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The Journal of Immunology, 2006, 177: 3012-3018.
Copyright © 2006 by The American Association of Immunologists, Inc.

T Cell Conditioning Explains Early Disappearance of the Memory CD8 T Cell Response to Infection1

Ali Jabbari*, Kevin L. Legge*,{dagger} and John T. Harty2,*,{ddagger}

* Interdisciplinary Graduate Program in Immunology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242; {dagger} Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242; and {ddagger} Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory CD8 T cells respond more rapidly to acute intracellular infections than naive CD8 T cells. An understanding of the biological processes involved in memory CD8 T cell recognition of Ag and up-regulation of effector mechanism necessitates analyzing memory CD8 T cells at early time points after infection. In the current study, we show that memory CD8 T cells ostensibly disappear from the spleens, blood, and peripheral organs of mice early after infection with Listeria monocytogenes. This disappearance is critically dependent on Ag, and cell-associated Ag alone can mediate this phenomenon. Further investigations, however, suggest that this disappearance is secondary to T cell-APC interactions, also known as T cell conditioning, and disruption of these putative interactions during splenic processing improves recovery of Ag-specific memory CD8 T cell populations after immunization. Conventional analyses of memory CD8 T cell populations early after infection and possibly in the presence of low levels of Ag (as during chronic infections) may exclude significant numbers of the responding CD8 T cell population.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8 T cells compose an arm of the adaptive immune response responsible for the defense against intracellular pathogens. CD8 T cells that respond to a peptide presented in the context of MHC class I undergo a differentiation pattern that results in the formation of a population of memory cells (1), a hallmark of the adaptive immune system. Secondary responses by memory CD8 T cells often reach higher peak numbers than primary responses and, in the case of secondary infections, are usually associated with enhanced clearance and decreased morbidity and mortality (2).

The increased level of protection conferred by memory CD8 T cells is most likely due to several factors. Naive CD8 T cells that become activated undergo well-defined expansion and contraction programs (3, 4, 5, 6) in which the net result is a substantially increased number of Ag-specific T cells; the increased number of Ag-specific memory cells compared with their naive counterparts contributes to their enhanced protective capacity. Memory CD8 T cells also can elaborate effector functions more quickly than naive CD8 T cells (7), although this may not be universally true (8). Also, there is some evidence that memory CD8 T cells undergo cell division faster than naive CD8 T cells and that memory CD8 T cells appear to generally require less lag time between the activating stimulus and the initiation of cell division (9). In total, the enhanced protection afforded by memory CD8 T cells is most likely not just a consequence of their increased frequency but also the result of the rapidity by which they respond to Ag.

We were therefore interested in examining early events in memory CD8 T cell responses after an infection. We have previously observed a transient drop in memory Ag-specific CD8 T cells after pathogenic challenge in a mouse model of familial hemophagocytic lymphohistiocytosis (10) and herein describe further studies in the setting of wild-type host mice after infection. We found that Ag-specific memory CD8 T cells undergo an apparent decline in numbers from the spleens of mice at early time points after infection with Listeria monocytogenes, as assessed by FACS. This effect, which was limited to memory CD8 T cells that were specific for a pathogen-expressed Ag, contrasts with reports by others of massive bystander attrition of nonspecific CD8 T cells (11, 12, 13) or dilution of bystander memory CD8 T cell populations due to the emergence of memory specific for other Ags (14, 15). Interestingly, our results indicate that this substantial early decline in Ag-specific memory CD8 T cells detected by FACS is not corroborated by histological analyses, consistent with a T cell clonal conditioning mechanism (16).

T cell conditioning has been proposed to be due to interactions with APCs during which T cells can be programmed either to become deleted or to survive depending on the status of the innate immune system. It has been proposed (16) that T cell conditioning may account for earlier reports in which superantigen challenge led to the disappearance of specific T cell populations (17, 18), but has to date not been addressed in the context of infections or memory T cell responses. Here, our studies describe memory CD8 T cell conditioning in the context of L. monocytogenes infection and that protease digestion can disrupt these aggregations.


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

We obtained C57BL/6 (B6) from the National Cancer Institute. OT-I TCR transgenic (Tg)3 mice have been previously described (19). All animal protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. L. monocytogenes strain 10403S has been previously described. L. monocytegenes expressing the OVA gene (LM-OVA) was obtained from Dr. H. Shen (University of Pennsylvania, Philadelphia, PA) and Dr. L. Lefrancois (University of Connecticut, Hartford, CT), and the attenuated strain of LM-OVA (actA– LM-OVA) has been previously described (20). The number of CFU injected in each experiment was confirmed by plating dilutions on selective medium containing streptomycin.

