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


     
 

The Journal of Immunology, 2008, 181, 2644 -2650
Copyright © 2008 by The American Association of Immunologists, Inc.
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 Bixby, L. M.
Right arrow Articles by Tarleton, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bixby, L. M.
Right arrow Articles by Tarleton, R. L.

Stable CD8+ T Cell Memory during Persistent Trypanosoma cruzi Infection1

Lisa M. Bixby and Rick L. Tarleton2

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8+ T cell responses to persistent infections caused by intracellular pathogens are dominated by resting T effectors and T effector memory cells, with little evidence suggesting that a T central memory (TCM) population is generated. Using a model of Trypanosoma cruzi infection, we demonstrate that in contrast to the T effector/T effector memory phenotype of the majority of T. cruzi-specific CD8+ T cells, a population of cells displaying hallmark characteristics of TCM cells is also present during long-term persistent infection. This population expressed the TCM marker CD127 and a subset expressed one or more of three other TCM markers: CD62L, CCR7, and CD122. Additionally, the majority of CD127high cells were KLRG1low, indicating that they have not been repetitively activated through TCR stimulation. These CD127high cells were better maintained than their CD127low counterparts following transfer into naive mice, consistent with their observed surface expression of CD127 and CD122, which confer the ability to self-renew in response to IL-7 and IL-15. CD127high cells were capable of IFN-{gamma} production upon peptide restimulation and expanded in response to challenge infection, indicating that these cells are functionally responsive upon Ag re-encounter. These results are in contrast to what is typically observed during many persistent infections and indicate that a stable population of parasite-specific CD8+ T cells capable of Ag-independent survival is maintained in mice despite the presence of persistent Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The high antigenic load and ongoing TCR stimulation that often characterize persistent viral infections have been shown to negatively impact the antiviral CD8+ T cell response leading to immune exhaustion. Antiviral CD8+ T cells during chronic lymphocytic choriomeningitis virus infection, for example, show signs of functional impairment including loss of cytokine production and proliferation and susceptibility to physical deletion (1). In contrast, studies in acute resolving bacterial and viral infection models have demonstrated that once established, stable CD8+ T cell memory is maintained independently of Ag (2, 3) by the homeostatic turnover of Ag-specific CD8+ T cells mediated by the cytokines IL-7 and IL-15 (4, 5, 6). CD8+ T cell memory during persistent infections, however, is less easily defined, in part due to the numerous infections which differ in regard to duration of chronic infection, antigenic load, and pathogen tropism.

CD8+ memory cells have been broadly classified into T central memory (TCM)3 and T effector memory (TEM) subsets. Although these subsets were initially defined based on high (TCM) and low (TEM) expression of the lymph node-homing molecules CD62L and CCR7 (7), a variety of additional markers have subsequently been used to discriminate between these two subsets of cells (8). Surface expression of IL-7R{alpha} (CD127) and IL-15Rβ (CD122), receptors that confer responsiveness to IL-7 and IL-15, respectively, is a hallmark of stable CD8+ T cell memory (3) and identify memory CD8+ T cell precursors destined to become long-lived TCM cells (9) capable of self renewal and rapid recall to Ag (10). In contrast, CD8+ T cells in chronic infections are maintained primarily as Ag-dependent, short-lived TE cells (3, 11, 12, 13, 14) generally fail to express CD127 and CD122 and therefore proliferate poorly in response to IL-7 and IL-15 (3, 11, 12).

We investigated the effects of persistent parasite Ag on the maintenance of CD8+ T cells during chronic infection with the protozoan parasite Trypanosoma cruzi. T. cruzi is the causative agent of Chagas’ disease and is responsible for >50,000 deaths a year in Latin America (15). Although acute infection is controlled by host immune responses, parasites persist at very low levels in certain tissues (16, 17). The use of tools such as MHC class I tetramers has allowed for the ex vivo detection and characterization of parasite-specific CD8+ T cells during T. cruzi infection (18). This infection therefore provides an opportunity to study the effects of persistent Ag on the parasite-specific CD8+ T cell response and, more specifically, to determine whether stable memory cells capable of Ag-independent persistence are generated during this infection. We report that consistent with the persistent nature of T. cruzi infection, the majority of parasite-specific CD8+ T cells during chronic infection are CD62LlowCD127lowCD122low and proliferate poorly to homeostatic cytokines. However, a minor population of parasite-specific CD8+ T cells also exists that displays characteristics of TCM cells. These CD127high Ag-specific CD8+ T cells produce IFN-{gamma} following peptide restimulation, are better maintained in the absence of Ag than their CD127low counterparts, and expand substantially following challenge. These data demonstrate that a stable memory CD8+ T cell population capable of Ag-independent survival is maintained for >1 year in an environment where Ag persists.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, parasites, and infections

