Sep 8, 1992 - AND ROBERT L. MODLIN*§. *Division of Dermatology ...... Schurr, E., Morgan, K.,Gros, P. & Skamene, E. (1991) Am. J. Trop. Med. Hyg. 44, 4-11. ... Fink, P. J., Matis, L. A., McElligot, D. L., Bookman, M. &. Hedrick, S. M. (1986) ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 188-192, January 1993 Immunology
Selection of T lymphocytes bearing limited T-cell receptor fJ chains in the response to a human pathogen (cell-mediated immunity/delayed-type hypersensitivity/major histocompatibility complex/leprosy)
XIAo-HONG WANG*, JEFFREY D. OHMEN*, KOICHI UYEMURA*, THOMAS H. REAt, MITCHELL KRONENBERGt, AND ROBERT L. MODLIN*§ *Division of Dermatology and tDepartment of Microbiology and Immunology, University of California School of Medicine, Los Angeles, CA 90024; and tSection of Dermatology, University of Southern California School of Medicine, Los Angeles, CA 90033
Communicated by Barry R. Bloom, September 8, 1992 (received for review January 27, 1992)
presentation associated with rapid fluctuations in the level of cell-mediated immunity. Of particular interest are reversal reactions, which are generally known to be naturally occurring delayed-type hypersensitivity (DTH) responses to M. leprae, associated with clearance of bacilli from lesions (8-12). The lesions are readily accessible to immunologic study and are characterized by an influx of CD4+ T cells and production of type 1-like cytokines including interferon y (13-15). Examination of the TCR (-chain repertoire in these lesions was undertaken to provide clues as to the nature and diversity of the set of antigens recognized by T cells mediating DTH in man. Because resistance to M. leprae infection is associated with specific major histocompatibility complex (MHC) class II alleles (16, 17), we explored the influence of MHC class II alleles in shaping the TCR repertoire at the site of infection.
ABSTRACT Delayed-type hypersensitivity (DTH) is a classic measure of T-cell responsiveness to foreign antigen. To estimate the extent of the T-cell repertoire in the DTH response to a human pathogen, we measured T-cell receptor (TCR) (3-chain variable-region (Vp) gene usage in reversal reactions in leprosy. Reversal reactions represent naturally occurring DTH responses in leprosy, in which augmentation of T-cell responses to Mycobacterium kprae is concomitant with clearance of bacilli from lesions. T cells using the Vp6-, Vp12-, Vp14-, and Vp19-encoded TCRs were strikingly overrepresented in the lesions of patients as compared to blood and pre-DTH lesions from the same individuals. Furthermore, these data indicate a possible association between the predominant expression of a Vp gene segment in lesions and the major histocompatibility complex class II haplotype of the individual. Vp6 was prominent in the lesions of four patients who were DR15, a marker of resistance in leprosy infection. Sequence analysis of Vp6 TCRs showed frequent use of Vp6.1 and Jp2.7 gene segments and a conserved amino acid motif in the V-J junction in a reversal-reaction lesion, but not in blood from the same patient. The limited TCR repertoire expressed by the infiltrating T cells suggests that a limited set of antigens is recognized in the DTH response to a human pathogen. We suggest that the mechanism by which major histocompatibility complex haplotype influences DTH in this disease involves the presentation of specific peptides, with subsequent selection of specific TCRs followed by local oligoclonal expansion.