Abs and reagents

Abs of the indicated specificities and with the appropriate combination of conjugated fluorophores were used in these studies: Thy1.1, CD8 (BD Pharmingen). Anti-IgM Ab, clone B76, was obtained from Dr. T. Waldschmidt (University of Iowa, Iowa City, IA). CFSE (Molecular Probes) was used at 0.5 µM unless indicated otherwise to label cells. In some cases, the PKH26 red fluorescent cell linker kit was used as per the manufacturer’s instructions (Sigma-Aldrich) to labeled cells before adoptive transfer. Synthetic peptide OVA257–264 has been previously described (19).

Adoptive transfer of OT-I CD8 T cells

For the generation of memory OT-I CD8 T cells, naive OT-I/Thy1.1 splenocytes were enriched for CD8+ cells by negative selection (Miltenyi Biotec), and 5 x 104 cells were adoptively transferred into naive Thy1.2 B6 mice. Recipient mice were infected i.v. with ~1 x 107 actA-LM-OVA. More than 6 wk later, splenocytes were harvested and adoptively transferred (21) into new recipient mice as indicated. In some cases, OT-I cells were purified using anti-Thy1.1-PE and anti-PE magnetic beads with AutoMACS before adoptive transfer.

Lymphocyte isolation from peripheral tissues

Organs and tissues were harvested after cardiac perfusion with PBS and heparin (70 U/ml). Livers and lungs were cut into small pieces and treated with collagenase D (150 U/ml) for 30 min at 37°C. Spleen and remains from liver and lung digestions were forced through metal meshes. Liver lymphocytes were isolated on a single-step 35% Percoll gradient. To correct for cell loss during gradient purification, a known number of CFSE-labeled splenocytes was mixed with the liver lymphocytes, and the composition of labeled to unlabeled cells was assessed by flow cytometry. RBC were lysed with ammonium-chloride-potassium buffer.

Peptide-coated splenocyte immunization

Spleen cells from B6 mice were coated with the indicated concentration of OVA257–264 peptide for 1 h at 37°C and washed three times before i.v. injection.

Immunofluorescent histology

Thy1.1-purified memory OT-I/Thy1.1 cells (4 x 106) were stained with CFSE to label the transferred T cells and adoptively transferred to Thy1.2 B6 hosts. One day later, recipients of memory OT-I/Thy1.1 cells were immunized with either 107 uncoated or 1 µM OVA257–264 peptide-coated splenocytes. Spleens were harvested 1 day postimmunization, and a portion of each spleen was frozen in OCT and sectioned on a microtome. The remaining portions of the spleens were used for flow cytometric analysis. Sections were stained with biotinylated anti-IgM primary Ab followed by an avidin-Cy5 secondary to demarcate splenic T cell zones. Sections were visualized on an Olympus BX-51 microscope.

Protease mixture digestion of spleens

Harvested spleens were digested with 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I-s, and 60 U/ml DNase I in HBSS for 30 min at 37°C before forcing remaining tissue through a metal mesh.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Decline in Ag-specific memory CD8 T cells in the spleen early after infection

