H2-M3 Major Histocompatibility Complex Class Ib-Restricted CD8 T Cells Induced by Salmonella enterica Serovar Typhimurium Infection Recogniz
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感染与免疫杂志 2005年第12期
Department of Microbiology and Immunology, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom
Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom
ABSTRACT
Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.
INTRODUCTION
Salmonella species are intracellular gram-negative pathogens that cause a range of diseases, including typhoid fever, which, despite widespread use of antibiotics, represents a significant health problem in a number in developing nations. The recent emergence of multidrug-resistant Salmonella strains and the variable efficacy of existing vaccines increase the need for better understanding of the mechanisms of protective immunity to salmonellae and improvement of vaccination strategies (11, 30).
Infection of susceptible strains of inbred mice with Salmonella enterica serovar Typhimurium provides a murine model for typhoid fever which bears many similarities to human serovar Typhi infection (10). After oral uptake in mice, S. enterica serovar Typhimurium crosses the intestinal epithelium and spreads within phagocytes via the bloodstream to the spleen and liver, where the bacteria replicate (28). Innate immune mechanisms, including Slc11a1 (formerly Nramp1) gene function, and production of nitric oxide and reactive oxygen intermediates are important in controlling initial bacterial growth. However, induction of the acquired immune response is necessary for clearance of infection (11).
In a primary infection, the clearance of salmonellae from infected tissues is controlled by the acquisition of specific T-cell immunity. In addition, both T and B lymphocytes are involved in protection against a secondary infection (11, 18, 19, 26). However, despite the firm evidence that T cells are indispensable in controlling Salmonella infection, understanding which T-cell subsets participate during distinct stages of infection is less clear. Major histocompatibility complex (MHC) class II-deficient mice fail to clear infection with an attenuated strain of S. enterica serovar Typhimurium and develop chronic infection, suggesting an essential role for CD4 T cells (9). However, it has also been demonstrated that beta-2-microglobulin (2m)-deficient mice surviving primary infection with an attenuated S. enterica serovar Typhimurium strain suffer from impaired resistance to challenge infection with a virulent strain (16), implicating CD8 T cells in protection against Salmonella infection. More recently, it has been shown that CD8 T cells that secrete gamma interferon and kill S. enterica serovar Typhi-infected cells are elicited by immunization of volunteers with an attenuated Salmonella vaccine strain (27). The same group have demonstrated the concomitant induction of both CD4 and CD8 T-cell responses after vaccination with another oral typhoid vaccine candidate, CVD 908-htrA.
Limited information is available on the specificity and MHC restriction phenotype of T cells induced by Salmonella infection. Salmonella infection of mice has been shown to induce MHC class II-restricted CD4 T cells specific for the flagellar antigen FliC, and immunization with recombinant FliC protects mice against infection with a low dose of a virulent strain of S. enterica serovar Typhimurium (3, 20). CD4 T cells from infected mice also recognize the Salmonella invasion protein SipC, a type III secretion product (21), but a role for SipC in protection has not been reported. Similarly, the only Salmonella antigen so far identified that is recognized by CD8 T cells is the heat shock protein GroEL, a response that is MHC class 1b (Qa-1) restricted (16, 17). A recent report shows that a subset of human S. enterica serovar Typhi-specific CD8 T cells are also MHC class 1b and HLA-E restricted, but the antigen recognized has not been identified (25).
In this study, we demonstrate that cytotoxic T-cell lines from mice infected with S. enterica serovar Typhimurium recognize Salmonella proteins presented by the nonclassical MHC class Ib molecule H2-M3.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains aro attenuated S. enterica serovar Typhimurium SL3261, virulent S. enterica serovar Typhimurium C5, the flagellum-defective variant S. enterica serovar Typhimurium C5-FliA–, and Salmonella enterica serovar Enteritidis SE795 were used in this study and grown as described elsewhere (2). S. enterica serovar Typhimurium C5-FliA– was grown in the presence of 25 μg/ml of tetracycline. Heat-killed S. enterica serovar Typhimurium SL3261 was prepared by washing bacteria in phosphate-buffered saline followed by incubation at 60°C for 3 h and washing again. Effective killing was confirmed by pour plating on LB agar plates.
Preparation of proteins released by S. enterica serovar Typhimurium. For the preparation of proteins, S. enterica serovar Typhimurium strain C5 FliA– was grown in LB medium as described above until late-exponential phase was reached. Bacteria were pelleted and proteins from filtered culture supernatant were precipitated overnight at 5°C with 40% ammonium sulfate, centrifuged at 43,000 x g, incubated at 4°C for 1 h, and dissolved in phosphate-buffered saline. The protein preparation was desalted by 10DG column passage and dialyzed according to the manufacture's protocol (Bio-Rad Laboratories Ltd., United Kingdom). Fractions were collected and assayed for protein concentration using the bicinchoninic acid protein assay kit (Pierce & Warriner, United Kingdom). Where indicated, Salmonella proteins were treated with 1 mg/ml of proteinase K (Sigma) for 1 h at 60°C, and dialyzed for 24 h in phosphate-buffered saline. For loading targets, cells were incubated with S. enterica serovar Typhimurium proteins (10 μg/ml) for 3 h at 37°C followed by washing to remove unbound proteins and proteinase K in experiments in which it was used.
