INFECTION AND IMMUNITY, Sept. 1986, p. 491-497 0019-9567/86/090491-07$02.00/0 Copyright 1986, American Society for Microbiology Vol. 53, No. 3 Mycobacterium bovis BCG-Induced Human T-Cell Clones from BCG-Vaccinated Healthy Subjects: Antigen Specificity and Lymphokine Production ABU SALIM MUSTAFA,l* GUNNAR KVALHEIM,2 MIKLOS DEGRE,3 AND TORE GODAL' Laboratory for Immunology, Department of Pathology' Department of Biochemistry,2 and The Norwegian Cancer Society, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo 3, and National Institute for Public Health, Postuttak, Geitmyrsveien 75, N-0038 Oslo 1,3 Norway Received 12 November 1985/Accepted 19 May 1986 A total of 121 human T-cell clones were raised from nine Mycobacterium bovis BCG-vaccinated healthy individuals. Three clones were autoreactive, 74 responded to BCG in the presence of antigen-presenting cells, and the others required in addition exogenous interleukin 2. Only one clone was CD8+ CD4-, and the rest were CD4+ CD8-. Testing with a panel of mycobacteria suggested that the clones were recognizing epitopes of varied specificity. Out of 44 clones tested, 15 were specific to BCG and Mycobacterium tuberculosis, 22 showed limited cross-reactivity, and 8 were broadly cross-reactive. None of the 22 BCG responder clones could differentiate between Danish, French, Prague, and Moreau strains of BCG. BCG and M. tuberculosis H37Rv also paralleled very closely; however, 6 of 18 BCG- and M. tuberculosis H37Rv-responding clones did not proliferate to Mycobacterium afnicanum. BCG- and M. tuberculosis H37Rv-specific as well as cross-reactive T-cell clones could be induced to produce interleukin 2, gamma interferon, and granulocyte-macrophage colony-stimulating factor activity. Mycobacterium bovis BCG vaccination against tuberculosis is practiced in many countries. It was shown to give very good protection in certain parts of the world, but was poor in other regions (4, 11, 17, 22). Many explanations have been offered for the apparently conflicting results (Editorial, Lancet i:309-310, 1981), i.e., (i) differences in the strains of BCG used in terms of antigens recognized by T cells, (ii) differences among substrains of Mycobacterium tuberculosis prevalent in different regions so that immunization with BCG would protect against some but not other substrains, (iii) the presence of environmental mycobacteria positively or negatively modulating the immune response to BCG (20), and (iv) the induction of suppressor cells (13, 14). We have addressed the two first questions by generating T-cell clones from BCG-immunized healthy subjects. The clones were also tested for antigen specificity and their ability to secrete biologically important lymphokines like interleukin 2 (IL-2), gamma interferon (IFN--y), and granulocyte-macrophage colony-stimulating factor (CSF). MATERIALS AND METHODS Isolation of PBMC and T cells. Peripheral blood mononuclear cells (PBMC) were obtained from the blood of nine BCG-vaccinated healthy blood donors by flotation on sodium metrizoate-ficoll gradient (lymphoprep; Nyegaard & Co., Oslo, Norway). Adherent cells were removed by adherence to plastic, and the T cells from the nonadherent pool were obtained by E rosetting (15). T-cell cloning and propagation of clones. PBMC or T cells with 10% adherent cells (2 x 106/ml) in complete medium (RPMI 1640, 15% AB serum, 1% penicillin-streptomycin) were stimulated with the Danish strain of BCG (20,ug/ml) in a total volume of 10 ml in 25-ml plastic flasks (Costar, Cambridge, Mass.) and incubated for 6 days at 37 C in a humidified atmosphere of 5% CO2 and 95% air. On day 6, the * Corresponding author. 