Evidence for Clonal Selection of -Y/B T Cells in Response to a Human Pathogen by Koichi Uyemura," Robert J. Deans,§ Hamid

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Evidence for Clonal Selection of -y/b T Cells in Response to a Human Pathogen By Koichi Uyemura," Robert J . Deans,§ Hamid Band,1 Jeffrey Ohmen," Govindaswamy Panchamoorthy,1 Craig T. Morita,l Thomas H . Rea,11 and Robert L. Modlin"$

From the *Division ofDermatology and #Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California 90024; the DDppartment of Neurology and Il Section of Dermatology, USC School of Medicine, Los Angeles, California 90033; and the (Laboratory of Immunochemistry, Dana Farber Cancer Institute, and Department of Rheumatology and Immunology, Harvard Medical School, Boston, Massachusetts, 02115 Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021

Summary T cells bearing y/b antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection . An important unsolved question about antigen-driven y/b T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR S gene expression among human y/b T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae . We show that the vast majority of'y/b cells from M. leprae lesions use either V61-J61 or V62 -J61 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of y/b T cells from peripheral blood of these same individuals, as well as skin from normal donors . These results indicate that they/b response to M. leprae involves the selection of a limited number ofclones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens .

ne of the fundamental aspects of cellular immunology Much experimental evidence indicates that y/b T cells con- is the selection and clonal expression of T cells bearing tribute to the granuloma formation in response to mycobac- specificO receptors by antigens. While the selection of ci/(3 terial infection (7) . First, TCR y/b cells comprise a strik- T cells has been studied in detail, little is known about the ingly high percentage of the T cell population in infectious selection ofy/b T cells by antigen . The genetic diversity of disease lesions that contain recently formed granulomas. These the TCR provides a measure of the scope of the T cell reper- include lepromin skin tests (Mitsuda reactions), which are toire. In contrast to the a/a TCR, the germline gene seg- experimental DTH reactions induced by intradermal injec- ment diversity for both the TCR y and S chains is small . tion ofMycobacterium lepme, reversal reactions in leprosy, which The further limitation of this diversity by the preferential represent a naturally occurring DTH response to M. leprae, usage of only a few variable (V) genes or V gene pairs at and localized American cutaneous leishmaniasis. Second, the specific anatomical locations (1-3) suggests a particularly y/b T cells from these infectious lesions proliferate to mycobac- narrow TCR repertoire, likely reflecting recognition of a terial antigens in vitro. Third, the y/b T cells from lesions limited number of ligands . On the other hand, there is un- appear to release a lymphokine(s) that synergizes with gran- precedented junctional diversity, particular in the b chains ulocyte/macrophage (GM)-CSF to induce macrophage adhe- of this receptor (4-6) . We reasoned that analysis of the TCR sion, aggregation, and proliferation, cellular events that would y/b repertoire of a specific immune response would provide be necessary for the granulomatous response. clues about the set of antigens recognized : whether diverse The present study was undertaken to determine the distri- or limited, conventional, or superantigen-like. The presence bution and TCR diversity of y/b T cell subpopulations in of antigen-reactive TCR -y/6-bearing cells in leprosy skin lepromin skin tests . This was accomplished by immunopatho- lesions (7) provides a unique in vivo model to examine the logic analysis according to VS chain expression and molec- y/b receptor repertoire at the site of immunopathologic ular analysis of Vb, Jb, and junctional elements. The reper- reaction . toire in these infectious lesions was compared with the

683 J. Exp. Med. ® The Rockefeller University Press " 0022-1007/91/09/0683/10 $2 .00 Volume 174 September 1991 683-692 Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021

684 Clonal Selection of ,y/S T Cells in Response to a Human Pathogen Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021

Figure 1 A-F . Immunohistochemical analysis of VS phenotypes in lesions and peripheral blood . Both V61 (A) and V62 (B) -bearing T cells were present in the dermal granulomas of lepromin skin tests. In contrast, V61 cells P were present in the epidermis in greater numbers than V62 cells (D), which were virtually absent . A more extensive view of the epidermis (E) shows V61 cells throughout . The percentages of V61- and V62-bearing T cells (± SD) are shown in F.

