Human T-Cell Clones with Reactivity to Mycobacterium leprae as Tools for the Characterization of Potential Vaccines against Leprosy

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1 INFECTION AND IMMUNITY, Mar. 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 51, No. 3 Human T-Cell Clones with Reactivity to Mycobacterium leprae as Tools for the Characterization of Potential Vaccines against Leprosy FRANK EMMRICH* AND STEFAN H. E. KAUFMANN Max-Planck-Institut fur Immunbiologie, D-7800 Freiburg, Federal Republic of Germany Received 10 September 1985/Accepted 10 December 1985 T-cell clones with the T4 phenotype were established from patients with tuberculoid leprosy. The antigen reactivity of these clones ranged from stringent specificity for Mycobacterium leprae to broad cross-reactivity with other mycobacteria. Killed M. leprae had a weak stimulatory capacity which could be enhanced by ultrasonication. Among the three candidate antileprosy vaccines, M. leprae, M. bovis BCG, and the ICRC (Indian Cancer Research Center) strain, the last was superior in stimulating cross-reactive T4 clones. This finding argues for a differential masking of similar or identical membrane antigens in various mycobacterial species. T-cell clones with defined reactivity patterns for mycobacterial antigens could be helpful tools for the characterization of an antileprosy vaccine. Leprosy is a chronic infectious disease caused by the intracellularly growing bacterium Mycobacteriuim leprae (3, 6, 12). It has been estimated that 15 million cases exist worldwide; this is considered a major health problem, particularly because of the extreme stigma and fear associated with this disease. Although leprosy can be treated chemotherapeutically, it has become clear that a satisfactory control of the disease will ultimately depend on active vaccination (3; T. H. Maugh II, Science 215:1083, 1982). The rational design of a potent antileprosy vaccine will depend on prior identification of microorganisms or proteins sharing protective epitopes with M. leprae. Identification of relevant antigens must be accomplished with T cells, owing to the exclusive role of T cells in protection (3, 6, 12). This has now become possible at the single-cell level because recent advances in tissue culture technology have allowed the propagation of monoclonal T cells which retain antigen specificity, phenotype, and biological function. Using this technology, we established and characterized T-cell clones with the helper/inducer phenotype from patients with tuberculoid leprosy and used these T-cell clones as tools in the functional analysis of antigenic relatedness at the T-cell level between various mycobacterial strains. MATERIALS AND METHODS Bacterial and antigen preparations. Purified protein derivative (PPD) of M. tuberc-ulosis was obtained from the Statens Serum Institute, Kopenhagen, Denmark. M. leprae proteins and cobalt-irradiated armadillo-derived M. leprae were kindly supplied by R. J. W. Rees through the World Health Organization-Immunology of Leprosy program and had been prepared in accordance with World Health Organization protocol 1/79, document Tropical Disease Research, Immunology of Leprosy-Scientific Working Group (S) Irradiated ICRC strain (1) was kindly provided by M. G. Deo, Cancer Research Institute, Bombay, India. An M. leprae cell wall preparation (batch IV) was kindly provided by P. Brennan, Colorado State University, Fort Collins, and had been extracted from lyophilized M. leprae with chloroform-methanol. M. bovis BCG was originally obtained from R. J. North, Trudeau Institute, Saranac Lake, N.Y., and M. tuberculosis was obtained from J. K. Seydel, * Corresponding author. 879 Forschunginstitut Borstel, Borstel, Federal Republic of Germany. The other mycobacteria were a kind gift from W. Brehmer, Robert Koch Institute, Berlin, Federal Republic of Germany. Cultivable mycobacteria were grown in Dubos broth (Difco Laboratories, Detroit, Mich.) supplemented with bovine serum albumin and Tween 80 at 37 C with shaking. The cultures were centrifuged and suspended in phosphate-buffered saline, and the numbers of viable organisms were determined by plating 1:10 dilutions on Middlebrook-Dubos agar (Difco Laboratories). Washed mycobacteria were heat killed and ultrasonicated three times for 3 min each time at 60 W with a Branson B12 Sonifier equipped with a microtip. Generation of human T-cell clones. Patients were classified on the basis of clinical and histopathological criteria at an Indian leprosy research institute (The