491 cells were washed and stimulated with 106 autologous irradiated (2,500 R) PBMC per ml as feeders plus BCG (20,ug/ml) for 3 more days. Afterward, viable cells were recovered on lymphoprep gradients. These were.60% blasts and were cloned by limiting dilution in 60-well Falcon microtest trays (Becton Dickinson Labware, Oxnard, Calif.) by standard procedures (16). In brief, 0.3 cells were added to each well with 104 irradiated autologous PBMC as feeders plus BCG (20,ug/ml) plus 20% lymphocult T (Biotest, Frankfurt, Federal Republic of Germany) as a source of IL-2 or 100 U of recombinant IL-2 (gift from Cetus Corp., Emeryville, Calif.) per ml. After 7 to 10 days of incubation at 37 C in a humidified atmosphere of 5% CO2 and 95% air, the cells from growing wells were transferred to 96-well flat-bottom trays (Costar) and added to 105 irradiated feeders plus BCG (20,ug/ml) plus IL-2. The plates were incubated further for 7 to 10 days; the growing clones were then transferred to 24-well plates (Costar) and added to 106 Ir feeders plus BCG (20,ug/ml) plus IL-2 and to IL-2 alone 4 days later. This cycle for propagation of the clones was repeated every seventh day. Cultures were split when the cell number reached.1 x 106 cells per well and reseeded at 0.2 x 106 cells per well. Mycobacterial antigens. Soluble antigens from a panel of fast- and slow-growing mycobacteria were kindly provided by J. L. Stanford and G. Rook (London). These were prepared by ultrasonication of whole bacilli and sterilized by membrane filtration (19). Mycobacterium kansasii, M. scrofulaceum, M. xeonpi, M. vaccae, M. nonchromogenicum, M. flavescens, M. duvalii, M. neoaurum, M. avium A, M. avium C, M. tuberculosis, and BCG were used to make soluble preparations. Armadillo-derived, killed whole Mycobaterium leprae and sonicated preparations were obtained through the IMMLEP component of the World Health Organization-United Nations Development Program-World Bank Special Programme for Research and Training in Tropical Diseases by R. J. W. Rees (London). Irradiated suspensions of M.
492 MUSTAFA ET AL. TABLE 1. Characteristics of T-cell clones raised from nine BCG-vaccinated healthy individuals" Subject No. of Responders Nonresponders Autoclones raised to BCG to BCG reactive 115/83 2 1 1 0 130/83 11 5 5 1 141/83 15 9 6 0 184/83 1 0 1 0 428/83 9 8 1 0 BC-7 7 3 3 1 BC-10 21 6 15 0 BC-24 21 16 4 1 24/85 34 26 8 0 a BCG reactivity of the clones was determined as described in Materials and Methods. A clone with Acpm 2 1,000 and TIC > 2 was considered a responder (positive response) to BCG. See the text for formulas. tuberculosis H37Rv, Mycobacterium phlei, M. avium (serotype-2, ATCC 15789), four strains of freeze-dried BCG vaccines (French strain 1173P2, Danish strain 1331, Prague strain 725, Brazilian strain Moreau), and purified protein derivative were obtained from J. Bennedsen and K. Bunch- Christensen (Copenhagen). Bacillary suspensions of Mycobacterium gordonae, M. flavescens, M. szulgai, M. triviale, M. terrae, M. gastri, M. abscessius, M. xenopi, M. africanum, M. asiaticum, M. simiae, M. smegmatis, and M. fortluitum were kindly supplied by Otto Closs (Oslo). Doseresponse experiments were done with both soluble preparations and whole bacilli. Soluble antigens were used at three different concentrations: 0.4, 2, and 10,ug/ml. None of the five clones tested responded to vaccine in this dose range; however, the clones and their respective PBMC showed an optimal response to tuberculin at 2 to 10 p.g/ml. Therefore, these doses of soluble antigens from mycobacterial sonicates were used in the experiments. Bacillary preparations of cultivable mycobacteria have been used at 20,ug of wet weight per ml, and M. leprae has been used at 5 x 107 bacilli per ml. Surface phenotype analysis. OKT4 and OKT8 