MATERIALS AND METHODS Patients. Patients with leprosy were classified according to the clinical and pathological criteria of Ridley (18). All specimens were obtained with informed consent from patients at the Hansen's Disease Clinic of Los Angeles County/ University of Southern California Medical Center. HLA typing was performed by PCR at the University of California, Los Angeles Tissue Typing Laboratory. Skin biopsy specimens were embedded in OCT medium (Ames, Elkhart, IN), frozen in liquid nitrogen, and stored at - 196C. Isolation of RNA and cDNA Synthesis. Total RNA was isolated from tissue sections and peripheral blood lymphocytes (19). Fifty 5-pum sections were lysed in guanidinium isothiocynate. Peripheral blood lymphocytes (5 x 106) were
Diversity in antigen recognition by the T-cell receptor (TCR) is conferred by the choice of variable (V) and joining (J) segments, as well as nucleotide (N) and diversity (D) segments at the V-J junction. The availability of nucleotide sequences for many of the V gene segments, and the use of PCR, has facilitated the investigation of the TCR repertoire at the site of disease. Such analysis of TCR a-chain and (3-chain V (Va and Vat) gene usage has been performed in diseases of autoimmune etiology: multiple sclerosis (1-4), rheumatoid arthritis (5), and autoimmune thyroiditis (6). The finding of a limited TCR repertoire in these diseases is thought to indicate the recognition of a limited number of autoantigens. The nature of the TCR repertoire in the immune response to foreign antigens in humans has been largely unexplored. Leprosy provides an ideal model to investigate the role of T-cell populations mediating such a response. The disease forms a spectrum, in which the clinical manifestations parallel the level of cell-mediated immunity to the pathogen, Mycobacterium leprae (7). The spectrum of leprosy is not static, but rather dynamic, with alterations in the clinical
isolated by Ficoll/Hypaque density gradient centrifugation and dissolved directly in guanidinium isothiocyanate solution. The samples were treated with DNase I (Promega) for 30 min at 370C. RNase inhibitor (Boehringer Mannheim) was present during all enzymatic manipulations of RNA. cDNA was synthesized from 1-10 pug of total RNA by reverse transcription (20). PCR. PCR analysis of the TCR VP repertoire was accomplished by using oligonucleotide primers to amplify specific Vi gene segments. Each reaction mixture contained 1 of 22 oligonucleotides, specific for a particular Vi family or subfamily, paired with a consensus (-chain constant-region (Ca) primer which yields products of =260 base pairs (bp). Sequences of oligonucleotide primers have been previously reported (21). The Vp6 primer recognizes V,6.1, -6.2, -6.3, and -6.4 subfamilies. PCR amplification was performed using Abbreviations: DTH, delayed-type hypersensitivity; TCR, T-cell receptor; MHC, major histocompatibility complex. §To whom reprint requests should be addressed at: University of California, Los Angeles, Division of Dermatology, 52-121 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90024-1750.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 188
Proc. Natl. Acad. Sci. USA 90 (1993)
Immunology: Wang et A a Perkin-Elmer DNA thermocycler employing a 35-cycle program consisting of 45 sec of denaturation at 940C, followed by 1 min of annealing/extension at 650C. The annealing/extension time was increased by adding 2 sec per cycle. The reagent concentrations and precautions to avoid PCR artifacts have been described (22). PCR was found to be reproducible, with repeat experiments on the same cDNA yielding comparable results. Quantification of PCR Product by Radioactive Hybridization. PCR products were transferred to Hybond-N nylon membranes (Amersham) following electrophoresis and hybridized with a radioactive Cal probe. The oligonucleotide probe, complementary to sequences internal to the sequence recognized by the Cal oligonucleotide used for PCR amplification, was labeled at the 5' end by T4 polynucleotide kinase (Boehringer Mannheim) and [y-32P]ATP (7000 Ci/mmol, ICN; 1 Ci = 37 GBq). PCR products were quantified using an AMBIS radioanalytic imaging system (Automated Microbiology Systems, San Diego). The amounts of radioactivity bound by all the Va amplified material, the background, and the total present in any one lane were determined. The relative intensity of individual bands was expressed as the number of counts present in any one Va family divided by the total counts present in the repertoire surveyed. Sequencing of PCR Product. For sequencing of V.36-Cp PCR products, oligonucleotide primers were designed to include appropriate restriction sites and PCR products were cloned and sequenced as described (22).
