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The Journal of Immunology, 2002, 169: 2545-2552.
Copyright © 2002 by The American Association of Immunologists

Role of IL-12-Independent and IL-12-Dependent Pathways in Regulating Generation of the IFN-{gamma} Component of T Cell Responses to Salmonella typhimurium1

Beena John, Deepa Rajagopal, Achal Pashine, Satyajit Rath, Anna George and Vineeta Bal2

National Institute of Immunology, New Delhi, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Clearance of facultative intracellular pathogens such as Salmonella requires IFN-{gamma} from CD4 T cells. Mechanisms linking intracellular pathogen recognition with induction of IFN-{gamma}-producing T cells are still poorly understood. We show in this study that IL-12 is not required for commitment to the IFN-{gamma}-producing T cell response in infection with Salmonella typhimurium, but is needed for its maintenance. The IL-12-independent signals required for commitment depend on events during the first hour of infection and are related to Ag presentation. Even transient attenuation of Ag presentation early during infection specifically abrogates the IFN-{gamma} component of the resulting CD4 T cell response. The IL-12 needed for maintenance is also better induced by live rather than dead bacteria in vivo, and this difference is due to specific suppression of IL-12 induction by dead bacteria. Presence of exogenous IL-4 down-modulates IL-12 production by macrophages activated in vitro. Furthermore, macrophages from IL-4-null mice secrete high levels of both IL-12 and IL-18 in response to stimulation in vivo even with dead bacteria, but this does not lead to induction of IFN-{gamma}-secreting T cells in response to immunization with dead S. typhimurium. Early IL-4 is contributed by triggering of CD4 NK T cells by dead, but not live, bacteria. Thus, Ag presentation-related IL-12-independent events and IL-4-sensitive IL-12-dependent events play crucial complementary roles in the generation of the IFN-{gamma}-committed CD4 T cell component of the immune response in Salmonella infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phagocytic cells first encounter most infectious agents, act as APCs to regulate quantities and qualities of resultant T cell responses, and mediate ultimate clearance of infection. Pathogen location determines the quality of effective T cell responses needed; thus, clearance of macrophage endocytic vesicle-resident facultative intracellular pathogens (FIPs)3 requires CD4 T cells activating macrophages through IFN-{gamma} (1, 2, 3). Commitment of responding CD4 T cells to production of cytokines such as IFN-{gamma}, IL-4, IL-5, IL-10, and others can be controlled by Ag-nonspecific signals such as CD80/CD86, IL-12/IL-18, IL-6, IL-4, or IFN-{gamma} (2, 4, 5, 6), as well as by peptide-MHC class II (MHCII) complex availability (7, 8). Pathogenic products can modulate many of these signals, and induce secretion of cytokines both from APCs (9) and from NK lineage cells (10, 11, 12). FIPs such as Salmonella, Mycobacterium, Leishmania, or Listeria use a variety of strategies to find safe niches inside phagocytic cells (13, 14, 15), which may influence Ag processing and presentation. However, it is unclear which pathways are actually used in regulating cytokine balance commitment in response to a given infecting pathogen so as to trigger the most optimal effector T cell cytokine profile for achieving clearance of infection.

We have used the FIP Salmonella typhimurium (Stm), a natural mouse pathogen, to examine how triggering of IFN-{gamma}-making T (IFN-{gamma}-T) cells against FIPs is regulated. We have shown earlier that immunization of mice with live Stm activates an IFN-{gamma}-T cell response (16), even when an auxotrophic mutant, Stm-aroA, which cannot multiply in vivo, is used (17), whereas injection of dead Stm triggers an equivalent magnitude of T cell responses that cannot, however, secrete IFN-{gamma}. We have now further analyzed signals required for inducing IFN-{gamma}-T cell responses to live Stm, and we demonstrate in this study that while IL-12-independent signals required for commitment of the T cell response to IFN-{gamma} are mediated through MHCII-restricted Ag presentation, the IL-12 induction needed for maintenance of the IFN-{gamma}-T cell response is controlled by presentation to NK T cells, illustrating the complex role Ag presentation plays in qualitative regulation of the T cell response to infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and immunizations

Mouse strains from The Jackson Laboratory (Bar Harbor, ME) or as a gift from L. van Kaer (Vanderbilt University, Nashville, TN), and maintained in the Small Animal Facility of the National Institute of Immunology (New Delhi, India) were used with the approval of the Institutional Animal Ethics Committee (two to five mice per group). For engineered mutant mouse strains, syngeneic mice served as wild-type (WT) controls. A metabolic mutant strain of Stm, Stm-aroA, was used for immunization either live or after heat killing in a boiling waterbath (17) at ~1 x 106 Stm-aroA i.p. per mouse. Parenteral gentamicin (GM) and tetracycline (TC) (1 mg each/mouse/day) treatment was initiated at various times to kill both extracellular and intracellular Stm, and was continued daily until euthanasia. Parenteral chloroquine (CQ) was given as a single dose (0.5 mg/mouse) at the times indicated.

Measurement of CFUs of intracellular Stm

Stm-infected mice were given GM (1 mg/mouse/day) at indicated times, and euthanized 48 h later. Splenic cells were isolated, washed, and lysed in 1% taurocholate in PBS, and dilutions were plated on Luria Bertani agar plates for overnight incubation at 37°C for CFU enumeration.

