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The Journal of Immunology, 2007, 179, 2457-2466
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Listeria Infections

A Role for IFN-{gamma} from Antigen-Specific CD8+ T Cells in Protective Immunity to Listeria monocytogenes1

Kelly A. N. Messingham*, Vladimir P. Badovinac*, Ali Jabbari* and John T. Harty2,*,{dagger}

* Department of Microbiology and {dagger} Interdisciplinary Program in Immunology, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Whether IFN-{gamma} contributes to the per-cell protective capacity of memory CD8+ T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8+ T cells via immunization of wild-type (WT) and IFN-{gamma}-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8+ T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8+ T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8+ T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8+ T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8+ T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8+ T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8+ T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-{gamma} production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-{gamma}-deficient LM-specific memory CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ T cell is critical for protective immunity to a variety of intracellular pathogens (1). This immunity is largely dependent on the diverse array of effector functions at the disposal of activated CD8+ T cells, including the secretion of cytokines, such as IFN-{gamma} and TNF (2, 3), and Ag-specific cytolysis of infected cells (3, 4, 5). In combination, it is thought that these effector functions produce a potent CD8+ T cell response resulting in pathogen clearance. Historically, it has been difficult to estimate the contribution of each effector mechanism independently because of the complexity of the CD8+ T cell response; however, experimental infection of gene knockout (KO)3 mice has proven useful for this purpose (3, 4).

Initial studies examining protective immunity in IFN-{gamma}-deficient KO (GKO) mice (6) determined that IFN-{gamma} is critical for resistance to primary infection with virulent Listeria monocytogenes (LM). However, vaccination of GKO mice with an attenuated (actA-deficient (7)) strain of LM generated protective CD8+ T cell immunity that was equivalent to that observed in vaccinated wild-type (WT) mice (6). This translates into a ~20,000-fold increase in resistance to challenge with virulent bacteria when compared with naive GKO mice (6). The conclusion drawn from these studies was that IFN-{gamma} is not required for the development or elaboration of protective CD8+ T cell-mediated immunity to LM. The caveat to this interpretation is that the protective immunity observed in vaccinated GKO mice occurred in the presence of dramatically elevated numbers of LM-specific memory CD8+ T cells (9). Subsequent studies have defined a role for IFN-{gamma} as a regulator of T cell homeostasis and attribute the increased memory CD8+ T cell numbers in vaccinated GKO mice to a markedly protracted and reduced contraction phase of the T cell response observed in the absence of IFN-{gamma} (8). Additionally, immunized GKO mice also display altered immunodominance hierarchies that have been attributed to the disparate influence of IFN-{gamma} on presentation of discrete bacterial peptides by MHC class I molecules (9). Thus, it remains to be determined whether the protective immunity provided by IFN-{gamma}-deficient CD8+ T cells is equivalent to WT CD8+ T cells on a per-cell basis or whether the protective immunity observed in LM-vaccinated GKO mice is due to the increased numbers of Ag-specific memory CD8+ T cells.

The goal of the current study was to determine whether IFN-{gamma} production by Ag-specific CD8+ T cells contributes to protective immunity to LM. To address this question, we transferred LM Ag-specific CD8+ T cells from dendritic cell (DC) peptide-immunized WT or GKO mice, into naive WT recipients and compared bacterial CFU reduction after challenge with virulent LM. In summary, memory CD8+ T cells generated in the WT or GKO mice were similar when functional avidity, TCR repertoire use, and cell surface phenotype were compared. However, a decrease in the protective immunity provided by memory CD8+ T cells from GKO mice was observed as early as day 3 after LM challenge, and was exacerbated when fewer Ag-specific CD8+ T cells were transferred or when a higher bacterial challenge dose was given. Further functional analysis revealed diminished Ag-specific cytolysis in vivo by the IFN-{gamma}-deficient memory CD8+ T cells as a mechanism for their decreased per-cell protective capacity. Finally, our studies suggest that IFN-{gamma} signals received by target cells from the memory CD8+ T cells is essential for the most efficient killing in vivo. Thus, the absence of IFN-{gamma} as a CD8 T cell effector molecule results in decreased killing efficiency and less effective protective immunity provided by these cells. We conclude that although IFN-{gamma} does not appear to be required for the development or elaboration of CD8+ T cell-mediated protective immunity, memory CD8+ T cell production of IFN-{gamma} contributes to optimal secondary resistance at early time points after LM infection of a vaccinated host.


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

Eight- to 10-wk-old female BALB/c (Thy1.2, H-2d MHC, WT) and C57BL/6 (Thy1.2, H-2b) mice were purchased from the National Cancer Institute (Frederick, MD). BALB/c IFN-{gamma}-deficient (Thy 1.2, H-2d MHC, GKO) mice (6), BALB/c Thy1.1 mice (provided by R. Dutton, Trudeau Institute, Saranac Lake, NY), OT-I (10) (C57BL/6 Tg(TcraTcrb)1100Mjb/j, Thy1.1; provided by Dr. T. L. Ratliff, University of Iowa, Iowa City, IA), and C57BL/6 IFN-{gamma}R KO (purchased from The Jackson Laboratory) mice were maintained by brother-sister mating and housed under specific pathogen-free conditions at the University of Iowa Animal Care Unit until initiation of experiments.

These studies used virulent and attenuated (actA-deficient) LM strains derived from XFL 303 (11), which expresses a secreted fusion protein containing the lymphocytic choriomeningitis virus (LCMV) NP118–126 epitope (LM-nuclear proteins (NPs); Ref. 12), or actA-deficient LM-NPs (7) for immunization of BALB/c mice and actA-deficient LM expressing the OVA protein (LM-OVA) (13, 14) for immunization of C57BL/6 mice. Growth and maintenance of LM strains was as described (6). CFU of LM per spleen or per gram of liver were determined by plating serial dilutions of organ homogenates as described (4). Pathogen-infected mice were housed at the appropriate biosafety level. Experimental protocols were approved by the University of Iowa Animal Care and Use Committee.

DC immunization

CD11c+ bone marrow-derived DCs were generated as previously described (15, 16). Briefly, RBC-depleted bone marrow was subjected to complement depletion after incubation with 50 µg/ml mAbs 3.168 (anti-CD8), 34-5-3 (anti-I-Ad), RL-172 (anti-CD4), and RA3-3A1/6.1 (anti-B220). Remaining cells were cultured with 1000 U/ml rGM-CSF (BD Pharmingen) and 25 U/ml rIL-4 (PeproTech) (DC medium) for 6 days with 80% medium replacement every other day. Next, cells were harvested and contaminating neutrophils were removed through complement depletion after incubation with the Ly-6-G-specific Ab RB6.8C5. The remaining cells were cultured overnight with in DC medium with LPS (500 ng/ml; Sigma-Aldrich) and 1 µM peptide was added to cultures 2 h before harvest, washing, and injection. The resulting cell populations consisted of 75–85% CD11c+ and H-2Ld+, B7.1+, B7.2+, CD8{alpha}–, CD4–, I-Ad+, and CD11bhigh. For vaccination, ~2.5 x 105 mature (CD11c+) DCs were injected i.v. per mouse. After 35 days, a booster immunization (0.8–1.0 x 106 DCs) was given. All experiments were conducted at least 35 days after the booster immunization.

