The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Messingham, K. A. N.
Right arrow Articles by Harty, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Messingham, K. A. N.
Right arrow Articles by Harty, J. T.
The Journal of Immunology, 2003, 171: 4254-4262.
Copyright © 2003 by The American Association of Immunologists

Deficient Anti-Listerial Immunity in the Absence of Perforin Can Be Restored by Increasing Memory CD8+ T Cell Numbers 1

Kelly A. Nordyke Messingham*, Vladimir P. Badovinac* 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
 References
 
Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8+ T cell memory, but exhibit reduced levels of protection against virulent LM. In this study, Ag-specific CD8+ T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM. Perforin deficiency resulted in an ~5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8+ T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time. However, perforin-deficient CD8+ T cells exhibited reduced in vivo cytotoxic function compared to WT CD8+ T cells. Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice. Thus, increasing the number of memory CD8+ T cells can overcome diminished per-cell protective immunity in the absence of perforin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD8+ T cells are critical for protective immunity to the intracellular bacterium Listeria monocytogenes (LM) 3 (1, 2, 3). Activated CD8+ T cells elaborate a variety of antimicrobial effector mechanisms, including perforin-dependent cytolysis and production of cytokines, which function to stimulate or activate elements of the innate immune response (4). Compared with wild type (WT), H-2b MHC perforin-deficient (PKO) mice demonstrated similar resistance (LD50) to primary LM infection, but reduced CD8+ T cell-mediated immunity to secondary challenge (5). Subsequent studies showed delayed clearance of primary LM infection from the spleen, but not liver, of PKO mice (6). Recently, perforin was shown to be required for CD8+ T cell immunity to secondary challenge with virulent LM, but not an attenuated (actA-deficient) LM that is deficient in cell-cell spread (7). These results suggested that perforin-dependent cytolysis was an essential effector function in CD8+ T cell-mediated secondary resistance to LM.

In contrast, adoptive transfer of LM Ag-specific CD8+ T cell lines from H-2d MHC PKO mice provided substantial and specific immunity against LM challenge (8). Similarly, adoptive transfer of CD4+ T cell-depleted LM immune splenocytes from H-2d MHC PKO mice provided anti-listerial immunity (9). In agreement, vaccination of H-2d MHC PKO mice with actA-deficient LM generated a degree of secondary resistance to subsequent challenge with virulent LM (J. T. Harty, unpublished data). Interestingly, vaccinated H-2d PKO mice exhibited higher numbers of LM Ag-specific memory CD8+ T cells than vaccinated WT mice (10), a factor that may contribute to secondary resistance in the absence of perforin. These results suggested the existence of perforin-independent pathways for CD8+ T cell immunity to LM.

Taken together, the results from various experimental approaches suggest that perforin-dependent cytolysis participates in, but is not absolutely essential for, CD8+ T cell immunity to LM infection. However, the precise contribution of perforin-dependent cytolysis remains to be determined. To address this issue, we chose to determine the per-cell protective capacity of Ag-specific memory CD8+ T cells from LM-vaccinated H-2d MHC PKO and WT mice. LM-specific CD8+ T cells from each host were analyzed in adoptive transfer experiments for the ability to provide anti-listerial immunity, by flow cytometry for CD62L/CD27 expression, repertoire use and functional avidity, and for in vivo cytotoxic function. The results demonstrate that the absence of perforin results in a 5-fold reduction in the per-cell protective capacity of LM Ag-specific memory CD8+ T cells, despite similar expansion upon reinfection. Importantly, the deficiency in antilisterial immunity observed in the PKO mice can be overcome by increasing the number of Ag-specific CD8+ T cells through repeated vaccination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, bacteria, and immunization

Eight- to 10-wk-old female BALB/c (Thy1.2, H-2d MHC, WT) mice were purchased from the National Cancer Institute (Frederick, MD). Female BALB/c perforin-deficient (Thy1.2, H-2d MHC, PKO) mice (previously described, Ref.8) and BALB/c Thy1.1 mice, obtained from R. Dutton (Trudeau Institute, Saranac Lake, NY) were maintained by brother-sister mating and housed under specific pathogen-free conditions at the University of Iowa (Iowa City, IA) animal care unit until the initiation of experiments. These studies used virulent and attenuated (actA deficient) LM strains derived from XFL 303 (11), which express a secreted fusion protein containing the lymphocytic choriomeningitis virus (LCMV) nuclear protein (NP)118–126 (NP118) epitope (LM-NPs, Ref.12 ; or actA- LM-NPs, Ref.13). Growth and maintenance of LM strains were as described (14). Naive WT or PKO mice were infected with 2 x 106 actA- LM-NPs (primary memory). After 30 days, mice were challenged with two to three LD50 of virulent LM-NPs (one LD50 = 1 x 104 bacteria) (secondary memory). All experiments used mice or memory cells obtained >30 days after challenge. Pathogen-infected mice were housed at the appropriate biosafety level.

Abs, peptides and MHC class I tetramers

mAbs with the following specificities were used: IFN-{gamma}–PE (clone XMG1.2; eBioscience, San Diego, CA), {alpha}CD8-FITC or CyChrome (clone MP6-XT22), CD62L-APC (clone MEL-14), CD27-biotin (clone LG.3A10), and streptavidin-FITC, V{beta} 2, 4,5.1/5.2, 8.1/8.2, and 10 (clones B20.6, KT4, MR9-4, MR5-2, and B21.5, respectively), and Thy1.2 (clone 53-6.7). All Abs were from BD PharMingen (San Jose, CA), unless otherwise noted. Synthetic peptides representing defined ((listeriolysin O (LLO)91–99 (LLO91) and p60217–225 (p60217) (15, 16), p60449–457 (p60449) (17, 18); all H-2Kd restricted), and the H2-M3 restricted f-MIGWIIA (19)) or recombinant LCMV NP118–126. H-2Ld-restricted (11, 13)) LM epitopes were obtained from Biosynthesis (Lewisville, TX). MHC class I tetramers specific for NP118, LLO91, or p60217 were obtained from the National Institute of Allergy and Infectious Diseases tetramer core (Atlanta, GA) (8).

