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The Journal of Immunology, 2005, 175: 1751-1757.
Copyright © 2005 by The American Association of Immunologists

Relative Contributions of NK and CD8 T Cells to IFN-{gamma} Mediated Innate Immune Protection against Listeria monocytogenes1

Rance E. Berg, Emily Crossley, Sean Murray and James Forman2

Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN-{gamma} is crucial in controlling bacterial numbers. We have shown recently that CD8 T cells have the ability to rapidly secrete IFN-{gamma} independent of Ag, in response to IL-12 and IL-18, during a LM infection. In the current study, we compared the relative abilities of NK and CD8 T cells to provide innate immune protection. Upon transfer of either NK or memory OT-I T cells (specific for the OVA protein) into IFN-{gamma}-deficient hosts that were infected subsequently with wild-type LM, both cell types were found in the spleen and had the ability to secrete IFN-{gamma}. However, the OT-I T cells were more effective at providing innate immune protection as determined by spleen and liver LM burdens. We used immunocytochemistry to demonstrate that upon infection with LM, marginal zone macrophages were localized to the T cell area of the splenic follicle. Transferred memory OT-I T cells were also found in the T cell area of the spleen, colocalizing with the LM and macrophages. In sharp contrast, NK cells were found predominantly in the red pulp region of the spleen. In addition, memory OT-I T cells were also found to be associated with LM lesions in the liver. These results highlight the importance of CD8 T cells in innate immune responses to LM and suggest that their increased protective ability compared with NK cells is the result of their colocalization with LM and macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune response to the Gram-positive intracellular bacterium, Listeria monocytogenes (LM)3, is a complex network involving cytokines, bactericidal effector mechanisms, and multiple cell types such as neutrophils, macrophages, and NK cells (1, 2). TLR recognition of bacterial products results in secretion of cytokines that recruit other cells, as well as directly activate innate effectors. Specifically, TLR2 and TLR5 recognize LM through interactions with bacterial products such as lipoteichoic acid, peptidoglycans, and flagellin (3, 4). Downstream signaling through TLRs is mediated through the adapter molecule, myeloid differentiation factor 88 (MyD88), and mice deficient in this molecule are highly susceptible to LM infection (5, 6). However, MyD88-independent innate and adaptive immune responses do exist (7, 8).

IL-12 and IL-18, produced mainly by activated macrophages, are both important mediators in the immune response against LM, with the primary function of inducing IFN-{gamma} secretion from responding immune cells (9, 10, 11, 12). However, an IFN-{gamma}-independent role in controlling LM has also been proposed for IL-18 (13). IL-12-deficient mice show an early impaired resistance to LM infection but are able to eliminate low doses of the bacteria, suggesting that IL-12 is less important in generating adaptive immunity (14, 15). In addition, other recently described IFN-{gamma}-inducing cytokines such as IL-21, IL-23, and IL-27 could play overlapping and/or redundant roles in the innate immune response against LM (16, 17, 18).

IFN-{gamma} secretion plays an important role during both innate and acquired immunity to intracellular bacteria by enhancing Th1-type immune responses through the activation of macrophages, the increase of MHC class I and class II expression, and the inhibition of proliferation of Th2 cells (19, 20, 21). IFN-{gamma} is produced by multiple cell types in response to LM or a combination of IL-12 and IL-18, including NK cells (22), NKT cells (23), macrophages (24), B cells (25), dendritic cells (26), {gamma}{delta} T cells (27), CD8 T cells (28), and primed CD4 T cells of the Th1 phenotype (29, 30). Evidence that IFN-{gamma} is critically important in the innate immune response comes from experiments using mice deficient in either the cytokine or its receptor. These animals rapidly succumb when infected with extremely low doses of LM (31, 32). SCID mice, which lack T and B lymphocytes, show increased susceptibility to LM when they are depleted of either IFN-{gamma} or IL-12 (9, 19). Providing IFN-{gamma} to IL-12-depleted SCID mice can reverse this effect, thereby indicating the importance of IL-12 for IFN-{gamma} production in response to LM (9). Impaired macrophage activity is one probable mechanism for the increased susceptibility of animals lacking the IFN-{gamma}R (21).