RESULTS Compartmentalization of the TCR Vp Response in ReversalReaction Skin Lesions. To identify whether specific Vat subpopulations accumulate at the site of disease activity in reversal reactions, PCR was utilized to determine V13 gene usage within lesions and blood from three individuals. Simultaneous measurement of TCR repertoires in lesions compared with blood minimized some of the inaccuracies in absolute quantification of each particular V13 family related to possible differences in primer efficiency in the PCR. Distinct compartmentalization of the immune response was evident in the overrepresentation of specific V13 families in lesions relative to blood (Fig. 1). For example, in patient A, Vpl, and Vp7 TCRs were preferentially used by T cells in V,06, lesions compared to blood, by at least 2-fold. In patient B we identified Vp6, Vp13, and V,019 gene families as frequently utilized in lesions. In the lesions from patient C we found relative overrepresentation of T cells using Vp6, V112, and V114 gene families. These data indicate that specific TCR V's populations accumulate at the site of disease activity in reversal-reaction lesions. V13 Gene Usage in Microanatomic Regions of a ReversalReaction Skin Lesion. To determine whether TCR V13 usage was consistent throughout the lesion, we measured the TCR V13 repertoire in four adjacent regions of a single lesion (patient B) (Fig. 2). We expressed the data as the relative percentage of each Va family within the lesion versus the peripheral blood of the same individual. Several V13 families were overrepresented about 2- to 7-fold throughout the lesion compared to the blood. For example, V113 and V,919 were similarly overrepresented throughout the different areas studied. Some V13 gene families-for example, V,06-were overrepresented in some but not all regions of the biopsy specimen. Several V13 families, including V,02, V311, V,912, and V18, were underrepresented by at least a factor of 2 throughout the lesion compared to the blood of this patient. Finally, the majority of V13 families were equally represented throughout the lesion compared to the blood of the same patient (data not shown). In summary, TCR V13 gene families were identified which were generally overrepresented, focally overrepresented, or underrepresented throughout the
189
PATIENT A
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FIG. 1. Compartmentalization of V1 gene usage in lesions. Va-C1 PCR products from the skin lesion and peripheral blood mononuclear cells (PBMC) of patients with reversal reaction were electrophoresed, transferred to nylon membrane, and hybridized with a 32p_ labeled Cal probe. Comparisons of the V13 repertoires in lesions and blood of three patients with reversal reaction (patients A, B, and C) are shown.
specimen. Furthermore, examination of TCR V13 repertoire from two distinct anatomic sites from the same patient with lepromatous leprosy yielded similar results, indicating that the V13 repertoire was relatively consistent from lesion to lesion (data not shown). Dynamics of the Local T-CeHl Response in Reversal Reaction. The dynamic changes in TCR V13 repertoire associated with the DTH response were studied in two patients in which specimens were obtained before the onset and during upgrading of reversal reactions. Striking changes in TCR V13 repertoire were observed in these DTH reactions (Fig. 3). Several V13 gene families were >2-fold increased in reversalreaction compared to pre-reversal-reaction lesions. In patient D, the onset of reversal reaction was associated with an influx of T cells expressing V,93, V,66, V,98, V13.1, V,919, and V,320 gene families. In patient E, T cells expressing TCRs belonging to the V,06, V,98, and Vp12 families were more prominent in the reversal-reaction lesion. These data indicate that the onset of DTH against M. leprae in reversal reactions is 7 °6 -
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FIG. 2. V13 gene usage in four adjacent areas of a reversal-reaction biopsy specimen (patient B). Data are represented as the ratio of VP gene usage in the regions of a lesion versus blood of the same donor. Each adjacent region of tissue represents 50 x 5 Am of tissue. (A) V13 gene families overrepresented in the biopsy specimen. (B) V13 gene families underrepresented throughout the lesion.
190
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the lesion compared to blood (Fig. 5; Table 1). Vp6.1 predominated in the lesion, expressed in 12 of 21 clones, compared to 2 of 10 in blood. There was a frequent use of a leucine in the fourth position after the second conserved cysteine of the Vp-encoded segment in 10 of 21 sequences from the lesion, but in only 2 of 10 from blood. A striking finding was the extensive involvement of the J1p2.7 gene segment, found in 14 of 21 lesion-derived but 0 of 10 bloodderived clones. The Jp2.7 gene segment encodes a unique Glu-Gln-Tyr sequence at its N terminus. The predicted amino acid sequences indicated conservation of a glutamine (17 of 21) followed by a tyrosine (17 of 21) encoded by different J gene segments. Only 6 of 13 of known germ-line Jp gene segments encode glutamine, and 5 of 13 encode tyrosine at these positions. In the blood of this patient, where no particular Jp selection was observed, the glutamine was present in 6 of 10 and the tyrosine in 5 of 10 clones, reflecting the germ-line frequencies (data not shown). Although the predicted amino acid sequence of the V-J junctions-differed from sequence to sequence, a glycine was present in position 8 after the second conserved cysteine of the in 11 of 21 lesion sequences. This glycine Vp-encoded segment originated six times from Dp-encoded segments and was contributed in part or fully by N segments in five instances. In the sequences derived from blood, glycine occurred 0 of 10 times in this position (data not shown). Interestingly, Jp2.7 was present in 7 of 11 lesion sequences in which glycine was present at position 8 and in 9 of 10 instances in which leucine was present in position 4. Overall, the data indicate clonal selection at the protein level of Vp, Jp, and V-J junctional elements in the DTH response to M. leprae. In analyzing the nucleotide sequences of the V-J junctions, we found that of the sequences derived from lesions, 2 sequences repeated three times, 1 repeated twice, and the remaining 14 sequences were unique. No repeat sequences were found in the blood of the same patient. The repeated junctional nucleotide sequences derived from lesions provide evidence for local oligoclonal expansion (22).
