Jun 3, 1986 - Corresponding author. centrifugation for 15 minat 2,000 x g, and membranes were ..... Allen, P. M., D. J. Strydom, and E. R. Unanue. 1984. Processing ... 109:76-86. 5. Hartzman, R. J., F. H. Bach, G. B. Thurman, and K. W. Seil.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1987, p. 641-644 0095-1137/87/040641-04$02.00/0 Copyright C 1987, American Society for Microbiology
Vol. 25, No. 4
Immunospecific T-Lymphocyte Stimulation by Membrane Proteins from Francisella tularensis G. SANDSTROM,l* A. TARNVIK,2 AND H. WOLF-WATZ' National Defence Research Institute (FOA 4), Umea, S-901 82 Umeà,l and Department of Clinical Bacteriology, University of Umea, S-901 85 Umea,2 Sweden Received 3 June 1986/Accepted 24 December 1986
Membranes from a capsule-deficient mutant of the live-vaccine strain of Francisella tularensis (F. tularensis LVS) were treated with N-lauroyl sarcosinate (Sarkosyl; CIBA-GEIGY Corp., Summit, N.J.). When the Sarkosyl-insoluble fraction was heated in the presence of sodium dodecyl sulfate and mercaptoethanol and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several polypeptides were distinguished. Four major polypeptides were eluted from the gel, each of which stimulated lymphocytes from tularemia-vaccinated individuals but not from nonvaccinated individuals. The stimulation occurred mainly in T lymphocytes. Radioactive labeling of surface proteins of the capsule-deficient bacteria indicated that at least two of the four polypeptides originated from outer membrane proteins. The results suggest that several membrane proteins of F. tularensis LVS induce a specific T-lymphocyte response.
centrifugation for 15 min at 2,000 x g, and membranes were pelleted by centrifugation for 1 h at 100,000 x g. The membranes were suspended in 5 ml of a solution of 0.5% N-lauroylsarcosinate (Sarkosyl; CIBA-GEIGY Corp., Summit, N.J.) in distilled water, incubated for 16 h (in some cases for 30 min only) at room temperature, and pelleted by centrifugation for 1 h at 100,000 x g. Analysis of membrane proteins by SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed essentially as described by Laemmli (8). Gradient gels contained 10 to 17.5% acrylamide and 0.20 to 0.46% bisacrylamide. Membranes were suspended in sample buffer (62.5 mM Tris hydrochloride [pH 6.8], 1% SDS, 0.5% ,B-mercaptoethanol, 10% glycerol), at a protein concentration of 0.5 to 2.5 mg/ml as estimated by the technique of Lowry et al. (9). In some experiments, ,Bmercaptoethanol was excluded. Samples (30 ,ul) were heated at 95°C for 5 min and applied to SDS-PAGE. After overnight electrophoresis, proteins were fixed by immersing the gel for 10 min in a mixture of 45% methanol and 9% acetic acid. The proteins were stained for 30 min in 0.2% Coomassie brilliant blue in 7% methanol-5% acetic acid and destained in 7% methanol-5% acetic acid with several changes. All steps were carried out at 37°C. The following protein standards were used to estimate molecular weight: phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and ot-lactalbumin (14,400) (Pharmacia, Uppsala, Sweden). Preparation of membrane polypeptides. Bacterial membranes were suspended in sample buffer at a protein concentration of 1.0 mg/ml. Samples (100 pul) were heated at 95°C for 5 min and applied to SDS-PAGE. After overnight electrophoresis, the polypeptides were visualized by treating the gel with 0.25 M KCI as described by Hager and Burgess (4). After 5 min of treatment, the polypeptides were clearly visible. Polypeptide bands were excised from the gel and eluted overnight in a solution of 0.05 M Tris hydrochloride, pH 7.9, containing 0.1% SDS, 0.1 mM EDTA, 5 mM dithiothreitol, and 0.2 M NaCI. The polypeptides were precipitated from the solution with 5 volumes of acetone by incubation for 1 h at -20°C. After centrifugation for 10 min