Mouse strains and cell lines. We maintained 6- to 8-week-old female BALB/c (H-2d), BALB.B (H-2b), BALB.K (H-2k), C3H/He (H-2k), CBA/J (H-2k), and B10.BR (H-2k) mice (Harlan Olac Ltd., Bicester, United Kingdom) at the University of Newcastle upon Tyne. Cell lines J774 (H-2d), IC-21 (H-2b), YAC (H-2d), EL-4 (H-2b), RMA (H-2b), and RMA-S (H-2b, TAP–/–) were obtained from the American Type Culture Collection. We acknowledge the kind gifts of the L cell fibroblast line (H-2k) and L cells transfected with Qa-1b (L-Qa-1b) from J. Dyson, Clinical Sciences Centre, Hammersmith Hospital, London, United Kingdom; the 2m-deficient C4.4 cells (H-2b) from R. Glas, Karolinska Institute, Stockholm, and the B10.CAS2 fibroblast cell line (H-2w17) and TR8.4a, a variant of B10.CAS2 transfected with H2-M3wt from C. R. Wang, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 μM 2-mercapthoethanol (RF-10). All tissue culture reagents were obtained from Sigma (United Kingdom) unless otherwise stated.
Bone marrow macrophages. Bone marrow-derived macrophages (referred to as cultured macrophages) were prepared as described elsewhere (4). Briefly, bone marrow cells were extracted from femurs and tibias and cultured for 6 days at 3 x 105 cells/ml in bacteriological-grade petri dishes containing RF-10 supplemented with 5% horse serum, 1 mM sodium pyruvate (Sigma), 40 μg/ml gentamicin (Sigma), and 20% conditioned medium from L929 cells as a source of macrophage colony-stimulating factor. Adherent cells were washed with phosphate-buffered saline (PBS), detached from the plastic with cold PBS containing 0.1% EDTA, washed twice with PBS, and used on days 6 to 14 of culture.
For infection cell lines were seeded at a density of 2 x 106 cells per well in 6 well cell culture plates in RF-10 without antibiotics. The following day the medium was removed and 1 ml of medium containing 100 x 106 CFU/ml of S. enterica serovar Typhimurium was added to each well. To facilitate infection, the plates were centrifuged at 400 x g for 10 min at room temperature, and the plates were incubated for 2 h at 37°C in 5% CO2. The cultures were washed twice with warmed RF-10 to remove unattached bacteria and incubated for 1 h in the presence of 100 μg/ml gentamicin to kill extracellular bacteria. Cells were removed from plates by using cold PBS containing 0.1% EDTA for use in experiments.
Establishment and maintenance of T-cell lines. Female 8-week-old BALB/c mice were immunized intravenously with 106 SL3261 in a tail vein as described previously (19), and challenged after 5 or 8 weeks with 5 x 1010 CFU of S. enterica serovar Typhimurium C5 by the intragastric route under mild anesthesia as described previously (10). Mice were sacrificed between 1 and 5 weeks after challenge and immune splenocytes were harvested to generate T-cell lines in vitro. CD8 cells were positively selected using magnetic beads according to the manufacturer's instructions (Miltenyi Biotec, GmbH, Bergish-Gladbach, Germany). The purity of the separated cell populations was determined by flow cytometry using the following monoclonal antibodies: anti-CD3-fluorescein isothiocyanate, anti-T-cell receptor -phycoerythrin, anti-T-cell receptor (TCR) -phycoerythrin, anti-CD8-fluorescein isothiocyanate, and anti-CD4-phycoerythrin (PharMingen, San Diego, CA).
We stimulated 2 x 106 CD8 T cells (>95% pure) with 105 irradiated (50 Gy) SL3261-infected J774 cells and 4 x 106 irradiated (20 Gy) syngeneic spleen cells in the presence of 40 μg/ml gentamicin. Recombinant human interleukin-2 (Cetus) was used at 100 IU/ml in the first week and at 10 IU/ml in the second week. Subsequent stimulations were performed fortnightly under the same conditions. Cytotoxicity assay was performed after restimulation and expansion in vitro at least three times. The phenotype of cultured T-cell lines was assessed by flow cytometry using the monoclonal antibodies described above and a mouse V T-cell receptor screening panel (PharMingen). The majority of T-cell lines were CD3+4–8+ TCR++ V8+. The cytotoxic T-cell activity of lines was assayed in a standard 4-h 51Cr-release assay and 19 of 25 T-cell lines were cytotoxic for J774 macrophages infected with S. enterica serovar Typhimurium SL3261. We selected five CD3+4–8+ TCR++ V8+ T-cell lines that were cytotoxic for SL3261-infected J774 to generate the data shown in the figures.
Cytotoxicity assay. Infected and uninfected target cells or target cells pulsed with Salmonella protein preparations were incubated for 1 h at 37°C with 3.7 MBq of [51Cr]sodium chromate (Amersham, United Kingdom). The targets were washed, resuspended at 5 x 104/ml, and 100-μl aliquots were added to 100-μl serial dilutions of T cells in round-bottomed 96-well plates. After 4 h of incubation, supernatants were collected and 51Cr activity was determined by gamma-scintillation spectroscopy. The percentage specific lysis was determined as follows: [(experimental release minus spontaneous release)/(maximum release minus spontaneous release)] x 100. Spontaneous release (incubation of target cells in the absence of T cells) was less than 20% of maximum release caused by Triton X detergent in all experiments except for infected J774 and cultured macrophages, in which spontaneous release was as high as 30%, presumably due to apoptosis induced by salmonellae.
RESULTS
Infection with S. enterica serovar Typhimurium primes cytotoxic T-cell responses in mice. Previous studies have demonstrated a role for CD8 T cells in immunity to S. enterica serovar Typhimurium (16, 18), but little is known about MHC restriction and specificity of the response. We generated T-cell lines from BALB/c mice that were infected with S. enterica serovar Typhimurium SL3261 and subsequently challenged with the virulent C5 strain. The T-cell lines used in this study were CD3+4–8+ TCR++ V8+ and cytotoxic for J774 macrophages infected with S. enterica serovar Typhimurium SL3261. However, T-cell lines did not lyse uninfected J774, J774 cells treated with heat-killed S. enterica serovar Typhimurium SL3261, or NK-sensitive YAC cells (Fig. 1a). The data are consistent with recognition of antigens synthesized and released by viable salmonellae. T-cell lines also lysed J774 macrophages infected with either Salmonella enterica serovar Enteritidis or S. enterica serovar Typhimurium with equal efficacy indicating shared specificity between the two Salmonella serovars (Fig. 1b).