Abs, peptides, and MHC class I tetramers

mAbs with the following specificities were used: TNF-{alpha}-PE or allophycocyanin (MP6-XT22), IL-2-PE (JES6-5H4), CD8-FITC or CyChrome (53-6.7), CD62L-PE (MEL-14), CD127-PE (A7R34), CD27-PE (LG.7F9), CD44-PE (Pgp-1), FITC-conjugated Vbeta 2, 4,5.1/5.2, 8.1/8.2, and 10 (B20.6, KT4, MR9-4, MR5-2 and B21.5, respectively), Thy 1.2-FITC (53-6.7), CD107a-FITC (1D4B; BD Pharmingen), anti-human granzyme B-PE (Caltag Laboratories) and appropriately labeled IgG2a, IgG2b, and IgG1 isotype controls. All Abs were obtained from eBioscience unless noted. Synthetic LLO91–99, p60217–226 (both H-2Kd), OVA257–264 (H-2Kb), and NP118–126 (H-2Ld) peptides were obtained from BioSynthesis. MHC class I tetramers specific for NP118–126, LLO91–99, or p60217–225 were obtained from the National Institute of Allergy and Infectious Diseases Tetramer Core (Atlanta, GA) or prepared using published protocols (17, 18).

T cell enrichment and adoptive transfer

Splenocyte cell suspensions were enriched for CD8+ T cells by negative selection (Stem Cell Technologies) (4). Briefly, 8 x 107 splenic leukocytes per ml were labeled with a CD8+ enrichment Ab mixture followed by conjugation to magnetic beads. CD8+ T cells were obtained in the flow-through after magnetic separation (Macs LS Separation Column; Miltenyi Biotec). Alternatively, memory OT-1 cells were generated by transferring 5 x 104 naive OT-I (Thy1.1) CD8+ T cells, enriched by negative selection, into naive B6 mice followed by infection with 106 actA-deficient LM-OVA (19). After ~35 days, mice were infected with 107 actA-deficient LM-OVA. Secondary memory OT-I T cells were purified with PE-conjugated anti-Thy1.1 (BD Pharmingen) and anti-PE magnetic beads (Miltenyi Biotec). All experiments used mice or memory cells obtained >35 days after secondary immunization. Enriched cells, routinely >93% pure by flow cytometry, were washed three times in sterile saline and adoptively transferred by i.v. injection.

To evaluate the percentage of NP118–126, LLO91–99, or p60217–225-specific CD8+ T cells present in the enriched population, a fraction of each donor cell pool (WT or GKO) was costained CD8 and LM peptide-specific MHC class I tetramers. The number of enriched cells from WT or GKO donors was adjusted so that equal numbers (noted in figure legends) of Ag-specific CD8+ T cells were transferred i.v. into naive female BALB/c (Thy1.2 or Thy1.1) recipient mice. After transfer (24 h), an equal number (~10% of the transferred cells) of WT or GKO cells were observed in the spleens of recipient mice (data not shown). One to 2 days after adoptive transfer, T cell-recipient or naive control mice were challenged with virulent LM-NPs (1 LD50 = ~10,000 bacteria) as indicated.

Detection of Ag-specific CD8+ T cells, T cell repertoire

The number of CD8+ T cells specific for specific for NP118–126, LLO91–99, or p60217–225 was determined using intracellular cytokine staining (ICS) for TNF-{alpha} after 5.5-h incubation with or without 200 nM peptide in brefeldin A (BD Pharmingen) (20). To detect CD62L on TNF+ CD8+ T cells, TAPI-2 (100 µM; a TACE inhibitor; Peptides International) was added 30 min before peptide addition (21). Nonspecific cytokine production (≤0.5%; no peptide control) was subtracted when the total number of Ag-specific CD8+ T cells per spleen was calculated. The T cell repertoire was determined by flow cytometry after staining with LM peptide-specific MHC class I tetramers and Abs specific for various Vbeta gene families (BD Pharmingen) (4).

In vivo cytotoxicity assay

The ability of WT or GKO memory CD8+ T cells to lyse CFSE-labeled target cells was evaluated in the spleens of T cell-recipient mice by flow cytometry. Equal numbers (20 x 106 total) of peptide (1 µM LLO91–99) coated or uncoated naive syngeneic splenocytes were labeled with 2.5 µM CFSE (CFSEhigh) or 0.25 µM (CFSElow), respectively, and injected into experimental mice i.v. (4).

To evaluate cytolysis of WT C57BL/6 vs IFN-{gamma}R-deficient (IFN-{gamma}R KO) targets, naive splenocytes of each type were incubated with or without OVA257–264 peptide (1 µM) and the resulting populations—WT with OVA, WT without OVA, IFN-{gamma}R KO with OVA, IFN-{gamma}R KO without OVA—were washed and labeled with serial dilutions (1, 0.1, 0.01, or 0.001 µM, respectively) of CFSE for 12 min. Next, 1 ml of FCS was added for 1 min and then cells were washed extensively. All target cells were labeled uniformly (4 min) at 2 x 107 cells/ml with red cell linker in diluent C (PKH26; Sigma-Aldrich), an equal volume of FCS was added (1 min), and cells were washed three times in medium containing 10% FCS. The labeled cells were combined so that 2 x 106 of each target type (8 x 106 total) was injected i.v. into naive C57BL/6 mice or OT-I memory T cell (1.5 x 106) recipient mice. OVA peptide/TCR complexes on target cell populations were evaluated with optimized concentrations of mAb (25-D1.16) and strepavidin-FITC (22). In all cases, the percent killing was calculated as follows: 100 – (((percentage of peptide pulsed in T cell-recipient (or memory) mice/percentage of unpulsed in T cell-recipient (or memory) mice)/percentage of peptide pulsed in naive mice/percentage of unpulsed in naive mice) x 100) (5).

Degranulation assay

Spleen cells were stimulated in vitro with or without 100 ng/ml LLO91–99 peptide in 500 µl of RPMI 1640 containing 10% FCS and penicillin/streptomycin. FITC-conjugated anti-mouse CD107a or isotype control and monensin A (Sigma-Aldrich) was added to cells during the stimulation period (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DC-LM peptide vaccination of WT and GKO mice

Although IFN-{gamma} is not required for protective CD8+ T cell immunity to LM (6), this cytokine plays a critical role in T cell homeostasis that is clearly evidenced by the altered immunodominance hierarchy and elevated numbers of memory CD8+ T cells in LM-vaccinated GKO mice (8). The disparity in number, and altered hierarchy of, memory CD8+ T cells in vaccinated GKO compared with WT mice precludes direct comparison of these mice to evaluate the role of CD8+ T cell derived IFN-{gamma} in protective immunity. The most valid approach to this question is comparison of the protective capacity of equal numbers of memory CD8+ T cells, of identical Ag specificity, from WT or GKO mice. However, evidence from others suggests that sorting of Ag-specific CD8+ T cells with conventional MHC class I-peptide tetramers can reduce protection by LM-specific CD8+ T cells (24). To avoid this problem, we generated single epitope-specific memory CD8+ T cells in WT and GKO mice using LM peptide-coated DCs for immunization.