Tularemia or vaccination with the live-vaccine strain of Francisella tularensis (F. tularensis LVS) results in specific antimicrobial resistance. Cell-mediated immunity is a prerequisite to this resistance, whereas humoral immunity seems to be of minor importance (7). Cell-mediated immunity toward microbial antigens can be demonstrated by the lymphocyte stimulation test. This is valid for F. tularensisinduced stimulation, since most of the cells which respond in the test have the characteristics of T lymphocytes (14, 15). Determinants of F. tularensis responsible for the reactivity with lymphocytes from tularemia-vaccinated individuals reside in protein, whereas the reactivity with immune serum depends mainly on carbohydrate determinants (13). We now present data indicating that various membrane proteins of F. tularensis may induce immunospecific T-lymphocyte stimulation. A capsule-deficient mutant of F. tularensis LVS was used for preparation of the membranes, because the absence of capsular material enabled a higher resolution of separated membrane proteins. MATERIALS AND METHODS Bacteria. F. tularensis LVS was supplied by the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Md. A capsule-deficient mutant of F. tularensis LVS was obtained by acridine orange treatment (manuscript in preparation). Briefly, LVS bacteria were suspended at a density of 1010 célls per ml in phosphate-buffered saline, pH 7.4 (12.6 mM KH2PO4, 54.0 mM Na2HPO4, and 85.5 mM NaCl), and acridine orange was added at a concentration of 125 ,ug/ml. Rough-colony mutants were induced at a frequency of 10-4. Bacteria were cultivated for 24 h on modified Thayer-Martin agar (13). Preparation of bacterial membranes. Colonies of the mutant strain of F. tularensis LVS were harvested from the surface of agar plates and suspended in 10 ml of 1 mM EDTA in 10 mM Tris hydrochloride, pH 8.0, at a density of 1010 cells per ml. The cells were disrupted by four 15-s bursts at full power of an ultrasonic disintegrator (Branson Sonic Power Co., Danbury, Ct.). Cell debris was removed by *
Corresponding author. 641
642
J. CLIN. MICROBIOL.
SANDSTROM ET AL.
at 5,000 x g, precipitated material was dried under vacuum. Before being used in the lymphocyte stimulation test, polypeptides were suspended in and dialyzed against RPMI 1640 with 20 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (RPMI-HEPES; GIBCO Laboratories, Grand Island, N.Y.). Cell surface iodination. F. tularensis LVS and its roughcolony mutant were grown overnight on modified ThayerMartin agar plates and suspended in phosphate-buffered saline at a density of 108 cells per ml. Bacteria from 1 ml of each suspension were washed three times in phosphatebuffered saline and suspended in 50 pl of 0.1 M sodium borate buffer, pH 8.5. 1251 (100 ,uCi; Bolton and Hunter reagent; Amersham Corp., Arlington Heights, Ill.) was added to each bacterial suspension, and iodination was performed according to instruction given by the manufacturer. The bacterial suspensions were then washed five times with 1 ml of phosphate-buffered saline containing 0.01 M potassium iodide. The radioactivities of iodinated bacteria and Sarkosyl-insoluble membranes were measured with a liquid scintillation counter. Radiolabeled polypeptides were revealed by autoradiography of dried polyacrylamide gels (slab gel dryer; LKB Instruments, Inc., Rockville, Md.) with X-ray film (Kodak X-Qmat XAR-5 film; Eastman Kodak Co., Rochester, N.Y.). Blood donors. Blood samples were obtained from eight healthy adults vaccinated 3 to 11 years previously with F. tularensis LVS and from four healthy adults who denied previous tularemia or tularemia vaccination. Lymphocyte