C57BL/6J and C57BL/6-Tg(ACTB-EGFP)1Osb/J mice were purchased from The Jackson Laboratory and C57B6-Ly5.2 mice were purchased from the National Cancer Institute at Frederick (Frederick, MD). Mice were maintained at the University of Georgia animal facility in microisolator cages under specific pathogen-free conditions. For the T. cruzi infections, tissue culture trypomastigotes (TCT) of the Brazil strain or CL strain of T. cruzi were obtained from passage through Vero cells. Mice were infected either i.p. with 1000 TCT or with 2000 TCT via footpad injection and sacrificed by CO2 inhalation. In some experiments, animals were challenged with 10,000 Brazil strain TCT. All animal protocols were approved by the University of Georgia Institutional Animal Care and Use Committee.

T cell phenotyping

RBCs in single-cell suspensions of spleen cells (SC) were lysed in a hypotonic ammonium chloride solution and washed in staining buffer (2% BSA and 0.02% azide in PBS (PAB)). In some cases, mouse peripheral blood was obtained by retro-orbital venipuncture, collected in sodium citrate solution, and washed in PAB. SC and whole blood were incubated with tetramer-PE and the following labeled Abs: anti-CD62L biotin, anti-CD8 allophycocyanin-Cy7 (BD Biosciences), anti-CD127 allophycocyanin (eBioscience), anti-CD122 Alexa Fluor 488, and streptavidin Alexa Fluor 405 (Caltag Laboratories/Invitrogen). Cells were also stained with anti-CD4, anti-CD11b, and anti-B220 Tricolor (Caltag Laboratories/Invitrogen) for use as an exclusion channel. Cells were stained for 45 min at 4°C in the dark, washed twice in PAB, and fixed in 2% formaldehyde. The ELC-Ig chimera was used for detecting CCR7 expression and was a gift from Dr. K. Klonowski (University of Georgia, Athens, GA). For CCR7 detection, cells were incubated at 37°C for 1 h, stained with ELC-Ig for 45 min, and washed. Cells were then stained with goat anti-human IgG Alexa Fluor 488 (Invitrogen) for 30 min, washed, and stained with surface markers as indicated above. For whole blood, RBCs were lysed after surface staining in a hypotonic ammonium chloride solution and washed twice in PAB. At least 500,000 lymphocytes were acquired using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). MHC I tetramers TSKB20 (ANYKFTLV/Kb) and TSKB74 (VNYDFTLV/Kb) were synthesized at the Tetramer Core Facility (Emory University, Atlanta, GA).

Lymphocyte proliferation

Splenocytes isolated from chronically infected mice were incubated with CFSE (Molecular Probes/Invitrogen) at a final concentration of 1 µM for 3 min at room temperature. CFSE labeling was quenched with an equal volume of horse serum and the cells were washed three times with RPMI 1640 containing 10% FBS. CFSE-labeled cells were cultured in RPMI containing 10% FBS for 72 h at 37°C with IL-7 (15 ng/ml; BD Biosciences), IL-15 (15 ng/ml; eBioscience), or medium alone. An anti-IL-15 Ab (eBioscience) was added to some cell cultures to block endogenous IL-15 activity. Following culture, splenocytes were washed twice in PAB and stained with TSKB20/Kb PE, anti-CD8 Alexa Fluor 405 (Caltag Laboratories/Invitrogen), and exclusion channel Abs.

Intracellular cytokine staining

Splenocytes isolated from naive or chronically infected mice were sorted as described below, cultured with feeder cells from GFP-expressing mice [C57BL/6-Tg(ACTB-EGFP)1Osb/J], and stimulated with T. cruzi peptides (1 µM) for 5 h at 37°C in the presence of brefeldin A (GolgiPlug; BD Biosciences). T. cruzi peptides used in this study were TSKB20 (ANYKFTLV), TSKB18 (ANYDTLV), CRZP5 (PSVRSSVPL), CRZP9 (VPLNKCNRL), GFT 16 (AAMSRHPPL), and GFT 17 (RGFDEGNGL) (18). Cells were surface stained with anti-CD8 Alexa Fluor 405 (Caltag Laboratories/Invitrogen) and intracellular cytokine staining was performed with anti-IFN-{gamma} allophycocyanin with a Cytofix/Cytoperm kit (BD Biosciences) in accordance with the manufacturer’s instructions. At least 250,000 lymphocyte events were acquired and analyzed as above.