Memory CD8 T cells substantially reduce the magnitude and duration of infections in many host-pathogen experimental models, and this enhanced immunity is thought to be at least partially a consequence of the rapidity by which memory CD8 T cells respond and elaborate effector functions (2). Additionally, although early events in naive CD8 T cell activation have been intensively studied (3, 4, 5, 6), little has been described about this critical time early after stimulation for memory CD8 T cells. We were therefore interested in determining the status of Ag-specific memory CD8 T cells at early time points after infection. Others have reported a transient decline in naive TCR Tg CD8 T cells in the blood after peptide immunization (22), and we have previously shown a similar phenomenon for memory CD8 T cells after lymphocytic choriomeningitis virus infection in the spleen of previously vaccinated perforin-knockout mice (10). We adopted a TCR Tg model to address these findings in the context of infection in an otherwise wild-type setting. We adoptively transferred memory OT-I/Thy1.1 cells, generated as outlined in Materials and Methods, into Thy1.2-expressing B6 mice. Groups of these mice were then either infected 1 day later with virulent L. monocytogenes or LM-OVA or were not infected. Because LM-OVA is less virulent than wild-type L. monocytogenes (data not shown), an 8-fold higher inoculum of LM-OVA was administered to approximate the bacterial burden of wild-type L. monocytogenes-infected mice; however, the bacterial burden of L. monocytogenes-infected mice was still significantly higher at day 1 than that of LM-OVA-infected mice (Fig. 1A). One day postinfection, the spleens of these mice were harvested, and the numbers of OT-I cells per spleen were determined by staining for CD8 and Thy1.1 and assessment by flow cytometry. Detection of OT-I cells by Thy1.1 staining does not rely on TCR expression and TCR-based signaling, which was a possible confounding factor in our prior studies because T cells may down-regulate surface TCR in response to Ag (23). We found that infection with LM-OVA resulted in a decrease in the number of OT-I cells, whereas infection with L. monocytogenes not expressing the OVA Ag resulted in no decline in splenic OT-I numbers compared with the uninfected controls (Fig. 1B). Other studies have indicated that the population of OT-I cells rebounds by day 2 after LM-OVA infection, correlating with observed cell division as assessed by CFSE dilution (data not shown). These data demonstrate that Ag-specific memory CD8 T cells detectable by FACS decline in number in the spleen early after infection. The observation of no decrease in OVA-specific memory CD8 T cells early during infection in the absence of OVA indicates that this early decline of Ag-specific cells does not also occur in bystander memory CD8 T cells. Additionally, these data demonstrate that the decline in Ag-specific memory CD8 T cells was not a consequence of TCR down-regulation or an inability to produce IFN-{gamma} (24).


Figure 1
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  		FIGURE 1. Decline in Ag-specific CD8 T cells in the spleen after infection. Approximately 6 x 105 memory OT-I-Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. One day later, mice were infected with 5.4 x 103 CFU L. monocytogenes or 4.2 x 104 CFU LM-OVA or were not infected (No Inf.). A, One day postinfection, spleens were harvested, and bacterial burden was assessed by plating serial dilutions of homogenized spleens (mean + SD). B, Memory OT-I-Thy1.1 cells were enumerated by flow cytometric analysis of CD8+Thy1.1+ cells (mean + SD). Statistical significance indicated. Data are indicative of three mice per group (mean + SD), representative of three experiments.

 
Early decline in splenic Ag-specific memory CD8 T cells is not due to peripheral dissemination

Others have previously shown (25) that CD8 T cells alter the array of homing or trafficking molecules decorating their cell surface upon activation. The rapid decline in splenic Ag-specific memory CD8 T cells detected early after infection may have been due to peripheral dissemination of this population after infection. We therefore determined whether this early Ag-specific decline in the spleen after infection could be explained by migration to peripheral organs, as read out by increased numbers of Ag-specific CD8 T cells at these sites. We examined the liver, a peripheral organ that is a site of infection for L. monocytogenes, as well as the lung, a peripheral organ that harbors high numbers of lymphocytes but is not a site associated with L. monocytogenes infection. Memory OT-I-Thy1.1 cells were adoptively transferred into Thy1.2-expressing B6 mice, and these recipient mice were subsequently infected with LM-OVA or left uninfected. One day later, lymphocytes were isolated from spleen, liver, lung, and blood, and the numbers of OT-I cells were determined by staining for CD8 and Thy1.1 and by flow cytometry. Whereas the number of OT-I cells was significantly diminished in the spleen of mice infected with LM-OVA, there was no compensatory increase in OT-I cells in the liver, lungs, or blood (Fig. 2) that accounted for the observed cell loss in the spleen; in fact, the number of OT-I T cells was significantly decreased in the blood and exhibited decreasing trends in the liver and lungs. These data indicate that the decline in splenic Ag-specific memory CD8 T cells was not due to generalized dissemination into the periphery.


Figure 2
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  		FIGURE 2. Decline in splenic Ag-specific CD8 T cells is not due to peripheral dissemination. Approximately 7.5 x 105 memory OT-I-Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. One day later, mice were either infected with 9.65 x 105 CFUs of actA– LM-OVA or not infected (No Inf.). One day postinfection, spleens were harvested and stained for CD8 and Thy1.1. A, Representative dot plots from spleen, blood, lung, and liver. Values are the percent of OT-I cells of total cells. B, Total numbers of memory OT-I-Thy1.1 cells per organ for spleen, lung, and liver and percent OT-I of total CD8s for blood (mean + SD). Statistical significance indicated. Data are indicative of three mice per group (mean + SD), representative of three experiments.

