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The in vitro inhibition of growth of intracellular Listeria monocytogenes by lymphocyte products
(‘ELLULAR
11IMUNOLOGY
The
9,
353-362 (1973)
in Vitro Inhibition monocyfogenes
of Growth of Intracellular by Lymphocyte Products
Wisteria
Tour JONES’ ANI) Guy P. YOUMANS Drpartment
of Microbiology
Northwrsteru Received
Uni~tersity Medical Lkccrr~bev
School
Clricago, Illinoir
60611
6,197Z
The listeria-inhibiting activity of culture supernatants from listeria-immune and nonimmune lymphocytes was assessed on listeria-infected macrophage cultures. It was found that supernatant from listeria-immune lymphocyte cultures stimulated in V&O with antigen was markedly inhibitory to the multiplication of intracellular listeria. Some inhibitory activity was also evident in supernatant from antigen-stimulated nonimmune lymphocyte cultures. Supernatant from listeria-immune lymphocytes stimulated in z&o with antigen was capable of inhibiting listeria. Some inhibitory activity was still evident upon dilution of active supernatant at 1: 100.
INTRODUCTION Cell-mediated immunity to Listeria monocytogenes has been shown to involve both macrophages and lymphocytes (l-3). While the immune or activated macrophage is ultimately responsible for the fate of the parasite, immunologically committed lymphocytes act as mediators. The role of the latter cell type in the specific immune response to listeria has been demonstrated only in viva; the means by which lymphocytes mediate immunity to listeria has not been determined. In a study with another facultative intracellular parasite, Mycobacterium tuberculosis, it has been shown that product(s) generated during the culture of lymphocytes from mice immunized with attenuated viable tubercle bacilli could inhibit the intracellular growth of virulent mycobacteria (4). Although it has been observed that the growth of listeria in cultured macrophages was nonspecifically inhibited by supernatants from cultures of spleen cells taken from mice immunized with Toxoplasvna gondii cultured with macrophages and soluble toxoplasma antigen (5), it has not been established that a similar inhibition of intracellular growth of listeria can be achieved by supernatants from cultures of lymphocytes taken from animals immunized with listeria. It is the purpose of this paper to demonstrate that the intracellular growth of listeria can be inhibited by lymphocyte culture products obtained from listeria-immune lymphocytes. MATERIALS
AND METHODS
Mice. Male inbred C57B1/6 (ARS/Sprague-Dawley) mice weighing 18-22 g were housed ten per cage in air-conditioned quarters and fed pellets and water nd libitum. 1 Present address : Department
of Pathology, Queen’s University, 353
Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
Kingston,
Ontario,
Canada.
354
JONES
AND
YOUMANS
Listeria. The attenuated, streptomycin-sensitive ATl6Ry strain of L. monocytogenes was obtained from Dr. Sydney Silverman, Fort Detrick, Maryland, and was used as the immunizing agent. The virulent 452 and 19115 (American Type Culture Collection) strains of L. monocytogenes were obtained from Dr. John Osebold, University of California, Davis, and Dr. Harvey Simon, NIH, Bethesda, Maryland, respectively. Streptomycin-resistant mutants of the virulent L. vnonocytogenes strains were isolated by plating 1.0 ml of a BHI-broth culture on BHI agar containing 0.1 mg per ml streptomycin. Mutants were as virulent for mice as the streptomycinsensitive parent strains and were resistant to 100 pg per ml streptomycin. This was the concentration of streptomycin used in all tissue culture media. Streptomycinresistant strains, termed 452SM and 19115SM, were used as infecting agents in all tissue culture work since streptomycin was used in all tissue culture medium to prevent the growth of contaminants. It has been demonstrated that extracellular streptomycin does enter cultured macrophages and will affect the growth of intracellular parasites (1, 7). All strains were passed at frequent intervals through mice to maintain virulence. Saline-washed single cell suspensions of 452SM or 19115SM were prepared from 8-12 hr BHI broth cultures grown at 37°C for the infection of macrophages in vitro. The number of organisms per milliliter was determined by optical density. Immunization. Mice were immunized intraperitoneally with approximately 5X lo6 living ATl6Ry cells suspended in 0.2 ml saline, obtained from a 15-18 hr BHI broth culture. Mice were boosted intraperitoneally with a comparable AT16Ry suspension after a two-week interval. Two weeks after boosting, 90-10070 listeriaimmunized mice and O-15% nonimmune mice survived challenge with one minimal lethal dose of 452SM, as previously described (8). Collection and C&we of Macrophages. A modification of the procedure of Chang (9) was used for the collection of murine peritoneal cells (7). Mice were killed by cervical dislocation, and peritoneal cells were withdrawn following the injection of 4 ml of a solution of 75% RPM1 (Roswell Park Memorial Institute j 1640 and 25% horse serum. After washing pooled cell suspensions 2-3 times in RPM1 1640, the cell concentration was adjusted to 2.0-2.5 X lo6 per ml. Twotenths milliliter of the cell suspension was pipetted onto sterile 10.5 X 22 mm glass coverslips placed in petri dishes, and incubated for 1-3 hr at 37°C in a humified CO2 incubator. Nonadherent cells were removed by the addition of sterile saline, and the coverslips were transferred to petri dishes containing fresh culture medium. Complete culture medium was composed of 55% RPM1 1640, 40% horse serum, 5% beef embryo extract, 2 pg per ml fungizone, and 50-100 pg per ml streptomycin. The cells were incubated for 48-72 hr at 37°C in a CO2 incubator to adjust to the conditions of tissue culture. The number of cells adhering per coverslip (l-2 X 105) represented 30-35% of the original suspension applied. Culture of Lymphocytes. The method of Adler et al (10) for culturing murine lymphocytes was used with modifications. Briefly, a single cell suspension of spleen cells was made by pressing whole spleens through 120 x mesh sterile stainless steel screen into a petri dish containing RPM1 1640. The screen and petri dish were rinsed with medium and the rinsings added to the cell suspension. Cells were sedimented by centrifugation, resuspended in a small volume of RPM1 1640 and counted. Cells were cultured at 5 x 10” or 1 X 10’ cells per ml in medium
