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The Journal of Immunology, 2006, 177: 138-146.
Copyright © 2006 by The American Association of Immunologists

Self-Antigen Maintains the Innate Antibacterial Function of Self-Specific CD8 T Cells In Vivo1

Salim Dhanji, Michael T. Chow and Hung-Sia Teh2

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Self-specific CD8 T cells, which are selected by high-affinity interactions with self-Ags, develop into a lineage distinct from conventional CD8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. In this study, we show that these self-specific cells have the ability to produce large amounts of IFN-{gamma} in response to infection with Listeria monocytogenes in a bystander fashion. The rapid production of IFN-{gamma} is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific CD8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific CD8 T cells are also more efficient in the production of IFN-{gamma} and TNF-{alpha}, thus providing more cytokine-dependent protection against bacterial infection when compared with NK cells. These findings illustrate that self-reactive CD8 T cells can play an important innate function in the early defense against bacterial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Successful immune protection against pathogens relies on cooperation between the innate and adaptive arms of the immune system. Cells of the innate immune system provide the first line of defense against pathogens and are usually able to clear infections before they spread. Innate cells use germline-encoded receptors, which either recognize pathogens directly or recognize changes on host cells as a result of infection. In cases where the innate immune system is overwhelmed, the adaptive immune system is usually able to provide the additional immune responses that are required for the elimination of the pathogen. However, the response of adaptive immune cells is slow and requires the expansion of T cells and B cells that express rearranged receptors specific for pathogen-derived epitopes. Another distinguishing feature of the adaptive immune system is the production of memory T and B lymphocytes, which are very effective in protection against subsequent infections by the same pathogen.

The distinction between innate and adaptive immunity is not absolute. Several adaptive lymphocytes have been shown to express Ag-specific receptors of limited diversity in conjunction with the expression of receptors associated with innate cells. For instance, B1-B cells express rearranged BCRs that display limited diversity and show specificity for self-Ags such as oxidized phospholipids (1). T cells expressing {gamma}{delta} TCRs also show limited diversity in their TCR rearrangements and some of these cells express innate system receptors which are specific for stress-induced Ags (2). Intraepithelial lymphocytes (IELs)3 can express TCRs specific for self-Ags and this interaction is crucial for their function (3, 4). NKT cells restricted to the nonclassical MHC class I molecule, CD1d, also express invariant TCRs (V{alpha}14i in mice) (5). This cell type is abundant in the spleen and liver where the cells are thought to become activated in response to inflammatory cytokines with concurrent recognition of lipids in the context of CD1d. They are then able to rapidly secrete large amounts of cytokines such as IFN-{gamma} or IL-4, which play a role in biasing the immune response toward the Th1 or Th2 lineage, respectively (6).

IFN-{gamma} is crucial for protection against various intracellular pathogens (7). Binding of IFN-{gamma} to its receptor on macrophages induces bactericidal activity in infected macrophages resulting in the production of reactive oxygen and nitrogen intermediates as well as the efficient fusion of lysosomes with phagosomes containing pathogens (8). IFN-{gamma} also has an impact on shaping the adaptive immune response and leads to the expression of host genes involved in Ag-processing and presentation (9). In addition, this cytokine plays an important role in the polarization of CD4 T cells to the Th1 lineage which can then activate macrophages (10). IFN-{gamma} signaling early during infection enhances CD4 and CD8 T cell responses (11, 12) and also programs contraction of Ag-specific CD8+ T cells thus controlling the level of immunological memory (13).

Listeria monocytogenes (LM) is a Gram-positive intracellular pathogen which is the cause of listeriosis in humans (14). Efficient protection against LM infection relies heavily on early IFN-{gamma} production by innate cells (15). IFN-{gamma} production during infection with LM was thought to come mainly from NK cells but recent studies suggest that memory-phenotype CD8+ T cells are also a major contributor (16, 17, 18, 19, 20). Memory-phenotype CD8+ T cells include those that are specific for foreign-Ags and which have been generated during a previous encounter with these Ags. These bona fide foreign Ag-specific memory CD8+ T cells have been shown to provide an innate source of IFN-{gamma} in the absence of cognate Ag through IL-12 and IL-18 signaling (17). Interestingly, memory CD8+ T cells have been shown to be more protective than NK cells during LM infection (18). However, not all memory-phenotype CD8+ T cells are specific for foreign Ags. CD8+ T cells that are restricted to nonclassical MHC class Ib molecules also have a memory phenotype (21). Some MHC class Ib-restricted CD8+ T cells have been shown to be selected by hemopoietic cells and can respond to LM infection in both an Ag-dependent and Ag-independent fashion by producing IFN-{gamma} (22, 23). Thus, memory-phenotype CD8+ T cells have been shown to be a very significant source of early IFN-{gamma} production during infections.

We have shown that memory-phenotype CD8+ T cells in normal mice contain a subset of T cells that demonstrate a very high reactivity for self-peptide/MHC (24). By virtue of high expression of CD122 (IL-2Rbeta), these cells respond to IL-2 and IL-15 both in vitro and in vivo. Upon activation, they express several NK receptors including CD16, NKG2D, and the adaptor protein DAP12. CD16 engagement on these cells results in both the production of inflammatory cytokines and the lysis of Ab-coated target cells (25). NKG2D engagement also results in the lysis of NKG2D-ligand expressing target cells. These cells comprise ~10% of peripheral CD8+ T cells in normal mice and they demonstrate specificity for syngeneic tumor cells.