Intravenous injection of relatively low numbers (~2 x 105) of LPS-matured LM peptide-coated DC into WT mice results in generation of Ag-specific memory CD8+ T cells of similar number and protective capacity as observed after sublethal infection with LM (15). Moreover, these DC-stimulated Ag-specific memory CD8+ T cell populations persist long-term and can be increased using a booster DC-peptide immunization (data not shown). To determine whether the absence of IFN-{gamma} altered CD8+ T cell responses to DC-peptide vaccination, we immunized WT and GKO mice i.v. with 2.5 x 105 WT DC coated with the defined CD8+ T cell epitopes LLO91–99, NP118–126, or p60217–226 followed 35 days later by a second immunization (0.8–1.0 x 106 peptide-coated WT DCs) to increase memory cell numbers. LLO91–99 and p60217–226 are well-characterized CD8+ T cell epitopes from LM (18) and rLM have been generated that express the LCMV-derived NP118–126 epitope as a secreted fusion protein (LM-NPs; Ref. 12). Forty-five days after the booster immunization, the number of Ag-specific memory CD8+ T cells in the spleens of vaccinated mice was evaluated using peptide-stimulated ICS for TNF-{alpha} (Fig. 1, A and B). DC-peptide vaccination of WT or GKO mice resulted in similar numbers of NP118–126, LLO91–99, or p60217–226- specific memory CD8+ T cells. As previously shown for primary DC-peptide immunization of WT mice (15), the number of LLO91–99-specific memory CD8+ T cells was highest of the three epitope specificities examined; therefore, some of the current studies will focus on comparison of memory cells of this specificity.


Figure 1
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  		FIGURE 1. DC-peptide vaccination generates LM-specific memory CD8+ T cells in GKO mice. CD8+ T cell memory was generated in WT and GKO mice through i.v. immunization with DCs (2.5 x 105) coated with LM peptides (LLO91–99, NP118–127, or p60217–225), and, 37 days later, followed by a booster injection (1 x 106 DCs coated with same peptide). Forty-five days after immunization, Ag-specific memory CD8+ T cell levels were determined using ICS for TNF-{alpha}. Representative contour plots (A) are gated on CD8+ T cells and show the percent Ag-specific Thy1.2+TNF+ after incubation with or without (number in parentheses) peptide (LLO91–99 in plots shown) followed by ICS. The number of LLO91–99, NP118–127, or p60217–225-specific memory CD8+ T cells/spleen (B) in immunized WT or GKO mice. Functional avidity (C) was evaluated by incubation (5.5 h) of splenocytes obtained from immunized WT or GKO mice with serial dilutions of cognate Ag followed by ICS for TNF-{alpha}. The Ag-specific CD8+ TCR repertoire (D) was evaluated using MHC tetramer staining and Vbeta-specific mAbs. Data shown are mean ± SD of three to six mice per group and are representative of two or more experiments.

 
Functional avidity, TCR repertoire use, and phenotypic analysis of LM-specific CD8+ T cells from immunized WT and GKO mice

After Ag encounter, peptide sensitivity is modulated in responding T cells resulting in increased sensitivity to low-dose Ag when compared with naive cells (26). Likewise, the diversity of the TCR repertoire is diminished (termed focusing) after secondary infection due to preferential outgrowth of highly responsive T cell clones (25). It is important to know whether Ag-specific CD8+ T cells generated in the absence of IFN-{gamma} exhibit similar functional avidity and/or repertoire use as those generated in WT hosts as these parameters could affect the protective capacity of the CD8+ T cell populations. To evaluate functional avidity of Ag-specific memory CD8+ T cells, splenocytes from immunized WT and GKO mice were incubated with 10-fold serial dilutions of LM peptides and the fraction of cells producing peptide-stimulated intracellular TNF-{alpha} was determined (Fig. 1C) (4, 26, 27). No differences in T cell functional avidity were observed when WT and GKO memory CD8+ T cells were compared for all three (LLO91–99, NP118–126, or p60217–225) epitopes examined. Depending on the Ag specificity, from 10–1 to 10–2 nM peptide was required to stimulate 50% of the maximum number of Ag-specific memory CD8+ T cells from immunized WT or GKO mice to produce TNF-{alpha}.

To evaluate TCR repertoire use by Ag-specific CD8+ T cells from WT and GKO mice, MHC class I tetramer+ CD8+ splenocytes were costained with a panel of Vbeta-specific mAbs known to represent the majority of T cells responding to LM infection in WT BALB/c mice (28). No difference in Vbeta segment usage was observed when WT and GKO memory CD8+ T cells were compared within each Ag specificity where the majority of the LLO91–99, NP118–126, or p60217–225 MHC class I tetramer+CD8+ cells expressed the Vbeta8.1/8.2 and Vbeta10 TCR chains (Fig. 1D). Thus, the absence of IFN-{gamma} does not influence functional avidity or TCR repertoire use of secondary memory CD8+ T cells specific for LM Ags.

The hallmarks of Ag-specific CD8+ T cell memory are increased numbers, long-term persistence, vigorous expansion upon Ag re-exposure, and the ability to mediate protective immunity against pathogen rechallenge (29). Phenotypically, memory CD8+ T cells remain a heterogeneous population that cannot be identified by a single cell surface marker. Recently, several markers indicative of Ag experience (CD44), homing potential (CD62L), costimulatory capacity (CD27), and cytokine responsiveness or expression (CD127 or IL-2/TNF/IFN-{gamma}) have been used to distinguish and further characterize memory cell subsets (16, 19, 30). Expression of these surface markers is modulated after Ag stimulation as CD8+ T cells progress from naive to memory cells, and a subset of these markers (CD62L for example) is differentially expressed on effector and central memory subsets (30). Therefore, combinations of these markers have been used to identify memory CD8+ T cells with the highest proliferative potential and protective capacity (16, 19, 30). Flow cytometric evaluation of memory markers on Ag-specific CD8+ T cells from WT and GKO mice (Fig. 2) 43 days after booster DC immunization revealed similar proportions of Ag-specific CD8+ T cells that produce both TNF and IL-2 after stimulation with cognate peptide, and similar proportions of Ag-specific cells with expression of CD44, CD127, CD62L, and CD27 (TCM phenotype). These finding indicate that the absence of IFN-{gamma} during DC immunization does not alter the phenotype of the resulting memory CD8+ T cells.


Figure 2
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  		FIGURE 2. The absence of IFN-{gamma} during DC immunization does not alter memory CD8+ T cell phenotype. The frequency of Ag-specific memory CD8+ T cells was determined 43 days after immunization in WT and GKO mice by ICS for TNF-{alpha} and memory cell phenotype was examined by costain for the indicated surface markers. Contour plots (A) are gated on CD8+ cells and numbers indicate the percentage of TNF-{alpha}+Thy1.2+ cells/spleen after incubation of splenocytes with or without (number in parentheses) LLO91–99 peptide. Histograms (B) representative of three independent experiments are gated on CD8+TNF-{alpha}+Thy1.2+ cells and show the percent positive (open histogram) for the indicated marker based on isotype control staining (shaded histogram) of representative mice.