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consisting of 95% RPM1 1640, 5% fresh, heat-inactivated human serum, 2 pg per ml fungizone, and 100 pg per ml streptomycin. Three milliliters of the suspension were cultured in plastic tissue culture tubes and incubated 48-72 hr at 37°C in a CO2 incubator. Lymphocytes were stimulated with antigen either in vitro or in z&o. In vitro, viable attenuated AT16Ry cells were added to the cultured lymphocytes in a ratio of 1: 1. This streptomycin-sensitive strain of listeria did not grow in the cultures becauseof the presence of streptomycin. The viability of in vitro antigen-stimulated lymphocytes was no different than the viability of unstimulated lymphocytes. For in tivo stimulation, spleen donors were injected intravenously with approximately 5 X lo6 viable AT16Ry in 0.2 ml saline, 45 hr prior to the removal of the spleens. Following incubation, lymphocytes were sedimented by centrifugation, and the supernatants were decanted and pooled. When lymphocytes were cultured in vitro with listeria, the supernatants were filtered through 0.45 pm Millipore membrane filters to remove residual bacteria. Ten milliliters of supernatant were supplemented with horse serum and beef embryo extract to a volume of 15 ml to give approximate concentrations of the components of complete culture medium for macrophages. The supplemented supernatant was then added to listeria-infected macrophages immediately following the infection period. Infection of lMacrophages In Vitro. Suspensions of cells of L. monocytogenes cultures 45,ZSM or 19115SM were prepared and 2 x lo6 organisms per ml were added to the infecting medium which consisted of 75% RPM1 1640 and 25% horse serum. The medium was aspirated from the macrophage cultures and 10 ml of infecting medium was added to each petri dish. This infecting dose gave a low but reliable level of infection of 5-20 listeria per 10,000 macrophages. Phagocytosis of listeria was allowed to proceed at 37°C in a COz incubator for 1.5 hr. The medium was removed by aspiration and the cultures were washed 2-3 times with lo-20 ml warm sterile saline to remove nonphagocytosed bacteria. Supplemented supernatants were added to each petri dish and the cultures were incubated at 37°C in a COz incubator over the course of the experiment. During this time period, listeria did not multiply extracellularly, since growth in the complete tissue culture medium was completely inhibited by the 40% horse serum. Because of the low level of infection, the loss of macrophages from coverslips over the 24-hr infection did not exceed lo-15% of the original number of cells, and the numbers of remaining macrophages did not vary significantly between the different treatments. Measurement of the Nwnber of Listeria per Macrophuge. The mean number of macrophages per coverslip was determined from Giemsa-stained preparations as previously described (7). The number of viable organisms per macrophage was determined by plate count. Three coverslips were removed, gently rinsed in sterile saline, and placed in 13 X 100 mm sterile glass tubes containing 2.5 ml sterile distilled water. Complete lysis of the macrophages occurred after a period of 30 min at room temperature. Appropriate dilutions in saline were made from the tubes and plated on BHI agar using the drop-plate technique of Miles and Misra (11). An average of the colonies from replicate drops was determined. The number of listeria per macrophage was calculated by dividing the mean number of listeria per coverslip by the mean number of macrophages per coverslip. Presentation and Analysis of Data. The data will be presented as listeria per
356
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TABLE
1
'I'HF. EFFECT OF SUPJXNATANTS FKOM CULTURES OF NONIMMUNII AND IMMUNIZ LYMI~HOCSTISS ONTHE GROWTHOF INTRACELLULAR LISTERIA Experiment
1
2
3
Source
of supernatant
Nonimmune Antigen-stimulated Immune Antigen-stimulated Nonimmune Antigen-stimulated Immune Antigen-stimulated Nonimmune Antigen-stimulated Immune Antigen-stimulated
Listeria per macrophage
nonimmune immune
nonimmune immune nonimmune
(1 Compare values of listeria per macrophage * Significantly different from antigen-stimulated c N. D. = Not done.
Significance”
4.53 1.90 3.87 0.51
0 56 15 89
+ + + ‘I
13.94 10.09 12.60 1.87
0 28 10 87
+ + +”
2.04 0.55 N. D.c
0 73 N. 1).
+
96
+*
0.08
immune
Decrease in listeria per macrophage (%I
of this group to nonimmune group ; + = nonimmune group at P = .05.
P = .05.
macrophage and percent decrease in listeria per macrophage, as calculated according to the following general formula : Percent decrease = 100 -
Listeria per macrophage, treatment No. 1 x 100. Listeria per macrophage, treatment No. 2 >
Treatment Nos. 1 and 2 represent the two different supernatants to be compared. The number of listeria per coverslip within an individual experiment, resulting from different treatments of infected macrophage populations, were compared using analysis of variance. The values for macrophages per coverslip under similar conditions were compared using analysis of variance. The values for macrophages per coverslip under similar conditions were compared using the Student’s T test. Conclusions regarding the number of listeria per macrophage, resulting from different treatments, were based on the separate statistical analysis of listeria and macrophagepopulations. RESULTS Supernatants from Cultures Stiwulated With Antigen In Vitro. Lymphocytes from immune and nonimmune animals were cultured in the presence or absence of antigen. The supernatants were added to cultures of infected macrophages, and the growth of listeria was measured after 24 hr of infection. The data are presented in Table 1, both as listeria per macrophage and percent decrease in listeria per macrophage. The values from different lymphocyte culture supernatants were compared to values from cultures treated with unstimulated nonimmune lymphocyte culture supernatant. As can be seen, the growth of listeria was markedly inhibited
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Infection
FIG. 1. The effect of supernatants from cultured nonimmune and immunelymphocyteson the
growth of intracellular listeria. A, Nonimmune ; Immune; and 0, Antigen-stimulated immune.
