Vol. 11, No. 1 Printed in U.S.A.
INFECrION AND IMMUNrrY, Jan. 197f5 D. 1-7
Copyright i 1975 American Society for Microbiology
Delayed Hypersensitivity and Acquired Cellular Resistance in Guinea Pigs Infected with Listeria monocytogenes BARBARA L. HALLIBURTON AND A. A. BLAZKOVEC* Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 16 August 1974
Randomly bred pigs of both sexes were injected intracardially with one-half of 50% lethal dose of Listeria monocytogenes. When infected animals were skin tested with 30 Ag of a water-soluble extract of sonically disrupted Listeria, both males and females had uniformly detectable levels of delayed hypersensitivity (DH) 4 days after infection. In males, cutaneous hypersensitivity to Listeria antigens reached a peak on day 5 or 6 of infection, and high levels of DH persisted through the 7th week. In females, DH reached a peak on day 6 or 7, remained at this level through the 4th week, and then dropped sharply. Cutaneous reactivity was usually higher for males than for females, and differences between the sexes were statistically significant 5, 6, and 7 weeks after infection. Low levels of DH were still present 41 weeks (females) or 46 weeks (males) after infection. Assays to determine the number of viable Listeria present in spleen homogenates indicated that bacterial multiplication occurred only during the first 24 h of infection. The number of Listeria declined steadily thereafter, and by day 13 no bacteria could be recovered from the spleens of infected animals. Spleen assays indicated that Listeria-infected animals of both sexes were resistant to a small challenge dose of Listeria given 48 h, 7 days, or 2 weeks after the primary infection. Resistance to re-infection was absent in females challenged at 41 weeks and in males challenged at 46 weeks. a
Mackaness and co-workers (2, 19, 20) believe that the development of acquired cellular resistance (ACR) depends upon the development of delayed hypersensitivity (DH). Others (23, 25, 26) disagree, however, and the exact relationship between DH and ACR is not known. Listeria monocytogenes infections in mice (13, 17, 18, 21, 22, 24) and rats (12, 14-16) have been used extensively in studies on the development and mechanisms of ACR. On the other hand, guinea pigs have been widely used in studies on the production and characterization of lymphokines, the soluble mediators of DH (7, 8). The feasibility of using a Listeria infection in guinea pigs for in vivo and in vitro investigations of both ACR and DH has not been explored. This paper reports the results of in vivo studies on the development and persistence of DH and ACR in Listeria-infected guinea pigs. (The research described in this paper was submitted in part to the Graduate School of the University of Wisconsin, Madison, by B. L. H. in partial "l-fillmpnt of the requirements for the Ph.D. ciegrep in Medical Microbioloev.) MATERIALS AND METHODS Animals. Randomly bred albino guinea pigs of
both sexes were obtained from local suppliers. The animals weighed 500 to 700 g at the time of infection. Bacteria. L. monocytogenes was provided by D. W. Smith (Department of Medical Microbiology, University of Wisconsin, Madison). The culture was guinea pig-passaged four times, and a fresh isolate was recovered from the spleen of an infected animal. A subculture of the isolate was grown up on a brain heart infusion (BHI) agar slant (Difco Laboratories, Detroit, Mich.) and used to inoculate 75 ml of BHI broth. The broth culture was incubated on a shaker at 37 C until the bacteria were in late log phase (optical density, 0.3 at 615 nm). The culture was then centrifuged at 1,000 x g for 15 min and resuspended in 75 ml of BHI broth. Samples (1 ml) of this suspension were stored at -70 C (frozen stock cultures). The 50% lethal dose for intracardially injected Listeria was 1.2 x 106. Immunizations. BHI broth was inoculated with a BHI agar slant subculture of the frozen stock. When the broth culture was in late log phase, a portion of it was centrifuged at 1,000 x g for 15 min. The pellet was washed once and then diluted with sterile saline. Approximately one-half of a 50% lethal dose in a total volume of 2 ml was injected intracardially into each guinea pig. The exact number of viable Listeria injected was determined by plate counts of the immunizing suspension. Antigen. The water-soluble extract of sonically disrupted Listeria was prepared by using a modification of the method of Hinsdill and Berman (11). Six
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HALLIBURTON AND BLAZKOVEC
liters of BHI broth (500 mi/flask) was inoculated with agar slant subcultures of the frozen stock and incubated on a shaker at 37 C. After 18 h, phenol was added at a final concentration of 5% (wt/vol), and the flasks were incubated for a further 8 h. The killed bacteria were collected by centrifugation at 7,000 x g for 30 min at 4 C and washed once with sterile saline and three times with sterile distilled water. The washed cells were suspended in enough distilled water to give a final volume of 50 ml and disrupted by sonic oscillation. The disrupted cells were centrifuged at 18,400 x g for 30 min at 4 C, and the supernatant was set aside. The cellular debris was suspended in 50 ml of distilled water and centrifuged as above. The two supernatants were combined and filtered (Whatman no. 1 filter paper). Samples (5 ml) were placed in small vials and lyophilized. The vials were sealed while under vacuum and stored in a desiccator at 4 C. Skin tests. Guinea pigs were injected intradermally on the shaved dorsal flank with 30 qg of the water-soluble extract in 0.1 ml of saline. The skin tests were read at 2, 4, 8, 12, 24, and 48 h. Cutaneous reactivity was measured by using three parameters: (i) degree of erythema, (ii) mean diameter of erythema, and (iii) changes in double skin thickness at the reaction site. The degree of erythema was scored arbitrarily as described in Table 1. Induration was measured using a "Schnelltester" (System Kr6plin, Type A, 2T, H. C. Kr6plin, Schliictern, Hessen, Germany). Each value given in the text represents the double skin thickness of the reaction site minus the mean of normal skin measured on either side of the test site. Spleen assays. The number of viable Listeria in BHI broth homogenates of spleen was determined by the method of Mackaness (17). Appropriate decimal dilutions were plated in duplicate on well-dried BHI plates. Colonies were counted after 24 to 36 h of incubation at 37 C. Experimental design. For studies on DH, two separate experiments were run. In one, 50 male guinea pigs were infected with Listeria. Groups of six were skin tested at 24-h intervals for the first 7 days and then at weekly intervals for the next 8 weeks. These animals were also skin tested at 12, 16, 33, and 46 weeks after infection. In the second experiment, 50 females were infected, and groups of six were skin tested at 24-h intervals for the first week and then at weekly intervals for the next 6 weeks. These same females were also skin tested at 12, 16, 33, and 41 weeks. The values for animals skin tested 7 or 10 days
INFECT. IMMUN.
after infection include pooled data from other experiments. For challenge experiments, guinea pigs were infected, and then at various times after immunization the infected animals and an equal number of uninfected control animals were challenged with one-half of a 50% lethal dose of Listeria. Spleen counts were run at 0, 24, and 48 h on five experimentals and five controls. For zero time counts, guinea pigs were killed 30 to 60 min after the challenge infection. In general, animals of one sex were assayed one week, and animals of the other sex were assayed the following week.