stimulation. Lymphocytes were prepared from heparinized blood by centrifugation on Lymphoprep (2), and five repeated cultures were established as previously described (16). Each culture (200 ptl) contained 3 x 105 lymphocytes and membrane polypeptide antigen (1 ,ug/ml) or purified protein derivate (PPD) of Mycobacterium tuberculosis (Statens Seruminstitut, Copenhagen, Denmark; 10 ,ug/ml) in RPMI-HEPES supplemented with 10% pooled normal human serum. The concentration of membrane polypeptide antigen used had been found to induce an optimal response in stimulation of lymphocytes from tularemiavaccinated individuals. After incubation at 37°C for 6 days, the cultures were pulsed with ['4C]thymidine and harvested (5). For control purposes, the molecular-weight standard (Pharmacia) was applied to SDS-PAGE, after which the polypeptides were stained with KCl, excised from the gel, and tested for lymphocyte-stimulating activity. Assay of T-lymphocyte and non-T-lymphocyte stimulation. Lymphocytes were incubated in tubes for 6 days in the presence of antigen and pulsed with ['4C]thymidine for 6 h. They were then mixed with sheep erythrocytes to allow rosette formation, and the mixture was centrifuged on Lymphoprep as previously described (14). The cell suspension of the interphase between serum and Lymphoprep (non-T lymphocytes) was washed once in saline, distributed in microplates, collected onto glass-fiber filters, and assayed for radioactivity. The T lymphocytes that had formed rosettes with sheep erythrocytes were collected at the bottom of the tube and assayed for radioactivity. RESULTS Preparation of membrane polypeptides from the capsuledeficient mutant of F. tularensis LVS. When Sarkosylinsoluble membranes of the capsule-deficient strain were heated in the presence of SDS and ,B-mercaptoethanol and subjected to SDS-PAGE, several polypeptides were distin-
1
2
3 ilib
4
5
6
',
4-I
.*
.0
m-ll
r.
4b
4* ._
FIG. 1. SDS-PAGE profile of Sarkosyl-insoluble membranes and prepared proteins of the capsule-deficient mutant of F. tularensis LVS. Lanes: 1, protein 1; 2, protein Il; 3, protein III; 4, protein IV; 5, molecular-weight markers phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), a-lactalbumin (14,400); 6, Sarkosyl-insoluble membranes. Arrows indicate proteins that were excised from gels.
guished (Fig. 1, lane 6). There were four major polypeptides (designated I to IV) showing relative molecular weights of 61,000, 37,000, 32,000, and 17,500, respectively (Fig. 1). At least two of these (III and IV) seemed to be localized at the bacterial surface, since protein iodination of intact bacteria resulted in radioactivity in the gel at positions corresponding to the polypeptides (Fig. 2). Gel pieces containing each of the four major polypeptides were excised and eluted in buffer solution. When samples of the four eluates .were heated in the presence of SDS and P-mercaptoethanol and applied to SDS-PAGE, the major polypeptides were preserved insofar as they showed the relative molecular weights expected (Fig. 1). All major polypeptides were separated from one another with one exception: the eluate containing polypeptide Il also contained traces of polypeptide III. The eluate of polypeptide IV showed homogeneity in SDSPAGE, whereas the other eluates contained one or a few minor polypeptides besides the major polypeptide. When the original strain of F. tularensis LVS was substituted for the capsule-deficient mutant, the SDS-PAGE showed a lower resolution of polypeptides (data not shown). Omission of P-mercaptoethanol resulted in a poor migration of polypeptides into the gel, irrespective of the bacterial strain used (data not shown). Lymphocyte response to membrane polypeptides. Each of the four polypeptide-containing eluates induced incorporation of ['4C]thymidine into lymphocytes from most of the tularemia-vaccinated individuals but did not induce any