Lymphocyte proliferation assays

Splenic cells from immunized mice were cultured in the presence of titrated amounts of sonicated Stm extract (Stm sonicate) in Click’s medium (Irvine Scientific, Irvine, CA) containing 10% FBS (Biological Industries, Rehovot, Israel), glutamine, and antibiotics. After 4 days of incubation, cultures were pulsed with 0.5 µCi [3H]thymidine (New England Nuclear, Boston, MA), harvested, and counted (Betaplate; LKB-Pharmacia, Turku, Finland). Counts from triplicate cultures are expressed as mean ± SEM.

Cytokine estimation

For cytokine estimation in T cell activation assays, culture supernatants (CS) were harvested at 60 h. For estimating cytokine induction from macrophages ex vivo, plastic-adherent peritoneal cells from mice given dead or live Stm-aroA i.p. 24 h earlier were cultured in titrating numbers for 24 h, and CS collected. For triggering cytokine secretion from peritoneal cells in vitro, live or dead Stm were added to wells with titrating cell numbers in presence or absence of rIL-4 (10 ng/ml; R&D Systems, Minneapolis, MN), where appropriate, and CS collected at 48 h. Cytokines were measured using commercial enzyme immunoassay systems (R&D Systems), with recombinant cytokine standards run in parallel for quantitation.

Flow cytometry

For measuring phagocytosis, FITC-labeled live or dead Stm-aroA were used. The viability of labeled live Stm-aroA was 90%. Peritoneal cells from mice receiving equal numbers of live or dead fluorescein-labeled Stm-aroA 15–240 min earlier were harvested and analyzed for cell-associated bacteria by flow cytometry (Bryte; Bio-Rad, Hemel Hampstead, U.K.). For detecting intracellular IL-4 in NK T cells, spleen cells from mice given live or dead Stm 24 h previously were stained for CD4 and NK1.1 in two separate colors, and then permeabilized and stained for IL-4, according to manufacturers’ instructions (BD PharMingen, San Diego, CA), before being subjected to flow cytometry (LSR; BD Biosciences, San Jose, CA). Flow cytometric data were analyzed with FlowJo software (Treestar, San Jose, CA).

Statistical analysis

Wherever appropriate, Student’s t test was applied to calculate significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Live, but not dead, Stm trigger IL-12 from macrophages as well as IFN-{gamma}-T cell responses in vivo

When we immunized C57BL/6 mice with either live or dead Stm-aroA i.p., the proliferative responses (Fig. 1GoA) of their splenic cells to Stm Ags in vitro at day 7 postimmunization (p.i.) were similar, as were the levels of IL-10 induced in CS (Fig. 1GoC). However, high IFN-{gamma} levels were seen in CS from live Stm-immune cells, while practically no IFN-{gamma} was seen in CS from dead Stm-immune cells (Fig. 1GoB). The ratio of IFN-{gamma}:IL-10 levels in T cell CS from live Stm-immune mice was significantly different from those from dead Stm-immune mice (p < 0.01). Spleen cells from unimmunized mice did not respond to Stm sonicate in vitro (data not shown). If MHCII-null (MHCII-/-) mice were similarly immunized with live Stm-aroA, their spleen cells did not generate IFN-{gamma} responses (Fig. 1GoD), while TAP-1-null mice generated excellent responses (Fig. 1GoE), demonstrating that the response being read out was mediated by MHCII-restricted T cells.



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  		FIGURE 1. Live Stm trigger IFN-{gamma}-T cell responses and IL-12/IL-18 secretion. Stm sonicate-induced responses of splenic cells from WT mice 7 days p.i. with either live or dead Stm showing proliferation (A), IFN-{gamma} levels (B), or IL-10 levels (C). D and E, Show Stm sonicate-induced IFN-{gamma} levels from splenic cells of live Stm-immunized MHCII-null or TAP-1-null mice, respectively, in comparison with WT mice. F, Shows IL-12 and IL-18 levels secreted by macrophages from WT mice given either live or dead Stm. Data represent three to nine separate experiments.

 
Because peritoneal macrophages are responsible for uptake of Stm injected i.p., and IL-12 and IL-18 are involved in IFN-{gamma} induction in T cells (18), we measured IL-12 and IL-18 levels secreted by peritoneal macrophages from mice given live or dead Stm in vivo. Macrophages from mice given live Stm secreted higher levels of both IL-12 and IL-18 than those from mice given dead Stm (Fig. 1GoF), suggesting an association between the ability of live Stm to induce IL-12 secretion from APCs in vivo and its potential to generate IFN-{gamma} in the T cell responses triggered.