 
Decline in Ag-specific memory CD8 T cells is temporally distinct from bystander CD8 T cell decline

Others have described (11, 12, 13) that total bystander CD8 T cells become reduced in number at relatively early time points after bacterial infection; however, this phenomenon did not occur for Ag-specific CD8 T cells (11). To confirm that the numeric decline in Ag-specific cells observed here was distinct from a global decline in CD8 T cells, as described by others, we adoptively transferred memory OT-I-Thy1.1 CD8 T cells into Thy1.2-expressing B6 mice. These mice were subsequently infected with L. monocytogenes or LM-OVA or were not infected. As before, a higher LM-OVA inoculum was administered relative to L. monocytogenes such that the day 1 bacterial burden was matched between these groups (Fig. 3A). At days 0, 1, and 3 postinfection, spleens were harvested from these mice, the bacterial burden was assessed, and OT-I cells were enumerated. As expected, memory CD8 T cells effectively reduced the bacterial load in the spleens of mice infected with LM-OVA between day 1 and day 3 compared with mice infected with L. monocytogenes. As observed previously, memory OT-I cells declined in the spleen at day 1 post-LM-OVA, but not LM, infection (Fig. 3B). Interestingly, memory CD8 T cells exhibited a decline in the spleens of recipient mice 3 days after infection by L. monocytogenes that does not express OVA (Fig. 3B), paralleling observations previously made by others (11, 26). This day 3 decline correlated with a decline in total CD8 T cells in recipient mice (data not shown), whereas the decline observed at day 1 for memory CD8 T cells after LM-OVA infection was specific for the memory OT-I T cell population. These data indicate that Ag-specific memory CD8 T cells exhibit a transient decline in the spleen early after infection and that this phenomenon is distinct from an Ag-independent global decline in CD8 T cells after L. monocytogenes infection described by others (11, 12, 13).


Figure 3
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  		FIGURE 3. Temporally distinct Ag-dependent and Ag-independent decline. Approximately 1 x 106 memory OT-I-Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. One day later, mice were infected with 2.5 x 104 CFU L. monocytogenes or 1.0 x 105 CFUs LM-OVA or were not infected (No Inf.). A, At 0, 1, and 3 days postinfection, spleens were harvested, and bacterial burden was assessed by plating serial dilutions of homogenized spleens (mean + SD). Statistical significance indicated. B, OT-I cells were enumerated by flow cytometric analysis of CD8+Thy1.1+ cells (mean + SD). Statistical signficance assessed with respect to no infection group of the corresponding day. Data are indicative of three mice per group (mean + SD), representative of three experiments.

 
Cell-associated Ag alone is sufficient to induce a decline in splenic Ag-specific memory CD8 T cells

These studies demonstrated that Ag in the context of infection was sufficient to effect a transient drop in Ag-specific memory CD8 T cells in the spleen. We next determined whether cell-associated Ag in the absence of infection was sufficient to induce this drop. Thy1.2-expressing recipients of memory OT-I/Thy1.1 CD8 T cells were infected with LM-OVA, not infected, or immunized with either 5 x 106 OVA-coated (1 µM) splenocytes or 5 x 106 uncoated splenocytes. One day later, the numbers of OT-I cells in spleens were assessed by flow cytometry. Similarly to LM-OVA-infected mice, mice immunized with OVA-coated splenocytes exhibited a drop in the number of splenic memory OT-I cells 1 day after immunization (Fig. 4A). Additionally, this effect was observed for Ag-experienced CD8 T cells with markedly different compositions of central and effector memory CD8 T cells subsets as distinguished by CD62L expression (Fig. 4B); similar declines were observed for CD8 T cells that had recently experienced Ag (day 14 postinfection) as well as CD8 T cells analyzed much later after antigenic stimulation (day 190 postinfection; Fig. 4C). In total, these data indicate that cell-associated Ag potently induces a decline in Ag-specific memory CD8 T cells in the spleen and that cell-associated Ag is both sufficient (Fig. 4) and necessary (Fig. 1) to induce this decline.