VOL.; 25,.1987
F. TULARENSIS-INDUCED T-LYMPHOCYTE STIMULATION
incorporation into lymphocytes from nonvaccinated individuals (Table 1). To exclude the possibility that any polypeptide separated by SDS-PAGE induced lymphocyte stimulation, polypeptides of the molecular standards were excised from the gel and tested for activity. No stimulation occurred (data not shown). Using lymphocytes from four tularemia-vaccinated individuals, we estimated the proportions of DNA synthesis occurring in T lymphocytes. Of the radioactivity, 74 to 96% was recovered in the T-cell fraction (Table 2). Failure to detect proteins of the Sarkosyl-insoluble membranes at the surface of encapsulated F. tularensis LVS. The possible extension of proteins of the Sarkosyl-insoluble membranes at the surface of encapsulated F. tularensis LVS was studied. After protein iodination of the surface of such bacteria, only 2% of the radioactivity was recovered in the Sarkosyl-insoluble membrane preparation. No radioactively labeled bands were observed by autoradiography of gels after SDS-PAGE of these membranes (data not shown). When bacteria of the capsule-deficient mutant were protein iodinated to a similar total activity, 15% of the radioactivity was recovered in the Sarkosyl-insoluble membrane fraction, and the radioactivity was well disclosed by autoradiography (Fig. 2). Thus, the membrane proteins were not detected with certainty at the surface of encapsulated F. tularensis LVS. DISCUSSION F. tularensis LVS has previously been reported to induce T-lymphocyte stimulation by protease-sensitive antigen bound in a high-molecular-weight complex (13). The present data suggest that membrane proteins can act as stimulatory agents. A capsule-deficient mutant of the vaccine strain was used in the preparatory work, thereby avoiding the lipid-rich macromolecular material (6) of the capsule. Four major
1 2
T~
-
FIG. 2. Autoradiograph of gel from SDS-PAGE on Sarkosylinsoluble membranes of l251-labeled mutant bacteria. Visualization by: lane 1, Coomassie brilliant blue; lane 2, autoradiography. Arrows indicate proteins used in the lymphocyte stimulation test.
643
TABLE 1. Response of lymphocytes from tularemia-vaccinated and nonimmunized individuals to membrane polypeptides of
F. tularensisa Radioactivity of lymphocytes with test antigen'
Subject
Polypeptide fraction II III
no.
I
1 2 3 4 5 6 7 8
0.6 ± 1.0 ± 0.5 ± 0.0 ± 1.7 ± 0.7 ± 0.3 ± 0.8 ±
9 10 il 12
0.0 0.0 0.0 0.0
0.3 0.9 0.5 0.0 0.8 0.5 0.4 0.8
0.8 0.1 2.0 ± 1.3 0.4 0.5 0.4 ± 0.2 1.5 + 1.5 1.5 ± 0.9 0.3 0.4 0.0 ± 0.0 2.7 0.3 1.4 ± 0.8 1.7 0.4 0.3 ± 0.4 0.2 0.2 0.0 ± 0.1 2.7 0.9 0.4 ± 0.6
PPD IV
0.5 ± 1.1 ± 4.2 ± 0.3 ± 2.4 ± 1.8 ± 0.1 ± 2.1 ±
0.1 0.5 5.1 0.2 0.8 2.0 0.1 0.6
1.2 0.5 3.3 0.8 0.3 ±0.2 1.1 0.4 3.7 0.2 2.0 ± 1.3 0.2 ± 0.1 0.1 ± 0.1
0.1 0.2 0.0 ± 0.1 0.0 ± 0.1 4.6 ± 0.1 + 0.1 0.0 ± 0.1 0.1 ± 0.1 2.2 ± ± 0.0 0.0 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 1.0 ± ± 0.0 0.1 ± 0.2 0.0 ± 0.1 0.0 ± 0.1 0.8 ±
± 0.1 ± 0.0
1.2 0.5 0.1 0.3
a Lymphocytes from eight tularemia-vaccinated (subjects 1 to 8) and four nonimmunized (subjects 9 to 12) individuals were pulsed with [14C]thymidine after 6 days of incubation with antigen. b Protein-containing gel pieces were excised and eluted after SDS-PAGE of Sarkosyl-insoluble membranes of capsule-deficient bacteria of F. tularensis LVS and used in doses of 1 ,ug/ml. The roman numerals refer to numbers indicated in Fig. 1. PPD (10 ±tg/ml) was used as a control. Data are expressed as counts per minute (103) (mean ± standard deviation) of five repeated cultures. Background values from cultures without antigen have been subtracted.
