD is associated with a decrease in T cells, primarily of the CD8+ phenotype, in the inflammatory infiltrate in muscle (14, 15). Taken together, these data suggest that an immune response to damaged muscle is occurring in the course of DMD, and that attenuating this response might have ben- eficial effects. To study whether this immune response is indeed mediated by specific mononuclear cells, we characterized the gene rearrangements of the TCR in the T cells infiltrating the muscle of DMD patients.

MateriaIs and Methods Patients and samples

Muscle biopsies of DMD patients no. 4, 5, 6, and 8 (ages ranging from 6 to 8 yr) who participated in a clinical trial (22) testing myoblast transfer as a gene replacement therapeutic strategy (23-26) were performed under local anesthesia. Patients no. 5 , 6, and 8 had shown the presence of dystropbin transcripts derived from donor cells 1 mo after transplant in the myoblast-injected leg ( 2 3 , but not patient no. 4. In patient no. 5 , normal dystrophin transcripts were detected after 6 mo. These patients had been immunosuppressed with cyclosporin A (5 mgkgiday) for a period of 6 mo, beginning 2 wk before myoblast transplantation. Muscle

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The Journal of Immunology 4799

biopsies from three other DMD patients, no. 84, 85, and 86 (age 6 yr) not related to the clinical trial were also performed as above. These patients were not immunosuppressed.

Control biopsies were taken from a 67-yr-old patient with myopathy caused by colchicine toxicity and from a 2-yr-old patient with a severe gait abnormality and with surgical correction of a congenital hip malfor- mation. Other control samples were taken postmortem and within 48 h from death: two muscle biopsies, one taken from a normal subject and another from a patient affected by sarcoma, a tumor sample from the same patient with sarcoma, and spinal cord tissue from two patients affected by amyotrophic lateral sclerosis (ALS).

Quantitative reverse transcriptase-PCR

Tissue was washed in cold sterile PBS before freezing and storage at -70°C. Each sample was homogenized, the total RNA was extracted and then reverse transcribed in cDNA as previously described (27). cDNA mix (15 pl), derived from 0.15 pg of total RNA, was then combined in a 45.~1 reaction mix with 3.5 pl 1OX PCR buffer (100 mM Tris-HCI, pH 8.3, 500 mM KCI, 15 mM MgCI,, 0.01% (wiv) gelatin; Perkin-Elmer Corp., Norwalk, a), 0.3 p M each of a 5’-Ca primer in conjunction with a 3’-Ca primer (28) and a 5’-Vp primer specific for each of 20 known V p gene families (27, 28) in conjunction with a C p primer (5’”ITCT GATGGCTCAAACAC-3’) (28). The C a primers served as a positive control for the integrity of the cDNA and the success of the PCR reaction as well as the internal control necessary for quantitation (28). A 5-p1 reaction mix containing 4.5 p1 of 2.5 mM deoxynucleotide triphosphates (Perkin-Elmer) and 1.25 U Tu4 polymerase (AmpliTuq; Perkin-Elmer) was added after the samples were brought to 80°C. The PCR profile used was: 93°C for 60 s, 55°C for 60 s , and 72°C for 60 s for 25 cycles in a DNA Thermal Cycler (Perkin-Elmer).

Fifteen microliters of each amplified product was run on agarose gel and then transferred by the method of Southern onto a nylon membrane. Filters were then hybridized with 1 pmol/ml each of a nonradioactive oligonucleotide probe labeled with horseradish peroxidase and specific for C a and Cp, as described (27). Intensity of the hybridized PCR products was quantitated with the use of a Molecular Dynamics (Sunnyvale, CA) computing densitometer with ImageQuant (Molecular Dynamics) software. The amount of TCR V p transcripts was then expressed as a percentage of the amount of amplified Ca-region transcripts.