peripheral blood of the same individual and the repertoire of an entire tissue section (-5 x 5 mm) were enumerated and in normal human skin . expressed as the percentage of total TCR61' cells in each micro- au.atomic location . Peripheral blood was obtained from the same individuals and analyzed by flow cytometry. Materials and Methods PCR Analysis . 20 5-P.m. sections from each of five lepromin Immunoperoxidase Staining ofBiopsies. Lepromin skin tests (3-wk skin tests were placed in 0.5 ml of 4 M guanidinium isothiocyanate Mitsuda reactions) were studied from 14 tuberculoid leprosy pa- buffer. Additionally, 106 PBMC were obtained by Ficoll-Hypaque tients (8) . All specimens were obtained with informed consent . density centrifugation . Genomic DNA was isolated by phenol/chlo- The patients were distributed among the different diagnostic groups roform extraction and ethanol precipitation with yields of 1-3 wg showing no segregation according to sex, race, or age. Skin biopsy of DNA. For each PCR reaction, -100 ng of DNA was used in specimens were obtained by punch or scalpel technique at the time a 25-w1 reaction . By calculating the numbers of y/b cells/mm2 of of diagnosis, embedded in OCT medium (Ames Co., Elkhart, IN), granuloma as detailed previously (10), and knowing the volume and snap frozen in liquid nitrogen . The tissues were stored at -70°C of tissue utilized, we determined that each PCR reaction of lesions until sectioning. Sections (3-5 P.m) were acetone fixed before un- contained DNA from -500 y/b T cells . Similar calculations for dergoing incubations with the mAbs for 45 min. mAbs included peripheral blood indicated identical numbers of y/b T cells per each the Cb-specific antibody antiTCR61 (9) (1:100, T-cell Sciences, reaction . Titration of VBJS containing plasmid confirmed that the Boston, MA), the V61-J61-specific S antibody, 6TCS1 (1 :10, Tcell sensitivity of our PCR amplification was on the order of 102 or Sciences), and the V62-specific antibody, BB3 (1 :3,000 ; a generous 103 copies. PCR amplification mixtures contained 10 pmol of each gift of Dr. L . Moretta, Istituto Nazionale per la Ricerca sul Cancro, oligonucleotide primer, 2.5 U Taq polymerase (Promega Corp., Genova, Italy) . The results obtained with 6TCS1 were corrobo- Madison, WI), and 2.5 MM M9C12 in PCR buffer (Promega rated with the V61-reactive antibody A13 (1:1,000 ; kindly provided Corp.) . Oligonucleotide primers were tested on a panel of y/6 T by Dr. S. Ferrini, Istituto Nazionale per la Ricerca sul Cancro, cell clones with known rearrangements. Sequences of oligonucleo- Genova, Italy) . The primary antibody was visualized using the alka- tide primers were selected to yield -300 by products (11) . 30 cycles line phosphatase anti-alkaline phosphatase technique (Dako Corp., of PCR amplification were performed using the following condi- Carpinteria, CA). Slides were sequentially incubated with rabbit tions : 1 min of denaturation at 94°C and 2 min of annealing/exten- anti-mouse antibody for 30 min, alkaline phosphatase mouse anti- sion at 65°C . Amplified products were subjected to electropho- alkaline phosphatase immune complex for 30 min, and then devel- resis on 1.5% agarose gels and visualized as single bands byethidium oped with Vector Red substrate for 30 min (Vector Laboratories, bromide. Verification of product was accomplished by nucleotide Burlingame, CA). Slides were washed in Tris buffer between incu- sequencing . Two skin biopsy specimens were obtained from normal bations . Endogenous alkaline phosphatase was blocked using 125 donors after informed consent . DNA was extracted from the en- mM levamisole (Vector Laboratories) . Slides were counterstained tire 4-mm punch biopsy and 100 ng used for PCR amplification with hematoxylin and then mounted in glycerine-gelatin . Some followed by sequencing of the V-J junctions. slides were stained using the ABC Elite system (Vector Laborato- Nucleotide Sequence Analysis of TLCR S VJJunctions. To facili- ries) . The numbers of VSpositive cells in the epidermis and dermis tate cloning into sequencing vector, oligonucleotide primers for 685 Uyemura et al .

PCR amplification were designed to contain a Sall or EcoRl restriction site in VS andJb primers, respectively. Amplified product was digested with both restriction enzymes and gel purified. Purified products were ligated into pUC18 vector and used to transform DH5a (BRL Laboratories, Gaithersburg, MD) and selected for am- picillin resistance. Plasmid DNA from insert containing colonies was sequenced (Sequenase 2 .0 Kit ; U .S. Biochemicals, Cleveland, OH) using the method of Sanger et al. (12) . Precautions taken to avoid PCR contamination artifact included the use of positive dis- placement pipettes, assembling ofPCR reactions in a laminar flow hood in a separate room from plasmid preparations, and inclusion of negative and well-characterized controls in the PCR reactions.