Foundation for Medical Research, Bombay, India) as polar tuberculoid. They were under chemotherapeutic treatment. Peripheral blood lymphocytes (PBL) were prepared with Ficoll-metrizoate (4). T lymphocytes specific for mycobacterial proteins were stimulated in cultures of 1.5 x 106 PBL per ml of RPMI 1640 medium supplemented with 10% screened human A or AB serum, 2 mm L-glutamine, 25 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 2 x 10-5 M 2-mercaptoethanol, 100 U of penicillin per ml, and 100,ug of streptomycin per ml in the presence of 10,ug of M. leprae protein and 10 Fig of PPD per ml in 24-well flat-bottomed culture trays (Nunc, Roskilde, Denmark). High-pressure liquid chromatography-purified lectin-free interleukin 2 (IL-2) was added (15 U/ml; Biotest-Seruminstitut GmbH, Frankfurt, Federal Republic of Germany) for a short period to cultures which showed vigorous growth. Subsequently, viable cells were separated on a Ficoll-metrizoate gradient and plated at one cell or five cells per microculture well of 96-well round-bottomed trays (Nunc) in medium containing 60 U of IL-2 per ml, 5 x 104 HLA-DR,-DQ-matched PBL (irradiated at 4,000 rads), and 10,ug of mycobacterial protein antigen per ml in a total volume of 100 pal. For accessory cells, only PBL from normal donors which had been selected from an HLA-typed panel for optimal antigen presentation in the absence of background stimulation were used. After incubation at 37 C in a humidified 5% CO-air atmosphere for 10 to 14 days, growing clones were transferred in succession to 96-well round-bottomed trays, 24-well flat-

2 880 EMMRICH AND KAUFMANN INFECT. IMMUN. TABLE 1. Antigen Reactivity patterns of representative T4 clones established from peripheral blood in tuberculoid leprosy Proliferative response of indicated T-cell clone (cpm ± SD): 2C11 lh1l 2G2 2D1 3B3 8C9 Control 156± 85 85± ± 4 93 ± ± 7 36 ± 7 Ovalbumin (10 p.giml) 149 ± ± ± ± ± ± 10 PPD (10,ug/ml) 43,309 ± 1,741 7,807 ± 2, ± ,443 ± 1, ± ± 32 M. Ieprae protein (10,ug/ml) 75,529 ± 269 4,841 ± ,839 ± ± 37 2,722 ± 81 36,546 ± 1,952 M. leprae (106) 202 ± ± ± ± ± 77 3,713 ± 84 Sonicated M. leprae (106) 13,665 ± 615 3,017 ± 42 1,118 ± ± ± 41 11,638 ± 1,491 M. leprae cell walls (10,ug/ml) 8,671 ± 652 5,751 ± 1,150 1,920 ± ± ± 32 10,933 ± 104 bottomed trays (Nunc), and 6-well tissue culture trays (Costar, Cambridge, Mass.). Subclones were obtained by recloning. Fresh IL-2 was added every 3 to 4 days, and irradiated pooled feeder cells were added every 7 days. Before use in assay systems, T-cell clones, were rested for 6 to 7 days after the last addition of feeder cells. Phenotypic analysis of differentiation antigens. Cells were analyzed cytofluorometrically (ORTHO systems 30/50; Ortho Instruments, Westwood, Mass.) after being stained with monoclonal antibodies OKT3, OKT4, and OKT8 (Ortho) and fluoresceinated F(ab')2 goat anti-mouse immunoglobulin G (Tago Inc., Burlingame, Calif.). Proliferation assays. Cloned T cells (104) were incubated in 200-,Il triplicate cultures with 5 x 104 irradiated (4,000 rads) HLA-DR,-DQ-matched PBL and antigen in round-bottomed microculture wells (Nunc). Either mycobacterial proteins or killed mycobacteria were used as the antigen at the concentrations indicated in Tables 1 through 3. After 72 h of in vitro incubation at 37 C in a humidified 5% CO2 atmosphere, cultures were labeled for 16 h with 1,uCi of [3Hlthymidine per well and harvested into glass fiber filters. [3H]thymidine incorporation was measured in a liquid scintillation spectrometer. TABLE 2. Lymphokine production by representative T4 clones reactive with M. leprae protein Recombinant Productionb of: Clone Antigen human IL-2 IL-2 IFN Specificity' (10 ng/ml)r (U/mI) (titer) 2C11 M. leprae protein M. leprae protein + 9 PPD PPD + 6 lh1l M. leprae protein M. leprae protein + 6 PPD PPD + 9 2G2 M. leprae protein M. leprae protein + 9 3B3 M. leprae protein M. leprae protein + 6 8C9 M. leprae protein M. leprae protein + NT PPD PPD + NT +, Present; -, absent. b 0, <0.5 U of IL-2 per ml;-, IFN titer <3; NT, not tested. ' As determined repeatedly by proliferation assays. +, reactive; -, not reactive. Lymphokine assays for IL-2 and IFN. For determination of human IL-2, triplicate cultures of 2 x 104 T cells were stimulated with antigen and 5 x 104 irradiated HLA-DR,- DQ-matched PBL for 48 h in round-bottomed microculture wells. IL-2 activity in the culture supernatants was determined by standard titrations with the IL-2-dependent mouse T-cell line CTLL-2 (10). For calibration, the BRMP (Biological