We have also characterized self-specific CD8+ T cells in H-Y TCR-transgenic (tg) mice (26). The H-Y TCR is specific for the male (H-Y) peptide presented by H-2Db. In female H-2b mice CD8+ T cells expressing the H-Y TCR are positively selected. In male H-2b H-Y TCR-tg mice, the deletion of virtually all of the double-positive thymocytes greatly affected the development of conventional CD8 as well as CD4 T cells (27, 28). However, large numbers of T cells which expressed low levels of CD8 and exclusively the H-Y TCR are present in the peripheral lymphoid organs of these male mice (29). More interestingly, these cells have a similar memory phenotype and functional properties as the self-specific CD8+ T cells that are found in normal mice. They also express NK receptors that function in cooperation with the TCR. Most importantly, we have shown that these cells become activated in vivo in response to LM infection (26).

In this report, we determined the role of self-specific CD8+ T cells in protection against LM infection and assessed the role of self-Ag interactions in the maintenance and function of this cell type. We show that these cells are protective immediately ex vivo but more so after IL-15 activation. We also demonstrate that self-Ag interactions are crucial for the early protection of IFN-{gamma}-deficient mice from LM infection and for maintaining the memory phenotype and cytokine responsiveness of these self-specific cells. Finally, we show that activated self-specific CD8+ T cells produce more IFN-{gamma} and TNF-{alpha} than NK cells and are more protective than NK cells during infection with LM in IFN-{gamma}–/– mice.


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

Breeders for C57BL/6 (B6), B6.foxn1nu (athymic nude), B6.Thy1.1, and B6.IFN-{gamma}–/– were obtained from The Jackson Laboratory. The H-Y TCR tg mice were bred to the B6 or B6.foxn1nu background. Mice 8–12 wk of age were used for the experiments described.

Abs and flow cytometry

The following mAbs specific for the indicated molecule were used: CD8{alpha} (53-6.7), CD8beta (53.58), CD44 (Pgp-1), H-Y TCR{alpha} (T3.70), CD94 (18D3), NK1.1 (PK136), NKG2D (CX5), CD122 (TM-beta1), CD127 (A7R34), CD132 (4G3), IFN-{gamma} (XMG1.2), and TNF-{alpha} (MP6-XT22). All mAbs were purchased from eBioscience, except anti-CD132 (BD Pharmingen). For intracellular flow cytometry, cells were stained for surface markers, washed, fixed with 2% paraformaldehyde and 0.2% Tween 20 in PBS for 20 min followed by one wash with PBS. The cells were then stained with mAbs specific for the intracellular cytokine in 0.2% Tween 20 in PBS. The CellQuest software program (BD Biosciences) was used for data acquisition and analysis.

CD8+ T cell purification

Single-cell suspensions from the lymph nodes (LNs) of H-Y male mice were treated with biotinylated anti-CD8beta mAb followed by positive selection using the MiniMACS system (Miltenyi Biotec), according to the manufacturer’s specifications. The resulting cells were >95% pure CD8{alpha}beta+ H-Y TCR{alpha}+ T cells. For purification of B6 CD8+CD44high and CD8+CD44low T cells, CD8beta+ T cells from B6 LN were first enriched by MiniMACS. MiniMACS purified B6 CD8+ T cells were then stained with anti-CD8{alpha}-FITC and anti-CD44-PE and sorted on a BD Biosciences FACS Vantage SE Turbo sort cell sorter. Cell sorting was performed by A. Johnson (University of British Columbia, Vancouver, British Columbia, Canada) and the sorted CD8+CD44high or CD8+CD44low cells were >98% pure (24).

NK cell purification

Single-cell suspensions from the spleens of B6 mice were treated with mAbs against CD4, CD8, and TCRbeta, followed by depletion of Ab-coated cells using Dynabeads M-450 sheep anti-mouse IgG (Dynal Biotech), according to manufacturer’s instructions. The resulting cells were ~50–60% CD3–NK1.1+ before culture and >95% CD3–NK1.1+ after culture in IL-15.

Adoptive transfers, infections, and bacterial load measurement

Purified H-Y CD8 T cells or sorted B6 CD8 T cells were injected into the lateral tail vein of IFN-{gamma}–/– or B6-Thy1.1 mice. For IL-2- or IL-15-cultured CD8 T cells and for IL-15-cultured NK cells, the cells were cultured in cytokine (100 ng/ml) for 4 days before transfer. The number of cells adoptively transferred is stated in the figure legends. For infection experiments, mice were infected with ~10,000 CFU of wild-type LM (strain 10403s) via the lateral tail vein 1 day after receiving adoptively transferred cells. On day 3 postinfection, mice were sacrificed and their spleen and liver were homogenized in PBS. The resulting cell suspensions were mixed with an equal volume of 1% Triton X-100 in PBS and plated on brain-heart infusion agar plates in serial 10-fold dilutions and incubated at 37°C overnight before counting. Fold reduction was used to assess relative protection in some experiments and was calculated as (CFU control mouse/CFU experimental mouse).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Self-specific CD8+ T cells in H-Y TCR-tg male mice can develop in the absence of a functional thymus