IL-12-null mice can generate an IFN-{gamma}-T cell response but cannot maintain it

To examine the role of IL-12 in anti-Stm T cell responses, we immunized WT or IL-12p40-null mice with live or dead Stm-aroA. Anti-Stm proliferative responses of splenic cells in vitro were similar between the two groups at both 3 and 7 days p.i. (Fig. 2Go, A and B), as were the levels of IL-10 induced (Fig. 2Go, C and D). In WT mice, live Stm immunization led to induction of detectable IFN-{gamma} levels even at 3 days p.i. (Fig. 2GoE), and these IFN-{gamma} levels increased significantly (p < 0.05) at 7 days p.i. (Fig. 2GoF), while no such induction was seen in T cells from dead Stm-immune mice. Interestingly, live Stm-mediated induction of IFN-{gamma}-T cells was almost unaffected at 3 days p.i. in IL-12p40-null mice (Fig. 2GoE). However, the levels of IFN-{gamma} induced did not go up further at 7 days p.i. in IL-12-null mice, unlike in WT mice (Fig. 2GoF). These data showed that even in the absence of IL-12, live Stm immunization could induce commitment to an IFN-{gamma}-T cell response, but its maintenance required IL-12.



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  		FIGURE 2. Live Stm induce, but cannot sustain IFN-{gamma}-T cell responses in IL-12-null mice. A, C, and E, Show responses on day 3; B, D, and F, show responses on day 7 p.i. Stm sonicate-induced responses of splenic cells from WT or IL-12-null mice immunized with either live or dead Stm are shown as proliferation (A and B), IL-10 levels (C and D), and IFN-{gamma} levels (C and F). Data represent three separate experiments.

 
Control of IFN-{gamma}-T cell commitment in response to live Stm involves Ag presentation

We next examined the possible role of Ag presentation-related events in the induction of IFN-{gamma}-T cell responses to Stm. The H-2 M (human HLA-DM) molecule catalyzes peptide loading onto MHCII in endolysosomal compartments (19). We immunized WT or H-2 M{alpha}-null mice with live or heat-killed Stm-aroA. Seven days p.i., proliferative responses of spleen cells were comparable in all groups (Fig. 3GoA). However, H-2 M-null mice generated no IFN-{gamma}-T cells in response to live Stm (Fig. 3GoB). We confirmed that the proliferative responses being detected were from CD4 T cells by adding anti-CD4 mAb to the recall cultures, which blocked the proliferative responses (Fig. 3GoC). Although H-2 M-null mice could not generate any IFN-{gamma} component in their T cell responses to live Stm, their macrophages secreted unchanged levels of IL-12 in response to live Stm (Fig. 3GoD), suggesting that IL-12-independent events relevant for IFN-{gamma} commitment of CD4 T cells against live Stm involve Ag presentation.



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  		FIGURE 3. CD4 T cells in H-2 M-null mice cannot mount IFN-{gamma}-T cell responses to live Stm. Stm sonicate-induced responses of splenic cells from WT or H-2 M-null mice 7 days p.i. with either live or dead Stm showing proliferation (A) and IFN-{gamma} levels (B). C, Shows blockade of the Stm-specific proliferative responses of splenic cells of immunized H-2 M-null mice by anti-CD4 mAb (a.CD4). D, Shows the levels of IL-12 secreted by macrophages from WT or H-2 M-null mice given either live or dead Stm. Data represent three independent experiments.

 
Induction of IFN-{gamma}-T cell responses depends on early events during Stm infection

To extrapolate from H-2 M-null mice to normal WT mice, it was necessary to compromise Ag presentation transiently during Stm immunization in WT mice. For this, we first established the time window over which Stm had to be alive in vivo to induce IFN-{gamma}-T cell responses. GM + TC treatment to kill Stm in vivo was either initiated just before live Stm injection, or 30 min later. One week p.i., the magnitude of splenic cell proliferative responses was comparable in all groups of immunized mice (Fig. 4GoA). However, if antibiotic treatment was initiated along with live Stm injection, IFN-{gamma}-T cell induction was prevented, while treatment begun 30 min after bacterial injection allowed efficient commitment to IFN-{gamma}-T cell pathways (Fig. 4GoB). Thus, live Stm are needed for only a short period after infection for induction of T cells committed to making IFN-{gamma}.



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  		FIGURE 4. A 30-min window of exposure to live Stm is sufficient for triggering IFN-{gamma}-T cell response associated with rapid macrophage entry. Stm sonicate-induced responses of splenic cells from WT mice 7 days p.i. with either live or dead Stm along with the antibiotic treatment indicated showing proliferation (A) and IFN-{gamma} levels (B). C, CFUs recovered per spleen from unimmunized mice when live Stm-aroA injection was followed by GM treatment begun at times indicated. D, Flow cytometric profiles of peritoneal cells from mice given live or killed fluorescein-labeled Stm-aroA at various times earlier, as indicated, with profiles of cells from mice not given bacteria, shown as shaded curves. Data represent five to six independent experiments.

 
FIPs such as Stm are known to promote their own phagocytosis (20), and we confirmed that more live Stm got into APCs in the 30-min time window than did dead bacteria. GM treatment was initiated at various times after live Stm injection for selective clearance of extracellular bacteria, and surviving CFUs were estimated 48 h later. Most live bacteria were in GM-inaccessible compartments 30–60 min postinjection, while earlier initiation of GM treatment decreased the surviving bacterial load (Fig. 4GoC). We also compared the efficiency of uptake of fluorescein-labeled live or dead Stm into peritoneal macrophages upon i.p. injection. Live Stm were associated with peritoneal APCs within 15–30 min after injection, while dead Stm were not (Fig. 4GoD). Thus, at early times after bacterial injection, more live Stm enter APCs, thereby increasing the early Ag load in APCs.