Results Distinct Microanatomic Localization of Vb T Cell Popula- tions . The microanatomic distribution of 'y/b T cell sub-

populations within lepromin skin tests was investigated by Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021 immunohistologic staining of frozen sections with mAbs directed against Vb-encoded determinants (Fig . 1) . Within the dermal granulomas, Vbl- and V62-bearing cells accounted for the majority of infiltrating y/b cells, with a V62/V61 ratio -2 :1 compared to 9 :1 in the peripheral blood of these same individuals . Unexpectedly, in contrast to the predomi- nance of V62-bearing cells in the peripheral blood and in the dermis, TCR6 * cells infiltrating the epidermis primarily expressed the V61-encoded TCRs . V82+ cells were rarely detected in the epidermal layer. Preferential Usage ofJ61 by y/b T Cells in Lesional Skin. To further characterize the repertoire of the y/b T cells in response to mycobacterial stimulation in vivo, we performed PCR amplification oftissue-derived vs . peripheral blood-derived DNA using paired V and joining (J) oligonucleotide primers . PCR conditions were established such that each PCR reaction included DNA derived from -500 y/b T cells. PCR analysis confirmed that Vbl and Vb2 were used by y/b T cells in these lesions, as amplified product was obtained from all five lesions for V61-J61 and V62 -J61 rearrangements (Fig . 2) . In contrast, V62-J63 rearrangements were detected in 1/5 lesions, but 5/5 blood samples (Figure 2) . These data suggest a selective localization of the V62-J61 subpopulation of VS2+ cells to lesions . PCR using other VS and Jb combinations did not reveal additional gene rearrangements (data not shown) . Limited functional Diversity of y/6 TCRs in Leprosy Skin Lesions. The junctional diversity of y/b T cells in lepromin skin tests was determined by cloning and sequencing PCR amplified products . We chose to focus on TCR b chain diver- Figure 2 . PCR analysis of S chain V-J gene segment usage in lesions sity for two reasons : it encodes the greatest amount of diver- and peripheral blood . VJ junctions were PCR amplified from DNA ex- sity in the y/b receptor ; and it is specific for y/b T cells since tracted from five lepromin skin tests (lanes A-E) and the peripheral blood of the same individuals. PCR-amplified product was detected in all five the b locus is deleted during rearrangement of the TCR a skin lesions and blood for Vbl Jbl (A) and VS2 JSl (B) . In all five blood chain . By using DNA rather than mRNA, bias towards ac- samples, but only one of five skin lesions, V62-J63 rearrangements could tivated lymphoblasts containing abundant mRNA is elimi- be amplified (C) . No rearrangements were detected for any other VBJb nated as each y/b T cell contains only one copy of its func- pairs (data not shown) . tionally rearranged b gene. Strikingly, in each of the three lepromin skin tests subjected to nucleotide sequencing analysis, the majority ofVblJb1 and V62 -J61 junctional sequences were found to be identical, but distinct for each patient (Fig . 3) . This was clearly different from peripheral blood of these

686 Clonal Selection of y/b T Cells in Response to a Human Pathogen

A V61-J61 JUNCTIONAL NUCLEOTIDE SEQUENCES FROM BIOPSY OF LEPROMIN SKIN TESTS Val P/N D61 P/N "6 P/N Qf3 P/N JJ61 FREQUENCY germline CTCTTGGGGAC GAAATAGT CCTTCCTAC ACTGGGGGATACG ACACCGATAA

PT A CTCTTGGGGAC AT CTA ACCCCGACA GGGAT TA ACCGATAA (5/8)- CTCT C TCCTA AGGGTAG CTGGGG TTCCC ACACCGATAA 13/8)

PT B CTCTTGGGGA TGCG CTA TTTGA GGGGGATACG CGAAATA ACACCGATAA (9/9)

PT D CTCTTGG TGACCCC GAA GTGAC TCC AG ACACCGATAA (8/8)

V62-J61 JUNCTIONAL NUCLEOTIDE SEQUENCES FROM BIOPSY OF LEPRONIN SKIM TESTS B P/N 261 P/N 62 P/N D63_ P/N J61 FREQUENCY germline GTGACACC GAAATAGT CCTTCCTAC ACTGGGGGATACG ACACCGATAA

PT A GTGACACC TTGC ACTGGGGGA GACGGG ACACCGATAA (6/6)

PT B GTGA TCG ACTGGGGG GCCCGT ACACCGATAA (B/B)