Salmonella-induced T-cell lines recognize proteins released by salmonellae. To assess the role of Salmonella secreted antigens in immunity to Salmonella infection, targets were sensitized with precipitated supernatants from cultured salmonellae and tested in cytotoxicity assays. T-cell lines lysed J774 cells sensitized with supernatant preparations from Salmonella C5FliA which lack flagellin (Fig. 2a), compared with control precipitates from LB medium alone. Furthermore, proteinase K treatment of the supernatant preparations before sensitizing target cells abrogated cytotoxicity (Fig. 2b), indicating that recognition was directed towards protein antigens.
Salmonella -induced T cells are not MHC class Ia-restricted. BALB/c T-cell lines killed both J774 (H-2d) and IC-21 (H-2b) target cells infected with S. enterica serovar Typhimurium or pulsed with Salmonella supernatant preparations (Fig. 3a). T-cell lines from BALB/c mice also killed antigen-pulsed bone marrow-derived macrophages from both H-2k and H-2d MHC haplotypes (Fig. 3b-c). Similar results were obtained when cultured macrophages were infected with SL3261 (data not shown). Antigen-pulsed BALB.B (H-2b) macrophages were not killed for reasons that are not yet clear. However, these data clearly indicate that the cytotoxic activity of T-cell lines described here is not MHC class Ia restricted.
The TAP and 2m dependence of antigen presentation in our system was investigated. RMA lymphoma cells pulsed with Salmonella supernatant preparations were lysed efficiently, whereas antigen pulsed TAP-defective RMA-S cells (17) were lysed to a much lesser extent (Fig. 2c), suggesting that presentation of Salmonella antigens was largely TAP dependent. The same pattern of CD8 T-cell lysis was observed when TAP+/+ and TAP–/– target cells were infected with S. enterica serovar Typhimurium SL3261 (data not shown). T-cell lines also recognized infected or Salmonella antigen-pulsed EL-4 but not the 2m-deficient counterpart C4.4 (5) (Fig. 3d), showing that presentation of Salmonella antigens to the T-cell lines described here was 2m-dependent.
Salmonella-induced T cells are H2-M3 restricted. The B10.CAS2-transfected fibroblast line was derived from the H2-M3 deficient mouse strain B10.CAS2, and TR8.4a is a variant of the B10.CAS2 line transfected with H2-M3wt (29). Our BALB/c T-cell lines lysed antigen-pulsed H2-M3 transfected TR8.4a cells while failing to lyse antigen-pulsed H2-M3-deficient B10.CAS2 cells (Fig. 4), strongly implicating the MHC class 1b molecule H2-M3 in restriction of the responses we describe here. It has previously been reported that CD8 T cells from Salmonella infected mice predominantly recognized a peptide representing amino acids 192 to 200 from the Salmonella heat shock protein GroEL presented by Qa-1b (16, 17). In addition, these CD8 T cells cross-reacted with peptide 216 to 224 from the endogenous mouse heat shock protein hsp60 (16, 17). The BALB/c T-cell lines generated in our system failed to lyse J774 treated with either the GroEL or hsp60 peptide whereas the a2d T-cell clone that recognizes Qa-1/Qdm (17), efficiently killed hsp peptide-pulsed J774 cells (Fig. 5a), confirming expression of functional Qa-1b molecules. It is noteworthy that our T-cell lines lysed infected or antigen-pulsed L cells that lack Qa-1 as efficiently as antigen-pulsed Qa-1b transfected L cells (Fig. 5b-c). Taken together, these data suggest that CD8 T cells in our system are H2-M3-, and not Qa-1b-restricted.
DISCUSSION
We investigated memory CD8 T cells induced by infection and challenge of mice with S. enterica serovar Typhimurium. CD8 T-cell lines were generated from BALB/c mice following infection with the attenuated S. enterica serovar Typhimurium strain SL3261 and challenge with the virulent strain C5. T-cell lines lysed J774 macrophages infected with viable S. enterica serovar Typhimurium, but not J774 treated with heat-killed S. enterica serovar Typhimurium. Responses cross-reacted between S. enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis. The T-cell lines also efficiently lysed macrophages treated with precipitates from Salmonella culture supernatants, but not supernatants digested with proteinase K. The data strongly implicated proteins synthesized and released by viable salmonellae as the principal antigens recognized by CD8 T cells in our system. Proteins released by salmonellae have not been implicated in recognition by Salmonella-specific CD8 T-cell responses previously, although secreted antigens have been shown to be superior carriers for the delivery of exogenous vaccine epitopes expressed by attenuated S. enterica serovar Typhimurium (7, 8, 23, 24).
We examined a range of wild-type and transfected target cells to determine the route of presentation and the antigen presenting molecules required for recognition of salmonellae antigens by CD8 T-cell lines. Salmonella-specific T-cell lines efficiently lysed the TAP-expressing RMA lymphoma cell line pulsed with Salmonella proteins or infected with S. enterica serovar Typhimurium, whereas similarly treated TAP-defective RMA-S cells were lysed only weakly. Thus, CD8 T-cell responses to viable salmonellae or proteins released from them were largely TAP dependent. However, the low level of cytotoxicity of T-cell lines against TAP-defective RMA-S targets suggest that our T-cell lines include other, TAP-independent specificities.
We further showed that presentation was 2m dependent using the paired cell lines EL-4 and the 2m-deficient counterpart C4.4 as targets. Consistent with these findings, a previous report has shown that 2m knockout mice show a dramatic increase in susceptibility to infection with S. enterica serovar Typhimurium (16), although an earlier report demonstrated that 2m knockout mice were resistant (9). The discrepancy is probably related to differences in both the mouse and Salmonella strains used in the two studies.