Early events mediating commitment to IFN-{gamma}-T cell responses to live Stm are sensitive to chloroquine

To block Ag presentation transiently during this critical early time window, we used a single dose of CQ, a lysosomotropic agent known to block MHCII-restricted presentation (21). CQ was given in a single dose either along with live Stm, or 2 h later. GM + TC treatment was initiated 1 h postinfection. CQ treatment at either time point had no effect on the magnitude of proliferative spleen cell responses triggered (Fig. 5GoA). If CQ treatment was initiated at 2 h postinfection, normal IFN-{gamma}-T cell responses were triggered (Fig. 5GoB), establishing that CQ did not abrogate anti-Stm T cell responses. However, if CQ was given along with live Stm, T cell commitment to IFN-{gamma} was inhibited by 10- to 30-fold (Fig. 5GoB). Macrophages from live Stm-immunized mice given CQ secreted levels of IL-12 indistinguishable from mice given live Stm alone (Fig. 5GoC), showing that the CQ-inhibitable early event responsible for triggering IFN-{gamma}-T cells was IL-12 independent.



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  		FIGURE 5. Early events mediating induction of IFN-{gamma}-T cell responses to live Stm are CQ sensitive. Stm sonicate-induced responses of splenic cells from WT mice 7 days p.i. with either live or dead Stm along with the CQ treatment indicated showing proliferation (A) or IFN-{gamma} levels (B). C, Levels of IL-12 secreted by macrophages from mice given either live or dead Stm along with CQ treatment, as indicated. Data represent two to four independent experiments.

 
Induction of IL-12 and IL-18 from macrophages in vivo is actively suppressed by dead, but not live, Stm

We also examined the control of induction of IL-12 by live, but not dead Stm in vivo. When live or dead Stm were used to stimulate macrophages in vitro, equivalent levels of IL-12 were generated (Fig. 6GoA), consistent with previous reports (22). Also, when peritoneal macrophages were harvested 24 h after injection of live or dead Stm and cultured in vitro for a further 24-h period, and cytokines secreted by them were measured, it was evident that, while macrophages from mice given live Stm showed higher levels of IL-12 and IL-18 as compared with cells from dead Stm-immune mice (Fig. 6Go, B and C), the levels of IL-1{beta}, TNF-{alpha}, and IL-10 secreted were not different (Fig. 6Go, D, E, and F), suggesting that induction of IL-12 and IL-18 by dead Stm is specifically suppressed in vivo.



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  		FIGURE 6. Live and dead Stm regulate cytokine secretion differentially. A, IL-12 levels secreted by peritoneal macrophages when stimulated with live or dead Stm in vitro. B–F, Levels of IL-12, IL-18, IL-1{beta}, TNF-{alpha}, and IL-10 secreted in culture by macrophages from mice receiving live or dead Stm in vivo 24 h earlier. Data represent three to nine separate experiments.

 
IL-12 induction from macrophages is regulated by IL-4

We tested whether IL-4, claimed to be an inhibitor of IL-12 induction (23), could suppress IL-12 induction. The addition of rIL-4 into macrophage cultures stimulated with Stm efficiently suppressed IL-12 induction in vitro (Fig. 7GoA). We therefore tested whether IL-4 played a role in the induction of IL-12 in vivo. IL-4-null or WT mice were given either live or dead Stm, and IL-12 and IL-18 secreted by their peritoneal macrophages were estimated. Although live Stm triggered equivalent levels of IL-12 and IL-18 from both IL-4-null and WT mice, dead Stm could evoke a similar induction of IL-12 and IL-18 in IL-4-null mice, but not in WT mice (Fig. 7Go, B and C), suggesting that in WT mice administration of dead Stm specifically induced IL-4-mediated suppression of IL-12 and IL-18 production.



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  		FIGURE 7. IL-4 negatively regulates IL-12 and IL-18 secretion, but not IFN-{gamma} commitment to live Stm immunization. A, IL-12 levels secreted by peritoneal macrophages when stimulated with live or dead Stm in vitro in the presence or absence of rIL-4. B and C, IL-12 and IL-18 levels secreted by macrophages from WT or IL-4-null mice receiving live or dead Stm 24 h earlier. D, Proliferative responses and E, IFN-{gamma} induction in Stm sonicate-activated cultures of splenocytes from WT or IL-4-null mice immunized with live or killed Stm i.p. Data are representative of three to eight independent experiments.

 
We further tested IFN-{gamma}-T cell responses in WT and IL-4-null mice. Although anti-Stm proliferative responses of immune T cells from all groups were comparable (Fig. 7GoD), live Stm induced high levels of IFN-{gamma}, while dead Stm did not (Fig. 7GoE). Thus, T cells from IL-4-null mice immunized with dead Stm still produced very low levels of IFN-{gamma}, despite triggering high levels of IL-12 and IL-18.