RESULTS Delayed hypersensitivity. (i) General trends. Figure 1 shows the cutaneous reactivity present 24 h after skin testing. According to all three parameters, both males and females had uniformly detectable DH to Listeria antigens 4 days after infection. In males, the response peaked at 5 to 6 days, dropped at 7 and 10 days, and then remained fairly steady through 7 weeks. The readings taken at 8 weeks and later were generally lower and fluctuated more than the previous readings. DH reactions in females were generally lower than those in males from day 4 to day 7, and the peak response in females occurred 1 to 2 days later, on day 6 or 7. The degree and mean diameter of erythema remained fairly constant from 7 days to 4 weeks, whereas the mean increase in double skin thickness varied considerably. However, at 5 weeks, according to all three parameters, the DH response dropped sharply and generally remained at very low levels for the remaining skin tests. (ii) Sexual dimorphism. During the first 4
TABLE 1. Scoring for degree of erythema Score
0 1 2 3 4 5 6
Degree of erythema
Trace Light pink Pink Dark pink + Erythema + + Erythema
+ Hemorrhage
FIG. 1. Development and persistence of DH in Listeria-infected guinea pigs. Responses 24 h after skin testing: *, males; 0, females. Each point represents the mean of 6 to 28 guinea pigs. Bars indicate standard error.
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IMMUNITY TO LISTERIA
weeks of skin testing, the differences between the responses of males and females were not significantly different when analyzed using the Student's t test. At 5 weeks, the males were significantly more responsive in terms of degree of erythema, P < 0.001, for diameter of erythema, P < 0.01, and for mean increase in double skin thickness, P < 0.001. At 6 weeks P values were likewise significantly in favor of the males, with P < 0.001, P < 0.01, and P < 0.01, respectively; at 7 weeks P values were P < 0.01, P < 0.02, and P < 0.01, respectively, with males still responding more strongly than females. At 12 weeks the females were significantly higher for degree of erythema, P < 0.001, and mean increase in double skin thickness, P < 0.01. (iii) Development of the peak response. Figure 2 shows the development of cutaneous reactivity in males' skin tested 5 days after infection and in females' skin tested 7 days after
HOURS AFTER SKIN TESTING
FIG. 2. Development of cutaneous reactivity in Listeria-infected guinea pigs. Symbols: 0, males skin tested 5 days after infection (mean of six guinea pigs per point); 0, females skin tested 7 days after infection (mean of 13 to 28 guinea pigs per point). Bars indicate standard error.
3
infection. The 5- and 7-day readings were chosen to represent the peak DH response to Listeria antigens. If one considers all three parameters, cutaneous reactivity was minimal 2 and 4 h after skin testing and then rose to a peak at 24 h. Except for the mean increase in double skin thickness at 4 h, all values for the males were consistently higher than values for the females. Significant differences in the degree of erythema (Fig. 2, top) occurred at 2 h, P < 0.001; 8 h, P < 0.02; 24 h, P < 0.05; and 48 h, P < 0.02. The mean diameter of erythema (Fig. 2, middle) was significantly greater in males at 12 h, P < 0.05, and at 48 h, P < 0.01. There were no significant differences between males and females in the mean increase in double skin thickness (Fig. 2, bottom) at any time. (iv) Persistence of DH. The longest time interval between infection and skin testing was 46 weeks for males and 41 weeks for females. Figure 3 shows the development of cutaneous reactivity in animals skin tested at these times. The results indicate that a very low or minimal level of DH to Listeria antigens persisted almost 1 year after infection. There were no significant differences between males and females for any parameter 24 h after skin testing. Females, however, had significantly higher cutaneous reactivity 4 h after skin testing. The P values were P < 0.05 for degree of erythema, P < 0.01 for mean diameter of erythema, and P < 0.001 for mean increase in double skin thickness. This higher cutaneous reactivity measurable so soon after skin testing probably represents an Arthus component. Although no antibody titrations were carried out, these results suggest that antibody levels directed toward Listeria antigens were higher in females than in males. ACR. (i) Growth of Listeria in the spleens of normal guinea pigs. Figure 4 shows the combined results of four separate experiments. In two experiments, spleen assays on five animals per group were run at zero time and then at daily intervals for the next 5 days. In two other experiments, assays were run at zero time and then at 3-day intervals beginning 3 or 4 days after infection. Multiplication of Listeria occurred only within the first 24 h of infection. Thereafter, the number of bacteria steadily decreased, and by day 13 none could be detected. (ii) Growth of an homologous challenge given 48 h or 7 days after primary immunization. The rapid inhibition of Listeria multiplication in vivo (Fig. 4) suggests that ACR may develop within the first 48 h of infection, whereas the data in Fig. 1 indicate that DH is
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INFECTr. IMMUN .
HALLIBURTON AND BLAZKOVEC
Fig. 5. For both sexes, the differences between experimentals and controls 24 h after challenge are significant with P < 0.001. At 48 h, previously infected females had significantly lower numbers of Listeria, P < 0.01; for males, however, no significant difference was shown to exist between the controls and experimentals, P
0.50. (iii) Growth of an homologous challenge
FIG. 3. Development of cutaneous reactivity in Listeria-infected guinea pigs. Symbols: *, males skin tested 46 weeks after infection (mean of 12 to 21 guinea pigs per point); 0, females skin tested 41 weeks after infection (mean of 14 to 17 guinea pigs per point). Bars indicate standard error.
I
DAYS AFTER INFECTION
FIG. 4. Growth of L. monocytogenes in the spleens of normal guinea pigs. Each point represents the geometric mean of five to ten guinea pigs. Bars indicate standard error. HOURS AFTER CHALLENGE first apparent on the 4th day. Therefore, we 5. Growth of a challenge dose of Listeria given wished to challenge animals before or after they 48 FIG. h or 7 days after a primary Listeria infection. developed DH. Symbols: 0, uninfected controls; 0, Listeria-infected The results for guinea pigs challenged 7-days guinea pigs. Each point represents the geometric after infection are shown in the bottom half of mean offive guinea pigs. Bars indicate standard error.
