- Email: [email protected]
Salmonella enterica infection
IMMUNITY
TO INTRACELLULAR
Saunders, B.M. & Cheers, C. (1996), Intranasal infection of beige mice with Mycobacterium avium complex: role of neutrophils and natural killer cells. Infect. Immun., 64,4236-4241. Schluger, N.W. et al. (1996), Principles of therapy of tuberculosis in the modern era in “Tuberculosis” (Rom, W.N. and Garay, S.M.) (pp. 751-761). Lippincott-Raven, New York. Shafer, R.W. & Edlin, B.R. (1996), Tuberculosis in patients infected with human immunodeficiency virus. perspective on the past decade. Clin. Infect. Dis., 22, 683-704. Shiratsuchi, H. et al. (1991), Bidirectional effects of cytokines on the growth of Mycobacterium avium within human monocytes. J. Immunol., 146,3165-3170. Sison, J.P. et al. (1996a), Treatment of Mycobacterium avium complex infection: do the result of in vitro susceptibility tests predict therapeutic outcome in humans? J. Infect. Dis., 173, 677-683. Sison, J.P. et al. (1996b), Treatment of Mycobacterium avium complex infection: does the beige mouse model predict therapeutic outcome in humans? J. Infect. Dis., 173, 750-753. Squires, K.E. et al. (1989). Interferon gamma and Mycobacterium avium-intracellulare infection. J. Infect. Dis., 159, 599-600. Squires, K.E. et al. (1992). Interferon gamma treatment for Mycobacterium avium-intracellulare complex bacillemia in patients with AIDS [Letter]. J. Infect. Dis., 166, 686-687. Stanford, J.L. et al. (1990). Mycobacterium vaccae in immunoprophylaxis and immunotherapy of leprosy and tuberculosis. Vaccine, 8, 525-530.
BACTERIA
Stanford, J.L. & Stanford, C.A. (1994), Immunotherapy of tuberculosis with Mycobacterium vaccae NCTC 11659. Immunobiology, 19 1, 555-563. Sundar, S. & Murray, H.W. (1995), Effect of treatment with interferon-y alone in visceral leishmaniasis. J. Infect. Dis., 172, 1627-1629. Toba, H. et al. (1989), pathogenicity of Mycobacterium avium for human monocytes: absence of macrophage-activating factor activity of gamma interferon. Infect. Immun., 57, 239-244. Tramontana, J.M., Utaipat, U., Molloy, A. Akarasewi, P., Burroughs, M., Makonkawkeyoon, S., Johnson, B., Klausner, J.D., Rorn, W. & Kaplan, G. (1995), Thalidomide treatment reduces tumor necrosis factor alpha production and enhances weight gain in patients with pulmonary tuberculosis. Mol. Med., 1, 384-397. Trudeau, E.L. (1907), Tuberculin immunization in the treatment of pulmonary tuberculosis. Am. J. Med. Sci., 133, 813-829. Wallis, R.S. et al. (1996), Pentoxifylline therapy in human immunodeficiency virus-seropositive persons with tuberculosis: a randomized, controlled trial. J. Infect. Dis., 174, 727-733. Wormser, G.P. et al. (1994), Low dose dexamethasone as adjunctive therapy for disseminated Mycobacterium avium complex infections in AIDS patients. Antimicrab. Agents Chemother., 38, 2215-2217. Wyser, C. et al. (1996), Corticosteroids in the treatment of tuberculous pleurisy : a double-blind, placebo-controlled, randomized study. Chest, 110, 333-338. Zaheer, S.A. et al. (1995), Addition of immunotherapy with Mycobacterium w vaccine to multi-dxug therapy benefits multibacillary leprosy patients. Vaccine, 13, 1102-l 110.
Salmonella enterica infection J. Hess (‘) (*) and S.H.E.
Kaufmann
(l. 2,
(I) De artment of Immunology, University of Vim, D-89081 Ulm (Germany), and (2PMax-Planck-Institute for Infection Biology, D-1011 7 Berlin (Germany)
Introduction
spectrum.
Salmonella enterica is an extremely
diverse
spe-
ties encompassing 2,324 serovars. The salmonellae inhabit various mammals, reptiles, and birds, some of them having a broad and some a rather narrow host
Received December (*) For comspondence:
The intestine
represents
the port of entry as
well as the major site of replication of salmonellae (Finlay and Falkow, 1989). In the susceptible host, intestinal colonization results in disease ranging from mild enterocolitis to severe diarrhoea. Some salmonellae are not restricted to the intestinal tract and
13, 1996. Dr. J. Hess, Department
of Immunology,
University
of Ulm,
Albert-Einstein-Allee
I I, D-89070,
Germany.
582
67th FORUM
enter the host, causing bacteraemia, sometimes resulting in focal infection or enteric fever - the most severe form of salmonellosis (Eisenstein et af., 1996). In humans, this latter disease, typhoid fever, is caused by S. enterica serova Typhi. In mice, a similar disease evolves after infection with S. enterica serovar Typhimurium. Although human typhoid fever is now well controlled in many countries, the enterocolitis and diarrhoea caused by various serovars represent an enormous threat to public health and economy. Because Typhi fails to cause enteric fever in mice, Typhimurium is frequently used for understanding the mechanisms responsible for control of typhoid. Salmonellae are intracellular bacteria for the host in which they cause enteric fever. Immunity to intracellular bacteria has been carefully analysed in experimental mouse models using Listeria monocytogenes (Kaufmann, 1993). These experiments have revealed exclusive dependency on T lymphocytes, and little, if any, contribution by antibodies from B cells. Yet a vast body of literature has provided compelling evidence for a major contribution of antibodies in vaccine-induced protection against Typhimurium (Eisenstein et al., 1996 ; Mastroeni et al., 1993). L. monocyfogenes is capable of leaving the macrophage phagosome and entering the cytosolic compartment (Gaillard et al., 1987). Because of its residence both in the phagosome and the cytosol, L. monocyfogenes stimulates both CD4 and CD8 T lymphocytes (Lade1 et al., 1994). Indeed, CD8 T cells appear most central to acquired resistance against listeriosis. In contrast, Typhimurium remains in the phagosome where it survives (Finlay and Falkow, 1989). Hence, this pathogen should primarily activate CD4 T cells (Nauciel, 1990). Yet evidence has been presented that, in addition to CD4 T cells, CD8 T cells are also required for optimum protection against mouse typhoid (Mastroeni et af., 1993; Pope and Kotlarski, 1994 ; Pope et al., 1994). Finally, listeriosis is an acute infection which is controlled by rapidly arising T lymphocytes, and it is generally overcome within less than 2 weeks. In contrast, Typhimurium can establish a more chronic infection which may last for many weeks. Evidence exists to suggest that in mm-me typhoid, the activation of T cells ensues two weeks postinfection (p.i.) and that a relatively long phase of resistance is T-cell-independent (Eisenstein et al., 1996). Even after T-cell activation, sterile eradication is delayed. Thus, the experiences gathered with experimental listeriosis cannot be simply translated to murine typhoid. We therefore decided to characterize immune mechanisms involved in control of Typhimurium in vivo, using a similar approach as has been performed in this laboratory with L. monocytogenes (Mombaerts et al., 1993 ; Lade1 et al., 1994). Our studies involved gene deletion mutant mice with defined immunodeficiencies in the T-cell compartment (Mombaerts et aZ., 199 1; Itohara et al., 1993; Zijlstra et al., 1989; Cosgrove et al., 1991).