It is generally thought that NK cells play a crucial role in the innate control of LM infection due to secretion of IFN-{gamma} induced by IL-12 and IL-18 (22). Indeed, depletion of NK cells before s.c. infection with LM led to higher burdens of bacteria in both the hind footpad and the draining lymph node (33). In contrast, other studies have found that depleting NK cells led to decreased splenic and liver LM burdens in B6 mice after either i.v. (34) or i.p. (35) infection. Studies using mice deficient in the common cytokine receptor {gamma}-chain (lacking NK cells) indicated that IFN-{gamma} was produced early after LM infection and that the most probable source of this cytokine was {alpha}{beta} T cells (36, 37). We and others (28, 38, 39) have shown that CD8 T cells respond to cytokines, primarily IL-12 and IL-18, by secreting IFN-{gamma} rapidly after infection with intracellular bacteria, including LM. These reports suggest that CD8 T cells are capable of contributing to the innate immune response to LM, especially with respect to IFN-{gamma} production. In addition, we have shown that memory CD8 T cells can provide innate immune protection from a LM infection independent of cognate Ag (39). The present study was performed to determine the relative abilities of both NK and CD8 T cells to provide innate immune protection against a LM infection.


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

C57BL/6J (B6), C57BL/6.PL-Thy1a/Cy (B6.Thy1.1), B6.129S7-Ifngtm1Ts (IFN-{gamma}–/–), B6.129S7-Ifngr1tm1Agt/J (IFN-{gamma}R–/–), B6.129S7-Rag1tm1Mom/J (RAG-1–/–), and OT-I TCR transgenic mice were either purchased from The Jackson Laboratory or bred and maintained at the University of Texas Southwestern Medical Center animal facility under the approval of the Institutional Animal Care and Use Committee.

Bacteria and viruses

For infection of mice, log-phase cultures of LM 10403 serotype 1 were washed twice and diluted in PBS to the desired concentration. LM was injected in the lateral tail vein at the indicated dosage. Vaccinia virus expressing full-length OVA protein (VV/OVA) was injected in the lateral tail vein at a dosage of 106 PFU for a primary response. Vesicular stomatitis virus expressing full-length OVA protein (VSV/OVA) was injected in the lateral tail vein at a dosage of 106 PFU for a primary response.

Abs and cell staining for flow cytometry

For cell staining experiments, the following Abs from BD Pharmingen were used: anti-CD8{alpha} (53-6.7), anti-CD90.2 (Thy1.2) (53-2.1), anti-NK1.1 (PK136), and anti-IFN-{gamma} (XMG1.2). Secondary streptavidin-conjugated reagents were used to reveal biotinylated primary Abs. In experiments designed to test the direct ex vivo activity of NK and T cells, splenocytes were cultured for 3 h in complete RPMI 1640 medium supplemented with 10% FCS (without added cytokines or Ags). Intracellular staining, data acquisition, and data analysis were performed as described previously (28).

NK and T cell transfers

For generation of the memory OT-I T cell populations, RBC-depleted splenocytes from OT-I TCR transgenic mice were passed over nylon wool columns to enrich for T cells. Approximately 2 x 106 cells were then injected i.v. into the lateral tail vein of B6.Thy1.1-recipient mice, which were challenged subsequently with VV/OVA or VSV/OVA. At >4 wk postinfection, the mice were sacrificed, and RBC-depleted splenocytes were purified on nylon wool columns. The resulting cells were stained for CD8 and Thy1.2 and sorted using a MoFlo high-speed sorter (DakoCytomation) for expression of these molecules. For the NK transfer experiments, RBC-depleted splenocytes from RAG-1–/– mice were stained for NK1.1 and sorted for expression of this molecule. For each of the sorted populations, the purity of the cells was >95% as determined by flow cytometry postsorting (data not shown). CFSE (Molecular Probes) labeling of splenocytes was performed at a final concentration of 1 µM. After CFSE labeling, the indicated numbers of sorted cells were injected i.v. into IFN-{gamma}–/– or IFN-{gamma}R–/– hosts, which were immediately infected with ~10,000–20,000 wild-type LM.