VOL. 11, 1975
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IMMUNITY TO LISTERIA
given 2 weeks after primary immunization. In the previous two experiments, one could not determine if the bacteria detected in the spleens of the experimental animals represented Listeria from the challenge dose or were residual bacteria from the primary infection. Therefore, we challenged guinea pigs 2 weeks after a primary immunization when no Listeria can be detected in the spleens of infected animals (Fig. 4). For both sexes, the experimental animals had significantly lower numbers of Listeria 24 and 48 h after challenge (Fig. 6, top). For females, the P values were P < 0.01 at 24 h and P < 0.05 at 48 h; for males, they were P < 0.05 at 24 h and P < 0.01 at 48 h. (iv) Growth of an homologous challenge given 41 weeks or 46 weeks after primary immunization. Figure 3 indicates that only low levels of DH were measurable 41 weeks (females) or 46 weeks (males) after infection. Resistance to re-infection in these animals was determined by challenging them 24 h after the last skin test. The results (Fig. 6, bottom) show that none of the infected animals were resistant to re-infection. DISCUSSION The growth of Listeria in the spleens of intracardially infected guinea pigs (Fig. 4) differs from that reported for intravenously infected rats (16) or mice (29). Although there is a change in the rate of multiplication between 24 and 48 h postinfection in Listeria-infected rats and mice, there is no sudden drop in the number of Listeria like that observed in infected guinea pigs. The temporal relationship between the development of ACR and DH also appears to be different from that reported for other infections (3-5, 17, 18, 21) in which the onset of DH precedes or parallels the expression of ACR. The results shown in Fig. 5 (top) suggest that guinea pigs infected with Listeria may develop ACR by day 2 of infection, whereas the results of Fig. 1 indicate that DH is first apparent on day 4 of infection. Since ACR and DH do not parallel each other in the guinea pig system, we are led to conclude that ACR does not depend upon the development of DH in this species. Our interpretation of these data must, however, be viewed with caution; DH appears only 2 days after onset of ACR, and there remains the possibility that this temporal difference may only reflect differences in terms of sensitivity between skin testing hnd bacterial inactivation as measures of cell-mediated immunity. Unpublished findings obtained in this laboratory have shown that purified peritoneal lymphocytes harvested from Listeria-infected
HOURS AFTER CHALLENGE
FIG. 6. Growth of a challenge dose of Listeria given 2, 41, or 46 weeks after a primary Listeria infection. Symbols: 0, uninfected controls; 0, Listeria-infected guinea pigs. Each point represents the geometric mean offive guinea pigs. Bars indicate standard error. guinea pigs, as early as 3 days postinfection, are capable of releasing mitogenic factor or undergoing blastogenesis, in the presence of Listeria antigen. Peritoneal lymphocytes from infected animals did not, however, uniformly undergo blastogenesis as measured by incorporation of tritiated thymidine until day 7 postinfection, at which time DH was likewise uniformly demonstrable. Use of this relatively sensitive in vitro assay to establish onset of cell-mediated immunity appears to be inadequate for use in attempting to further dissect in a definitive manner the relationship or lack thereof between ACR and DH. Additional in vitro studies utilizing intracellular killing of Listeria by activated macrophages have been initiated with the hope of establishing the onset of in vivo macrophage activation, which might be correlated with the relatively early in vivo development of ACR measurable by day 2 postinfection. Although there is a definite difference between infected and control animals 24 h after challenge (Fig. 5, top), one may ask if the lower number of Listeria detected in the infected
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HALLIBURTON AND BLAZKOVEC
animals is really an indication of ACR. The number of Listeria found in the spleens of infected animals 24 h after challenge, a time equivalent to 3 days after the primary infection, is no lower than the number of Listeria found in infected animals which were not challenged (Fig. 4). Likewise, when animals were challenged 7 days after infection, the number of Listeria recovered 24 h later (Fig. 5, bottom) is not less than the number of bacteria detected 9 days after a primary infection (Fig. 4). It is possible that the lower number of Listeria found in the spleens of infected animals reflects uptake of the bacteria by other organs or a blockade of the reticuloendothelial system. Another explanation is suggested by the work of Collins (2), who infected mice with Salmonella typhimurium C5 and then challenged them with S. typhimurium SM" at various times after infection. In every case, the re-infecting organisms SMH were inactivated even though the residual C5 organisms were not. A similar inactivation of the challenge organisms, but not the residual infecting organisms, may occur in guinea pigs infected with Listeria and then challenged with the same organism. In this study, a low dose of Listeria was used both for infection and for challenge, and no distinction could be made between the challenge population and the residual infecting population. Perhaps a more definitive test for the presence of ACR would be to use a heterologous organism, an antibiotic-resistant strain, or a much higher dose of Listeria as the challenge organism. These studies are presently in progress. The preliminary findings presented in this paper, in addition to experimental studies presently in progress, strongly suggest major differences between species in terms of host resistance to Listeria. Results obtained using the guinea-pig Listeria system cannot be directly correlated and compared with findings obtained with the mouse system, which has been the most commonly used species for studies of this nature. Although results obtained from passive transfer studies carried out with mice provide evidence that the nonspecific aspect of ACR is indeed dependent upon interaction between sensitized lymphocytes and specific antigen (21), similar transfer studies must likewise be done with the guinea pig model. Studies of this nature would serve to establish whether our present findings pertaining to rapid inactivation of Listeria in animals infected 48 h prior to challenge constitute evidence for a lymphocytemediated phenomenon and that rapid inactivation within the spleen cannot be ascribed to
INFECT. IMMUN.