Figure 4
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  		FIGURE 4. Ag in the absence of infection can mediate memory CD8 T cell decline. Approximately 7.5 x 105 memory OT-I-Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. One day later, mice were either infected with 2.8 x 105 CFUs LM-OVA or immunized with either 5 x 106 splenocytes coated with 1 µM OVA-coated splenocytes or uncoated splenocytes. No Imm, Not immunized. A, One day later, spleens were harvested and memory OT-I cells were enumerated by flow cytometric analysis of CD8+Thy1.1+ cells (mean + SD). B, Memory OT-I recipients were immunized with the 5 x 106 splenocytes coated with 0, 0.075, 0.3, or 1000 nM OVA peptide. One day later, spleens were harvested, and memory OT-I cells were enumerated (mean + SD). C, Ag-experienced OT-I-Thy1.1 cells were purified either 14 or 190 days postinfection (p.i.) as indicated in Materials and Methods. Approximately 7.5 x 105 cells of either type were adoptively transferred into Thy1.2 recipients. Shown are the CD62L expression profiles of the two memory OT-I CD8 T cell populations assessed before adoptive transfer. D, One day later, recipient mice were immunized with splenocytes either coated with 1 µM OVA peptide or uncoated, and splenic OT-I cells were enumerated 1 day after immunization (mean + SD). Statistical significance indicated (three mice/group).

 
Ag-specific CD8 T cell deline is not apparent by histological analysis

Although this is the first report of a decline in Ag-specific memory CD8 T cells in the context of infection of a wild-type animal, others have previously reported an analogous phenomenon of a decline in naive CD4 T cells after peptide immunization (16). The flow cytometry-based assessments in that study, however, was found to contrast with histological examinations at the same time points, indicating that these CD4 T cells actually remained in the spleen. This discrepancy was proposed to be due to T cell-APC aggregation that prevented extraction of the splenocytes for flow cytometric analysis. Before testing whether the memory CD8 T cells in our model were undergoing a similar phenomenon, we first confirmed that we were able to histologically detect CFSE-labeled, adoptively transferred CD8 T cells in the T cell zones, identified as areas surrounded by IgM+-staining B cells, of splenic cryosections (Fig. 5A). To test whether the observed memory CD8 T cell decline early after immunization could be confirmed histologically, spleens of memory OT-I-Thy1.1 CD8 T cell recipients were harvested 1 day after immunization with either OVA-coated or uncoated splenocytes. Flow cytometric analysis of a portion of these spleens indicated that the number of OT-I cells was greatly reduced in those mice that received OVA-coated splenocytes relative to those mice immunized with uncoated splenocytes (Fig. 5B). Analysis of the same spleens by immunofluorescent histology indicated, however, that those mice immunized with OVA-coated splenocytes retained a number of OT-I cells similar to that of mice immunized with uncoated splenocytes (Fig. 5C), and enumeration of OT-I cells relative to the number of T cell zones, as defined by a region enclosed by IgM+-staining cells, revealed no diminishment in the frequency of OT-I cells in mice immunized with OVA-coated splenocytes (Fig. 5D). These data indicate that Ag-specific memory CD8 T cells do not, in fact, undergo a transient decline in the spleen early after Ag recognition but rather may undergo T cell conditioning. Furthermore, this is consistent with our data demonstrating that peripheral dissemination does not explain the early decline in memory T cell numbers (Fig. 2) as well as with our preliminary studies indicating that memory CD8 T cells do not undergo Ag-induced cell death in vitro under conditions in which effector CD8 T cells do (data not shown).


Figure 5
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  		FIGURE 5. Decline in splenic Ag-specific CD8 T cells is not apparent by immunofluorescent histology and is consistent with a conditioning model. A, Splenic sections from control B6 (top) or B6 mice that received ~4 x 106 CFSE-coated negatively-selected CD8+ B6 splenocytes (bottom) were stained with biotinylated anti-IgM Abs and visualized by light microscopy, as indicated in Materials and Methods. B, Approximately 4 x 106 CFSE-labeled, magnetic bead-purified memory OT-I-Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. One day later, mice were immunized with 1 x 107 splenocytes either coated with 1 µM OVA peptide or uncoated. One day later, spleens were harvested. A small portion of the spleen was processed for flow cytometry and stained for CD8 and Thy1.1. C, The harvested spleen was processed for immunofluorescent histology, stained with anti-IgM Abs, and visualized as indicated in Materials and Methods. D, The number of CFSE+ cells was averaged over the number of T cell zones (mean + SD; 20–25 T cell zones/group).