CD8+ T cell enrichment and adoptive transfer

Splenocyte cell suspensions were enriched for CD8+ T cells by negative selection (Stem Cell Technologies, Vancouver, Canada). Briefly, 8 x 107 splenic leukocytes/milliliter 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, Auburn, CA). CD8+-enriched cells were washed three times in sterile saline and used for adoptive transfer as described in the figure legends. Recovered cells were routinely >93% CD8+ by flow cytometry, and enrichment did not alter the percentage of Ag-specific cells as determined by MHC class I/peptide tetramer staining or intracellular cytokine staining (ICS) (data not shown).

CD8+ T cell purity and the percentage of Ag-specific CD8+ T cells specific for NP118, LLO, or p60217 was determined by costain for CD8 and MHC class I tetramers, and was confirmed by ICS. The number of Ag-specific cells transferred was based on preliminary experiments that showed a dramatic reduction in bacterial load after challenge with approximately one LD50 LM-NPs in the presence of >=3 x 105 WT Ag-specific CD8+ T cells (data not shown). Cells were transferred i.v. into naive female BALB/c (Thy1.2 or Thy1.1) recipient mice. Two to three days after adoptive transfer, recipient or naive control mice were challenged with 1.2–1.5 LD50 virulent LM-NPs as indicated in the figure legends.

In some instances the CD8+ T cell-enriched populations were used for FACS after costain with {alpha}CD8-FITC and MHC class I tetramers specific for NP118-APC and LLO91-PE. The recovered single epitope-specific cells were combined at an NP:LLO ratio of 2:1, and 2.5 x 105 Ag-specific CD8+ T cells were adoptively transferred into naive BALB/c Thy1.1 recipient mice before challenge.

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

The number of CD8+ T cells specific for NP118, LLO91, or p60217 in recipient mice (or donor mice for functional avidity/repertoire studies) was determined by ICS for IFN-{gamma} after a 5.5-h incubation with or without 200 nM peptide in brefeldin A (BD PharMingen) (20). Nonspecific cytokine production (<=0.5%, no peptide control) has been subtracted. The total number of epitope-specific CD8+ T cells per spleen was calculated from this frequency, the percentage of CD8+ T cells in each sample, and the total number of cells per spleen. T cell repertoire was determined by flow cytometry using Abs specific for various v{beta} gene families (BD PharMingen) with and without costaining with MHC tetramers. CFU of LM/spleen or gram liver were determined at 3 and/or 5 days after challenge (8).

In vivo cytotoxicity assay

Target cells were prepared by incubating equal numbers of naive BALB/c splenocytes with or without NP118 and LLO91 peptides (both at 1 µM) for 1 h. After washing the plus peptide and minus peptide (to control for Ag specificity), fractions were labeled at 37°C for 16 min with 2.5 µM CFSE (CFSE high) or 0.25 µM CFSE (CFSE low), respectively. The p60217 peptide was not used in these studies to eliminate competition for binding of the H-2Kd molecule (21). Cells were washed, combined, and ~20 x 106 total cells were transferred into mice i.v. The disappearance of the peptide-coated (CFSE high) population, or increase in the CFSE low/high ratio, served as an index of in vivo cytotoxicity (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD8+ T cell response of vaccinated perforin-deficient and WT mice

Vaccination of H-2d MHC, WT, or PKO mice with actA-LM generates CD8+ T cell memory levels that can be enhanced by a subsequent infection. In this study, naive WT BALB/c or PKO mice were immunized i.v. with 2 x 106 actA-LM-NPs, and after 30 days were challenged with two LD50 virulent LM-NPs to boost memory levels. At least 30 days after secondary challenge, the number of memory CD8+ T cells/spleen specific for five LM Ags was determined (Fig. 1A).



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 1. CD8+ T cell response and anti-listerial immunity in vaccinated perforin-deficient and WT mice. WT BALB/c and PKO mice were immunized i.v. with 2 x 106 actA- LM-NPs, and after 30 days, mice were challenged with two LD50 LM-NPs. A, Representative FACS plots showing the level of Ag-specific CD8+ T cell memory >30 days after secondary challenge. Numbers represent the percentage of CD8+ T cells and Ag-specific memory CD8+ T cells (bold) as determined by ICS. B, The total number of Ag-specific CD8+ T cells/spleen. C, Mice were challenged with four LD50 LM-NPs, and portions of the spleen and liver were plated to determine CFU of LM at 3 days after challenge. Data are expressed as total CFU/spleen or CFU/gram liver. Data shown are mean ± SD of three to six mice per group and are representative of two or more experiments. Line depicts limit of detection for this asay. *, Bar represents two of four mice; two mice were below the limit of detection.

 
CD8+ T cells specific for LLO91 and p60217 comprise the dominant responses after LM infection of BALB/c mice (23). The use of recombinant LM-NPs allowed us to generate another CD8+ T cell response (NP118) that is similar in magnitude to the LLO91 response. It has been shown that single immunization with actA- LM-NPs results in detectable memory levels of NP118, LLO91, and p60217-specific CD8+ T cells in WT mice (13), and a reproducible immunodominance hierarchy with the percentage of CD8+ T cells specific for NP118 > LLO91 > p60217 (ratio of ~1.0:0.7:0.3). By comparison, the number of Ag-specific cells is increased (~2-fold) in single-vaccinated PKO mice; however, the immunodominance hierarchy is maintained (10). Double-vaccinated WT and PKO mice had high levels of NP118, LLO91, and p60217-specific CD8+ T cells (henceforth referred to as Ag-specific cells) in the spleen (Fig. 1, A and B), and displayed the predicted immunodominance hierarchy. Epitope-specific cells were not detectable in naive control mice (data not shown).

Responses to other LM epitopes (p60449, mpl84, f-MIGWIIA) constitute subdominant responses that are generally ~10-fold lower than the dominant responses in BALB/c mice. To further characterize the patterns of immunodominance in vaccinated WT and PKO mice, the number of CD8+ T cells specific for p60449 and f-MIGWIIA were also evaluated (Fig. 1, A and B). Compared to the CD8+ T cell responses to the dominant LM epitopes, responses to these subdominant epitopes were decreased in magnitude in both WT and PKO mice. Overall, PKO mice had 2- to 2.4-fold higher levels of CD8+ T cell memory for each epitope. However, no differences in hierarchy were observed between WT and PKO mice, suggesting that perforin-deficiency does not influence immunodominance hierarchies after LM infection. For this reason, the remaining experiments focus on the readily detectable dominant responses.