Response Modifier Programme) IL-2 standard was used (Letter, Lymphokine Res. 3:227, 1984). Interferon (IFN) was determined in the same culture supernatants as well as in parallel cultures to which exogeneous recombinant IL-2 had been added. Recombinant IL-2 was generously provided by E. Liehl (Sandoz Forschungsinstitut GmbH, Vienna, Austria). For determination of the IFN titers, serial threefold dilutions of supernatants were added to 104 HEp-2 cells in flat-bottomed microculture wells (14). After 18 h, the cells were washed, and 103 U of vesicular stomatitis virus was added. After another 48 h, cytopathic effects were determined by visual inspection. Control cultures with recombinant human IFN-y of known activity were tested in parallel. No IFN activity was found in the recombinant IL-2 preparation (unpublished observation). RESULTS Establishment of M. leprae-reactive human T-cell clones. The antigen specificity of 105 T-cell clones obtained from a patient with tuberculoid leprosy was determined. This patient was selected from other leprosy patients because of the TABLE 3. Fine specificity of representative T-cell clones for different mycobacterial species' Proliferative response of indicated T-cell clone Antigen (stimulation index): 2C11 lh1l 2G2 2D1 3B3 8C9 PPD M. leprae protein M. leprae M. tuberculosis M. bovis BCG M. smegmatis NT M. intracellulare NT M. avium NT M. ulcerans NT M. vaccae NT ICRC strain N. asteroides NT a Proliferation assays were carried out with 106 ultrasonicated killed microorganisms. The ICRC strain was not ultrasonicated. Stimulation indices were calculated as the quotient of the test counts per minute divided by the ovalbumin control counts per minute, which ranged from 51 to 168 cpm. NT, Not tested.

3 VOL. 51, 1986 HUMAN T-CELL CLONES WITH REACTIVITY TO M. LEPRAE 881 considerable proliferative response of blood lymphocyte cultures stimulated with M. leprae protein. With the exception of six clones which showed autoreactive behavior, all clones were reactive with mycobacterial antigens presented by HLA-DR,-DQ-matched accessory cells. Fourteen clones with high proliferative responses to the certain antigens were analyzed in detail; the reactivity patterns of six representative T-cell clones are shown in Table 1. Clones with selective reactivity for M. leprae protein (2G2, 8C9, and 3B3) or PPD (2D1) as well as clones cross-reactive with both antigens (2C11 and lhil) were identified. Whole irradiated M. leprae failed to stimulate all but 1 of the 14 T-cell clones and was only antigenic after disruption by ultrasonication. Clones reactive with M. leprae protein could also be stimulated by a cell wall-enriched preparation. These findings indicate that the antigenic moieties are hidden in undegraded M. leprae. There was no suppressive effect of unsonicated M. leprae cocultured with the protein antigen (data not shown). Alternatively, the possibility remains that intact organisms cannot be processed or presented appropriately by normal human PBL in vitro. However, this interpretation seems unlikely because one clone (8C9) was stimulated by unsonicated M. leprae presented by normal human PBL. It should be noted that the degradation and processing of particulate M. leprae in vivo seems to be more efficient because priming by the M. leprae protein has obviously occurred, since a considerable proliferative response was observed in PBL bulk cultures from leprosy patients as compared with normal individuals. Lymphokine secretion by M. Ieprae-reactive T-cell clones. Analysis with the fluorescence-activated cell sorter revealed that all T-cell clones had the phenotype of mature helper/inducer T cells (T3+, T4+, T8-). In cultures, only a few of our clones maintained their initial capacity to produce IL-2, although all clones repeatedly became susceptible to exogeneous IL-2 after antigen stimulation. However, upon appropriate stimulation, all cultures produced IFN (Table 2). In the case of clone 3B3, exogenous IL-2 had to be added to yield IFN production. In general, maximal IFN-y secretion has been reported to depend on stimulation with IL-2 (20), which can be provided by an exogenous source or by the T-cell clone itself. Specificity patterns of M.leprae-reactive T-cell clones. For a detailed analysis of the reactivity patterns of the T-cell clones, they were stimulated by killed organisms of different mycobacterial species. Clone 2C11 cross-reacted with several but not all mycobacterial species (Table 3). Although clone ihil cross-reacted with PPD, M. leprae