Suppression of IL-12 induction in vivo in mice given dead Stm is mediated by nonclassical MHC class I-restricted NK T cells

Because the IL-4-mediated suppression of IL-12 in vivo in response to dead Stm was an early event mediated in the first 24 h after injection, we looked at the most likely candidate component of the innate immune system capable of secreting IL-4 early after exposure, the NK T cells. Spleen cells from mice receiving either live or dead Stm 24 h before euthanasia were stained for CD4 (Fig. 8GoA), NK1.1 (Fig. 8GoB), and intracellular IL-4 (Fig. 8GoD). NK T cells from dead Stm-immunized mice showed substantial increase in size (Fig. 8GoC) as well as induction of intracellular IL-4 (Fig. 8GoD), while cells from live Stm-immunized mice did not, showing that dead Stm triggered NK T cells better than live Stm could. NK T cells from IL-4-null mice given live or dead Stm showed no staining for IL-4 (data not shown), confirming the specificity of detection.



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  		FIGURE 8. Dead, but not live, Stm trigger activation and IL-4 production in NK T cells in a nonclassical MHC class I-restricted fashion. A and B, The gate used for CD4 cells and NK1.1-expressing CD4 (NK T) cells, respectively. C, Forward scatter; D, intracellular staining for IL-4 on gated NK T cells from pooled splenocytes of mice given dead (thick lines) or live (thin lines) Stm. E, Levels of IL-12 secreted in culture by macrophages from WT, TAP-null, or {beta}2m-null mice receiving live or dead Stm in vivo 24 h earlier. Data represent three to five separate experiments.

 
We next examined the MHC restriction of suppression of IL-12 in response to dead Stm in vivo, using TAP-1-null and {beta}2-microglobulin ({beta}2m)-null mice in comparison with WT mice, because TAP-1-null mice have normal CD1-restricted NK T cells, while {beta}2m-null mice lack them (24, 25). All three strains showed similar induction of IL-12 levels in response to live Stm (Fig. 8GoE). However, while WT and TAP-1-null mice showed low levels of IL-12 induction in response to dead Stm, high levels of IL-12 were induced by dead Stm in {beta}2m-null mice (Fig. 8GoE), suggesting restriction of IL-4 production by nonclassical MHC class I.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Upon infection, FIPs actively attempt to enter their favorite intracellular niches, while the immune system needs to sense the presence of these intracellular pathogens early during infection to generate effective IFN-{gamma}-T cells for protective immunity. However, the mechanisms by which detection of the intracellular nature of FIP infection is connected to induction of IFN-{gamma}-T cell responses are not yet clear. Our data in this study suggest that rapid ingress of live Stm into APCs and associated early Ag presentation-related events culminate in specific commitment to IFN-{gamma}-T cell responses in an IL-12-independent fashion. The induction of IL-12 is crucially needed for maintaining immune response to live Stm, and is ensured by the absence of its IL-4-mediated suppression that is normally seen in response to dead Stm via activation of NK T cells.

All proliferative and IFN-{gamma} responses we have reported in this work require MHCII and are inhibited by anti-CD4 mAb in culture, ensuring that selective induction of IFN-{gamma} in responding MHCII-restricted CD4 T cells is being measured. We have also estimated a non-IFN-{gamma} cytokine in all these experiments, and the levels of IL-10 induced follow the proliferative responses in magnitude, again confirming that the modulatory effects observed are specific to IFN-{gamma} induction. We have also observed that immunization with dead Stm induces greater levels of IL-13, a Th2 cytokine, than immunization with live Stm does (data not shown), confirming the radical cytokine profile shift observed between the two modes of immunization.

It has been reported that IL-12 induces IFN-{gamma} responses in activated T cells (2, 3) and IL-12p40-null mice are susceptible to FIP infections (18, 23). However, we show in this work that live Stm induce a IFN-{gamma}-T cell response quite well in IL-12p40-null mice early after immunization. Thus, IL-12 is not essential for selective commitment to IFN-{gamma} in live Stm-induced T cell responses. In support of other reports (26, 27), our results also show that the absence of IL-12 does cause lack of maintenance of IFN-{gamma}-T cell response, so that at 7 days p.i., cells from immunized IL-12-null mice make relatively little IFN-{gamma} in their responses. By day 20, there is no detectable IFN-{gamma}-T cell response in live Stm-immune IL-12-null mice (data not shown). We find normal IL-18 levels in IL-12-null mice receiving live Stm (data not shown). Thus, IL-12 is not necessary to induce IFN-{gamma}-T cell responses, but is essential (and nonreplaceable by IL-18) for maintaining them.

We have examined Ag-nonspecific signals reported to be involved in IFN-{gamma}-T cell commitment, and have not observed any differences in induction of the cell surface molecules CD40, CD80, CD86, ICAM-1, or LFA-1 by flow cytometry, or in mRNA levels of TGF-{beta}, ligand for chemokine receptor CCR4, or ligand for chemokine receptor CXCR3 by real-time quantitative RT-PCR (Taqman) analysis, in APCs from mice recently given live or killed Stm (data not shown). No differential modulation of Ag-nonspecific signals by live vs killed Stm can therefore be invoked to explain the induction of IFN-{gamma}-T cell responses by live Stm.

In contrast, our data do show that Ag presentation-related events play a crucial role in IFN-{gamma}-T cell induction by live Stm. APCs from H-2 M-null mice show defective presentation of many Ags on MHCII, although they present exogenous peptides well, but these mice can still generate CD4 T cell responses to some immunogens (28). The Stm sonicate used in this study for recall responses contains sufficient degraded peptidic moieties to be well presented without processing by fixed APCs (data not shown), ensuring its efficient presentation by H-2 M-null APCs during recall assays. In H-2 M-null mice, live Stm did not generate any IFN-{gamma}-T cell response, although the magnitude of T cell priming was unaffected, as indicated by proliferative responses and induction of IL-10 (data not shown), suggesting a role for Ag presentation in the IFN-{gamma}-T cell response induced by live Stm. However, it remained possible that the known changes found in the positively selected TCR repertoires of H-2 M-null mice (19, 28) may have contributed to the phenomenon observed, necessitating transient inhibition of Ag processing in WT mice.

The antibiotic treatment-based data show that Stm are required to be live only for the first 30 min in vivo for the eventual induction of IFN-{gamma}-T cell responses. This time window correlates with the more rapid entry of live than killed Stm into APCs. FIPs such as Stm efficiently enter phagocytes (20) and persist in early endosomal compartments by redirecting their intracellular transport pathways (29). Parasitophorous vacuoles can contain H-2 M (30), and peptide-MHC complexes can be formed efficiently in phagosomes (31). We (8) and others (7, 32) have shown that increased efficiency of Ag presentation can enhance the IFN-{gamma}-T cell responses, raising this possibility in the present case as well. Furthermore, recent evidence suggests that Ag presentation for as short a period as 2 h may be enough to induce commitment in responding T cells (33), providing one possible explanation for the data we report in this work.

Such an explanation is supported by our findings using CQ, a known inhibitor of endolysosomal acidification that inhibits presentation of Stm Ags to Stm-immune T cells in vitro (data not shown). The dose of CQ used is within the clinical range, and CQ did not show any toxicity against Stm (data not shown). The use of a single dose of CQ to block Ag presentation on MHCII transiently in vivo did not change the magnitude of the T cell responses. However, CQ treatment inhibited the IFN-{gamma}-T cell response to live Stm only if treatment was initiated along with immunization, and not if it was started 2 h after immunization. The data with CQ treatment and H-2 M-null mice together suggest strongly that, rather than any repertoire restriction in H-2 M-null mice, early Ag presentation-related events associated with presence of live Stm are likely to be essential, despite being themselves transient, for mediating long-range commitment of responding T cells to IFN-{gamma}. Early events may mediate identification of the FIP nature of an incoming infection so that responding T cells may commit themselves quickly to IFN-{gamma}-T cell pathway, and it is surprising and significant that these early events may function through differential Ag presentation.

The ability of H-2 M-null mice or CQ-treated WT mice to produce IL-12 in vivo is not compromised, despite inefficient commitment to IFN-{gamma}-T cell responses against live Stm. High levels of IL-12 and IL-18 by themselves, as found in IL-4-null mice receiving dead Stm, are not sufficient to trigger IFN-{gamma}-T cell responses. Thus, IL-12 appears to have little influence on the IFN-{gamma}-T cell commitment during Stm infection. It must be noted that these findings may be Stm specific, since IL-12 is a major crucial factor for preferential IFN-{gamma}-T cell responses during infection by Leishmania (34).

On the other hand, IL-12 is clearly essential for the maintenance of the IFN-{gamma}-T cell response against live Stm, which is initially triggered in IL-12-independent fashion. Our data show that live and dead Stm trigger IL-12 and IL-18 differentially, while they induce equivalent levels of other APC-derived cytokines such as IL-1{beta}, TNF-{alpha}, or IL-10. This indicates the possibility that there may be specific suppression of IL-12 induction in response to dead Stm. This was also supported by the IL-4-mediated inhibition of IL-12 production in vitro, and by the ability of macrophages from IL-4-null mice given dead Stm to secrete high levels of IL-12 and IL-18.

The role of early IL-4 in naive CD4 T cell commitment has been extensively studied. Although most reports favor for a role of IL-4 in polarizing T cells to Th2 pathway (1, 2, 3), some reports indicate a role for IL-4 even in Th1 responses when used for DC maturation and priming along with CpG DNA (35). Our data show that IL-4 suppresses IL-12 and IL-18 induction from macrophages, and the disparity between our results and those previously reported (35) may be due either to the different cell types and/or receptors studied and/or the mouse strain used (36).

Because the IL-12 induction responses being read out are within 24–48 h of immunization, it is likely that elements of the innate immune system are the source of the regulatory IL-4 triggered by dead, but not live Stm. NK T cells are known to respond rapidly to microbial infection and secrete cytokines, including IL-4 (10, 12). Our data show that dead Stm, but not live Stm, activate NK T cells and induce IL-4 in them, providing strong support for their role in regulating IL-12 induction by dead, but not live Stm. CD1-mediated Ag presentation plays a prominent role in activating NK T cells in situations such as mycobacterial infections (11), although their ability to recognize Stm Ags is as yet unexplored. CD1-mediated Ag presentation is TAP independent (24), but CD1 expression is {beta}2m dependent (25). Therefore, the finding that {beta}2m, but not TAP-1, is essential for suppression of IL-12 in dead Stm-injected mice provides an indication that preferential CD1-mediated presentation of dead Stm and not live Stm may be responsible for the differential induction of IL-12 observed. Thus, the induction of IL-12, the cytokine essential for maintenance of the IFN-{gamma}-T cell response, also appears to be regulated by early Ag presentation-related events, albeit through an entirely separate MHC-TCR interaction.

The model for explaining the generation of IFN-{gamma}-T cell responses against live Stm, but not dead Stm, thus has two elements. In the first, IL-12-independent phase of commitment, Ag-presentation-related early stimuli provided by live, but not dead Stm would lead to IFN-{gamma}-T cell commitment. Simultaneously, dead Stm would be presented to NK T cells, leading to induction of IL-4 and suppression of IL-12. Presentation of live Stm to NK T cells would be prevented, thus leaving IL-12 induction unaffected, thereby ensuring that the IFN-{gamma}-T cell response triggered in the first phase is maintained well. The major factors determining the immune response outcome in favor of IFN-{gamma} following Stm infection thus appear to be associated with the presentation of Stm Ags by priming APCs, on classical MHCII for the IL-12-independent events responsible for commitment to IFN-{gamma}, and on nonclassical MHC for the regulation of IL-12, which is essential for maintenance of the IFN-{gamma} response. Thus, Ag presentation appears to be complexly and crucially involved in the early immune sensing of live Stm as an FIP.


   Acknowledgments
 
We thank Inderjit Singh and Drs. R. K. Anand and R. K. Juyal for help with maintaining the mouse strains used.


   Footnotes
 
1 This work was supported in part by grants from Department of Science and Technology, Government of India (to A.G., S.R., and V.B.); Department of Biotechnology, Government of India (to S.R.); and the Wellcome Trust (to V.B.). The National Institute of Immunology is funded by Department of Biotechnology, Government of India. Back

2 Address correspondence and reprint requests to Dr. Vineeta Bal, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India. E-mail address: vineeta{at}nii.res.in Back

3 Abbreviations used in this paper: FIP, facultative intracellular parasite; {beta}2m, {beta}2-microglobulin; CQ, chloroquine; CS, culture supernatant; GM, gentamycin; IFN-{gamma}-T, IFN-{gamma}-making T; p.i., postimmunization; Stm, Salmonella typhimurium; TC, tetracycline; WT, wild type. Back