Immunocytochemistry and microscopy

Immunocytochemistry of spleens was performed by making 5-µm sections of frozen spleens from B6, IFN-{gamma}–/–, and IFN-{gamma}–/– mice transferred with CFSE-labeled NK or memory OT-I T cells using a Leica CM 1850 cryostat. Five-micrometer liver sections were made from OT-I-transferred, VSV/OVA-primed mice that were >4 wk post primary infection. Spleen and liver sections were then acetone fixed before staining. The Ab combinations used to stain splenic sections were as follows: purified anti-CD3{epsilon} (145-2C11), purified CD45R/B220 (RA3-6B2), purified CD11b (M1/70) (all from BD Pharmingen), and Difco Listeria O polyserum (Fisher Scientific). The CD3{epsilon} was developed with anti-hamster biotin (Jackson ImmunoResearch Laboratories) followed by Streptavidin Alexafluor 594 (Molecular Probes). The CD45R/B220 and CD11b Abs were developed with anti-rat Alexafluor 350 (Molecular Probes). The Difco Listeria O polyserum was developed with anti-rabbit biotin (BD Pharmingen) followed by Streptavidin Alexafluor 594. The liver sections were stained with the following Ab combinations: purified anti-CD90.2 (Thy1.2) (53-2.1) and Difco Listeria O polyserum. The Thy1.2 was developed with anti-rat Alexafluor 488 (Molecular Probes), and the Listeria O polyserum was developed as above. All experiments were also performed with isotype control Abs to assure there was no background staining. Stained spleen and liver sections were then visualized on a Zeiss Axiovert 100M digital light microscope. Pictures were taken with a Hamamatsu Orca digital gray scale camera. Image J Software, from the National Institutes of Health, was used to analyze the data and give false colors to the images shown. The distribution of Thy1.2+ T cells in the liver sections in relation to LM lesions was determined by dividing microscopic fields with ~100 grids, each ~37 x 37 µm. T cells were scored as being in the same grid as LM, a grid adjacent to LM, or a grid not associated with LM.

Statistical analyses

Statistical significance for Figs. 1 and 2 was measured using a Student’s two-tailed t test. Statistical significance for Table I was measured using Fisher’s exact test. Statistical significance for Fig. 5d was measured using a goodness-of-fit {chi}2 analysis.



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  		FIGURE 1. Innate IFN-{gamma} production by NK and memory OT-I T cells. Memory OT-I T cells were sorted from spleens of OT-I transferred, VV/OVA-primed B6.Thy1.1 mice based on expression of CD8 and Thy1.2. NK cells were sorted from spleens of RAG-1–/– mice based on expression of NK1.1. The indicated numbers of purified cells were then labeled with CFSE and transferred into IFN-{gamma}–/– recipients, which were infected with ~20,000 wild-type LM. At day 3 postinfection, the mice were sacrificed, and splenocytes were cultured in vitro for 3 h in the presence of GolgiPlug containing brefeldin A to inhibit protein transport. The cultures were harvested and stained for CD8, NK1.1, and intracellular IFN-{gamma}. In addition, the transferred cells were identified by their expression of CFSE. The number of cells was determined by multiplying the percentage of transferred cells or transferred cells secreting IFN-{gamma} by the total number of splenocytes. Data presented are derived from two independent experiments with two to three mice in each group per experiment. *, p < 0.05.

 


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  		FIGURE 2. Protection from LM infection by memory OT-I T cells is more efficient than NK cells. The same mice analyzed in Fig. 1 for the presence of transferred IFN-{gamma}-secreting cells were also analyzed for the presence of LM in both spleen and liver at day 3 postinfection with ~20,000 wild-type LM. In addition, B6 and IFN-{gamma}–/– mice that were infected at the same time were also analyzed for LM burdens in their spleens and livers. Homogenates from the organs of the infected mice were diluted and then plated on brain-heart infusion medium plates. One day later, the number of bacterial colonies was determined by counting plates and multiplying by the dilution factor. Log CFU protection was calculated by subtracting the average log bacterial burden in the spleens and livers of transferred IFN-{gamma}–/– mice from the average log bacterial burden in the same organs of untransferred IFN-{gamma}–/– mice. Data presented are derived from two independent experiments with two to three mice in each group per experiment. *, p < 0.05.