leukocytosis, i.e., recruitment of polymorphonuclear leukocytes and normal macrophages into inflammatory foci. The loss of resistance to re-infection seen in males challenged at 46 weeks and in females challenged at 41 weeks (Fig. 6, bottom) is not surprising. This loss is similar to the decay of immunity reported in other infections (1, 6, 10, 17). Persistence of ACR is thought to depend upon the persistence of antigen, and no Listeria could be detected in the spleens of infected animals after the first 2 weeks of infection. Loss of resistance probably occurs earlier than 41 or 46 weeks, and this possibility could be investigated by challenging animals at other intervals after infection. Sexual dimorphism in both antibodymediated and cell-mediated immune responses has been shown for a number of species (9). In most cases females produce higher and more sustained responses than males. One of the major purposes of characterizing the onset and development of cell-mediated immunity to Listeria in the guinea pig is to utilize ACR and delayed-type cutaneous hypersensitivity induced with Listeria for systemic and local immunopotentiation of tumor immunity. For this reason, in most experiments which constitute the present study, equal numbers of males and females were used. Although the onset of DH was the same for both males and females, males had consistently higher levels of cutaneous reactivity (Fig. 1). Differences between the sexes were statistically significant 5, 6, and 7 weeks after infection. Tolderlund et al. (27, 28) found that high levels of tuberculin sensitivity could be maintained in male guinea pigs infected with BCG if the animals were repeatedly skin tested. In the present study, DH reactions 3 or more weeks after infection were measured by using animals which had been skin tested at least once before. Thus, whereas repeated skin testing might account for the persistence of high levels of DH in males, it did not have the same effect on females. ACKNOWLEDGMENTS This investigation was supported by grant no. 5-R22-AI08608 from the United States-Japan Cooperative Medical Science Program administered by the U.S. Department of Health, Education, and Welfare; by funds from General Research Support Grant no. G-386-17 to the University of Wisconsin Medical School from the Public Health Service, Division of Research Facilities and Resources, Bethesda, Md.: by funds from Training Grant no. A100451 from the Public Health Service, National Institute of Allergy and Infectious Diseases; and by Public Health Service Predoctoral Fellowship 5-FO1-GM-41315 from the National Institute of General Medical Sciences.
IMMUNITY TO LISTERIA
VOL. 11, 1975 LITERATURE CITED
1: Blanden, R. V., G. B. Mackaness, and F. M. Collins.
2.
3.
4.
5.
6.
7.
8.
9. 10. 11.
12.
13. 14.
1966. Mechanisms of acquired resistance in mouse typhoid. J. Exp. Med. 124:55-600. Collins, F. M. 1971. Mechanisms in antimicrobial immunity. J. Reticuloendothel. Soc. 10:58-99. Collins, F. M., and G. B. Mackaness. 1968. Delayed hypersensitivity and Arthus reactivity in relation to host resistance in salmonella-infected mice. J. Immunol. 101:830-845. Collins, F. M., and G. B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antituberculous immunity. I. Tuberculin sensitivity and resistance to reinfection in BCG-vaccinated mice. Cell. Immunol. 1:253-265. Collins, F. M., and G; B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antituberculous immunity. II. Effect of adjuvant on the allergenicity and immunogenicity of heat-killed tubercle bacilli. Cell. Immunol. 1:266-275. Collins, F. M., G. B. Mackaness, and R. V. Blanden. 1966. Infection-immunity in experimental salmonellosis. J. Exp. Med. 124:601-619. David, J. R., and R. R. David. 1972. Cellular hypersensitivity and immunity. Inhibition of macrophage migration and the lymphocyte mediators. Progr. Allergy 16:300-449. Dumonde, D. C., D. A. Page, M. Matthew, and R. A. Wolstencroft. 1972. Role of lymphocyte activation products (LAP) in cell-mediated immunity. I. Preparation and partial purification of guinea-pig LAP. Clin. Exp. Immunol. 10:25-47. Goble, F. C., and E. A. Konopka. 1973. Sex as a factor in infectious disease. Trans. N.Y. Acad. Sci. 35:325-346. Halliburton, B. L., and R. D. Hinsdill. 1972. Recall of acquired cellular resistance in mice by antigens from killed Brucella. Infect. Immunity 5:42-47. Hinsdill, R. D., and D. T. Berman. 1967. Antigens of Brucella abortus. I. Chemical and immunoelectrophoretic characterization. J. Bacteriol. 93:544-549. Koster, F. T., D. D. McGregor, and G. B. Mackaness. 1971. The mediator of cellular immunity. II. The migration of immunologically committed lymphocytes into inflammatory exudates. J. Exp. Med. 133:400-409. Lane, F. C., and E. R. Unanue. 1972. Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135:1104-1112. McGregor, D. D., H. H. Hahn, and G. B. Mackaness. 1973. The mediator of cellular immunity. V. Development of cellular resistance to infection in thymecto-
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mized irradiated rats. Cell. Immunol. 6:186-199. 15. McGregor, D. D., and F. T. Koster. 1971. The mediator of cellular immunity. IV. Cooperation between lymphocytes and mononuclear phagocytes. Cell. Immunol. 2:317-325. 16. McGregor, D. D., F. T. Koster, and G. B. Mackaness. 1971. The mediator of cellular immunity. I. The life-span and circulation dynamics of the immunologically committed lymphocyte. J. Exp. Med. 133:389-399. 17. Mackaness, G. B. 1962. Cellular resistance to infection. J. Exp. Med. 116:381-406. 18. Mackaness, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105-119. 19. Mackaness, G. B. 1967. The relationship of delayed hypersensitivity to acquired cellular resistance. Brit. Med. Bull. 23:52-54. 20. Mackaness, G. B. 1968. The immunology of antituberculosis immunity. Amer. Rev. Resp. Dis. 97:337-344. 21. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973-992. 22. Miki, K., and G. B. Mackaness. 1964. The passive transfer of acquired resistance to Listeria monocytogenes. J. Exp. Med. 120:93-103. 23. Neiburger, R. G., G. P. Youmans, and A. S. Youmans. 1973. Relationship between tuberculin hypersensitivity and cellular immunity to infection in mice vaccinated with viable attenuated mycobacterial cells or with mycobacterial ribonucleic acid preparations. Infect. Immunity 8:42-47. 24. North, R. J. 1972. The action of cortisone acetate on cell-mediated immunity to infection; histogenesis of the lymphoid cell response and selective elimination of committed lymphocytes. Cell. Immunol. 3:501-515. 25. Raffel, S. 1948. The components of the tubercle bacillus responsible for the delayed type of "infectious" allergy. J. Infect. Dis. 82:267-293. 26. Rich, A. R. 1951. The pathogenesis of tuberculosis. Charles C Thomas, Springfield, Ill. 27. Tolderlund, K., M. W. Bentzon, K. Bunch-Christensen, B. Mackeprang, J. Guld, and J. Waaler. 1967. BCGinduced allergy and immunity in guinea-pigs during the first year after vaccination. Bull. W.H.O. 36:747-758. 28. Tolderlund, L., K. Bunch-Christensen, and J. Guld. 1967. Duration of allergy and immunity in BCG-vaccinated guinea pigs. Bull. W.H.O. 36:759-769. 29. Tripathy, S. P., and G. B. Mackaness. 1969. The effect of cytotoxic agents on the primary immune response to Listeria monocytogenes. J. Exp. Med. 130:1-16.