Our T-cell lines were cytotoxic for a variety of antigen-pulsed or infected targets of four MHC haplotypes, H-2d (J774, IC21), H-2b (EL-4, RMA), H-2wt17 (B10.CAS2) and H-2k (L cells), clearly showing that the T-cell specificity is not MHC class Ia restricted. Previous studies have also uniformly failed to demonstrate a major contribution of MHC class Ia-restricted responses among the CD8 T cells induced by S. enterica serovar Typhimurium infection (11). One explanation is the recently demonstrated interference with peptide presentation by MHC class Ia molecules by-products of the Salmonella yej operon, the first such virulence mechanism to be identified in bacteria, although a common mechanism of viral immune evasion (23).
MHC class Ia-restricted T-cell responses may not be completely lacking as anti-murine MHC class Ia antibodies partially inhibited cytoxicity in the CD8 T-cell responses reported by Lo, et al. (16). Thus, MHC class Ib-restricted CD8 T-cell responses may predominate in the immune response during S. enterica serovar Typhimurium infection. However, S. enterica serovar Typhi has been shown to induce MHC-restricted cytotoxic CD8 T-cell responses in BALB/c mice that were largely inhibited by monoclonal antibodies to H-2Ld (22). Also, anti-HLA MHC class Ia antibodies inhibited Salmonella enterica serovar Typhi-specific cytotoxicity in human attenuated S. enterica serovar Typhi vaccinees (27). It remains to be determined whether S. enterica serovar Typhi lacks a homologue of the yej-mediated immune evasion mechanism of S. enterica serovar Typhimurium, which would explain the apparent difference in the predominant cytotoxic T-lymphocyte specificity between these two distinct S. enterica serovars.
In view of the lack of MHC class Ia restriction but 2m and TAP dependence of T-cell cytotoxicity in our system, we examined the possibility of MHC class Ib-restricted responses. We compared the lytic activity of CD8 T-cell lines for antigen-pulsed B10.CAS2 fibroblast cells with and without transfection with M3wt, and the results clearly showed an H2-M3 dependence for the majority of the cytotoxic activity. The MHC class Ib molecule H2-M3 has been implicated in restriction of CD8 TCR cytotoxic T-cell responses for a number of N-formylated hydrophobic peptides derived from mitochondria and two bacterial species, Listeria monocytogenes and Mycobacterium tuberculosis (13, 15). Restriction of T-cell recognition to a nonformylated peptide bound to H2-M3 has also been reported (1).
H2-M3 is associated with 2m (15), consistent with the 2m dependence of the cytotoxicity shown here. We demonstrated that cytotoxicity was TAP dependent when either viable salmonellae or Salmonella protein preparations were used to sensitize target cells. Previously identified H2-M3 ligands vary in their dependence on TAP-mediated transport, responses to Listeria antigens being TAP-independent (13, 14), whereas mitochondrial ligands for H2-M3 require TAP-dependent transport (15). Our data suggests that Salmonella antigens delivered in the form of viable salmonellae or as released proteins are presented via a cytosolic route requiring TAP-mediated transport into the endoplasmic reticulum, although we have not investigated the mechanisms of antigen processing and presentation any further.
We examined whether our T-cell lines exhibited Qa-1-restricted cytotoxicity. The T-cell lines we generated were equally cytotoxic for L cells whether or not transfected with the Qa-1b molecule. Thus, most T cells in our lines were not Qa-1-restricted, in contrast to previous reports (16, 17). We further eliminated the possibility of Qa-1-restricted responses by showing that our lines were not cytotoxic for target cells treated with the peptide epitopes from the Salmonella heat shock protein GroEL or endogenous mouse hsp60 identified in the previous studies (16, 17). The discrepancy in results may be due to the different mouse strain (CB6F1) and Salmonella strains (SL3235 infected and C5 challenged) used in the experiments used to demonstrate Qa-1 restriction.
H2-M3-restricted, Listeria-specific cytotoxic T cells have been shown to provide protective immunity against listerial infection, indicating that these MHC class Ib-restricted responses can play a role in host defense against intracellular bacterial infection (13, 15). However, the antilisterial function of the H2-M3-restricted CD8 T-cell response appears to be limited to resistance against primary infection, indicating that MHC class Ib-restricted CD8 T cells serve as a bridge between the very rapid innate immune response and the slower developing MHC class Ia-restricted T-cell response (13, 15). Indeed, during Listeria infection it has been shown that expansion of MHC class Ib-restricted memory T cells is suppressed by the development of MHC class Ia-restricted memory T cells (6, 12).
We have generated T-cell lines from mice both infected and challenged with salmonellae, suggesting that H2-M3-restricted responses are detectable in the spleens of mice during the induction of a memory T-cell response. We suggest that MHC class Ib-restricted T cells, whether H2-M3 or Qa-1 restricted, may predominate in Salmonella infection due to the poor induction of MHC class Ia-restricted T cells and their consequent regulatory function upon challenge. However, we do not know if H2-M3-restricted T cells play any role in the protective immune response during Salmonella infection.
The antigen or peptide recognized by T-cell lines in this study remains unknown. However, cytotoxicity of macrophages infected with viable, but not heat-killed, S. enterica serovar Typhimurium, and the lack of lysis of macrophages incubated with supernatant proteins treated with proteinase K suggest antigens that are secreted proteins that would be largely removed by washing prior to the heat treatment. Alternatively, phagocytosis and processing of heat killed bacteria may not generate sufficient amounts of antigenic peptides to enter the MHC class I pathway. Otherwise, we have no information on which Salmonella protein(s) is recognized by CD8 T cells in our system, although a formylated Salmonella peptide is a likely candidate, by analogy with the other known ligands for H2-M3. However, our demonstration of H2-M3-restricted T-cell recognition induced by Salmonella infection extends the range of previously reported H2-M3-restricted responses to include a member of the Enterobacteriaceae.