l
, Antigen-stimulatednomimmune ; 0,
by supernatants from antigen-stimulated immune lymphocyte cultures, and to a significant degree by supernatants from antigen-stimulated nonimmune lymphocyte cultures. Slight inhibition was evident in cultures treated with supernatants from unstimulated immune lymphocyte cultures. Inhibition was also observed at 12 hr after infection but it was not as marked as after 24 hr of infection. The typical experiment plotted in ,Fig. 1 provides an example of the observations made on the effect of supernatants from lymphocyte cultures on the growth of intracellular listeria. The events can be summarized as follows: (1) a substantial inhibition of growth of listeria by the supernatant from antigen.-stimulated immune lymphocyte cultures was evident after 24 hr of infection ; (2) a significant degree of inhibition by the samesupernatant was evident at 12 hr, but was not as great as at 24 hr; and (3) the supernatant from antigen-stimulated nonimmune lymphocyte cultures was significantly inhibitory at 24 hr, but was not apparent at 12 hr. Supernatants frona Cztltures Stimulated with Antigen In Vivo. It was felt that if lymphocytes could produce an active product in vitro following stimulation with antigen in viva, as would occur with an infection, this approach might offer further support for the possibility that this assay actually reflects cellular immunity to infection. Forty-eight hours prior to the removal of spleen, donor animals were injected intravenously with whole attenuated listeria. Lymphocytes from the spleen of injected animals and unstimulated animals were cultured without further additions of listeria and the supernatants were added to infected macrophages. The growth of listeria was measured after 24 hr. In Table 2, experiments in which immune lymphocytes were stimulated in zfivo are presented along with control experiments in which nonimmune lymphocytes were stimulated in z&o. The listeria per macrophage values from cultures treated with supernatant from antigenstimulated lymphocyte cultures were compared to values from cultures treated with supernatant from unstimulated lymphocyte cultures. In all cases, listeria per macrophage in cultures treated with supernatant from antigen-stimulated immune lymphocyte cultures were significantly less than listeria per macrophage in cultures treated with supernatant from unstimulated immune lymphocyte cultures. Thus, immune lymphocytes stimulated in viz’0 with antigen were as capable of generating
35s
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TABLE THE EFFECT OF ANTIGEN STIMULATION ON THE INHIBITORY ACTIVITY
Experiment
1 2 3 4 5 6 7
Source of supernatant
Immune Antigen-stimulated Immune Antigen-stimulated Immune Antigen-stimulated Immune Antigen-stimulated Immune Antigen-stimulated Nonimmune Antigen-stimulated Nonimmune Antigen-stimulated
immune immune immune immune immune nonimmune nonimmune
2 OF LYMPHOCYTES OF SUPERNATANTS
Listeria per macrophage
9.38 5.98 6.74 0.65 0.37 0.11 43.30 19.10 0.97 0.10 14.50 7.30 1.25 0.64
in
Vitm
Decrease in listeria per macrophage (%I
Significance”
36
+
90
t
70
t
56
+
90
t
50
+
50
+
G Compare values of listeria per macrophage of antigen stimulated group to unstimulated
group.
a supernatant that could inhibit the growth of listeria as immune lymphocytes stimulated in vitro with antigen (Table 1). Nonimmune lymphocytes were also stimulated with antigen to a significant degree, as was the case with in vitro stimulation. In 3 out of 5 casesthe inhibition observed with supernatant from antigen-stimulated immune lymphocytes was substantially greater than that observed with supernatant from antigen-stimulated nonimmune lymphocytes. If the multiplication of listeria can be inhibited by active supernatants added directly to macrophages after infection, it was considered possible that macrophages exposed to a lymphocyte culture product prior to infection might also inhibit listeria growth during a subsequent infection. In a preliminary experiment supernatants were obtained from immune lymphocytes stimulated in viva with antigen and added to infected and uninfected macrophage cultures. It was observed that macrophages incubated in active supernatant 48 hr prior to infection were able to inhibit the growth of listeria to the samedegree as macrophages exposed to supernatant after infection. The effect of dilution on supernata?zt activity. In all previous experiments with lymphocyte culture supernatants, the supernatants were used undiluted as collected from the lymphocyte cultures. It was of interest to determine how much inhibitory activity could be retained upon dilution of the supernatant from antigen-stimulated immune lymphocyte cultures. Lymphocytes were stimulated in &JO prior to removal of the spleen and then cultured. Supernatants were collected and diluted with either fresh RPM 1640 or supernatant from nonimmune lymphocyte cultures and were added to infected macrophages. The growth of listeria was measured
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TABLE THE
EFFECT IMMUNE
Experi-
OF DILUTION LYMPHOCYTE Source
ON THE CULTURES
of supernatant
ment
CAPACITY
350
PRODUCTS
3 OF SUPERNATANTS
TO INHIBIT Dilution supernatant
THE GROWTH of
FROM
ANTIGEN-STIMULATED
OF INTRACELLULAR
Listeria
per
macrophage
LISTERIA
Decrease listeria
in per
macrophage
1
Significance’ pared
comwith
(%‘L)
undiluted supernatant
+
Immune
Undiluted
6.74
0
Antigen-stimulated immune
Undiluted
0.65
90
1:s
1.67
75
+
1:lO
1.80
73
+
1:50
3.73
45
f
1:lOO
N. D.‘J
N. D.
N. D.
Antigen-stimulated immune Antigen-stimulated immune
Antigen-stimulated immune Antigen-stimulated immune
2
Immune
Undiluted
0.97
0
Antigen-stimulated immune
Undiluted
0.10
89
Antigen-stimulated immune
1:s
0.27
72
-
Antigen-stimulated immune
1:lO
N. D.
N. D.
N. D.
Antigen-stimulated immune
1:50
0.26
73
-I-
Antigen-stimulated immune
1: 100
0.52
44
-I
+
,J + = P = 0.0s. b N. D.
= Not
Done.
after 24 hr of infection. Table 3 presents the results of these experiments as listeria per macrophage and percent decrease in listeria per macrophage, comparing values from cultures treated with supernatant from antigen-stimulated immune lymphocyte cultures to values from cultures treated with supernatant from unstimulated immune lymphocyte cultures. As can be seen from the table, some inhibitory activity was lost by a dilution of 1 : 5, but this loss was significant only in one case. There was significantly less inhibition of listeria at dilutions of 1: 50 and 1 : 100, but in both cases some inhibitory activity remained. A basic question to be asked concerning the activity of supernatants from lymphocyte cultures is whether or not the supernatants have any direct inhibitory effect on the growth of listeria. When lo3 listeria were added directly to active supernatants, no inhibition of growth was observed over a 24-hr period, suggesting that the inhibitory effect of lymphocyte supernatants on the intracellular growth of listeria was probably not due to the direct action of the supernatant on the bacteria.