INFECrION AND IMMUNrrY, Jan. 197f5 D. 1-7
Copyright i 1975 American Society for Microbiology
Delayed Hypersensitivity and Acquired Cellular Resistance in Guinea Pigs Infected with Listeria monocytogenes BARBARA L. HALLIBURTON AND A. A. BLAZKOVEC* Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 16 August 1974
Randomly bred pigs of both sexes were injected intracardially with one-half of 50% lethal dose of Listeria monocytogenes. When infected animals were skin tested with 30 Ag of a water-soluble extract of sonically disrupted Listeria, both males and females had uniformly detectable levels of delayed hypersensitivity (DH) 4 days after infection. In males, cutaneous hypersensitivity to Listeria antigens reached a peak on day 5 or 6 of infection, and high levels of DH persisted through the 7th week. In females, DH reached a peak on day 6 or 7, remained at this level through the 4th week, and then dropped sharply. Cutaneous reactivity was usually higher for males than for females, and differences between the sexes were statistically significant 5, 6, and 7 weeks after infection. Low levels of DH were still present 41 weeks (females) or 46 weeks (males) after infection. Assays to determine the number of viable Listeria present in spleen homogenates indicated that bacterial multiplication occurred only during the first 24 h of infection. The number of Listeria declined steadily thereafter, and by day 13 no bacteria could be recovered from the spleens of infected animals. Spleen assays indicated that Listeria-infected animals of both sexes were resistant to a small challenge dose of Listeria given 48 h, 7 days, or 2 weeks after the primary infection. Resistance to re-infection was absent in females challenged at 41 weeks and in males challenged at 46 weeks. a
Mackaness and co-workers (2, 19, 20) believe that the development of acquired cellular resistance (ACR) depends upon the development of delayed hypersensitivity (DH). Others (23, 25, 26) disagree, however, and the exact relationship between DH and ACR is not known. Listeria monocytogenes infections in mice (13, 17, 18, 21, 22, 24) and rats (12, 14-16) have been used extensively in studies on the development and mechanisms of ACR. On the other hand, guinea pigs have been widely used in studies on the production and characterization of lymphokines, the soluble mediators of DH (7, 8). The feasibility of using a Listeria infection in guinea pigs for in vivo and in vitro investigations of both ACR and DH has not been explored. This paper reports the results of in vivo studies on the development and persistence of DH and ACR in Listeria-infected guinea pigs. (The research described in this paper was submitted in part to the Graduate School of the University of Wisconsin, Madison, by B. L. H. in partial "l-fillmpnt of the requirements for the Ph.D. ciegrep in Medical Microbioloev.) MATERIALS AND METHODS Animals. Randomly bred albino guinea pigs of
both sexes were obtained from local suppliers. The animals weighed 500 to 700 g at the time of infection. Bacteria. L. monocytogenes was provided by D. W. Smith (Department of Medical Microbiology, University of Wisconsin, Madison). The culture was guinea pig-passaged four times, and a fresh isolate was recovered from the spleen of an infected animal. A subculture of the isolate was grown up on a brain heart infusion (BHI) agar slant (Difco Laboratories, Detroit, Mich.) and used to inoculate 75 ml of BHI broth. The broth culture was incubated on a shaker at 37 C until the bacteria were in late log phase (optical density, 0.3 at 615 nm). The culture was then centrifuged at 1,000 x g for 15 min and resuspended in 75 ml of BHI broth. Samples (1 ml) of this suspension were stored at -70 C (frozen stock cultures). The 50% lethal dose for intracardially injected Listeria was 1.2 x 106. Immunizations. BHI broth was inoculated with a BHI agar slant subculture of the frozen stock. When the broth culture was in late log phase, a portion of it was centrifuged at 1,000 x g for 15 min. The pellet was washed once and then diluted with sterile saline. Approximately one-half of a 50% lethal dose in a total volume of 2 ml was injected intracardially into each guinea pig. The exact number of viable Listeria injected was determined by plate counts of the immunizing suspension. Antigen. The water-soluble extract of sonically disrupted Listeria was prepared by using a modification of the method of Hinsdill and Berman (11). Six
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HALLIBURTON AND BLAZKOVEC
liters of BHI broth (500 mi/flask) was inoculated with agar slant subcultures of the frozen stock and incubated on a shaker at 37 C. After 18 h, phenol was added at a final concentration of 5% (wt/vol), and the flasks were incubated for a further 8 h. The killed bacteria were collected by centrifugation at 7,000 x g for 30 min at 4 C and washed once with sterile saline and three times with sterile distilled water. The washed cells were suspended in enough distilled water to give a final volume of 50 ml and disrupted by sonic oscillation. The disrupted cells were centrifuged at 18,400 x g for 30 min at 4 C, and the supernatant was set aside. The cellular debris was suspended in 50 ml of distilled water and centrifuged as above. The two supernatants were combined and filtered (Whatman no. 1 filter paper). Samples (5 ml) were placed in small vials and lyophilized. The vials were sealed while under vacuum and stored in a desiccator at 4 C. Skin tests. Guinea pigs were injected intradermally on the shaved dorsal flank with 30 qg of the water-soluble extract in 0.1 ml of saline. The skin tests were read at 2, 4, 8, 12, 24, and 48 h. Cutaneous reactivity was measured by using three parameters: (i) degree of erythema, (ii) mean diameter of erythema, and (iii) changes in double skin thickness at the reaction site. The degree of erythema was scored arbitrarily as described in Table 1. Induration was measured using a "Schnelltester" (System Kr6plin, Type A, 2T, H. C. Kr6plin, Schliictern, Hessen, Germany). Each value given in the text represents the double skin thickness of the reaction site minus the mean of normal skin measured on either side of the test site. Spleen assays. The number of viable Listeria in BHI broth homogenates of spleen was determined by the method of Mackaness (17). Appropriate decimal dilutions were plated in duplicate on well-dried BHI plates. Colonies were counted after 24 to 36 h of incubation at 37 C. Experimental design. For studies on DH, two separate experiments were run. In one, 50 male guinea pigs were infected with Listeria. Groups of six were skin tested at 24-h intervals for the first 7 days and then at weekly intervals for the next 8 weeks. These animals were also skin tested at 12, 16, 33, and 46 weeks after infection. In the second experiment, 50 females were infected, and groups of six were skin tested at 24-h intervals for the first week and then at weekly intervals for the next 6 weeks. These same females were also skin tested at 12, 16, 33, and 41 weeks. The values for animals skin tested 7 or 10 days
INFECT. IMMUN.
after infection include pooled data from other experiments. For challenge experiments, guinea pigs were infected, and then at various times after immunization the infected animals and an equal number of uninfected control animals were challenged with one-half of a 50% lethal dose of Listeria. Spleen counts were run at 0, 24, and 48 h on five experimentals and five controls. For zero time counts, guinea pigs were killed 30 to 60 min after the challenge infection. In general, animals of one sex were assayed one week, and animals of the other sex were assayed the following week.