IN IMMUNOLOGY Because of the chronicity of infection and disease caused by aroA- Typhimurium, we considered application of mouse mutants with stable deficiencies particularly helpful. By purpose, aroA- strains of Typhimurium were employed which offer the possibility to analyse the chronic course of infection (Hoiseth and Stocker, 1981). Moreover, the aroA- strains are promising vaccine candidates both for the control of salmonellae and as carriers of antigens from unrelated pathogens (Hormaeche and Khan, 1996). Finally, we decided to begin with the systemic infection model, although Typhimurium as well as other salmonellae generally enter the host through the gut. Whilst gene disruption affects lymphocytes in all organs, the mucosal and the central immune response differ markedly. To avoid interferences between mucosal and central immunity, the mucosal immune system was bypassed and the bacteria were introduced to the central immune system directly.
Role of CK~ and y6 T cells in primary rium amA- infection
Typhimu-
To analyse the contribution of fl and a/3 T cells in primary Typhimurium infection, TCRP’- or TCRG? deficient mice and their heterozygous littermates were infected i.v. The bacterial burdens in spleens of these animals were determined at various time points (fig. 1). Homozygous TCRG? and heterozygous TCRS+‘- or TCRP+‘- mice cleared infection successfully, whereas TCRP-‘- mice succumbed to Typhimurium aroA-, slrain SL7207 after day 60 p-i. (Hess et al., 1996a). Thus, our findings provide formal proof for the central role of I$ T cells in acquired resistance against murine typhoid and argue against a critical role of $5 T cells in control of systemic Typhimurium infection. The crucial dependency on ap T cells is not surprising. The finding, however, that bacterial counts in TCRP’- and TCRP’I- mice were almost equal during the first three weeks of infection deserves attention, because it suggests a rather long period of T-cellindependent control of Typhimurium.
Importance infection
of IFNy in early Typhimurium
amA-
Evidence has been presented that early control of Typhimurium is a function of IFNy-secreting NK cells (Schafer and Eisenstein, 1992). To gain deeper insights into the role of IFNy, we employed IFN’yreceptor-deficient mouse mutants (IFNy R-/-) which are unresponsive to IFNy (Huang et al., 1993). Consistent with the role of T-cell-independent IFNy in control of early Typhimurium infection (fig. 2), IFNy R-/- mouse mutants suffered from elevated bacterial numbers already one week after infection. Moreover, the IFNy R-/- mutants succumbed to infection
IMMUNITY
TO INTRACELLULAR
Contribution Typhimurium
at around week 3 (Hess et al., 1996a) (fig. 2). We take these findings as evidence for an important role of alp T-cell-independent II?Ny in control of Typhimurium infection.
1
583
of CD4 T cells to the control of amA- infection
In CD4-deficient AP” mouse mutants, a persistent infection was established when a sublethal inoculum of 5 x lo5 Typhimurium bacteria was administered i.v. (fig. 3). Higher bacterial doses of Typhimurium SL7207 could not be controlled by Afi-/- mice; they died of salmonellosisbetween days 15 and 19 p.i. (Hess et al., 1996a). These findings not only emphasize the essentialrole of CD4 T cells in control of typhoid. They also suggest that aroA
A a
BACTERIA
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7
A
20
40
60
Days p.i. 01
I 0
5
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Typhimurium SL7207 infection R-/- and IF’Ny R+‘- mice.
of IFNy
Days
1’5 p.i.
Fig. 1. Course of aroA- Typhimurium SL7207 infection in TCRP-‘-, TCRp’-, TCRP+‘-, TCR&‘and TCRPmutant mice.
Fig. 2. AroA-
Mice were infected i.v. with 5 x l@ Typhimurium SL7207 and CFU in spleens were determined on various days p.i. (A) (m) TCRP-/-, (A) TCRP+‘-; (B) (0) TCRS-/and (A) TCR6+‘- mice. The symbol (+) represents mortality of all mice per group beyond this time point. Means f SD of five animals per time point. Statistically significant difference (p < 0.05) between TCRPA- and TCRP+‘- mice at day 40 (Student’s t test). Data reproduced with permission from Journal of Immunology (Hess et al., 156, 3321. 3326, 1996), Copyright 1996. The American Association of Immunologists.
(A) Course of CFU in spleens of Ty himurium SL7207-infected IFNy R-/- (B) and IFNy R+-P (A) mice. Means + SD of five animals per time point are shown. (B) Mice were infected i.v. with 5 x l@ Typhimurium SL7207 bacteria (10 mice/group). Survival was recorded daily and is given as the percentage of live animals per time point. + represents the mortality of all five mice per group beyond this time point. Data reproduced with permission from Journal of Immunology (Hess et al., 156, 3321-3326, 1996), Copyright 1996. The American Association of Immunologists.
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IN IMMUNOLOGY
gene deficiency does not inevitably lead to sterile eradication of Typhimurium in the immunocompromised host. Probably, sufficient concentrations of aromatic components were provided by the diet, so that the aroA- mutation crippled Typhimurium, but was not lethal for it. Sterile eradication even of the auxotrophic strain depended on a competent immune system. This finding illustrates the risk of viable vaccines for immunocompromised patients.