 

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  		Table I. Location of transferred memory OT-I T cells and NK cells 1 day postinfection with LMa

 


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  		FIGURE 5. Memory OT-I T cells preferentially localize with LM lesions in the liver. OT-I T cell-transferred B6.Thy1.1 mice were infected with VSV/OVA and allowed to rest for >4 wk to generate memory OT-I T cells. Liver sections day 3 postinfection with LM (a and b) or from uninfected memory OT-I mice (c) are shown with LM identified with red and the memory OT-I T cells identified with green. b, The inset box shown in a. d, Cumulative data assigning memory OT-I T cells to one of three areas: within a grid containing LM, within a grid that has LM in a surrounding grid, or within a grid neither containing nor surrounded by LM. Data are representative of two liver sections. Original magnification was x100. A value of p < 0.001 as determined by {chi}2 test for a random assortment of T cells in the three defined areas.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Memory OT-I T cells and NK cells secrete IFN-{gamma} in response to wild-type LM when transferred into IFN-{gamma}-deficient hosts

Our previous results have shown that memory CD8 T cells specific for OVA (OT-I T cells) have the ability to provide innate immune protection from a wild-type LM infection when transferred into IFN-{gamma}–/– hosts (39). This protection is mediated by the secretion of IFN-{gamma} in response to IL-12 and IL-18, which is produced during the LM infection. Other studies have suggested a protective role for NK cells during the innate immune response to LM infection, once again citing their ability to rapidly secrete IFN-{gamma} (22, 33). Therefore, our first set of experiments was designed to ascertain the IFN-{gamma}-secreting potential of both NK and memory CD8 T cells in response to wild-type LM in an IFN-{gamma}–/– setting. The NK cells were isolated from spleens of RAG-1–/– mice by sorting on the NK1.1 molecule. The majority (~90%) of the NK1.1+ NK cells also expressed DX5 before sorting (data not shown). Therefore, this population of NK1.1+ cells isolated from mice deficient in NKT or T cells represents a pure population of NK cells. The memory OT-I T cells were isolated from OT-I transferred, VV/OVA-primed B6.Thy1.1 mice by sorting on Thy1.2 and CD8 molecules. Our previous data established that these cells represent memory CD8 T cells (39). Memory OT-I T or NK cells, which were transferred into IFN-{gamma}–/– mice that were infected subsequently with wild-type LM, could be identified in the spleen (Fig. 1). These transferred cells were capable of secreting IFN-{gamma} at day 3 postinfection. In addition, our previously published data showed that both NK and CD8 T cells were capable of secreting IFN-{gamma} at day 1 postinfection with LM (28, 39).

Memory OT-I T cells provide more efficient innate immune protection against LM than NK cells

The results presented in Fig. 1 suggest that both NK and memory OT-I T cells should have the ability to provide protection against a LM infection due to their abilities to secrete IFN-{gamma}. However, when we determined spleen and liver LM counts in transferred IFN-{gamma}–/– recipients, we found that the memory OT-I T cells provided more efficient protection when compared with the NK cells (Fig. 2). In fact, when we transferred four times more NK cells than T cells, which resulted in a greater number of IFN-{gamma}-secreting NK cells compared with memory OT-I T cells, the T cells still provided more innate immune protection against the LM infection. We chose day 3 to analyze because our previously published data indicated that before this time point there was no difference in LM counts in B6 and IFN-{gamma}–/– mice (39). However, our previous data (28, 39), as well as unpublished results, indicate that both NK and CD8 T cells are actively secreting IFN-{gamma} as early as day 1 postinfection with LM. To show that the IFN-{gamma} that is produced by the transferred OT-I T cells is indeed the effector molecule responsible for the protection from the LM infection, we used IFN-{gamma}R–/– mice as recipients for the transfer of memory OT-I T cells. Three days postinfection with ~15,000 wild-type LM, the LM burdens were measured in the spleens and livers (three mice per group). The IFN-{gamma}R–/– mice that did not receive memory OT-I T cells had an average of 1.87 x 109 LM/spleen and 9.96 x 108 LM/liver. The memory OT-I-transferred IFN-{gamma}R–/– mice contained an average of 3.99 x 109 LM/spleen and 1.31 x 109 LM/liver. Although there was no protection offered by the memory OT-I T cells, these transferred cells were visible (CFSE+) and were actively secreting IFN-{gamma} in the spleen (data not shown).