To evaluate anti-listerial immunity, vaccinated WT and PKO mice or naive controls were challenged with four LD50 LM-NPs (Fig. 1C). The high levels of CD8+ T cell memory achieved with vaccination of WT and PKO mice were associated with substantial anti-listerial immunity in the spleens and livers after challenge (Fig. 1C). Despite a 2-fold increase in Ag-specific CD8+ T cell memory, and maintenance of the immunodominance hierarchy (Fig. 1, A and B), double-vaccinated PKO mice exhibited ~100-fold more bacteria at day 3 after LM challenge than similarly vaccinated WT mice.

Qualitative evaluation of CD8+ T cell memory in the absence of perforin

Studies have shown that memory CD8+ T cells can be divided into "central" (TCM) or "effector" (TEM) memory subsets. These subsets are defined primarily by expression of surface molecules considered key for homing to lymph nodes with TCM expressing high levels of CD62L, CD27, and CCR7, allowing them to enter all lymphoid tissue, whereas TEM cells express low levels of these molecules and do not enter lymph nodes (24, 25). Recently, Wherry and colleagues (26) have shown that TCM provides better protective immunity against certain infections on a per-cell basis than TEM. To determine whether generation of memory CD8+ T cells in the absence of perforin resulted in alteration of the proportion of TCM vs TEM cells at 40 days after secondary challenge, splenocytes from vaccinated WT and PKO mice were enriched for CD8+ T cells and then evaluated for costain with MHC class I tetramers specific for the immunodominant LM epitopes NP118 or LLO91, and mAbs specific for CD62L and CD27 (Fig. 2A). Approximately 50% of the Ag-specific memory CD8+ T cells from both WT and PKO mice were CD62LhighCD27high at this time; thus, it is unlikely that differences in the proportion of TEM to TCM CD8+ T cells can account for the decreased protective immunity observed in vaccinated PKO mice compared to WT.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 2. Characterization of Ag-specific CD8+ T cells generated in the absence of perforin. WT BALB/c and PKO mice were immunized i.v. with 2 x 106 actA- LM-NPs, and after 30 days, mice were challenged with two LD50 LM-NPs. A, After >30 days, splenic CD8+ T cells were enriched through negative selection, and CD62L and CD27 expression on Ag-specific CD8+ T cells was evaluated using MHC class I tetramers, immunofluorescent staining, and flow cytometry. Representative dot-plots from two experiments are shown. B, The Ag-specific CD8+ TCR repertoire was evaluated using MHC tetramer staining and V{beta} mAbs. C, Functional avidity was evaluated by incubation of memory WT or PKO splenocytes with serial dilutions of each peptide (NP118, LLO91, or p60217) followed by ICS for IFN-{gamma}. B and C, Data shown are mean ± SD of three to four mice per group and are representative of two or more experiments.

 
The possibility remained that the epitope-specific CD8+ T cells that develop in the absence of perforin are qualitatively different from those that develop in WT mice. Such qualitative differences in CD8+ T cell activity could occur via an altered repertoire, or decreased functional avidity, of memory CD8+ T cells in perforin-deficient mice compared to WT mice. We examined the TCR repertoire of CD8+ T cells in LM-vaccinated WT or PKO mice using a panel of V{beta}-specific mAb shown to represent the majority (>80%) of NP118 (27), LLO91, and p60217-specific CD8+ T cells (28). Evaluation of the TCR repertoire revealed diverse, but similar, V{beta} segment use by CD8+ T cells from WT and PKO mice (data not shown). Further examination of the NP118, LLO91, and p60217 epitope-specific CD8+ T cells by costain for V{beta} segment use and specific MHC class I tetramers revealed some differences in the distribution of receptor usage for each epitope, but no differences between CD8+ T cells from WT and PKO mice within each epitope (Fig. 2B).

Functional avidity of epitope-specific CD8+ T cells was assessed by incubation of splenocytes from LM-vaccinated WT or PKO mice with 10-fold dilutions (1 pM to 10 µM) of NP118, LLO91, or p60217 peptides, and the percentage maximal response at each peptide concentration was determined by intracellular IFN-{gamma} production (29). No differences in functional avidity of Ag-specific CD8+ T cells from vaccinated WT or PKO mice were observed (Fig. 2C). Thus, when taken together, these studies indicate that it is unlikely that any qualitative differences in the memory CD8+ T cells generated in the absence of perforin can account for their diminished protective capacity.

Perforin deficiency results in decreased per-cell protective capacity

Previous studies identified a key role for perforin in the regulation of CD8+ T cell expansion after LM infection, such that PKO mice have elevated levels of memory CD8+ T cells after vaccination (10). Therefore, the existence of detectable anti-listerial immunity generated in the absence of perforin (Fig. 1C) may result from increased numbers of memory CD8+ T cells. To determine the contribution of perforin-dependent cytolysis to anti-listerial immunity, we wished to compare protection by equal numbers of Ag-specific memory CD8+ T cells from vaccinated WT and PKO mice.

To address this issue, we initially chose to transfer CD8+ T cells that were enriched by negative selection to avoid possible alterations in function, such as TCR stimulation, during the enrichment phase. The number of Ag-specific cells transferred was determined by evaluation of a subset of the purified CD8+ T cells specific for the dominant LM epitopes (NP118, LLO91, and p60217) using MHC class I tetramers. Because the hierarchy of the dominant and subdominant (p60449, f-MIGWIIA) epitopes and the quality of Ag-specific CD8+ T cell memory were the same (Fig. 2A) in double-vaccinated PKO and WT mice, we assumed that the responses to all other (unidentified) LM epitopes would also be proportional in WT and PKO hosts. Under this assumption, it was possible to deliver equal numbers of Ag-specific CD8+ T cells by first determining the frequency of cells specific for each dominant epitope. The total number of CD8+ T cells transferred was adjusted so the recipient animals received equivalent numbers (~3.5 x 105 in total) of Ag-specific (NP118, LLO91, and p60217) cells from either WT or PKO donors.