6
1
MHC class I processing of antigen secreted by Typhimurium amAThe stimulation of CDS T cells by “endosomal microbes” has been reported (Kaufmann, 1993 ; Pfeifer et al., 1993 ; Pope et al., 1994). Moreover, recent studies from our laboratory suggest that the Typhimurium strains used are capable of inducing protective CD8 T cells. This assumption is based on the finding that recombinant strains of Typhimurium SL7207 p6Ossecreting the listerial antigen p60 apparently stimulate CD8 T cells which contribute to antilisterial protection (Hess et al., 1996b). It therefore appearsthat antigens secretedby Typhimurium within the phagosomehave accessto MHC class I processing. In order to analyse antigen delivery by Typhimurium for MHC classI presentationunder defined conditions, we took advantage of the availability of the Typhirnurium SL7207 p6Os strain secreting the listerial antigen p60 and of a p60-specific cytotoxic T-cell lymphocyte (CTL) line. Analogous to the experimental settings originally described by Harty and Pamer (1995), we infected 5774 macrophage-like cells with Typhimurium SL7207 or SL7207 ~60s and determined target cell lysis causedby CTL specific for the H-2Kd p60-epitope (217-225). As shown in figure 4, Typhimurium SL7207 p60s-infected target cells and the P815 p60-transfectant, pP60.3, were recognized by the anti-p60 T cells. This finding clearly underlines the capability of aroA- Typhimurium carriers to introduce antigens into the MHC class I processing and presentation pathway.
CD8 T cells in primary infection
Typhimurium
0
20
40
60
60
Days
p.i.
Fig. 3. Courseof aroA- Typhimurium SL7207 infection in APJ-- and AP+‘--deficient mice. Mice were infected i.v. with 5 x IO5 Typhimurium SL7207 and CFU in spleensof Ap-‘- (I) and Ap+‘- (A) mice weredeterminedon variousdaysp.i. Means* SD of five animalsper time point. Statistically significantdifference(p < 0.05) betweenAP” and ApI- mice at day 60 (Student’st test). Data reproducedwith permissionfrom Journal of Immunology (Hess et al., 156, 3321-3326, 1996), Copyright 1996. The American Association of Immunologists.
-.- .I774 +A SL7207 wSL7207 pPSo.3
pSOr
amA-
Knowing that Typhimurium is capable of introducing antigens into the MHC class I pathway, we sought to assessthe contribution of MHC class Idependent immune mechanismsto protection against Typhimurium infection. The p,rnA- mutant mice and their heterozygous littermates were infected i.v. with 5 x lo5 bacteria, and the course of infection was followed (Hess et al., 1996a) (fig. 5). Interestingly, both mouse mutants cleared their bacterial load with equal efficiency. Thus, our findings do not provide evidence for a significant role of CD8 T cells in control
30:1
1O:l
3:l
1:l
E:T
Fig. 4. p60-specificCD8 T cells recognizearoA- TyphimuriumSL7207 p60s-infectedmacrophages. The p60-specificCD8 T-cell line was assayed for its ability to lyse uninfected J774 cells (0), Typhimurium SL7207 p60s-infected 5774 cells (W). Typhimurium SL7207 control infected 5774 targets (0) or p60-transfectant pP60.3 cells (A) in a standard5’ chromium release assay.
IMMUNITY
TO INTRACELLULAR
of primary mouse typhoid. Lack of CD8 T-cell contribution is in contrast to reports by others describing an auxiliary function of CD8 T cells in protection (Mastroeni et al., 1993). It is, however, consistent with the endosomal residence of Typhimurium in macrophages. What happens if one renders Typhimurium capable of egressing into the cytosol? Are CD8 T cells important for control of such “cytosolic” Typhimurium? To address this question, the r-Typhimurium SL7207 Hlys strain was constructed. This strain secretes biologically active listeriolysin which is responsible for listerial egression from the endosome into the cytosol (Gentschev et al., 1995). Indeed, this Typhimurium SL7207 Hlys strain can be found in the cytosolic compartment of J774 macrophages (Hess and Kaufmann, 1997). To assess whether the dominance of MHC class II-dependent over MHC class I-dependent immune mechanisms in Typhimurium infection is directly related to the endosomal localization of the pathogen, we infected p rnA- and &m+‘- mutant mice with the Typhimur&m SL7207 Hlys strain. By day 21 p.i., CFU were slightly increased in Typhimurium SL7207 Hlysinfected mice compared to the SL7207 controlinfected animals independently of p m-deficiency (Hess et d., 1996a) (fig. 5). Importan~y, the overall course of infection and time point of sterile clearance were virtually identical for the Typhimurium stains SL7207 and SL7207 Hlys in p,m’- and &m+‘- mice. These findings suggest that the capacity to egress into the cytosol does not significantly increase virulence of Typhimurium amA-. We assume that egression into the cytosol does not provide an additional advantage for survival of Typhimurium amA- which has adopted maximally to intraphagosomal living. This markedly contrasts with the strategy employed by L,. monocytogenes, which is strictly dependent on escape from the phagosome for its survival within macrophages. Concluding
remarks
In the early fifties, studies on experimental salmonellosis and listeriosis in mice revealed marked differences in the immune response to these pathogens, which caused remarkable controversies about the contribution of humoral immunity against intracellular pathogens (Eisenstein et al., 1996). In subsequent years, analyses employing L. monocytogenes by far outnumbered studies with S. entericu, resulting in the dogma of crucial T-cell dependence of acquired immunity against intracellular bacteria. Indeed, the elegance of experimental listeriosis of mice has provided unparalleled insights into the acquired immune response against intracellular bacteria. At the same time, however, it has also biased our views about the underlining mechanisms, for example, by emphasizing the role of CD8 T cells. This
585
BACTERIA
A
I I
I
I
I
I
0
20
40
60
60
Days
I
pi.
B 6 1
0
20
60
40 Days
60
p.i.
Fig. 5. Courseof aroA- Typhimurium SL7207andSL7207 Hlys infection in p,m’-- and P2rnq-deficientmice. Five mice per group were infected i.v. with 5 x lo5 (A) Typhimurium SL7207 or (B) SL7207 Hlys. CFU (meansf SD) in spleensof p?rn’- (B) and &m+‘- (A) mice were determinedon varrous days p.i. Data reproduced with permission from Journal of Immunology (Hesset al., 156, 3321-3326,1996). Copyright 19%. The American Associationof immunologists.
bias was further supported by findings that acquired immunity againts M. tubercdosis crucially depends on CD8 T cells as well. The model of murine Typhimurium infection clearly emphasizes the heterogeneity of intracellular bacterial pathogens by demonstrating the virtual irrelevance of CD8 T cells and the central role of CD4 T cells. Therefore, infections with S. entericu ssp.deserve further interest.