Splenic architecture and macrophage migration in LM-infected IFN-{gamma}–/– and B6 mice are similar

To begin to dissect why memory OT-I T cells provide more efficient innate immune protection from a LM infection in IFN-{gamma}–/– mice, we sought to analyze the splenic architecture of these mice. To this end, 5-µm frozen sections from B6 (Fig. 3a) and IFN-{gamma}–/– (Fig. 3b) spleens were stained with Abs to visualize T cells, B cells, and macrophages. The microscopy data reveals that in spleen sections from both mice there are well-defined white pulp zones that are surrounded by macrophages in the marginal zones. In the next set of experiments, we infected B6 (Fig. 3c) and IFN-{gamma}–/– (Fig. 3d) mice with wild-type LM and then analyzed the location of T cells, B cells, macrophages, and LM at day 1 postinfection. After infection with LM, most macrophages are detected in the T cell area of the periarteriolar lymphoid sheath (PALS) region of the spleen, rather than the marginal zone in both strains of mice. Previous studies have shown that LM are trapped by macrophages in the marginal zone of the spleen and that these LM-infected macrophages then migrate into the white pulp area within 12–24 h postinfection (40, 41).



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  		FIGURE 3. Immunocytochemistry of uninfected and LM-infected B6 and IFN-{gamma}–/– mice. B6 (a), IFN-{gamma}–/– (b), day 1 LM-infected B6 (c), and day 1 LM-infected IFN-{gamma}–/– (d) mice were sacrificed, and 5-µm splenic sections were stained for T cells, B cells, macrophages, and LM. The mice in c and d were infected with ~20,000 wild-type LM and were sacrificed at 16–20 h postinfection. For each of the slides, B cells are shown in blue, T cells in red, macrophages in cyan, and LM in yellow. Original magnification was x100. Data are representative of at least 10 sections from two to three independent mice.

 
Splenic localization of transferred NK and memory OT-I T cells in LM-infected IFN-{gamma}–/– mice

To establish a mechanism to explain why transferred NK and memory OT-I T cells provide differential protective ability, experiments were performed to visualize the localization of these cells in LM-infected IFN-{gamma}–/– mice. A representative splenic section shows that the transferred memory OT-I T cells reside in a T cell area of the PALS at day 1 after LM infection (Fig. 4a) and that the LM and macrophages are also found in this area (Fig. 4b). Previous results from T cell transfer experiments have shown that bulk CD8 splenocytes (42), as well as memory CD8 T cells (43), preferentially localize in white pulp regions of uninfected spleens. In contrast to the OT-I T cells, transferred NK cells did not localize into the T cell areas of LM-infected IFN-{gamma}–/– mice (Fig. 4c). The NK cells are found mainly in the red pulp, which is not where the LM and macrophages are found in a consecutive spleen section (Fig. 4d). In support of this result, previous studies analyzing the location of endogenous NK cells or transferred NK cells have shown that they are predominantly found in the red pulp of uninfected spleens (44, 45, 46). Our data suggest that even during a LM infection, NK cells are not induced to migrate toward the infected foci in the spleen. Therefore, in combination, the previous results suggest that memory CD8 T cells preferentially home to T cell areas of the spleen, which is also the area where LM and macrophages migrate to during infection (Fig. 4, a and b). NK cells, in contrast, do not localize to the T cell areas containing LM and macrophages (Fig. 4, c and d). Table I summarizes data from experiments analyzing the location of transferred NK and memory OT-I T cells. At day 1 after LM infection, the location of the transferred cells was determined easily due to the intact splenic architecture at this time. The data once again demonstrates that at day 1 the transferred memory OT-I T cells are found in the T cell areas of the spleen, whereas the NK cells are found predominantly in the red pulp distant from the foci of LM. However, at day 3 postinfection, which is the time at which differences in spleen and liver LM counts are seen between B6 and IFN-{gamma}–/– mice, the LM infection has progressed to the point where the conventional anatomy of the spleen is altered (data not shown). Therefore, it was difficult to assign exact locations to the identifiable transferred populations of NK and memory OT-I T cells. Nonetheless, as previously demonstrated, the transferred memory OT-I T cells did provide more efficient innate immune protection at day 3 postinfection compared with the NK cells (Fig. 2).



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  		FIGURE 4. Memory OT-I T cells preferentially localize with macrophages and LM in the T cell areas of the spleen whereas NK cells do not. Sorted memory OT-I T cells (7 x 105) (a) and NK cells (2 x 106) (c) were purified as previously described, CFSE labeled, and transferred into IFN-{gamma}–/– mice, which were infected subsequently with ~20,000 wild-type LM. At day 1 postinfection, the mice were sacrificed, and 5-µm spleen sections were stained for B cells (blue) and T cells (red). CFSE-labeled cells were identified as well (green). Consecutive sections showing the macrophages (magenta) and LM (yellow) are shown for the transferred memory OT-I T cell experiment (b) and the transferred NK cell experiment (d). Green arrows identify either the transferred memory OT-I T cells (a) or the transferred NK cells (c). Yellow arrows identify the LM. Original magnification was x100. Data are representative of at least four sections from two independent transfer experiments.