There is some controversy regarding whether the thymus is essential for the development of self-specific CD8 T cells in the H-Y model. In the first description of the H-Y TCR+ CD8+ cells in H-Y male mice, it was suggested that these cells were conventional CD8+ T cells that had escaped negative selection by decreasing the expression of the CD8+ coreceptor and thus lowering their avidity for self-peptide/MHC (27, 29). Consistent with the requirement for the thymus for their development was the observation that these cells failed to develop in athymic nude mice (29). However, this conclusion was challenged by a later report, which showed using adult thymectomy followed by bone marrow reconstitution that self-specific H-Y CD8+ T cells can develop in the absence of a functional thymus (30). In view of our findings that these self-specific CD8+ T cells are functionally distinct from conventional CD8+ T cells, we decided to re-evaluate whether self-specific CD8 T cells can in fact develop in athymic nude (foxn1nu) mice. We first compared the CD4 and CD8 populations in the LNs of euthymic H-Y male, athymic H-Y male, euthymic H-Y female, and athymic H-Y female mice. The top panel in Fig. 1A shows that euthymic H-Y female mice have the largest population of both CD4 and CD8 cells making up 19.0 and 30.7% of the LNs, respectively. Consistent with previous reports (27, 29) LNs from euthymic H-Y male mice contain very few peripheral CD4 T cells (3.1%) but possess larger numbers of CD8low cells (16.7%). By contrast, the LNs of athymic H-Y male mice contain fewer numbers of these CD8low cells (3.7%) and possess virtually no CD4 T cells (0.6%). Consistent with previous reports (27, 29) virtually all of the CD8low cells (96.7%) in euthymic H-Y male mice express exclusively the H-Y TCR, as indicated by the same level of staining with either a mAb specific for the TCR beta-chain or the H-Y TCR (Fig. 1A, lower panel). Also consistent with previous reports these CD8low T cells express exclusively the CD8{alpha}beta heterodimer (Ref. 29 and data not shown). By contrast, only 34.9% of the CD8+ T cells in euthymic H-Y female mice express the H-Y TCR; this is consistent with previous reports that the majority of peripheral CD8+ T cells from euthymic female H-Y TCR mice use endogenous TCR {alpha}-chains for their positive selection (31). The majority (62.6%) of CD8low cells in athymic male H-Y mice also express the H-Y TCR. It is noted that about one-third of the CD8low cells from athymic H-Y male mice did not express the TCR beta-chain suggesting that these cells are unlikely to be of the TCR{alpha}beta lineage. In athymic H-Y female mice there is very poor development of CD8low cells (1.0%) consistent with our previous observation that the presence of self-Ag is required for their development (26). In terms of absolute cell numbers, there is a 26-fold increase in the frequency of CD8low TCRbeta+ H-Y TCR{alpha}+ cells in athymic nude H-Y male mice compared with athymic nude H-Y female mice. There is also a 7-fold reduction in the recovery of CD8low H-Y TCR+ cells in athymic nude H-Y male mice relative to euthymic H-Y male mice. This suggests that additional cells of this lineage may develop in the thymus or that some thymus-dependent cell(s) may provide selection or survival cues for these cells. We also noted that the H-Y TCR+CD8low T cells that developed extrathymically in athymic H-Y male mice expressed high levels of the memory markers CD44 and CD122 as well as the NK receptors NKG2D and CD94 (Fig. 1B). These observations support the following conclusions: 1) self-specific CD8low H-Y TCR+ T cells can develop in the absence of the thymus, 2) thymus-independent development of these cells is relatively inefficient as indicated by a seven-fold reduction in absolute numbers relative to euthymic mice, 3) the development of self-specific CD8 T cells in athymic mice is also dependent on their interaction with self-Ags, and 4) the self-specific CD8 T cells that develop in athymic mice express high levels of memory markers (CD44 and CD122) as well as NK receptors (NKG2D and CD94).


Figure 1
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  		FIGURE 1. Limited development of self-specific CD8 T cells in athymic (nude) H-Y TCR-tg male mice. A, LN cells from age-matched male and female H-Y TCR-tg mice on either a wild-type B6 or athymic nude B6 background were stained with mAbs against CD4, CD8, TCRbeta, and the H-Y TCR{alpha}. The TCRbeta vs H-Y TCR{alpha} plots in the lower panel are for gated CD8+CD4– cells from the upper panel. The cell recovery was similar for all of the mice and the data shown is representative of at least four mice per group. B, Expression of memory markers and NK receptors on gated self-specific male H-Y TCR+ CD8+ cells from euthymic (black histogram) or athymic H-Y male mice (gray line).

 
Activated self-specific CD8 T cells produce proinflammatory cytokines

V{alpha}14i NKT cells are an innate T cell type which has been shown to produce both Th1 (IFN-{gamma}) and Th2 (IL-4) cytokines upon activation (6). Because self-specific CD8 T cells can produce cytokines immediately ex vivo, we determine whether these cells were similar to NKT cells in their cytokine production profile (Fig. 2). We compared the cytokine production of self-specific CD8 T cells from H-Y male mice to conventional Ag-specific CD8 T cells from H-Y female mice. CD8low cells from euthymic H-Y male mice were used as a source of self-specific CD8 T cells because they provide a more convenient and larger source of these cells. We also included memory phenotype (containing self-specific CD8 T cells of unknown specificity) and naive CD8 T cells from normal B6 mice in these analyses. Splenocytes from H-Y male, H-Y female, and B6 mice were stimulated with PMA and ionomycin for 4 h. We then measured the production of IFN-{gamma}, TNF-{alpha}, IL-2, and IL-4 by intracellular flow cytometry after gating on the cell type of interest. It is clear that self-specific H-Y male CD8 T cells were only capable of producing the proinflammatory Th1 cytokines IFN-{gamma}, TNF-{alpha}, and IL-2 but not IL-4 (Fig. 2). The naive H-Y female CD8 T cells were only capable of producing TNF-{alpha} and a small amount of IL-2. These results were mirrored by those obtained from the non-tg CD8 T cells from normal B6 mice where the memory-phenotype CD8+CD44high T cells were capable of producing IFN-{gamma}, TNF-{alpha}, and IL-2 during a short stimulation and the naive CD8+CD44low T cells could only produce TNF-{alpha} and IL-2 (Fig. 2). In addition, IFN-{gamma} production by self-specific H-Y and memory-phenotype B6 CD8+CD44high T cells was maintained even at 24-h poststimulation whereas the naive H-Y female and naive B6 CD8 T cells still could not produce IFN-{gamma} (data not shown). IL-4 production by all the cell types was never detected even at 24-h poststimulation. These results further emphasize the functional similarity of self-specific CD8low T cells from H-Y male mice and the CD8+CD44high cells from B6 mice.