67th FORUM
Not only does the immune responseto S. enter& ssp.dramatically differ from that against other intracellular bacteria such as L. monocytogenes but diseasescaused by S. enterica also cause major problems worldwide and S. enterica ssp. provide fascinating opportunities as viable vaccine carriers for heterologous antigens. We believe that the availability of gene deletion mutant mice with defined immune deficiencies provide the appropriate tools for elucidating the cells and cytokines which participate in immunity to salmonellosis.Because the species S. enterica is extremely heterogenouswith different serovars having adopted strategies which allow them to survive in a variety of host organisms,we may eventually find that differences may exist even amongst serovarsof a single specieswith regard to their survival strategy and the type of protective immunity. Acknowledgements We thank A. Dreher, R. Mahmoudi and D. Miko for expert support. Many thanks to Dr. C.H. Lade1 for several experimental contributions to this study. We are grateful to Drs. Aguet, R. Jaen&h, D. Mathis, and S. Tonegawa for breeding pairs of knockout mice; to Dr. B. Stocker for Typhimurium SL7207 strain; to Dr. J.T. Harty for the p&specific CI’L line and p60-transfectant, and to Drs. I. Gentschev and W. Goebel for Typhimurium SL7207 Hiys.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ka 573/3-l/2). References Cosgrove, D., Gray, D., Dierich, A., Kaufman, J., Lemeur, M., Benoist, C. & Mathis, D. (1991), Mice lacking mHC class II molecules. Cell, 66, 1051-1058. Eisenstein, T.K., Huang, D. & Schwacha, M.G. (1996). Immunity to Salmonella infections, in “Enteric Infections and Immunity” (L.J. Paradise et al.) (pp. 57-78). Plenum Press, New York. Finlay, B.B. & Falkow, S. (1989), Salmonella as an intracellular parasite. Mol. Microbial., 3, 1833-1841. Gaillard, J.-L., Berche, P., Mounier, Richard, S. & Sansonetti, P. (1987), In vitro model of penetration and intracellular growth of Listeriu monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun., 55, 2822-2829. Gentschev, I., Sokolovic, Z., Mollenkopf, H.-J., Hess, J., Kaufmann, S.H.E. & Goebel, W. (1995), Salmonella secreting active listeriolysin changes its intracellular localization. Infect. Immun., 63, 4202-4204. Harty, J.T. & Pamer, E.G. (1995), CD8 T lymphocytes specific for the secreted p60 antigen protect against L.&e& rrwrwcytogenes infection. J. Immurwl., 154,4642-4650. Hess, J., Ladel, C., Mike, D. & Kaufmann, S.H.E. (1996a), Salmonella typhimurium aroA- infection in gene-targeted immunhficient mice: Major role of CD4+ TCR ap cells and IFN-y in bacterial clearance independent from intracellular location. J. Immunol,, 156,3321-3326. Hess, J., Gentschev, I., Miko, D., Welzel, M., Ladel, C.H., Goebel, W. & Kaufmann, S.H.E. (1996b), Superior efficacy of secreted over somatic antigen display in
IN IMMUNOLOGY Salmonella vaccine induced protection against listeriosis. Proc. Natl. Acad. Sci. USA, 93, 1458-1463. Hess, J. & Kaufmann, S.H.E. (1997), Principles of cellmediated immunity underlying vaccination strategies against intracellular pathogens, in “Host response to intracellular pathogens” (S.H.E. Kaufmann). R.G. Landes Company, Austin (in press). Hoiseth, S.K. & Stocker, B.A.D. (1981), Aromatic-dependent Salmonella typhimurium are non-virulent and effective as a vaccine. Nature (Lond.) 291, 238-241. Huang. S., Hendriks, W., Althage, A., Hemmi, S., Bluethmann, H., Kamijo, R., Vlcek, J., Zinkemagel, R.M. & Aguet, M. (1993), Immune response in mice that lack the interferon-y receptor. Science, 259, 1742- 1748. Itohara, S., Mombaerts, P. Lafaille, J., Lacomini, J., Nelson, A., Clarke, A.R., Hooper, M.L., Farr, A. & Tonegawa, S. (1993), T cell receptor 6 gene mutant mice: independent generation of ap T cells and programmed rearrangements of @ TCR genes. Cell, 72,337-443. Kaufmann, S.H.E. (1993), Immunity to intracellular bacteria. Annu. Rev. Immun., 11, 129- 163. Hormaeche, C.E. & Khan, C.M.A. (1996), Recombinant bacteria as vaccine carriers of heterologous antigens, in “Concept in Vaccine Development” (S.H.E. Kaufmann). Walter de Gruyter, Berlin and New York, 327-349. Ladel, C.H., Flesch, I.E.A., Arnoldi, J. & Kaufmann, S.H.E. (1994), Studies with MHC-deficient knockout mice reveal impact of both MHC I- and MHC IIdependent T cell responses in Listeria monocytogenes. J. Immunol., 153, 3116-3122. Mastroeni, P., Villarreal-Ramos, B. & Hormaeche, C.E. (1993), Adoptive transfer of immunity to oral challenge with virulent Salmonellae in innately susceptible BALB/c mice requires both serum and T cells. Infect. Immun , 6 1, 398 l-3984. Mombaerts, P., Clarke, A.R., Hooper, M.L. & Tonegawa, S. (1991), Creation of a large genomic deletion at the T-cell antigen receptor P-subunit locus in mouse embryonic stem cells by gene targeting. Proc. Natl. Acad. Sci. USA 88,3084-3041. Mombaerts, P., Arnoldi, J., Russ, F., Tonegawa, S. 8c Kaufmann, S.H.E. (1993). Different roles of ap and yS T cells in immunity against an intracellular bacterial pathogen. Nature (Lond.), 365, 53-56. Nauciel, C. (1990). Role of CD4’ T cells and T-independent mechanisms in squired resitance to Salmonella typhimurium infection. J. Immurwl., 145, 12651269. Pfeifer, J.D., Wick, M.J., Roberts, R.L., Findlay, K., Normark, S.J. & Harding, C.V. (1993), Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature (Land.) 361, 359-362. Pope, M. & Kotlarski, I. (1994), Detection of Salmonellaspecific L3T4+ and Lyt-2+ T cells which can proliferate in vitro and mediate delayed-type hypersensitivity reactivity. Immunology, 81, 183-191. Pope, M., Kotlarski, I. & Doherty, K (1994), Induction of Lyt2+ cytotoxic T lymphocytes following primary and secondary SalmonelIa infection Immurw logy, 81, 177-183. Schafer, R. & Eisenstein, T.K. (1992), Natural killer cells mediate protection induced by a Salmonella aroA mutant. Infect. Immun., 60, 791-797. Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. & Jaenisch, R. (1989). Germ-line transmission of a disrupted P2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature (Land.) 342,435-438.