In vivo BrdU incorporation assay

BrdU (Sigma-Aldrich) solution (0.8 mg/ml) was made fresh every 2 days and given to mice for 12 days starting on the day of challenge. SC from mice were RBC lysed and washed two times in PAB. Cells were surface stained for 45 min at 4°C in the dark with TSKB20/Kb PE and the following labeled Abs: anti-CD8 Alexa Fluor 405 (Caltag Laboratories/Invitrogen), anti-CD45.2 allophycocyanin, and anti-CD127 PE-Cy7 (eBioscience). An exclusion channel was used as described above. Cells were permeabilized and stained with a BrdU flow kit according to the manufacturer’s instructions (BD Biosciences). Briefly, cells were permeabilized twice and treated with DNase to expose incorporated BrdU. Cells were then stained intracellularly with anti-BrdU FITC, washed twice, and fixed in 2% formaldehyde. At least 2 x 106 lymphocyte events were acquired as above.

CD8+ T cell sorting and adoptive transfers

SC from naive or chronically T. cruzi-infected mice were washed twice in PBS following RBC lysis. CD8+ T cells were negatively selected through magnetic sorting (CD8a+ T cell isolation kit; Miltenyi). For in vitro peptide restimulation experiments, single-cell suspensions enriched for CD8+ T cells were stained with anti-CD127 PE and cells were sorted into CD127low and CD127high populations. For adoptive transfer experiments, single-cell suspensions enriched for CD8+ T cells were stained with anti-CD127 PE (eBioscience) and anti-CD44 allophycocyanin (BD Biosciences) and cells were sorted into CD44highCD127high and CD44highCD127low populations. Sorting was performed on a MoFlo cell sorter (DakoCytomation). In brief, 1.3 x 106 CD44highCD127high or CD44highCD127low cells were transferred i.v. into naive congenic mice. Mice were infected 21 days after transfer with 10,000 Brazil strain T. cruzi parasites and administered BrdU in drinking water for 12 days to assess proliferation rates in vivo. Mice were sacrificed 12 days postchallenge infection and splenocytes were stained for BrdU and surface markers as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Presence of a heterogeneous CD127high T. cruzi-specific CD8+ T cell population during chronic infection

We have recently shown that T. cruzi infection elicits a strong CD8+ T cell response that is highly focused on epitopes encoded by genes belonging to the trans-sialidase (ts) family (18). To examine the phenotypic characteristics of parasite-specific CD8+ T cells, we analyzed the longitudinal expression of markers of Ag-independent memory on TSKB20-specific CD8+ T cells following infection of C57BL/6 mice with the Brazil strain of T. cruzi (Fig. 1A). As previously described (18), TSKB20-specific CD8+ T cells expanded and contracted during acute infection reflecting the peak parasitemia observed at ~30 dpi in this model and these cells were maintained during the chronic phase. A significant fraction of TSKB20-specific T cells expressed CD127 early in the acute infection, but this population represented <2% of all the TSKB20-specific cells by day 64 postinfection. From this low point, the fraction of TSKB20-specific cells expressing CD127 increased steadily to nearly 24% by ~1 year postinfection (Fig. 1, A and C). The frequency of TSKB20-specific cells expressing CD122 (Fig. 1A) and CCR7 (Fig. 1B) also increased as the infection progressed, but remained at <5% even at 400 days postinfection.


Figure 1
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 1. Emergence of a parasite-specific CD127high population during chronic T. cruzi infection. Splenocytes from naive or Brazil strain-infected mice were isolated and stained with the TSKB20/Kb or TSKB74/Kb tetramer and for surface expression of CD127, CD122, CCR7, or KLRG1 and non-CD8 lineage abs (anti-CD4, anti-CD11b, anti-B220) at various times following infection. Gates for the distribution of CD127, CD122, and CCR7 staining on tetramer+ cells were set using an unstained negative control (FMO control) and the staining pattern of each marker on bulk naive and chronic CD8+ T cells. A, Expression of CD127 and CD122 on TSKB20/Kb+ cells. Cells shown in top panel are gated on CD4–CD11b–B220– T cells. Numbers indicate the percentage of CD8+ T cells that are TSKB20 tetramer+. Lower panels, Closed histograms indicate the TSKB20-specific population and open histograms indicate CD8+ T cells from age-matched naive (uninfected) controls. Numbers indicate the percentage of tetramer+CD8+ T cells that express each given marker. Data are representative of two independent experiments. B, Expression of CCR7 on TSKB20/Kb+ cells. Filled histograms indicate the TSKB20-specific population and open histograms indicate naive CD8+ T cells. Numbers indicate the percentage of tetramer+CD8+ T cells that express CCR7. C, Expression of memory markers on TSKB20-specific or TSKB74-specific CD8+ T cells from mice at 327 dpi. Filled histograms indicate the tetramer+ population while open histograms indicate naive CD8+ T cells. D, Coexpression of memory markers on TSKB20-specific CD8+ T cells from mice at 327 dpi. Dot plots are gated on CD127highTSKB20/Kb+ cells. Numbers indicate the percentage of CD127high cells that express each of the given memory markers. For all data, the percentages indicated are from an average of two to five animals per experimental group.