Present address: Department of Clinical Biochemistry and Immunology, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom.
Deceased March 2005.
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Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom
ABSTRACT
Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.
INTRODUCTION
Salmonella species are intracellular gram-negative pathogens that cause a range of diseases, including typhoid fever, which, despite widespread use of antibiotics, represents a significant health problem in a number in developing nations. The recent emergence of multidrug-resistant Salmonella strains and the variable efficacy of existing vaccines increase the need for better understanding of the mechanisms of protective immunity to salmonellae and improvement of vaccination strategies (11, 30).
Infection of susceptible strains of inbred mice with Salmonella enterica serovar Typhimurium provides a murine model for typhoid fever which bears many similarities to human serovar Typhi infection (10). After oral uptake in mice, S. enterica serovar Typhimurium crosses the intestinal epithelium and spreads within phagocytes via the bloodstream to the spleen and liver, where the bacteria replicate (28). Innate immune mechanisms, including Slc11a1 (formerly Nramp1) gene function, and production of nitric oxide and reactive oxygen intermediates are important in controlling initial bacterial growth. However, induction of the acquired immune response is necessary for clearance of infection (11).
In a primary infection, the clearance of salmonellae from infected tissues is controlled by the acquisition of specific T-cell immunity. In addition, both T and B lymphocytes are involved in protection against a secondary infection (11, 18, 19, 26). However, despite the firm evidence that T cells are indispensable in controlling Salmonella infection, understanding which T-cell subsets participate during distinct stages of infection is less clear. Major histocompatibility complex (MHC) class II-deficient mice fail to clear infection with an attenuated strain of S. enterica serovar Typhimurium and develop chronic infection, suggesting an essential role for CD4 T cells (9). However, it has also been demonstrated that beta-2-microglobulin (2m)-deficient mice surviving primary infection with an attenuated S. enterica serovar Typhimurium strain suffer from impaired resistance to challenge infection with a virulent strain (16), implicating CD8 T cells in protection against Salmonella infection. More recently, it has been shown that CD8 T cells that secrete gamma interferon and kill S. enterica serovar Typhi-infected cells are elicited by immunization of volunteers with an attenuated Salmonella vaccine strain (27). The same group have demonstrated the concomitant induction of both CD4 and CD8 T-cell responses after vaccination with another oral typhoid vaccine candidate, CVD 908-htrA.
Limited information is available on the specificity and MHC restriction phenotype of T cells induced by Salmonella infection. Salmonella infection of mice has been shown to induce MHC class II-restricted CD4 T cells specific for the flagellar antigen FliC, and immunization with recombinant FliC protects mice against infection with a low dose of a virulent strain of S. enterica serovar Typhimurium (3, 20). CD4 T cells from infected mice also recognize the Salmonella invasion protein SipC, a type III secretion product (21), but a role for SipC in protection has not been reported. Similarly, the only Salmonella antigen so far identified that is recognized by CD8 T cells is the heat shock protein GroEL, a response that is MHC class 1b (Qa-1) restricted (16, 17). A recent report shows that a subset of human S. enterica serovar Typhi-specific CD8 T cells are also MHC class 1b and HLA-E restricted, but the antigen recognized has not been identified (25).
In this study, we demonstrate that cytotoxic T-cell lines from mice infected with S. enterica serovar Typhimurium recognize Salmonella proteins presented by the nonclassical MHC class Ib molecule H2-M3.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains aro attenuated S. enterica serovar Typhimurium SL3261, virulent S. enterica serovar Typhimurium C5, the flagellum-defective variant S. enterica serovar Typhimurium C5-FliA–, and Salmonella enterica serovar Enteritidis SE795 were used in this study and grown as described elsewhere (2). S. enterica serovar Typhimurium C5-FliA– was grown in the presence of 25 μg/ml of tetracycline. Heat-killed S. enterica serovar Typhimurium SL3261 was prepared by washing bacteria in phosphate-buffered saline followed by incubation at 60°C for 3 h and washing again. Effective killing was confirmed by pour plating on LB agar plates.
Preparation of proteins released by S. enterica serovar Typhimurium. For the preparation of proteins, S. enterica serovar Typhimurium strain C5 FliA– was grown in LB medium as described above until late-exponential phase was reached. Bacteria were pelleted and proteins from filtered culture supernatant were precipitated overnight at 5°C with 40% ammonium sulfate, centrifuged at 43,000 x g, incubated at 4°C for 1 h, and dissolved in phosphate-buffered saline. The protein preparation was desalted by 10DG column passage and dialyzed according to the manufacture's protocol (Bio-Rad Laboratories Ltd., United Kingdom). Fractions were collected and assayed for protein concentration using the bicinchoninic acid protein assay kit (Pierce & Warriner, United Kingdom). Where indicated, Salmonella proteins were treated with 1 mg/ml of proteinase K (Sigma) for 1 h at 60°C, and dialyzed for 24 h in phosphate-buffered saline. For loading targets, cells were incubated with S. enterica serovar Typhimurium proteins (10 μg/ml) for 3 h at 37°C followed by washing to remove unbound proteins and proteinase K in experiments in which it was used.
Mouse strains and cell lines. We maintained 6- to 8-week-old female BALB/c (H-2d), BALB.B (H-2b), BALB.K (H-2k), C3H/He (H-2k), CBA/J (H-2k), and B10.BR (H-2k) mice (Harlan Olac Ltd., Bicester, United Kingdom) at the University of Newcastle upon Tyne. Cell lines J774 (H-2d), IC-21 (H-2b), YAC (H-2d), EL-4 (H-2b), RMA (H-2b), and RMA-S (H-2b, TAP–/–) were obtained from the American Type Culture Collection. We acknowledge the kind gifts of the L cell fibroblast line (H-2k) and L cells transfected with Qa-1b (L-Qa-1b) from J. Dyson, Clinical Sciences Centre, Hammersmith Hospital, London, United Kingdom; the 2m-deficient C4.4 cells (H-2b) from R. Glas, Karolinska Institute, Stockholm, and the B10.CAS2 fibroblast cell line (H-2w17) and TR8.4a, a variant of B10.CAS2 transfected with H2-M3wt from C. R. Wang, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 μM 2-mercapthoethanol (RF-10). All tissue culture reagents were obtained from Sigma (United Kingdom) unless otherwise stated.