Figure 2
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  		FIGURE 2. Self-specific CD8 T cells rapidly produce inflammatory cytokines upon activation. Splenocytes from euthymic H-Y male, H-Y female, or B6 mice were stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 h in the presence of a Golgi inhibitor. The histograms depict intracellular cytokine staining on gated CD8+ H-Y TCR+ cells from H-Y male and female mice and from CD8+CD44high and CD8+CD44low T cells from B6 mice. Cytokine production by unstimulated cells (black line) was compared with PMA and ionomycin-stimulated cells (gray line) with the numbers in the plots representing the percentage of cytokine-positive cells after subtracting the background cytokine production.

 
IFN-{gamma} production by self-specific CD8 T cell provides protection against bacterial infection

Memory-phenotype CD8 T cells have been shown to produce large amounts of IFN-{gamma} early during infection with Listeria and these cells have been shown to be protective during infection (16, 17, 18, 19, 20). To test the ability of self-specific CD8 T cells to produce IFN-{gamma} in vivo and to address whether this cytokine production has biological significance, we measured the ability of this cell type to protect IFN-{gamma}–/– mice against LM infection. Two previous studies have used this approach to demonstrate that CD8 T cells and NK cells capable of producing IFN-{gamma} during infection can protect IFN-{gamma}–/– mice against LM infection due to their ability to provide a source of IFN-{gamma} (17, 18). We transferred purified self-specific CD8 T cells into male IFN-{gamma}–/– mice, which were infected with 104 CFU of Listeria 1-day posttransfer. At day 3 postinfection, we sacrificed the animals and assessed cytokine production by the transferred cells in the absence of any restimulation. Fig. 3A depicts IFN-{gamma} production by gated self-specific CD8 T cells recovered from the spleens of infected or uninfected IFN-{gamma}–/– mice. It is clear that a significant proportion (~15%) of the transferred cells were actively producing IFN-{gamma} and that this cytokine production was a consequence of infection. We also determined whether IFN-{gamma} production by self-specific CD8+ T cells was associated with a decrease in bacterial burden in the spleens of infected IFN-{gamma}–/– mice. Fig. 3B clearly shows that IFN-{gamma}–/– mice that had received self-specific CD8 T cells before infection had at least a 10-fold reduction in bacterial load in the spleen relative to mice which did not. Therefore, the ability of self-specific CD8 T cells to protect against Listeria infection is directly associated with their ability to produce IFN-{gamma}.


Figure 3
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  		FIGURE 3. Self-specific CD8 T cells provide an innate source of IFN-{gamma} during infection. A total of 3 x 106 purified self-specific CD8 T cells were transferred into B6 male IFN-{gamma}–/– recipients, which were infected the following day with wild-type LM. A, Intracellular IFN-{gamma} staining in the absence of restimulation on gated donor cells (CD8+ H-Y TCR+) isolated 3 days postinfection from IFN-{gamma}–/– recipients infected with LM or left uninfected. The cells were cultured for 5 h in vitro in the presence of a Golgi inhibitor before staining. B, Bacterial load in the spleens of IFN-{gamma}–/– mice on day 3 postinfection that had received 3 x 106 self-specific CD8 cells (black) or that did not receive any cells (white). Error bars represent the SD from three to four mice per group.

 
Memory-phenotype CD8+CD44high cells and self-specific H-Y male CD8 T cells provide similar innate protection during infection

After determining the ability of self-specific CD8 T cells from H-Y male mice to protect IFN-{gamma}–/– mice from infection with LM, we decided to test the relative efficacy of self-specific CD8 T cells from H-Y male mice, memory-phenotype CD8+CD44high T cells from B6 mice and naive CD8+CD44low T cells from B6 mice to confer protection against LM infection in IFN-{gamma}–/– mice. It is noted that memory-phenotype CD8 T cells from B6 mice, like self-specific CD8 T cells from H-Y male mice, have the ability to produce IFN-{gamma} rapidly upon activation whereas naive CD8 T cells do not (Fig. 2). We sorted memory and naive-phenotype CD8 T cells from B6 mice and we purified self-specific CD8 T cells from H-Y male mice. We then transferred 9 x 105 cells into male IFN-{gamma}–/– mice and infected the mice the following day with LM. The results in Fig. 4 demonstrate that both self-specific H-Y male CD8 T cells and memory-phenotype B6 CD8 T cells, but not naive B6 CD8 T cells, provide a small but significant degree of protection against LM infection in IFN-{gamma}–/– mice. This protection was observed in both the spleen and liver of IFN-{gamma}–/– mice that had received relatively few transferred cells (9 x 105), with both the H-Y male and B6 CD8+CD44high cells reducing the bacterial load by >3-fold. This result indicates that self-specific CD8 T cells from H-Y male mice and CD8+CD44high cells from B6 mice are functionally similar in terms of their ability to confer protection against LM infection in IFN-{gamma}–/– mice.


Figure 4
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  		FIGURE 4. Self-specific H-Y male CD8 T cells and memory-phenotype CD8 T cells from normal B6 mice provide similar protection against LM infection. A total of 9 x 105 sorted B6 CD8+CD44high, B6 CD8+CD44low, and male H-Y TCR+ CD8+ cells were transferred into male IFN-{gamma}–/– mice 1 day before infection with wild-type LM (104 CFU). Bacterial load in the spleen and liver of infected mice that received either no cells (black bar), B6 CD8+CD44low (white bar), B6 CD8+CD44high (dark gray bar), or male H-Y TCR+ CD8+ (light gray) cells is shown. The error bars represent the SD from three mice per group.