360
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DISCUSSION It is evident from the results of this study that intracellular growth of L. monocytogenes can be inhibited by a product or products elaborated by listeriaimmune spleen cells in vitro following stimulation with antigen. The nature of this product has not yet been established. A possible role for a lymphocyte product as a mediator of cellular immunity to listeria was proposed by Mackaness (2). The present observations agree with those made in a nonspecific system in which intracellular listeria was inhibited from growing by a product from Toxoplasmaimmune spleen cells (5). In the present experiments, it is apparent that the production of an active supernatant from immune lymphocytes requires antigen stimulation of the lymphocyte population. This observation is in accord with the studies on other lymphocyte products (12-15). The antigen dependency of this reaction reflects the antigen dependent nature of cellular immunity in z&o (16, 17). The nature of the stimulation of nonimmune lymphocytes by whole listeria is not known; endotoxinlike activity has not been described for listeria. The inhibition of listeria growth produced by such supernatants was not as great as that produced by supernatants from antigen-stimulated immune lymphocyte cultures. The fact that active supernatants were obtained not only from immune lymphocytes stimulated with antigen in vitro but also from immune lymphocytes stimulated in viva suggests that the present observations are not merely artifacts of in vitro culture methods. It is possible that the lymphocyte product may function in the in z&o immune response. Another lymphocyte product, macrophage inhibitory factor (MIF), has been produced in vivo and in vitro from lymphocytes stimulated with antigen in viva (18, 19). Furthermore, extracts from biopsies of delayed hypersensitive skin reaction sites have been shown to have chemotactic activity for monocytes in vitro, and possess skin reactive factorlike activity when injected in the skin of nonimmune animals (20). Thus, the gap between the in vitro demonstrations of lymphocyte products and their existence and action in viva is lessening, but their actual function in tivo remains to be determined. In the experiments utilizing different dilutions of an active supernatant, it was apparent that activity was still present at a dilution of 1 : 100, although the inhibtion was only about 50% of that observed with undilute supernatant. While the mechanisms of action of other lymphocyte products are probably different than in the present system, it is of interest to note that mouse MIF and mouse lymphotoxin activities are completely lost by dilutions of 1 : 8 and 1 : 20, respectively (18, 21). This suggests that supernatant in the present study is either more active in terms of amount of active product present. or that the inhibition of multiplication of intracellular listeria can be caused by a much smaller amount of the active material. Except for the work of Krahenbuhl and Remington, (5) other investigators have not been able to inhibit the growth of intracellular listeria with lymphocyte culture products. Leibowitch and David (22) incubated macrophage monolayers in supernatants from 0-chloryl benzoyl-bovine gamma globulin-immune lymph node cell cultures for 24 hr; these cultures did not inhibit the growth of listeria upon subsequent infection. After a 72-hr incubation in MIF-rich Sephadex fractions, destruction of the macrophage monolayer by the subsequent listeria infection was not as great, but no actual inhibition of listeria growth was observed. The possi-
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bility exists that MIF is not the same as that product which causes macrophages to inhibit the growth of an intracellular parasite. It has been shown in this laboratory that the capacity of lymphocyte supernatants to inhibit the growth of intracellular virulent mycobacteria or to inhibit the migration of macrophages depended greatly on the composition of the lymphocyte culture medium; both activities were not present at the same time under given culture conditions (18, 23). Thus, the lack of listeria-inhibiting capacity of a supernatant may be due to culture conditions for lymphocytes which do not favor production of an inhibitory product, but do allow production of MIF. Although supernatants in the present study did not have any direct effect on listeria, it cannot be positively concluded that the product acts only on the macrophage. Listeria sm-viving and multiplying in an intracellular environment (macrophage) may be altered and thus susceptible to the action of the lymphocyte product, whereas listeria growing in a test tube may not display such alterations. It must also be considered that the present observations could be the result of ilz vitro culture conditions; that is, inhibition of listeria could be due to increased intracellular serum concentration as the result of increased pinocytosis of serum by activated macrophages. Listeria did not grow at high concentrations of horse serum, and the complete culture medium did contain 405% horse serum. This, however, is not a likely explanation when one considers the preliminary experiment in which macrophages were incubated in active supernatant 48 hr prior to infection. If increased serum concentration in the intracellular milieu were the explanation for the inhibition of listeria, then much greater and earlier inhibition should have been observed in cultures treated in this fashion. However, inhibition of listeria was not observed until 12 hr after infection, and the degree of inhibition at 12 and 24 hr was the same as that observed when supernatant was added to macrophages after infection. It appears that the inhibition of growth was not just the result of a high intracellular serum concentration. The possibility must be considered, then, that the lymphocyte product does act only on the macrophage to initiate the inhibition of growth of a parasite. If this assay reflects some events that occur in ziz~ following the development of cellular immunity, it offers a means of studying in z&o the macrophage-lymphocyte interactions that occur as a result of the immune response and of studying the possible role that lymphocyte products play in cell-mediated immunity to infection. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Grant Al-01636 from the National Institute of Allergy and Infectious Diseases. Tobi Jones was a predoctoral trainee supported by Microbiology Training Grant 5-Tl-GM-724 from the National Institute of General Medical Sciences.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Mackaness, G. B., J. Exp. Med. 116, 381, 1962. Mackaness, G. B., J. Exp. Med. 129, 973, 1969. McGregor, D. D., and Koster, F. T., Cell. Immunol. 2, 317, 1971. Patterson, R. J., and Youmans, G. P., Infec. Immunity 1, 600, 1970. Krahenbuhl, J. L., and Remington, J. S., Infer. Immzlnity 4, 337, 1971. Chang, Y. T., Appl. Microbial. 17, 750, 1969. Patterson, R. J., and Youmans, G. P., Infec. Immunity 1, 30, 1970.
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8. Coppel, S., and Youmans, G. P., J. Bacterial. 97, 114, 1970. 9. Chang, Y. T., J. Natl. Cancer Inst. 32, 19, 1964. 10. Adler, W. H., Takaguichi, T., Marsh, B., and Smith, R. T., J. Exp. Med. 131, 1049, 1970. 11. Miles, A. A., and Misra, S. S., J. Hyg. 38, 732, 1938. 12. David, J. R., Proc. Natl. Acad. Sci. U.S.A. 56, 72, 1966. 13. Ward, P. A., and David, J. R., Fed. Proc. 28,630, 1968. 14. Williams, T. W., and Granger, G. A., J. Iwznzunol. 102, 911, 1969. 1.5. Lebowitz, A., and Lawrence, H. S., Fed. Proc. 28, 630, 1968. 16. Mackaness, G. B., J. Exp. Med. 120, 105, 1964. 17. Blanden, R. V., Lefford, M. J., and Mackaness, G. B., J. Exp. Med. 129, 1079, 1969. 18. Neiburger, R., Ph.D. Thesis, Northwestern University, 1972. 19. Salvin, S. B., and Youngner, J. S., Fed. Proc. 31, 754, 1972. 20. Cohen, S., and Ward, P. A., Fed. Proc. 31, 7.54, 1972. 21. Kolb, W. P., and Granger, G. A., Cell. Immunol. 1, 122, 1970. 22. Leibowitch, J., and David, J. R., Fed. Proc. 31, 610, 1972. 23. Klun, C., Ph.D. Thesis, Northwestern University, 1972.