PATIENT D
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Proc. Natl. Acad. Sci. USA 90 (1993)
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associated with the increase of specific T-cell populations locally. Patterns of Vp Usage in Reversal-Reaction Lesions. To identify any overall patterns of Vi gene usage in the DTH response to M. leprae, we compared the TCR Vp repertoire in reversal-reaction lesions from the five patients reported above and two additional patients (Fig. 4). An unexpected finding was the overrepresentation of several Vp families in the majority of lesions studied, including Vp6, Vp12, V,14, and Vp19. The use ofparticular V genes in the T-cell response to a foreign antigen may be influenced by the MHC of the individual. In comparing Vp gene usage to HLA-DR haplotype, we found that VP6 was prominent in all four DR15 individuals, accounting for 7-15% of the total repertoire in lesions. Vp6 also was expressed in lesions of non-DR15 individuals; therefore V,96 may be selected by several MHC class II alleles. Strikingly, in both DR9 individuals, V86, in lesions, and in both V,12, and Vp14 were overrepresented DR13 individuals, V,14 and Vp19 were predominant. These data suggest a role for the MHC in selecting TCRs of specific in the response to foreign antigen. Vp families of Diversity Vp6 T Cels In Lesions. The selection of the TCR by peptide antigen has been hypothesized to occur at the level of the V-J junction (23). To determine the diversity of these junctions in the DTH response to M. leprae, we cloned and sequenced the junction region for the V,6 PCR product derived from a reversal-reaction lesion and peripheral blood lymphocytes from the same patient (patient D). We chose V,96 because this Vi family was consistently implicated in the local T-cell response, being preferentially localized to lesions, found to increase during the reversal reaction, and predominant in lesions in a majority of patients. Analysis of the nucleotide and predicted amino acid sequences of V,96 clones indicated selection of specific TCRs in
DISCUSSION Although T lymphocytes play an integral role in mediating DTH against a variety of pathogens, it is not known whether these responses involve a limited or diverse array of antigenspecific T cells. To assess the extent of the TCR repertoire in DTH reactions, we performed quantitative PCR to determine diversity in the Vp gene usage. toWeM.chose to measure TCR DTH response leprae by studying the compartmentalization and dynamics of the T-cell response in "reversal reactions," naturally occurring DTH reactions in leprosy. The results suggest selection of particular Vp, Jp, and V-J junctional elements of the TCR by a small set of antigens and MHC class II determinants. V: family
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Proc. Natl. Acad. Sci. USA 90 (1993)
Immunology: Wang et al.
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191
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FIG. 5. Nucleotide and predicted amino acid sequences of P-chain V-D-J region of V,6 T cells from a reversal-reaction lesion. A minimum of four consecutive nucleotides was required to assign coding to a D. gene segment. We assigned the maximum number of nucleotides possible to Vp and Jp gene segments. The conserved glycine residues and encoding nucleotides are underlined. P/N, palindromic/nucleotide sequences.