Bone marrow macrophages. Bone marrow-derived macrophages (referred to as cultured macrophages) were prepared as described elsewhere (4). Briefly, bone marrow cells were extracted from femurs and tibias and cultured for 6 days at 3 x 105 cells/ml in bacteriological-grade petri dishes containing RF-10 supplemented with 5% horse serum, 1 mM sodium pyruvate (Sigma), 40 μg/ml gentamicin (Sigma), and 20% conditioned medium from L929 cells as a source of macrophage colony-stimulating factor. Adherent cells were washed with phosphate-buffered saline (PBS), detached from the plastic with cold PBS containing 0.1% EDTA, washed twice with PBS, and used on days 6 to 14 of culture.
For infection cell lines were seeded at a density of 2 x 106 cells per well in 6 well cell culture plates in RF-10 without antibiotics. The following day the medium was removed and 1 ml of medium containing 100 x 106 CFU/ml of S. enterica serovar Typhimurium was added to each well. To facilitate infection, the plates were centrifuged at 400 x g for 10 min at room temperature, and the plates were incubated for 2 h at 37°C in 5% CO2. The cultures were washed twice with warmed RF-10 to remove unattached bacteria and incubated for 1 h in the presence of 100 μg/ml gentamicin to kill extracellular bacteria. Cells were removed from plates by using cold PBS containing 0.1% EDTA for use in experiments.
Establishment and maintenance of T-cell lines. Female 8-week-old BALB/c mice were immunized intravenously with 106 SL3261 in a tail vein as described previously (19), and challenged after 5 or 8 weeks with 5 x 1010 CFU of S. enterica serovar Typhimurium C5 by the intragastric route under mild anesthesia as described previously (10). Mice were sacrificed between 1 and 5 weeks after challenge and immune splenocytes were harvested to generate T-cell lines in vitro. CD8 cells were positively selected using magnetic beads according to the manufacturer's instructions (Miltenyi Biotec, GmbH, Bergish-Gladbach, Germany). The purity of the separated cell populations was determined by flow cytometry using the following monoclonal antibodies: anti-CD3-fluorescein isothiocyanate, anti-T-cell receptor -phycoerythrin, anti-T-cell receptor (TCR) -phycoerythrin, anti-CD8-fluorescein isothiocyanate, and anti-CD4-phycoerythrin (PharMingen, San Diego, CA).
We stimulated 2 x 106 CD8 T cells (>95% pure) with 105 irradiated (50 Gy) SL3261-infected J774 cells and 4 x 106 irradiated (20 Gy) syngeneic spleen cells in the presence of 40 μg/ml gentamicin. Recombinant human interleukin-2 (Cetus) was used at 100 IU/ml in the first week and at 10 IU/ml in the second week. Subsequent stimulations were performed fortnightly under the same conditions. Cytotoxicity assay was performed after restimulation and expansion in vitro at least three times. The phenotype of cultured T-cell lines was assessed by flow cytometry using the monoclonal antibodies described above and a mouse V T-cell receptor screening panel (PharMingen). The majority of T-cell lines were CD3+4–8+ TCR++ V8+. The cytotoxic T-cell activity of lines was assayed in a standard 4-h 51Cr-release assay and 19 of 25 T-cell lines were cytotoxic for J774 macrophages infected with S. enterica serovar Typhimurium SL3261. We selected five CD3+4–8+ TCR++ V8+ T-cell lines that were cytotoxic for SL3261-infected J774 to generate the data shown in the figures.
Cytotoxicity assay. Infected and uninfected target cells or target cells pulsed with Salmonella protein preparations were incubated for 1 h at 37°C with 3.7 MBq of [51Cr]sodium chromate (Amersham, United Kingdom). The targets were washed, resuspended at 5 x 104/ml, and 100-μl aliquots were added to 100-μl serial dilutions of T cells in round-bottomed 96-well plates. After 4 h of incubation, supernatants were collected and 51Cr activity was determined by gamma-scintillation spectroscopy. The percentage specific lysis was determined as follows: [(experimental release minus spontaneous release)/(maximum release minus spontaneous release)] x 100. Spontaneous release (incubation of target cells in the absence of T cells) was less than 20% of maximum release caused by Triton X detergent in all experiments except for infected J774 and cultured macrophages, in which spontaneous release was as high as 30%, presumably due to apoptosis induced by salmonellae.
RESULTS
Infection with S. enterica serovar Typhimurium primes cytotoxic T-cell responses in mice. Previous studies have demonstrated a role for CD8 T cells in immunity to S. enterica serovar Typhimurium (16, 18), but little is known about MHC restriction and specificity of the response. We generated T-cell lines from BALB/c mice that were infected with S. enterica serovar Typhimurium SL3261 and subsequently challenged with the virulent C5 strain. The T-cell lines used in this study were CD3+4–8+ TCR++ V8+ and cytotoxic for J774 macrophages infected with S. enterica serovar Typhimurium SL3261. However, T-cell lines did not lyse uninfected J774, J774 cells treated with heat-killed S. enterica serovar Typhimurium SL3261, or NK-sensitive YAC cells (Fig. 1a). The data are consistent with recognition of antigens synthesized and released by viable salmonellae. T-cell lines also lysed J774 macrophages infected with either Salmonella enterica serovar Enteritidis or S. enterica serovar Typhimurium with equal efficacy indicating shared specificity between the two Salmonella serovars (Fig. 1b).
