INFECTION AND IMMUNITY, Mar. 1990,
Vol. 58, No. 3
p. 654-658
0019-9567/90/030654-05$02.00/0 Copyright C) 1990, American Society for Microbiology
Expression of Systemic Protection and Delayed-Type Hypersensitivity to Listeria monocytogenes Is Mediated by Different T-Cell Subsets JORY R. BALDRIDGE,' RONALD A. BARRY,"2 AND DAVID J. HINRICHSl2* Chiles Research Institute, Providence Medical Center, Portland, Oregon 97213,1 and Veterans Administration Medical Center, Portland, Oregon 972072 Received 1 August 1989/Accepted 1 December 1989
The relationship between acquired cellular resistance and delayed-type hypersensitivity (DTH) during the immune response to Listeria monocytogenes was investigated. Treatment of concanavalin A-stimulated Listeria-immune spleen cells with anti-CD8 antibody plus complement abrogated the adoptive transfer of systemic antilisterial immunity but had no effect on the transfer of DTH. In contrast, in vitro depletion of the CD4+ T-cell subset eliminated the ability of culture-activated cells to transfer DTH reactivity but did not interfere with the adoptive transfer of protection. In vivo, the infusion of anti-CD8 antibody inhibited the expression of both actively and adoptively transferred protection but did not influence the development of DTH skin test reactivity to L. monocytogenes antigens. In vivo depletion of the CD4+ T-cell subset eradicated the DTH response, with only minor influence of the protective anti-Listeria response. The apparent functional dissociation of the CD4+ (DTH) and CD8+ (protection) T-cell populations was further emphasized by our findings that the adoptive transfer of protection was dependent on a cyclophosphamide-sensitive cell population, whereas DTH reactivity was mediated by a cyclophosphamide-resistant population.
of the CD4+ and the CD8+ T-cell subsets in the expression of these two independent responses.
The association of acquired cellular resistance (ACR) and delayed-type hypersensitivity (DTH) remains controversial. The initial arguments which proposed that these two phenomena are direct manifestations of the same cell population were based on observations which showed an apparent inseparability of ACR and DTH during the immune response (17-19). This association is highlighted by the need for an active infection to induce both ACR and DTH, unless special immunizing techniques are applied (4, 14, 21, 29, 31-33). Also, the kinetics of development and longevity of these responses are similar (17, 24, 26). Furthermore, both ACR and DTH are T-cell-mediated events which rely, for full expression, on the involvement and activation of macrophages (17, 19). However, the opposing view asserts that ACR and DTH are separate entities of the cell-mediated response (16, 34). This view has been strengthened in some studies by the use of specific T-cell subpopulations in adoptive transfer (7, 28) and by in vivo use of subset-specific anti-T-cell antibodies (3, 9, 20). Previous results from this laboratory established that antilisterial immunity could be adoptively transferred by the CD8+ T-cell subset. These observations were obtained by using activated immune spleen cells recovered from concanavalin A (Con A)-stimulated cultures (5). Immune spleen cells recovered from these cultures have been shown to transfer greatly enhanced and long-lived levels of immunity (1, 5); however, our initial observations did not show an increase in the level of adoptively transferred DTH reactivity (2). Collectively these findings prompted this further investigation of the association between cells mediating protection and DTH. In all experiments with adoptive transfer techniques, as well as analysis of actively immunized mice, we found that the immune response leading to DTH reactivity is completely distinct from a protective response. The distinction can be attributed to the separate involvement *
MATERIALS AND METHODS
Bacteria. Listeria monocytogenes 10403 serotype 1 was used throughout this study. It was maintained in a virulent state by repeated passage in mice and had a 50% lethal dose of approximately 4.5 x 103 bacteria for BALB/c mice. Mice. BALB/c female mice of matched ages, 5 to 9 weeks, were purchased from the Fred Hutchison Cancer Research Center, Seattle, Wash., and Jackson Laboratories, Bar Harbor, Maine. MAbs. All monoclonal antibodies (MAbs) were obtained either from concentrated hybridoma culture supernatants or from mouse ascites fluids. The hybridomas were purchased from the American Type Culture Collection. Hybridoma 19-198 produces a mouse immunoglobulin G2a (IgG2a) antibody specific for the Lyt 2.2 (CD8) glycoprotein. Hybridoma GK1.5 produces a rat IgG2b antibody specific for the L3T4 (CD4) glycoprotein. The hybridoma 30-H12 produces a rat IgG2b antibody which is specific for the mouse Thy 1.2 antigen. The hybridoma 7D2.1.4.1.5 produces a rat IgG2a antibody specific for mouse immunoglobulin heavy chains. The antibody concentrations of supernatants and ascites were determined by enzyme-linked immunosorbent assay titration and compared with known mouse or rat IgG standards. Immunizations. Mice were immunized with approximately a 0.1 50% lethal dose of viable L. monocytogenes injected in volumes of 0.2 ml into the lateral tail vein. Cell culture. Spleen cells from immunized mice were cultured for 72 h in RPMI 1640 supplemented with 5% fetal calf serum, 100 U of penicillin per ml, 100 ,ug of streptomycin sulfate per ml, 5 x 10-5 M 2-mercaptoethanol, and 1 ,ug of ConA per ml. A total of 1 x 108 cells were grown in 75-cm2 flasks with 50 ml of medium. The cells were maintained at
Corresponding author. 654
VOL. 58, 1990
DISSOCIATION OF PROTECTION AND DTH
0
INCREASED FOOTPAD THICKNESS (mm) 0.10 0.05 0.15 0.20
TABLE 1. The influence of T-cell-specific antibody in vivo on the expression of antilisterial resistance and DTH'
0.25
I, ., I, , I,,
I. NORMAL MICE
Treatment
DTH b response (SD)
Logb protection
Day 6 active Day 6 active Day 6 active
None Anti-CD4 Anti-CD8
0.37 (0.02)c 0.05 (0.02) 0.32 (0.09)c
5.46 3.17 3.57
Day 10 active Day 10 active Day 10 active
None Anti-CD4 Anti-CD8
0.19 (0.08)d 0.02 (0.01) 0.14 (0.04)d
4.78 3.78 2.24
Immune spleen cell recipient Immune spleen cell
None
0.15 (0.05)e
2.17
Immune status
Group
C' CONTROL ANTI-CD4
A
ANTi-CD8
A
ANTI-THY 1.2
C' CONTROL ANTI-CD4
~.