The simultaneous comparison of the Vq gene usage in lesions compared with blood permitted us to determine whether specific TCR-bearing cells comprise a relatively larger part of the repertoire at the site of disease activity. We found that in each example several Vp gene families were 2to 7-fold overrepresented in lesions compared to the blood of the same individual. This indicates that several specific T-cell populations accumulate in lesions, concurrent with the DTH response against M. leprae. Several Vp families were preferentially overrepresented, although sometimes focally, and some underrepresented in adjacent regions of the lesion as compared to blood. Exploration of the dynamics of the cellular response clearly showed that the increase in DTH against M. leprae was temporally associated with increased representation of select Vp populations. It could be argued that the changes in the TCRB-chain repertoire reflect the shift in T-cell subsets
from CD8 to CD4 in these lesions, since the repertoires of CD4 and CD8 cells are known to differ (21, 24). However, the same Vq families found to be predominant dynamically in lesions were predominant compared to blood. The CD4/CD8 ratio is similar in blood and reversal-reactions lesions; therefore the dynamic alterations in repertoire in lesions most likely reflect an influx or accumulation of disease-specific T cells. Vp-bearing The most striking finding of the present study was the prevalence of T cells bearing TCRs encoded by Vp6, Vp12, Vp14, and Vp19 in the majority of lesions studied. Most often these Vp families were overrepresented in DTH lesions compared to blood and the pre-DTH lesions from the same patient. Although limited TCR V-gene usage has been found in autoimmune conditions, it is particularly surprising to find a limited TCR repertoire in response to an infectious organism which contains a large array of potential antigenic determinants.
Table 1. Frequency of Vp and Jp gene usage in a patient with reversal reaction
J;p1.1 Vp6.1
Vp6.2 Vp6.3 Vp6.4 Total
Skin Blood Skin Blood Skin Blood Skin Blood Skin Blood
Jp1.3
Jp1.5
Jp2.1
Jp2.3
Jp2.5
1
Jp32.7 9
1
1
1
1 1
Jp2.2 1
1 1 1
1
1 1
2/21 2/10
1/21
2/10
1 1 1/21
2/10
1/10
2/21 1/10
1 1 1 1/21 2/10
4
14/21
Total 12/21 2/10 2/21 2/10 1/21 3/10 6/21 3/10
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The selection of murine V regions in the response to peptide antigen is dictated by MHC class II (25, 26). Similarly, our data suggests common 3-chain usage among individuals of the same HLA-DR type. MHC class II alleles have been correlated with resistance versus susceptibility to M. leprae infection (16, 17). The resistant or tuberculoid form is associated with DR2 and DR3. Our patients began at the lepromatous region of the spectrum and upgraded toward the tuberculoid pole. It was of interest that four of our patients with DTH reactions were DR15 (a subset of DR2) and one was DR17 (a subset ofDR3), markers ofresistance in leprosy. It is thought that such immune-response genes mediate resistance by efficiently presenting a specific set of peptides to T cells bearing TCRs selected by the antigen-MHC complex. The finding of a limited Va repertoire in reversal reaction lesions, relative to blood or pre-DTH lesions, suggests that a limited set of peptides are presented by such class II antigens in response to M. leprae. That Vp6 was prominent in the lesions of DR15 individuals suggests that the same limited set of peptides is recognized by different individuals. Clearly, a larger number of individuals need to be studied to definitively correlate Vat usage with MHC haplotype in the context of M. leprae recognition. The analysis of the nucleotide and predicted amino acid sequence of Vp6 TCRs indicated selection of limited TCRs in lesions but not blood of the same patient, implicating these TCR-bearing cells in the pathogenesis of the DTH response to M. leprae. The prominence of V,6.1 and J,2.7 geneencoded elements in the lesion studied, but not in blood, indicates clonal selection of these TCRs. The frequency of the Vp6.1-Jp2.7 TCR is probably not due to preferential recombination, since analysis of Vp6.1 sequences derived from human gut indicated that Jp2.7 was not preferentially