TO INTRACELLULAR
Saunders, B.M. & Cheers, C. (1996), Intranasal infection of beige mice with Mycobacterium avium complex: role of neutrophils and natural killer cells. Infect. Immun., 64,4236-4241. Schluger, N.W. et al. (1996), Principles of therapy of tuberculosis in the modern era in “Tuberculosis” (Rom, W.N. and Garay, S.M.) (pp. 751-761). Lippincott-Raven, New York. Shafer, R.W. & Edlin, B.R. (1996), Tuberculosis in patients infected with human immunodeficiency virus. perspective on the past decade. Clin. Infect. Dis., 22, 683-704. Shiratsuchi, H. et al. (1991), Bidirectional effects of cytokines on the growth of Mycobacterium avium within human monocytes. J. Immunol., 146,3165-3170. Sison, J.P. et al. (1996a), Treatment of Mycobacterium avium complex infection: do the result of in vitro susceptibility tests predict therapeutic outcome in humans? J. Infect. Dis., 173, 677-683. Sison, J.P. et al. (1996b), Treatment of Mycobacterium avium complex infection: does the beige mouse model predict therapeutic outcome in humans? J. Infect. Dis., 173, 750-753. Squires, K.E. et al. (1989). Interferon gamma and Mycobacterium avium-intracellulare infection. J. Infect. Dis., 159, 599-600. Squires, K.E. et al. (1992). Interferon gamma treatment for Mycobacterium avium-intracellulare complex bacillemia in patients with AIDS [Letter]. J. Infect. Dis., 166, 686-687. Stanford, J.L. et al. (1990). Mycobacterium vaccae in immunoprophylaxis and immunotherapy of leprosy and tuberculosis. Vaccine, 8, 525-530.
BACTERIA
Stanford, J.L. & Stanford, C.A. (1994), Immunotherapy of tuberculosis with Mycobacterium vaccae NCTC 11659. Immunobiology, 19 1, 555-563. Sundar, S. & Murray, H.W. (1995), Effect of treatment with interferon-y alone in visceral leishmaniasis. J. Infect. Dis., 172, 1627-1629. Toba, H. et al. (1989), pathogenicity of Mycobacterium avium for human monocytes: absence of macrophage-activating factor activity of gamma interferon. Infect. Immun., 57, 239-244. Tramontana, J.M., Utaipat, U., Molloy, A. Akarasewi, P., Burroughs, M., Makonkawkeyoon, S., Johnson, B., Klausner, J.D., Rorn, W. & Kaplan, G. (1995), Thalidomide treatment reduces tumor necrosis factor alpha production and enhances weight gain in patients with pulmonary tuberculosis. Mol. Med., 1, 384-397. Trudeau, E.L. (1907), Tuberculin immunization in the treatment of pulmonary tuberculosis. Am. J. Med. Sci., 133, 813-829. Wallis, R.S. et al. (1996), Pentoxifylline therapy in human immunodeficiency virus-seropositive persons with tuberculosis: a randomized, controlled trial. J. Infect. Dis., 174, 727-733. Wormser, G.P. et al. (1994), Low dose dexamethasone as adjunctive therapy for disseminated Mycobacterium avium complex infections in AIDS patients. Antimicrab. Agents Chemother., 38, 2215-2217. Wyser, C. et al. (1996), Corticosteroids in the treatment of tuberculous pleurisy : a double-blind, placebo-controlled, randomized study. Chest, 110, 333-338. Zaheer, S.A. et al. (1995), Addition of immunotherapy with Mycobacterium w vaccine to multi-dxug therapy benefits multibacillary leprosy patients. Vaccine, 13, 1102-l 110.
Salmonella enterica infection J. Hess (‘) (*) and S.H.E.
Kaufmann
(l. 2,
(I) De artment of Immunology, University of Vim, D-89081 Ulm (Germany), and (2PMax-Planck-Institute for Infection Biology, D-1011 7 Berlin (Germany)
Introduction
spectrum.
Salmonella enterica is an extremely
diverse
spe-
ties encompassing 2,324 serovars. The salmonellae inhabit various mammals, reptiles, and birds, some of them having a broad and some a rather narrow host
Received December (*) For comspondence:
The intestine
represents
the port of entry as
well as the major site of replication of salmonellae (Finlay and Falkow, 1989). In the susceptible host, intestinal colonization results in disease ranging from mild enterocolitis to severe diarrhoea. Some salmonellae are not restricted to the intestinal tract and
13, 1996. Dr. J. Hess, Department
of Immunology,
University
of Ulm,
Albert-Einstein-Allee
I I, D-89070,
Germany.
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enter the host, causing bacteraemia, sometimes resulting in focal infection or enteric fever - the most severe form of salmonellosis (Eisenstein et af., 1996). In humans, this latter disease, typhoid fever, is caused by S. enterica serova Typhi. In mice, a similar disease evolves after infection with S. enterica serovar Typhimurium. Although human typhoid fever is now well controlled in many countries, the enterocolitis and diarrhoea caused by various serovars represent an enormous threat to public health and economy. Because Typhi fails to cause enteric fever in mice, Typhimurium is frequently used for understanding the mechanisms responsible for control of typhoid. Salmonellae are intracellular bacteria for the host in which they cause enteric fever. Immunity to intracellular bacteria has been carefully analysed in experimental mouse models using Listeria monocytogenes (Kaufmann, 1993). These experiments have revealed exclusive dependency on T lymphocytes, and little, if any, contribution by antibodies from B cells. Yet a vast body of literature has provided compelling evidence for a major contribution of antibodies in vaccine-induced protection against Typhimurium (Eisenstein et al., 1996 ; Mastroeni et al., 1993). L. monocyfogenes is capable of leaving the macrophage phagosome and entering the cytosolic compartment (Gaillard et al., 1987). Because of its residence both in the phagosome and the cytosol, L. monocyfogenes stimulates both CD4 and CD8 T lymphocytes (Lade1 et al., 1994). Indeed, CD8 T cells appear most central to acquired resistance against listeriosis. In contrast, Typhimurium remains in the phagosome where it survives (Finlay and Falkow, 1989). Hence, this pathogen should primarily activate CD4 T cells (Nauciel, 1990). Yet evidence has been presented that, in addition to CD4 T cells, CD8 T cells are also required for optimum protection against mouse typhoid (Mastroeni et af., 1993; Pope and Kotlarski, 1994 ; Pope et al., 1994). Finally, listeriosis is an acute infection which is controlled by rapidly arising T lymphocytes, and it is generally overcome within less than 2 weeks. In contrast, Typhimurium can establish a more chronic infection which may last for many weeks. Evidence exists to suggest that in mm-me typhoid, the activation of T cells ensues two weeks postinfection (p.i.) and that a relatively long phase of resistance is T-cell-independent (Eisenstein et al., 1996). Even after T-cell activation, sterile eradication is delayed. Thus, the experiences gathered with experimental listeriosis cannot be simply translated to murine typhoid. We therefore decided to characterize immune mechanisms involved in control of Typhimurium in vivo, using a similar approach as has been performed in this laboratory with L. monocytogenes (Mombaerts et al., 1993 ; Lade1 et al., 1994). Our studies involved gene deletion mutant mice with defined immunodeficiencies in the T-cell compartment (Mombaerts et aZ., 199 1; Itohara et al., 1993; Zijlstra et al., 1989; Cosgrove et al., 1991).