 
Protective immunity and expansion of LM-specific CD8+ T cells from WT and GKO mice

DC-peptide immunization of WT mice results in CD8+ T cell memory capable of providing sterilizing immunity to previously lethal doses of LM (15, 31). We next wished to evaluate the contribution of CD8+ T cell-derived IFN-{gamma} to protection against LM infection. However, challenge of the DC-immunized WT and GKO mouse strains was problematic due to potential variation in the number of Ag-specific CD8+ T cells (Fig. 1B) and differences in innate resistance of the two strains (32). To overcome these limitations and evaluate the contribution of CD8+ T cell-derived IFN-{gamma} in protective immunity, equal numbers (3 x 105, determined by MHC class I tetramer staining) of LM Ag-specific (LLO91–99, NP118–126, or p60217–226) memory CD8+ T cells from the spleens of vaccinated WT or GKO mice (Thy 1.2) were transferred i.v. into naive WT-recipient mice (Thy1.1). One day later, T cell-recipient mice and naive controls were challenged with 2 x 104 (2 LD50 for naive mice) LM-NPs and protective immunity was evaluated on days 3 and 5 postinfection (p.i.) by determining CFU of LM in the spleens and livers (Table I). Regardless of epitope specificity, Ag-specific CD8+ T cells from WT mice provided robust antilisterial immunity, as shown by a 2 log10 reduction in the bacterial counts in the spleens and 3 log10 reduction of CFU in the livers of recipient mice at 3 days after infection (Table I). In contrast, IFN-{gamma}-deficient memory CD8+ T cells exhibited decreased protective capacity resulting in only 1 log10 reduction in bacterial numbers in the spleens and 2 log10 reduction in CFU in the livers of GKO CD8+ T cell-recipient mice. Thus, mice that received GKO memory CD8+ T cells had ~1 log10 more bacteria in their organs 3 days after LM challenge than mice that received an equal number of WT memory CD8+ T cells. Therefore, Ag-specific CD8+ T cell-derived IFN-{gamma} contributes to protective immunity mediated by memory CD8+ T cells against LM infection. However, it seems that the role of IFN-{gamma} is more critical very early after infection or the GKO cells can somehow compensate for their decreased protective function because the difference in number of bacterial CFU in the organs between groups is less pronounced by day 5 p.i.


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  		Table I. Log10 CFU protection from challenge with LM in WT or GKO T cell-recipient micea

 
The remaining experiments will focus on a more detailed comparison of memory CD8+ T cells from WT and GKO mice specific for the LLO91–99 epitope because this response is the largest (vs NP118–126 or p60217–225) after DC-peptide immunization, and protective immunity and expansion was similar for CD8+ T cells specific for all three LM epitopes (Table I).

Ag-specifc CD8+ T cells from GKO mice provide decreased protective immunity to high-dose challenge

Thus far, our data indicate that Ag-specific CD8+ T cells from vaccinated GKO mice provide decreased early protection on a per-cell basis against a specific LM challenge dose. To extend this finding, we evaluated the protective immunity under conditions where we varied the number of memory cells transferred or the challenge dose of bacteria. First, we adoptively transferred fewer (1 x 105) Ag-specific CD8+ T cells from WT and GKO donors and examined the rate of bacterial clearance in spleens and livers of naive and T cell-recipient mice challenged with LM (Fig. 3). Similar to our previous findings, transfer of CD8+ T cells from WT mice resulted in 1–2 log10 reduction in bacterial numbers early after infection (day 3) compared with infected naive mice whereas equal numbers of GKO-derived Ag-specific CD8+ T cells provided reduced protection. In contrast to the previous experiment in which more (3-fold) Ag-specific CD8+ T cells were transferred, increased bacterial numbers were observed in the spleens and livers of GKO CD8+ T cell-recipient mice on days 5 and 7 p.i. compared with WT T cell-recipient mice (Fig. 3). Over the course of 9 days, the infected naive mice were unable to clear LM while both WT and GKO CD8+ T cell-recipient mice were able to clear the infection. The reduced bacterial clearance in GKO CD8+ T cell-recipient mice confirms that loss of CD8+ T cell-derived IFN-{gamma} contributes to CD8+ T cell-mediated protective immunity.


Figure 3
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  		FIGURE 3. Adoptive transfer of IFN-{gamma}-deficient memory CD8+ T cells results in delayed bacterial clearance. LLO91–99-specific memory CD8+ T cells (1 x 105) were obtained from immunized WT or GKO donor mice and transferred i.v. into naive recipient mice. One day later, T cell-recipient and naive control mice were challenged with 2 x 104 LM-NPs. At various days after challenge, portions of the spleen (A) and liver (B) were plated to determine CFU of LM/spleen or /gram liver. The total number of Ag-specific CD8+ T cells/spleen (C) was determined by ICS for TNF-{alpha} on day 5 p.i. Data are represented as mean ± SD of three to four mice/group; 1/3 indicates that one mouse had CFU below the LOD for this assay; *, p ≤ 0.05 compared with GKO cell-recipient mice.

 
The infectious course of L. monocytogenes in an immune host is dependent on a balance between the number and function of Ag-specific CD8+ T cells and the number of bacteria present (4). Successful sterilizing immunity occurs only when the potency of the Ag-specific CD8 T cell response is greater than the bacterial survival and proliferation. Therefore, we evaluated the number of LLO91–99-specific CD8+ T cells in the spleens of recipient mice 5 days after bacterial challenge, the peak of the memory response to this Ag (Fig. 3C) and identified a 3-fold increase in the number of LLO91–99-specific CD8+ T cells in the spleens of mice that received equal numbers Ag-specific cells from WT and GKO donors. A similar increase in the number of Ag-specific CD8+ T cells in the spleens of recipient mice was observed when donor (Thy1.2) CD8+ T cells specific for NP118–126 or p60217–226 LM epitopes were adoptively transferred into naive Thy 1.1 mice (Table I and data not shown). Thus, the transferred memory CD8+ T cells from GKO mice are capable of robust Ag-induced proliferation. Additionally, it is likely that in some experimental scenarios the increased proliferation of IFN-{gamma}-deficient CD8+ T cells could compensate for reduced per-cell protective capacity, as has been shown in the absence of other effector molecules (4).

Next, we examined the protective immunity provided by adoptively transferred T cells to a 2.5-fold higher dose of bacteria (5 x 104 LM-NPs) and evaluated the kinetics of bacterial clearance on days 1–4 p.i. (Fig. 4A). Naive control mice had very high numbers (107–108) of bacteria in both organs throughout the experiment. The adoptive transfer of WT memory CD8+ T cells resulted in 1–2 log10 fewer bacteria in both organs on days 1 and 2 p.i., whereas GKO memory CD8+ T cell-recipient mice had intermediate levels of bacteria in both organs assayed at early time points. The deficiency in protective immunity exhibited by GKO CD8+ T cells is most evident on days 3 and 4 p.i. when the number of bacteria in the organs of WT CD8+ T cell-recipient mice declines but bacterial numbers in GKO CD8+ T cell-recipient mice increase to levels similar to those observed in the naive control mice. Of additional interest is the observation that by day 4 p.i., some mortality was observed in both the naive (three dead) and GKO CD8+ T cell-recipient (two dead) mice but all WT CD8+ T cell-recipient mice survived this high-dose LM challenge dose.