Cloning of Vp2 PCR products

Reverse-transcribed total RNA (0.2 pg; cDNA) was amplified in a 100-p1 PCR reaction by using primers specific for the Vp2 (5’-CUAC UACUACUATCATCAACCATGCAAGCCTGACCT) T cell receptor family and for the C p (5’-CAUCAUCAUCAUTTCTGATGGCTCM CAC) constant p-chain region (27) and also suitable for cloning in pAMP 1 vector (Life Technologies, Inc., Grand Island, NY). cDNA was ampli- tied for 35 cycles in a DNA Thermal Cycler (Perkin-Elmer) under the following conditions: 93°C for 60 s, 55°C for 60 s, and 72°C for 60 s. PCR products were then washed in deionized sterile water and concentrated in a Centricon 100 microconcentrator (Amicon, Beverly, MA). Before cloning, a small aliquot (1 to 2 p1) of the concentrated PCR product was run on a 2% agarose gel, subjected to the Southern blot, and then hybridized with the use of a C p probe as described above. Fifty nanograms of PCR product was digested and ligated to 50 ng of pAMP 1 vector. DH5a-competent cells (Life Technologies) were then trans- formed with 30 ng of ligation reaction according to the manufacturer’s instructions. Single colonies were picked and grown in 2 ml of Terrific Broth medium (29) and plasmid DNA was extracted by using the alkaline lysis method (29). Purified plasmid DNA was run on a 0.7% agarose gel and transferred to a nylon membrane. Filters were hybridized with 1 pmol/ml C p probe as described above to verify the presence of the de- sired insert.

Sequencing reactions

Double-stranded plasmid DNA (5 pg) was sequenced according to the protocol included in Sequenase version 2.0 T7 DNA Polymerase kit (United States Biochemical, Cleveland, OH). Three microliters of each sequencing reaction was run on a 6% denaturing acrylamide gel (6% Gene Page; Amresco; Solon, Ohio) with the use of a Bio-Rad Sequi-Gen Sequencing Cell. The gel was dried at 80°C and then ex-

posed overnight at room temperature to Hyperfilm-MP (Amersham, Arlington Heights, IL).

Immunohistochemistry

Muscle biopsies were frozen in freezing isopentane, and IO-pm serial sections cut at -20°C. Nonspecific Ab binding was blocked by incuba- tion of the sections for 1 h in PBS and 10% horse serum. Vp2 T cells were identitied by staining for-l h with a mAb that recognizes all mem- bers of the Vp2 family (0.5 pgisection; AMAC, Inc., Westbrook, ME). After washing in PBS plus 0.2% Tween-20 three times for 5 min each, the sections were reacted for an additional 1 h with biotinylated anti- mouse IgM (li200; Vector Labs, Burlingame, CA), washed, and subse- quently treated for 30 min with avidin-biotin complex horseradish peroxidase (Vectastain ABC kit; Vector Labs) and visualized by using 0.5 mgiml diaminobenzidine, 0.03% H,O, in 50 mM Tris-HC1 (pH 7.4). The slides were mounted in Gelvatol (Monsanto, St. Louis, MO), and examined with the use of a Zeiss Axiophot microscope. Normal human tonsil sections were used as a positive control for Vp2 T cell staining. All Abs were diluted in PBS plus 0.2% Tween-20 plus 5% horse serum. All incu- bations were performed at room temperature in a humidified chamber.

Results Quantitative PCR analysis of TCR transcripts

Muscle biopsies of DMD patients (no. 5 , 6, and 8) who participated in a myoblast transfer clinical trial (22), other DMD patients (no. 84, 85, and 86), and controls were analyzed by reverse transcriptase-PCR for the presence and relative amounts of 20 of the known TCR V p families (Table I). For DMD patients no. 5, 6, and 8, pre- and post-implant (1 and 6 mo) muscle tissue from the myoblast-injected leg was tested. Rearranged transcripts from different Vp families were amplified, but Vp2 was the only family consistently found in all of the 12 muscle biopsies analyzed (Table I). For patient no. 86, the relative percentage of Vp2 was 30% of the coamplified Ca-Ca band, whereas in pre-implant biopsies of patients no. 5 and 6 this value was 28.3 and 39.6%, respectively. For patient no. 8 the relative amount of Vp2 transcripts in pre-implant tissue was 6.3% of the co-amplified Ca region. With few exceptions, similar Vp families were detected in pre-implant and post-transplant specimens of DMD patients no. 5, 6, and 8.