IN IMMUNOLOGY Because of the chronicity of infection and disease caused by aroA- Typhimurium, we considered application of mouse mutants with stable deficiencies particularly helpful. By purpose, aroA- strains of Typhimurium were employed which offer the possibility to analyse the chronic course of infection (Hoiseth and Stocker, 1981). Moreover, the aroA- strains are promising vaccine candidates both for the control of salmonellae and as carriers of antigens from unrelated pathogens (Hormaeche and Khan, 1996). Finally, we decided to begin with the systemic infection model, although Typhimurium as well as other salmonellae generally enter the host through the gut. Whilst gene disruption affects lymphocytes in all organs, the mucosal and the central immune response differ markedly. To avoid interferences between mucosal and central immunity, the mucosal immune system was bypassed and the bacteria were introduced to the central immune system directly.
Role of CK~ and y6 T cells in primary rium amA- infection
Typhimu-
To analyse the contribution of fl and a/3 T cells in primary Typhimurium infection, TCRP’- or TCRG? deficient mice and their heterozygous littermates were infected i.v. The bacterial burdens in spleens of these animals were determined at various time points (fig. 1). Homozygous TCRG? and heterozygous TCRS+‘- or TCRP+‘- mice cleared infection successfully, whereas TCRP-‘- mice succumbed to Typhimurium aroA-, slrain SL7207 after day 60 p-i. (Hess et al., 1996a). Thus, our findings provide formal proof for the central role of I$ T cells in acquired resistance against murine typhoid and argue against a critical role of $5 T cells in control of systemic Typhimurium infection. The crucial dependency on ap T cells is not surprising. The finding, however, that bacterial counts in TCRP’- and TCRP’I- mice were almost equal during the first three weeks of infection deserves attention, because it suggests a rather long period of T-cellindependent control of Typhimurium.
Importance infection
of IFNy in early Typhimurium
amA-
Evidence has been presented that early control of Typhimurium is a function of IFNy-secreting NK cells (Schafer and Eisenstein, 1992). To gain deeper insights into the role of IFNy, we employed IFN’yreceptor-deficient mouse mutants (IFNy R-/-) which are unresponsive to IFNy (Huang et al., 1993). Consistent with the role of T-cell-independent IFNy in control of early Typhimurium infection (fig. 2), IFNy R-/- mouse mutants suffered from elevated bacterial numbers already one week after infection. Moreover, the IFNy R-/- mutants succumbed to infection
IMMUNITY
TO INTRACELLULAR
Contribution Typhimurium
at around week 3 (Hess et al., 1996a) (fig. 2). We take these findings as evidence for an important role of alp T-cell-independent II?Ny in control of Typhimurium infection.
1
583
of CD4 T cells to the control of amA- infection
In CD4-deficient AP” mouse mutants, a persistent infection was established when a sublethal inoculum of 5 x lo5 Typhimurium bacteria was administered i.v. (fig. 3). Higher bacterial doses of Typhimurium SL7207 could not be controlled by Afi-/- mice; they died of salmonellosisbetween days 15 and 19 p.i. (Hess et al., 1996a). These findings not only emphasize the essentialrole of CD4 T cells in control of typhoid. They also suggest that aroA
A a
BACTERIA
T
7
A
20
40
60
Days p.i. 01
I 0
5
10
15
20
25
Days p.i. B ------------
60z * ‘5
60-
20I
I
0
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1
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\
,
I
40 p.i.
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i
60
1’0
i0
i5
Typhimurium SL7207 infection R-/- and IF’Ny R+‘- mice.
of IFNy
Days
1’5 p.i.
Fig. 1. Course of aroA- Typhimurium SL7207 infection in TCRP-‘-, TCRp’-, TCRP+‘-, TCR&‘and TCRPmutant mice.
Fig. 2. AroA-
Mice were infected i.v. with 5 x l@ Typhimurium SL7207 and CFU in spleens were determined on various days p.i. (A) (m) TCRP-/-, (A) TCRP+‘-; (B) (0) TCRS-/and (A) TCR6+‘- mice. The symbol (+) represents mortality of all mice per group beyond this time point. Means f SD of five animals per time point. Statistically significant difference (p < 0.05) between TCRPA- and TCRP+‘- mice at day 40 (Student’s t test). Data reproduced with permission from Journal of Immunology (Hess et al., 156, 3321. 3326, 1996), Copyright 1996. The American Association of Immunologists.
(A) Course of CFU in spleens of Ty himurium SL7207-infected IFNy R-/- (B) and IFNy R+-P (A) mice. Means + SD of five animals per time point are shown. (B) Mice were infected i.v. with 5 x l@ Typhimurium SL7207 bacteria (10 mice/group). Survival was recorded daily and is given as the percentage of live animals per time point. + represents the mortality of all five mice per group beyond this time point. Data reproduced with permission from Journal of Immunology (Hess et al., 156, 3321-3326, 1996), Copyright 1996. The American Association of Immunologists.
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IN IMMUNOLOGY
gene deficiency does not inevitably lead to sterile eradication of Typhimurium in the immunocompromised host. Probably, sufficient concentrations of aromatic components were provided by the diet, so that the aroA- mutation crippled Typhimurium, but was not lethal for it. Sterile eradication even of the auxotrophic strain depended on a competent immune system. This finding illustrates the risk of viable vaccines for immunocompromised patients.
6
1
MHC class I processing of antigen secreted by Typhimurium amAThe stimulation of CDS T cells by “endosomal microbes” has been reported (Kaufmann, 1993 ; Pfeifer et al., 1993 ; Pope et al., 1994). Moreover, recent studies from our laboratory suggest that the Typhimurium strains used are capable of inducing protective CD8 T cells. This assumption is based on the finding that recombinant strains of Typhimurium SL7207 p6Ossecreting the listerial antigen p60 apparently stimulate CD8 T cells which contribute to antilisterial protection (Hess et al., 1996b). It therefore appearsthat antigens secretedby Typhimurium within the phagosomehave accessto MHC class I processing. In order to analyse antigen delivery by Typhimurium for MHC classI presentationunder defined conditions, we took advantage of the availability of the Typhirnurium SL7207 p6Os strain secreting the listerial antigen p60 and of a p60-specific cytotoxic T-cell lymphocyte (CTL) line. Analogous to the experimental settings originally described by Harty and Pamer (1995), we infected 5774 macrophage-like cells with Typhimurium SL7207 or SL7207 ~60s and determined target cell lysis causedby CTL specific for the H-2Kd p60-epitope (217-225). As shown in figure 4, Typhimurium SL7207 p60s-infected target cells and the P815 p60-transfectant, pP60.3, were recognized by the anti-p60 T cells. This finding clearly underlines the capability of aroA- Typhimurium carriers to introduce antigens into the MHC class I processing and presentation pathway.
CD8 T cells in primary infection
Typhimurium
0
20
40
60
60
Days
p.i.
Fig. 3. Courseof aroA- Typhimurium SL7207 infection in APJ-- and AP+‘--deficient mice. Mice were infected i.v. with 5 x IO5 Typhimurium SL7207 and CFU in spleensof Ap-‘- (I) and Ap+‘- (A) mice weredeterminedon variousdaysp.i. Means* SD of five animalsper time point. Statistically significantdifference(p < 0.05) betweenAP” and ApI- mice at day 60 (Student’st test). Data reproducedwith permissionfrom Journal of Immunology (Hess et al., 156, 3321-3326, 1996), Copyright 1996. The American Association of Immunologists.
-.- .I774 +A SL7207 wSL7207 pPSo.3
pSOr
amA-
Knowing that Typhimurium is capable of introducing antigens into the MHC class I pathway, we sought to assessthe contribution of MHC class Idependent immune mechanismsto protection against Typhimurium infection. The p,rnA- mutant mice and their heterozygous littermates were infected i.v. with 5 x lo5 bacteria, and the course of infection was followed (Hess et al., 1996a) (fig. 5). Interestingly, both mouse mutants cleared their bacterial load with equal efficiency. Thus, our findings do not provide evidence for a significant role of CD8 T cells in control
30:1
1O:l
3:l
1:l
E:T
Fig. 4. p60-specificCD8 T cells recognizearoA- TyphimuriumSL7207 p60s-infectedmacrophages. The p60-specificCD8 T-cell line was assayed for its ability to lyse uninfected J774 cells (0), Typhimurium SL7207 p60s-infected 5774 cells (W). Typhimurium SL7207 control infected 5774 targets (0) or p60-transfectant pP60.3 cells (A) in a standard5’ chromium release assay.
IMMUNITY
TO INTRACELLULAR
of primary mouse typhoid. Lack of CD8 T-cell contribution is in contrast to reports by others describing an auxiliary function of CD8 T cells in protection (Mastroeni et al., 1993). It is, however, consistent with the endosomal residence of Typhimurium in macrophages. What happens if one renders Typhimurium capable of egressing into the cytosol? Are CD8 T cells important for control of such “cytosolic” Typhimurium? To address this question, the r-Typhimurium SL7207 Hlys strain was constructed. This strain secretes biologically active listeriolysin which is responsible for listerial egression from the endosome into the cytosol (Gentschev et al., 1995). Indeed, this Typhimurium SL7207 Hlys strain can be found in the cytosolic compartment of J774 macrophages (Hess and Kaufmann, 1997). To assess whether the dominance of MHC class II-dependent over MHC class I-dependent immune mechanisms in Typhimurium infection is directly related to the endosomal localization of the pathogen, we infected p rnA- and &m+‘- mutant mice with the Typhimur&m SL7207 Hlys strain. By day 21 p.i., CFU were slightly increased in Typhimurium SL7207 Hlysinfected mice compared to the SL7207 controlinfected animals independently of p m-deficiency (Hess et d., 1996a) (fig. 5). Importan~y, the overall course of infection and time point of sterile clearance were virtually identical for the Typhimurium stains SL7207 and SL7207 Hlys in p,m’- and &m+‘- mice. These findings suggest that the capacity to egress into the cytosol does not significantly increase virulence of Typhimurium amA-. We assume that egression into the cytosol does not provide an additional advantage for survival of Typhimurium amA- which has adopted maximally to intraphagosomal living. This markedly contrasts with the strategy employed by L,. monocytogenes, which is strictly dependent on escape from the phagosome for its survival within macrophages. Concluding
remarks
In the early fifties, studies on experimental salmonellosis and listeriosis in mice revealed marked differences in the immune response to these pathogens, which caused remarkable controversies about the contribution of humoral immunity against intracellular pathogens (Eisenstein et al., 1996). In subsequent years, analyses employing L. monocytogenes by far outnumbered studies with S. entericu, resulting in the dogma of crucial T-cell dependence of acquired immunity against intracellular bacteria. Indeed, the elegance of experimental listeriosis of mice has provided unparalleled insights into the acquired immune response against intracellular bacteria. At the same time, however, it has also biased our views about the underlining mechanisms, for example, by emphasizing the role of CD8 T cells. This
585
BACTERIA
A
I I
I
I
I
I
0
20
40
60
60
Days
I
pi.
B 6 1
0
20
60
40 Days
60
p.i.
Fig. 5. Courseof aroA- Typhimurium SL7207andSL7207 Hlys infection in p,m’-- and P2rnq-deficientmice. Five mice per group were infected i.v. with 5 x lo5 (A) Typhimurium SL7207 or (B) SL7207 Hlys. CFU (meansf SD) in spleensof p?rn’- (B) and &m+‘- (A) mice were determinedon varrous days p.i. Data reproduced with permission from Journal of Immunology (Hesset al., 156, 3321-3326,1996). Copyright 19%. The American Associationof immunologists.
bias was further supported by findings that acquired immunity againts M. tubercdosis crucially depends on CD8 T cells as well. The model of murine Typhimurium infection clearly emphasizes the heterogeneity of intracellular bacterial pathogens by demonstrating the virtual irrelevance of CD8 T cells and the central role of CD4 T cells. Therefore, infections with S. entericu ssp.deserve further interest.
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Not only does the immune responseto S. enter& ssp.dramatically differ from that against other intracellular bacteria such as L. monocytogenes but diseasescaused by S. enterica also cause major problems worldwide and S. enterica ssp. provide fascinating opportunities as viable vaccine carriers for heterologous antigens. We believe that the availability of gene deletion mutant mice with defined immune deficiencies provide the appropriate tools for elucidating the cells and cytokines which participate in immunity to salmonellosis.Because the species S. enterica is extremely heterogenouswith different serovars having adopted strategies which allow them to survive in a variety of host organisms,we may eventually find that differences may exist even amongst serovarsof a single specieswith regard to their survival strategy and the type of protective immunity. Acknowledgements We thank A. Dreher, R. Mahmoudi and D. Miko for expert support. Many thanks to Dr. C.H. Lade1 for several experimental contributions to this study. We are grateful to Drs. Aguet, R. Jaen&h, D. Mathis, and S. Tonegawa for breeding pairs of knockout mice; to Dr. B. Stocker for Typhimurium SL7207 strain; to Dr. J.T. Harty for the p&specific CI’L line and p60-transfectant, and to Drs. I. Gentschev and W. Goebel for Typhimurium SL7207 Hiys.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ka 573/3-l/2). References Cosgrove, D., Gray, D., Dierich, A., Kaufman, J., Lemeur, M., Benoist, C. & Mathis, D. (1991), Mice lacking mHC class II molecules. Cell, 66, 1051-1058. Eisenstein, T.K., Huang, D. & Schwacha, M.G. (1996). Immunity to Salmonella infections, in “Enteric Infections and Immunity” (L.J. Paradise et al.) (pp. 57-78). Plenum Press, New York. Finlay, B.B. & Falkow, S. (1989), Salmonella as an intracellular parasite. Mol. Microbial., 3, 1833-1841. Gaillard, J.-L., Berche, P., Mounier, Richard, S. & Sansonetti, P. (1987), In vitro model of penetration and intracellular growth of Listeriu monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun., 55, 2822-2829. Gentschev, I., Sokolovic, Z., Mollenkopf, H.-J., Hess, J., Kaufmann, S.H.E. & Goebel, W. (1995), Salmonella secreting active listeriolysin changes its intracellular localization. Infect. Immun., 63, 4202-4204. Harty, J.T. & Pamer, E.G. (1995), CD8 T lymphocytes specific for the secreted p60 antigen protect against L.&e& rrwrwcytogenes infection. J. Immurwl., 154,4642-4650. Hess, J., Ladel, C., Mike, D. & Kaufmann, S.H.E. (1996a), Salmonella typhimurium aroA- infection in gene-targeted immunhficient mice: Major role of CD4+ TCR ap cells and IFN-y in bacterial clearance independent from intracellular location. J. Immunol,, 156,3321-3326. Hess, J., Gentschev, I., Miko, D., Welzel, M., Ladel, C.H., Goebel, W. & Kaufmann, S.H.E. (1996b), Superior efficacy of secreted over somatic antigen display in
IN IMMUNOLOGY Salmonella vaccine induced protection against listeriosis. Proc. Natl. Acad. Sci. USA, 93, 1458-1463. Hess, J. & Kaufmann, S.H.E. (1997), Principles of cellmediated immunity underlying vaccination strategies against intracellular pathogens, in “Host response to intracellular pathogens” (S.H.E. Kaufmann). R.G. Landes Company, Austin (in press). Hoiseth, S.K. & Stocker, B.A.D. (1981), Aromatic-dependent Salmonella typhimurium are non-virulent and effective as a vaccine. Nature (Lond.) 291, 238-241. Huang. S., Hendriks, W., Althage, A., Hemmi, S., Bluethmann, H., Kamijo, R., Vlcek, J., Zinkemagel, R.M. & Aguet, M. (1993), Immune response in mice that lack the interferon-y receptor. Science, 259, 1742- 1748. Itohara, S., Mombaerts, P. Lafaille, J., Lacomini, J., Nelson, A., Clarke, A.R., Hooper, M.L., Farr, A. & Tonegawa, S. (1993), T cell receptor 6 gene mutant mice: independent generation of ap T cells and programmed rearrangements of @ TCR genes. Cell, 72,337-443. Kaufmann, S.H.E. (1993), Immunity to intracellular bacteria. Annu. Rev. Immun., 11, 129- 163. Hormaeche, C.E. & Khan, C.M.A. (1996), Recombinant bacteria as vaccine carriers of heterologous antigens, in “Concept in Vaccine Development” (S.H.E. Kaufmann). Walter de Gruyter, Berlin and New York, 327-349. Ladel, C.H., Flesch, I.E.A., Arnoldi, J. & Kaufmann, S.H.E. (1994), Studies with MHC-deficient knockout mice reveal impact of both MHC I- and MHC IIdependent T cell responses in Listeria monocytogenes. J. Immunol., 153, 3116-3122. Mastroeni, P., Villarreal-Ramos, B. & Hormaeche, C.E. (1993), Adoptive transfer of immunity to oral challenge with virulent Salmonellae in innately susceptible BALB/c mice requires both serum and T cells. Infect. Immun , 6 1, 398 l-3984. Mombaerts, P., Clarke, A.R., Hooper, M.L. & Tonegawa, S. (1991), Creation of a large genomic deletion at the T-cell antigen receptor P-subunit locus in mouse embryonic stem cells by gene targeting. Proc. Natl. Acad. Sci. USA 88,3084-3041. Mombaerts, P., Arnoldi, J., Russ, F., Tonegawa, S. 8c Kaufmann, S.H.E. (1993). Different roles of ap and yS T cells in immunity against an intracellular bacterial pathogen. Nature (Lond.), 365, 53-56. Nauciel, C. (1990). Role of CD4’ T cells and T-independent mechanisms in squired resitance to Salmonella typhimurium infection. J. Immurwl., 145, 12651269. Pfeifer, J.D., Wick, M.J., Roberts, R.L., Findlay, K., Normark, S.J. & Harding, C.V. (1993), Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature (Land.) 361, 359-362. Pope, M. & Kotlarski, I. (1994), Detection of Salmonellaspecific L3T4+ and Lyt-2+ T cells which can proliferate in vitro and mediate delayed-type hypersensitivity reactivity. Immunology, 81, 183-191. Pope, M., Kotlarski, I. & Doherty, K (1994), Induction of Lyt2+ cytotoxic T lymphocytes following primary and secondary SalmonelIa infection Immurw logy, 81, 177-183. Schafer, R. & Eisenstein, T.K. (1992), Natural killer cells mediate protection induced by a Salmonella aroA mutant. Infect. Immun., 60, 791-797. Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. & Jaenisch, R. (1989). Germ-line transmission of a disrupted P2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature (Land.) 342,435-438.