monoclonal antibodies (Ortho Pharmaceutical Corp., Raritan, N.J.) were used to identify T-cell subsets of clones. Monoclonal antibodies D11-2 and D4-22 (provided by R. S. Accolla, Lausanne, Switzerland) recognize two different subsets of human leukocyte antigen (HLA) DR designated as NG1 and NG2 coexpressed at the single-cell level (1). MHM4 (a kind gift from A. McMichael, Oxford, United Kingdom) is related to the DP locus (10), and BT 3/4 monoclonal antibody (kindly provided by R. Corte, Milano, Italy) recognized the DQ locus (5), of class II major histocompatibility complex (MHC) molecules. These antibodies were used at saturating concentrations to determine the expression of different class II molecules on the clones. The cells were stained with fluorescein-conjugated sheep anti-mouse immunoglobulin in the second layer as described by Kval0y et al. for other cell types (9). The percent positive cells was scored with a fluorescence microscope. In vitro proliferation. Samples of 105 irradiated PBMC in complete medium were added to each well of 96-well flatbottom microtiter plates and incubated for 1 h in an atmosphere of 5% CO2 and 95% air. Nonadherent cells were washed, and adherent cells were used as antigen-presenting cells (APC). Samples of 104 T-cell clones were added to the wells and stimulated with optimal concentrations of antigens in triplicate. The total culture volume was kept at 200,ul. The plates were incubated for 72 h at 37 C in a humidified INFECT. IMMUN. atmosphere of 5% CO2 and 95% air. At 4 h before harvesting, cultures were pulsed with 0.045 MBq of [3H]thymidine (specific activity, 185 x 103 MBq/mmol). The cultures were harvested, and the radioactivity incorporated was determined by standard procedures (13). The median values of counts per minute (cpm) have been used to express results. A clone to a given antigen was considered a responder when Acpm was.1,000 and T/C was >2, where Acpm = [(cpm in culture with clone + APC + antigen) - (cpm in cultures with clone + APC)], and TIC = [(cpm in cultures with clone + APC + antigen)/(cpm in cultures with clone + APC)]. Lymphokine production. Samples of 2 x 105 T-cell clones in 1 ml of complete medium were added to the wells of 24-well Costar plates with adherent cells from 106 irradiated autologous PBMC plus 20 plg of BCG. Plates were incubated at 37 C in a humidified atmosphere of 5% CO2 and 95% air. Cell-free supernatants were collected after different time intervals and stored at -20 C until assayed for lymphokine activities. IL-2 activity assay. IL-2 activity in the supernatants was determined by their ability to induce the proliferation among IL-2-dependent T-cell clones (14). In brief, 104 IL-2- dependent T-cell clones in 100 p.1 of complete medium plus 100 p.l of supernatants were added to the wells of 96-well flat-bottom trays. Plates were incubated at 37 C in an atmosphere of 5% CO2 and 95% air. On day 2, 0.045 MBq of [3H]thymidine was added to the cultures during the last 4 h of incubation. The cultures were harvested, and incorporated radioactivity was determined by liquid scintillation spectroscopy. Granulocyte-MCSF assay. Mononuclear cells were separated on a lymphoprep gradient from bone marrow cells of subjects undergoing marrow aspiration. The cells were suspended in medium containing 0.3% agar, 15% fetal calf serum, 10% supernatants, and McCoy medium to a final concentration of 2 x 105 cells per ml. Samples of 1 ml were cultured in triplicate in 35-mm plastic dishes at 37 C in an atmosphere of 5% CO2 and 95% air. After 14 days of incubation, colonies of more than 40 cells were counted (3). The lineage of cells constituting the colonies was identified by light microscopy after staining with May