paired (27). Although it is generally believed that the V-J junction is critically involved in the T-cell response to peptide, there is only limited evidence which indicates selection of mouse J regions by antigen (28). The preferential use of in V,6 TCRs found in the leprosy lesion but not the Jp2.7 blood likely reflects selection for a particular amino acid motif at the N terminus of the J region. A second line of evidence for clonal selection of these TCRs in DTH responses was the conservation of a glycine, which was frequently encoded by N segments, in a specific position within the V-J junction of clones derived from the lesion but not the blood. Finally, the identification of repeated junction sequences in the lesion indicated local oligoclonal expansion of T cells expressing af3 TCRs. Similarly, T cells expressing y5 TCRs in leprosy lesions appear to undergo clonal selection and local oligoclonal expansion (22). A fundamental issue in understanding the mechanism(s) of protective immunity to human pathogens is the nature of the T-cell population which mediates the effective response. In designing recombinant vaccines against infectious diseases of developing countries, it will be necessary to define the number of antigens required to engender protection against a given agent (29). The overall findings of locally limited Va response suggest that a limited number of antigens are recognized by ad T cells in the DTH response to M. leprae, and perhaps recognition of these antigens is sufficient to mediate protective immunity. It should now be possible to identify these antigens by using T-cell clones with specific VP TCRs. The further characterization of these dominant TCR subpopulations according to antigen reactivity and function should elucidate the interplay among antigen, TCRs, and MHC in the generation of DTH. We thank Paul Terasaki and Richard Tonei for performing HLA typing of our patients. We express our sincere gratitude to Harout DerSimonian and Michael B. Brenner for their assistance with the
Proc. Natl. Acad. Sci. USA 90 (1993) molecular strategies we employed. This work was supported by grants from the National Institutes of Health (A122553 and AR40312), the United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (IMMLEP and THELEP), the Heiser Trust, the Leonard Wood Memorial/American Leprosy Mission, the American Lung Association of California, and the Dermatology Research Foundation of California, Inc. 1. Oksenberg, J. R., Stuart, S., Begovich, A. B., Bell, R. B., Erlich, H. A., Steinman, L. & Bernard, C. C. (1990) Nature (London) 345, 344-346. 2. Wucherpfennig, K. W., Ota, K., Endo, N., Seidman, J. G., Rosenzweig, A., Weiner, H. L. & Hafler, D. A. (1990) Science 248, 1016-1019. 3. Ben Nun, A., Liblau, R. S., Cohen, L., Lehmann, D., Tournier Lasserve, E., Rosenzweig, A., Zhang, J. W., Raus, J. C. & Bach, M. A. (1991) Proc. Natl. Acad. Sci. USA 88,2466-2470. 4. Kotzin, B. L., Karuturi, S., Chou, Y. K., Lafferty, J., Forrester, J. M., Better, M., Nedwin, G. E., Offner, H. & Vandenbark, A. A. (1991) Proc. Nati. Acad. Sci. USA 88, 91619165. 5. Paliard, X., West, S. G., Lafferty, J. A., Clements, J. R., Kappler, J. W., Marrack, P. & Kotzin, B. L. (1991) Science 253, 325-329. 6. Davies, T. F., Martin, A., Concepcion, E. S., Graves, P., Cohen, L. & Ben Nun, A. (1991) N. Engl. J. Med. 325,238-244. 7. Bloom, B. R. & Mehra, V. (1984) Immunol. Rev. 80, 5-28. 8. Waters, M. F. R., Turk, J. L. & Wemambu, S. N. C. (1971) Int. J. Lepr. 39, 417-428. 9. Godal, T., Myrvang, B., Samuel, D. R., Ross, W. F. & Lofgren, M. (1973) Acta Pathol. Microbiol. Scand. 236, (Suppl.), 45-53. 10. Barnetson, R. S., Bjune, G., Pearson, J. M. H. & Kronvall, G. (1976) Int. J. Lepr. 44, 267-273. 11. Bjune, G., Barnetson, R. S., Ridley, D. S. & Kronvall, G. (1976) Clin.- Exp. Immunol. 25, 85-94. 12. Rea, T. H. & Taylor, C. R. (1977) Infect. Immun. 18, 847-856. 13. Modlin, R. L., Gebhard, J. F., Taylor, C. R. & Rea, T. H. (1983) Clin. Exp. Immunol. 53, 17-24. 14. Cooper, C. L., Mueller, C., Sinchaisri, T.-A., Pirmez, C., Chan, J., Kaplan, G., Young, S. M. M., Weissman, 1. L., Bloom, B. R., Rea, T. H. & Modlin, R. L. (1989) J. Exp. Med.
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