PT D GTGACACC G TGGGGG GAACCTCGT ACACCGATAA (11/13) GTGACA GACC CCTT GAC CTGGGGGA GACGGGGAGG AA (1/13) GTGACAC CC "CTGGGG CCCACAT ACACCGATAA (1/13) Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021

V61-JSI JUNCTIONAL NUCLEOTIDE SEQUENCES FROM PATIENT PBMC

VVal P/N 26-1 P/N 62 P/N D6 P/N J61 FREQUENCY

germline CTCTTGGGGAC GAAATAGT CCTTCCTAC ACTGGGGGATACG ACACCGATAA

PT A CTCTTGGGG CC GGGATAC GAG ACACCGATAA (1/5) CTCTTGGGGA TCA TGGGG ACT ACACCGATAA (1/5) CTCTTGGGGA T PLAT CTGGGGG CCA ACACCGATAA (1/5) CTCTTGGGGAC CTT ATGA GGGGAT T ACACCGATAA (1/5) " CTCTTGGGGAC TGG CCTAC TGGGGG T ACACCGATAA (1/5) "

PT B CTCTTGGGGAC A TCCT T ACTGGGGG GT ACACCGATAA (1/7) CTCTTGGGGAC GA TCCT TAGCC ACACCGATAA (1/7)* CTCTTGGGGAC TCC ACTGGGGG CCGT ACACCGATAA (1/7)w CTCTTGGGGAC CACG TGGGGG G ACACCGATAA (1/7) CTCTTGGGGAC ACGCGC GGGGA GA ACACCGATAA (1/7) CTCTTGGGGAC TGCG TCCT TGTG TGGGG CC ACACCGATAA (1/7) CTCTTGGGGA TTGCGT TGGGGG TO ACACCGATAA (1/7)"

PT D CTCTTGGGGAC CCCGGTG TGGGGG CATCC ACCGATAA (1/6) CTCTTGGGGAC TCGAGGG TTCC A CTGGG AT ACACCGATAA (1/6) CTCTTGGGGAC GGGACGAGG ACTGGG ACACCGATAA (1/6) " CTCTTGGGGAC AACCTGTCG TCCT GCT ACACCGATAA (1/6) CTCTTGGGGAC G TGGGG C ACACCGATAA (1/6) CTC AAT CT ACTG CCAGCTGC ACACCGATAA (1/6)

D V62-J61 JUNCTIONAL NUCLEOTIDE SEQUENCES FROM PATIENT PEMC

P/N D_61 P/N D62 P/N D63 P/N J61 FREQUENCY germline GTGACACC GAAATAGT CCTTCCTAC ACTGGGGGATACG ACACCCATAA

PT A GTGACACC G ATAG G CCTT GCAAT ACCGATAA (1/13) GTGAC CCGGCC CTGGGG CCCACAT ACACCGATAA (1/13) GTGACAC CCGGCCCG ACTGGGG CCCACATCACT CGATAA (1/13) " GTGACACC TGAC AAT GC TCCT CCT ACACCGATAA (1/13) GTGACACC C TACT GAAGG CTT ACC ACACCGATAA (1/13) GTGAC CT ACTGGGGGAT T ACACCGATAA (1/13) GTGA ATCGCTT TGGGGG T ACACCGATAA (1/13) GTGACACC GTG CTGGGG CC ACACCCATAA (1/13)- GTGAC CCCTTA GAAATA CGCT ACACCGATAA (1/13) GTGAC TCAA TGGGGGATACG CAGG CCGATAA (1/13) GTGAC CC ACTGGGGGAC CCAG ACACCGATAA (1/13) GTGAC CAG CTGGGGG TTACTGAGG ACACCGATAA 11/13) GTGACACC CTTTCGAGGG TCC CAG ACACCCATAA (1/13) Figure 3. Nucleotide sequence