Grunwald Giemsa and peroxidase. IFN-y assay. IFN--y activity in the supernatants was determined by the method of Dahl and Degre (6), employing TABLE 2. Restoration of BCG-induced proliferation among nonresponder clones by exogenous recombinant IL-2 and enhancement of the proliferation among responder clonesa cpm (103) in clones with the following: Clones APC, IL-2 APC, APC, BCG, IL-2 APC (5 U/ml) BCG (5 U/ml) BCG nonresponders (1) 184/83 0.1 0.3 0.3 3.6 (4) 141/83 0.3 0.9 0.4 1.6 (7) 141/83 0.2 1.0 0.4 2.7 BCG responders (1) 115/83 0.2 0.5 12.3 20.5 (1) 130/83 0.2 1.5 5.9 14.4 (9) 130/83 0.1 0.8 3.0 11.9 " The clones were cultured in IL-2 alone for 10 to 14 days before the effect of recombinant IL-2 on the BCG-induced proliferation of T-cell clones was determined. This had probably resulted in loss of IL-2 receptors and diminished proliferation to IL-2 alone. cpm values showing positive response are indicated in boldface type.
VOL. 53, 1986 BCG-INDUCED HUMAN T-CELL CLONES 493 TABLE 3. Proliferation of antigen-specific T-cell clones to mycobacterial antigensa Clone None BCG M. tuberculosis M. avium M. gordonae M. phlei M. vaccae M. leprae (1) 130/83 0.1 26.3 28.5 0.1 0.4 0.3 0.1 0.6 (4) 130/83 0.2 1.4 0.7 0.1 NT NT 0.1 0.1 (9) 130/83 0.1 3.0 3.9 0.2 0.2 0.1 0.1 0.1 (5) 141/83 0.7 3.6 1.7 0.7 0.5 0.1 0.1 0.1 (26) 141/83 0.6 2.1 7.3 0.9 0.2 0.2 1.3 0.2 (13) BC-7 0.7 44.0 49.5 0.8 0.1 0.2 0.1 0.2 (1) BC-24 0.4 14.3 10.5 0.2 0.4 0.3 0.2 0.4 (3) BC-24 0.1 52.5 40.2 0.1 0.2 0.5 0.1 0.1 (7) BC-24 0.3 27.6 28.0 0.1 0.2 0.6 0.1 0.1 (10) BC-24 0.2 40.6 24.4 0.1 0.1 0.2 0.1 0.4 (13) BC-24 0.3 28.6 35.8 0.2 0.2 0.1 0.1 0.2 (14) BC-24 0.2 11.2 4.7 0.2 0.2 0.1 0.1 0.1 (15) BC-24 0.6 49.4 31.7 0.2 0.2 0.3 0.2 0.5 (17) BC-24 0.1 9.4 8.6 0.1 0.6 0.2 0.2 0.4 (36) 84/85 0.4 22.0 5.1 0.6 0.5 0.4 0.5 1.0 a The numbers of responders/total were as follows: 15/15 for BCG, 14/15 for M. tuberculosis H37Rv, and 0/15 for all other mycobacteria tested. NT, Not tested. human embryonic lung fibroblast cells and vesicular stomatitis virus as the challenge virus. The antiviral activity was calculated as the reciprocal of the highest dilution of the samples preventing development of cytopathogenic effect in 50% of the cells at the time when control cells were completely destroyed. One milliliter of the international standard preparation G-023-901-527 was equivalent to 1 to 1.3 U in our assay system. Treatment at ph 2 abolished the antiviral activity of all supernatants. RESULTS Antigen reactivity and surface phenotype of T-cell clones. A total of 121 T-cell clones were raised from nine BCGvaccinated healthy subjects and were propagated to the extent that their antigen responsiveness was determined. Clone TABLE 4. Three of them were autoreactive; they proliferated to autologous adherent cells alone. The proliferation of 74 clones required BCG as antigen and autologous adherent cells as APC. These were termed responder clones (Table 1). The stimulation of the remaining 44 clones with BCG plus APC was not sufficient for their proliferation. These clones were designated as nonresponders (Table 1). However, the addition of exogenous recombinant IL-2 triggered such clones to proliferate in an antigen-dependent fashion (Table 2). Exogenous recombinant IL-2 could also enhance the BCGdependent proliferation of responder clones (Table 2). The clones from BCG responder as well as nonresponder groups proliferated to mitogens like phytohemagglutinin, concanavalin A, and monoclonal antibody UCHT1, but, failed to proliferate in response to nonmycobacterial antigens, i.e., tetanus toxoid, diphtheria toxoid, and streptokinase- Proliferation of limited cross-reactive T-cell clones to mycobacterial antigensa M. tuberculosis None