membrane polypeptides were identified by SDS-PAGE, and each of them was found to induce stimulation of T lymphocytes exclusively from presensitized individuals. Two of eight vaccinated individuals responded poorly to the polypeptides. This is in accordance with previous studies (15, 17) indicating that there is a wide variation between tularemia-vaccinated individuals in the lymphocyte response to membranes of the vaccine bacteria. At least two of the four major polypeptides seemed to be exposed at the surface of the mutant bacteria. When the Sarkosyl-insoluble membranes of the mutant strain of F. tularensis LVS were subjected to SDS-PAGE in the absence of mercaptoethanol, polypeptides migrated poorly. This indicates that the membrane proteins of F. tularensis are bound in macromolecular complexes by disulfide linkages. Disulfide-linked macromolecules have been recognized in Legionella pneumophila (3), Chlamydia psittaci, and Chlamydia trachomatis (11), but not in Escherichia coli (10), and have been suggested to be typical of facultatively intracellular bacteria (3). Accessory cells such as mononuclear phagocytes are generally required in antigen-induced stimulation of T lymphocytes. They ingest and process antigen and present antigen at the phagocyte surface together with phagoçyte proteins encoded by the major histocompatibility complex (1). This seems true also for F. tularensis-induced stimulation, since the lymphocyte response to F. tularensis is strictly accessory cell dependent (14). According to the present results, processing would involve the intracellular release of membrane proteins from the bacteria. Then, the bacterial capsule may have to become uncovered, since our data indicate that the membrane proteins did not extend through the capsule of the bacteria. Our data indicate that more than one outer membrane protein of F. tularensis LVS induces T lymphocyte stimulation. It is unknown whether these proteins contain identical or different lymphocyte-stimulating epitopes. Studies of My-
644
J. CLIN. MICROBIOL.
SANDSTROM ET AL.
TABLE 2. Proportions of newly synthesized DNA in T-cell and non-T-cell fractions of lymphocytes stimulated with membrane polypeptides from F. tularensisa Subject no.
1
2
Radioactivity'
Test
antigen
Dose (p.g/ml)
T-lymphocyte fraction
Non-T-lymphocyte fraction
I II IlI IV PPD I II III IV PPD I Il III IV
1.0 1.0 1.0 1.0 10.0 1.0 1.0 1.0 1.0 10.0 1.0 1.0 1.0 1.0 10.0 1.0 1.0 1.0 1.0 10.0
2.9 (74) 3.4 (79) 4.7 (90) 1.7 (74) 6.3 (83) 5.0 (89) 2.1 (81) 6.1 (91) 3.3 (78)
1.0 (26) 0.9 (21) 0.5 (10) 0.6 (26) 1.3 (17) 0.6 (11) 0.5 (19) 0.6 (9) 0.9 (22)
NTd
NTd
2.0 (12) 2.1 (17) 1.4 (11) 2.3 (21) 0.6 (4) PFD 4 0.6 (7) I 0.9 (9) II 1.0 (12) III 0.7 (11) IV 0.3 (5) PPD a Lymphocytes from tularemia-vaccinated individuals were pulsed with 3
14.5 (88) 10.1 (83) 11.4 (89) 8.9 (79) 16.4 (96) 7.5 (93) 8.1 (90) 7.2 (88) 5.4 (89) 5.8 (95)
['4C]thymidine after 6 days of incubation with antigen and thereafter fractionated into T and non-T lymphocytes. b Protein-containing gel pieces were excised and eluted after SDS-PAGE on Sarkosyl-insoluble membranes of capsule-deficient mutant bacteria of F. tularensis LVS. The roman numbers refer to protein designations indicated in Fig. 1. PPD was used as a control. ' Incorporation of ["Cithymidine into DNA was calculated as counts per minute (103) after subtraction of values obtained in the absence of antigen. Numbers in parentheses represent percent radioactivity in the two fractions. d NT, Not tested.