11IMUNOLOGY
The
9,
353-362 (1973)
in Vitro Inhibition monocyfogenes
of Growth of Intracellular by Lymphocyte Products
Wisteria
Tour JONES’ ANI) Guy P. YOUMANS Drpartment
of Microbiology
Northwrsteru Received
Uni~tersity Medical Lkccrr~bev
School
Clricago, Illinoir
60611
6,197Z
The listeria-inhibiting activity of culture supernatants from listeria-immune and nonimmune lymphocytes was assessed on listeria-infected macrophage cultures. It was found that supernatant from listeria-immune lymphocyte cultures stimulated in V&O with antigen was markedly inhibitory to the multiplication of intracellular listeria. Some inhibitory activity was also evident in supernatant from antigen-stimulated nonimmune lymphocyte cultures. Supernatant from listeria-immune lymphocytes stimulated in z&o with antigen was capable of inhibiting listeria. Some inhibitory activity was still evident upon dilution of active supernatant at 1: 100.
INTRODUCTION Cell-mediated immunity to Listeria monocytogenes has been shown to involve both macrophages and lymphocytes (l-3). While the immune or activated macrophage is ultimately responsible for the fate of the parasite, immunologically committed lymphocytes act as mediators. The role of the latter cell type in the specific immune response to listeria has been demonstrated only in viva; the means by which lymphocytes mediate immunity to listeria has not been determined. In a study with another facultative intracellular parasite, Mycobacterium tuberculosis, it has been shown that product(s) generated during the culture of lymphocytes from mice immunized with attenuated viable tubercle bacilli could inhibit the intracellular growth of virulent mycobacteria (4). Although it has been observed that the growth of listeria in cultured macrophages was nonspecifically inhibited by supernatants from cultures of spleen cells taken from mice immunized with Toxoplasvna gondii cultured with macrophages and soluble toxoplasma antigen (5), it has not been established that a similar inhibition of intracellular growth of listeria can be achieved by supernatants from cultures of lymphocytes taken from animals immunized with listeria. It is the purpose of this paper to demonstrate that the intracellular growth of listeria can be inhibited by lymphocyte culture products obtained from listeria-immune lymphocytes. MATERIALS
AND METHODS
Mice. Male inbred C57B1/6 (ARS/Sprague-Dawley) mice weighing 18-22 g were housed ten per cage in air-conditioned quarters and fed pellets and water nd libitum. 1 Present address : Department
of Pathology, Queen’s University, 353
Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
Kingston,
Ontario,
Canada.
354
JONES
AND
YOUMANS
Listeria. The attenuated, streptomycin-sensitive ATl6Ry strain of L. monocytogenes was obtained from Dr. Sydney Silverman, Fort Detrick, Maryland, and was used as the immunizing agent. The virulent 452 and 19115 (American Type Culture Collection) strains of L. monocytogenes were obtained from Dr. John Osebold, University of California, Davis, and Dr. Harvey Simon, NIH, Bethesda, Maryland, respectively. Streptomycin-resistant mutants of the virulent L. vnonocytogenes strains were isolated by plating 1.0 ml of a BHI-broth culture on BHI agar containing 0.1 mg per ml streptomycin. Mutants were as virulent for mice as the streptomycinsensitive parent strains and were resistant to 100 pg per ml streptomycin. This was the concentration of streptomycin used in all tissue culture media. Streptomycinresistant strains, termed 452SM and 19115SM, were used as infecting agents in all tissue culture work since streptomycin was used in all tissue culture medium to prevent the growth of contaminants. It has been demonstrated that extracellular streptomycin does enter cultured macrophages and will affect the growth of intracellular parasites (1, 7). All strains were passed at frequent intervals through mice to maintain virulence. Saline-washed single cell suspensions of 452SM or 19115SM were prepared from 8-12 hr BHI broth cultures grown at 37°C for the infection of macrophages in vitro. The number of organisms per milliliter was determined by optical density. Immunization. Mice were immunized intraperitoneally with approximately 5X lo6 living ATl6Ry cells suspended in 0.2 ml saline, obtained from a 15-18 hr BHI broth culture. Mice were boosted intraperitoneally with a comparable AT16Ry suspension after a two-week interval. Two weeks after boosting, 90-10070 listeriaimmunized mice and O-15% nonimmune mice survived challenge with one minimal lethal dose of 452SM, as previously described (8). Collection and C&we of Macrophages. A modification of the procedure of Chang (9) was used for the collection of murine peritoneal cells (7). Mice were killed by cervical dislocation, and peritoneal cells were withdrawn following the injection of 4 ml of a solution of 75% RPM1 (Roswell Park Memorial Institute j 1640 and 25% horse serum. After washing pooled cell suspensions 2-3 times in RPM1 1640, the cell concentration was adjusted to 2.0-2.5 X lo6 per ml. Twotenths milliliter of the cell suspension was pipetted onto sterile 10.5 X 22 mm glass coverslips placed in petri dishes, and incubated for 1-3 hr at 37°C in a humified CO2 incubator. Nonadherent cells were removed by the addition of sterile saline, and the coverslips were transferred to petri dishes containing fresh culture medium. Complete culture medium was composed of 55% RPM1 1640, 40% horse serum, 5% beef embryo extract, 2 pg per ml fungizone, and 50-100 pg per ml streptomycin. The cells were incubated for 48-72 hr at 37°C in a CO2 incubator to adjust to the conditions of tissue culture. The number of cells adhering per coverslip (l-2 X 105) represented 30-35% of the original suspension applied. Culture of Lymphocytes. The method of Adler et al (10) for culturing murine lymphocytes was used with modifications. Briefly, a single cell suspension of spleen cells was made by pressing whole spleens through 120 x mesh sterile stainless steel screen into a petri dish containing RPM1 1640. The screen and petri dish were rinsed with medium and the rinsings added to the cell suspension. Cells were sedimented by centrifugation, resuspended in a small volume of RPM1 1640 and counted. Cells were cultured at 5 x 10” or 1 X 10’ cells per ml in medium