PT B GTGAC CCCTGT ACTGGGGG TACGTGT ACACCGATAA (2/12) analysis of TCR6 VJjunctions of GTGACACC GCAACCACC GGGGG T ACACCGATAA (1/12) skin test lesions and blood. Se- GTGAC C CCT CTCGGTTG TGGGG TC ACACCGATAA (1/121 GTGAC CTAG TGGGGGA CC ACACCGATAA (1/12) quencing of PCR products was ETGAC GCGT ACTGGGGG GGAAAG ACACCGATAA (1/12)+ performed to analyze the diversity GTGACACC G TCCTAC G TGGGGG CCCTC ACACCGATAA (1/12) at the VJjunction . Limited diver- GTGACAC COG ATAG AAGCACAA GGGGGAT ACACCGATAA (1/12) GTGACACC TT ACTGGGGG CACCGA T ACACCGATAA (1/12) " sity was found in both the V61- GTGAC GGC CTGGGGG GTATAAAGCGTG ACACCGATAA (1/12) J61(A) andV62JSl (B)junctions GTGACA A ACTGGGGG GTGCCTTT ACACCGATAA (1/12) " OTC TGAC GGGGG TTCAGGAGTTTTT AA (1/12) " of y/6 T cells from lesions in patients A, B, and D. In contrast, PT D GTGAC CCCCG TCC CT ACTGGGGG CTACCGGGCG CACCGATAA (1/9)" GT CTGGTGCATGCCCTG TACT CCCAG CTAC TC ACACCGATAA (1/9) there was marked genetic diversity GT CATCC ACTGGGGG GCCCGT ACACCGATAA (1/9) in both the V61-J61 (C) and V62- GTGAC GGCGC ACTGGGGG CGAAGA ACACCGATAA (1/9) GTGAC CCTTGCTCCGGGGC ACT ACTGGGGGAT GCCGT ACACCGATAA (1/91 . J61(D) junctions from the blood GTGAC CCAC ACTGG AGGCGAGCCCCAG ACACCGATAA (1/9) of the same individuals . Nonfunc- GTGACACC GTC GGGGA CA ACACCGATAA (1/9) GTGAC CAGGTC CTGGGGGATACG CGGGGAC CCGATAA (1/91 tional sequences are marked with GTGACACC ACT ACTGGGGGAT TCCTCGT ACACCGATAA (1/9) . an asterisk.

687 Uyernura et al.

same individuals, which exhibited extensive diversity (Fig. We wanted to determine whether the limited TCR diversity 3) . The data indicate clonal selection by antigen with among -y1b T cells from leprosy lesions reflected a limited oligoclonal expansion within the lesion. The occurrence of diversity within the resident y/b population in skin or rather identical sequences that are nonproductive in a given sample clonal expansion of specific TCRs in response to antigenic is likely to represent the nonfunctional allele in the cell un- selection . The y/b TCR repertoire analysis of normal skin dergoing clonal expansion . indicated the presence of V62-J61 rearrangements, with minor To determine the extent of oligoclonal expansion through- V61Jb1 products and undetectable V62-J63 gene rearrange- out the lesion, spatially separated regions ofone biopsy spec- ments (Fig. 5) . This indicates that in diseased or normal skin, imen were studied . Within each region of the biopsy spec- there is selective localization of y/b T cells expressing the imen, there was limited diversity of the VJ junctions, although J61 gene product . Lack ofb chain-bearing J63 rearrangements the predominant sequence differed from site to site (Fig. 4) . in DNA derived from normal skin further supports our con- Given the limited diversity observed in these lesions, we ad- clusion that the PCR products are derived from a resident dressed the possibility of a PCR artifact producing the sur- y/b population, not from T cells within blood vessel lumina. prising results. Extensive precautions were undertaken to avoid In contrast to diseased skin, the analysis of TCR junctional PCR contaminations as outlined in the Materials and Methods. sequences derived from normal skin indicates extensive diversity Simultaneous PCR and cloning was performed on lesions equivalent to peripheral blood (Fig. 5) . and blood, so that finding of limited diversity in lesions was Conserved Motif in Predicted Amino Acid Sequences of V,62- Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021 accompanied by finding of expected diversity in blood. The J61 Junctions . The predicted amino acid sequences of pre- finding of different predominant sequences in separate areas dominant lesional V62-J61 gene junctions indicated con- of the biopsy argues against sample to sample contamina- servation of amino acids from site to site within a biopsy tion. The finding of limited diversity was obtained by com- and from patient to patient (Fig . 6) . The conserved motif plete workup of tissue samples by three separate investigators comprised the 5' end of the VJ junction : T .L/V.G.G/D, in three separate laboratories . We are confident that the PCR beginning in the third amino acid position after the second reaction was performed on DNA from -500 cells and not cysteine of V62. This motif results primarily from the con- on a single cell based upon : (a) calculation of the number tribution of V62, P, and D63 gene segments. A similar motif of y1b cells in the biopsy specimen ; (b) comparison of PCR is seen in the peripheral blood of these individuals, but only product to PCR titration ofknown amounts of plasmid DNA in those clones that solely use the D63 gene segment (Fig. containing 6 gene segments; (c) appearance of several unique 6) . This motif was rarely observed in normal skin (Fig . 6) . sequences along with the predominant sequence in many of In addition, the length of the VJjunction was relatively con- the clonings of lesional skin ; and (d) derivation of the same served in the lesional sequences, as compared to the periph- sequence by separate PCR reactions and cloning of the tissue eral blood . sample. To ascertain whether the conserved motif we observed in Extensive Diversity of y/b TCRs in Normal Human Skin. the V62-J61 junctions of leprosy lesions was present in other The resident y/b population in normal human skin is small, granulomatous diseases, we translated the V62-J61 nucleo- with few cells identified by immunohistologic techniques (13) . tide sequences of y/6 T cells from the lungs of patients with