BCG H37Rv M. avium M. gordonae M. phlei M. vaccae M. leprae (1) 115/83 0.1 18.9 13.4 19.0 0.1 0.2 0.2 0.2 (2) 130/85 0.2 43.5 32.1 0.1 4.2 0.3 0.2 0.5 (8) 130/83 0.3 2.6 3.4 0.2 0.4 1.5 0.4 0.2 (1) 141/83 0.3 17.8 14.0 2.8 1.0 3.4 0.1 0.1 (3) 141/83 0.2 4.2 3.2 1.6 NT 3.5 NT 1.0 (6) 141/83 0.2 1.6 1.6 0.9 NT 3.1 NT 0.1 (1) BC-7 0.2 8.3 0.3 20.5 24.9 0.2 0.1 0.1 (5) BC-10 0.1 4.3 8.4 2.6 1.6 0.1 0.7 0.1 (12) BC-10 0.3 4.4 7.4 2.3 0.3 3.1 0.1 0.2 (2) BC-24 0.1 39.7 36.7 0.2 5.0 0.7 0.1 0.2 (4) BC-24 0.3 20.5 25.9 8.2 18.0 0.1 0.1 0.2 (6) BC-24 0.1 12.4 10.3 3.1 0.2 0.2 0.1 0.1 (8) BC-24 0.1 37.9 35.4 0.1 23.2 0.2 0.1 0.1 (11) BC-24 0.7 5.5 8.3 0.5 3.1 0.1 0.2 0.2 (12) BC-24 0.2 5.9 3.5 1.2 3.8 0.1 0.2 0.6 (16) BC-24 0.3 21.4 15.6 7.1 3.8 0.3 0.4 0.2 (7) 84/85 1.1 15.7 9.2 7.6 0.2 0.1 0.4 0.3 (9) 84/85 5.5 12.6 12.5 14.0 6.1 0.2 7.5 0.4 (12) 84/85 0.1 19.5 21.0 15.9 0.4 0.5 0.2 0.3 (13) 84/85 12.1 30.4 30.0 7.0 6.7 0.9 0.2 26.8 (15) 84/85 0.6 4.4 2.6 1.7 1.7 0.7 0.4 0.7 a The numbers of responders/total were as follows: 21/21 for BCG, 20/21 for M. tuberculosis H37Rv, 14/21 for M. avium, 10/19 for M. gordonae, 5/21 for M. phlei, 0/19 for M. vaccae, and 1/21 for M. leprae. NT, not tested.
494 MUSTAFA ET AL. INFECT. IMMUN. TABLE 5. Proliferation of broadly cross-reactive T-cell clones to mycobacterial antigensa Clone M. tuberculosis M. avium M. gordonae M. phl M. vaccae M. leprae None BCG H37RvM.aim Mgodne Mphe M.vce M.lre (2) 141/83 0.2 4.3 2.9 2.0 0.1 7.8 4.8 0.8 (9) BC-7 0.2 5.5 9.8 2.7 2.0 0.9 1.3 0.8 (1) BC-10 6.4 50.0 52.9 16.6 46.3 35.0 13.3 0.9 (2) BC-10 4.4 46.5 35.4 36.2 47.2 27.0 28.7 19.2 (3) BC-10 0.4 12.1 10.6 6.6 3.2 0.2 1.9 0.2 (9) BC-24 0.3 27.7 28.4 19.0 20.1 20.4 13.2 19.7 (8) 84/85 0.2 12.3 15.1 18.2 11.3 1.4 2.6 0.8 (30) 84/85 5.9 28.5 29.8 28.2 32.5 3.9 6.1 12.8 a The numbers of responders/total were as follows: 8/8 for BCG, M. tuberculosis H37Rv, and M. avium; 7/8 for M. gordonae; 5/8 for M. phlei; 7/8 for M. vaccae; and 3/8 for M. leparae. streptodornase, although their respective PBMC responded to these antigens (data not shown). Phenotypically, 120 clones were CD4+ CD8- and only 1 clone was CD8+ CD4-; of the 120 CD4+ CD8- clones, 74 were BCG responders, 43 were nonresponders, and 3 were autoreactive. All of the 11 clones tested for the expression of class II MHC molecules reacted with the monoclonal antibodies with specificity for DR, DQ, and DP-related loci. Antigen specificity of T-cell clones. To determine the antigen specificity of T-cell clones, 44 BCG responder clones were tested with M. tuberculosis H37Rv, M. avium, M. gordonae, M. vaccae, M. phlei, and M. leprae. Depending upon their reactivity, the clones could be classified into three groups. (i) BCG- and M. tuberculosis H37Rv-specific clones. Fifteen clones responded to BCG and M. tuberculosis H37Rv, but not to other mycobacteria (Table 3). In this group, one of the clones, (4) 130/83, a weak responder to BCG, did not show significant proliferation to M. tuberculosis H37Rv. TABLE 6. (ii) Limited cross-reactive clones. Twenty-one clones, in addition to responding BCG and M. tuberculosis H37Rv, responded to -2 preparations of the mycobacteria tested (Table 4). One clone of this group, (1) BC-7, responding to BCG, M. avium, and M. gordonae, failed to proliferate to M. tuberculosis H37Rv. M. avium and M. gordonae stimulated 14 and 10 clones, respectively. Six clones proliferated to both of these preparations. Five clones proliferated to M. phlei, one proliferated to M. leprae, and none proliferated to M. vaccae (Table 4). (iii) Broadly cross-reactive clones. Eight clones responded to.3 preparations of the non BCG-M. tuberculosis complex mycobacteria. M. avium and M. gordonae stimulated eight and