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consisting of 95% RPM1 1640, 5% fresh, heat-inactivated human serum, 2 pg per ml fungizone, and 100 pg per ml streptomycin. Three milliliters of the suspension were cultured in plastic tissue culture tubes and incubated 48-72 hr at 37°C in a CO2 incubator. Lymphocytes were stimulated with antigen either in vitro or in z&o. In vitro, viable attenuated AT16Ry cells were added to the cultured lymphocytes in a ratio of 1: 1. This streptomycin-sensitive strain of listeria did not grow in the cultures becauseof the presence of streptomycin. The viability of in vitro antigen-stimulated lymphocytes was no different than the viability of unstimulated lymphocytes. For in tivo stimulation, spleen donors were injected intravenously with approximately 5 X lo6 viable AT16Ry in 0.2 ml saline, 45 hr prior to the removal of the spleens. Following incubation, lymphocytes were sedimented by centrifugation, and the supernatants were decanted and pooled. When lymphocytes were cultured in vitro with listeria, the supernatants were filtered through 0.45 pm Millipore membrane filters to remove residual bacteria. Ten milliliters of supernatant were supplemented with horse serum and beef embryo extract to a volume of 15 ml to give approximate concentrations of the components of complete culture medium for macrophages. The supplemented supernatant was then added to listeria-infected macrophages immediately following the infection period. Infection of lMacrophages In Vitro. Suspensions of cells of L. monocytogenes cultures 45,ZSM or 19115SM were prepared and 2 x lo6 organisms per ml were added to the infecting medium which consisted of 75% RPM1 1640 and 25% horse serum. The medium was aspirated from the macrophage cultures and 10 ml of infecting medium was added to each petri dish. This infecting dose gave a low but reliable level of infection of 5-20 listeria per 10,000 macrophages. Phagocytosis of listeria was allowed to proceed at 37°C in a COz incubator for 1.5 hr. The medium was removed by aspiration and the cultures were washed 2-3 times with lo-20 ml warm sterile saline to remove nonphagocytosed bacteria. Supplemented supernatants were added to each petri dish and the cultures were incubated at 37°C in a COz incubator over the course of the experiment. During this time period, listeria did not multiply extracellularly, since growth in the complete tissue culture medium was completely inhibited by the 40% horse serum. Because of the low level of infection, the loss of macrophages from coverslips over the 24-hr infection did not exceed lo-15% of the original number of cells, and the numbers of remaining macrophages did not vary significantly between the different treatments. Measurement of the Nwnber of Listeria per Macrophuge. The mean number of macrophages per coverslip was determined from Giemsa-stained preparations as previously described (7). The number of viable organisms per macrophage was determined by plate count. Three coverslips were removed, gently rinsed in sterile saline, and placed in 13 X 100 mm sterile glass tubes containing 2.5 ml sterile distilled water. Complete lysis of the macrophages occurred after a period of 30 min at room temperature. Appropriate dilutions in saline were made from the tubes and plated on BHI agar using the drop-plate technique of Miles and Misra (11). An average of the colonies from replicate drops was determined. The number of listeria per macrophage was calculated by dividing the mean number of listeria per coverslip by the mean number of macrophages per coverslip. Presentation and Analysis of Data. The data will be presented as listeria per
356
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TABLE
1
'I'HF. EFFECT OF SUPJXNATANTS FKOM CULTURES OF NONIMMUNII AND IMMUNIZ LYMI~HOCSTISS ONTHE GROWTHOF INTRACELLULAR LISTERIA Experiment
1
2
3
Source
of supernatant
Nonimmune Antigen-stimulated Immune Antigen-stimulated Nonimmune Antigen-stimulated Immune Antigen-stimulated Nonimmune Antigen-stimulated Immune Antigen-stimulated
Listeria per macrophage
nonimmune immune
nonimmune immune nonimmune
(1 Compare values of listeria per macrophage * Significantly different from antigen-stimulated c N. D. = Not done.
Significance”
4.53 1.90 3.87 0.51
0 56 15 89
+ + + ‘I
13.94 10.09 12.60 1.87
0 28 10 87
+ + +”
2.04 0.55 N. D.c
0 73 N. 1).
+
96
+*
0.08
immune
Decrease in listeria per macrophage (%I
of this group to nonimmune group ; + = nonimmune group at P = .05.
P = .05.
macrophage and percent decrease in listeria per macrophage, as calculated according to the following general formula : Percent decrease = 100 -
Listeria per macrophage, treatment No. 1 x 100. Listeria per macrophage, treatment No. 2 >
Treatment Nos. 1 and 2 represent the two different supernatants to be compared. The number of listeria per coverslip within an individual experiment, resulting from different treatments of infected macrophage populations, were compared using analysis of variance. The values for macrophages per coverslip under similar conditions were compared using analysis of variance. The values for macrophages per coverslip under similar conditions were compared using the Student’s T test. Conclusions regarding the number of listeria per macrophage, resulting from different treatments, were based on the separate statistical analysis of listeria and macrophagepopulations. RESULTS Supernatants from Cultures Stiwulated With Antigen In Vitro. Lymphocytes from immune and nonimmune animals were cultured in the presence or absence of antigen. The supernatants were added to cultures of infected macrophages, and the growth of listeria was measured after 24 hr of infection. The data are presented in Table 1, both as listeria per macrophage and percent decrease in listeria per macrophage. The values from different lymphocyte culture supernatants were compared to values from cultures treated with unstimulated nonimmune lymphocyte culture supernatant. As can be seen, the growth of listeria was markedly inhibited
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Infection
FIG. 1. The effect of supernatants from cultured nonimmune and immunelymphocyteson the
growth of intracellular listeria. A, Nonimmune ; Immune; and 0, Antigen-stimulated immune.