 
Localization of memory OT-I T cells in the livers of LM-infected mice

Previous studies have shown that T cells can be found within and around LM lesions in the livers of LM-infected mice (47). We sought to determine whether memory OT-I T cells that were not specific for wild-type LM could be found in the livers of infected mice. Our previous data suggests that there is a preferential localization of memory T cells to peripheral organs such as the liver and importantly that these T cells can respond to the LM-induced cytokines IL-12 and IL-18 (39). Therefore, we used B6.Thy1.1 mice that had been transferred with OT-I T cells and primed >4 wk previously with VSV/OVA. Upon challenging these mice with ~20,000 LM, we were easily able to identify LM lesions at day 3 postinfection (Fig. 5, a and b). Importantly, memory OT-I T cells could be visualized and were preferentially located in or surrounding the LM lesions. Analysis of memory OT-I T cells located in the livers of mice that were not rechallenged with LM revealed cells that were scattered randomly throughout the liver section (Fig. 5c). Similar results were observed on day 1 before lesions of LM were detectable in the liver at the LM dosage used for infection (data not shown). To enumerate the localization of the memory OT-I T cells in the liver of day 3 infected mice, we used a grid system that divided the microscopic fields into ~100 grids that were ~37 x 37 µm in size. For each T cell counted, a designation of the proximity of LM was assigned. Indeed, the vast majority of the T cells counted at day 3 after LM infection either had LM residing within the grid they were located within or in a surrounding grid (Fig. 5d). Very few memory OT-I T cells were found to be independent of LM lesions in the liver. Therefore, this data strongly suggests that in the liver, as well as the spleen, memory CD8 T cells that are not LM specific can preferentially localize with LM lesions and provide a protective effect by secreting IFN-{gamma}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that IFN-{gamma} is required during the innate immune response to LM (31, 32, 39, 48). However, the population of cells responsible for secreting IFN-{gamma} is open for debate. Reports suggest that multiple cell types have the ability to rapidly secrete IFN-{gamma} in response to infection with LM. Therefore, dissecting which cell or cells are actually able to control the LM by secreting IFN-{gamma} has been difficult. Our recent data has shown that memory CD8 T cells secrete IFN-{gamma} in response to IL-12 and IL-18, which are produced by LM-infected macrophages (28, 39). Indeed, when small numbers of memory OT-I T cells (specific for the OVA-derived peptide, SIINFEKL) are transferred into IFN-{gamma}-deficient mice, these lymphocytes can provide innate protection from wild-type LM in the absence of cognate Ag (Fig. 2) (39). Our current report now determines the relative contribution of NK and memory CD8 T cells in providing protection from a LM infection. Surprisingly, we have found that memory OT-I T cells are more efficient at providing protection against a LM infection than are NK cells.

Why were the memory OT-I T cells more effective than NK cells at reducing LM burdens? Several hypotheses were considered to explain this finding, including: 1) increased survival of the memory OT-I T cells compared with the NK cells; 2) increased production of IFN-{gamma} by the memory OT-I T cells; or 3) localization of the memory OT-I T cells, but not the NK cells, with the LM-infected areas of the spleen and liver. An experiment designed to count the number of viable cells remaining in the spleen at days 1 and 3 postinfection with LM revealed that both NK cells and memory OT-I T cells suffer considerable losses (unpublished results). In addition, Figs. 1 and 2 suggest that even when more NK cells are present during the LM infection, they are still less efficient at reducing the bacterial burden. Therefore, we believe that increased survival of the memory OT-I T cells does not account for their increased protective ability. Our previous data (28), as well as data presented here (Fig. 1), indicate that both NK and memory CD8 T cells have the ability to rapidly secrete IFN-{gamma} after infection with LM. Therefore, a difference in IFN-{gamma}-secreting ability is unlikely to account for the difference in protective ability between the two cell types.