To assess protective immunity, T cell-recipient and naive control mice were challenged with 1.5 LD50 virulent LM-NPs. WT cells provided robust anti-listerial immunity, as shown by a dramatic reduction in bacterial counts in the spleens and livers at 3 and 5 days after challenge (Fig. 3A). In contrast, the same number of PKO-derived CTL did not reduce bacterial numbers compared with controls. Evaluation of the CD8+ T cell response revealed an ~5-fold increase in Ag-specific cells/spleen in both WT and PKO T cell recipient mice compared to naive controls, suggesting memory CD8+ T cell expansion, rather than a primary response, although the transferred cells in these experiments were not allelically marked. Comparison of the CD8+ T cell response in mice that received either WT or PKO donor cells shows similar expansion of NP118, LLO91, and p60217-specific cells on day 5 after challenge, despite the absence of protective immunity in the mice that received perforin-deficient memory CD8+ T cells (Fig. 3B). These findings suggest that loss of effector function in transferred PKO cells, compared to WT, results in decreased per-cell protective capacity.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 3. Perforin-deficient CD8+ T cells have decreased protective capacity despite similar expansion of Ag-specific cells. The number of Ag-specific cells in CD8+ T cell-enriched immune splenocytes from LM-vaccinated mice was determined using MHC tetramer staining. A total of 4 x 105 WT or PKO Ag-specific cells were transferred i.v. into naive recipient mice. Two days later, mice were challenged with 1.2 LD50 LM-NPs. A, Three and five days after challenge, portions of the spleen and liver were plated to determine CFU of LM. B, ICS for IFN-{gamma} was performed 5 days after challenge. The total number of Ag-specific cells in the spleen is indicated. C, A total of 3.5 x 105 WT or 3.5 x 105 (1x), 8.75 x 105 (2.5x), or 1.75 x 106 (5x) PKO Ag-specific cells were transferred i.v. into naive recipient mice. Two days after cell transfer, mice were challenged with 1.5 LD50 LM-NPs. Three and five days after challenge, portions of the spleen and liver were plated to determine CFU of LM. Data are expressed as total CFU/spleen or CFU/gram liver. Data are represented as mean ± SD of three to four mice per group from one (A and B) or two (C) independent experiments.

 
Increased CD8+ T cell memory can restore immunity in the absence of perforin

If the defect in effector function in PKO mice decreases, but does not eliminate, CD8+ T cell-mediated anti-listerial immunity on a per-cell basis, then increasing the number of perforin-deficient cells should restore protection to the level achieved with WT cell transfer. To address this, 3.5 x 105 Ag-specific CD8+ T cells from WT or PKO (1x) memory mice, or 2.5- or 5-fold higher numbers of PKO cells were adoptively transferred i.v. into naive recipient mice (Fig. 3C). Two days later, mice were challenged with 1.5 LD50 LM-NPs to evaluate protective immunity. Three and five days after challenge, portions of the spleen and liver were plated to determine CFU of LM. WT CD8+ T cells provided a 2–4 log10 reduction in CFU, whereas a similar number of perforin-deficient cells failed to protect the recipients. Approximately 5-fold more PKO cells were needed to generate equivalent protection against virulent LM-NPs as observed with WT cells (Fig. 3C).

Although unlikely, the possibility remained that undetected differences in the hierarchy or quality of the CD8+ T cells specific for undefined LM epitopes could account for the deficient protective immunity afforded by perforin-deficient memory CD8+ T cells. To address this issue, we chose to sort purify single epitope-specific cells from vaccinated Thy1.2 WT or PKO mice based on MHC class I tetramer staining. For efficiency, CD8+ enriched splenocytes were costained for NP118 and LLO91. Sorted single epitope-specific CD8+ T cells were recombined at an NP:LLO ratio of 2:1, and 2.5 x 105 total Ag-specific CD8+ T cells were adoptively transferred into Thy1.1 recipients. Recipient mice were challenged with one LD50 LM-NPs and evaluated on day 5 for protective immunity (Fig. 4A) and expansion (Fig. 4B) of the adoptively transferred (Thy1.2) cells. In agreement with the enriched CD8+ T cell transfer experiments, transfer of sort-purified Ag-specific CD8+ T cells from WT mice afforded substantial (2.5 log10) protective immunity to recipient mice. By comparison, transfer of the same number of perforin-deficient CD8+ T cells did not provide any protective immunity. This result recurred despite similar expansion of both WT and PKO donor memory CD8+ T cells (Fig. 4B). These findings support the enriched CD8+ cell transfer experiments and are consistent with the notion that loss of perforin-mediated cytolysis is responsible for the decreased protective immunity afforded by memory CD8+ T cells from PKO mice.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4. Decreased protective capacity of epitope-specific CD8+ T cells from PKO mice. Splenocytes from LM-vaccinated Thy1.2 WT or PKO mice were enriched for CD8+ T cells and stained using {alpha}-CD8{alpha} and MHC class I tetramers specific for NP118 and LLO91. Ag-specific CD8+ T cells were sort purified, and 2.5 x 105 cells were recombined at an NP:LLO ratio of 2:1 and adoptively transferred into naive Thy1.1 recipient mice. Two days later, mice were challenged with 1.2 LD50 LM-NPs. A and B, Five days after challenge, portions of the spleen and liver were plated to determine CFU of LM, and ICS for IFN-{gamma} was performed. B, The total number of Ag-specific cells in the spleen is indicated. Data shown are mean ± SD of three to four mice per group. BLD indicates below the level of detection for this assay.