Received for publication January 14, 2002. Accepted for publication June 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lichtman, A. H., A. K. Abbas. 1997. T-cell subsets: recruiting the right kind of help. Curr. Biol. 7:R242.[Medline]
  2. Murphy, K. M.. 1998. T lymphocyte differentiation in the periphery. Curr. Opin. Immunol. 10:226.[Medline]
  3. Infante-Duarte, C., T. Kamradt. 1999. Th1/Th2 balance in infection. Springer Semin. Immunopathol. 21:317.[Medline]
  4. Kuchroo, V. K., M. P. Das, J. A. Brown, A. M. Ranger, S. S. Zamvil, R. A. Sobel, H. L. Weiner, N. Nabavi, L. H. Glimcher. 1995. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707.[Medline]
  5. Rincon, M., J. Anguita, T. Nakamura, E. Fikrig, R. A. Flavell. 1997. Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4+ T cells. J. Exp. Med. 185:461.[Abstract/Free Full Text]
  6. Kim, S., K. Iizuka, H. L. Aguila, I. L. Weissman, W. M. Yokoyama. 2000. In vivo natural killer cell activities revealed by natural killer cell-deficient mice. Proc. Natl. Acad. Sci. USA 97:2731.[Abstract/Free Full Text]
  7. Constant, S., C. Pfeiffer, A. Woodard, T. Pasqualini, K. Bottomly. 1995. Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells. J. Exp. Med. 182:1591.[Abstract/Free Full Text]
  8. Singh, N., S. Bhatia, R. Abraham, S. K. Basu, A. George, V. Bal, S. Rath. 1998. Modulation of T cell cytokine profiles and peptide-MHC complex availability in vivo by delivery to scavenger receptors via antigen maleylation. J. Immunol. 160:4869.[Abstract/Free Full Text]
  9. Trinchieri, G.. 1997. Cytokines acting on or secreted by macrophages during intracellular infection (IL-10, IL-12, IFN-{gamma}). Curr. Opin. Immunol. 9:17.[Medline]
  10. Chen, Y. H., N. M. Chiu, M. Mandel, N. Wang, C. R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1 deficient mice. Immunity 6:459.[Medline]
  11. Emoto, M., Y. Emoto, I. B. Buchwalow, S. H. Kaufmann. 1999. Induction of IFN-{gamma}-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guerin. Eur. J. Immunol. 29:650.[Medline]
  12. Apostolou, I., Y. Takahama, C. Belmant, T. Kawano, M. Huerre, G. Marchal, J. Cui, M. Taniguchi, H. Nakauchi, J. J. Fournie, et al 1999. Murine natural killer T (NKT) cells contribute to the granulomatous reaction caused by mycobacterial cell walls. Proc. Natl. Acad. Sci. USA 96:5141.[Abstract/Free Full Text]
  13. Teitelbaum, R., M. Crammer, M. L. Maitland, N. E. Freitag, J. Condeelis, B. R. Bloom. 1999. Mycobacterial infection of macrophages results in membrane permeable phagosomes. Proc. Natl. Acad. Sci. USA 96:15190.[Abstract/Free Full Text]
  14. Rathman, M., M. D. Sjaastad, S. Falkow. 1996. Acidification of phagosomes containing Salmonella typhimurium in murine macrophages. Infect. Immun. 64:2765.[Abstract]
  15. Duclos, S., M. Desjardins. 2000. Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell. Microbiol. 2:365.[Medline]
  16. Thatte, J., S. Rath, V. Bal. 1993. Immunization with live versus killed Salmonella typhimurium leads to the generation of an interferon-{gamma}-dominant versus an interleukin-4-dominant immune response. Int. Immunol. 5:1431.[Abstract/Free Full Text]
  17. Pashine, A., B. John, S. Rath, A. George, V. Bal. 1999. Th1-dominance in the immune response to live Salmonella typhimurium requires bacterial invasiveness but not persistence. Int. Immunol. 11:481.[Abstract/Free Full Text]
  18. Jouanguy, E., R. Doffinger, S. Dupuis, A. Pallier, F. Altare, J. L. Casanova. 1999. IL-12 and IFN{gamma} in host defense against mycobacteria and Salmonella in mice and men. Curr. Opin. Immunol. 11:346.[Medline]
  19. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, L. Van Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection. Cell 84:543.[Medline]
  20. Alpuche-Aranda, C. M., E. L. Racoosin, J. A. Swanson, S. I. Miller. 1994. Salmonella stimulate macrophage macropinocytosis and persist within phagosomes. J. Exp. Med. 179:601.[Abstract/Free Full Text]
  21. Harding, C. V., D. S. Collins, J. W. Slot, H. J. Geuze, E. R. Unanue. 1991. Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell 64:393.[Medline]
  22. Cheers, C., Y. Zhan. 1996. How do macrophages distinguish the living from the dead?. Trends Microbiol. 4:453.[Medline]
  23. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN{gamma} production and type 1 cytokine responses. Immunity 4:471.[Medline]
  24. Hanau, D., D. Fricker, T. Bieber, M. E. Esposito-Farese, H. Bausinger, J. P. Cazenave, L. Donato, M. M. Tongio, H. de la Salle. 1994. CD1 expression is not affected by human peptide transporter deficiency. Hum. Immunol. 41:61.[Medline]
  25. Rolph, M. S., B. Raupach, H. H. Kobernick, H. L. Collins, B. Perarnau, F. A. Lemonnier, S. H. Kaufmann. 2001. MHC class Ia-restricted T cells partially account for {beta}2-microglobulin-dependent resistance to Mycobacterium tuberculosis. Eur. J. Immunol. 31:1944.[Medline]
  26. Stobie, L., S. Gurunathan, C. Prussin, D. L. Sacks, N. Glaichenhaus, C. Y. Wu, R. A. Seder. 2000. The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge. Proc. Natl. Acad. Sci. USA 97:8427.[Abstract/Free Full Text]
  27. Park, A. Y., B. D. Hondowicz, P. Scott. 2000. IL-12 is required to maintain a Th1 response during Leishmania major infection. J. Immunol. 165:896.[Abstract/Free Full Text]
  28. Wolf, P. R., S. Tourne, T. Miyazaki, C. Benoist, D. Mathis, H. L. Ploegh. 1998. The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed. Eur. J. Immunol. 28:2605.[Medline]
  29. Mukherjee, K., S. A. Siddiqi, S. Hashim, M. Raje, S. K. Basu, A. Mukhopadhyay. 2000. Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome. J. Cell Biol. 148:741.[Abstract/Free Full Text]
  30. Antoine, J. C., T. Lang, E. Prina, N. Courret, R. J. Hellio. 1999. H-2M molecules, like MHC class II molecules, are targeted to parasitophorous vacuoles of Leishmania infected macrophages and internalized by the amastigotes of L. amazonensis and L. mexicana. J. Cell Sci. 112:2559.[Abstract]
  31. Ramachandra, L., R. Song, C. V. Harding. 1999. Phagosomes are fully competent antigen processing organelles that mediate the formation of peptide:class II MHC complexes. J. Immunol. 162:3263.[Abstract/Free Full Text]
  32. Ruedl, C., M. F. Bachmann, M. Kopf. 2000. The antigen dose determines T helper subset development by regulation of CD40 ligand. Eur. J. Immunol. 30:2056.[Medline]
  33. van Stipdonk, M. J. B., E. E. Lemmens, S. P. Schoenberger. 2001. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nat. Immun. 2:423.
  34. Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, M. K. Gately, J. A. Louis, G. Alber. 1996. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26:1553.[Medline]
  35. Biedermann, T., S. Zimmermann, H. Himmelrich, A. Gumy, O. Egeter, A. K. Sakrauski, I. Seegmuller, H. Voigt, P. Launois, A. D. Levine, et al 2001. IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nat. Immun. 2:1054.
  36. Hsieh, C.-S., S. E. Macatonia, A. O’Garra, K. M. Murphy. 1995. T cell genetic background determines default T helper phenotype development in vitro. J. Exp. Med. 181:713.[Abstract/Free Full Text]



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