RESULTS Delayed hypersensitivity. (i) General trends. Figure 1 shows the cutaneous reactivity present 24 h after skin testing. According to all three parameters, both males and females had uniformly detectable DH to Listeria antigens 4 days after infection. In males, the response peaked at 5 to 6 days, dropped at 7 and 10 days, and then remained fairly steady through 7 weeks. The readings taken at 8 weeks and later were generally lower and fluctuated more than the previous readings. DH reactions in females were generally lower than those in males from day 4 to day 7, and the peak response in females occurred 1 to 2 days later, on day 6 or 7. The degree and mean diameter of erythema remained fairly constant from 7 days to 4 weeks, whereas the mean increase in double skin thickness varied considerably. However, at 5 weeks, according to all three parameters, the DH response dropped sharply and generally remained at very low levels for the remaining skin tests. (ii) Sexual dimorphism. During the first 4
TABLE 1. Scoring for degree of erythema Score
0 1 2 3 4 5 6
Degree of erythema
Trace Light pink Pink Dark pink + Erythema + + Erythema
+ Hemorrhage
FIG. 1. Development and persistence of DH in Listeria-infected guinea pigs. Responses 24 h after skin testing: *, males; 0, females. Each point represents the mean of 6 to 28 guinea pigs. Bars indicate standard error.
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IMMUNITY TO LISTERIA
weeks of skin testing, the differences between the responses of males and females were not significantly different when analyzed using the Student's t test. At 5 weeks, the males were significantly more responsive in terms of degree of erythema, P < 0.001, for diameter of erythema, P < 0.01, and for mean increase in double skin thickness, P < 0.001. At 6 weeks P values were likewise significantly in favor of the males, with P < 0.001, P < 0.01, and P < 0.01, respectively; at 7 weeks P values were P < 0.01, P < 0.02, and P < 0.01, respectively, with males still responding more strongly than females. At 12 weeks the females were significantly higher for degree of erythema, P < 0.001, and mean increase in double skin thickness, P < 0.01. (iii) Development of the peak response. Figure 2 shows the development of cutaneous reactivity in males' skin tested 5 days after infection and in females' skin tested 7 days after
HOURS AFTER SKIN TESTING
FIG. 2. Development of cutaneous reactivity in Listeria-infected guinea pigs. Symbols: 0, males skin tested 5 days after infection (mean of six guinea pigs per point); 0, females skin tested 7 days after infection (mean of 13 to 28 guinea pigs per point). Bars indicate standard error.
3
infection. The 5- and 7-day readings were chosen to represent the peak DH response to Listeria antigens. If one considers all three parameters, cutaneous reactivity was minimal 2 and 4 h after skin testing and then rose to a peak at 24 h. Except for the mean increase in double skin thickness at 4 h, all values for the males were consistently higher than values for the females. Significant differences in the degree of erythema (Fig. 2, top) occurred at 2 h, P < 0.001; 8 h, P < 0.02; 24 h, P < 0.05; and 48 h, P < 0.02. The mean diameter of erythema (Fig. 2, middle) was significantly greater in males at 12 h, P < 0.05, and at 48 h, P < 0.01. There were no significant differences between males and females in the mean increase in double skin thickness (Fig. 2, bottom) at any time. (iv) Persistence of DH. The longest time interval between infection and skin testing was 46 weeks for males and 41 weeks for females. Figure 3 shows the development of cutaneous reactivity in animals skin tested at these times. The results indicate that a very low or minimal level of DH to Listeria antigens persisted almost 1 year after infection. There were no significant differences between males and females for any parameter 24 h after skin testing. Females, however, had significantly higher cutaneous reactivity 4 h after skin testing. The P values were P < 0.05 for degree of erythema, P < 0.01 for mean diameter of erythema, and P < 0.001 for mean increase in double skin thickness. This higher cutaneous reactivity measurable so soon after skin testing probably represents an Arthus component. Although no antibody titrations were carried out, these results suggest that antibody levels directed toward Listeria antigens were higher in females than in males. ACR. (i) Growth of Listeria in the spleens of normal guinea pigs. Figure 4 shows the combined results of four separate experiments. In two experiments, spleen assays on five animals per group were run at zero time and then at daily intervals for the next 5 days. In two other experiments, assays were run at zero time and then at 3-day intervals beginning 3 or 4 days after infection. Multiplication of Listeria occurred only within the first 24 h of infection. Thereafter, the number of bacteria steadily decreased, and by day 13 none could be detected. (ii) Growth of an homologous challenge given 48 h or 7 days after primary immunization. The rapid inhibition of Listeria multiplication in vivo (Fig. 4) suggests that ACR may develop within the first 48 h of infection, whereas the data in Fig. 1 indicate that DH is
4
INFECTr. IMMUN .
HALLIBURTON AND BLAZKOVEC
Fig. 5. For both sexes, the differences between experimentals and controls 24 h after challenge are significant with P < 0.001. At 48 h, previously infected females had significantly lower numbers of Listeria, P < 0.01; for males, however, no significant difference was shown to exist between the controls and experimentals, P
0.50. (iii) Growth of an homologous challenge
FIG. 3. Development of cutaneous reactivity in Listeria-infected guinea pigs. Symbols: *, males skin tested 46 weeks after infection (mean of 12 to 21 guinea pigs per point); 0, females skin tested 41 weeks after infection (mean of 14 to 17 guinea pigs per point). Bars indicate standard error.
I
DAYS AFTER INFECTION
FIG. 4. Growth of L. monocytogenes in the spleens of normal guinea pigs. Each point represents the geometric mean of five to ten guinea pigs. Bars indicate standard error. HOURS AFTER CHALLENGE first apparent on the 4th day. Therefore, we 5. Growth of a challenge dose of Listeria given wished to challenge animals before or after they 48 FIG. h or 7 days after a primary Listeria infection. developed DH. Symbols: 0, uninfected controls; 0, Listeria-infected The results for guinea pigs challenged 7-days guinea pigs. Each point represents the geometric after infection are shown in the bottom half of mean offive guinea pigs. Bars indicate standard error.