seven clones. M. phlei, M. vaccae, and M. leprae induced proliferation among five, seven, and three clones of this group, respectively. Two clones, (2) BC-10 and (9) BC-24, responded to all preparations (Table 5). To establish the specificity and cross-reactivity of the clones on a broader base, seven specific, seven limited, and Proliferation of BCG-responder T-cell clones to 12 additional strains of mycobacteriaa Clone M. M. M. M. M. M. M. M. M. smeg- M. for- M. None BCG africanum asiaticum szulgai triviale terrae gastri abscessus xenopi matis tuitum simiae flavescens BCG and M. tuberculosis specific (1) 130/83 0.9 7.4 8.2 0.2 0.2 0.1 0.1 0.1 0.3 0.3 0.2 0.3 0.5 0.5 (9) 130/83 0.4 2.3 1.2 0.1 0.1 0.1 0.1 0.4 0.1 0.1 0.1 0.1 0.1 0.1 (5) 141/83 0.2 3.7 0.7 0.5 0.2 0.1 0.1 0.4 0.3 0.2 0.1 0.1 0.5 0.1 (26) 141/83 0.3 2.1 5.2 0.1 0.1 0.1 0.1 0.4 0.3 0.2 0.1 0.1 0.4 0.3 (13) BC-7 0.7 44.0 30.4 0.5 0.2 0.1 0.1 0.5 0.1 0.1 0.1 0.2 0.1 0.1 (10) BC-24 0.3 69.6 23.3 0.1 0.1 0.3 0.2 0.1 0.1 0.2 0.4 0.6 0.1 0.2 (13) BC-24 0.3 28.7 0.5 0.2 0.2 0.3 0.4 0.8 0.2 0.7 0.7 0.5 0.2 0.4 Limited cross-reactive (1) 115/83 0.1 18.9 6.7 0.1 1.1 6.9 0.3 0.4 2.6 0.5 0.7 0.4 2.0 0.1 (2) 130/83 0.2 7.0 2.9 1.2 4.5 0.1 1.9 5.2 0.1 0.1 0.2 0.2 0.2 0.1 (8) 130/83 0.3 2.6 0.6 0.1 0.1 0.1 0.2 0.3 0.2 0.2 1.3 0.6 0.2 0.4 (1) 141/83 0.6 19.4 3.4 0.2 0.1 6.4 0.3 0.4 2.6 0.5 0.7 5.5 1.9 (2) 141/83 0.2 4.3 1.0 0.2 1.0 0.1 0.5 1.0 0.1 0.2 0.7 3.7 0.4 0.9 (1) BC-7 0.2 8.3 0.2 21.9 0.3 0.1 0.5 20.5 0.1 2.4 0.4 0.1 6.5 0.1 (12) BC-10 0.3 4.4 0.5 0.4 0.5 1.1 0.2 0.6 0.9 0.2 0.2 0.7 0.9 0.2 Broad cross-reactive (1) BC-10 6.4 50.0 28.9 40.7 73.1 10.2 45.3 75.1 9.8 9.4 25.8 53.5 11.9 2.3 (2) BC-10 4.4 46.5 47.3 50.4 52.2 0.5 38.3 44.2 1.1 33.9 37.8 40.0 32.6 10.7 (3) BC-10 0.4 12.1 14.2 1.5 7.4 1.2 1.9 6.4 1.4 1.3 5.6 9.8 2.4 0.4 (5) BC-10 0.1 4.3 2.9 0.6 6.5 0.9 2.0 3.6 1.6 1.4 3.3 4.9 1.6 0.4 (9) BC-7 0.2 5.5 2.2 1.7 1.4 0.5 1.2 1.8 0.8 0.1 2.3 1.2 0.9 0.6 a cpm values of clones that are responders to the antigens are indicated in boldface type. M.
VOL. 53, 1986 BCG-INDUCED HUMAN T-CELL CLONES 495 Clone TABLE 7. Proliferation of T-cell clones to different BCG strains None French Danish Prague Moreau BCG and M. tuberculosis specific (1) 130/83 1.3 9.3 11.3 12.9 11.4 (5) 141/83 0.7 2.9 4.5 2.8 4.7 (13) BC-7 0.7 31.0 40.3 34.0 33.4 (10) BC-24 0.3 64.5 70.4 63.5 60.4 (13) BC-24 0.3 54.3 28.6 23.4 19.9 (14) BC-24 2.2 21.6 11.6 14.1 12.9 Limited cross-reactive (1) 115/83 0.1 15.1 9.2 8.0 10.7 (2) 130/83 0.5 15.1 14.5 17.5 14.5 (1) 141/83 5.2 19.2 24.3 24.5 14.0 (2) 141/83 3.5 14.1 12.2 11.2 15.1 (3) 141/83 0.2 4.1 4.2 3.0 3.0 (6) 141/83 0.2 3.0 1.6 1.5 2.7 (1) BC-7 0.2 4.5 3.6 5.9 6.0 (12) BC-10 0.3 1.6 2.0 3.0 1.2 Broad cross-reactive (9) BC-7 0.5 3.0 4.6 4.5 4.0 (1) BC-10 6.4 56.7 40.7 49.8 42.8 (2) BC-10 4.4 16.3 8.5 21.1 11.9 (3) BC-10 0.4 14.0 18.3 9.0 7.1 (5) BC-10 0.1 8.3 4.9 5.8 5.0 Unknown specificity (11) 141/83 0.8 2.9 4.0 4.0 3.6 (17) 141/83 0.7 1.8 2.4 2.9 2.8 (23) 141/83 1.8 14.0 12.6 13.9 11.8 five broadly cross-reactive clones were further tested with 12 additional preparations of whole mycobacteria, including M. africanum, a member of the BCG-M. tuberculosis complex. Even after further testing, the pattern of reactivity of specific and cross-reactive clones was preserved. However, 6 of 18 BCG- and M. tuberculosis-responding clones did not proliferate to M. africanum (Table 6). Five selected clones of specific and cross-reactive types were tested with soluble preparations from 13 species of mycobacteria. The specific clones responded to BCGin and tuberculin only, whereas the cross-reactive clones responded to other soluble preparations as well (data not shown). Response of T-cell clones to different BCG strains. The results presented above suggest that the T-cell clones were recognizing epitopes of varied specificity. There were also clones that responded to BCG but not to other members of tuberculosis complex, especially M. africanum. To determine whether these clones would differentiate among different BCG strains, 22 clones were tested with French, Danish, Prague, and Moreau strains of BCG. However, irrespective of their specificity, all clones responded to these BCG strains (Table 7). Lymphokine production. The conditions were standardized for the production of IL-2 and CSF by the clones. The kinetics of their production revealed that detectable quantities were produced within 6 h of activation and peaked by 12 to 24 h. Thereafter, IL-2 levels dropped sharply, whereas CSF levels were maintained. Antigen and adherent APCs were required for the production of IL-2, CSF, and IFN--y. The addition of IL-2 to the clones at the stage when these were fully proliferating to IL-2 did not induce CSF or IFN--y production in the absence of BCG and APC (data not shown). The results from 14 responder clones and 5 nonresponder clones show that IL-2 production correlated well with antigen-induced proliferation. However, CSF and IFN--y activities were detected also in the supernatants of the clones which did not have detectable IL-2 activity and antigen-induced proliferation. Multiple lymphokines were produced by both BCG- and M. tuberculosis-specific clones and cross-reactive clones (Table 8). DISCUSSION A total of 121 T-cell clones from 9 BCG (Danish strain)- vaccinated healthy subjects were raised against BCG and maintained in vitro to the extent that their antigen responsiveness was determined. Cloning efficiency varied from person to person and ranged from 5 to 30%. Some of the BCG-reactive clones have been continuously maintained in vitro for >4 months, but most of them were frozen when sufficient numbers of cells (5 x 106 to 10 x 106) were available at about 5 to 8 weeks of cloning and thawed whenever required. The BCG responsiveness of the clones was determined as soon as sufficient cells from each clone were available. Although all of the clones were growing in the presence of BCG, adherent cells, and exogenous IL-2, in the absence of exogenous IL-2 only 74 clones proliferated to BCG plus APC, whereas 44 clones did not proliferate. This nonresponsiveness was due to their inability to produce IL-2 in response to BCG, and antigen-specific proliferation was restored by exogenous IL-2. These results suggest that vaccination with BCG can lead into activation of both IL-2-producing and IL-2-nonproducing T cells in the pres-
496 MUSTAFA ET AL. INFECT. IMMUN. Clone TABLE 8. BCG-induced proliferation and lymphokine production by the T-cell clones" BCG-induced Lymphokine activities in the supernatants proliferation (Acpm, 103) IL-2 (U/ml) CSF (cfu/ml) IFN--y (IU/mi) BCG and M. tuberculosis specific (1) 130/83 6.5 1 993 18 (4) 130/83 3.9 0.5 460 89 (9) 130/83 1.9 0.4 100 56 (5) 141/83 3.5 0.8 NT 56 (26) 141/83 1.5 0.3 NT 28 (13) BC-7 43.7 >10.0 1150 28 Cross reactive (1) 115/83 17.6 8.0 420 178 (2)130/83 6.8 1.0 20 NT (8) 130/83 1.4 0.1 70 36 (1) 141/83 18.5 4.0 356 89 (3) 141/83 3.5 0.7 NT 71 (6) 141/83 6.2 1.0 NT 45 (1) BC-7 8.1 2.0 1,060 22 (9) BC-7 5.0 1.0 1,050 18 Nonresponders (5) 130/83 0.1 <0. 1 NT 28 (6) 130/83 0.4 <0. 1 220 NT (4) 141/83 0.1 <0. 1 NT 71 (7) 141/83 0.3 <0. 