 
IL-15-activated cells provide protection against LM infection

Previous reports have shown that self-specific CD8 T cells can proliferate and become activated in vitro in response to IL-2 or IL-15 (32). The expression of IL-2Rbeta (CD122) in conjunction with common {gamma} chain (CD132) is sufficient to confer lymphocytes with the ability to respond to both IL-2 and IL-15 (33, 34). Activation of NK cells or memory-phenotype CD8 T cells with either of these cytokines is able to enhance both the cytotoxicity as well as cytokine production by these cells (35, 36, 37). Because immediately ex vivo self-specific CD8 T cells provide protection against LM infection in IFN-{gamma}–/– mice, we wanted to see whether activation of the cells with IL-2 or IL-15 could enhance this function. We cultured purified and CFSE-labeled self-specific CD8 T cells with either 100 ng/ml IL-2 or IL-15 for 4 days. Both IL-2 and IL-15 induced the same amount of proliferation of these cells (Fig. 5A). We also determined the potential of cells cultured in either IL-2 or IL-15 to produce IFN-{gamma}, TNF-{alpha} and the cytolytic marker, granzyme B. We found that PMA and ionomycin activated IL-2- and IL-15-cultured cells produced equivalent amounts of IFN-{gamma}. However, activated IL-2-cultured cells produced a greater amount of granzyme B than did IL-15-cultured cells. By contrast, IL-15-cultured cells produced more TNF-{alpha} than IL-2 cultured cells. We next determined the ability of IL-2 or IL-15-cultured cells to protect IFN-{gamma}–/– mice from LM infection. IL-2 or IL-15 cultured cells were transferred into IFN-{gamma}–/– mice and the mice were infected 1 day later with LM. At day 3 postinfection, we assessed the total numbers of transferred cells recovered as well as the bacterial load in the spleens of infected mice. Fig. 5B clearly shows that self-specific CD8 T cells cultured in IL-2 were much less efficient at homing to and/or surviving in the spleens of infected animals as we recovered only ~105 IL-2 cultured cells 3 days postinfection. In addition, the few IL-2 cultured cells that were present were completely ineffective in protecting IFN-{gamma}–/– mice from LM infection, causing no reduction in bacterial load (Fig. 5C). By contrast, IL-15-cultured cells did either home to and/or survive better in the spleen as we recovered ~7 x 105 cells and the cells were extremely protective against infection, reducing the bacterial load by greater than 100-fold compared with control IFN-{gamma}–/– mice. The protection by IL-15 cultured cells was greater than that achieved with immediately ex vivo cells (Fig. 2B) even though protection was observed in both cases. Therefore consistent with results using IL-15 tg mice (38) culturing self-specific CD8 T cells with IL-15 provides a novel method for the expansion of cells with increase effectiveness in providing protection against bacterial infection.


Figure 5
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  		FIGURE 5. Self-specific CD8 T cells expanded in IL-15 are protective in vivo whereas IL-2-expanded cells are not. A, Purified self-specific CD8 T cells were CFSE labeled and cultured in either IL-2 (100 ng/ml) or IL-15 (100 ng/ml) for 4 days. The CFSE profiles of the cultured cells on day 4 are as indicated. The CFSE-labeled cells cultured in IL-2 (filled histogram) or IL-15 (black line) were stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 h in the presence of a Golgi inhibitor. The cells were then stained intracellularly for IFN-{gamma}, TNF-{alpha}, and granzyme. B, A total of 3 x 106 day 4 IL-2 or IL-15 cultured cells was transferred into male IFN-{gamma}–/– mice that were infected the next day with LM. Absolute numbers of transferred cells previously expanded in IL-2 ({square}) or IL-15 ({blacksquare}) and recovered from the spleen of recipient mice at day 3 postinfection are indicated. C, Bacterial load on day 3 postinfection in spleen of IFN-{gamma}–/– mice that had received IL-2 ({square}) or IL-15 ({blacksquare}) cultured cells 1 day before infection compared with IFN-{gamma}–/– mice that did not receive any cells ({cjs2108}). Error bars represent the SD from three to four mice per group.

 
Self-Ag interactions are important for innate protection by self-specific CD8 T cells

We next determined whether the effectiveness of self-specific CD8 T cells in controlling LM infection is dependent on interaction with self-Ag. IL-15-cultured cells were used because they were more protective than immediately ex vivo cells. We transferred 1 x 106 IL-15 cultured self-specific CD8 T cells into either male (self-Ag+) or female (self-Ag–) IFN-{gamma}–/– recipients and then infected the mice with 104 CFU of LM the following day. We then compared the relative reduction in bacterial load in mice that had either received or not received cells. This was done to eliminate any differences in bacterial load due to the sex of the recipient mice. Fig. 6 shows that self-specific CD8 T cells were protective in both male and female recipients. However, protection offered by the transferred cells in female recipients was modest, resulting in only a 2-fold reduction in bacterial load in the spleen and liver of female IFN-{gamma}–/– mice receiving self-specific CD8 T cells relative to female IFN-{gamma}–/– mice that did not. By contrast, the transferred cells offered much greater protection in male IFN-{gamma}–/– recipients, resulting in about a 10-fold reduction in bacteria in the spleen and about a 100-fold reduction in bacteria in the liver relative to male IFN-{gamma}–/– mice that did not receive any cells. This result indicates that the presence of self-Ag is required for efficient protection against LM infection.