 
Protease mixture digestion increases extraction of Ag-specific memory CD8 T cells at early time points after immunization

It has been proposed that mature, naive T cells undergo a process termed T cell conditioning, thought to take place after their initial stimulation and before clonal expansion. This process, which has been described for naive CD4 T cells (16), is hypothesized to be due to interactions between Ag-specific T cells and Ag-laden APCs. These interactions are thought to complicate the extraction of these populations; protease digestion of spleens before forcing through a metal mesh, however, has been met with some success in increasing the yield of naive Ag-specific T cells after primary stimulation (16). We addressed whether this same treatment would result in an increase in the number of Ag-specific memory cells detectable by flow cytometry at day 1 post secondary immunization. Recipients of memory OT-I cells were immunized with OVA-loaded splenocytes, and 1 day postimmunization spleens were harvested and processed either by forcing through a metal mesh or by treating with a protease mixture of hyaluronidase, collagenase, and DNase before forcing through a metal mesh. We found that protease mixture treatment resulted in increased recovery of Ag-specific memory CD8 T cells 1 day postimmunization with cell-associated Ag (Fig. 6A). Protease treatment resulted in little to no increase in recovery of Ag-specific memory cells after immunization with uncoated splenocytes. These results indicate that disruption of protein-protein interactions by a protease mixture enhances the recovery of Ag-specific memory CD8 T cells at early time points postimmunization. Further phenotypic and functional analyses failed to identify a distinguishing feature of splenic Ag-specific CD8 T cells liberated by protease digestion vs Ag-specific CD8 T cells from untreated spleens 1 day after immunization with cell-associated peptide (Fig. 6B). Additionally, the levels of Thy1.1 expression by protease-liberated Ag-specific CD8 T cells 1 day postimmunization with cell-associated Ag were not diminished compared with CD8 T cells from unimmunized mice (data not shown), confirming the utility of this marker for our initial experiments (Fig. 1).


Figure 6
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  		FIGURE 6. Protease treatment of splenocytes results in increased recovery of Ag-specific CD8 T cells at day 1 postimmunization. Approximately 8 x 105 memory OT-I cells were adoptively transferred into naive B6 mice. One day after adoptive transfer, mice were immunized with 8 x 106 splenocytes either coated with 1 mM OVA peptide or left uncoated. One day after immunization, spleens were harvested and either forced through a metal mesh or digested with a protease cocktail, as indicated in Materials and Methods, to extract a single-splenocyte suspension. One day postimmunization, splenocytes were stained for CD8 and Thy1.1. A, Total numbers of OT-I cells per spleen. Data are indicative of three mice per group (mean + SD), representative of two experiments. B, Approximately 1 x 106 memory OT-I cells were adoptively transferred into naive B6 mice. One day after adoptive transfer, mice were immunized with 20 x 106 splenocytes either coated with 1 mM OVA peptide or left uncoated. One day after immunization, spleens were harvested and either forced through a metal mesh or digested with a protease mixture, as before, to extract a single-splenocyte suspension. Splenocytes were surface stained for CD8, Thy1.1, and the indicated marker or underwent 5 h in vitro incubation in brefeldin A with (black-lined histograms) or without (shaded histograms) OVA peptide before being stained for intracellular IFN-{gamma} or IL-2.

 
Naive CD8 T cells undergo T cell conditioning after immunization with cell-associated Ag but not after L. monocytogenes infection

T cell conditioning has been observed for naive T cells after in vivo injection of superantigens or synthetic peptide epitopes (16, 17, 18, 22). To determine whether naive CD8 T cells also undergo conditioning after infection, we transferred naive or memory OT-I-Thy1.1 CD8 T cells into Thy1.2 mice. One day later, mice were either left uninfected or infected with LM-OVA, and the numbers of OT-I cells were determined 1 day postinfection. To our surprise, naive CD8 T cells did not undergo T cell conditioning under these conditions, because their numbers did not decrease using conventional extraction methods, whereas the number of memory CD8 T cells did decrease (Fig. 7A). However, immunization of naive OT-I recipients with cell-associated Ag did result in observable T cell conditioning by the naive CD8 T cell population, similarly to memory CD8 T cells (Fig. 7B).