V6-J6 JUNCTIONAL NUCLEOTIDE SEQUENCES FROM PATIENT B BIOPSY OF LEPROMIN SKIN TEST

Vdl-J61

V61 P/N D61 P/N Db2 P/N D63 P/N J61 FREQUENCY

germline CTCTTGGGGAC GAAATAGT CCTTCCTAC ACTGGGGGATACG ACACCGATAA

AREA 1 CTCTTGGGGA TGCG CIA TTTGA GGGGGATACG CGAAATA ACACCGATAA (9/9)

AREA 3 CTCTTGGG ACGGGGGTGA GGGGA CTCCAGG ACACCGATAA (12/12)

V62-J61

Vb2 P/N D61 P/N Db2 P/N D63 P/N J61 FREQUENCY

'germline GTGACACC GAAATAGT CCTTCCTAC ACTGGGGGATACG ACACCGATAA

AREA 1 GTGA TCG ACTGGGGG GCCCGT ACACCGATAA (8/e) Figure 4. Microheterogeneity AREA 2 GTGAC GOO CTGGGGGATAC TA ACACCGATAA (10/17) of y/8 T cell receptors in lesions. GTGACACC GTA GGGGG TCGGT ACACCGATAA (6/17) GTGAC CCCC CTGGGG CCCACAT ACACCGATAA (1/17)- Within different regions of a bi- GTGAC TCC ATT ACTGGGGGA GAACAGT ACACCGATAA (1/17) opsy specimen, distinct clonal junctional nucleotide sequences AREA 4 GTGACACC GT CTGG A ACACCGATAA (15/30) f GTGACACC A TGGGGGA CCCACGG GATAA (8/30) were identified . Nonfunctional GTGACACC GTGAC ACTGGGGG GTACGCTGAG CGATAA (1/30) sequences are marked with an GTGACACC OTC CCTA CTGGGGG GGCCCT CACCGATAA (1/30) GTGA TCC C CTGGGGG GGAGGG ACCGATAA (1/30)' asterisk . Sequences marked by GTGAC GG ACTGGG CACCGATAA (1/30) a pound sign were obtained by GTGACACC TGTA GGGGG G ACACCGATAA (1/30) GTCACA TO CCTT G GGGGGATACG G ACACCOATAA (1/30) three separate PCR reactions and GTGAC CG CCT GGGGG GCG ACACCGATAA (1/30) cloning .

688 Clonal Selection of y/b T Cells in Response to a Human Pathogen Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021

Figure 5 . PCR amplification and 'PCR V-J junctional sequences in normal skin . Analysis of V6 and J6 gene usage by PCR amplification revealed product for both V61-J61 and V62-J61 but not other Vb-J5 combinations (A shows the results for normal donor F; donor G gave identical results) . Nucleotide sequences of Vb2J61 junctions revealed extensive nucleotide diversity for specimens from normal donors F and G (B) . Nonfunctional sequences are marked with an asterisk .