 
The pattern of expression of CD127 and CD122 on CD8+ T cells specific for the subdominant TSKB74 epitope was similar to that for TSKB20/Kb+ cells (Fig. 1C), indicating that the memory phenotype does not vary among different T cell populations within the dominance hierarchy. Of the TSKB20-specifc CD127high cells, nearly one-third coexpressed CD62L and ~60% of the CD127high TSKB20/Kb+ cells lacked expression of the killer cell lectin-like receptor G1 (KLRG1) (Fig. 1D), a molecule induced by repetitive Ag stimulation that marks CD8+ T cells with impaired proliferative capacities (19). Collectively, these results indicate a heterogeneous population of parasite-specific CD8+ T cells with the phenotype of long-lived, Ag-independent TCM cells persists in mice with chronic T. cruzi infection.

A subset of T. cruzi-specific CD8+ T cells from chronically infected mice proliferate in response to IL-7 or IL-15

The observed frequencies of CD127high and CD122high Ag-specific CD8+ T cells from chronically T. cruzi-infected mice predicts that only a subset of the T. cruzi-specific T cell population should respond to IL-7 or IL-15. To test this prediction, splenocytes were cultured with IL-7 and IL-15 and proliferation was measured on the basis of dilution of CFSE stain. Because we have previously observed that endogenous IL-15 in these spleen cell cultures contributes to background proliferation in the negative control (medium only) conditions, graded doses of an anti-IL-15 Ab were added to block endogenous IL-15 in cultures. The effects of endogenous IL-15 on the proliferation of TSKB20-specific cells from chronically infected mice could be blocked in a dose-dependent manner with anti-IL-15-blocking Ab, and this blocking effect could be partially overcome by addition of exogenous IL-15 (Fig. 2). Likewise, a fraction of the TSKB-specific cells in chronically infected mice responded to IL-7 (Fig. 2), thus confirming that CD127 and CD122 expression correlated with the ability of these T cells to functionally respond to the respective ligands.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2. A subset of TSKB20 tetramer+CD8+ T cells from chronically infected mice proliferate in response to the cytokines IL-7 and IL-15. Splenocytes from mice chronically infected with T. cruzi at 154 dpi were labeled with CFSE and cultured in the presence of 15 ng/ml IL-7 or IL-15 or with medium alone for 72 h. An anti-IL-15-blocking Ab was added to cultures at the indicated concentrations. The concentration of 0.78 µg/ml was previously shown to block 100% of 50 ng/ml IL-15 activity. Cells were stained with the TSKB20/Kb tetramer and for surface expression of CD8 and non-CD8 lineage Abs (anti-CD4, anti-CD11b, anti-B220). Proliferation of CD4–CD11b–B220– TSKB20-specific CD8+ T cells was assessed by CFSE dilution. Numbers indicate the percentage of TSKB20-specific cells that had not divided by the end of the culture period. Data are representative of two independent experiments.

 
CD127high and CD127low T cells produce IFN-{gamma} in response to T. cruzi peptides encoding both immunodominant and subdominant epitopes

Although CD8+ T cells specific for the immunodominant TSKB20 and TSKB74 epitopes (18) in chronically infected mice contain a relatively low number of CD127high cells, we hypothesized that CD8+ T cells specific for subdominant epitopes might contain a higher fraction of CD127high cells. Immunodominant responses may achieve their respective frequencies due to a greater amount and presentation of cognate Ag throughout the infection, and the abundance of Ag likely contributes to the maintenance of these cells as primarily TE/TEM populations. Lower frequency CD8+ T cells specific for scarce or sparsely presented Ags may encounter Ag less frequently and thus contain a higher proportion of stable TCM-like cells. To test this hypothesis, we examined the responses of the relatively rarer T. cruzi-specific CD8+ T cells specific for non-ts peptides. These peptides are present in proteins encoded by the cruzipain (CRZP) and β-galactofuranosyltransferase gene families and individually elicit a CD8+ T cell response that collectively represents less than one-third of that observed to the TSKB20 peptide during chronic infection (18). Sorted CD127high and CD127low CD8+ T cells from chronically T. cruzi-infected mice were stimulated with a pool of ts or CRZP/GFT T. cruzi peptides and were examined for IFN-{gamma} production (Fig. 3). Naive sorted controls did not produce IFN-{gamma} in response to any of the T. cruzi peptides. Surprisingly, the majority of responding CD8+ T cells to the CRZP/GFT pool of peptides was in the CD127low population, as was also the case for the ts pool of peptides. However, we did observe the presence of a small population of IFN-{gamma}-producing CD8+ T cells in both peptide pools in the CD127high fraction, indicating that regardless of the epitope specificity and possibly the level of Ag exposure, the majority of T. cruzi-specific cells in persistently infected mice are of the CD127low phenotype. Importantly, these data also confirm that T. cruzi-specific CD127high T cells are functionally responsive and capable of being activated to effector function.