Figure 4
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  		FIGURE 4. IFN-{gamma} participates in protective immunity early after high-dose LM challenge. LLO91–99-specific memory CD8+ T cells (3 x 105 Thy 1.2+) were obtained from immunized WT or GKO donor mice and transferred i.v. into naive recipient mice (Thy 1.1+). One day later, T cell-recipient mice were challenged with ~5 x 104 LM-NPs. On various days after challenge, portions of the spleen and liver were plated to determine CFU of LM (A). In a separate experiment, bacterial clearance and survival was examined in T cell-recipient mice after i.v. transfer of (3 x 105 Thy 1.2+) LLO91–99-specific memory CD8+ T cells and challenge with 5 x 104 LM-NPs. On day 3 after challenge, portions of the spleen and liver were plated to determine CFU of LM (B). Survival was evaluated until day 12 (C), when remaining mice were sacrificed and portions of the spleens and livers were plated for CFU of LM (D). Data are represented as mean ± SD of 3–4 (A, B, and D) and 10–12 (C) mice/group. Data are expressed as total CFU/spleen or CFU/gram liver. BLD, Below the level of detection for this assay; *, p ≤ 0.05 from GKO T cell-recipient mice; ***, p ≤ 0.0001 (log-rank test for trend) from WT T cell-recipient mice.

 
Finally, we repeated a high-dose challenge (5 x 104 LM-NPs) experiment and evaluated bacterial numbers on days 3 and 12 p.i. in naive and T cell-recipient mice (Fig. 4, B and D). As previously observed on day 3 p.i., WT CD8+ T cell-recipient mice had significantly fewer bacteria when compared with infected naive control mice whereas an equal number (3 x 105) of Ag-specific CD8+ T cells from GKO mice did not provide any protection (Fig. 4B). Challenge with this dose of LM resulted in 0% survival of naive mice day 7 p.i. compared with 78 and 18% survival for WT and GKO CD8+ T cell-recipient mice, respectively (Fig. 4C). When CFUs were evaluated on day 12 p.i. in the organs of remaining T cell-recipient mice (Fig. 4D), WT CD8+ T cell-recipient mice had cleared bacteria from their spleens and livers to a level below the limit of detection (LOD). In contrast, adoptive transfer of an equal number of IFN-{gamma}-deficient CD8+ T cells were unable to effectively clear the infection and had nearly 5 log10 bacteria remaining in their organs on day 12 p.i.

Taken together, the data clearly demonstrate that the Ag-specific CD8+ T cells obtained from vaccinated GKO mice exhibit decreased CD8+ T cell-dependent protective immunity when compared with WT CD8+ T cells on a per cell basis.

In vivo cytotoxicity

One of the main mechanisms by which Ag-specific memory CD8+ T cells mediate protective immunity against LM is through lysis of infected target cells (4, 33). A deficiency in cytotoxicity could result in higher bacterial numbers within the infected host as we have observed when protective immunity mediated by memory CD8+ T cells from GKO mice is compared with mice that received WT CD8+ T cells. To address potential differences in cytolysis between WT and GKO-derived CD8+ T cells, we used an in vivo cytotoxicity assay (5). Equal numbers (3 x 105) of LLO91–99-specific memory CD8+ T cells from DC-vaccinated WT and GKO mice were transferred into naive WT-recipient mice (Fig. 5). Next, a 1:1 ratio of LLO91–99-coated (CFSEhigh) and uncoated (CFSElow) targets (syngeneic spleen cells) were injected i.v. into naive controls or the T cell-recipient mice and the relative elimination of the CFSEhigh peak (Ag-specific killing) was monitored in the spleens using flow cytometry. At 8, 22, and 44 h after target cell transfer, 24, 48, and 63% of the peptide-pulsed targets had been eliminated in mice that received WT-derived CD8+ T cells (Fig. 5, A and B). In contrast, Ag-specific killing by an equal number of GKO-derived CD8+ T cells was reduced at all time points, with a peak level of 38% cytotoxicity at 44 h post-target cell transfer. Further change in the percent cytotoxicity was not observed at later time points (data not shown) and may reflect loss of peptide/MHC stability on the target cells. In this experiment, memory CD8+ T cells from WT or GKO mice were adoptively transferred into WT T cell-recipient mice so the deficiency is not due to a complete absence of IFN-{gamma} in the system, but results from an inherent difference in function of memory CD8+ T cells from GKO mice. Regardless, our studies are the first to identify a role for IFN-{gamma} in protective immunity to LM and suggest that the absence of this cytokine results in inefficient Ag-specific cytolysis.


Figure 5
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  		FIGURE 5. IFN-{gamma}-deficient memory CD8+ T cells exhibit decreased Ag-specific target cell lysis. In vivo cytolysis of peptide-coated (CFSEhigh) or uncoated (CFSElow) naive syngeneic lymphocytes was evaluated in WT or GKO memory CD8+ T cell-recipient (4 x 105 LLO91–99-specific) mice. Histograms are gated on CFSE+ cells (A) and numbers represent the percentage of targets killed 44 h after target cell transfer. Percent killing over time (B). Granzyme B (C) or CD107a expression (D and E) on CD8+ T cells from immunized WT and GKO mice after LLO91–99 peptide stimulation. Histograms (C) are gated on CD8+TNF+ cells and numbers indicate the MFI of stained cells (open histogram) compared with the isotype control (in parentheses; shaded histogram). Histograms (D) are gated on CD8+TNF+ cells and numbers indicate the percent positive (open histogram) based on isotype control staining (shaded histogram). Mean percentage ± SD CD107a (E) staining from four mice per group and is representative of two independent experiments.

 
The primary mechanism of CD8+ T cell-mediated target cell killing is through directed release of stored lytic granules containing perforin and granzymes into the target cell (34). To assess potential differences in cytotoxic potential, we evaluated level of granzyme B expression (Fig. 5C) in memory CD8+ T cells from WT and GKO mice by flow cytometry. As is characteristic of memory cells (23), a low level of intracellular granzyme B was detected in Ag-specific CD8+ T cells from vaccinated WT (mean fluorescence intensity (MFI) = 17.1 ± 1.1/isotype = 8.9 ± 0.5) and GKO mice (MFI = 23.2 ± 0.9/isotype = 9.0 ± 0.4) (Fig. 5C). Thus, the decreased target cell killing by memory CD8+ T cells from GKO mice cannot be attributed to differences in granzyme B levels in stable memory populations.