Identification of specific TCR V-0-J gene rearrangements

We investigated the possibility that T cells, expressing a similar receptor and targeting a specific Ag bound to MHC, were selected in DMD muscle. We sequenced the rearranged Vp-Dp-Jp transcripts amplified from different DMD samples and controls. We chose to sequence Vp2 transcripts because it was a V p family consistently found in all DMD samples analyzed. Vp2 PCR products were cloned, sequenced, and the rearranged V-D-J regions iden- tified in 121 transcripts derived from muscle samples of seven DMD patients. The deduced amino acid data sequences are listed in Tables I1 and 111. One common amino acid motif, RVSG, was found in the CDR3 region of V/32 transcripts amplified from pre-implant-, placebo-, and myoblast-injected leg biopsies of patients no. 5 , 6, and 8


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4800 CHARACTERIZATION OF MUSCLE-INFILTRATING T LYMPHOCYTES IN DMD

Table 1. Relative amounts of TCR Vp families in DMD patientsa

DMD Patients V p l Vp2 Vp3 Vp4 Vp5.l Vp5.2 Vp6 Vp7 Vp8 Vp9 Vpl0 Vpl l Vp12 Vp13 Vp14 Vp15 Vp16 Vpl7 Vp18 Vp19 Vp20

84 - + " - 85 - + " - + - + " " " - - - - _ " 86 + + + - - + + + - - - - - -

5 + " " "

pre-implant - + + - - + - + - - - - - + - - + - " - 1 mo post - + + - - + - - - - - - - - - - - - - - -

6mopost - + + - + + - - - - - - - - - - - - - - -

- - - " - - " " _ " "

6 pre-implant - + - - - + + + - - - - - - - - - - - - - 1 mo post - + - - -

6 mo post - + - - -

+ " f " " " _ " " "

pre-implant -

1 mo post + + + - - + " - - - + + - - - - + - - - - - - -

" + + " - " + " " " 6mopost + + + - - + - - - - - - - - + " " "

8 - - - - - - - - - - - - - - - -

comitantly with a TCR p-chain transcript, to assess the relative amount of each of 20 known TCR Vp families. Quantity of TCR Vp transcripts was expressed as Quantitation of TCR transcripts wa5 accomplished through amplification of a Ca-Ca region a5 an internal control for variation among samples (28), con-

a percentage of the quantity of the co-amplified Ca-Ca transcripts. In this table, the symbol + indicates values greater than 5%, and - indicates values 4 % .

and was also detected in clones derived from a muscle biopsy of patient no. 86, (Table 111) who was neither a participant in the myoblast transfer trial, nor immune suppressed.

To investigate whether the RVSG rearrangement was selectively expressed by DMD muscle-infiltrating lymphocytes, Vp2 transcripts amplified from the PBLs taken at the time of the pre-implant biopsy of patient no. 8 were analyzed under the same conditions. No similarities were observed with the Vp2 amino acid sequences found in the muscle (Table 11).

The HLA phenotype of DMD patients no. 5 , 6, and 8 had no common HLA class I1 phenotype, but all shared the class I allele A2. Given this common HLA, we chose to study TCR Vp2 transcripts in the muscle of another patient from the trial (no. 4), who was also known to be HLA-A2. The RVSG rearrangement was found in the CDR3 region of transcripts sequenced from this patient (Table 11). The HLA class I phenotype of patient no. 86 was also studied, and he had A l , suggesting that the expression of the RVSG motif is related to the disease rather than a particular HLA phenotype (A2). The peptide rec- ognized by the RVSG motif might bind to various HLA class I molecules in a redundant manner (30).