With the above information in mind, we focused on the localization of the two different cell types to ascertain why T cells were more effective at providing innate immune protection from LM when compared with the NK cells. Prior studies showed that the majority of NK cells reside in the red pulp region of the spleen in uninfected mice (44, 45, 46). T cells, in contrast, reside in the PALS region of the spleen in uninfected mice (42, 43). Upon infection with LM, the splenic marginal zone macrophages are responsible for the uptake of the bacteria (40). At ~6–24 h postinfection (depending upon the dose of LM), the LM can be found within the white pulp area of the PALS (Refs. 41 and 49 ; Fig. 3). However, the molecular basis for this macrophage migration is unknown. CCL21 and CCL19 play a role in the maintenance of the localization of macrophages in the marginal zone of the spleen, and furthermore, infection with Leishmania donovani results in the loss of stromal cells secreting CCL21 and CCL19 (50). Infections that deplete the stromal cell subset responsible for secreting CCL21 and CCL19 were suggested to result in selective loss of the marginal zone macrophages. Future studies will be required to determine whether this scenario controls the loss of marginal zone macrophages during LM infection. However, even if the loss of CCL21 and CCL19 does occur, this does not explain why the LM, and presumably the marginal zone macrophages, migrate to the white pulp.

Taken as a whole, the above data suggest that LM initially infect marginal zone macrophages, which then migrate to the T cell areas of the white pulp. Our data shows that transferred memory OT-I T cells can be found localizing in the same areas of the spleen at day 1 postinfection where the macrophages and LM are found (Fig. 4, a and b). In contrast, the NK cells are not found in this same area of the spleen but are instead found in the red pulp away from the LM lesions (Fig. 4, c and d). So even though both the NK cells and the memory OT-I T cells secrete IFN-{gamma}, the T cells have a selective advantage in contributing to the clearance of the LM. Interestingly, the ability of these memory OT-I T cells to assist in controlling the numbers of LM can only be visualized at day 3 postinfection. However, even differences in susceptibility of B6 and IFN-{gamma}-deficient mice can only be visualized at day 3 postinfection (39). At this time point, the splenic morphology is so altered that it is difficult to assign locations to the transferred lymphocytes (data not shown). The secretion of IFN-{gamma} at day 1 postinfection seems to be crucial in controlling the proliferation of LM.

The localization of memory OT-I T cells within the livers of LM infected mice was determined by using mice that contained high frequencies of endogenous memory T cells that preferentially localize to peripheral organs such as the liver. Our data demonstrates that in the liver, just as in the spleen, memory CD8 T cells are found in and adjacent to LM lesions at day 3 after LM infection. It is likely that the T cells are induced to migrate toward the LM lesions. It seems possible that both resident liver memory T cells and circulating T cells will be recruited to the area where the LM lesion begins, and this recruitment may be the result of chemokine secretion induced by the inflammation (51). Our previously published data has shown that memory OT-I T cells found within peripheral organs such as the lung and liver are very efficient producers of IFN-{gamma} when stimulated with the LM-induced cytokines IL-12 and IL-18. It is likely that Kupffer cells that reside in the liver are capable of secreting IL-12 and IL-18 to induce IFN-{gamma} production from T cells, but this has not been formally established (52).

In conclusion, our data strongly suggest that T cells, and not NK cells, may be the most important IFN-{gamma}-secreting population of cells during the early stages of a LM infection. In most settings, the redundancy of IFN-{gamma}-secreting cell types masks the intricacies of the system. However, by using mice deficient in IFN-{gamma}, we were able to clearly show that memory CD8 T cells, which are not specific for LM, effectively reduce LM burdens by way of secreting IFN-{gamma} in the proximity of the LM-infected areas of the spleen and liver.


   Acknowledgments
 
We thank Angie Mobley of the Dallas Cell Analysis Facility for cell sorting and J. Marshall Haynie for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI45764 (to J.F.) and a National Institutes of Health postdoctoral fellowship (to R.E.B.). Back

2 Address correspondence and reprint requests to Dr. James Forman, Center for Immunology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail address: James.Forman{at}UTSouthwestern.edu Back

3 Abbreviations used in this paper: LM, Listeria monocytogenes; MyD88, myeloid differentiation factor 88; VV/OVA, vaccinia virus expressing full-length OVA protein; VSV/OVA, vesicular stomatitis virus expressing full-length OVA protein; PALS, periarteriolar lymphoid sheath. Back

Received for publication November 13, 2004. Accepted for publication May 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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