 
Decreased Ag-specific in vivo cytotoxicity in the absence of perforin

In light of the decreased protective capacity demonstrated by Ag-specific CD8+ T cells from PKO mice (Fig. 3), the cytotoxic capacity of Ag-specific memory CD8+ T cells was examined in vivo (22). A 1:1 ratio of peptide-coated (CFSE high) and uncoated (CFSE low) target cells were injected i.v. into naive controls or mice that had received 3.5 x 105 Ag-specific (NP118, LLO91) memory CD8+ T cells from vaccinated WT or PKO donors. At various time points after target cell transfer, spleen cells of recipient mice were analyzed for the presence of CFSE high and CFSE low populations, and the relative elimination of the CFSE high population (ratio CFSE low/high) was used as an index of in vivo cytotoxicity (22). A loss of the CFSE high peak was evident as early as 20 h after target cell transfer in mice that received WT LM-specific CD8+ T cells (Fig. 5, A and B) (average low/high = 1.45), which became maximal by 44 h (44 h low/high = 2.51, 68 h low/high = 2.43). In comparison, loss of the CFSE high peak in the PKO cell recipient mice was decreased in rate and magnitude. Further change in the CFSE low/high ratios was not observed in any group after 68 h (data not shown), which may be a consequence of peptide/MHC stability. The selective loss of the peptide-pulsed (CFSE high) targets indicates Ag-specific cytotoxicity based on the maintenance of the CFSE low (no peptide) and the presence of a low/high ratio of ~1 in the naive control mice throughout the experiment. Delayed cytolysis in the absence of perforin is consistent with previous studies that have documented the importance of perforin in cytolytic assays in vitro (5, 8, 9, 30, 31, 32). The protracted time course of target cell killing in PKO CD8+ T cell recipient mice is consistent with previous findings that identified a perforin-independent, CD95-dependent pathway of cytolysis (6, 8, 33, 34).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 5. CD8+ T cells from PKO mice, compared to WT, exhibit decreased Ag-specific cytolysis in vivo. The number of NP118 and LLO91-specific cells in CD8+ T cell-enriched immune splenocytes from LM-vaccinated mice was determined using MHC tetramer staining. A total of 3.5 x 105 Ag-specific cells from WT or PKO mice were adoptively transferred into naive recipient mice. One day later, naive syngeneic splenic leukocytes were incubated with or without 1 µM NP118 and LLO91 peptide and then labeled with high or low CFSE, respectively (target cells). The target cells were combined at a 1:1 ratio and transferred into mice i.v. The ratio of CFSE low/high was determined by flow cytometry. A, Fluorescence histograms indicate the low/high ratio in the spleen at 44 h after target cell transfer. B, Target cell low/high ratios over time. Each point represents an individual mouse. C, The number of Ag-specific cells in the peripheral blood of naive or LM vaccinated WT or PKO mice was determined using MHC tetramer staining. Representative FACS profiles are shown with numbers indicating the frequency of Ag-specific CD8+ T cells. D, To assess cytotoxicity, splenic leukocytes from naive mice were incubated with or without 1 µM NP118 and LLO91 peptide, and then labeled with high or low CFSE, respectively (target cells). The target cells were combined at a 1:1 ratio and transferred into mice i.v. The ratio of CFSE low/high in the blood (D) and spleen (E) of immune mice was determined by flow cytometry at 22 h after target cell transfer. Each point represents an individual mouse.

 
Next, we determined whether the increased levels of Ag-specific CD8+ T cell memory in vaccinated PKO mice (Fig. 1, A and B) could compensate for the decrease in per-cell protective capacity (Figs. 1C and 4A) and cytotoxicity (Fig. 5, A and B) compared to WT mice. First, the level of Ag-specific CD8+ T cell memory was evaluated in the peripheral blood of vaccinated WT and PKO mice (or naive controls) using MHC class I tetramers specific for NP118 and LLO91. The higher percentage of Ag-specific CD8+ T cells in the peripheral blood (1.45-fold difference (WT = 12.2%, PKO = 17.8%)) approaches a 2.5-fold increase in the total number of Ag-specific cells when the difference in total CD8+ T cell number is taken into account (1.7-fold difference (WT = 9.35%, PKO = 16.1%)) (Fig. 5C). This 2.5-fold increase in memory levels in PKO mice is maintained when the number of Ag-specific CD8+ T cells/10,000 peripheral blood leukocytes is calculated (data not shown).

Despite the >2-fold increase in memory levels in vaccinated PKO mice, elimination of peptide-coated target cells was still delayed compared to WT mice. A dramatic loss of the CFSE high peak was evident in both the peripheral blood and the spleens (Fig. 5, D and E) of vaccinated WT mice at 22 h (low/high = 87.9 ± 18.0 for spleen, and 77.5 ± 19.1 for blood), which was substantially diminished in the vaccinated PKO mice (low/high = 21.7 ± 6.01 for spleen, and 20.7 ± 3.2 for blood). Naive mice maintained both CFSE peaks (0.9 ± 0.0 for spleen, and 1.1 ± 0.1 for blood), indicating that the killing in vaccinated mice was Ag-specific. These results show that while the increased level of CD8+ T cell memory likely contributes to the protective immunity in PKO mice, the absence of perforin dramatically hinders the Ag-specific cytolysis, which translates into increased levels of infection upon re-challenge with LM (Fig. 1C).

Repeated vaccination increases anti-listerial immunity in perforin-deficient mice

Double-vaccinated PKO mice have decreased anti-listerial immunity (Fig. 1B) compared to double-vaccinated WT mice, despite having increased levels of CD8+ T cell memory (Fig. 1A). Moreover, anti-listerial immunity in the absence of perforin can be improved if the level of Ag-specific CD8+ T cell memory is increased (Fig. 3C). Therefore, we determined whether better protective immunity could be improved by repeated vaccination to boost memory CD8+ T cell numbers. Anti-listerial immunity was examined in single- and double-vaccinated PKO mice challenged with ~3.5 LD50 LM-NPs. Single-vaccinated WT mice were used for comparison.

A single immunization resulted in detectable levels of Ag-specific CD8+ T cell memory in WT mice that was increased ~2-fold in single-vaccinated PKO mice (Fig. 6A). A single vaccination in WT mice resulted in a 3.5–4 log10 reduction in the number of LM, whereas similarly vaccinated PKO mice had a 2 log10 reduction in bacterial number (Fig. 6B). A second vaccination of PKO mice resulted in a >5-fold increase in Ag-specific CD8+ T cell numbers over single-vaccinated WT mice and similar levels of anti-listerial immunity (Fig. 6, A and B). Thus, it is possible to improve protective immunity in the absence of perforin by increasing the level of CD8+ T cell memory through repeated vaccination.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 6. Repeated vaccination increases anti-listerial immunity in the absence of perforin. WT and PKO mice were immunized i.v. with 2 x 106 actA- LM-NPs, and after 30 days, a subset of PKO mice were challenged with two LD50 LM-NPs. A, The number of Ag-specific memory CD8+ T cells was determined in unchallenged mice by ICS. The total number of Ag-specific cells in the spleen is indicated. Data shown are mean ± SD of three mice per group and are representative of two experiments. BLD indicates below the level of detection for this assay. B, Primary and secondary memory mice were challenged with approximately three LD50 LM-NPs, and 3 days after challenge portions of the spleen and liver were plated to determine CFU of LM. N indicates naive mouse, 1° indicates primary immune, 2° indicates secondary immune. Data are expressed as total CFU/spleen or CFU/gram liver. Data are represented as mean ± SD of six to seven mice per group. *, p < 0.05 as determined by Student’s t test.