An additional characteristic that can distinguish memory cell populations (23) is the rate of degranulation of Ag-specific CD8+ T cells. We considered the possibility that delayed Ag-induced degranulation by GKO-derived memory CD8+ T cells could contribute to reduced target cell killing in vivo. Recently, the presence of lysosomal-associated glycoproteins, molecules normally confined to the membrane of stored lytic granule, on the plasma membrane of T cells has been used as an indicator of degranulation (35). We evaluated the kinetics of degranulation as indicated by CD107a (lysosomal-associated glycoprotein-1) surface staining on the surface of memory CD8+ T cells early after Ag stimulation (Fig. 5, D and E). Because 4 h of peptide stimulation was required to detect the maximal number of Ag-specific cells using ICS for TNF-{alpha}, we evaluated CD107a staining in memory CD8+ T cells from vaccinated WT and GKO mice at 4 and 6 h after stimulation with LLO91–99 peptide. A small decrease in the percentage of degranulated GKO cells was observed 4 h after Ag stimulation (GKO = 72.6 ± 3.4, WT = 80.5 ± 1.2), while no difference was observed by 6 h (GKO = 80.9 ± 5.1, WT = 85.2 ± 3.8) (Fig. 5, D and E). Although it is possible that a decrease in the rate of degranulation could influence the rate of cytolysis early after infection, a similar maximal degranulation is achieved within 6 h after Ag stimulation, so it is unlikely that inefficient degranulation could wholly account for the diminished protective immunity provided by memory CD8+ T cells from GKO mice.

Ag-specific CD8+ T cell-derived IFN-{gamma} improves in vivo target cell killing

In a variety of in vitro experimental systems, IFN-{gamma} can sensitize target cells to CD8+ T cell-mediated lysis via the granule exocytosis pathway or death receptor engagement (36, 37). To determine whether IFN-{gamma} production by memory CD8+ T cells could influence cytolysis by directly acting on target cells in vivo, we used a C57BL/6 OT-1 adoptive transfer system in which the transferred WT OVA-specific memory CD8+ T cells were evaluated for their ability to kill WT or IFN-{gamma}R-deficient (IFN-{gamma}R KO) targets. In this experiment, the sensitivity of the target cells to the direct effect of IFN-{gamma} differs while the source of memory CD8+ T cells remains the same. Each target cell population (WT or IFN-{gamma}R KO) was coated with (Ag specific) or without (nonspecific) 1 µM OVA peptide (Fig. 6A), and the resulting target cell populations (WT without OVA, WT with OVA, IFN-{gamma}R KO without OVA and IFN-{gamma}R KO with OVA) were labeled with 10-fold dilutions of CFSE (populations labeled 1–4 in Fig. 6B). Uniform identification of all target cell populations was achieved by combining equal numbers of each target cell population labeling with cell linker red dye (Fig. 6B). Targets (8 x 106 total/mouse) were injected i.v. into naive C57BL/6 mice or OT-I memory CD8+ T cell-recipient mice (1.5 x 106 cells/mouse, i.v.) (Fig. 6B). Injection of all target cell populations into each experimental (and naive) mouse controlled for any possible mouse to mouse variations. The disappearance of the peptide-coated population relative to its uncoated control was monitored in the spleens of target cell-recipient mice and the percent killing was calculated.


Figure 6
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  		FIGURE 6. T cell-derived IFN-{gamma} increases in vivo Ag-specific cytolysis. Lysis of peptide-coated WT or IFN-{gamma}R-deficient (IFN-{gamma}R KO) target cells was evaluated in naive B6 or memory OT-I (1.5 x 106) recipient mice. Histograms (A) indicate uniform peptide coating of WT or IFN-{gamma}R KO targets with Kb-OVA mAb (open histogram) or isotype control (shaded histogram). Target cells (B) were stained with PKH26 for uniform identification and serial dilutions of CFSE to identify target subpopulations as indicated and injected into naive or memory OT-I T cell-recipient mice. Percent killing (C) was determined 4 h post-target cell transfer by relative CFSE fluorescence of the PKH26+ target cell population. *, p ≤ 0.05 from WT targets.

 
At 4 h post-target cell transfer, significantly (p < 0.05) more (~48%) of the WT target cells had been lysed compared with the (~25%) IFN-{gamma}R KO target cells (WT 48.5 ± 8.06; GKO 23 ± 6.27) (Fig. 6, B and C). The difference was maintained 8 h post-target cell transfer and was not altered by incubation of target cells with 10-fold more peptide (data not shown). These findings suggest that IFN-{gamma} derived from Ag-specific memory CD8+ T cells can increase the susceptibility of target cells to lysis in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Activated CD8+ T cells possess an array of antimicrobial effector mechanisms capable of controlling pathogenic microbes, including production of proinflammatory cytokines (IFN-{gamma} and TNF) and killing infected cells via the perforin-dependent granule exocytosis pathway. Interestingly, studies with GKO mice show that pathogen biology appears to dictate which effector pathways dominate in resistance to specific infection (reviewed in Ref. 2). For example, clearance of LCMV, a nonlytic viral infection, is absolutely dependent on perforin-mediated killing by CD8+ T cells. In contrast, IFN-{gamma} is essential for clearance of vaccinia, a rapidly lytic viral infection. Furthermore, granzyme A, but not granzyme B, is essential for clearance of ectromelia virus. Importantly, these above mentioned effector pathways are not unique to CD8+ T cells, but are also deployed by other immune cells types (i.e., cytokine production by Th1 and NK cells, perforin-mediated killing by NK cells). This complicates interpretation of vaccine studies with GKO mice. In addition, the absence of at least some of these effector pathways can alter the homeostasis of Ag-specific CD8+ T cells stimulated by infection or vaccination (8). Thus, determining the contribution of a specific effector function to CD8+ T cell immunity is technically challenging. Additionally, these data suggest that the relevant CD8+ T cell effector pathways for individual pathogens cannot be assumed but must be identified for each pathogen. This information is critical to vaccine design so that T cells with the correct array of effector mechanisms can be targeted.

LM is a facultative intracellular bacterium capable of infecting a wide array of eukaryotic cells and tissues and is extensively used as a model organism to evaluate innate and T cell immunity to infection. Previous studies from our laboratory revealed that perforin-dependent cytolysis is a major effector pathway for memory CD8+ T cell-mediated protective immunity against LM infection (4). However, while perforin-deficient CD8+ T cells exhibited only one-fifth the per-cell protective capacity of WT CD8 T cells, increasing the number of perforin-deficient CD8+ T cells restored protective immunity against LM infection. Thus, other effector pathways contribute to CD8+ T cell immunity against LM infection. In the current study, we used adoptive transfer of single Ag-specific CD8+ T cells from WT and GKO mice to show that IFN-{gamma} derived from memory CD8+ T cells also contributes to protective immunity against LM infection.

IFN-{gamma} plays a critical role in innate resistance to primary LM infection such that GKO mice succumb to as few as 10 virulent bacteria (6). Because activated CD8+ T cells produce IFN-{gamma} and secondary resistance to LM is dependent on CD8+ T cells, it was assumed that this cytokine was also involved in adaptive protective immunity (38); however, the generation of protective immunity in vaccinated GKO mice proved otherwise (6). Careful examination of the LM Ag-specific CD8+ T cell responses in vaccinated GKO mice revealed alterations in immunodominance hierarchies that persisted into the memory phase of the response and a substantial increase in the number of memory CD8+ T cells due to lack of contraction (8, 39). Because of the changes in the number and hierarchy of memory CD8+ T cells the previous studies were unable to determine whether CD8+ T cell-derived IFN-{gamma} contributed to CD8+ T cell-mediated protective immunity against LM infection.