1 NT 71 (16) 141/83 0.3 <0.1 NT 89 a Supematants for IL-2 and CSF activities were harvested after 16 h and those for IFN--y were harvested after 48 h from cultures of the clones with APC plus BCG as described in Materials and Methods. ence of a specific stimulus. The reason why autoreactive clones were'raised in our system is not clear. Could it be due to antigenic cross-reactivity between BCG and some of the host antigens? Evidence for common epitopes between mycobacteria and host tissue antigens at the antibody (21) and T-cell level (7) has recently been published. Alternatively, they may be responding to allotypes present in the serum. The phenotype analysis of the clones showed that all but one of the clones were CD4+. Predominance of CD4+ clones has also been reported with' other antigens like tetanus toxoid (18), influenza virus (8), and Chlamydia trachomatis (16). The clones'were also expressing NG1 and NG2 subsets of HLA-DR, DP-associated antigens, and DQ locus antigens. However, functional relevance of the expression of class II MHC molecules on activated T cells of CD4 type is not known. Under certain situations activated T cells have been shown to present the antigen in the context of class II MHC molecules (2). The results show that vaccination of humans with BCG activates T cells of varied specificity. No major difference were found among four different BCG strains in their ability to stimulate the tested T-cell clones. Thus, the Danish strain does not appear to have major epitopes recognized by T cells that are not present on the other three BCG strains. With minor exceptions, BCG and M. tuberculosis H37Rv also overlapped with regard to antigenic composition. However, a type strain of M. africanum which also belongs to the M. tuberculosis family did not stimulate 33% of the T-cell clones tested. This suggests that this type strain of M. africanum is antigenically not as closely related to BCG as M. tuberculosis H37Rv. As we have tested only single strains of M. tuberculosis and M. africanum, the results reported cannot be generalized. These studies are now being expanded to substantiate the findings by testing a number of M. tuberculosis and M. africanum isolates from Africa. IL-2, CSF, and IFN--y are among the lymphokines which may have direct or indirect effects on cell-mediated immunity functions. These lymphokines were produced by BCGand M. tuberculosis-specific as well as cross-reactive clones. This may suggest that both specific and cross-reactive determinants may play important roles in relation to protective immunity. However, functional inference from T-cell clones cultured in vitro for many generations with regard to in vivo function should be cautiously interpreted. It was of interest to find that clones that did not respond by proliferation and did not secrete detectable IL-2 in the supernatants nevertheless were producing IFN--y and CSF in response to BCG. Thus the proliferation and IL-2 production may not be sufficient criteria for functional assessment of T-cell clones. The T-cell clones from BCG-vaccinated individuals can be used to identify antigens and epitopes of M. tuberculosis produced by DNA technology (23).We have already employed such an approach to identify recombinant M. leprae antigens stimulating human T-cell clones (12). The antigens recognized by BCG- and M. tuberculosis-specific T-cell clones, if active in delayed-type hypersensitivity, might be a source of more specific skin test reagents, and the ones recognized by helper T cells could be the tools to construct new recombinant vaccines. ACKNOWLEDGMENTS We thank J. L. Stanford, G. Rook, R. J. W. Rees, 0. Closs, J. Bennedsen, and K. Bunch-Christensen for mycobacterial antigens. The technical assistance provided by Monica Laukas and Inger Natvig is greatfully acknowledged. We thank Heidi Andersen for expert secretarial assistance. This work was supported by grants from the United Nations Development Program-World Bank-World Health Organization
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