 
Persistence of Ag-specific CD8+ T cell memory in the absence of perforin

Persistence of a stable memory population is critical for protective immunity. Although repeated vaccination increased immunity in the absence of perforin, an inability to maintain adequate memory levels would result in a loss of immunity over time. To evaluate whether Ag-specific CD8+ T cells from vaccinated PKO mice were maintained at levels similar to WT, 5 x 105 CD8+-enriched Thy1.2 epitope-specific (NP118 and LLO91) CD8+ T cells were adoptively transferred into naive Thy1.1 recipient mice, and 233 days later, mice were challenged with 1.2 LD50 LM-NPs. Protective immunity and Ag-specific CD8+ T cell expansion were evaluated on day 5 after challenge. As previously observed, mice that received memory CD8+ T cells from perforin-deficient donors had diminished protective immunity compared to mice that received WT cells (2 vs 5 log10 reduction, Fig. 7A), despite similar expansion of the adoptively transferred cells (Fig. 7, B and C). Likewise, no differences in the endogenous CD8+ T cell response were observed, suggesting that the host cells do not increase expansion to compensate for the deficient anti-listerial immunity in the absence of perforin.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 7. Persistence of Ag-specific CD8+ T cell memory. The number of Ag-specific cells in CD8+ T cell-enriched immune splenocytes from LM-vaccinated mice was determined using MHC tetramer staining. A total of 5 x 105 Thy1.2 WT or PKO Ag-specific cells were transferred i.v. into naive Thy1.1 recipient mice. Then, 233 days later, mice were challenged with 1.2 LD50 LM-NPs. A, Five days after challenge, portions of the spleen and liver were plated to determine CFU of LM. 2/4, indicates that two mice cleared the infection. B, ICS for IFN-{gamma} was performed 5 days after challenge, and representative dot-plots are shown. C, The total number of Ag-specific cells in the spleen is indicated. Data shown are mean ± SD of three to four mice per group, representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Perforin is an important CTL effector mechanism against many pathogens (4); however, the degree to which a vaccine induced secondary response depends on perforin in any system has not been determined. The goal of our studies was to quantify the contribution of perforin as an effector molecule in CD8+ T cell resistance to LM. In summary, the absence of perforin in LM-epitope-specific memory CD8+ T cells results in a 5-fold reduction in anti-listerial immunity on a per-cell basis, which is in agreement with decreased cytotoxic capacity of PKO cells in vivo. The defect in immunity observed in the absence of perforin was not due to altered memory cell quality or decreased expansion of Ag-specific memory cells upon infection. Moreover, the low level of immunity achieved through vaccination of perforin-deficient mice could be effectively boosted to attain a stable memory population. Although no single effector molecule has been shown to be absolutely required for effective CD8+ T cell immunity to LM (9, 33, 35), our results demonstrate that perforin-dependent cytotoxicity constitutes the major means by which CD8+ T cells protect against LM.

In the current studies, adoptive transfer of equal numbers of WT and PKO memory CD8+ T cells provided ~3 log10 reduction in CFU in WT cell recipient mice, and no detectable immunity in PKO cell recipient mice. This deficiency is likely caused by the loss of perforin as an effector molecule, because no differences in quality or hierarchy of the memory CD8+ populations were observed between vaccinated WT and PKO mice. Although NP118, LLO91, and p60217 appear to be the dominant epitopes after infection with LM-NPs, it is possible that the number of LM-epitopes could actually be quite large when the size of the Listeria genome (~2800 genes, Ref.36) is considered. In this study, the adoptive transfer experiments with CD8-enriched spleen cells were based on the assumption that the CD8+ T cell responses to all other (unidentified) LM epitopes would be proportional in WT and PKO mice because the hierarchy (Fig. 1A) of CD8+ T cells specific for the dominant and subdominant epitopes (five in total) was the same in double-vaccinated PKO and WT mice (Fig. 1). Under this assumption, equal numbers of Ag-specific cells were transferred after the frequency of cells specific for the dominant epitopes was determined. Similarly, a reduction in the per-cell protective capacity of tetramer-sorted WT NP118/LLO91 memory CD8+ T cells lacking perforin was also observed. It is unclear whether tetramer-sorted cells have similar Ag-specific per-cell protective capacity as the negatively selected CD8+ population. A recent study suggested that tetramer-stained LLO91-specific CD8+ T cell lines provided reduced anti-listerial immunity (compared to unstained) in adoptive transfer assays because of functional alterations resulting from the persistence of TCR-MHC class I interactions (37). Although it is possible that tetramer-sorted memory CD8+ T cells exhibit reduced protective capacity compared to the enriched CD8+ T cell population, it is reasonable that any functional alterations induced by tetramer-sorting of WT or PKO cells would be equal. Regardless, the lack of anti-listerial immunity provided by PKO-derived Ag-specific cells was observed with both negatively selected or tetramer-sorted memory CTL, demonstrating that perforin deficiency decreases per-cell protection.

It has been suggested that the quality, rather than quantity, of memory CD8+ T cells is critical for protective immunity (24, 26, 38). In bacterial and viral infection models, repeated Ag exposure with re-infection leads to increased Ag affinity and a focusing of TCR expression (29, 39). More recently, a functional distinction between TEM and TCM CD8+ T cell subsets suggests that the quality of immunological memory depends on the type of CD8+ T cell memory and the rate of conversion from TEM to TCM (26). In this report, the quality of CD8+ T cell memory generated in the presence or absence of perforin was assessed through the evaluation of TEM and TCM populations (CD62L and CD27 expression), TCR repertoire use, and functional avidity. Based on these criteria, perforin deficiency did not influence the quality of CD8+ T cell memory LM-vaccinated mice. Approximately 50% of the Ag-specific CD8+ T cells isolated from the spleens of vaccinated WT and PKO mice at 40 days after infection were CD62Lhigh CD27high, which is consistent with previous findings (26). Although inefficient homing of TCM to the lymph nodes (dependent on CCR7) could contribute to the decreased anti-listerial immunity in vaccinated PKO mice, it is unlikely because CCR7 expression on memory CD8+ T cells appears to be coordinate with CD27 (26). Moreover, whereas these studies do not preclude a role for "affinity maturation" or focusing of LM epitope-specific CD8+ T cells after vaccination (29, 39), they suggest that perforin deficiency does not overtly influence the epitope-specific CD8+ T cell repertoire, similar to results obtained in previous studies of immunodominance in bacterial (10) and viral (40) infection of PKO mice. When taken together, the current studies suggest that perforin deficiency does not influence the quality of CD8+ T cell memory to LM.