Seventy-one percent of the TCR Vp2 RVSG clones from DMD patients were found to be rearranged with the J region Jp 1.3. The 'SG' portion of the RVSG motif found in DMD muscle represents the first two amino acids of Jp 1.3. However, the RVSG motif was found in the CDR3 of clones from patient no. 8, in which it is rearranged with J P 1.4, and in patient no. 4 with Jp 2.1 (Tables 11, 111, and IV). This reinforces further the idea that selection for a particular sequence, RVSG, occurs, and that both use of N-region addition and Jp genes are employed in different DMD patients to encode this disease-specific motif.

Two related motifs, KVSG and HVSG, were seen in the pre-implant biopsy of patient no. 5, and they were rearranged with Jp 2.1 and Jp 2.4, respectively. This might imply additional selection for a particular CDR3 motif, in which the first amino acid (R, K, or H) has a basic side chain, followed by VSG, independent of the J region.

Table IV shows the nucleotide sequence of the RVSG rearrangements found in each DMD sample. Differences are present at the DNA nucleotide level, whereas the amino acids encoded by these codons are conserved. These data suggest that selection for the RVSG CDR3 sequence occurred at the protein level.

We investigated whether Vp2 T cells with CDR3 rearrangements similar to the ones seen in the DMD muscle were present in normal muscle or in other circum- stances in which muscle injury is known io occur. As controls, rearranged TCR transcripts were analyzed from freshly removed surgical biopsies of muscle. We examined 1) a biopsy from a 67-yr-old patient with muscle necrosis caused by colchicine toxicity; 2) a biopsy from a muscle crushed in the hip socket of a 2-yr- old toddler with an abnormal gait caused by congenital hip disease; and 3) a muscle sample taken freshly postmortem from a normal individual. We compared the TCR sequences with those we previously reported in a study of TCR usage in polymyositis (31) (Table V). The skeletal muscle from the colchicine myopathy revealed vacuolation and type I and type I1 fiber atrophy. Vacu- oles contained electron-dense, lamellar structures. Sar- comeric ultrastructure was perturbed with Z-band streaming. In the case of the 2-yr-old with congenital hip disease, the psoas muscle was stuck into the anterior aspect of the hip capsule. Muscle tightening was observed, and a surgical release was performed. In these


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Table I I . VpZ-Dp-jp rearrangements in DMD patients participating in a myoblast transfer clinical trial”

D M D Pre-implant Placebo-injected Leg (1 mo.) Myoblast-injected Leg (1 mo.) Myoblast-injected Leg (6 mo.)

SAQRVSGNTI Jpl . 3 [CSA-NTI Jpl .3 I ICSAS-NTI Jpl .3 1 no. 5 (CSAQRVSGNTI CSALKVSGGEQF CSA-NIQ C S A m S Y E

CSVJKNI CSAAQGLSQPQ CSARDHGLEPQH CSASESGN C S A m S Y E

Jpl. 31 Jp2.1

Jp2.4 Jp2. 7

Jp2.4 Jpl. 5 Jpl. 5

Jpl. 3 Jp2.7

no. 6 ICSAS-NTIY Jpl. 3) CSANQGAGETQ Jp2.5 C S A R W S N Q Jpl .5

CSASEGAN Jp2.6 CSARDTQLSYGY Jpl .2

CSARDYSGGILNGYT Jp1.2 CSARRQSGGEQY Jp2.7 CSARGQRHTQY Jp2.3 CSAMYVSGNTI Jpl. 3

CSAHRTSGVKEQY Jp2.7 no. 8 Muscle

CSAS-EKL Jpl .4] C S A R W N E Q Jp2.1 CSAKIRQATTEA Jp1.1 CSARVNGVPGETQ Jp2.5

CSARmSNQ Jpl .5

PBLS

CSALPRQAVSYE Jp2.7 CSARDWRIVRETQ Jp2.5 CSAITGGDTDT Jp2.3

C S A R m T E A Jpl. 1 CSAVSGTQWVTEA Jpl .1 CSASPPLGQGYQPQ Jp1.5

no. 4

(CSAGRVSGNTI JPI. 3 1 S A R W N T I Jpl . 3

CSARDLGQGTQPQ Jp1.5

CSVKGGQLDNEQ Jp2.1 CSARGTGQASSYE Jp2.7

C S A A A M K L Jpl. 4 C S A R C A T E J p l . 1 C S A R W T D T Jp2.3 CSALKVSGGEQF Jp2.1 CSASPSMVGNIQ Jp2.4 CSAS-NTI ~p1.31 CSAR-TEA Jpl .1 CSANQGAGETQ Jp2.5 C S A R a P L H Jpl .6

[CSAQRVSGTKLF J ~ I .4 I CSARPRLAGGHSTD Jp2.3 CSAR-SYE Jp2.7

C S A R W E Q Y Jp2.7 CSARWNQP Jpl .5 CSATAPQRGPYEQ Jp2.7 CSAERTSGGAGEQF Jp2.1 CSARDPRGVGGLYFG Jp2.3

CSAL-NE4 Jp2.1

CSASWGQGFHEQY Jp2.7 CSARRGQGFHEQY Jp2.7 C S A R W T Q Y Jp2.5

~

CSARGTADNEQF Jp2.1

CSARGTADNEQF Jp2.1 CSAmQET Jp2.5 CSALKVSGGEQF Jp2.1

CSAVpSNQ Jp1.5 CSANQGAGETQ Jp2.5 CSASESYE Jp2. 7

CSATTQGAGNQP Jp1.5

CSARDRGFDEQY C S A S a S Y E

CSARZSNQ

CSARWGNT CGASWYEQ CSASPRPATSNQ

CSARESNQ C S A R B Y E Q

Jp2.7

Jp2.7 Jpl .5

Jpl .3 Jp2.7 jp1.5 Jpl .5 JB2.7

CSARDKTGNGYT JP1.2

CSAK-TQY Jp2 - 3

CSA-EQF Jp2.1 C S A S B N E Q Jp2.1 C S A R m N T E A Jpl. 1

CSAR-PQH Jpl. 5

~CSANRVSGNTI ~p1.31 CSARDPREGATEA Jp1.1 CSAHRTSGVKEQY Jp2.7

CSAS-YNE Jp2.1

I CSAQRVSGTKLF Jpl .4 I CSARZSNQ Jpl .5 CSARDRGFDEQY Jp2.7

CSAS-TDT Jp2.3 CSAR=SYE Jp2. 7

C S A R W T E A Jpl. 1

CSASEETQ Jp2.5 CSARmGYT Jpl .2

C SARWNQP Jp 1 .5 C S A S W Y N E Jp2.1 CSARWNTE Jpl .1

C S A R W Q P Jpl. 5

CSAREPQGEYFG Jp2.7

a In each sequence listed, the CDR3 motif is underlined, and the first three amino acids of each J region following the CFR3 motif are reported. Transcripts sharing the RVSC motif rearrangement are outlined by a box.

muscles with secondary damage, no CDR3 sequences sim- in one normal sample when amplified for 35 cycles before ilar to those seen in DMD patients were noted. cloning. No Vp2 transcripts were seen in the polymyositis

No Vp2 transcripts were detected in two normal muscle samples (31). The nucleotide sequence of these Vp2 tran- samples by quantitative PCR analysis after 25 cycles of scripts from normal muscle were determined (Table V). No amplification, but low levels of this PCR product were found similarities were seen with sequences found in DMD muscle.


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Table 111. VpZ-Dp-Jp rearrangements in DMD patients not immunosuppressed”

D M D Patient

No. 84 No. 85 No. 86

CSAR=EQY Jp2.7 CSASmTEA Jpl. 1 SAS-NTI Jp1.3 I CSASADWSGDYNE Jp2.1 CSATTQAGGSNIQ CSAFSGTSVSNEQ Jp2.1

Jp2.4 CSA-NTI C S A R W T E A

Jpl .3 Jpl. 1 CSAREQEHGLEQY Jp2.7

CSADALGGGFTDT Jp2.3 CSARPHGNRAYEQ Jp2.7 CSASSPGLGWSNEQ Jp2.l CSARDTRGYTDT Jp2.3 CSARGEAGGRTDT Jp2.3 CSAR-SYN Jp2.1 CSAPRGQGNYNE Jp2.1 CSARGDRASGWADTQ Jp2.3 CSALRTSGGKEFFG Jp2.l CSAEGSGQGYEQ Jp2.7 CSAR-SYE Jp2.7 C S A S U N T I Jpl. 3 CSAEDGGVQET Jp2.5 CSAKYQGWGEAF Jpl . 1 In each sequence listed, the CDR3 motif is underlined, and the first three amino acids of each J region following the CDR3 motif are reported. Transcripts

sharing the RVSG motif rearrangement are outlined by a box.