.
ANTI-CDS
B
ANT.THY 1.2
B. 0
1.0
2.0
3.0
655
4.0
LOG PROTECnON
FIG. 1. CD4+ culture-activated immune spleen cells adoptively transfer DTH, and CD8+ culture-activated immune spleen cells adoptively transfer protection. Six days after primary immunization, donor spleen cells were stimulated in culture with ConA for 72 h. Complement-mediated depletion of culture-activated T-cell subsets was performed with subset-specific antibody prior to adoptive transfer. Recipients were infused with 2 x 107 cells and then assessed for DTH reactivity (A) or systemic protection (B) (see Materials and Methods). The mice in this experiment were challenged with 1 x 108 HKLM in a 25-ml volume for DTH analysis. The standard deviation for mean log1o CFU per spleen for all groups was less than 0.4.
37°C in a humidified incubator with a mixture of 6% CO2 and 94% air. Determination of log protection. Groups of mice (5 mice per group) were challenged with approximately 10 50% lethal doses of viable L. monocytogenes by intravenous injection into the lateral tail vein. The spleens were removed 48 h after challenge and were individually homogenized in 4.5 ml of 1% proteose-peptone in phosphate-buffered saline. The homogenate was serially diluted, and the appropriate dilutions were plated on brain heart infusion agar. Following a 24- to 48-h incubation, the CFU were counted and the log10 CFU per spleen was determined. Protection (log protection) was assessed by subtracting the mean log10 CFU per spleen of the test groups from the mean log1o CFU per spleen of the normal control group. Delayed-type hypersensitivity response. Standard footpad skin tests were used to assay DTH responsiveness. The right hind footpads of test mice were measured with dial calipers at time 0 and then injected with approximately 1 x 108 heat-killed L. monocytogenes (HKLM) in a volume of 25 ,ul (Fig. 1) or 50 p.l (Table 1; Fig. 2) of saline. The increase in footpad thickness was measured 24 h later. CY treatment. Cyclophosphamide (CY) (Sigma Chemical Co. St. Louis, Mo.) was reconstituted in distilled H20 immediately prior to use. Donor mice received a 150-mg/kg of body weight dose of CY by intravenous injection 1 h prior to removal of their spleens. In vivo MAb treatment. MAbs were injected intravenously into mice at concentrations of 500 to 600 p.g per injection on the day of L. monocytogenes challenge and again the following day for groups of mice being assessed for systemic protection. In groups of mice being assessed for DTH responsiveness, MAbs were infused only once, at the time of HKLM challenge. Control groups were infused with ascites containing rat IgG (from hybridoma 7D2.1.4.1.5) specific for mouse immunoglobulin heavy chains. The infusion of this
C
Anti-CD4
0.07 (0.02)
0.58
recipient Immune spleen cell recipient
Anti-CD8
0.12 (0.02)d
0.35
EISC recipient EISC recipient EISC recipient
None Anti-CD4 Anti-CD8
NT NT NT
4.78 3.05 0.13
a Data are representative of three or more separate experiments. b Assayed as described in Materials and Methods; NT = not tested. C P < 0.001 versus untreated normal mice. d p < 0.02 versus untreated normal mice. e P < 0.01 versus untreated normal mice.
ascites had no significant effect on the expression of DTH or protection (data not shown). In vitro complement-mediated cytotoxicity. Culture-activated immune spleen cells were suspended in hybridoma supernatants in an ice bath for 1 h. The MAb-treated cells were pelleted and suspended in Low-Tox-M rabbit complement (Cedar Lane Laboratories, Hornby, Ontario, Canada) diluted 1:12 in RPMI 1640 with 0.3% bovine serum albumin. Cells were then incubated for 1 h in a 37°C water bath with INCREASED FOOTPAD THICKNESS
0.05
0
_
NORMAL CONTROL
(mm)
0.15
0.10
0.20
0.25
~~~~~A
_
No CY
CY
No CY
lB
CY I
0
0.5
1.0
II 1.5
2.0
2.5
3.0
3.5
LOG PROTECTION
FIG. 2. CY treatment of donor mice inhibits adoptive transfer of protection but does not interfere with transfer of DTH. Donor mice were immunized with L. monocytogenes 6 days prior to infusion with CY. One hour after CY treatment, the spleens were removed and the immune spleen cells (1 x 108) were transferred to naive recipients which were concomitantly injected with HKLM in the footpad (A) or challenged intravenously with 10 50%o lethal doses of L. monocytogenes (B) (see Materials and Methods). The standard deviation for mean log1o CFU per spleen for all groups was less than 0.4.
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BALDRIDGE ET AL.