cobacterium leprae have indicated that presensitization may lead to the appearance of T-cell clones responding to various epitopes of bacterial proteins (12). A possible heterogeneity in immunogenic epitopes would favor the ability of the host to develop cell-mediated immunity, pot only because the magnitude of the lymphocyte response would increase with a variety of activating epitopes, but also because of differences in the capacity of distinct HLA-DR antigens to serve as restricting elements in the cellular interactions occurring in reactions to distinct epitopes (18). Recent data have indicated that tularemia-vaccinated individuals respond to different structures of the vaccine bacteria with humoral and cell-mediated immunity (13). For preparation of subcellular agents inducing cell-mediated immunity, the lymphocyte stimulation test seems to be the in vitro assay of choice, especially if modified (15) to determine more specifically the activation of T lymphocytes. In focusing on membrane proteins as tools in the inducement of cell-mediated immunity towards F. tularensis, one task will be to explore the possible heterogeneity in epitopes among the different proteins. ACKNOWLEDGMENTS We are grateful to Lena Ohlund and Torsten Johansson for excellent technical assistance.
This work was supported by the National Defence Research Institute. LITERATURE CITED D. P. J. Strydom, and E. R. Unanue. 1984. Processing 1. Allen, M., of lysozyme by macrophages: identification of the determinant recognized by two T cell hybridomas. Proc. Natl. Acad. Sci.
USA 81:2489-2493. 2. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation. Scançd. J. Clin. Lab. Invest. Suppl. 21:27-50. 3. Gabay, J. E., M. Blake, W. D. Niles, and M. A. Horwitz. 1985. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. J. Bacteriol. 162:85-91. 4. Hager, D. A., and R. Burgess. 1980. Elution of proteins from sodium dodecyl sulphate polyacrylamide gels, removal of sodium dodecyl sulphate and renaturation of enzymic activity, results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal. Biochem. 109:76-86. 5. Hartzman, R. J., F. H. Bach, G. B. Thurman, and K. W. Seil. 1972. Precipitation of radioactivity labeled samples: a semiautomatic multiple sample processor. Cell. Immunol. 4:182186. 6. Hood, A. M. 1977. Virulence factors of Francisella tularensis. J. Hyg. 79:47-60. 7. Kostiala, A. D. I., D. D. McGregor, and P. S. Logie. 1975. Tularemia in rat. I. The cellular basis of the host resistance to infection. Immunology 28:855-869. 8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
229:680-685. 9. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 10. Nakae, T., J. Ishii, and M. Tokunaga. 1979. Subunit structure of functional porin oligomers that form permeability channels in the outer membrane of Escherichia coli. J. Biol. Chem. 254:1457-1461. 11. Newhall, W. J., V, and R. B. Jones. 1983. Disulfide-linked oligomers of the major outer membrane protein of chlamydia. J. Bacteriol. 154:998-1001. 12. Ottenhoff, T. M. H., P. R. Klatser, J. Ivanyi, D. G. Elferink, M. Y. L. de Wit, and R. R. P. de Vries. 1986. Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells. Nature (London) 319:66-68. 13. Sandstrom, G., A. Tarnvik, H. Wolf-Watz, and S. Lofgren. 1984. Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions. Infect. Immun. 45:101-106. 14. Tarnvik, A., and S. E. Holm. 1978. Stimulation of subpopulations of human lymphocytes by a vaccine strain of Francisella tularensis. Infect. Immun. 20:698-704. 15. Tarnvik, A., M.-L. Lôfgren, S. Lofgren, G. Sandstrom, and H. Wolf-Watz. 1985. Long-lasting cell-mediated immunity induced by a live Francisella tularensis vaccine. J. Clin. Microbiol. 22:527-530. 16. Tarnvik, A., and S. Lofgren. 1975. Stimulation of human lymphocytes by a vaccine strain of Francisella tularensis. Infect. Immun. 12:951-957. 17. Tarnvik, A., G. Sandstrom, and S. Lofgren. 1979. Time of lymphocyte response after onset of tularemia and after tularemia vaccination. J. Clin. Microbiol. 10:854-860. 18. Van Eden, W., R. R. P. De Vries, J. L. Stanford, and G. A. W. Rook. 1983. HLA-DR3 associated genetic control of response to multiple skin tests with new tuberculins. Clin. Exp. Immunol. 52:287-292.