l
, Antigen-stimulatednomimmune ; 0,
by supernatants from antigen-stimulated immune lymphocyte cultures, and to a significant degree by supernatants from antigen-stimulated nonimmune lymphocyte cultures. Slight inhibition was evident in cultures treated with supernatants from unstimulated immune lymphocyte cultures. Inhibition was also observed at 12 hr after infection but it was not as marked as after 24 hr of infection. The typical experiment plotted in ,Fig. 1 provides an example of the observations made on the effect of supernatants from lymphocyte cultures on the growth of intracellular listeria. The events can be summarized as follows: (1) a substantial inhibition of growth of listeria by the supernatant from antigen.-stimulated immune lymphocyte cultures was evident after 24 hr of infection ; (2) a significant degree of inhibition by the samesupernatant was evident at 12 hr, but was not as great as at 24 hr; and (3) the supernatant from antigen-stimulated nonimmune lymphocyte cultures was significantly inhibitory at 24 hr, but was not apparent at 12 hr. Supernatants frona Cztltures Stimulated with Antigen In Vivo. It was felt that if lymphocytes could produce an active product in vitro following stimulation with antigen in viva, as would occur with an infection, this approach might offer further support for the possibility that this assay actually reflects cellular immunity to infection. Forty-eight hours prior to the removal of spleen, donor animals were injected intravenously with whole attenuated listeria. Lymphocytes from the spleen of injected animals and unstimulated animals were cultured without further additions of listeria and the supernatants were added to infected macrophages. The growth of listeria was measured after 24 hr. In Table 2, experiments in which immune lymphocytes were stimulated in zfivo are presented along with control experiments in which nonimmune lymphocytes were stimulated in z&o. The listeria per macrophage values from cultures treated with supernatant from antigenstimulated lymphocyte cultures were compared to values from cultures treated with supernatant from unstimulated lymphocyte cultures. In all cases, listeria per macrophage in cultures treated with supernatant from antigen-stimulated immune lymphocyte cultures were significantly less than listeria per macrophage in cultures treated with supernatant from unstimulated immune lymphocyte cultures. Thus, immune lymphocytes stimulated in viz’0 with antigen were as capable of generating
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TABLE THE EFFECT OF ANTIGEN STIMULATION ON THE INHIBITORY ACTIVITY
Experiment
1 2 3 4 5 6 7
Source of supernatant
Immune Antigen-stimulated Immune Antigen-stimulated Immune Antigen-stimulated Immune Antigen-stimulated Immune Antigen-stimulated Nonimmune Antigen-stimulated Nonimmune Antigen-stimulated
immune immune immune immune immune nonimmune nonimmune
2 OF LYMPHOCYTES OF SUPERNATANTS
Listeria per macrophage
9.38 5.98 6.74 0.65 0.37 0.11 43.30 19.10 0.97 0.10 14.50 7.30 1.25 0.64
in
Vitm
Decrease in listeria per macrophage (%I
Significance”
36
+
90
t
70
t
56
+
90
t
50
+
50
+
G Compare values of listeria per macrophage of antigen stimulated group to unstimulated
group.
a supernatant that could inhibit the growth of listeria as immune lymphocytes stimulated in vitro with antigen (Table 1). Nonimmune lymphocytes were also stimulated with antigen to a significant degree, as was the case with in vitro stimulation. In 3 out of 5 casesthe inhibition observed with supernatant from antigen-stimulated immune lymphocytes was substantially greater than that observed with supernatant from antigen-stimulated nonimmune lymphocytes. If the multiplication of listeria can be inhibited by active supernatants added directly to macrophages after infection, it was considered possible that macrophages exposed to a lymphocyte culture product prior to infection might also inhibit listeria growth during a subsequent infection. In a preliminary experiment supernatants were obtained from immune lymphocytes stimulated in viva with antigen and added to infected and uninfected macrophage cultures. It was observed that macrophages incubated in active supernatant 48 hr prior to infection were able to inhibit the growth of listeria to the samedegree as macrophages exposed to supernatant after infection. The effect of dilution on supernata?zt activity. In all previous experiments with lymphocyte culture supernatants, the supernatants were used undiluted as collected from the lymphocyte cultures. It was of interest to determine how much inhibitory activity could be retained upon dilution of the supernatant from antigen-stimulated immune lymphocyte cultures. Lymphocytes were stimulated in &JO prior to removal of the spleen and then cultured. Supernatants were collected and diluted with either fresh RPM 1640 or supernatant from nonimmune lymphocyte cultures and were added to infected macrophages. The growth of listeria was measured
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of supernatant
ment
CAPACITY
350
PRODUCTS
3 OF SUPERNATANTS
TO INHIBIT Dilution supernatant
THE GROWTH of
FROM
ANTIGEN-STIMULATED
OF INTRACELLULAR
Listeria
per
macrophage
LISTERIA
Decrease listeria
in per
macrophage
1
Significance’ pared
comwith
(%‘L)
undiluted supernatant
+
Immune
Undiluted
6.74
0
Antigen-stimulated immune
Undiluted
0.65
90
1:s
1.67
75
+
1:lO
1.80
73
+
1:50
3.73
45
f
1:lOO
N. D.‘J
N. D.
N. D.
Antigen-stimulated immune Antigen-stimulated immune
Antigen-stimulated immune Antigen-stimulated immune
2
Immune
Undiluted
0.97
0
Antigen-stimulated immune
Undiluted
0.10
89
Antigen-stimulated immune
1:s
0.27
72
-
Antigen-stimulated immune
1:lO
N. D.
N. D.
N. D.
Antigen-stimulated immune
1:50
0.26
73
-I-
Antigen-stimulated immune
1: 100
0.52
44
-I
+
,J + = P = 0.0s. b N. D.
= Not
Done.
after 24 hr of infection. Table 3 presents the results of these experiments as listeria per macrophage and percent decrease in listeria per macrophage, comparing values from cultures treated with supernatant from antigen-stimulated immune lymphocyte cultures to values from cultures treated with supernatant from unstimulated immune lymphocyte cultures. As can be seen from the table, some inhibitory activity was lost by a dilution of 1 : 5, but this loss was significant only in one case. There was significantly less inhibition of listeria at dilutions of 1: 50 and 1 : 100, but in both cases some inhibitory activity remained. A basic question to be asked concerning the activity of supernatants from lymphocyte cultures is whether or not the supernatants have any direct inhibitory effect on the growth of listeria. When lo3 listeria were added directly to active supernatants, no inhibition of growth was observed over a 24-hr period, suggesting that the inhibitory effect of lymphocyte supernatants on the intracellular growth of listeria was probably not due to the direct action of the supernatant on the bacteria.