protein, M. leprae, M. tuberculosis, M. bovis BCG, and the ICRC strain, it was not stimulated by the atypical mycobacteria M. intracellulare and M. avium. Clone 3B3 may be of particular was capable of differentiating M. leprae interest because it from the other mycobacteria tested so far. None of the T-cell clones was stimluated by Nocardia asteroides, although nocardiae are thought to share cross-reactive antigens with mycobacteria (24). Stimulation of M. leprae-reactive T-cell clones by three candidate vaccines. Currently, in vitro cultivable mycobacteria sharing cross-reactive epitopes with M. leprae (e.g., BCG or the ICRC strain), killed armadillo-derived M. leprae, or combinations of both are considered for vaccination against leprosy [2, 3, 5, 8, 9; T. H. Maugh II, Science 215:1083, 1982; N. Williams, Nature (London) 316:183, 1985]. We therefore compared T-cell stimulation by M. leprae protein and by the three candidate vaccines M. leprae, BCG, and the ICRC strain. Only 2 of 14 T-cell clones gave a significant response (stimulation index, >5) against C) 0. G- (-) mcd (-) ' t @@ l l09 * * I i 'o 0 - v- lb 0 1~~~~~ l i~~~~~~ L-- l * 09 * I I 0 ~~ SI, M.leprae protein 1000 FIG. 1. Stimulation of 14 representative mycobacterium-reactive T-cell clones by various candidate antileprosy vaccines. Fourteen independent T-cell clones reactive with M. leprae protein were tested with M. Ieprae (A), BCG (B), or the ICRC strain (C). Proliferation assays were carried out as described in Materials and Methods with 10 jig of M. leprae protein per ml or 106 killed unsonicated organisms per culture. Stimulation indices (SI) were calculated as described in Table 3, footnote a, and are shown in a logarithmic scale. Broken lines indicate an SI of 5, which was taken as the threshold value. M. leprae (Fig. 1). A better stimulation was observed when BCG was used as the antigen (6 responsive clones of 14). On the other hand, 10 of the 14 clones gave a significant response against the ICRC strain, and several of them reacted equally well with the ICRC strain and M. leprae protein, indicating a high relatedness between the ICRC strain and M. leprae proteins with respect to their antigenicity for T cells. In addition, our findings demonstrate, at least in vitro, that the ICRC strain antigens for our T-cell clones are more accessible for processing or presenting by normal human mononuclear cells than is M. leprae. DISCUSSION In the present study, a battery of human T-cell clones of the helper/inducer phenotype and with reactivity against M. leprae were established and used for the functional characterization of candidate antileprosy vaccines. The specificity - 0

4 882 EMMRICH AND KAUFMANN of our T-cell clones ranged from broad cross-reactivity with a variety of mycobacterial species to unique specificity for M. Ieprae. Earlier studies with T cells from leprosy patients failed to demonstrate M. leprae-specific proliferative T-cell responses (3, 11). Our clonal approach revealed that the T-cell response of leprosy patients is heterogeneous and encompasses not only cross-reactive but also M. leprae-specific T-cell populations. Therefore, the use of a panel of T-cell clones with various reactivity patterns will markedly improve the classification of mycobacterial species and strains according to their relatedness with M. leprae antigens at the T-cell level. Based on histopathological findings, leprosy can be divided into two main stages. At the one pole (tuberculoid leprosy), patients have efficient specific T-cell immunity capable of limiting bacterial growth, whereas at the other pole (lepromatous leprosy), specific T-cell immunity is markedly impaired and unable to control the multiplication of the microorganisms. Recent studies with T cells from leprosy patients have shed some light on the role of helper/inducer T4+ cells in this disease. In lepromatous, but not in tuberculoid, leprosy the following observations, which point to a quantitative defect in the T4-cell population, have been made: (i) in lesions, the T4/T8 cell ratio is drastically reduced (25); (ii) antigen-induced IL-2 and IFN--y secretion is markedly impaired (13, 18); and (iii) IFN--y secretion-at least partially-can be restored by exogenous IL-2 (13, 18). Taken together, these data indicate that T4+ lymphocytes capable of secreting IFN--y after antigenic stimulation play an important role in acquired resistance to M. Ieprae. Our T-cell clones expressed the T4+ phenotype, and produced IFN in cultures with antigen and accessory cells. Clone 3B3 produced IFN only in the presence of additional exogenous IL-2. Although not formally proven, the dependence of T cells on IL-2 indicates that IFN was produced by the T-cell clones and belonged to the IFN--y class, which is known to be a potent mediator of macrophage activation (e.g., reference 21). We therefore suggest that our clones were derived from a T-cell population relevant to protection against M. leprae. Consequently, these T-cell clones were used as tools for the functional characterization of candidate vaccines, since no other procedures are available at present to analyze the capacity of antigen preparations to stimulate T-cell immunity at the preclinical level. In developing a vaccination strategy against leprosy, the following approaches are currently being considered [2, 3, 5, 8, 9; T. H. Maugh II, Science 215:1083, 1982; N. Williams, Nature (London) 316:183, 1985]. One approach would be to use related cultivable mycobacteria sharing protective epitopes with M. leprae. In principle, the advantage of such vaccines would be the ability to supply large numbers of microorganisms and the presumptive lack of suppressive epitopes. On the other hand, it is not known to what degree these organisms cross-react with M. leprae at the level of protective T-cells and hence can stimulate resistance against subsequent infection. Vaccination trials with live M. bovis BCG hitherto yielded controversial results (2, 3, 5). Besides BCG, the ICRC strain is another in vitro cultivable candidate vaccine against leprosy. This strain has been found to convert lepromin skin test-negative individuals (lepromatous leprosy patients and household contacts) into skin testpositive cases (9) and is currently being tested in an antileprosy vaccination trial in India. Another approach to vaccination would be the use of killed M. leprae, since it has been shown that even in the absence of an adjuvant killed M. leprae can induce cellular immune responses in experimentally infected animals (15, 16, 19, 22). A comparison of the antigenicity of the three candidate vaccines, M. leprae, BCG, and the ICRC strain, with that of our M. leprae protein-reactive human T-cell clones revealed the following ranking order with respect to their capacity to stimulate human T cells in undegradated form: the ICRC strain > BCG > M. leprae. The ICRC vaccine seems to be processed or presented by normal human accessory cells in a more efficient way than the BCG and M. leprae vaccines. This finding would favor the use of the ICRC strain as an antileprosy vaccine to stimulate cross-reactive T cells. Since we detected T-cell clones with exclusive specificity for M. leprae, it might be interesting to evaluate their particular contribution to protection. It is anticipated that a functional analysis of antigenic relatedness with T-cell clones as probes will have a great potential for the rational design of vaccines against bacterial and parasitic diseases in which protection is predominantly governed by T cells. This approach will gain particular importance once recombinant proteins become available for the construction of a novel generation of vaccines (7, 26). We plan to use our panel of M. leprae-reactive T-cell clones to characterize distinct proteins or peptides with respect to their capacity to stimulate helper/inducer T cells. ACKNOWLEDGMENTS INFECT. IMMUN. We are grateful to N. H. Antia, The Foundation for Medical Research, Bombay, India, for providing the clinical characterization and HLA-DR typing of patients and for allowing us to use the facilities at his institute, T. J. Birdi for her great help, and M. G. Deo and S. G. Gangal, Cancer Research Institute, Bombay, India, for additional support during the initial phase of our studies. We gratefully acknowledge A. Mayerova, Institut fur Humangenetik, Freiburg, Federal Republic of Germany, for performing the HLA typing of PBL donors at our institute. We also thank B. Schilling, C. Riesterer, E. Hug, and U. Vath for helpful technical assistance and H. Kuttler for typing the manuscript. This investigation was supported in part by the Immunology of Leprosy component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. LITERATURE CITED 1. Bapat, C. V., K. J. Randive, and V. R. Khanolkar In vitro cultivation of an acid fast mycobacterium isolated from human lepromatous leprosy. J. Pathol. Bacteriol. 1: Bechelli, L. M., P. G. Garbajosa, M. M. Gye, K. Uemura, T. Sundaresan, W. M. Dominguez, M. Matejka, C. Tamondong, R. Quagliato, V. Engler, and M. 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