Figure 6
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  		FIGURE 6. Self-Ag interactions enhance the protection mediated by self-specific CD8 T cells during infection. Purified self-specific CD8 T cells were cultured with IL-15 (100 ng/ml) for 4 days and then 106 cells were transferred into either male or female IFN-{gamma}–/– mice which were infected the next day with 104 CFU wild-type LM. The data represent the relative reduction in bacterial load on day 3 postinfection in the spleens and livers of male (black) and female (white) IFN-{gamma}–/– mice that had received self-specific CD8 cells relative to male and female IFN-{gamma}–/– mice not receiving any cells. Error bars represent the SD from three to four mice per group.

 
Self-Ag interactions are crucial for maintaining the memory phenotype and cytokine responsiveness of self-specific CD8 T cells

Previous studies had shown that the expansion of self-specific CD8 T cells in response to either IL-2 or IL-15 in vitro was independent of self-Ag interactions (32). Our studies have suggested that self-Ag interactions played a role in the proliferation of self-specific CD8 T cells in vivo during LM infection (26). Because we had also shown that self-Ag interactions dictate the extent of the memory phenotype in these self-specific CD8 T cells (26), we decided to test the idea that self-Ag interactions control the expansion of these cells in vivo in part by helping to maintain a memory phenotype and high levels of the associated cytokine receptors. To address this possibility, we transferred self-specific CD8 T cells into congenic Thy-1.1 male or female recipient mice and determined their cell surface phenotype in the spleens of recipient mice 7 days posttransfer. There was no difference in the frequency of self-specific CD8 T cells in the spleen or livers of male or female recipients (Fig. 7A) suggesting that survival and homing are unaffected by self-Ag 7 days posttransfer. However, we noticed striking differences in the expression of the TCR and CD44 as well as the cytokine receptors CD122 and CD127. Self-Ag interactions in male recipients led to lower TCR expression and higher expression of CD44 (Fig. 7B). CD122 and CD127 were also maintained at higher levels in male recipients over female recipients suggesting that self-Ag interaction is crucial for the high expression of these receptors in self-specific CD8 T cells. The lower expression of CD122 on self-specific CD8 T cells that are maintained in female recipients likely leads to their decreased ability to respond to IL-15. To test this possibility more directly, we purified self-specific CD8 T cells which had been rested in either male or female recipients for 7 days, CFSE-labeled these cells and tested their ability to proliferate in response to IL-15 in vitro. Fig. 7C clearly demonstrates that self-specific CD8 T cells maintained in Ag– female recipients are less responsive to IL-15 as only 21% of the cells have undergone greater than four division events compared with ~51% of the cells maintained in male mice. These observations provide an explanation for the decreased proliferation of these cells in vivo in response to LM infection in female recipient mice.


Figure 7
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  		FIGURE 7. Self-Ag interactions maintain the memory phenotype and cytokine responsiveness of self-specific CD8 T cells in vivo. A total of 3 x 106 purified self-specific CD8 T cells (Thy-1.2+) were transferred into male and female B6-Thy-1.1 congenic mice. A, Seven days posttransfer, the frequency of donor cells (Thy-1.2+) was quantified in the spleen and liver of recipient mice. B, Surface marker expression on gated transferred CD8 cells (Thy-1.2+) 7 days posttransfer into male (black line) or female (filled histogram) mice. The numbers in the plots represent the MFI of cells from male (M) or female (F) mice. C, Seven days posttransfer into male or female mice self-specific CD8 T cells were enriched for, labeled with CFSE, and cultured in IL-15 (100 ng/ml) in vitro for 3 days. The histograms represent the cell division of donor self-specific CD8 T cells from male (black line) or female (filled histogram) recipients in response to IL-15.

 
Self-specific CD8 T cells provide greater IFN-{gamma}-dependent protection against LM infection than NK cells

Several recent reports have focused on the ability of memory-phenotype CD8 T cells to provide innate protection during infection. One report compared the ability of NK cells and foreign-Ag specific memory CD8 T cells to protect IFN-{gamma}–/– mice against infection with LM (18). This study found that memory CD8 T cells are more protective than NK cells, in part due to their ability to localize to areas of the spleen bearing LM-infected macrophages. We compared the relative efficacy of self-specific CD8 T cells and NK cells in providing protection against LM infection by first comparing the ability of self-specific CD8 T cells and NK cells to produce IFN-{gamma}, TNF-{alpha}, and granzyme B. This was done by culturing both cell types in IL-15 for 4 days and restimulating the cells with PMA and ionomycin for 4 h. We found that self-specific CD8 T cells were much more efficient producers of both IFN-{gamma} and TNF-{alpha} than NK cells; however, NK cells were much more efficient in producing granzyme B (Fig. 8A). These differences seen after IL-15 activation were also true of these cell types immediately ex vivo (data not shown). To compare the ability of self-specific CD8 T cells and NK cells in conferring protection against LM infection in IFN-{gamma}–/– mice equal numbers of day 4 IL-15-cultured self-specific CD8 T cells and NK cells were transferred into male IFN-{gamma}–/– recipients. We then infected the mice with LM the next day and measured the reduction in bacterial load in the spleens and livers of mice receiving self-specific CD8 cells or NK cells. The results in Fig. 8B indicate that NK cells offered a small degree of protection against LM infection, reducing the bacterial load in the spleen and liver by ~2- and 3-fold, respectively. By contrast, self-specific CD8 T cells were much more effective in reducing bacterial load in IFN-{gamma}–/– mice, reducing the bacterial load in spleen and liver by ~30- and 700-fold, respectively. These results indicate that self-specific CD8 T cells are more effective than NK cells in providing protection against LM infection in IFN-{gamma}–/– mice.