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5
IMMUNITY TO LISTERIA
given 2 weeks after primary immunization. In the previous two experiments, one could not determine if the bacteria detected in the spleens of the experimental animals represented Listeria from the challenge dose or were residual bacteria from the primary infection. Therefore, we challenged guinea pigs 2 weeks after a primary immunization when no Listeria can be detected in the spleens of infected animals (Fig. 4). For both sexes, the experimental animals had significantly lower numbers of Listeria 24 and 48 h after challenge (Fig. 6, top). For females, the P values were P < 0.01 at 24 h and P < 0.05 at 48 h; for males, they were P < 0.05 at 24 h and P < 0.01 at 48 h. (iv) Growth of an homologous challenge given 41 weeks or 46 weeks after primary immunization. Figure 3 indicates that only low levels of DH were measurable 41 weeks (females) or 46 weeks (males) after infection. Resistance to re-infection in these animals was determined by challenging them 24 h after the last skin test. The results (Fig. 6, bottom) show that none of the infected animals were resistant to re-infection. DISCUSSION The growth of Listeria in the spleens of intracardially infected guinea pigs (Fig. 4) differs from that reported for intravenously infected rats (16) or mice (29). Although there is a change in the rate of multiplication between 24 and 48 h postinfection in Listeria-infected rats and mice, there is no sudden drop in the number of Listeria like that observed in infected guinea pigs. The temporal relationship between the development of ACR and DH also appears to be different from that reported for other infections (3-5, 17, 18, 21) in which the onset of DH precedes or parallels the expression of ACR. The results shown in Fig. 5 (top) suggest that guinea pigs infected with Listeria may develop ACR by day 2 of infection, whereas the results of Fig. 1 indicate that DH is first apparent on day 4 of infection. Since ACR and DH do not parallel each other in the guinea pig system, we are led to conclude that ACR does not depend upon the development of DH in this species. Our interpretation of these data must, however, be viewed with caution; DH appears only 2 days after onset of ACR, and there remains the possibility that this temporal difference may only reflect differences in terms of sensitivity between skin testing hnd bacterial inactivation as measures of cell-mediated immunity. Unpublished findings obtained in this laboratory have shown that purified peritoneal lymphocytes harvested from Listeria-infected
HOURS AFTER CHALLENGE
FIG. 6. Growth of a challenge dose of Listeria given 2, 41, or 46 weeks after a primary Listeria infection. Symbols: 0, uninfected controls; 0, Listeria-infected guinea pigs. Each point represents the geometric mean offive guinea pigs. Bars indicate standard error. guinea pigs, as early as 3 days postinfection, are capable of releasing mitogenic factor or undergoing blastogenesis, in the presence of Listeria antigen. Peritoneal lymphocytes from infected animals did not, however, uniformly undergo blastogenesis as measured by incorporation of tritiated thymidine until day 7 postinfection, at which time DH was likewise uniformly demonstrable. Use of this relatively sensitive in vitro assay to establish onset of cell-mediated immunity appears to be inadequate for use in attempting to further dissect in a definitive manner the relationship or lack thereof between ACR and DH. Additional in vitro studies utilizing intracellular killing of Listeria by activated macrophages have been initiated with the hope of establishing the onset of in vivo macrophage activation, which might be correlated with the relatively early in vivo development of ACR measurable by day 2 postinfection. Although there is a definite difference between infected and control animals 24 h after challenge (Fig. 5, top), one may ask if the lower number of Listeria detected in the infected
6
HALLIBURTON AND BLAZKOVEC
animals is really an indication of ACR. The number of Listeria found in the spleens of infected animals 24 h after challenge, a time equivalent to 3 days after the primary infection, is no lower than the number of Listeria found in infected animals which were not challenged (Fig. 4). Likewise, when animals were challenged 7 days after infection, the number of Listeria recovered 24 h later (Fig. 5, bottom) is not less than the number of bacteria detected 9 days after a primary infection (Fig. 4). It is possible that the lower number of Listeria found in the spleens of infected animals reflects uptake of the bacteria by other organs or a blockade of the reticuloendothelial system. Another explanation is suggested by the work of Collins (2), who infected mice with Salmonella typhimurium C5 and then challenged them with S. typhimurium SM" at various times after infection. In every case, the re-infecting organisms SMH were inactivated even though the residual C5 organisms were not. A similar inactivation of the challenge organisms, but not the residual infecting organisms, may occur in guinea pigs infected with Listeria and then challenged with the same organism. In this study, a low dose of Listeria was used both for infection and for challenge, and no distinction could be made between the challenge population and the residual infecting population. Perhaps a more definitive test for the presence of ACR would be to use a heterologous organism, an antibiotic-resistant strain, or a much higher dose of Listeria as the challenge organism. These studies are presently in progress. The preliminary findings presented in this paper, in addition to experimental studies presently in progress, strongly suggest major differences between species in terms of host resistance to Listeria. Results obtained using the guinea-pig Listeria system cannot be directly correlated and compared with findings obtained with the mouse system, which has been the most commonly used species for studies of this nature. Although results obtained from passive transfer studies carried out with mice provide evidence that the nonspecific aspect of ACR is indeed dependent upon interaction between sensitized lymphocytes and specific antigen (21), similar transfer studies must likewise be done with the guinea pig model. Studies of this nature would serve to establish whether our present findings pertaining to rapid inactivation of Listeria in animals infected 48 h prior to challenge constitute evidence for a lymphocytemediated phenomenon and that rapid inactivation within the spleen cannot be ascribed to
INFECT. IMMUN.