As additional controls, we have also examined tissues other than muscle from other conditions in which inflammatory infiltrates containing Vp2 T cells are present. Thus, Vp2 PCR products from infiltrating T cells in a sarcoma sample as well as two spinal cord specimens from patients affected by ALS were analyzed (Table V). Infiltrating T cells are seen in the spinal cord of ALS patients (32,33). No similarities in the CDR3 rearrangements were observed between Vp2 T cells infiltrating DMD muscle and any of these control samples listed (Table V).

Immunohistochemistry

Immunohistochemical analyses of placebo-injected muscle biopsies 1 mo after myoblast transfer from patient no. 8 were performed to confirm the presence of Vp2 T cells in DMD muscle (Fig. 1). Quantitation of Vp2-positive T cells over the total number of T cells was not attempted. Focal endomysia1 inflammatory infiltrates were present in these biopsies. Every section analyzed contained Vp2 T cells both within and surrounding normal and large diameter fibers typical of mature muscle. In one muscle biopsy analyzed, an average of 25 Vp2-positive T cells was observed in single transverse sections of approximately 650 muscle fibers each. A total of 317 Vp2 T cells was observed over a longitudinal distance of 350 p m in this biopsy. In a biopsy taken from a deeper site in the same muscle, an average of four positive cells was found in multiple transverse sections of approximately 340 muscle fibers. A total of 86 cells was observed over a longitudinal distance of 350 pm. Small diameter regenerating fibers were negative for the presence of these Vp2 T cells. These findings suggest that an immune reaction may be specifi- cally targeting mature muscle fibers.

Discussion The pathogenesis of DMD is a result of the lack of func- tional dystrophin, arising from mutations in the gene on the X chromosome encoding this protein (1, 2). In DMD patients, several lines of evidence indicate that the immune

Table IV. Selection for the RVSC rearrangement: DNA sequences a

Patient #5 Pre-implant TGC AGT GCC CAG CGT GTG TCT GGA AAC C S A Q R V S G N

Myoblast-injected leg 1 mo after transplant TGC AGT GCC CAG CGT GTG TCT GGA AAC C S A Q R V S G N

Placebo-injected leg 1 mo after transplant TGC AGT GCC CAG CGT GTG TCT GGA AAC C S A Q R V S G N TGC AGT GCA GGG AGG GTC TCT GGA AAC C S A G R V S G N TGC AGT GCT AGG AGG GTG TCT GGA AAC C S A R R V S G N

Myoblast-injected leg 6 mo after transplant TGC AGT GCT AGC CGA GTA TCT GGA AAC C S A S R V S G N

Patient #6 Pre-implant TGC AGT GCT TCT CGG GTC TCT GGA AAC C S A S R V S G N

Placebo-injected leg 1 mo after transdant TGC AGT ’GCT T& CGG GTC TCT GGA AAC C S A S R V S G N

Myoblast-injected leg 6 mo after transplant ~~

TGC AGT GCT AAC AGG GTC TCT GGA AAC C S A N R V S G N

Patient #8 Pre-implant TGC AGT GCT AGT AGG GTG TCC GGT GAA C S A S R V S G E

Placebo-injected leg 1 rno after transplant TGC AGT GCT CAG AGG GTG TCG GGA ACA C S A Q R V S G T

Myoblast-injected leg 6 mo after transplant TGC AGT GCT CAG AGG GTG TCG GGA ACA C S A Q R V S G T

Patient #4 Placebo-injected leg 1 mo after transplant TGC AGT GCC TTG AGG GTG TCG GGC ATT C S A L R V S G N

Patient #86 TGC AGT GCT AGT AGG GTT TCT GGA AAC C S A S R V S G N

Jp 1.3 ( Y )

Jp 1.3

Jp 1.3 ( Y )

Jp 1.3

Jp 1.3

Jp 1.3

Jp 1.3 ( Y )

Jp 1.3

Jp 1.3

Jp 1.4

Jp 1.4

Jp 1.4

Jp 2.1 (Y)

JB 1 .3 (91)

a Nucleotide and amino acid sequences of the Vf32 T cells expressing the RVSG CDR3 motif. In some samples, clones with an identical nucleotide sequence were found more than one time. The symbol (Y) designates clones found two times, and (41) designates clones found five times in the same sample.


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Table V. Vp2-Dp-43 rearrangements in controls

Colchicine-induced myopathy Psoas from congenital hip disorder Normal muscle

CSA-NTEAFFGQGTRLTW Jpl . 1 CSATQETQYFGPGTRLLVL Jp2 .5 C S m Y E Q Y F G P G T R L T V T CSA-NQFQHFGDGTRLSIL Jp1.5 CSASDYRDRGPALAETQYFGPGTRLLVL Jp2.5 CSAPRPRTSGRDSYJTQYFGPGTRLTVT Jp2.7

Jp2 . 7 CSAVSQDSQVQPQHFGLXTRLSIL Jp1.5 CSADPSGGNNEQFFGPGTRLTGL Jp2 . 1 C S B Y N E Q F F G P G T R L T V L CSASNpTGAAFFGaGTRLTW Jpl . 1 CSAR-QYFGPGTRLT

Jp2.1 Jp2 .I CSAARARYJTQYFGPGTRLTVT

C S m m T D T Q Y F G P G T R L T V L Jp2 - 3 CSAR-NEQFFGPGTRLTGL Jp2.7

Jp2.l C S A R B E K L F F G S G T Q L S V L CSAREGAGAGVTGELFFGEGSRLTVL Jp2.2 C S A P m E K L F F G S G T Q L S V L

Jpl .4 Jpl .4 CSA=YEQYFGPGTRLTVT

CSASQTSYGYTFGSGTRLTW Jp1.2 CSARDP-VNSNQPQHFGLXTRLSIL Jp1.5 Jp2.7

CSAIWNIQYFGAGTPVSVL Jp2.4 CSASESYEQYFGPGTRLTVT Jp2. 7 CSASSGQGLWEQYLRAGTRLTVT CSARDHTDTQYFGPGTRLTVL Jp2.3 CSALDSRRWSPLHFGNGTRLTVT Jpl. 6 CSAKaSNQFQHFGDGTRLSIL Jpl -5 CSNSYNEQFFGPGTRLTVL Jp2 . 1 CSARDWSRRGAVFGEGSRLTVL Jp2.2 CSRQGNTEAFFGQGTRLTW Jpl. 1

JP

ALS spinal cord Sarcoma

CSAGTGTRGGEQFFGPGTRLTVL Jp2.1 CSAGGYKPGQPTDTQYFGPGTRLTVL Jp2.3 CSAFARDRDSNQFQHFGDGTRLSIL Jp1.5 CSASRSSGRNGEQFFGPGTRLTVL Jp2.1 CSARAYRDRETDTQYFGPGTRLTVL Jp2.3 C S A R S A K N I Q Y F G G T R L S V L CSA-LFFGSGTQLSVL

Jp2.4 Jpl - 4 CSARIWGVAGGEQFFGPGTRLTVL

CSASGLDINEQYFGPGTRLTVT Jp2.7 C S A P P E K L F F G S G T Q L S V L Jp2.1 Jpl. 4

CSARASASSYEQYFGPGTRLTVT Jp2.7 CSm-EQFFGPGTRLTVL Jp2.1 CSAR=SPLHFGNGTRLTVT Jpl .6 CSARDDMGGLRAYNEQFFGPGTRLTVL Jp2.1 C S A R m M Y E Q Y F G P G T R L T V T Jp2.7 CSALLPGLAGLGRSTDTQYFGPGTRLTVL Jp2.3

were analyzed from a patient with myopathy due to colchicine toxicity and a patient with a congenital hip disorder. Other control samples were taken postmortem As in Table II, each CDR3 motif is underlined. The complete amino acid sequence of the J region following the CDR3 motif is reported. Fresh muscle biopsies

from a normal control (muscle), from patients affected by ALS (spinal cord) and from a patient affected by sarcoma (tumor site). No similarities between sequence data from DMD patients and controls are detected.

FIGURE 1. Vp2 T cells in DMD muscle detected by immunohistochemistry. Vp2 T cells are localized both outside and inside muscle fibers, as indicated by arrows. Four fields are shown of transverse sections of 4 to 6 muscle fibers. Re- action with mAbs specific to T cells of Vp2 class reveal the presence of these lymphocytes (X780 magnification).

system may be involved as a secondary pathologic event. Immunohistochemistry has demonstrated the presence of infiltrating T cells in DMD muscle fibers undergoing degeneration (12-15). When steroids are given, the cellular infiltrate in muscle is diminished (14, 15), and boys with DMD remain ambulatory for a few additional years (17-20). Similarly, when cyclosporin A is administered to boys with DMD, muscle strength is improved (21).

Although prednisone and cyclosporin A possess multiple activities, both share the capacity to suppress the immune system.

Rearranged V-D-J segments of the TCR a- and p-chains generate TCR diversity and encode receptors with specificity for processed Ags associated with the MHC (34). In this study we show that T cells invading the muscle of DMD patients transcribe similar receptors. These data indicate that a selective T cell response di- rected to a specific Ag is occurring in DMD muscle. The nature of the Ag that triggers these T cells is unknown. However, it might be specific for an ongoing process in DMD muscle, as no similarities were found in infiltrating T cells from control samples including fresh samples from muscle degenerating as a result of other factors.

The analysis of the TCR-a/-p repertoire of muscle-infiltrating T lymphocytes in 15 samples from patients affected by polymyositis, an inflammatory myopathy, has recently been reported by R. Mantegazza et al. (31). It is not established whether polymyositis is primarily autoimmune, or whether it is a consequence of an immune response to an infectious agent (35). In this study, none of 15 polymyositis samples had detectable Vp2 transcripts, whereas Vpl5 transcripts were the predominant TCR V- gene family. Furthermore, the sequence data of Vpl5 clones from polymyositis samples showed CDR3 rearrangements that were different from the RVSG motif we


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found in DMD muscle. These results support a disease- specific role for these T cells in DMD muscle.

In other genetic diseases that are not usually thought to have a pathologic immune component, cellular infiltrates in the vicinity of degeneration have been observed. For example, in the X chromosome-linked recessive condition, adrenoleukodystrophy, which is characterized by a muta- tion in an AJ3C-transporter gene (36), lymphocytic infiltrates are seen in the white matter of the central nervous system. In atherosclerosis, CD4+ and CD8+ T cells are detected in the endothelial plaque in addition to increased MHC class I1 expression (37). In adrenoleukodystrophy and in atherosclerosis it is not entirely clear what role the inflammatory infiltrate is playing in the pathogenesis of the disease. More generally, in diseases in which mutant structural genes are the primary cause of the pathology the secondary autoimmune response might be playing a major role in the ultimate destruction of the target tissue. Cer- tainly in DMD, immune suppression prolongs the patient’s ability to walk for several years (17-20). These results might have clinical consequences. It may be possible to suppress particular T cells with specificity for degenerating muscle to slow the progression of the disease with drugs that are less toxic than corticosteroids or cyclosporine. Finally, in diseases thought to be primarily of autoimmune origin, a gene might encode an aberrant structural protein that might trigger a secondary autoimmune response in the diseased tissue.

Acknowledgments

The authors thank Drs. J. R. Oksenberg, F. Szafer, and C. M. Greco for helpful discussions.

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specific t cell receptor gene rearrangements at the …...specific t cell receptor gene...

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