These studies demonstrate a deficiency in the per-cell protective capacity afforded by Ag-specific CD8+ T cells from PKO mice compared to WT cells, despite similar levels of expansion early after infection (Figs. 1, 4, and 7). The current findings are in agreement with previous studies that ruled out inefficient priming of CD8+ T cells in PKO mice as a source of the inadequate protective immunity against LM (8), and suggest that the reduced anti-listerial immunity in the absence of perforin is caused by the defect in effector function in these cells.

The absence of protective immunity in both the spleen and the liver of perforin-deficient CD8+ T cell recipient mice differ from previous studies that identified a higher level of dependence on perforin in the spleen than the liver for protection (8, 9). It is likely that the liver is less dependent on the adaptive immune response to control LM, but when significantly fewer Ag-specific memory CD8+ T cells were transferred, as in the current study (3.5 x 105 vs 1–5 x 107), the defect in protective immunity afforded by PKO cells may be more pronounced. Also, some previous studies used whole splenocyte transfers rather than enriched CD8+ T cells, so it may be that other cell types can compensate for the loss of perforin more effectively in the liver microenvironment. In support of this, depletion of CD8+ T cells from the whole splenocyte transfers significantly reduced the anti-listerial immunity in the spleen, but not the liver (8).

Approximately 5-fold more perforin-deficient memory CD8+ T cells were needed to generate equivalent protection against virulent LM-NPs as observed with WT cells (Fig. 3C). Recent studies suggest that the role of perforin-dependent cytolysis in anti-listerial immunity is to limit cell-cell spread of bacteria (7). In this scenario, perforin-mediated cytolysis not only destroys infected cells, it also decreases the rate at which new cells become infected. Our results demonstrate that perforin-deficiency can be overcome by increasing the number of memory CD8+ T cells; thus, the requirement for perforin-mediated cytolysis in anti-listerial immunity is not absolute. Furthermore, these findings suggest that the protective immunity observed in LM-vaccinated PKO mice is caused, at least in part, by the elevated memory CD8+ T cell levels in these mice. It is likely that the number of memory CD8+ T cells required to compensate for perforin deficiency may depend on the level of infection or type of pathogen. In the case of LM and/or LCMV infection in WT mice, the level of CD8+ T cell memory is proportional to the level of protection against infection (41, 42). However, in the case of LCMV infection, where survival of PKO mice is dependent on deletion of NP118-specific CD8+ T cells in the primary response, generation and persistence of a stable memory population through vaccination with LM-NPs results in massive expansion of memory CD8+ T cells, and associated mortality, upon infection with LCMV (43). Thus, while vaccination generally enhances antimicrobial immunity through establishment of a memory population, it has the potential to be harmful under certain circumstances, so vaccination strategies will have to be tailored to the pathogen and/or host.

The current studies show that while the effector function of perforin is important for anti-bacterial immunity, the loss of this molecule can be overcome by increasing memory levels. It is likely that qualitative defects in CD8+ T cell memory can also be overcome in this manner. The central goal of vaccination remains the generation of immunological memory so that disease is reduced or absent after subsequent pathogen exposure. These studies suggest that it may be possible to achieve protective immunity despite a deficiency in anti-microbial immunity, such as the absence of an effector molecule (36) (i.e., perforin), through repeated vaccination to attain higher levels of immunological memory.


   Acknowledgments
 
We thank the National Institute of Allergy and Infectious Diseases tetramer core for tetramer reagents, Katherine L. Rensberger for her technical assistance, and S. Perlman for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI42767, AI46653, and AI50073 (to J.T.H.) and T32 A107260, the Interdisciplinary Immunology Training Grant (to K.A.N.M.), and by the Leukemia and Lymphoma Society Fellow Grant (to V.P.B.). 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: LM, Listeria monocytogenes; WT, wild type; LCMV, lymphocytic choriomeningitis virus; ICS, intracellular cytokine staining; LLO, listeriolysin-O; NP, nuclear protein; TCM, CD8+ T cell central memory subset; TEM, CD8+ T cell effector memory subset; PKO, perforin deficient. Back