leukocytosis, i.e., recruitment of polymorphonuclear leukocytes and normal macrophages into inflammatory foci. The loss of resistance to re-infection seen in males challenged at 46 weeks and in females challenged at 41 weeks (Fig. 6, bottom) is not surprising. This loss is similar to the decay of immunity reported in other infections (1, 6, 10, 17). Persistence of ACR is thought to depend upon the persistence of antigen, and no Listeria could be detected in the spleens of infected animals after the first 2 weeks of infection. Loss of resistance probably occurs earlier than 41 or 46 weeks, and this possibility could be investigated by challenging animals at other intervals after infection. Sexual dimorphism in both antibodymediated and cell-mediated immune responses has been shown for a number of species (9). In most cases females produce higher and more sustained responses than males. One of the major purposes of characterizing the onset and development of cell-mediated immunity to Listeria in the guinea pig is to utilize ACR and delayed-type cutaneous hypersensitivity induced with Listeria for systemic and local immunopotentiation of tumor immunity. For this reason, in most experiments which constitute the present study, equal numbers of males and females were used. Although the onset of DH was the same for both males and females, males had consistently higher levels of cutaneous reactivity (Fig. 1). Differences between the sexes were statistically significant 5, 6, and 7 weeks after infection. Tolderlund et al. (27, 28) found that high levels of tuberculin sensitivity could be maintained in male guinea pigs infected with BCG if the animals were repeatedly skin tested. In the present study, DH reactions 3 or more weeks after infection were measured by using animals which had been skin tested at least once before. Thus, whereas repeated skin testing might account for the persistence of high levels of DH in males, it did not have the same effect on females. ACKNOWLEDGMENTS This investigation was supported by grant no. 5-R22-AI08608 from the United States-Japan Cooperative Medical Science Program administered by the U.S. Department of Health, Education, and Welfare; by funds from General Research Support Grant no. G-386-17 to the University of Wisconsin Medical School from the Public Health Service, Division of Research Facilities and Resources, Bethesda, Md.: by funds from Training Grant no. A100451 from the Public Health Service, National Institute of Allergy and Infectious Diseases; and by Public Health Service Predoctoral Fellowship 5-FO1-GM-41315 from the National Institute of General Medical Sciences.
IMMUNITY TO LISTERIA
VOL. 11, 1975 LITERATURE CITED
1: Blanden, R. V., G. B. Mackaness, and F. M. Collins.
2.
3.
4.
5.
6.
7.
8.
9. 10. 11.
12.
13. 14.
1966. Mechanisms of acquired resistance in mouse typhoid. J. Exp. Med. 124:55-600. Collins, F. M. 1971. Mechanisms in antimicrobial immunity. J. Reticuloendothel. Soc. 10:58-99. Collins, F. M., and G. B. Mackaness. 1968. Delayed hypersensitivity and Arthus reactivity in relation to host resistance in salmonella-infected mice. J. Immunol. 101:830-845. Collins, F. M., and G. B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antituberculous immunity. I. Tuberculin sensitivity and resistance to reinfection in BCG-vaccinated mice. Cell. Immunol. 1:253-265. Collins, F. M., and G; B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antituberculous immunity. II. Effect of adjuvant on the allergenicity and immunogenicity of heat-killed tubercle bacilli. Cell. Immunol. 1:266-275. Collins, F. M., G. B. Mackaness, and R. V. Blanden. 1966. Infection-immunity in experimental salmonellosis. J. Exp. Med. 124:601-619. David, J. R., and R. R. David. 1972. Cellular hypersensitivity and immunity. Inhibition of macrophage migration and the lymphocyte mediators. Progr. Allergy 16:300-449. Dumonde, D. C., D. A. Page, M. Matthew, and R. A. Wolstencroft. 1972. Role of lymphocyte activation products (LAP) in cell-mediated immunity. I. Preparation and partial purification of guinea-pig LAP. Clin. Exp. Immunol. 10:25-47. Goble, F. C., and E. A. Konopka. 1973. Sex as a factor in infectious disease. Trans. N.Y. Acad. Sci. 35:325-346. Halliburton, B. L., and R. D. Hinsdill. 1972. Recall of acquired cellular resistance in mice by antigens from killed Brucella. Infect. Immunity 5:42-47. Hinsdill, R. D., and D. T. Berman. 1967. Antigens of Brucella abortus. I. Chemical and immunoelectrophoretic characterization. J. Bacteriol. 93:544-549. Koster, F. T., D. D. McGregor, and G. B. Mackaness. 1971. The mediator of cellular immunity. II. The migration of immunologically committed lymphocytes into inflammatory exudates. J. Exp. Med. 133:400-409. Lane, F. C., and E. R. Unanue. 1972. Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135:1104-1112. McGregor, D. D., H. H. Hahn, and G. B. Mackaness. 1973. The mediator of cellular immunity. V. Development of cellular resistance to infection in thymecto-
7
mized irradiated rats. Cell. Immunol. 6:186-199. 15. McGregor, D. D., and F. T. Koster. 1971. The mediator of cellular immunity. IV. Cooperation between lymphocytes and mononuclear phagocytes. Cell. Immunol. 2:317-325. 16. McGregor, D. D., F. T. Koster, and G. B. Mackaness. 1971. The mediator of cellular immunity. I. The life-span and circulation dynamics of the immunologically committed lymphocyte. J. Exp. Med. 133:389-399. 17. Mackaness, G. B. 1962. Cellular resistance to infection. J. Exp. Med. 116:381-406. 18. Mackaness, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105-119. 19. Mackaness, G. B. 1967. The relationship of delayed hypersensitivity to acquired cellular resistance. Brit. Med. Bull. 23:52-54. 20. Mackaness, G. B. 1968. The immunology of antituberculosis immunity. Amer. Rev. Resp. Dis. 97:337-344. 21. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973-992. 22. Miki, K., and G. B. Mackaness. 1964. The passive transfer of acquired resistance to Listeria monocytogenes. J. Exp. Med. 120:93-103. 23. Neiburger, R. G., G. P. Youmans, and A. S. Youmans. 1973. Relationship between tuberculin hypersensitivity and cellular immunity to infection in mice vaccinated with viable attenuated mycobacterial cells or with mycobacterial ribonucleic acid preparations. Infect. Immunity 8:42-47. 24. North, R. J. 1972. The action of cortisone acetate on cell-mediated immunity to infection; histogenesis of the lymphoid cell response and selective elimination of committed lymphocytes. Cell. Immunol. 3:501-515. 25. Raffel, S. 1948. The components of the tubercle bacillus responsible for the delayed type of "infectious" allergy. J. Infect. Dis. 82:267-293. 26. Rich, A. R. 1951. The pathogenesis of tuberculosis. Charles C Thomas, Springfield, Ill. 27. Tolderlund, K., M. W. Bentzon, K. Bunch-Christensen, B. Mackeprang, J. Guld, and J. Waaler. 1967. BCGinduced allergy and immunity in guinea-pigs during the first year after vaccination. Bull. W.H.O. 36:747-758. 28. Tolderlund, L., K. Bunch-Christensen, and J. Guld. 1967. Duration of allergy and immunity in BCG-vaccinated guinea pigs. Bull. W.H.O. 36:759-769. 29. Tripathy, S. P., and G. B. Mackaness. 1969. The effect of cytotoxic agents on the primary immune response to Listeria monocytogenes. J. Exp. Med. 130:1-16.