occasional mixing. Antibody-plus-complement treatments were conducted twice. Viable cell counts were then determined by trypan blue dye exclusion. Statistics. A two-tailed Student's t test was used to compare mean values of increased footpad thickness between control and experimental groups. RESULTS Phenotype of culture-activated cells responsible for the expression of protection versus DTH. As previously demonstrated, the adoptive transfer of protection but not DTH in antilisterial immunity can be enhanced after in vitro culture activation (2). By using this culture activation system, the phenotype of the culture-enhanced immune spleen cells (EISC) responsible for the adoptive transfer of protection and DTH was addressed. Six days postimmunization, spleen cells from immune donor mice were stimulated in culture with ConA. Following this 72-h culture period, the EISC were collected and depleted of selected T-cell subpopulations by antibody-dependent complement-mediated cytotoxicity prior to infusion into normal syngeneic recipients. The data displayed in Fig. 1 are representative of four experiments and indicate that recipients of EISC depleted of the CD8+ subset lack systemic protection yet are fully competent in their ability to express DTH. In contrast, mice receiving EISC depleted of the CD4+ subset are fully protected against systemic challenge but are unable to express DTH. Recipients of EISC depleted of all T cells were unable to resist systemic challenge or to manifest a DTH response. In vivo T-cell subset depletion by MAb infusion. To further address the phenotypes of the immune cell populations mediating protection and DTH, MAbs were directly infused into mice. Cytometric analysis of spleen cells from MAbtreated mice showed that T-cell-subset-specific depletion occurred within 24 h of MAb infusion and lasted for more than 8 days. Control spleen cell populations consisted of 80 to 85% Thy 1.2+, 43 to 47% CD4+, and 19 to 23% CD8+. Infusion of anti-Thy 1.2 MAb reduced the Thy 1.2+ population to 17.62% within 24 h and to 18.38% 8 days later. Similarly, after anti-CD4 (anti-L3T4) treatment, the CD4+ population in the spleen was reduced to 3.66% within 24 h and to 6.94% 8 days later. Treatment with MAb to the CD8 marker (anti-Lyt 2.2) decreased the CD8+ population in the spleen to 2.72% by 24 h and to 3.56% by 8 days. In the experiments presented in Table 1, actively immunized mice and recipients of immune spleen cells were infused with either anti-CD4 or anti-CD8 MAb to accomplish in vivo T-cell subset depletion. The mice in group A (Table 1) represent actively immunized test groups. Six or ten days after receiving a primary immunization (as indicated in the table), these actively immunized mice were injected with the appropriate MAb and immediately challenged with either (i) viable L. monocytogenes for assessment of systemic protection or (ii) HKLM for assessment of DTH reactivity. Similarly, recipient mice upon receiving either immune spleen cells (Table 1, group B) or EISC (Table 1, group C) were immediately infused with the appropriate MAb and challenged with either (i) viable L. monocytogenes for assessment of systemic protection or (ii) HKLM for assessment of DTH reactivity. All groups of mice that received anti-CD8 antibody exhibited a pronounced reduction in the protective immune response to challenge with viable L. monocytogenes. However, infusion of anti-CD8 MAb had no influence on the
INFECT. IMMUN.
expression of DTH reactivity for any group tested. In contrast, all immune groups treated with anti-CD4 MAb were unable to express a DTH response to HKLM. Depending on the test group, treatment with anti-CD4 antibody variably influenced the expression of protection. Inhibition of protection by treatment with anti-CD4 was pronounced in recipients of directly transferred spleen cells (Table 1, group B) and day 6 actively immunized mice (Table 1, group A). In addition, slight inhibition of protection was seen in actively immune mice 10 days after immunization (Table 1, group A) and in recipients of culture-activated spleen cells (Table 1, group C). CY treatment inhibits adoptive transfer of DTH but not protection. It has been reported that CY enhances DTH responsiveness and reduces CD8+ cell activity (15, 27). In order to determine the effect of CY on antilisterial immunity, we immunized mice with viable L. monocytogenes and 6 days later injected these mice with CY at a dose of 150 mg/kg of body weight. These CY-treated mice served as donors of spleen cells for adoptive transfer studies. The spleen cells were obtained from immune mice 1 h after CY injection and directly transferred to naive recipients. The results in Fig. 2 are representative of three separate experiments and show the DTH reactivity to HKLM (A) as well as the protective response seen in identical recipients (B). Immune spleen cells from CY-treated donors transferred identical levels of DTH responsiveness compared with immune spleen cells from untreated donors. However, immune spleen cells from CY-treated donors failed to transfer protection to naive
recipients. DISCUSSION Elimination of facultative intracellular parasites appears to depend on the development of activated macrophages (17, 19, 21, 24, 25) which emerge after stimulation with lymphocyte-derived macrophage-activating factors (10, 19). Lymphokine production by CD4+ lymphocytes is well established, and the involvement of this subset in macrophage activation and DTH reactions has led to the presumption that CD4+ lymphocytes play a significant role in the expression of protective antilisterial immunity. Although CD8+ lymphocytes have been recognized as cytotoxic cells with relevance in viral infections, a protective role for this lymphocyte subset against facultative intracellular parasites has only recently become apparent (5, 23, 27). One approach used to establish the importance of CD8+ lymphocytes in the expression of antilisterial immunity involves adoptive transfer of in vitro-selected T-cell subsets. In the rat model of listeriosis, Chen-Woan et al. (7) used peritoneal cells to show that the cellular mediators of systemic protection were W3/25-, OX8+(CD8+) T cells, while W3/25+, OX8- (CD4+) T cells were responsible for the expression of DTH reactivity. These investigators also reported that the protective activity of the CD8+ cells was enhanced when cotransferred with CD4+ cells. Previous studies in which mouse lymphocytes were used also indicated some role for the CD8+ (Lyt 2+) T-cell subset; however, these studies also showed an important, if not dominant, role for CD4+ (Lyt 1+2-) T cells (11, 13, 22). Our initial observation showing the requirement for CD8+ cells in antilisterial immunity in mice used immune spleen cells that had been stimulated in vitro prior to adoptive transfer (5). Within this transfer system, elimination of the CD4+ T-cell subset by anti-CD4 antibody plus complement treatment had no effect on the transfer of antilisterial resistance
VOL. 58, 1990
and the protective activity of the CD8+ T-cell population obtained from culture was not enhanced in the presence of CD4+ T cells. However, prior to culture, both the CD8+ and CD4+ T cells were shown to be required for full expression of adoptive protection in recipients subsequently challenged with L. monocytogenes (5). Since (i) both acquired cellular resistance and DTH are dependent on lymphokine-mediated macrophage activation and (ii) our previous results demonstrated the importance of CD8+ cells for protection, we evaluated recipients of CD8+ cells for expression of DTH to HKLM. In this study, T-cell subset depletion of EISC (Fig. 1) confirmed previous reports that the CD8+ cell population is critical for the expression of culture-enhanced adoptive protection and further showed that this same CD8+ subset does not contribute to the expression of adoptively transferred DTH. In contrast, the CD4+ population was shown to be essential for the expression of DTH but not required for the expression of adoptive protection with EISC. The second approach used to assess T-cell subset interaction in the expression of protection and DTH involved in vivo injection of MAbs specific for CD4+ or CD8+ T cells. The effect of such treatment is to cause a reduction in a specific T-cell subset. In Table 1 we again show the phenotypic dissociation of systemic protection and DTH in both adoptively transferred and actively induced immunity. In vivo administration of anti-CD8 antibody had no influence on DTH reactivity but reduced or eliminated the protective response to L. monocytogenes challenge in all cases. After infusion of anti-CD4 MAb, all DTH reactions were inhibited, while the effect on systemic protection was variable. AntiCD4 treatment only slightly inhibited adoptive transfer of protection with culture-activated immune spleen cells or actively immunized mice 10 days after primary immunization. However, this same anti-CD4 treatment significantly blocked adoptive protection in recipients of directly transferred immune spleen cells and day 6 actively immunized mice. This latter observation correlates with previous findings from this laboratory in which day 6 immune spleen cells transferred directly to naive recipients were used (5). The greatest amount of immunity directly transferred by this population of immune spleen cells is seen in the presence of both CD4+ and CD8+ T-cell populations (5; Table 1). Although cells of the CD8+ T-cell subset dominate the expression of systemic protection, CD4+ cells appear to be essential for DTH responsiveness and (perhaps relatedly) for the development of acquired cellular resistance. Similar conclusions were reached by Mielke et al. (20), who also suggested that when the numbers of immune CD8+ cells are low, the CD4+ cells are required for CD8+ cell amplification via interleukin production. Berche et al. (3) also found similar results, suggesting that the CD8+ subset was the dominant T-cell population in protection, while CD4+ T cells mediated the DTH response. Also, Czuprynski et al. (9) selectively depleted CD4+ T cells by MAb treatment of mice, which abolished the DTH response and also slightly inhibited resistance; they concluded that the CD4+ population participates in the development of acquired cellular resistance but is not the dominant subset involved. It would appear that the CD8+ T-cell subset is the major T-cell component responsible for the expression of antilisterial immunity (3, 5, 8, 20). In addition, this subset may be responsible for the expression of immunity directed against other intracellular parasites. Orme (27) has recently noted that CD8+ cells are highly sensitive to the effects of CY and that the adoptive transfer of protection to Mycobacterium
DISSOCIATION OF PROTECTION AND DTH
657
tuberculosis is severely inhibited after CY treatment, an observation that can also be extended to the Listeria system (Fig. 2). The report (12) of protection after adoptive transfer of large numbers of a CD4+ Listeria-specific T-cell clone may be reconciled with the observation of a need for the CD4+ subset during the early phase of the immune response. The suggestion by Mielke et al. (see above and reference 20) and our observation that the CD8+ T-cell population develops CD4+ independence only after in vitro stimulation (5) indicate that the development of immunity to L. monocytogenes requires both T-cell subsets. However, final expression of specific antilisterial immunity is the responsibility of the CD8+ T-cell population. This suggests that at least some of the required differentiation signals are derived from the CD4+ T-cell subset, which is also solely responsible for the DTH response. The most convincing evidence for CD8+ T-cell-subsetmediated antilisterial immunity is seen with immune spleen cells obtained after culture with ConA. The culture environment has yet to be defined; however, it is surmised that the CD4+ T-cell subset is required for CD8+ cell development during the culture period. Thus, the cooperative events during culture may lead to a differentiation of the CD8+ T cell and thus the CD4+ cell is not required in these adoptivetransfer experiments. Similar signals may be required for the expression of immunity by directly transferred immune spleen cells. Consequently, direct transfer of immunity requires CD4+ and CD8+ T-cell subsets for full expression of immunity. It is unlikely that the CD8+ T-cell subset expresses antilisterial immunity by virtue of direct cytotoxicity against Listeria-infected cells, since this facultative intracellular parasite does not enter into an eclipse phase during its intracellular existence. Thus, immunity is dependent on macrophage-activating lymphokines and we would propose that one source of these lymphokines is the CD8+ T cell. This subset has been shown to produce many lymphokines (30). The protective role of the CD8+ cell would also imply that Listeria-antigen presentation occurs in context with class I molecules of the major histocompatibility complex, a suggestion that is supported by some studies of adoptive transfer of antilisterial immunity between congenic strains of mice (6). ACKNOWLEDGMENTS We thank Susan Hendrickson and Carolyn Barney for their expert
technical assistance. This work was supported by Public Health Service grant A123455 from the National Institutes of Health and by the Medical Research Foundation of Oregon.
LITERATURE CITED 1. Barry, R. A., and D. J. Hinrichs. 1982. Enhanced adoptive transfer of immunity to Listeria monocytogenes after in vitro culture of murine spleen cells with concanavalin A. Infect.
Immun. 35:560-565. 2. Barry, R. A., and D. J. Hinrichs. 1983. Lack of correlative enhancement of passive transfer of delayed-type hypersensitivity and antilisterial resistance when using concanavalin Astimulated primed spleen cells. Infect. Immun. 39:1208-1212. 3. Berche, P., C. Decreusefond, I. Theodorou, and C. Stiffel. 1989. Impact of genetically regulated T cell proliferation on acquired resistance to Listeria monocytogenes. J. Immunol. 142:932-939. 4. Berche, P., J. Gaillard, and P. J. Sansonetti. 1987. Intracellular growth of Listeria monocytogenes as a prerequisite for in vivo induction of T cell-mediated immunity. J. Immunol. 138:22662271.
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5. Bishop, D. K., and D. J. Hinrichs. 1987. Adoptive transfer of immunity to Listeria monocytogenes: the influence of in vitro stimulation on lymphocyte subset requirements. J. Immunol. 139:2005-2009. 6. Cheers, C., and M. S. Sandrin. 1983. Restriction of adoptive transfer of resistance to Listeria monocytogenes. II. Use of congenic and mutant strains show transfer to be H-2K restricted. Cell. Immunol. 78:199-205. 7. Chen-Woan, M., D. H. Sajewski, and D. D. McGregor. 1985. T-cell co-operation in the mediation of acquired resistance to Listeria monocytogenes. Immunology 56:33-42. 8. Czuprynski, C. J., and J. F. Brown. 1987. Dual regulation of antibacterial resistance and inflammatory neutrophil and macrophage accumulation by L3T4+ and Lyt 2+ Listeria-immune T cells. Immunology 60:287-293. 9. Czuprynski, C. J., J. F. Brown, K. M. Young, and A. J. Cooley. 1989. Administration of purified anti-L3T4 monoclonal antibody impairs the resistance of mice to Listeria monocytogenes infection. Infect. Immun. 57:100-109. 10. Fowles, R. E., I. M. Fajardo, J. L. Leibowitch, and J. R. David. 1973. The enhancement of macrophage bacteriostasis by products of activated lymphocytes. J. Exp. Med. 138:952-964. 11. Kaufmann, S. H. E. 1982. Effective antibacterial protection induced by a Listeria monocytogenes-specific T cell clone and its lymphokines. Infect. Immun. 39:1265-1270. 12. Kaufmann, S. H. E. 1988. Listeria monocytogenes specific T-cell lines and clones. Infection 16(Suppl. 2):S128-S136. 13. Kaufmann, S. H. E., and V. Brinkmann. 1984. Attempts to characterize the T-cell population and lymphokine involved in the activation of macrophage oxygen metabolism in murine listeriosis. Cell. Immunol. 88:545-550. 14. Kearns, R. J., and D. J. Hinrichs. 1977. Kinetics and maintenance of acquired cellular resistance in mice to Listeria monocytogenes. Infect. Immun. 16:923-927. 15. Kerckhaert, J. A. M., F. M. A. Hofuis, and J. M. N. Willers. 1977. Influence of cyclophosphamide on delayed hypersensitivity and acquired cellular resistance to Listeria monocytogenes.
Immunology 32:1027-1032. 16. Lefford, M. J. 1975. Delayed hypersensitivity and immunity in tuberculosis. Am. Rev. Respir. Dis. 111:243-246. 17. Mackaness, G. B. 1962. Cellular resistance to infection. J. Exp. Med. 116:381-406. 18. Mackaness, G. B. 1967. The relationship of delayed hypersensitivity to acquired cellular resistance. Br. Med. Bull. 23:52-54. 19. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973-992. 20. Mielke, M. A., S. Ehlers, and H. Hahn. 1988. T-cell subsets in delayed-type hypersensitivity, protection, and granuloma formation in primary and secondary Listeria infection in mice:
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Vol. 58, No. 3
p. 654-658
0019-9567/90/030654-05$02.00/0 Copyright C) 1990, American Society for Microbiology
Expression of Systemic Protection and Delayed-Type Hypersensitivity to Listeria monocytogenes Is Mediated by Different T-Cell Subsets JORY R. BALDRIDGE,' RONALD A. BARRY,"2 AND DAVID J. HINRICHSl2* Chiles Research Institute, Providence Medical Center, Portland, Oregon 97213,1 and Veterans Administration Medical Center, Portland, Oregon 972072 Received 1 August 1989/Accepted 1 December 1989
The relationship between acquired cellular resistance and delayed-type hypersensitivity (DTH) during the immune response to Listeria monocytogenes was investigated. Treatment of concanavalin A-stimulated Listeria-immune spleen cells with anti-CD8 antibody plus complement abrogated the adoptive transfer of systemic antilisterial immunity but had no effect on the transfer of DTH. In contrast, in vitro depletion of the CD4+ T-cell subset eliminated the ability of culture-activated cells to transfer DTH reactivity but did not interfere with the adoptive transfer of protection. In vivo, the infusion of anti-CD8 antibody inhibited the expression of both actively and adoptively transferred protection but did not influence the development of DTH skin test reactivity to L. monocytogenes antigens. In vivo depletion of the CD4+ T-cell subset eradicated the DTH response, with only minor influence of the protective anti-Listeria response. The apparent functional dissociation of the CD4+ (DTH) and CD8+ (protection) T-cell populations was further emphasized by our findings that the adoptive transfer of protection was dependent on a cyclophosphamide-sensitive cell population, whereas DTH reactivity was mediated by a cyclophosphamide-resistant population.
of the CD4+ and the CD8+ T-cell subsets in the expression of these two independent responses.
The association of acquired cellular resistance (ACR) and delayed-type hypersensitivity (DTH) remains controversial. The initial arguments which proposed that these two phenomena are direct manifestations of the same cell population were based on observations which showed an apparent inseparability of ACR and DTH during the immune response (17-19). This association is highlighted by the need for an active infection to induce both ACR and DTH, unless special immunizing techniques are applied (4, 14, 21, 29, 31-33). Also, the kinetics of development and longevity of these responses are similar (17, 24, 26). Furthermore, both ACR and DTH are T-cell-mediated events which rely, for full expression, on the involvement and activation of macrophages (17, 19). However, the opposing view asserts that ACR and DTH are separate entities of the cell-mediated response (16, 34). This view has been strengthened in some studies by the use of specific T-cell subpopulations in adoptive transfer (7, 28) and by in vivo use of subset-specific anti-T-cell antibodies (3, 9, 20). Previous results from this laboratory established that antilisterial immunity could be adoptively transferred by the CD8+ T-cell subset. These observations were obtained by using activated immune spleen cells recovered from concanavalin A (Con A)-stimulated cultures (5). Immune spleen cells recovered from these cultures have been shown to transfer greatly enhanced and long-lived levels of immunity (1, 5); however, our initial observations did not show an increase in the level of adoptively transferred DTH reactivity (2). Collectively these findings prompted this further investigation of the association between cells mediating protection and DTH. In all experiments with adoptive transfer techniques, as well as analysis of actively immunized mice, we found that the immune response leading to DTH reactivity is completely distinct from a protective response. The distinction can be attributed to the separate involvement *
MATERIALS AND METHODS
Bacteria. Listeria monocytogenes 10403 serotype 1 was used throughout this study. It was maintained in a virulent state by repeated passage in mice and had a 50% lethal dose of approximately 4.5 x 103 bacteria for BALB/c mice. Mice. BALB/c female mice of matched ages, 5 to 9 weeks, were purchased from the Fred Hutchison Cancer Research Center, Seattle, Wash., and Jackson Laboratories, Bar Harbor, Maine. MAbs. All monoclonal antibodies (MAbs) were obtained either from concentrated hybridoma culture supernatants or from mouse ascites fluids. The hybridomas were purchased from the American Type Culture Collection. Hybridoma 19-198 produces a mouse immunoglobulin G2a (IgG2a) antibody specific for the Lyt 2.2 (CD8) glycoprotein. Hybridoma GK1.5 produces a rat IgG2b antibody specific for the L3T4 (CD4) glycoprotein. The hybridoma 30-H12 produces a rat IgG2b antibody which is specific for the mouse Thy 1.2 antigen. The hybridoma 7D2.1.4.1.5 produces a rat IgG2a antibody specific for mouse immunoglobulin heavy chains. The antibody concentrations of supernatants and ascites were determined by enzyme-linked immunosorbent assay titration and compared with known mouse or rat IgG standards. Immunizations. Mice were immunized with approximately a 0.1 50% lethal dose of viable L. monocytogenes injected in volumes of 0.2 ml into the lateral tail vein. Cell culture. Spleen cells from immunized mice were cultured for 72 h in RPMI 1640 supplemented with 5% fetal calf serum, 100 U of penicillin per ml, 100 ,ug of streptomycin sulfate per ml, 5 x 10-5 M 2-mercaptoethanol, and 1 ,ug of ConA per ml. A total of 1 x 108 cells were grown in 75-cm2 flasks with 50 ml of medium. The cells were maintained at
Corresponding author. 654
VOL. 58, 1990
DISSOCIATION OF PROTECTION AND DTH
0
INCREASED FOOTPAD THICKNESS (mm) 0.10 0.05 0.15 0.20
TABLE 1. The influence of T-cell-specific antibody in vivo on the expression of antilisterial resistance and DTH'
0.25
I, ., I, , I,,
I. NORMAL MICE
Treatment
DTH b response (SD)
Logb protection
Day 6 active Day 6 active Day 6 active
None Anti-CD4 Anti-CD8
0.37 (0.02)c 0.05 (0.02) 0.32 (0.09)c
5.46 3.17 3.57
Day 10 active Day 10 active Day 10 active
None Anti-CD4 Anti-CD8
0.19 (0.08)d 0.02 (0.01) 0.14 (0.04)d
4.78 3.78 2.24
Immune spleen cell recipient Immune spleen cell
None
0.15 (0.05)e
2.17
Immune status
Group
C' CONTROL ANTI-CD4
A
ANTi-CD8
A
ANTI-THY 1.2
C' CONTROL ANTI-CD4
~.
.
ANTI-CDS
B
ANT.THY 1.2
B. 0
1.0
2.0
3.0
655
4.0
LOG PROTECnON
FIG. 1. CD4+ culture-activated immune spleen cells adoptively transfer DTH, and CD8+ culture-activated immune spleen cells adoptively transfer protection. Six days after primary immunization, donor spleen cells were stimulated in culture with ConA for 72 h. Complement-mediated depletion of culture-activated T-cell subsets was performed with subset-specific antibody prior to adoptive transfer. Recipients were infused with 2 x 107 cells and then assessed for DTH reactivity (A) or systemic protection (B) (see Materials and Methods). The mice in this experiment were challenged with 1 x 108 HKLM in a 25-ml volume for DTH analysis. The standard deviation for mean log1o CFU per spleen for all groups was less than 0.4.
37°C in a humidified incubator with a mixture of 6% CO2 and 94% air. Determination of log protection. Groups of mice (5 mice per group) were challenged with approximately 10 50% lethal doses of viable L. monocytogenes by intravenous injection into the lateral tail vein. The spleens were removed 48 h after challenge and were individually homogenized in 4.5 ml of 1% proteose-peptone in phosphate-buffered saline. The homogenate was serially diluted, and the appropriate dilutions were plated on brain heart infusion agar. Following a 24- to 48-h incubation, the CFU were counted and the log10 CFU per spleen was determined. Protection (log protection) was assessed by subtracting the mean log10 CFU per spleen of the test groups from the mean log1o CFU per spleen of the normal control group. Delayed-type hypersensitivity response. Standard footpad skin tests were used to assay DTH responsiveness. The right hind footpads of test mice were measured with dial calipers at time 0 and then injected with approximately 1 x 108 heat-killed L. monocytogenes (HKLM) in a volume of 25 ,ul (Fig. 1) or 50 p.l (Table 1; Fig. 2) of saline. The increase in footpad thickness was measured 24 h later. CY treatment. Cyclophosphamide (CY) (Sigma Chemical Co. St. Louis, Mo.) was reconstituted in distilled H20 immediately prior to use. Donor mice received a 150-mg/kg of body weight dose of CY by intravenous injection 1 h prior to removal of their spleens. In vivo MAb treatment. MAbs were injected intravenously into mice at concentrations of 500 to 600 p.g per injection on the day of L. monocytogenes challenge and again the following day for groups of mice being assessed for systemic protection. In groups of mice being assessed for DTH responsiveness, MAbs were infused only once, at the time of HKLM challenge. Control groups were infused with ascites containing rat IgG (from hybridoma 7D2.1.4.1.5) specific for mouse immunoglobulin heavy chains. The infusion of this
C
Anti-CD4
0.07 (0.02)
0.58
recipient Immune spleen cell recipient
Anti-CD8
0.12 (0.02)d
0.35
EISC recipient EISC recipient EISC recipient
None Anti-CD4 Anti-CD8
NT NT NT
4.78 3.05 0.13
a Data are representative of three or more separate experiments. b Assayed as described in Materials and Methods; NT = not tested. C P < 0.001 versus untreated normal mice. d p < 0.02 versus untreated normal mice. e P < 0.01 versus untreated normal mice.
ascites had no significant effect on the expression of DTH or protection (data not shown). In vitro complement-mediated cytotoxicity. Culture-activated immune spleen cells were suspended in hybridoma supernatants in an ice bath for 1 h. The MAb-treated cells were pelleted and suspended in Low-Tox-M rabbit complement (Cedar Lane Laboratories, Hornby, Ontario, Canada) diluted 1:12 in RPMI 1640 with 0.3% bovine serum albumin. Cells were then incubated for 1 h in a 37°C water bath with INCREASED FOOTPAD THICKNESS
0.05
0
_
NORMAL CONTROL
(mm)
0.15
0.10
0.20
0.25
~~~~~A
_
No CY
CY
No CY
lB
CY I
0
0.5
1.0
II 1.5
2.0
2.5
3.0
3.5
LOG PROTECTION
FIG. 2. CY treatment of donor mice inhibits adoptive transfer of protection but does not interfere with transfer of DTH. Donor mice were immunized with L. monocytogenes 6 days prior to infusion with CY. One hour after CY treatment, the spleens were removed and the immune spleen cells (1 x 108) were transferred to naive recipients which were concomitantly injected with HKLM in the footpad (A) or challenged intravenously with 10 50%o lethal doses of L. monocytogenes (B) (see Materials and Methods). The standard deviation for mean log1o CFU per spleen for all groups was less than 0.4.
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occasional mixing. Antibody-plus-complement treatments were conducted twice. Viable cell counts were then determined by trypan blue dye exclusion. Statistics. A two-tailed Student's t test was used to compare mean values of increased footpad thickness between control and experimental groups. RESULTS Phenotype of culture-activated cells responsible for the expression of protection versus DTH. As previously demonstrated, the adoptive transfer of protection but not DTH in antilisterial immunity can be enhanced after in vitro culture activation (2). By using this culture activation system, the phenotype of the culture-enhanced immune spleen cells (EISC) responsible for the adoptive transfer of protection and DTH was addressed. Six days postimmunization, spleen cells from immune donor mice were stimulated in culture with ConA. Following this 72-h culture period, the EISC were collected and depleted of selected T-cell subpopulations by antibody-dependent complement-mediated cytotoxicity prior to infusion into normal syngeneic recipients. The data displayed in Fig. 1 are representative of four experiments and indicate that recipients of EISC depleted of the CD8+ subset lack systemic protection yet are fully competent in their ability to express DTH. In contrast, mice receiving EISC depleted of the CD4+ subset are fully protected against systemic challenge but are unable to express DTH. Recipients of EISC depleted of all T cells were unable to resist systemic challenge or to manifest a DTH response. In vivo T-cell subset depletion by MAb infusion. To further address the phenotypes of the immune cell populations mediating protection and DTH, MAbs were directly infused into mice. Cytometric analysis of spleen cells from MAbtreated mice showed that T-cell-subset-specific depletion occurred within 24 h of MAb infusion and lasted for more than 8 days. Control spleen cell populations consisted of 80 to 85% Thy 1.2+, 43 to 47% CD4+, and 19 to 23% CD8+. Infusion of anti-Thy 1.2 MAb reduced the Thy 1.2+ population to 17.62% within 24 h and to 18.38% 8 days later. Similarly, after anti-CD4 (anti-L3T4) treatment, the CD4+ population in the spleen was reduced to 3.66% within 24 h and to 6.94% 8 days later. Treatment with MAb to the CD8 marker (anti-Lyt 2.2) decreased the CD8+ population in the spleen to 2.72% by 24 h and to 3.56% by 8 days. In the experiments presented in Table 1, actively immunized mice and recipients of immune spleen cells were infused with either anti-CD4 or anti-CD8 MAb to accomplish in vivo T-cell subset depletion. The mice in group A (Table 1) represent actively immunized test groups. Six or ten days after receiving a primary immunization (as indicated in the table), these actively immunized mice were injected with the appropriate MAb and immediately challenged with either (i) viable L. monocytogenes for assessment of systemic protection or (ii) HKLM for assessment of DTH reactivity. Similarly, recipient mice upon receiving either immune spleen cells (Table 1, group B) or EISC (Table 1, group C) were immediately infused with the appropriate MAb and challenged with either (i) viable L. monocytogenes for assessment of systemic protection or (ii) HKLM for assessment of DTH reactivity. All groups of mice that received anti-CD8 antibody exhibited a pronounced reduction in the protective immune response to challenge with viable L. monocytogenes. However, infusion of anti-CD8 MAb had no influence on the
INFECT. IMMUN.
expression of DTH reactivity for any group tested. In contrast, all immune groups treated with anti-CD4 MAb were unable to express a DTH response to HKLM. Depending on the test group, treatment with anti-CD4 antibody variably influenced the expression of protection. Inhibition of protection by treatment with anti-CD4 was pronounced in recipients of directly transferred spleen cells (Table 1, group B) and day 6 actively immunized mice (Table 1, group A). In addition, slight inhibition of protection was seen in actively immune mice 10 days after immunization (Table 1, group A) and in recipients of culture-activated spleen cells (Table 1, group C). CY treatment inhibits adoptive transfer of DTH but not protection. It has been reported that CY enhances DTH responsiveness and reduces CD8+ cell activity (15, 27). In order to determine the effect of CY on antilisterial immunity, we immunized mice with viable L. monocytogenes and 6 days later injected these mice with CY at a dose of 150 mg/kg of body weight. These CY-treated mice served as donors of spleen cells for adoptive transfer studies. The spleen cells were obtained from immune mice 1 h after CY injection and directly transferred to naive recipients. The results in Fig. 2 are representative of three separate experiments and show the DTH reactivity to HKLM (A) as well as the protective response seen in identical recipients (B). Immune spleen cells from CY-treated donors transferred identical levels of DTH responsiveness compared with immune spleen cells from untreated donors. However, immune spleen cells from CY-treated donors failed to transfer protection to naive
recipients. DISCUSSION Elimination of facultative intracellular parasites appears to depend on the development of activated macrophages (17, 19, 21, 24, 25) which emerge after stimulation with lymphocyte-derived macrophage-activating factors (10, 19). Lymphokine production by CD4+ lymphocytes is well established, and the involvement of this subset in macrophage activation and DTH reactions has led to the presumption that CD4+ lymphocytes play a significant role in the expression of protective antilisterial immunity. Although CD8+ lymphocytes have been recognized as cytotoxic cells with relevance in viral infections, a protective role for this lymphocyte subset against facultative intracellular parasites has only recently become apparent (5, 23, 27). One approach used to establish the importance of CD8+ lymphocytes in the expression of antilisterial immunity involves adoptive transfer of in vitro-selected T-cell subsets. In the rat model of listeriosis, Chen-Woan et al. (7) used peritoneal cells to show that the cellular mediators of systemic protection were W3/25-, OX8+(CD8+) T cells, while W3/25+, OX8- (CD4+) T cells were responsible for the expression of DTH reactivity. These investigators also reported that the protective activity of the CD8+ cells was enhanced when cotransferred with CD4+ cells. Previous studies in which mouse lymphocytes were used also indicated some role for the CD8+ (Lyt 2+) T-cell subset; however, these studies also showed an important, if not dominant, role for CD4+ (Lyt 1+2-) T cells (11, 13, 22). Our initial observation showing the requirement for CD8+ cells in antilisterial immunity in mice used immune spleen cells that had been stimulated in vitro prior to adoptive transfer (5). Within this transfer system, elimination of the CD4+ T-cell subset by anti-CD4 antibody plus complement treatment had no effect on the transfer of antilisterial resistance
VOL. 58, 1990
and the protective activity of the CD8+ T-cell population obtained from culture was not enhanced in the presence of CD4+ T cells. However, prior to culture, both the CD8+ and CD4+ T cells were shown to be required for full expression of adoptive protection in recipients subsequently challenged with L. monocytogenes (5). Since (i) both acquired cellular resistance and DTH are dependent on lymphokine-mediated macrophage activation and (ii) our previous results demonstrated the importance of CD8+ cells for protection, we evaluated recipients of CD8+ cells for expression of DTH to HKLM. In this study, T-cell subset depletion of EISC (Fig. 1) confirmed previous reports that the CD8+ cell population is critical for the expression of culture-enhanced adoptive protection and further showed that this same CD8+ subset does not contribute to the expression of adoptively transferred DTH. In contrast, the CD4+ population was shown to be essential for the expression of DTH but not required for the expression of adoptive protection with EISC. The second approach used to assess T-cell subset interaction in the expression of protection and DTH involved in vivo injection of MAbs specific for CD4+ or CD8+ T cells. The effect of such treatment is to cause a reduction in a specific T-cell subset. In Table 1 we again show the phenotypic dissociation of systemic protection and DTH in both adoptively transferred and actively induced immunity. In vivo administration of anti-CD8 antibody had no influence on DTH reactivity but reduced or eliminated the protective response to L. monocytogenes challenge in all cases. After infusion of anti-CD4 MAb, all DTH reactions were inhibited, while the effect on systemic protection was variable. AntiCD4 treatment only slightly inhibited adoptive transfer of protection with culture-activated immune spleen cells or actively immunized mice 10 days after primary immunization. However, this same anti-CD4 treatment significantly blocked adoptive protection in recipients of directly transferred immune spleen cells and day 6 actively immunized mice. This latter observation correlates with previous findings from this laboratory in which day 6 immune spleen cells transferred directly to naive recipients were used (5). The greatest amount of immunity directly transferred by this population of immune spleen cells is seen in the presence of both CD4+ and CD8+ T-cell populations (5; Table 1). Although cells of the CD8+ T-cell subset dominate the expression of systemic protection, CD4+ cells appear to be essential for DTH responsiveness and (perhaps relatedly) for the development of acquired cellular resistance. Similar conclusions were reached by Mielke et al. (20), who also suggested that when the numbers of immune CD8+ cells are low, the CD4+ cells are required for CD8+ cell amplification via interleukin production. Berche et al. (3) also found similar results, suggesting that the CD8+ subset was the dominant T-cell population in protection, while CD4+ T cells mediated the DTH response. Also, Czuprynski et al. (9) selectively depleted CD4+ T cells by MAb treatment of mice, which abolished the DTH response and also slightly inhibited resistance; they concluded that the CD4+ population participates in the development of acquired cellular resistance but is not the dominant subset involved. It would appear that the CD8+ T-cell subset is the major T-cell component responsible for the expression of antilisterial immunity (3, 5, 8, 20). In addition, this subset may be responsible for the expression of immunity directed against other intracellular parasites. Orme (27) has recently noted that CD8+ cells are highly sensitive to the effects of CY and that the adoptive transfer of protection to Mycobacterium
DISSOCIATION OF PROTECTION AND DTH
657
tuberculosis is severely inhibited after CY treatment, an observation that can also be extended to the Listeria system (Fig. 2). The report (12) of protection after adoptive transfer of large numbers of a CD4+ Listeria-specific T-cell clone may be reconciled with the observation of a need for the CD4+ subset during the early phase of the immune response. The suggestion by Mielke et al. (see above and reference 20) and our observation that the CD8+ T-cell population develops CD4+ independence only after in vitro stimulation (5) indicate that the development of immunity to L. monocytogenes requires both T-cell subsets. However, final expression of specific antilisterial immunity is the responsibility of the CD8+ T-cell population. This suggests that at least some of the required differentiation signals are derived from the CD4+ T-cell subset, which is also solely responsible for the DTH response. The most convincing evidence for CD8+ T-cell-subsetmediated antilisterial immunity is seen with immune spleen cells obtained after culture with ConA. The culture environment has yet to be defined; however, it is surmised that the CD4+ T-cell subset is required for CD8+ cell development during the culture period. Thus, the cooperative events during culture may lead to a differentiation of the CD8+ T cell and thus the CD4+ cell is not required in these adoptivetransfer experiments. Similar signals may be required for the expression of immunity by directly transferred immune spleen cells. Consequently, direct transfer of immunity requires CD4+ and CD8+ T-cell subsets for full expression of immunity. It is unlikely that the CD8+ T-cell subset expresses antilisterial immunity by virtue of direct cytotoxicity against Listeria-infected cells, since this facultative intracellular parasite does not enter into an eclipse phase during its intracellular existence. Thus, immunity is dependent on macrophage-activating lymphokines and we would propose that one source of these lymphokines is the CD8+ T cell. This subset has been shown to produce many lymphokines (30). The protective role of the CD8+ cell would also imply that Listeria-antigen presentation occurs in context with class I molecules of the major histocompatibility complex, a suggestion that is supported by some studies of adoptive transfer of antilisterial immunity between congenic strains of mice (6). ACKNOWLEDGMENTS We thank Susan Hendrickson and Carolyn Barney for their expert
technical assistance. This work was supported by Public Health Service grant A123455 from the National Institutes of Health and by the Medical Research Foundation of Oregon.
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Immun. 35:560-565. 2. Barry, R. A., and D. J. Hinrichs. 1983. Lack of correlative enhancement of passive transfer of delayed-type hypersensitivity and antilisterial resistance when using concanavalin Astimulated primed spleen cells. Infect. Immun. 39:1208-1212. 3. Berche, P., C. Decreusefond, I. Theodorou, and C. Stiffel. 1989. Impact of genetically regulated T cell proliferation on acquired resistance to Listeria monocytogenes. J. Immunol. 142:932-939. 4. Berche, P., J. Gaillard, and P. J. Sansonetti. 1987. Intracellular growth of Listeria monocytogenes as a prerequisite for in vivo induction of T cell-mediated immunity. J. Immunol. 138:22662271.
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