sarcoidosis (14) . This comparison revealed a relatively high patients with Celiac disease, a gluten sensitive enteropathy, frequency of the T-L/V.G.G/D motifin V62-J61 junctional was found to contain V61 + cells (15) . sequences (data not shown) . This motifoccurred in the same The experimental evidence indicates that V61 and V62 amino acid positions as in sequences from leprosy lesions, populations may recognize distinct antigens and/or presen- but only when the D63 segment was the only D segment tation molecules. Some human V61 + cells appear to have used . The length of the VJjunction in the repeated sequences cytotoxic activity against allogeneic targets ; this cytotoxicity of the sarcoidosis patients was conserved, at a length similar is blocked by a mAb that recognizes a 43-kD protein termed to that observed in the leprosy lesion sequences. This was TCT 1 (16) . Also, V61 + cells have been reported to recog- in contrast to the variable length of the V62-J61 junction nize CD1 molecules (17, 18) . Langerhans cells express CD1 sequences of normal blood y/6 T cells reported (14) . molecules, can present antigen, and are increased in the epidermis and dermis of these lesions (8) . Perhaps, the expression of the TCT 1 molecule and/or CD1 molecules by Discussion diseased epithelia results in the stimulation of the V61 popu- Distinct Microanatomic Patterns of V8 Populations in Leprosy lation . In contrast, human VS2+ cells expand in response Lesions. Immunohistologic identification of VS T cell popu- to M. tuberculosis antigens in vitro. The ability of these same lations in leprosy lesions reveals distinct microanatomic pat- V62+ cells to proliferate to Daudi cell lines is blocked by a terns for V61- and V62-bearing cells : the dermal infiltrate polyclonal serum to a human HSP 58-kD protein (19) . containing both populations, but the epidermal y/6 T cells Limited Diversity ofy/S TCRs in Lesional Microenvironments preferentially expressing the V61 chain . The localization of Suggests Clonal Selection by Antigen . The most striking data V61' cells to epidermis in these mycobacterial lesions sug- presented here are the limited diversity of the VJjunctional gests either homing, retention, and/or in situ expansion of elements of y/6 T cells in leprosy lesions. The junctional V61 + cells during the immunopathogenic reaction . This lo- sequences of y/6 T cells from lesions were not found in the calization of V61+ cells to an epithelial surface may be char- respective individual's peripheral blood, indicating that the acteristic of the inflammatory process . The gut epithelia of lesional sequences do not represent an already existing clonal

689 Uyemura et al .

LEPROSY SKIN BIOPSY SPECIMENS heterogeneity implies that a very small number of y/6 clones PT A C DT L H W GR R D K initiate the oligoclonal expansion and that their progeny do not disperse homogeneously throughout the lesion . Studies PT B C DR L GGPY T DK C D G L GDTN T D K of murine -y/b T cells indicate the positive selection of y/6 C DT V GG R Y T D K T cells in the periphery (20) . The study of leprosy lesions CDT W N TD K CD T M GDPR TD K indicates that the clonal selection by foreign antigen occurs at the site ofinfection . Furthermore, y/b T cells in the lungs PT D CDT V GGTSY T D K and blood ofpatients with sarcoidosis (14) may similarly re- sult from the oligoclonal expansion to antigen . PBMC FROM LEPROSY PATIENTS The nucleotide sequence analysis of lesional y/6 T cells PT A CDTDTP C N TD K revealed that while V61J61 T cells utilize multiple Db seg- CDS M G D T Q AD K ments at the VJ junction, the V62-J61 T cells use only the CD PLG DPD TD K CDQLGV T ED T D K D63 gene segment in 98% of the isolates. This is in contrast CDT Q C S S YT D K to the 67% usage of D63 alone in sequences derived from CESL W G YTD K the blood of the same individuals, or the 33% usage in normal CD PL E I R YT D K CDT H W G I TP T D K skin . The differences in D6 usage suggest a fundamental dis- tinction in the V61 and V62 populations in skin lesions . The Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021 PT B C D PC T G G TC T D K C D PS R L W HG T D K exclusive use of the D63 gene segment with minimal use of C D L V G D HT D K N segments is a feature of y/6 T cells representing an early C D TV L R G GP H TD K fetal thymic wave (11) . Since V62-D63-J61 cells in leprosy C D GL G G I K R D TD K skin lesions were found to contain N segments, they are likely PT D CDP R P Y W GL P G AP K derived from a later developmental stage when the levels of C A GA C P VV P A TH T D K C HR L G GPYT DK terminal deoxynucleotide transferase are higher. The finding CD GA L G AK N T D K of conserved amino acid residues and length of the predomi- C D PC S G A V L G D A V D K nant lesion-derived V62-J61 junctions suggests that thejunc- CDP H W R R A P D TD K C D T V G EN T D K tional region, and specifically the D63-encoded sequence, par- CDQV L GDTR G PD K ticipates in antigen recognition (Fig. 4) . This is supported by murine studies indicating that the junctional sequences NORMAL SKIN influence the specificity of the ylb TCR (21) . When com- NL F CD T D PW K T D K pared to the limited TCR diversity displayed by y/b T cells CD H I VV T D K from leprosy lesions, the normal skin data suggests that the CDS LL YT D K selection from CD T SL L Y T D K y/6 T cells in leprosy lesions represent clonal CE M G D W G I Y T D K among a genetically diverse peripheral blood or resident skin CD T PS L H D TD K population . The reported diversity of VJ junctions in NL G CD TRVT G G CT D K mycobacterial-reactive murine y/6 T cells derived from CD R LGGP Y T D K antigen-unselected neonatal thymus indicates that the breadth Figure 6 . Predicted amino acid sequences based on V62 J61 nucleotide of the antigen-reactive repertoire is large (22) . sequences. The predicted amino acid sequences for the predominant le- Human vs. Murine Skin y/b T Cells. The normal murine sional V62-J61 junctions are shown . The conservation in amino acid se- epidermis contains a dendritic cell population that is Thy- quences, the T-L/V-G-G/D motif beginning in the third amino acid po- 1+ and bears y/6 TCRs . This resident population is of ex- sition after the second cysteine of V62, is primarily encoded by the V62, tremely limited genetic diversity, with virtually exclusive usage P, and the D63 gene segments. Although D63 gene segment usage is fre- quent in the peripheral blood, the conserved motif does not appear in the of a single Vy gene and single V6 gene and no junctional same location within the VJ junction with the high frequency of leprosy diversity (1) . Similar to the murine intraepidermal T cells, lesional sequences. In addition, the junctional amino acid lengths of pe- we find the normal human TCR y/6 skin population to be ripheral blood V62* cells are more variable, occasionally showing a greater encoded by a restricted set of V and J genes (V62-J61) . In length than the lesion-derived sequences. The amino acid sequences of response to infection, the epidermis is found to contain a normal skin show even greater heterogeneity. specific TCR 6 population (V61-J61), and all TCRs are of limited genetic diversity. expansion in the peripheral repertoire. It is noteworthy that Our findings indicate clear differences between human versus in each spatially separated region of the biopsy specimen, a murine skin y/6 T cells. The resident human skin popula- limited number ofjunctional nucleotide sequences were rep- tion, known to be significantly smaller than the murine resi- resented multiple times, although the predominant sequence dent population (13), displays extensive, rather than limited, differed from site to site. Since there was conservation in the genetic diversity ofjunctional gene elements . Even in human predicted amino acid sequence in various areas of the biopsy skin lesions of infectious etiology, the number of epidermal and among individuals, we hypothesize that they/6 T cell y/6 T cells was relatively few as compared to the resident expansion in lesions is selected by a limited set of antigens murine population . However, limited genetic diversity occurs within the tissue microenvironment . The limited micro- in y/6 T cells responding to infection and appears to be due

690 Clonal Selection of y/6 T Cells in Response to a Human Pathogen

to the oligoclonal expansion of specific TCR-bearing cells in early lesions of leprosy and leishmaniasis (7) . Studies of from a diverse pool. human y/S have indicated reactivity to a number of infec- Implications. y/S T cells appear to function as a first line tious agents, including mycobacteria (7, 31-33), listeria (34), ofdefense against infectious pathogens (23) . This hypothesis streptococcus (34, 35), staphylococcus (34-36), plasmodium is based on the finding of large numbers of y/b T cells at (37, 38), and herpes viruses (39) . The present data indicate peripheral interfaces, including normal murine skin (24), gut that the y/b T cell response to infection involves clonal se- (25, 26), and lungs (3) . y/S T cells derived from antigen- lection from a diverse TCR repertoire and further expansion unselected murine neonatal thymocytes (27) respond to by antigen in situ . The limitedjunctional diversity in leprosy mycobacterial antigens in vitro. Furthermore, the initial im- lesions is not consistent with current knowledge of superan- mune response to M. tuberculosis (28), Listeria monocytogenes tigen responses (40) . Our data suggest that VJjunctions are (29), and Trypanosoma cruzi (30) infection is characterized by critical for antigen recognition . expansion of y/S T cells . In humans, y/b T cells accumulate

Brenner, Kronenberg, and Barry R. Bloom for scientific We are grateful to Drs . Michael B . Mitchell Downloaded from http://rupress.org/jem/article-pdf/174/3/683/1267794/683.pdf by guest on 26 September 2021 discussions and encouragement . We are indebted to Drs. Shuzhuang Lu, Erica Nelson, and Florence Hofman for technical expertise .

This work was supported by grants from the National Institutes of Health (AI-22553, AR-40312, AI- 28508), the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (IMMLEP and THELEP), the Heiser Trust, and The Dermatologic Research Founda- tion of California, Inc. H. Band is a Leukemia Society of America Special Fellow and an Arthritis Founda- tion Investigator. Address correspondence to Robert L . Modlin, Division of Dermatology, 52-121 CHS, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90024 .

Received for publication 8 April 1991 .

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692 Clonal Selection of .y/S T Cells in Response to a Human Pathogen