To overcome these limitations, we used an adoptive transfer system to compare the per cell protective capacity of memory CD8+ T cells from WT or GKO mice of a single LM Ag specificity. The current studies determined that LM-epitope specific CD8+ T cells from immunized GKO mice provided significantly decreased protective immunity early (days 1–3) after infection when compared with equal numbers of Ag-matched WT CD8+ T cells. This finding was reproducible in a variety of experimental settings including those with fewer CD8+ T cells transferred, increased challenge doses of LM, and at later time points when survival and bacterial clearance was examined.

How does CD8+ T cell-derived IFN-{gamma} contribute to antilisterial immunity? Ag-specific CD8+ T cells generated by DC immunization in WT and GKO mice are phenotypically similar for a number of relevant memory cell markers (30) and produce similar amounts of important cytokines such as TNF and IL-2 after Ag stimulation. However, Ag-specific CD8+ T cells from GKO mice exhibit a decreased ability to kill Ag-coated target cells in vivo. Our data suggest that this deficiency is due to the loss of IFN-{gamma} as an effector molecule rather than a deficiency in the mechanisms of cytotoxicity such as granzyme B expression or a dramatic alteration in Ag-stimulated degranulation.

IFN-{gamma} is a pluripotent cytokine capable of regulating >1000 genes in a variety of cell types including monocytes/macrophages, B cells, NK cells, and T cells as well as nonimmune cells. In our studies, the inefficient killing of IFN-{gamma}-insensitive (IFN-{gamma}R–/–) peptide-coated targets identifies a need for this cytokine at the time of T cell engagement of the target cell. Several in vitro studies suggest that IFN-{gamma} can increase the sensitivity of target cells to CD8+ T cell lysis (36, 37). One potential mechanism by which IFN-{gamma} could control the sensitivity of target cell lysis is through up-regulation of MHC class I gene expression with concurrent increase in number of stimulatory peptide-MHC class I complexes on the surface of infected cells (31). In the case of LM infection, this would aid in the detection of LM, an intracellular pathogen, by Ag-specific CD8+ T cells.

Because the defect in cytolysis by the memory CD8+ T cells from GKO mice occurred relatively quickly (4 h) after Ag stimulation (target injection), it is unlikely that increased class I Ag presentation is the only mechanism involved. To this end, IFN-{gamma} has also been shown to exert apoptotic effects on immune cells. For example, IFN-{gamma} can increase expression of Fas, FasL (40, 41), and TNF (42) receptors on target cells within hours after Ag stimulation and also increases the production of death-associated proteins, such as DAPI-5 (43). Moreover, Ag-specific CD8+ T become rapidly insensitive to IFN-{gamma} through receptor down-regulation (13, 44). Thus, during LM infection, localized IFN-{gamma} production in the spleen or liver microenvironment could sensitize infected cells to Ag-specific destruction by activated CD8+ T cells while the effector cells would remain insensitive to the proapoptotic effects of IFN-{gamma} by virtue of their receptor down-regulation (13, 44). Even a small decrease in the efficiency of Ag-specific target cell killing in the early hours after infection would exponentially increase the number of infected cells (due in part to cell-cell bacterial spread) at later days after bacterial challenge (45).

Although perforin-dependent cytolysis is the major effector mechanism of Ag-specific CD8+ T cells in resistance to LM infection (46), we conclude that the production of IFN-{gamma} by effector T cells augments the efficiency of target cell killing. It remains to be determined whether other effector pathways such as TNF production also contribute to memory CD8+ T cell protection against LM infection.


   Acknowledgment
 
We thank R. Podyminogin for technical support.


   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 Grants AI42767, AI46653, AI50073, and AI59752 (to J.T.H.), Grant IRG-77-004-28 from the American Cancer Society, administered through The Holden Comprehensive Cancer Center at University of Iowa (to V.P.B.) and an American Cancer Society Postdoctoral Fellowship (to K.A.N.M.). Back

2 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, University of Iowa, 3-512 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu Back

3 Abbreviations used in this paper: KO, knockout; GKO, IFN-{gamma}-deficient KO; LM, Listeria monocytogenes; WT, wild type; DC, dendritic cell; LCMV, lymphocytic choriomeningitis virus; LLO, listeriolysin O; NP, nuclear protein; ICS, intracellular cytokine staining; p.i., postinfection; MFI, mean fluorescence intensity; LOD, limit of detection. Back