Figure 7
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  		FIGURE 7. Naive CD8 T cells undergo conditioning in the context of peptide immunization but not during L. monocytogenes infection. A, Approximately 6.4 x 105 naive or 6.8 x 105 memory OT-I-Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. One day later, mice were either infected with 1.027 x 105 CFUs LM-OVA or left uninfected. One day later, spleens were harvested and OT-I cells were enumerated by flow cytometric analysis of CD8+Thy1.1+ cells (mean + SD). B and C, Approximately 10.0 x 105 naive or 7.5 x 105 memory OT-I/Thy1.1 CD8 T cells were adoptively transferred into Thy1.2 recipient mice. B, One day later, mice were immunized with 10.0 x 106 splenocytes (S) either coated with 1 µM OVA peptide or left uncoated. One day later, spleens were harvested, and OT-I cells were enumerated by flow cytometric analysis of CD8+Thy1.1+ cells (mean + SD). C, Recipients of either naive or memory OT-I-Thy1.1 CD8 T cells were immunized with the 5.0 x 106 splenocytes coated with 0, 0.075, 0.3, or 1000 nM OVA peptide. One day later, spleens were harvested, and memory OT-I cells were enumerated (mean + SD).

 
Memory CD8 T cells possess a number of functional characteristics that distinguish them from naive CD8 T cells. One such example illustrated by others is that memory CD8 T cells have a higher functional avidity for Ag than naive CD8 T cells (27). Thus, we considered the possibility that memory CD8 T cells undergo T cell conditioning at much lower concentrations of Ag than naive CD8 T cells. This notion could explain why naive CD8 T cells undergo conditioning after immunization with cells coated with high concentrations of Ag but, discordant with memory cells, do not undergo conditioning in the context of L. monocytogenes infection where the levels of Ag on the cell surface may be much less. To test this idea, we immunized recipients of naive or memory OT-I CD8 T cells with cells coated with a graded amount of OVA peptide. One day postimmunization, the number of OT-I cells per spleen was assessed after conventional splenocyte extraction and FACS analysis. Although the observed trend was that naive CD8 T cells underwent less conditioning than memory CD8 T cells, we were unable to identify a concentration of Ag that would induce the majority of memory but not naive CD8 T cells to undergo conditioning. Therefore, although a reduced sensitivity by naive CD8 T cells may contribute, it appears that a simple difference in functional avidity does not fully account for the difference observed between naive and memory CD8 T cell conditioning in the context of infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have reported a transient decline in number, as assessed by flow cytometry, of Ag-specific memory CD8 T cells in the spleens of wild-type mice early after infection or secondary immunization. This decline, however, appears to be a consequence of the T cells undergoing a APC-dependent conditioning phase that renders them undetectable by flow cytometry, although they remain readily detectable by histological techniques. Although this process has been described for naive T cells after superantigen administration (17, 18) or naive CD4 T cells after peptide immunization (16), our studies are the first report of this process occurring 1) in the context of infection and 2) for a population of memory CD8 T cells.

Our initial experiments indicated that infection of mice leads to a drop in the number of Ag-specific memory CD8 T cells at an early time point postinfection. This drop did not occur during infection in the absence of Ag (Fig. 1), indicating that Ag is necessary for the disappearance and that this phenomenon does not extend to bystander memory CD8 T cells. These observations contrast with a separate phenomenon observed here (Fig. 3) and initially reported by others in which L. monocytogenes infection leads to apoptosis of a large proportion of CD8 T cells (11) and total lymphocytes in the spleen due to type I IFN- and/or TRAIL-mediated apoptosis (12, 13, 28). More precisely, whereas pathogen-specific memory CD8 T cells seem to disappear at day 1 postinfection, non-pathogen-specific CD8 T cells undergo a decline as observed at day 3 postinfection. Although not directly compared here, others have shown that the non-pathogen-specific CD8 T cell drop is also seen histologically, whereas, as further discussed below, the earlier decline in pathogen-specific CD8 T cells is not, implying differences in mechanism.

Our studies further suggested that the disappearance in the spleen of Ag-specific memory CD8 T cells could not be mechanistically explained by a dissemination of Ag-specific CD8 T cells into the periphery (Fig. 2). We did observe some up-regulation of early apoptotic markers in memory OT-I T cells after OVA-coated splenocyte immunization at a time point where the disappearance was incomplete (data not shown). However, many reports now describe the adoption of an early apoptotic phenotype (29) or the activation of some caspase species (30, 31) without significant apoptosis before T cell expansion. Additionally, preliminary in vitro experiments suggested that memory CD8 T cells did not undergo activation-induced cell death under stimulatory conditions that caused apoptosis of effector CD8 T cells (data not shown), further suggesting that these cells were not dying in response to secondary Ag exposure in vivo.