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DISCUSSION It is evident from the results of this study that intracellular growth of L. monocytogenes can be inhibited by a product or products elaborated by listeriaimmune spleen cells in vitro following stimulation with antigen. The nature of this product has not yet been established. A possible role for a lymphocyte product as a mediator of cellular immunity to listeria was proposed by Mackaness (2). The present observations agree with those made in a nonspecific system in which intracellular listeria was inhibited from growing by a product from Toxoplasmaimmune spleen cells (5). In the present experiments, it is apparent that the production of an active supernatant from immune lymphocytes requires antigen stimulation of the lymphocyte population. This observation is in accord with the studies on other lymphocyte products (12-15). The antigen dependency of this reaction reflects the antigen dependent nature of cellular immunity in z&o (16, 17). The nature of the stimulation of nonimmune lymphocytes by whole listeria is not known; endotoxinlike activity has not been described for listeria. The inhibition of listeria growth produced by such supernatants was not as great as that produced by supernatants from antigen-stimulated immune lymphocyte cultures. The fact that active supernatants were obtained not only from immune lymphocytes stimulated with antigen in vitro but also from immune lymphocytes stimulated in viva suggests that the present observations are not merely artifacts of in vitro culture methods. It is possible that the lymphocyte product may function in the in z&o immune response. Another lymphocyte product, macrophage inhibitory factor (MIF), has been produced in vivo and in vitro from lymphocytes stimulated with antigen in viva (18, 19). Furthermore, extracts from biopsies of delayed hypersensitive skin reaction sites have been shown to have chemotactic activity for monocytes in vitro, and possess skin reactive factorlike activity when injected in the skin of nonimmune animals (20). Thus, the gap between the in vitro demonstrations of lymphocyte products and their existence and action in viva is lessening, but their actual function in tivo remains to be determined. In the experiments utilizing different dilutions of an active supernatant, it was apparent that activity was still present at a dilution of 1 : 100, although the inhibtion was only about 50% of that observed with undilute supernatant. While the mechanisms of action of other lymphocyte products are probably different than in the present system, it is of interest to note that mouse MIF and mouse lymphotoxin activities are completely lost by dilutions of 1 : 8 and 1 : 20, respectively (18, 21). This suggests that supernatant in the present study is either more active in terms of amount of active product present. or that the inhibition of multiplication of intracellular listeria can be caused by a much smaller amount of the active material. Except for the work of Krahenbuhl and Remington, (5) other investigators have not been able to inhibit the growth of intracellular listeria with lymphocyte culture products. Leibowitch and David (22) incubated macrophage monolayers in supernatants from 0-chloryl benzoyl-bovine gamma globulin-immune lymph node cell cultures for 24 hr; these cultures did not inhibit the growth of listeria upon subsequent infection. After a 72-hr incubation in MIF-rich Sephadex fractions, destruction of the macrophage monolayer by the subsequent listeria infection was not as great, but no actual inhibition of listeria growth was observed. The possi-
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bility exists that MIF is not the same as that product which causes macrophages to inhibit the growth of an intracellular parasite. It has been shown in this laboratory that the capacity of lymphocyte supernatants to inhibit the growth of intracellular virulent mycobacteria or to inhibit the migration of macrophages depended greatly on the composition of the lymphocyte culture medium; both activities were not present at the same time under given culture conditions (18, 23). Thus, the lack of listeria-inhibiting capacity of a supernatant may be due to culture conditions for lymphocytes which do not favor production of an inhibitory product, but do allow production of MIF. Although supernatants in the present study did not have any direct effect on listeria, it cannot be positively concluded that the product acts only on the macrophage. Listeria sm-viving and multiplying in an intracellular environment (macrophage) may be altered and thus susceptible to the action of the lymphocyte product, whereas listeria growing in a test tube may not display such alterations. It must also be considered that the present observations could be the result of ilz vitro culture conditions; that is, inhibition of listeria could be due to increased intracellular serum concentration as the result of increased pinocytosis of serum by activated macrophages. Listeria did not grow at high concentrations of horse serum, and the complete culture medium did contain 405% horse serum. This, however, is not a likely explanation when one considers the preliminary experiment in which macrophages were incubated in active supernatant 48 hr prior to infection. If increased serum concentration in the intracellular milieu were the explanation for the inhibition of listeria, then much greater and earlier inhibition should have been observed in cultures treated in this fashion. However, inhibition of listeria was not observed until 12 hr after infection, and the degree of inhibition at 12 and 24 hr was the same as that observed when supernatant was added to macrophages after infection. It appears that the inhibition of growth was not just the result of a high intracellular serum concentration. The possibility must be considered, then, that the lymphocyte product does act only on the macrophage to initiate the inhibition of growth of a parasite. If this assay reflects some events that occur in ziz~ following the development of cellular immunity, it offers a means of studying in z&o the macrophage-lymphocyte interactions that occur as a result of the immune response and of studying the possible role that lymphocyte products play in cell-mediated immunity to infection. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Grant Al-01636 from the National Institute of Allergy and Infectious Diseases. Tobi Jones was a predoctoral trainee supported by Microbiology Training Grant 5-Tl-GM-724 from the National Institute of General Medical Sciences.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Mackaness, G. B., J. Exp. Med. 116, 381, 1962. Mackaness, G. B., J. Exp. Med. 129, 973, 1969. McGregor, D. D., and Koster, F. T., Cell. Immunol. 2, 317, 1971. Patterson, R. J., and Youmans, G. P., Infec. Immunity 1, 600, 1970. Krahenbuhl, J. L., and Remington, J. S., Infer. Immzlnity 4, 337, 1971. Chang, Y. T., Appl. Microbial. 17, 750, 1969. Patterson, R. J., and Youmans, G. P., Infec. Immunity 1, 30, 1970.
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8. Coppel, S., and Youmans, G. P., J. Bacterial. 97, 114, 1970. 9. Chang, Y. T., J. Natl. Cancer Inst. 32, 19, 1964. 10. Adler, W. H., Takaguichi, T., Marsh, B., and Smith, R. T., J. Exp. Med. 131, 1049, 1970. 11. Miles, A. A., and Misra, S. S., J. Hyg. 38, 732, 1938. 12. David, J. R., Proc. Natl. Acad. Sci. U.S.A. 56, 72, 1966. 13. Ward, P. A., and David, J. R., Fed. Proc. 28,630, 1968. 14. Williams, T. W., and Granger, G. A., J. Iwznzunol. 102, 911, 1969. 1.5. Lebowitz, A., and Lawrence, H. S., Fed. Proc. 28, 630, 1968. 16. Mackaness, G. B., J. Exp. Med. 120, 105, 1964. 17. Blanden, R. V., Lefford, M. J., and Mackaness, G. B., J. Exp. Med. 129, 1079, 1969. 18. Neiburger, R., Ph.D. Thesis, Northwestern University, 1972. 19. Salvin, S. B., and Youngner, J. S., Fed. Proc. 31, 754, 1972. 20. Cohen, S., and Ward, P. A., Fed. Proc. 31, 7.54, 1972. 21. Kolb, W. P., and Granger, G. A., Cell. Immunol. 1, 122, 1970. 22. Leibowitch, J., and David, J. R., Fed. Proc. 31, 610, 1972. 23. Klun, C., Ph.D. Thesis, Northwestern University, 1972.