Received for publication March 20, 2006. Accepted for publication June 7, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Harty, J. T., V. P. Badovinac. 2002. Influence of effector molecules on the CD8+ T cell response to infection. Curr. Opin. Immunol. 14: 360-365. [Medline]
  2. Harty, J. T., A. R. Tvinnereim, D. W. White. 2000. CD8+ T cell effector mechanisms in resistance to infection. Annu. Rev. Immunol. 18: 275-308. [Medline]
  3. Harty, J. T., D. White. 1999. A knockout approach to understanding CD8+ cell effector mechanisms in adaptive immunity to Listeria monocytogenes. Immunobiology 201: 196-204. [Medline]
  4. Messingham, K. A., V. P. Badovinac, J. T. Harty. 2003. Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers. J. Immunol. 171: 4254-4262. [Abstract/Free Full Text]
  5. Barber, D. L., E. J. Wherry, R. Ahmed. 2003. Cutting edge: rapid in vivo killing by memory CD8 T cells. J. Immunol. 171: 27-31. [Abstract/Free Full Text]
  6. Harty, J. T., M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN {gamma}. Immunity 3: 109-117. [Medline]
  7. Brundage, R. A., G. A. Smith, A. Camilli, J. A. Theriot, D. A. Portnoy. 1993. Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc. Natl. Acad. Sci. USA 90: 11890-11894. [Abstract/Free Full Text]
  8. Badovinac, V. P., A. R. Tvinnereim, J. T. Harty. 2000. Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-{gamma}. Science 290: 1354-1358. [Abstract/Free Full Text]
  9. Skoberne, M., G. Geginat. 2002. Efficient in vivo presentation of Listeria monocytogenes-derived CD4 and CD8 T cell epitopes in the absence of IFN-{gamma}. J. Immunol. 168: 1854-1860. [Abstract/Free Full Text]
  10. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76: 17-27. [Medline]
  11. Shen, H., C. M. Tato, X. Fan. 1998. Listeria monocytogenes as a probe to study cell-mediated immunity. Curr. Opin. Immunol. 10: 450-458. [Medline]
  12. Shen, H., J. F. Miller, X. Fan, D. Kolwyck, R. Ahmed, J. T. Harty. 1998. Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity. Cell 92: 535-545. [Medline]
  13. Haring, J. S., G. A. Corbin, J. T. Harty. 2005. Dynamic regulation of IFN-{gamma} signaling in antigen-specific CD8+ T cells responding to infection. J. Immunol. 174: 6791-6802. [Abstract/Free Full Text]
  14. Pope, C., S. K. Kim, A. Marzo, D. Masopust, K. Williams, J. Jiang, H. Shen, L. Lefrancois. 2001. Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J. Immunol. 166: 3402-3409. [Abstract/Free Full Text]
  15. Hamilton, S. E., J. T. Harty. 2002. Quantitation of CD8+ T cell expansion, memory, and protective immunity after immunization with peptide-coated dendritic cells. J. Immunol. 169: 4936-4944. [Abstract/Free Full Text]
  16. Badovinac, V. P., K. A. Messingham, A. Jabbari, J. S. Haring, J. T. Harty. 2005. Accelerated CD8+ T-cell memory and prime-boost response after dendritic-cell vaccination. Nat. Med. 11: 748-756. [Medline]
  17. Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, M. M. Davis. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274: 94-96. [Abstract/Free Full Text]
  18. Busch, D. H., I. M. Pilip, S. Vijh, E. G. Pamer. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8: 353-362. [Medline]
  19. Jabbari, A., J. T. Harty. 2006. Secondary memory cells are more protective but slower to acquire a central memory phenotype. J. Exp. Med. 203: 919-932. [Abstract/Free Full Text]
  20. Badovinac, V. P., J. T. Harty. 2000. Intracellular staining for TNF and IFN-{gamma} detects different frequencies of antigen-specific CD8+ T cells. J. Immunol. Methods 238: 107-117. [Medline]
  21. Jabbari, A., J. T. Harty. 2006. Simultaneous assessment of antigen-stimulated cytokine production and memory subset composition of memory CD8 T cells. J. Immunol. Methods 313: 161-168. [Medline]
  22. Jabbari, A., J. T. Harty. 2005. Cutting edge: differential self-peptide/MHC requirement for maintaining CD8 T cell function versus homeostatic proliferation. J. Immunol. 175: 4829-4833. [Abstract/Free Full Text]
  23. Wolint, P., M. R. Betts, R. A. Koup, A. Oxenius. 2004. Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells. J. Exp. Med. 199: 925-936. [Abstract/Free Full Text]
  24. Knabel, M., T. J. Franz, M. Schiemann, A. Wulf, B. Villmow, B. Schmidt, H. Bernhard, H. Wagner, D. H. Busch. 2002. Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nat. Med. 8: 631-637. [Medline]
  25. Blattman, J. N., D. J. Sourdive, K. Murali-Krishna, R. Ahmed, J. D. Altman. 2000. Evolution of the T cell repertoire during primary, memory, and recall responses to viral infection. J. Immunol. 165: 6081-6090. [Abstract/Free Full Text]
  26. Slifka, M. K., J. N. Blattman, D. J. Sourdive, F. Liu, D. L. Huffman, T. Wolfe, A. Hughes, M. B. Oldstone, R. Ahmed, M. G. Von Herrath. 2003. Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy. J. Immunol. 170: 1231-1239. [Abstract/Free Full Text]
  27. Slifka, M. K., J. L. Whitton. 2001. Functional avidity maturation of CD8+ T cells without selection of higher affinity TCR. Nat. Immunol. 2: 711-717. [Medline]
  28. Busch, D. H., I. Pilip, E. G. Pamer. 1998. Evolution of a complex T cell receptor repertoire during primary and recall bacterial infection. J. Exp. Med. 188: 61-70. [Abstract/Free Full Text]
  29. Badovinac, V. P., J. T. Harty. 2006. Programming, demarcating, and manipulating CD8+ T-cell memory. Immunol. Rev. 211: 67-80. [Medline]
  30. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225-234. [Medline]
  31. Hamilton, S. E., B. B. Porter, K. A. Messingham, V. P. Badovinac, J. T. Harty. 2004. MHC class Ia-restricted memory T cells inhibit expansion of a nonprotective MHC class Ib (H2-M3)-restricted memory response. Nat. Immunol. 5: 159-168. [Medline]
  32. Unanue, E. R.. 1997. Studies in listeriosis show the strong symbiosis between the innate cellular system and the T-cell response. Immunol. Rev. 158: 11-25. [Medline]
  33. White, D. W., J. T. Harty. 1998. Perforin-deficient CD8+ T cells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-{gamma} but requires TNF-{alpha}. J. Immunol. 160: 898-905. [Abstract/Free Full Text]
  34. Trambas, C. M., G. M. Griffiths. 2003. Delivering the kiss of death. Nat. Immunol. 4: 399-403. [Medline]
  35. Betts, M. R., J. M. Brenchley, D. A. Price, S. C. De Rosa, D. C. Douek, M. Roederer, R. A. Koup. 2003. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods 281: 65-78. [Medline]
  36. Refaeli, Y., L. Van Parijs, S. I. Alexander, A. K. Abbas. 2002. Interferon {gamma} is required for activation-induced death of T lymphocytes. J. Exp. Med. 196: 999-1005. [Abstract/Free Full Text]
  37. Boehm, U., T. Klamp, M. Groot, J. C. Howard. 1997. Cellular responses to interferon-{gamma}. Annu. Rev. Immunol. 15: 749-795. [Medline]
  38. Buchmeier, N. A., R. D. Schreiber. 1985. Requirement of endogenous interferon-{gamma} production for resolution of Listeria monocytogenes infection. Proc. Natl. Acad. Sci. USA 82: 7404-7408. [Abstract/Free Full Text]
  39. Badovinac, V. P., J. T. Harty. 2002. CD8+ T-cell homeostasis after infection: setting the "curve". Microbes Infect. 4: 441-447. [Medline]
  40. Deiss, L. P., H. Galinka, H. Berissi, O. Cohen, A. Kimchi. 1996. Cathepsin D protease mediates programmed cell death induced by interferon-{gamma}, Fas/APO-1 and TNF-{alpha}. EMBO J. 15: 3861-3870. [Medline]
  41. Xu, X., X. Y. Fu, J. Plate, A. S. Chong. 1998. IFN-{gamma} induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression. Cancer Res. 58: 2832-2837. [Abstract/Free Full Text]
  42. Tsujimoto, M., Y. K. Yip, J. Vilcek. 1986. Interferon-{gamma} enhances expression of cellular receptors for tumor necrosis factor. J. Immunol. 136: 2441-2444. [Abstract]
  43. Kissil, J. L., L. P. Deiss, M. Bayewitch, T. Raveh, G. Khaspekov, A. Kimchi. 1995. Isolation of DAP3, a novel mediator of interferon-{gamma}-induced cell death. J. Biol. Chem. 270: 27932-27936. [Abstract/Free Full Text]
  44. Tau, G. Z., S. N. Cowan, J. Weisburg, N. S. Braunstein, P. B. Rothman. 2001. Regulation of IFN-{gamma} signaling is essential for the cytotoxic activity of CD8+ T cells. J. Immunol. 167: 5574-5582. [Abstract/Free Full Text]
  45. San Mateo, L. R., M. M. Chua, S. R. Weiss, H. Shen. 2002. Perforin-mediated CTL cytolysis counteracts direct cell-cell spread of Listeria monocytogenes. J. Immunol. 169: 5202-5208. [Abstract/Free Full Text]
  46. Kagi, D., B. Ledermann, K. Burki, H. Hengartner, R. M. Zinkernagel. 1994. CD8+ T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur. J. Immunol. 24: 3068-3072. [Medline]



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C. J. Henry, D. A. Ornelles, L. M. Mitchell, K. L. Brzoza-Lewis, and E. M. Hiltbold
IL-12 Produced by Dendritic Cells Augments CD8+ T Cell Activation through the Production of the Chemokines CCL1 and CCL17
J. Immunol., December 15, 2008; 181(12): 8576 - 8584.
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