Figure 8
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  		FIGURE 8. Self-specific CD8 T cells provide more IFN-{gamma}-dependent protection in vivo against LM infection than NK cells. Purified self-specific CD8 T cells and NK cells were cultured in IL-15 (100 ng/ml) for 4 days. A, Intracellular staining for IFN-{gamma}, TNF-{alpha} and granzyme B of self-specific CD8 (black line) and NK (filled histogram) cells stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 h. Numbers in the plots are the MFI. B, Bacterial load reduction day 3 postinfection in the spleen and liver of IFN-{gamma}–/– mice receiving 1 x 106 activated self-specific CD8 ({blacksquare}) or NK ({square}) cells 1 day before infection relative to control IFN-{gamma}–/– mice not receiving any cells. Error bars represent the SD of data from three to four mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we have clearly demonstrated an important role for self-specific CD8 T cells in early protection of mice from LM infection. We have shown that these self-specific cells are potent producers of IFN-{gamma} and TNF-{alpha} immediately ex vivo. We also showed that self-specific CD8 T cells offered significant protection against LM infection in IFN-{gamma}–/– mice, particularly in Ag-expressing mice. This protection was greatly enhanced by culturing self-specific CD8 T cells in IL-15 before transfer. We also demonstrate an important role for self-Ag interactions in maintaining the memory phenotype of these cells and their responsiveness to IL-15. Finally, we found that self-specific CD8 T cells were much more effective than NK cells in protecting IFN-{gamma}–/– mice against LM infection.

We have also confirmed in this study that self-specific CD8 T cells from H-Y male mice can develop in the absence of a functional thymus. We did, however, notice a significant reduction in the frequency of H-Y CD8 T cells in athymic male mice compared with euthymic mice (Fig. 1A). This suggests that thymus-dependent mechanisms or thymus-dependent T cells can improve the survival, selection, or expansion of this cell type. The fact that these self-specific CD8 T cells can be selected by cognate self-Ag outside the thymus indicates that these cells constitute a lineage that is distinct from conventional CD8 T cells (29, 30). The memory markers CD44 and CD122 as well as the NK receptors CD94 and NKG2D are significantly higher on self-specific CD8 T cells from athymic nude mice relative to their euthymic counterparts. We have seen the expression of CD94 and NKG2D increase on self-specific CD8 T cells upon activation by Ag or IL-15 and thus the increased expression of these receptors on athymic CD8 T cells likely reflects their activation history (26). This may be due in part to increased availability of IL-15 due to the smaller numbers (one-seventh) of self-specific CD8 T cells in the athymic nude mice. Consistent with this hypothesis, IL-15 has been shown to play a role in the induction of both NKG2D and CD94 in CD8 T cells (39, 40). The significance of the high level of CD94 expression in athymic CD8 T cells is unknown but it may play a role in their survival as one study showed that CD94 expression is associated with protection from activation induced cell death and correlates with increased survival in CD8 T cells (41). Whether athymic CD8 T cells preferentially express CD94 to enhance their survival remains to be determined.

Although this current study focused mainly on the role of a homogenous population of self-specific CD8 T cells from male H-Y TCR-tg mice, we have previously shown that memory-phenotype CD8 T cells from normal B6 mice share many similarities to this cell type (24). In this study, we have provided further confirmation that memory-phenotype CD8 T cells are very similar to self-specific H-Y CD8 T cells in their cytokine production profiles as well as in their ability to provide innate protection to IFN-{gamma}–/– mice against LM infection. The main problem with non-TCR-tg mice for these studies is the heterogeneity of the memory-phenotype population, which makes it impossible to dissect the relative contributions of conventional memory CD8 T cells from self-specific CD8 T cells. The H-Y model is not unique when it comes to the development of memory-phenotype CD8 T cells specific for a self-Ag. For instance, in doubly tg mice, which express a tg TCR specific for the gag protein from Friend murine leukemia virus as well as the tg cognate Ag, Friend murine leukemia virus gag, CD8 T cells expressing the tg TCR still develop. Furthermore, these cells exhibit a memory-phenotype similar to the self-specific CD8 T cells from H-Y male mice; these mice also did not develop detectable autoimmune diseases even though the self-specific CD8 T cells retain effector function and are specific for the self-Ag (42). A recent report on these cells also demonstrated their ability to proliferate in response to IL-2 or IL-15 alone (43). Another TCR-tg model that shares similarities to ours is the P14 TCR tg in which CD8 T cells are specific for a peptide derived from the lymphocytic choriomeningitis virus glycoprotein. When P14 TCR-tg mice are crossed to mice ubiquitously expressing cognate Ag the developing P14 CD8 T cells share a nearly identical cell surface phenotype with the self-specific CD8 T cells from H-Y male mice (P. S. Ohashi, unpublished observation). Whether the CD8 T cells expressing these self-specific TCR’s behave the same way as those from H-Y male mice remains to be determined although based on their phenotype it is reasonable to believe that they will also share similarities in function. Together, these observations emphasize the importance of TCR/cognate self-Ag interactions for the development of self-specific CD8 T cells.

Self-reactivity is also characteristic of some other cell types of the adaptive immune cells. For instance, NK-T cells exhibit a high degree of self-reactivity and function through the recognition of self-lipid in the context of CD1d (44). These cells also have an unusual requirement for positive selection in the thymus in that they are selected by bone marrow derived double-positive thymocytes rather than cortical epithelial cells which select conventional T cells (5). CD8{alpha}{alpha} IELs are also selected by agonist self-peptides (3). Studies using H-Y TCR-tg mice demonstrate strong interaction with self-Ag is required for the development of CD8{alpha}{alpha} IELs whereas weaker selection leads to the development of CD8{alpha}beta T cells (45). Interestingly, the strength of selection also correlates with the expression of genes associated with innate immune cells. Although the self-specific CD8 T cells are unrelated to CD8{alpha}{alpha} IELs, it is conceivable that their unconventional functions may also be a consequence of their positive selection by agonist self-Ags.