We found that the decline in Ag-specific memory CD8 T cells was most likely explained mechanistically by T cell conditioning. T cell conditioning has been proposed as a process whereby APCs laden with Ag form aggregates with specific T cells, preventing them from being properly extracted or processed for flow cytometry. This is illustrated by the inability to detect a decline in the numbers of Ag-specific CD8 T cells after immunization with cell-associated Ag when assessed histologically in samples that show a marked decline in frequency and deduced total number when assessed by flow cytometry (Fig. 5). Disruption of T cell conditioning by proteases also resulted in the increased recovery of Ag-specific memory CD8 T cells at early time points after immunization with Ag (Fig. 6).

These studies may have some impact on how memory CD8 T cells and secondary responses are examined. In the context of infection or peptide administration, Ag-specific memory CD8 T cells are not detectable by conventional flow cytometric methods. Studies of early, Ag-specific memory CD8 T cell behavior that rely on flow cytometry-based technologies, therefore, may be limited to those cells that have not yet or are no longer involved in interactions with Ag-laden APCs. For example, some reports have concluded that memory CD8 T cells do not divide in vivo during the first 24 h after infection (9); if those cells that are interacting with APCs are not detectable by the conventionally used flow cytometry-based techniques, it is possible that those Ag-specific cells have in fact divided but are not able to be visualized. Additionally, studies of T cells in chronic, smoldering infection models may underestimate the number of Ag-specific T cells, and flow cytometric analysis of those cells may exclude a portion of this Ag-specific CD8 T cell population in these models.

Histological analysis, intravital imaging, or disruption of T cell-APC interactions may be therefore required at these early time points for accurate analysis of memory CD8 T cell responses. The potential drawbacks of direct ex vivo analyses highlighted here, furthermore, underscore the possible usefulness of in vivo imaging, especially two-photon imaging (32). This technology, which can track multiple populations of cells in lymph nodes of intact animals, has most recently been used to examine naive CD4 and CD8 T cell responses to immunization with peptide or peptide-loaded dendritic cells (DCs) (33, 34, 35). Interestingly, some studies have concluded that DCs form tight interactions with CD8 T cells for up to 20 h after immunization (36), which may correlate with the timing of T cell conditioning. Two-photon in vivo imaging, comparing naive and memory CD8 T cell responses early after infection, may elucidate biological differences that explain the observed findings derived from direct ex vivo, flow cytometry-based examinations. In addition to providing an avenue by which to study early memory T cell responses to DC immunization or pathogenic infection, this technology additionally provides a backdrop to cell-cell interactions unparalleled by direct ex vivo or in vitro analyses.

We observed a discrepancy between naive and memory CD8 T cell conditioning after L. monocytogenes infection. Naive CD8 T cells did not undergo conditioning after L. monocytogenes infection (Fig. 7A), but, like memory CD8 T cells, they did undergo conditioning after immunization with cell-associated peptide (Fig. 7B). Further, our data indicate that the observed difference is most likely not entirely due to differential sensitivity to Ag (Fig. 7C). One possibility has to do with the different contexts in which naive and memory CD8 T cells recognize Ag; naive CD8 T cells form stable contacts with Ag on DCs, whereas memory CD8 T cells theoretically recognize Ag on all MHC class I-expressing cells. Alternatively, the inflammatory environment stimulated by infection, which likely differs in naive and immune mice, may affect the complex formation involved in T cell conditioning.

Our studies suggest that Ag-specific memory CD8 T cells enter into a period of T cell conditioning after infection. The interactions that take place between T cells and APCs are most likely critical in engendering a robust secondary response (37). Studies of those interactions will be helpful in elucidating requirements for CD8 T cell responses and may be instructive in the design of vaccines and vaccination protocols. Furthermore, studies of this time period may require careful analysis and the use of non-flow cytometry-based techniques to study CD8 T cell biology.


   Acknowledgments
 
We thank Dr. Stanley Perlman for critiquing the manuscript and Rebecca Podyminogin for excellent technical assistance.


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


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

1 This work was supported by National Institutes of Health Grants AI42767, AI46653, AI50073, and AI059752 (to J.T.H.) and by American Heart Association Heartland Predoctoral Grant 0610047Z (to A.J.). 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: Tg, transgenic; LM-OVA, L. monocytgenes expressing the OVA gene; actA– LM-ova, the attenuated strain of LM-OVA; DC, dendritic cell. Back

Received for publication March 31, 2006. Accepted for publication June 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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