Salmonella-induced T-cell lines recognize proteins released by salmonellae. To assess the role of Salmonella secreted antigens in immunity to Salmonella infection, targets were sensitized with precipitated supernatants from cultured salmonellae and tested in cytotoxicity assays. T-cell lines lysed J774 cells sensitized with supernatant preparations from Salmonella C5FliA which lack flagellin (Fig. 2a), compared with control precipitates from LB medium alone. Furthermore, proteinase K treatment of the supernatant preparations before sensitizing target cells abrogated cytotoxicity (Fig. 2b), indicating that recognition was directed towards protein antigens.
Salmonella -induced T cells are not MHC class Ia-restricted. BALB/c T-cell lines killed both J774 (H-2d) and IC-21 (H-2b) target cells infected with S. enterica serovar Typhimurium or pulsed with Salmonella supernatant preparations (Fig. 3a). T-cell lines from BALB/c mice also killed antigen-pulsed bone marrow-derived macrophages from both H-2k and H-2d MHC haplotypes (Fig. 3b-c). Similar results were obtained when cultured macrophages were infected with SL3261 (data not shown). Antigen-pulsed BALB.B (H-2b) macrophages were not killed for reasons that are not yet clear. However, these data clearly indicate that the cytotoxic activity of T-cell lines described here is not MHC class Ia restricted.
The TAP and 2m dependence of antigen presentation in our system was investigated. RMA lymphoma cells pulsed with Salmonella supernatant preparations were lysed efficiently, whereas antigen pulsed TAP-defective RMA-S cells (17) were lysed to a much lesser extent (Fig. 2c), suggesting that presentation of Salmonella antigens was largely TAP dependent. The same pattern of CD8 T-cell lysis was observed when TAP+/+ and TAP–/– target cells were infected with S. enterica serovar Typhimurium SL3261 (data not shown). T-cell lines also recognized infected or Salmonella antigen-pulsed EL-4 but not the 2m-deficient counterpart C4.4 (5) (Fig. 3d), showing that presentation of Salmonella antigens to the T-cell lines described here was 2m-dependent.
Salmonella-induced T cells are H2-M3 restricted. The B10.CAS2-transfected fibroblast line was derived from the H2-M3 deficient mouse strain B10.CAS2, and TR8.4a is a variant of the B10.CAS2 line transfected with H2-M3wt (29). Our BALB/c T-cell lines lysed antigen-pulsed H2-M3 transfected TR8.4a cells while failing to lyse antigen-pulsed H2-M3-deficient B10.CAS2 cells (Fig. 4), strongly implicating the MHC class 1b molecule H2-M3 in restriction of the responses we describe here. It has previously been reported that CD8 T cells from Salmonella infected mice predominantly recognized a peptide representing amino acids 192 to 200 from the Salmonella heat shock protein GroEL presented by Qa-1b (16, 17). In addition, these CD8 T cells cross-reacted with peptide 216 to 224 from the endogenous mouse heat shock protein hsp60 (16, 17). The BALB/c T-cell lines generated in our system failed to lyse J774 treated with either the GroEL or hsp60 peptide whereas the a2d T-cell clone that recognizes Qa-1/Qdm (17), efficiently killed hsp peptide-pulsed J774 cells (Fig. 5a), confirming expression of functional Qa-1b molecules. It is noteworthy that our T-cell lines lysed infected or antigen-pulsed L cells that lack Qa-1 as efficiently as antigen-pulsed Qa-1b transfected L cells (Fig. 5b-c). Taken together, these data suggest that CD8 T cells in our system are H2-M3-, and not Qa-1b-restricted.
DISCUSSION
We investigated memory CD8 T cells induced by infection and challenge of mice with S. enterica serovar Typhimurium. CD8 T-cell lines were generated from BALB/c mice following infection with the attenuated S. enterica serovar Typhimurium strain SL3261 and challenge with the virulent strain C5. T-cell lines lysed J774 macrophages infected with viable S. enterica serovar Typhimurium, but not J774 treated with heat-killed S. enterica serovar Typhimurium. Responses cross-reacted between S. enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis. The T-cell lines also efficiently lysed macrophages treated with precipitates from Salmonella culture supernatants, but not supernatants digested with proteinase K. The data strongly implicated proteins synthesized and released by viable salmonellae as the principal antigens recognized by CD8 T cells in our system. Proteins released by salmonellae have not been implicated in recognition by Salmonella-specific CD8 T-cell responses previously, although secreted antigens have been shown to be superior carriers for the delivery of exogenous vaccine epitopes expressed by attenuated S. enterica serovar Typhimurium (7, 8, 23, 24).
We examined a range of wild-type and transfected target cells to determine the route of presentation and the antigen presenting molecules required for recognition of salmonellae antigens by CD8 T-cell lines. Salmonella-specific T-cell lines efficiently lysed the TAP-expressing RMA lymphoma cell line pulsed with Salmonella proteins or infected with S. enterica serovar Typhimurium, whereas similarly treated TAP-defective RMA-S cells were lysed only weakly. Thus, CD8 T-cell responses to viable salmonellae or proteins released from them were largely TAP dependent. However, the low level of cytotoxicity of T-cell lines against TAP-defective RMA-S targets suggest that our T-cell lines include other, TAP-independent specificities.
We further showed that presentation was 2m dependent using the paired cell lines EL-4 and the 2m-deficient counterpart C4.4 as targets. Consistent with these findings, a previous report has shown that 2m knockout mice show a dramatic increase in susceptibility to infection with S. enterica serovar Typhimurium (16), although an earlier report demonstrated that 2m knockout mice were resistant (9). The discrepancy is probably related to differences in both the mouse and Salmonella strains used in the two studies.
Our T-cell lines were cytotoxic for a variety of antigen-pulsed or infected targets of four MHC haplotypes, H-2d (J774, IC21), H-2b (EL-4, RMA), H-2wt17 (B10.CAS2) and H-2k (L cells), clearly showing that the T-cell specificity is not MHC class Ia restricted. Previous studies have also uniformly failed to demonstrate a major contribution of MHC class Ia-restricted responses among the CD8 T cells induced by S. enterica serovar Typhimurium infection (11). One explanation is the recently demonstrated interference with peptide presentation by MHC class Ia molecules by-products of the Salmonella yej operon, the first such virulence mechanism to be identified in bacteria, although a common mechanism of viral immune evasion (23).
MHC class Ia-restricted T-cell responses may not be completely lacking as anti-murine MHC class Ia antibodies partially inhibited cytoxicity in the CD8 T-cell responses reported by Lo, et al. (16). Thus, MHC class Ib-restricted CD8 T-cell responses may predominate in the immune response during S. enterica serovar Typhimurium infection. However, S. enterica serovar Typhi has been shown to induce MHC-restricted cytotoxic CD8 T-cell responses in BALB/c mice that were largely inhibited by monoclonal antibodies to H-2Ld (22). Also, anti-HLA MHC class Ia antibodies inhibited Salmonella enterica serovar Typhi-specific cytotoxicity in human attenuated S. enterica serovar Typhi vaccinees (27). It remains to be determined whether S. enterica serovar Typhi lacks a homologue of the yej-mediated immune evasion mechanism of S. enterica serovar Typhimurium, which would explain the apparent difference in the predominant cytotoxic T-lymphocyte specificity between these two distinct S. enterica serovars.
In view of the lack of MHC class Ia restriction but 2m and TAP dependence of T-cell cytotoxicity in our system, we examined the possibility of MHC class Ib-restricted responses. We compared the lytic activity of CD8 T-cell lines for antigen-pulsed B10.CAS2 fibroblast cells with and without transfection with M3wt, and the results clearly showed an H2-M3 dependence for the majority of the cytotoxic activity. The MHC class Ib molecule H2-M3 has been implicated in restriction of CD8 TCR cytotoxic T-cell responses for a number of N-formylated hydrophobic peptides derived from mitochondria and two bacterial species, Listeria monocytogenes and Mycobacterium tuberculosis (13, 15). Restriction of T-cell recognition to a nonformylated peptide bound to H2-M3 has also been reported (1).
H2-M3 is associated with 2m (15), consistent with the 2m dependence of the cytotoxicity shown here. We demonstrated that cytotoxicity was TAP dependent when either viable salmonellae or Salmonella protein preparations were used to sensitize target cells. Previously identified H2-M3 ligands vary in their dependence on TAP-mediated transport, responses to Listeria antigens being TAP-independent (13, 14), whereas mitochondrial ligands for H2-M3 require TAP-dependent transport (15). Our data suggests that Salmonella antigens delivered in the form of viable salmonellae or as released proteins are presented via a cytosolic route requiring TAP-mediated transport into the endoplasmic reticulum, although we have not investigated the mechanisms of antigen processing and presentation any further.
We examined whether our T-cell lines exhibited Qa-1-restricted cytotoxicity. The T-cell lines we generated were equally cytotoxic for L cells whether or not transfected with the Qa-1b molecule. Thus, most T cells in our lines were not Qa-1-restricted, in contrast to previous reports (16, 17). We further eliminated the possibility of Qa-1-restricted responses by showing that our lines were not cytotoxic for target cells treated with the peptide epitopes from the Salmonella heat shock protein GroEL or endogenous mouse hsp60 identified in the previous studies (16, 17). The discrepancy in results may be due to the different mouse strain (CB6F1) and Salmonella strains (SL3235 infected and C5 challenged) used in the experiments used to demonstrate Qa-1 restriction.
H2-M3-restricted, Listeria-specific cytotoxic T cells have been shown to provide protective immunity against listerial infection, indicating that these MHC class Ib-restricted responses can play a role in host defense against intracellular bacterial infection (13, 15). However, the antilisterial function of the H2-M3-restricted CD8 T-cell response appears to be limited to resistance against primary infection, indicating that MHC class Ib-restricted CD8 T cells serve as a bridge between the very rapid innate immune response and the slower developing MHC class Ia-restricted T-cell response (13, 15). Indeed, during Listeria infection it has been shown that expansion of MHC class Ib-restricted memory T cells is suppressed by the development of MHC class Ia-restricted memory T cells (6, 12).
We have generated T-cell lines from mice both infected and challenged with salmonellae, suggesting that H2-M3-restricted responses are detectable in the spleens of mice during the induction of a memory T-cell response. We suggest that MHC class Ib-restricted T cells, whether H2-M3 or Qa-1 restricted, may predominate in Salmonella infection due to the poor induction of MHC class Ia-restricted T cells and their consequent regulatory function upon challenge. However, we do not know if H2-M3-restricted T cells play any role in the protective immune response during Salmonella infection.
The antigen or peptide recognized by T-cell lines in this study remains unknown. However, cytotoxicity of macrophages infected with viable, but not heat-killed, S. enterica serovar Typhimurium, and the lack of lysis of macrophages incubated with supernatant proteins treated with proteinase K suggest antigens that are secreted proteins that would be largely removed by washing prior to the heat treatment. Alternatively, phagocytosis and processing of heat killed bacteria may not generate sufficient amounts of antigenic peptides to enter the MHC class I pathway. Otherwise, we have no information on which Salmonella protein(s) is recognized by CD8 T cells in our system, although a formylated Salmonella peptide is a likely candidate, by analogy with the other known ligands for H2-M3. However, our demonstration of H2-M3-restricted T-cell recognition induced by Salmonella infection extends the range of previously reported H2-M3-restricted responses to include a member of the Enterobacteriaceae.
Present address: Department of Clinical Biochemistry and Immunology, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom.
Deceased March 2005.
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