One phenotypic similarity between self-specific CD8 T cells and other innate T cells is their memory phenotype. This phenotype is associated with expression of high levels of CD122, which confers IL-2 and IL-15 responsiveness. IL-15 responsiveness seems to be crucial for the maintenance of NK, NKT, and memory phenotype CD8 T cells (which include self-specific CD8 T cells) as all of these cell types are either drastically reduced in number or virtually absent in mice lacking either IL-15 or its receptor (46, 47). An interesting finding of the present study is that self-specific CD8 T cells require self-Ag for the maintenance of high levels of CD122, which then makes them more responsive to cytokine stimulation. Two T-box family transcription factors T-bet and eomesodermin have recently been shown to be crucial for the maintenance of IL-15-dependent cell types including NK, NKT, and memory phenotype CD8 T cells (48). Notably, these transcription factors act directly on the CD122 promoter, inducing transcription and expression of CD122. It remains to be determined whether T-bet or eomesodermin expression by self-specific CD8 T cells is induced or maintained through self-Ag interactions.

We have compared the ability of IL-2 or IL-15 cultured self-specific CD8+ T cells to protect IFN{gamma}–/– mice from LM infection. The ineffectiveness of IL-2-cultured cells in conferring protection could be due to their inability to home to and/or survive in the spleen because IL-2 is limiting in vivo. IL-15 cultured cells, which are smaller and look more like naive cells compared with IL-2-cultured cells, either home more efficiently or survive better in the spleen of recipient mice. Furthermore, these cells offered superior protective function against LM infection, presumably as a consequence of their ability to respond to IL-15 produced in vivo upon infection. The ability of self-specific CD8 T cells to respond to IL-15 in vivo allows the cells to rapidly become activated during infection or inflammation and results in the production of cytokines such as IFN-{gamma} and TNF-{alpha}. We have demonstrated in this report that these self-specific CD8 T cells do produce IFN-{gamma} early during infection and provide protection against LM infection whether or not the cells were pretreated with IL-15. The mechanism for the induction of IFN-{gamma} by these cells likely involves responsiveness to IL-12 produced by infected macrophages. IL-12 in conjunction with IL-2, IL-15, or IL-18 has been shown to be sufficient to induce large amounts of IFN-{gamma} production from self-specific CD8 T cells (32). There is also the possibility that NKG2D may play a role in inducing IFN-{gamma} production by self-specific CD8 T cells. We have shown previously that these cells do in fact increase expression of NKG2D upon infection (26) and reports suggest that infected macrophages express the ligands for NKG2D (49). It is quite possible that NKG2D stimulation may play an important role in inducing IFN-{gamma} by these cells.

The requirement for interaction of self-specific CD8 T cells with their cognate Ag in conferring better protection against LM infection indicates that TCR interactions play a role in the function of these cells. We have shown in this study that continuous interaction with self-Ag in vivo is required for maintaining the high responsiveness of these cells to IL-15. Thus, the increased protective function of self-specific CD8 T cells in cognate Ag-expressing mice is likely due to the requirement for self-Ag in maintaining high expression of cytokine receptors such as CD122, which allows them to sense bacterial infection more effectively by responding to physiological levels of IL-15. The self-specific CD8 cells still retain function after being in an Ag– female recipient for 7 days. It is clear, however, that self-specific CD8 T cells are less efficient at responding to IL-15 in Ag– female recipients, but this inefficiency can be partially overcome by increasing the numbers of these cells.

NK cells are a source of early IFN-{gamma} and TNF-{alpha} that are involved in protection from pathogens as well as tumor cells (50). Our study clearly demonstrates that self-specific CD8 T cells are much more potent producers of both IFN-{gamma} and TNF-{alpha} when compared with NK cells. Consequently, self-specific CD8 T cells provide more protection than NK cells to IFN-{gamma}–/– mice during infection with LM. Another difference between self-specific CD8 T cells and NK cells is their localization. NK cells are relatively abundant in the spleen yet are virtually absent in the LNs of mice (51). Self-specific CD8 T cells in contrast are present in larger numbers in both the spleen and LNs and are more efficient than NK cells in homing to either location (our unpublished observations). NK cells have been shown to be involved in many steps of T cell priming either by acting on dendritic cells (52) or by producing IFN-{gamma} that can participate in CD4 T cell priming (51). Because T cell priming occurs primarily in secondary lymphoid organs where self-specific CD8 T cells are abundant it remains possible that these cells may also be involved in this aspect of immunity. Notably, memory phenotype CD8 T cells, which serve as a source of early IFN-{gamma}, were shown to polarize CD4 T cells to the Th1 lineage (38, 53). It remains to be determined whether priming of CD4 T cells toward the Th1 lineage is another function of self-specific CD8 T cells.


   Acknowledgments
 
We thank Soo-Jeet Teh for excellent technical assistance and John Priatel for helpful discussion.


   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 a grant from the Cancer Research Society (to H.-S.T.). S.D. is supported by the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research. Back

2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. E-mail address: teh{at}interchange.ubc.ca Back

3 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; LM, Listeria monocytogenes; tg, transgenic; LN, lymph node. Back

Received for publication January 24, 2006. Accepted for publication April 12, 2006.


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