Received for publication June 3, 2003. Accepted for publication August 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kaufmann, S. H., C. H. Ladel. 1994. Role of T cell subsets in immunity against intracellular bacteria: experimental infections of knock-out mice with Listeria monocytogenes and Mycobacterium bovis BCG. Immunobiology 191:509.[Medline]
  2. Roberts, A. D., D. J. Ordway, I. M. Orme. 1993. Listeria monocytogenes infection in {beta}2 microglobulin-deficient mice. Infect. Immun. 61:1113.[Abstract/Free Full Text]
  3. Ladel, C. H., I. E. Flesch, J. Arnoldi, S. H. Kaufmann. 1994. Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153:3116.[Abstract]
  4. Harty, J. T., A. R. Tvinnereim, D. W. White. 2000. CD8+ T cell effector mechanisms in resistance to infection. Annu. Rev. Immunol. 18:275.[Medline]
  5. 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.[Medline]
  6. Jensen, E. R., A. A. Glass, W. R. Clark, E. J. Wing, J. F. Miller, S. H. Gregory. 1998. Fas (CD95)-dependent cell-mediated immunity to Listeria monocytogenes. Infect. Immun. 66:4143.[Abstract/Free Full Text]
  7. 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.[Abstract/Free Full Text]
  8. White, D. W., A. MacNeil, D. H. Busch, I. M. Pilip, E. G. Pamer, J. T. Harty. 1999. Perforin-deficient CD8+ T cells: in vivo priming and antigen-specific immunity against Listeria monocytogenes. J. Immunol. 162:980.[Abstract/Free Full Text]
  9. 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.[Abstract/Free Full Text]
  10. 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.[Abstract/Free Full Text]
  11. Shen, H., C. M. Tato, X. Fan. 1998. Listeria monocytogenes as a probe to study cell-mediated immunity. Curr. Opin. Immunol. 10:450.[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.[Medline]
  13. Tvinnereim, A. R., J. T. Harty. 2000. CD8+ T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of {gamma} interferon. Infect. Immun. 68:2196.[Abstract/Free Full Text]
  14. Harty, J. T., M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN {gamma}. Immunity 3:109.[Medline]
  15. Harty, J. T., E. G. Pamer. 1995. CD8 T lymphocytes specific for the secreted p60 antigen protect against Listeria monocytogenes infection. J. Immunol. 154:4642.[Abstract]
  16. Pamer, E. G., J. T. Harty, M. J. Bevan. 1991. Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes. Nature 353:852.[Medline]
  17. Busch, D. H., H. G. Bouwer, D. Hinrichs, E. G. Pamer. 1997. A nonamer peptide derived from Listeria monocytogenes metalloprotease is presented to cytolytic T lymphocytes. Infect. Immun. 65:5326.[Abstract]
  18. Vijh, S., E. G. Pamer. 1997. Immunodominant and subdominant CTL responses to Listeria monocytogenes infection. J. Immunol. 158:3366.[Abstract]
  19. Lenz, L. L., B. Dere, M. J. Bevan. 1996. Identification of an H2–M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity 5:63.[Medline]
  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.[Medline]
  21. 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.[Abstract/Free Full Text]
  22. Coles, R. M., S. N. Mueller, W. R. Heath, F. R. Carbone, A. G. Brooks. 2002. Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1. J. Immunol. 168:834.[Abstract/Free Full Text]
  23. 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.[Medline]
  24. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708.[Medline]
  25. Masopust, D., V. Vezys, A. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413.[Abstract/Free Full Text]
  26. 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.[Medline]
  27. 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.[Abstract/Free Full Text]
  28. Huleatt, J. W., I. Pilip, K. Kerksiek, E. G. Pamer. 2001. Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes. J. Immunol. 166:4065.[Abstract/Free Full Text]
  29. Slifka, M. K., J. L. Whitton. 2001. Functional avidity maturation of CD8+ T cells without selection of higher affinity TCR. Nat. Immunol. 2:711.[Medline]
  30. Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. Huang, J. D. Young, R. Ahmed, W. R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91:10854.[Abstract/Free Full Text]
  31. Kojima, H., N. Shinohara, S. Hanaoka, Y. Someya-Shirota, Y. Takagaki, H. Ohno, T. Saito, T. Katayama, H. Yagita, K. Okumura, et al 1994. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity 1:357.[Medline]
  32. Lowin, B., M. Hahne, C. Mattmann, J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370:650.[Medline]
  33. White, D. W., V. P. Badovinac, G. Kollias, J. T. Harty. 2000. Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice. J. Immunol. 165:5.[Abstract/Free Full Text]
  34. Harty, J. T., D. White. 1999. A knockout approach to understanding CD8+ cell effector mechanisms in adaptive immunity to Listeria monocytogenes. Immunobiology 201:196.[Medline]
  35. Endres, R., A. Luz, H. Schulze, H. Neubauer, A. Futterer, S. M. Holland, H. Wagner, K. Pfeffer. 1997. Listeriosis in p47phox-/- and TRp55-/- mice: protection despite absence of ROI and susceptibility despite presence of RNI. Immunity 7:419.[Medline]
  36. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt, T. Chakraborty, et al 2001. Comparative genomics of Listeria species. Science 294:849.[Abstract/Free Full Text]
  37. 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.[Medline]
  38. Lauvau, G., S. Vijh, P. Kong, T. Horng, K. Kerksiek, N. Serbina, R. A. Tuma, E. G. Pamer. 2001. Priming of memory but not effector CD8+ T cells by a killed bacterial vaccine. Science 294:1735.[Abstract/Free Full Text]
  39. Busch, D. H., E. G. Pamer. 1999. T cell affinity maturation by selective expansion during infection. J. Exp. Med. 189:701.[Abstract/Free Full Text]
  40. Chen, W., J. R. Bennink, P. A. Morton, J. W. Yewdell. 2002. Mice deficient in perforin, CD4+ T cells, or CD28-mediated signaling maintain the typical immunodominance hierarchies of CD8+ T-cell responses to influenza virus. J. Virol. 76:10332.[Abstract/Free Full Text]
  41. Badovinac, V. P., K. N. M. Messingham, S. E. Hamilton, J. T. Harty. 2003. Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host. J. Immunol. 170:4933.[Abstract/Free Full Text]
  42. Badovinac, V. P., B. B. Porter, J. T. Harty. 2002. Programmed contraction of CD8+ T cells after infection. Nat. Immunol. 3:619.[Medline]
  43. Badovinac, V. P., S. E. Hamilton, J. T. Harty. 2003. Viral infection results in massive CD8+ T cell expansion and mortality in vaccinated perforin-deficient mice. Immunity 18:463.[Medline]



This article has been cited by other articles:


Home page
 JEMHome page
E. Narni-Mancinelli, L. Campisi, D. Bassand, J. Cazareth, P. Gounon, N. Glaichenhaus, and G. Lauvau
Memory CD8+ T cells mediate antibacterial immunity via CCL3 activation of TNF/ROI+ phagocytes
J. Exp. Med., September 3, 2007; 204(9): 2075 - 2087.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. A. N. Messingham, V. P. Badovinac, A. Jabbari, and J. T. Harty
A Role for IFN-{gamma} from Antigen-Specific CD8+ T Cells in Protective Immunity to Listeria monocytogenes
J. Immunol., August 15, 2007; 179(4): 2457 - 2466.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. E. Hamilton, V. P. Badovinac, A. Khanolkar, and J. T. Harty
Listeriolysin O-Deficient Listeria monocytogenes as a Vaccine Delivery Vehicle: Antigen-Specific CD8 T Cell Priming and Protective Immunity
J. Immunol., September 15, 2006; 177(6): 4012 - 4020.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
A. Jabbari and J. T. Harty
Secondary memory CD8+ T cells are more protective but slower to acquire a central-memory phenotype
J. Exp. Med., April 17, 2006; 203(4): 919 - 932.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M.-F. Pahl-Seibert, M. Juelch, J. Podlech, D. Thomas, P. Deegen, M. J. Reddehase, and R. Holtappels
Highly Protective In Vivo Function of Cytomegalovirus IE1 Epitope-Specific Memory CD8 T Cells Purified by T-Cell Receptor-Based Cell Sorting
J. Virol., May 1, 2005; 79(9): 5400 - 5413.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. K. Nussbaum and J. L. Whitton
The Contraction Phase of Virus-Specific CD8+ T Cells Is Unaffected by a Pan-Caspase Inhibitor
J. Immunol., December 1, 2004; 173(11): 6611 - 6618.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Messingham, K. A. N.
Right arrow Articles by Harty, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Messingham, K. A. N.
Right arrow Articles by Harty, J. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS