Clin. exp. Immunol. (1975) 22, 514-521.
Mitogenicity of Corynebacterium parvum for mouse lymphocytes H. Z 0 LA Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent
(Received 24 March 1975)
SUM MARY
Corynebacterium parvum, a powerful in vivo immunostimulant, is shown to stimulate lymphocyte proliferation in vitro. C. parvum is mitogenic for B lymphocytes and does not stimulate T cells. The mitogenicity is dependent on the presence of macrophages. C. diphtheriae and another strain of C. parvum, both of which are devoid of adjuvant, RES stimulation, and tumour-inhibiting activities in vivo are nevertheless mitogenic. A lipid-free fraction from C. parvum, which retains in vivo immunostimulatory properties, is not mitogenic. Thus the mitogenic property is not correlated with the in vivo properties of C. parvum. INTRODUCTION There is currently a great deal of interest in the use of C. parvum as an adjuvant, particularly in the immunotherapy of cancer (reviewed by Scott, 1974). Killed organisms stimulate macrophages (Halpern et al., 1964), suppress some T-cell functions (Asherson & Allwood, 1971; Scott, 1972; Castro, 1974) and act as an adjuvant in the antibody response (reviewed by Howard, Christie & Scott, 1973). Since C. parvum stimulates the antibody response to T-independent antigens (Howard et al., 1973) it may be regarded as a B-cell adjuvant (without implying that it does not stimulate T cells). Consideration of current theories which seek to interpret the response to T-independent antigens (Greaves et al., 1974; Feldmann, Howard & Desaymard, 1975) indicate that B mitogens might be expected to act as adjuvants for T-independent antigens. Indeed Escherichia coli lipopolysaccharide (LPS) is both a B-cell adjuvant and a B mitogen (Bullock & Anderson, 1973; Skidmore et al., 1975). It seemed relevant therefore to determine whether C. parvum functions as a mitogen in vitro. MATERIALS AND METHODS Mitogens and media. Purified PHA was obtained from Wellcome Reagents Ltd, Beckenham, as were RPMI 1640 medium, Hanks's balanced salt solution (BSS) and foetal calf serum (FCS). Levan was isolated from cultures of Corynebacterium levaniformis and purified by Dr C. Moreno of this Department, essentially as described by Miranda (1972). The standard preparation of C. parvum used in these experiments was a suspension of heat-killed organisms, Wellcome strain CN6134, batch PX 365B, prepared by Dr C. Adlam of these laboratories using the method of Adlam & Scott (1973). The suspension contained 7 mg dry weight/ml. In addition C. parvum Correspondence: Dr H. Zola, Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS.
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C. parvum as a B-cell mitogen
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CN5888 (NCTC 10387) and C. diphtheriae CN2000 (Park Williams No. 8), both of which are inactive in vivo (Adlam & Scott, 1973; Bomford & Olivotto, 1975; Bomford & Christie, 1975) were prepared in the same way. A lipid-free preparation was obtained from C. parvum CN6134 batch CA226 by phenol extraction followed by repeated solvent extraction and treatment with pronase, ribonuclease and trichoracetic acid (Adlam, Reid & Torkington, 1975). This material retained the in vivo activities of the starting material (Adlam et al., in preparation). Batch CA 274 was phenol extracted; the aqueous and phenol phases were separated, dialysed and freeze-dried. Filtrate from a culture of C. parvum CN 6134 after 11 days of growth was dialysed and freeze-dried, as also was unused culture medium. C. parvum acid polysaccharide (Dawes, Tuach & McBride, 1974) was a gift from Dr W. McBride, University of Edinburgh. Cell isolation procedures. Spleens were taken from 6- to 10-week-old normal male CBA-T6 mice and placed in cold Hanks's BSS containing 500 heat-inactivated FCS. The spleens were swollen by injecting the medium and gently disrupted by stroking with the plunger of a 1-ml disposable syringe. The cell suspension was decanted, leaving behind undispersed tissue. Erythrocytes were lysed by centrifuging the cells and resuspending them in Geys haemolytic medium (5 ml per spleen) for 5 min at room temperature. The cells were centrifuged again and washed once in Hanks's BSS. Adherent cells were removed by incubation on plastic Petri dishes for 1 hr at 370C. Cells were washed once in Hanks's BSS and resuspended in HEPESbuffered RPMI containing 0 5% CBA serum. The CBA serum was prepared on the day of the experiment, essentially as described by Peck & Bach (1973). Viable white cell counts were made using a haemocytometer and trypan blue, and cell concentrations adjusted to 5 x 106/ml. Viability was >90%. Cells prepared in this way are referred to as unfractionated cells. For T and B cell-enriched populations fractionation was carried out essentially as described by Julius, Simpson & Herzenberg (1973) and Trizio & Cudcowicz (1974). Nylon wool (0-5 g) (LP-1 Leuko-Pak, Fenwal Laboratories, Morton Grove, Illinois) was pressed into the barrel of a 10-ml polypropylene syringe, so that it occupied 6 ml. 'Brunswick' syringes supplied by Sherwood Medical Industries, Crawley, Sussex, were convenient for this purpose because they could be replaced in their containers, sealed with tape and autoclaved. Sterilized columns were washed with phosphate-buffered saline (50 ml) followed by Hanks's BSS/FCS (50 ml) and incubated, wet, for 1 hr at 370C. The columns were kept vertical in their holders during the incubation. Five millilitres of Hanks's BSS/FCS was then washed through the column and the cells (prepared as described above but suspended in Hanks's BSS/FCS) applied to the column. A column containing 0 5 g dry weight nylon wool was used to fractionate 108 cells, suspended in 1 ml. When larger quantities were needed several columns were used, rather than larger columns. The cells were washed into the column with 2 ml Hanks's BSS/FCS at 370C, the column was replaced in its holder and incubated at 370C for 45 min. The T cell-enriched fraction was eluted by gentle drop-wise addition of BSS/FCS at 370C, without building up a pressure-head. Twenty-five millilitres of BSS/FCS was used to collect the T cells and was followed by 100 ml of BSS/FCS washed through more rapidly and discarded. Finally, the B cell-enriched fraction was obtained by pressing and squeezing the nylon wool and washing through with a total of 15 ml of BSS/FCS. Eluted cells were washed once in RPMI containing 0 5% CBA serum and resuspended in the same medium, counted and adjusted to 5 x 106 viable white cells/ml. Macrophages. Macrophages were counted with the help of fluorescein-labelled C. parvum organisms, which are rapidly ingested. The method has been described by Bell & Shand (1972) and the conjugated bacteria were supplied by Mr F. Shand. Antimacrophage serum was a gift from Dr H. Gauchi of the Chester Beatty Research Institute and was prepared by immunizing rabbits with mouse macrophages and absorbing the serum with non-adherent cells. To deplete macrophages a spleen B-cell suspension prepared as described above was centrifuged and 107 cells resuspended in 2 ml of a 1/25 dilution of the antiserum. Two millilitres of a 1/4 dilution of guinea-pig serum was added as a source of complement and the suspension was incubated for 1 hr at 370C. The cells were centrifuged and washed three times in medium before being used in the mitogenicity assay. No phagocytic cells could be detected in this suspension by the fluorescent C. parvum test, indicating less than 0-2%0 residual phagocytic cells. Micro-culture technique. Cultures were set up in sterile flat-bottomed microplates (Cooke Microtiter, type 29ART, Dynatech Laboratories, Billingshurst, Sussex) fitted with lids. One hundred microlitre aliquots of cell suspension were dispensed using either a piston-type dispenser (Finnpipette, Jencons Ltd, Hemel Hempstead) or, where many replicates were required, a Hamilton syringe operating in a Repeating Dispenser (Hamilton, Reno, Nevada). Ten microlitres of mitogen was added using similar instruments. Cultures were incubated at 370C in a humidified incubator gased with 1000 CO2 in air. After various incubation periods
H. Zola
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10 pl of medium containing 1 pu Ci of [3H] thymidine at a specific activity of 21 Ci/mmol thymidine was added and incubation continued for a further 24 hr. Cultures were then transferred to 2-3 cm diameter paper discs (Whatman No. 3, H. Reeve Angel & Company, London) which were dried and then treated successively in: 10%Y TCA at + 40C for 10 min, twice in 500 TCA at + 40C for 10 min, twice in 1:1 methanol: diethylether mixture for 10 min, and finally once in ether for 5 min. The discs were dried in air and placed in scintillation counting vials with 2 ml of scintillation fluid consisting of 5%o Bio-solv BBS-3 (Beckman Instruments Incorporated, Fullerton, California) in AR grade toluene containing 0-8% butyl PBO (Ciba). Samples were counted in a Beckman liquid scintillation counter set to give counts with standard deviation of 2%. The stimulation index for a particular mitogen was obtained by dividing the mean background-corrected counts per minute (ct/min) for the quadruplicate cultures by the mean background-corrected ct/min for quadruplicate control cultures containing 10 ,ul medium instead of the mitogen suspension.
RESULTS cell populations of different Response Stimulation of unfractionated spleen leucocytes by PHA was strong, but stimulation by levan or C. parvurn was weak (Table 1). The results on fractionated cells in Table 1, which TABLE 1. Stimulation of unfractionated and T and B cells
Stimulation index Mitogen C. parvum Levan PHA
70 ug/ml 100 pg/ml 2 ,g/ml
Unfractionated cells
T cells
B cells
2-69 (n.s.) 1-39 (0 01) 50-1 (0 01)
0-98 (n.s.) 0-82 (n.s.) 45-8 (0 001)
6-70 (0 05) 2-10 (0 01) 0-88 (n.s.)
T cells + macrophages at:
0.50 C. parvum
70 fg/ml
085 (n.s.)
2%Y
4%
1-35 (n.s.)
092 (n.s.)
Quadruplicate cultures were pulsed after 3 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value. n.s. = Not significant.
is representative of several experiments, show that C. parvum is mitogenic for B cells but not for T cells. A test for immunoglobulin-bearing cells using rabbit anti-mouse immunoglobulin followed by fluorescein-conjugated sheep anti-rabbit immunoglobulin showed 4%0 immunoglobulin-bearing cells in the T-cell preparation and 10% non-immunoglobulin cells in the B-cell preparation. Treatment with AKR-anti-C3H serum and guinea-pig complement for 1 hr at 370C did not significantly reduce subsequent mitogenic responses of the B-cell preparation to C. parvum. The adequacy of the cell fractionation is demonstrated by the failure of levan (a B-cell mitogen) to stimulate the T-cell fraction and by the failure of
C. parvum as a B-cell mitogen
517
TABLE 2. Mitogenicity for spleen B lymphocytes of heat-killed C. diphtheriae and C. parvum strains CN 5888 and CN 6134
Mitogen concentration (,ug/ml) Activity in vivo
Mitogen
C. parvum CN 6134 + C. parvum CN 5888 C. diphtheriae CN 2000 Lipopolysaccharide 10 ,ug/ml PHA
700
70
2 8 (0 05) 0-9 (n.s.) 2-0 (0 001)
2-3 2-5 10 1 87 07
(0 02) (0 01) (0 001) (0 001) (n.s.)
7 1-7 (0-01) 1 6 (0 01) 5 5 (0 001)
Quadruplicate cultures were pulsed after 2 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value. n.s. = Not significant.
TABLE 3. Mitogenicity for spleen B lymphocytes of different batches of C. parvum and fractions obtained from the whole organisms
Mitogen
Stimulation index
P
C. parvum CN 6134/PX 365B
Heat-killed (70 ,ug/ml) C. parvum CN 6134/CA 226 Live (700 pg/ml) Live (70 pg/ml) Lipid-free residue prepared from CA 226 700 pg/ml 70 pg/ml C. parvum CN 6134/CA 274 Live (700 pg/ml) Phenol extraction: Solvent phase from CA 274 Aqueous phase Residue phase Culture medium, unused Culture supernatant (C. parvum CN 6134) Acid polysaccharide (McBride) PHA (T-cell control) E. coli lipopolysaccharide (B-cell control)
4-6
0 001
202 23
0001 005
08 1-4
> 0-05 > 0 05
15 8
0 01
1.9 14 0-86 18 07 09 1-2 4-4
0 01 005 > 0 05 > 005 > 0 05 > 0 05 > 0 05 0 05
Quadruplicate cultures were pulsed after 2 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value.
K
H. Zola
518
PHA to stimulate the B-cell fraction. Since the failure of T cells to respond to C. parvum might have resulted from a lack of macrophages rather than an intrinsic inability of T cells to respond to C. parvum, the effect of adding peritoneal macrophages at 0.04%0 to 40'( of the total cell population was tested. T cells were not stimulated by C. parvum even in the presence of macrophages (Table 1).
Kinetics, concentration dependence, and macrophage dependence of the B-cell mitogenicity of C. parvum Cultures of B cells with C. parvum were pulsed 1, 2, 3, 4 and 5 days after initiation, and harvested 18 hr after pulsing. Peak stimulation was obtained with cells pulsed after 2 days in culture. Cultures containing concentrations of heat-killed C. parvurn CN6134 ranging between 700 fig/ml and 7 ig/ml did not show pronounced variation (Table 2), although mitogenicity of live C. parvum was more concentration dependent (Table 3). Depletion of TABLE 4. Effects of macrophage depletion and restoration on B-cell mitogenicity of C. parvur
Cell preparation 1. 2. 3. 4. 5.
B lymphocytes 1 -macrophage (anti-macrophage serum)
2+peritoneal macrophages (0 04%) 2+peritoneal macrophages (0 4°0) 2 + peritoneal macrophages (4%)
Stimulation index 28 09 1-3 1-9 41
(0 05) (n.s.) (n.s.) (n.s.) (0 001)
Quadruplicate cultures were pulsed after 3 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value. n.s. = Not significant.
adherent cells on plastic Petri dishes left approximately 1% phagocytic cells in the suspension, when measured by the uptake of fluorescein-labelled C. parvum. The effect of removal of these residual macrophages with antimacrophage serum and reconstitution with peritoneal macrophages is shown in Table 4. Removal of macrophage eliminates the mitogenicity of C. parvum by B cells, and addition of macrophages restores stimulation. The actual number of macrophages required for optimal stimulation was not constant from experiment to experiment; in another test 2% peritoneal macrophages inhibited stimulation. This variation may be attributable to a variable degree of macrophage activation in our mouse colony.
Mitogenicity of different strains andfractions Three batches of C. parvum CN6 134, one heat-killed and the others live, were tested, and all were mitogenic (Table 3). Cell wall material prepared by phenol extraction followed by lipid extraction and treatment with pronase, ribonuclease, and TCA was inactive (Table 3). The material obtained from both the phenol and the aqueous phases of a phenolic extract had very little mitogenic activity, and the insoluble residue was inactive (Table 3). Acid polysaccharide of C. parvum (Dawes et al., 1974) was not mitogenic. Culture medium, before
C. parvum as a B-cell mitogen
519
and after 10 days growth of C. parvum CN6134 were not mitogenic (Table 3). Heat-killed C. parvum strain CN5888 showed mitogenic activity, and C. diphtheriae was more active (Table 2). DISCUSSION The results show that heat-killed or live C. parvum organisms are mitogenic for B lymphocytes. T cells are not stimulated, and do not have to be present in the culture for B-cell stimulation to occur. Macrophages, however, are needed, while an excessive proportion of macrophages can be suppressive. The macrophage requirement is interesting in view of the suggestion that the adjuvant action of C. parvum is macrophage-mediated (Wiener & Bandieri, 1975; Wiener, 1975). The mitogenic effect of C. parvum is not an artefact arising from contaminating endotoxin in the culture medium since the latter was not mitogenic, either before or after use. The superiority of live organisms to heat-killed bacteria probably reflects damage to the mitogenic substance during heating. Control cultures containing live C. parvum but no lymphocytes did not give significant counts after pulsing with [3H]thymidine. The nature of the mitogenic substance has not so far been determined. Activity was not recovered to a significant extent in the phenolic or aqueous phases or the residue of a phenol extraction, suggesting that the active material is phenol-sensitive. C. parvum is a Grampositive organism and does not make an endotoxin. The soluble polysaccharide described by Dawes et al. (1974) was not mitogenic. A comment on the microtechnique used in the experiments is needed. The micromethod was highly sensitive for PHA stimulation of T cells but relatively insensitive for the B-cell mitogens levan and lipopolysaccharide when compared with values obtained using a macromethod (Desaymard & Ivanyi, 1975). Comparative studies of micro- and macromitogenicity assays by Greaves et al. (1974) also showed lower sensitivity for levan in the microtechnique. In the present studies mouse serum was used in preference to foetal calf serum, several batches of which were tried and found to be mitogenic. It may be that the low values obtained for B-cell mitogens in the present work resulted from the use of mouse serum or from a non-optimal proportion of macrophages which are important in the mitogenicity of levan (Desaymard & Ivanyi, 1975) as well as C. parvum. In spite of the relatively low values the technique was sufficiently sensitive to demonstrate unequivocally the mitogenic effect of C. parvum and its specificity for B lymphocytes. The relevance of the B-cell mitogenicity of C. parvum to its in vivo properties as an adjuvant, reticuloendothelial stimulant, and anti-tumour agent was investigated by examining a number of preparations with different in vivo properties. C. diphtheriae and C. parvum (CN 5888) are both mitogenic, but are inactive as adjuvants for the antibody response to sheep erythrocytes or in stimulating large increases in the spleen weight, an index of reticuloendothelial stimulation (Adlam & Scott, 1973). C. parvum CN5888 failed to enhance carbon clearance, unlike the active strain CN 6134 (Adlam & Scott, 1973). C. dipltheriae and C. parvum CN5888 are also inactive in preventing the formation of lung metastases in a syngeneic tumour model (Bomford & Olivotto, 1975). Thus the mitogenic property of C. parvum is not sufficient to produce adjuvanticity, reticuloendothelial stimulation, or antitumour activity in vivo, but it may be one of several requisite properties. This aspect was examined using a phenol extracted, solvent extracted 'cell wall' material derived from C.
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parvum CN6134 which was not mitogenic in vitro, but which did prevent the formation of lung nodules in vivo, enhanced carbon clearance and was an adjuvant for antibody response to SRBC (Adlam et al., in preparation). Thus the mitogenic properties of C. parvum are irrelevant to its various in vivo effects. I should like to thank Dr Elizabeth Simpson, Clinical Research Centre, Northwick Park, for demonstrating the microculture technique and the nylon wool procedure for purifying T cells, Dr C. Adlam, Department of Bacteriology, Wellcome Research Laboratories, for supplying the strains and fractions of micro-organisms, and Mrs Susan Barnes for skilled assistance. REFERENCES ADLAM, C., REID, D.E. & TORKINGTON, P. (1975) isms. The Immune System-Genes, Receptors, The nature of the active principle of C. parvum. Signals (ed. by E. E. Sercarz and A. R. WilliamProceedings of the International Conference on son), p. 271. Academic Press, New York. C. parvum (ed. by B. Halpern). Springer Verlag, HALPERN, B.N., PREVOT, A.R., Biozzi, G., STIFFEL, Berlin. (In press.) C., MOUTON, D., MORARD, J.C., BOUTHILLIER, Y. ADLAM, C. & SCOTT, M. (1973) Lympho-reticular & DECREUSEFOND, C. (1964) Stimulation of the stimulatory properties of Corynebacterium parvum phagocytic activity of the reticuloendothelial and related bacteria. J. med. Microbiol. 6, 261. system provoked by Corynebacterium parvum. ASHERSON, G.L. & ALLWOOD, G.G. (1971) DepresJ. reticuloendothel. Soc. 1, 77. sion of delayed hypersensitivity by pretreatment HOWARD, J.G., CHRISTIE, G.H. & SCOTT, M.T. with Freund-type adjuvants. I. Description of the (1973) Biological effects of Corynebacterium phenomenon. Clin. exp. Immunol. 9, 249. parvum. IV. Adjuvant and inhibitory activities on BELL, E.B. & SHAND, F.L. (1972) A search for B lymphocytes. Cell. Immunol. 7, 290. lymphocyte-derived macrophages during xeno- HOWARD, J.G., SCOTT, M.T. & CHRISTIE, G.H. geneic graft-versus-host reactions induced by rat (1973) Cellular mechanisms underlying the adjuthoracic duct cells. Immunology, 22, 538. vant activity of Corynebacterium parvum: interBOMFORD, R. & CHRISTIE, G.H. (1975) Mechanisms actions of activated macrophages with T and B of macrophage activation by C. parvum. II. In lymphocytes. Immunopotentiation, Ciba Foundation Symposium 18 (ed. by J. Knight and G. E. H. vivo experiments. Cell. Immunol. 17, 150. BOMFORD, R. & OLIVOTTO, M. (1975) Inhibition of Wolstenholme), p. 101. Elsevier, Amsterdam. lung nodule formation by C. parvum. Proceedings JULIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A rapid method for the isolation of funcof the International Conference on C. parvum (ed. tional thymus-derived murine lymphocytes. Europ. by B. Halpern). Springer Verlag, Berlin. (In press.) J. Immunol. 3, 645. BULLOCK, W.C. & ANDERSON, F. (1973) Mitogens as probes for immunocyte regulation: specific and MIRANDA, J.J. (1972) Studies on immunological paralysis. IX. The immunogenicity and toleronon-specific suppression of B cell mitogenesis. genicity of levan (polyfructose) in mice. ImImmunopotentiation, Ciba Foundation Symposium munology, 23, 839. posium 18 (ed. by J. Knight and G. E. H. PECK, A.B. & BACH, F. (1973) A miniaturized mouse Wolstenholme), p. 173. Elsevier, Amsterdam. mixed leukocyte culture in serum-free and mouse CASTRO, J.E. (1974) The effect of Corynebacterium serum supplemented media. J. immunol. Meth. 3, parvum on the structure and function of the 147. lymphoid system in mice. Europ. J. Cancer, 10, 115. DAWES, J., TUACH, S.J. & MCBRIDE, W.H. (1974) SCOTT, M.T. (1972) Biological effects of the adjuvant Corynebacterium parvum. 1. Inhibition of PHA, Properties of an antigenic polysaccharide from mixed lymphocyte and GVH reactivity. Cell. Corynebacterium parvum. J. Bact., 120, 24. Immunol. 5, 459. DESAYMARD, C. & IVANYI, L. (1975) Comparison of in vitro immunogenicity, tolerogenicity and SKIDMORE, B.J., CHILLER, J.M., MORRISON, D.C. & WEIGLE, W.O. (1975) Immunologic properties of mitogenicity of dinitrophenyl levan conjugates with bacterial lipopolysaccharide (LPS): correlation varying epitope density. Cell. Immunol. (In press.) between the mitogenic, adjuvant, and immunoFELDMANN, M., HOWARD, J.G. & DESAYMARD, C. genic activities. J. Immunol. 114, 770. (1975) Role of antigen structure in the discrimination between tolerance and immunity by B cells. SCOTT, M.T. (1974) Corynebacterium parvum as an immunotherapeutic anticancer agent. Semin. Transplant. Rev. 23, 78. Oncol. 1, 367. GREAVES, M., JANOSSY, G., FELDMANN, M. & DOENHOFF, M. (1974) Polyclonal mitogens and TRIZIO, D. & CUDKOWICZ, G. (1974) Separation of T and B lymphocytes by nylon wool columns: the nature of B lymphocyte activation mechan-
C. parvum as a B-cell mitogen evaluation of efficacy by functional assays in vivo. J. Immunol. 113, 1093. WIENER, E. (1975) The role of macrophages in the amplified in vivo response to sheep red blood cells by spleen cells from Corynebacterium parvum treated mice. Cell. Immunol. (In press.)
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WIENER, E. & BANDIERI, A. (1975) Modifications in the handling in vitro of 125I labelled keyhole limpet hemocyanin by peritoneal macrophages from mice pretreated with the adjuvant Corynebacterium parvum. Immunology. (In press.)
Mitogenicity of Corynebacterium parvum for mouse lymphocytes H. Z 0 LA Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent
(Received 24 March 1975)
SUM MARY
Corynebacterium parvum, a powerful in vivo immunostimulant, is shown to stimulate lymphocyte proliferation in vitro. C. parvum is mitogenic for B lymphocytes and does not stimulate T cells. The mitogenicity is dependent on the presence of macrophages. C. diphtheriae and another strain of C. parvum, both of which are devoid of adjuvant, RES stimulation, and tumour-inhibiting activities in vivo are nevertheless mitogenic. A lipid-free fraction from C. parvum, which retains in vivo immunostimulatory properties, is not mitogenic. Thus the mitogenic property is not correlated with the in vivo properties of C. parvum. INTRODUCTION There is currently a great deal of interest in the use of C. parvum as an adjuvant, particularly in the immunotherapy of cancer (reviewed by Scott, 1974). Killed organisms stimulate macrophages (Halpern et al., 1964), suppress some T-cell functions (Asherson & Allwood, 1971; Scott, 1972; Castro, 1974) and act as an adjuvant in the antibody response (reviewed by Howard, Christie & Scott, 1973). Since C. parvum stimulates the antibody response to T-independent antigens (Howard et al., 1973) it may be regarded as a B-cell adjuvant (without implying that it does not stimulate T cells). Consideration of current theories which seek to interpret the response to T-independent antigens (Greaves et al., 1974; Feldmann, Howard & Desaymard, 1975) indicate that B mitogens might be expected to act as adjuvants for T-independent antigens. Indeed Escherichia coli lipopolysaccharide (LPS) is both a B-cell adjuvant and a B mitogen (Bullock & Anderson, 1973; Skidmore et al., 1975). It seemed relevant therefore to determine whether C. parvum functions as a mitogen in vitro. MATERIALS AND METHODS Mitogens and media. Purified PHA was obtained from Wellcome Reagents Ltd, Beckenham, as were RPMI 1640 medium, Hanks's balanced salt solution (BSS) and foetal calf serum (FCS). Levan was isolated from cultures of Corynebacterium levaniformis and purified by Dr C. Moreno of this Department, essentially as described by Miranda (1972). The standard preparation of C. parvum used in these experiments was a suspension of heat-killed organisms, Wellcome strain CN6134, batch PX 365B, prepared by Dr C. Adlam of these laboratories using the method of Adlam & Scott (1973). The suspension contained 7 mg dry weight/ml. In addition C. parvum Correspondence: Dr H. Zola, Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS.
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C. parvum as a B-cell mitogen
515
CN5888 (NCTC 10387) and C. diphtheriae CN2000 (Park Williams No. 8), both of which are inactive in vivo (Adlam & Scott, 1973; Bomford & Olivotto, 1975; Bomford & Christie, 1975) were prepared in the same way. A lipid-free preparation was obtained from C. parvum CN6134 batch CA226 by phenol extraction followed by repeated solvent extraction and treatment with pronase, ribonuclease and trichoracetic acid (Adlam, Reid & Torkington, 1975). This material retained the in vivo activities of the starting material (Adlam et al., in preparation). Batch CA 274 was phenol extracted; the aqueous and phenol phases were separated, dialysed and freeze-dried. Filtrate from a culture of C. parvum CN 6134 after 11 days of growth was dialysed and freeze-dried, as also was unused culture medium. C. parvum acid polysaccharide (Dawes, Tuach & McBride, 1974) was a gift from Dr W. McBride, University of Edinburgh. Cell isolation procedures. Spleens were taken from 6- to 10-week-old normal male CBA-T6 mice and placed in cold Hanks's BSS containing 500 heat-inactivated FCS. The spleens were swollen by injecting the medium and gently disrupted by stroking with the plunger of a 1-ml disposable syringe. The cell suspension was decanted, leaving behind undispersed tissue. Erythrocytes were lysed by centrifuging the cells and resuspending them in Geys haemolytic medium (5 ml per spleen) for 5 min at room temperature. The cells were centrifuged again and washed once in Hanks's BSS. Adherent cells were removed by incubation on plastic Petri dishes for 1 hr at 370C. Cells were washed once in Hanks's BSS and resuspended in HEPESbuffered RPMI containing 0 5% CBA serum. The CBA serum was prepared on the day of the experiment, essentially as described by Peck & Bach (1973). Viable white cell counts were made using a haemocytometer and trypan blue, and cell concentrations adjusted to 5 x 106/ml. Viability was >90%. Cells prepared in this way are referred to as unfractionated cells. For T and B cell-enriched populations fractionation was carried out essentially as described by Julius, Simpson & Herzenberg (1973) and Trizio & Cudcowicz (1974). Nylon wool (0-5 g) (LP-1 Leuko-Pak, Fenwal Laboratories, Morton Grove, Illinois) was pressed into the barrel of a 10-ml polypropylene syringe, so that it occupied 6 ml. 'Brunswick' syringes supplied by Sherwood Medical Industries, Crawley, Sussex, were convenient for this purpose because they could be replaced in their containers, sealed with tape and autoclaved. Sterilized columns were washed with phosphate-buffered saline (50 ml) followed by Hanks's BSS/FCS (50 ml) and incubated, wet, for 1 hr at 370C. The columns were kept vertical in their holders during the incubation. Five millilitres of Hanks's BSS/FCS was then washed through the column and the cells (prepared as described above but suspended in Hanks's BSS/FCS) applied to the column. A column containing 0 5 g dry weight nylon wool was used to fractionate 108 cells, suspended in 1 ml. When larger quantities were needed several columns were used, rather than larger columns. The cells were washed into the column with 2 ml Hanks's BSS/FCS at 370C, the column was replaced in its holder and incubated at 370C for 45 min. The T cell-enriched fraction was eluted by gentle drop-wise addition of BSS/FCS at 370C, without building up a pressure-head. Twenty-five millilitres of BSS/FCS was used to collect the T cells and was followed by 100 ml of BSS/FCS washed through more rapidly and discarded. Finally, the B cell-enriched fraction was obtained by pressing and squeezing the nylon wool and washing through with a total of 15 ml of BSS/FCS. Eluted cells were washed once in RPMI containing 0 5% CBA serum and resuspended in the same medium, counted and adjusted to 5 x 106 viable white cells/ml. Macrophages. Macrophages were counted with the help of fluorescein-labelled C. parvum organisms, which are rapidly ingested. The method has been described by Bell & Shand (1972) and the conjugated bacteria were supplied by Mr F. Shand. Antimacrophage serum was a gift from Dr H. Gauchi of the Chester Beatty Research Institute and was prepared by immunizing rabbits with mouse macrophages and absorbing the serum with non-adherent cells. To deplete macrophages a spleen B-cell suspension prepared as described above was centrifuged and 107 cells resuspended in 2 ml of a 1/25 dilution of the antiserum. Two millilitres of a 1/4 dilution of guinea-pig serum was added as a source of complement and the suspension was incubated for 1 hr at 370C. The cells were centrifuged and washed three times in medium before being used in the mitogenicity assay. No phagocytic cells could be detected in this suspension by the fluorescent C. parvum test, indicating less than 0-2%0 residual phagocytic cells. Micro-culture technique. Cultures were set up in sterile flat-bottomed microplates (Cooke Microtiter, type 29ART, Dynatech Laboratories, Billingshurst, Sussex) fitted with lids. One hundred microlitre aliquots of cell suspension were dispensed using either a piston-type dispenser (Finnpipette, Jencons Ltd, Hemel Hempstead) or, where many replicates were required, a Hamilton syringe operating in a Repeating Dispenser (Hamilton, Reno, Nevada). Ten microlitres of mitogen was added using similar instruments. Cultures were incubated at 370C in a humidified incubator gased with 1000 CO2 in air. After various incubation periods
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10 pl of medium containing 1 pu Ci of [3H] thymidine at a specific activity of 21 Ci/mmol thymidine was added and incubation continued for a further 24 hr. Cultures were then transferred to 2-3 cm diameter paper discs (Whatman No. 3, H. Reeve Angel & Company, London) which were dried and then treated successively in: 10%Y TCA at + 40C for 10 min, twice in 500 TCA at + 40C for 10 min, twice in 1:1 methanol: diethylether mixture for 10 min, and finally once in ether for 5 min. The discs were dried in air and placed in scintillation counting vials with 2 ml of scintillation fluid consisting of 5%o Bio-solv BBS-3 (Beckman Instruments Incorporated, Fullerton, California) in AR grade toluene containing 0-8% butyl PBO (Ciba). Samples were counted in a Beckman liquid scintillation counter set to give counts with standard deviation of 2%. The stimulation index for a particular mitogen was obtained by dividing the mean background-corrected counts per minute (ct/min) for the quadruplicate cultures by the mean background-corrected ct/min for quadruplicate control cultures containing 10 ,ul medium instead of the mitogen suspension.
RESULTS cell populations of different Response Stimulation of unfractionated spleen leucocytes by PHA was strong, but stimulation by levan or C. parvurn was weak (Table 1). The results on fractionated cells in Table 1, which TABLE 1. Stimulation of unfractionated and T and B cells
Stimulation index Mitogen C. parvum Levan PHA
70 ug/ml 100 pg/ml 2 ,g/ml
Unfractionated cells
T cells
B cells
2-69 (n.s.) 1-39 (0 01) 50-1 (0 01)
0-98 (n.s.) 0-82 (n.s.) 45-8 (0 001)
6-70 (0 05) 2-10 (0 01) 0-88 (n.s.)
T cells + macrophages at:
0.50 C. parvum
70 fg/ml
085 (n.s.)
2%Y
4%
1-35 (n.s.)
092 (n.s.)
Quadruplicate cultures were pulsed after 3 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value. n.s. = Not significant.
is representative of several experiments, show that C. parvum is mitogenic for B cells but not for T cells. A test for immunoglobulin-bearing cells using rabbit anti-mouse immunoglobulin followed by fluorescein-conjugated sheep anti-rabbit immunoglobulin showed 4%0 immunoglobulin-bearing cells in the T-cell preparation and 10% non-immunoglobulin cells in the B-cell preparation. Treatment with AKR-anti-C3H serum and guinea-pig complement for 1 hr at 370C did not significantly reduce subsequent mitogenic responses of the B-cell preparation to C. parvum. The adequacy of the cell fractionation is demonstrated by the failure of levan (a B-cell mitogen) to stimulate the T-cell fraction and by the failure of
C. parvum as a B-cell mitogen
517
TABLE 2. Mitogenicity for spleen B lymphocytes of heat-killed C. diphtheriae and C. parvum strains CN 5888 and CN 6134
Mitogen concentration (,ug/ml) Activity in vivo
Mitogen
C. parvum CN 6134 + C. parvum CN 5888 C. diphtheriae CN 2000 Lipopolysaccharide 10 ,ug/ml PHA
700
70
2 8 (0 05) 0-9 (n.s.) 2-0 (0 001)
2-3 2-5 10 1 87 07
(0 02) (0 01) (0 001) (0 001) (n.s.)
7 1-7 (0-01) 1 6 (0 01) 5 5 (0 001)
Quadruplicate cultures were pulsed after 2 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value. n.s. = Not significant.
TABLE 3. Mitogenicity for spleen B lymphocytes of different batches of C. parvum and fractions obtained from the whole organisms
Mitogen
Stimulation index
P
C. parvum CN 6134/PX 365B
Heat-killed (70 ,ug/ml) C. parvum CN 6134/CA 226 Live (700 pg/ml) Live (70 pg/ml) Lipid-free residue prepared from CA 226 700 pg/ml 70 pg/ml C. parvum CN 6134/CA 274 Live (700 pg/ml) Phenol extraction: Solvent phase from CA 274 Aqueous phase Residue phase Culture medium, unused Culture supernatant (C. parvum CN 6134) Acid polysaccharide (McBride) PHA (T-cell control) E. coli lipopolysaccharide (B-cell control)
4-6
0 001
202 23
0001 005
08 1-4
> 0-05 > 0 05
15 8
0 01
1.9 14 0-86 18 07 09 1-2 4-4
0 01 005 > 0 05 > 005 > 0 05 > 0 05 > 0 05 0 05
Quadruplicate cultures were pulsed after 2 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value.
K
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PHA to stimulate the B-cell fraction. Since the failure of T cells to respond to C. parvum might have resulted from a lack of macrophages rather than an intrinsic inability of T cells to respond to C. parvum, the effect of adding peritoneal macrophages at 0.04%0 to 40'( of the total cell population was tested. T cells were not stimulated by C. parvum even in the presence of macrophages (Table 1).
Kinetics, concentration dependence, and macrophage dependence of the B-cell mitogenicity of C. parvum Cultures of B cells with C. parvum were pulsed 1, 2, 3, 4 and 5 days after initiation, and harvested 18 hr after pulsing. Peak stimulation was obtained with cells pulsed after 2 days in culture. Cultures containing concentrations of heat-killed C. parvurn CN6134 ranging between 700 fig/ml and 7 ig/ml did not show pronounced variation (Table 2), although mitogenicity of live C. parvum was more concentration dependent (Table 3). Depletion of TABLE 4. Effects of macrophage depletion and restoration on B-cell mitogenicity of C. parvur
Cell preparation 1. 2. 3. 4. 5.
B lymphocytes 1 -macrophage (anti-macrophage serum)
2+peritoneal macrophages (0 04%) 2+peritoneal macrophages (0 4°0) 2 + peritoneal macrophages (4%)
Stimulation index 28 09 1-3 1-9 41
(0 05) (n.s.) (n.s.) (n.s.) (0 001)
Quadruplicate cultures were pulsed after 3 days. The significance of the difference between the experimental groups and control unstimulated cultures was determined by the Student's t-test and is expressed as a probability (P) value. n.s. = Not significant.
adherent cells on plastic Petri dishes left approximately 1% phagocytic cells in the suspension, when measured by the uptake of fluorescein-labelled C. parvum. The effect of removal of these residual macrophages with antimacrophage serum and reconstitution with peritoneal macrophages is shown in Table 4. Removal of macrophage eliminates the mitogenicity of C. parvum by B cells, and addition of macrophages restores stimulation. The actual number of macrophages required for optimal stimulation was not constant from experiment to experiment; in another test 2% peritoneal macrophages inhibited stimulation. This variation may be attributable to a variable degree of macrophage activation in our mouse colony.
Mitogenicity of different strains andfractions Three batches of C. parvum CN6 134, one heat-killed and the others live, were tested, and all were mitogenic (Table 3). Cell wall material prepared by phenol extraction followed by lipid extraction and treatment with pronase, ribonuclease, and TCA was inactive (Table 3). The material obtained from both the phenol and the aqueous phases of a phenolic extract had very little mitogenic activity, and the insoluble residue was inactive (Table 3). Acid polysaccharide of C. parvum (Dawes et al., 1974) was not mitogenic. Culture medium, before
C. parvum as a B-cell mitogen
519
and after 10 days growth of C. parvum CN6134 were not mitogenic (Table 3). Heat-killed C. parvum strain CN5888 showed mitogenic activity, and C. diphtheriae was more active (Table 2). DISCUSSION The results show that heat-killed or live C. parvum organisms are mitogenic for B lymphocytes. T cells are not stimulated, and do not have to be present in the culture for B-cell stimulation to occur. Macrophages, however, are needed, while an excessive proportion of macrophages can be suppressive. The macrophage requirement is interesting in view of the suggestion that the adjuvant action of C. parvum is macrophage-mediated (Wiener & Bandieri, 1975; Wiener, 1975). The mitogenic effect of C. parvum is not an artefact arising from contaminating endotoxin in the culture medium since the latter was not mitogenic, either before or after use. The superiority of live organisms to heat-killed bacteria probably reflects damage to the mitogenic substance during heating. Control cultures containing live C. parvum but no lymphocytes did not give significant counts after pulsing with [3H]thymidine. The nature of the mitogenic substance has not so far been determined. Activity was not recovered to a significant extent in the phenolic or aqueous phases or the residue of a phenol extraction, suggesting that the active material is phenol-sensitive. C. parvum is a Grampositive organism and does not make an endotoxin. The soluble polysaccharide described by Dawes et al. (1974) was not mitogenic. A comment on the microtechnique used in the experiments is needed. The micromethod was highly sensitive for PHA stimulation of T cells but relatively insensitive for the B-cell mitogens levan and lipopolysaccharide when compared with values obtained using a macromethod (Desaymard & Ivanyi, 1975). Comparative studies of micro- and macromitogenicity assays by Greaves et al. (1974) also showed lower sensitivity for levan in the microtechnique. In the present studies mouse serum was used in preference to foetal calf serum, several batches of which were tried and found to be mitogenic. It may be that the low values obtained for B-cell mitogens in the present work resulted from the use of mouse serum or from a non-optimal proportion of macrophages which are important in the mitogenicity of levan (Desaymard & Ivanyi, 1975) as well as C. parvum. In spite of the relatively low values the technique was sufficiently sensitive to demonstrate unequivocally the mitogenic effect of C. parvum and its specificity for B lymphocytes. The relevance of the B-cell mitogenicity of C. parvum to its in vivo properties as an adjuvant, reticuloendothelial stimulant, and anti-tumour agent was investigated by examining a number of preparations with different in vivo properties. C. diphtheriae and C. parvum (CN 5888) are both mitogenic, but are inactive as adjuvants for the antibody response to sheep erythrocytes or in stimulating large increases in the spleen weight, an index of reticuloendothelial stimulation (Adlam & Scott, 1973). C. parvum CN5888 failed to enhance carbon clearance, unlike the active strain CN 6134 (Adlam & Scott, 1973). C. dipltheriae and C. parvum CN5888 are also inactive in preventing the formation of lung metastases in a syngeneic tumour model (Bomford & Olivotto, 1975). Thus the mitogenic property of C. parvum is not sufficient to produce adjuvanticity, reticuloendothelial stimulation, or antitumour activity in vivo, but it may be one of several requisite properties. This aspect was examined using a phenol extracted, solvent extracted 'cell wall' material derived from C.
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parvum CN6134 which was not mitogenic in vitro, but which did prevent the formation of lung nodules in vivo, enhanced carbon clearance and was an adjuvant for antibody response to SRBC (Adlam et al., in preparation). Thus the mitogenic properties of C. parvum are irrelevant to its various in vivo effects. I should like to thank Dr Elizabeth Simpson, Clinical Research Centre, Northwick Park, for demonstrating the microculture technique and the nylon wool procedure for purifying T cells, Dr C. Adlam, Department of Bacteriology, Wellcome Research Laboratories, for supplying the strains and fractions of micro-organisms, and Mrs Susan Barnes for skilled assistance. REFERENCES ADLAM, C., REID, D.E. & TORKINGTON, P. (1975) isms. The Immune System-Genes, Receptors, The nature of the active principle of C. parvum. Signals (ed. by E. E. Sercarz and A. R. WilliamProceedings of the International Conference on son), p. 271. Academic Press, New York. C. parvum (ed. by B. Halpern). Springer Verlag, HALPERN, B.N., PREVOT, A.R., Biozzi, G., STIFFEL, Berlin. (In press.) C., MOUTON, D., MORARD, J.C., BOUTHILLIER, Y. ADLAM, C. & SCOTT, M. (1973) Lympho-reticular & DECREUSEFOND, C. (1964) Stimulation of the stimulatory properties of Corynebacterium parvum phagocytic activity of the reticuloendothelial and related bacteria. J. med. Microbiol. 6, 261. system provoked by Corynebacterium parvum. ASHERSON, G.L. & ALLWOOD, G.G. (1971) DepresJ. reticuloendothel. Soc. 1, 77. sion of delayed hypersensitivity by pretreatment HOWARD, J.G., CHRISTIE, G.H. & SCOTT, M.T. with Freund-type adjuvants. I. Description of the (1973) Biological effects of Corynebacterium phenomenon. Clin. exp. Immunol. 9, 249. parvum. IV. Adjuvant and inhibitory activities on BELL, E.B. & SHAND, F.L. (1972) A search for B lymphocytes. Cell. Immunol. 7, 290. lymphocyte-derived macrophages during xeno- HOWARD, J.G., SCOTT, M.T. & CHRISTIE, G.H. geneic graft-versus-host reactions induced by rat (1973) Cellular mechanisms underlying the adjuthoracic duct cells. Immunology, 22, 538. vant activity of Corynebacterium parvum: interBOMFORD, R. & CHRISTIE, G.H. (1975) Mechanisms actions of activated macrophages with T and B of macrophage activation by C. parvum. II. In lymphocytes. Immunopotentiation, Ciba Foundation Symposium 18 (ed. by J. Knight and G. E. H. vivo experiments. Cell. Immunol. 17, 150. BOMFORD, R. & OLIVOTTO, M. (1975) Inhibition of Wolstenholme), p. 101. Elsevier, Amsterdam. lung nodule formation by C. parvum. Proceedings JULIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A rapid method for the isolation of funcof the International Conference on C. parvum (ed. tional thymus-derived murine lymphocytes. Europ. by B. Halpern). Springer Verlag, Berlin. (In press.) J. Immunol. 3, 645. BULLOCK, W.C. & ANDERSON, F. (1973) Mitogens as probes for immunocyte regulation: specific and MIRANDA, J.J. (1972) Studies on immunological paralysis. IX. The immunogenicity and toleronon-specific suppression of B cell mitogenesis. genicity of levan (polyfructose) in mice. ImImmunopotentiation, Ciba Foundation Symposium munology, 23, 839. posium 18 (ed. by J. Knight and G. E. H. PECK, A.B. & BACH, F. (1973) A miniaturized mouse Wolstenholme), p. 173. Elsevier, Amsterdam. mixed leukocyte culture in serum-free and mouse CASTRO, J.E. (1974) The effect of Corynebacterium serum supplemented media. J. immunol. Meth. 3, parvum on the structure and function of the 147. lymphoid system in mice. Europ. J. Cancer, 10, 115. DAWES, J., TUACH, S.J. & MCBRIDE, W.H. (1974) SCOTT, M.T. (1972) Biological effects of the adjuvant Corynebacterium parvum. 1. Inhibition of PHA, Properties of an antigenic polysaccharide from mixed lymphocyte and GVH reactivity. Cell. Corynebacterium parvum. J. Bact., 120, 24. Immunol. 5, 459. DESAYMARD, C. & IVANYI, L. (1975) Comparison of in vitro immunogenicity, tolerogenicity and SKIDMORE, B.J., CHILLER, J.M., MORRISON, D.C. & WEIGLE, W.O. (1975) Immunologic properties of mitogenicity of dinitrophenyl levan conjugates with bacterial lipopolysaccharide (LPS): correlation varying epitope density. Cell. Immunol. (In press.) between the mitogenic, adjuvant, and immunoFELDMANN, M., HOWARD, J.G. & DESAYMARD, C. genic activities. J. Immunol. 114, 770. (1975) Role of antigen structure in the discrimination between tolerance and immunity by B cells. SCOTT, M.T. (1974) Corynebacterium parvum as an immunotherapeutic anticancer agent. Semin. Transplant. Rev. 23, 78. Oncol. 1, 367. GREAVES, M., JANOSSY, G., FELDMANN, M. & DOENHOFF, M. (1974) Polyclonal mitogens and TRIZIO, D. & CUDKOWICZ, G. (1974) Separation of T and B lymphocytes by nylon wool columns: the nature of B lymphocyte activation mechan-
C. parvum as a B-cell mitogen evaluation of efficacy by functional assays in vivo. J. Immunol. 113, 1093. WIENER, E. (1975) The role of macrophages in the amplified in vivo response to sheep red blood cells by spleen cells from Corynebacterium parvum treated mice. Cell. Immunol. (In press.)
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WIENER, E. & BANDIERI, A. (1975) Modifications in the handling in vitro of 125I labelled keyhole limpet hemocyanin by peritoneal macrophages from mice pretreated with the adjuvant Corynebacterium parvum. Immunology. (In press.)