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 3. T. cruzi-specific CD127high and CD127low T cells are functionally responsive. Splenocytes from T. cruzi-infected (200 dpi) or aged-matched naive mice were pooled, enriched for CD8+ cells, and sorted into CD127high and CD127low groups (sort purity was >97% for both populations). IFN-{gamma} production by purified CD127high and CD127low CD8+ T cells was assessed following a 5-h incubation with a pool of ts peptides (TSKB20, TSKB74) or a pool of non-ts peptides (CRZP5 and CRZP9, GFT16 and GFT17) in the presence of GFP+ feeder cells. Splenocyte cultures from naive (top panel) and chronically infected mice (bottom panel) were stained with anti-CD8 and anti-IFN-{gamma}. Numbers indicate the percentage of CD8+ IFN-{gamma}-producing cells for each condition. All plots are gated on GFP-negative cells. Data are representative of two independent experiments.

 
Maintenance and expansion of CD127highCD8+ T cells from chronically infected mice

Preservation following pathogen clearance and rapid expansion following secondary Ag exposure are hallmarks of Ag-independent memory T cells. To further examine these characteristics in the CD127highCD8+ T cell population from chronically infected mice, splenocytes from naive and chronically infected mice were enriched for CD8+ T cells and then sorted into CD44highCD127high and CD44highCD127low groups (Fig. 4A). We chose to sort on CD44high cells to focus on Ag-experienced CD8+ T cells. Equal numbers of sorted cells were transferred into CD45.1+ congenic naive mice, and mice were rested to determine the maintenance of these cells in the absence of Ag. Monitoring the peripheral blood of recipient mice for CD45.2+ donor cells at 20 days after transfer revealed that the CD127high donor populations from naive and chronically infected mice were better maintained at this time than the CD127low donor populations (Fig. 4B). Phenotypic analysis revealed that the CD127high transferred cells retained their original CD127high phenotype in the absence of Ag. In addition, the donor cells in the recipients of CD127low cells were now ~40% CD127high, indicating either the preferential retention or expansion of the minor contaminating CD127high population in these recipients or the conversion of CD127low into CD127high cells (Fig. 4B).


Figure 4
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 4. CD127highCD8+ T cells persist in the absence of Ag. A, CD45.2+ splenocytes from mice chronically infected (350 dpi) or from naive age-matched mice were enriched for CD8+ T cells and sorted into CD127highCD44high and CD127lowCD44high groups. Sort purity was ~85 and ~90% for the CD127high and CD127low populations, respectively. Equal numbers (1.3 x 106) of CD127highCD44high or CD127lowCD44lowCD45.2+ T cells were transferred into naive CD45.1+ recipients. B, Mouse peripheral blood cells were stained with the TSKB20/Kb tetramer and for surface expression of CD45.2, CD8, CD127, and non-CD8 lineage Abs (anti-CD4, anti-CD11b, and anti-B220) 20 days following transfer. The top panels are gated on CD4–CD11b–B220– cells. Numbers in the top panels represent the percentage of transferred CD8+ T cells of the total CD8+ T cell population. Bottom panels are gated on CD45.2+CD8+ T cells and numbers indicate the percentage of cells that are CD127high. Gating for the distribution of CD127 staining on CD45.2+CD8+ T cells was set using an unstained negative control (FMO control) and the staining pattern of CD127 on bulk naive CD8+ T cells. C, Mice receiving transfers were challenged with 10,000 Brazil strain parasites 21 days after transfer. Splenocytes from mice 12 days after challenge were stained with the TSKB20/Kb tetramer and for expression of CD8, CD127, CD45.2, and BrdU. Top panels are gated on CD4–CD11b–B220– cells and bottom panels are gated on the total CD8+ T cell population. Numbers in top panels indicate the percentage of transferred CD8+ T cells of the total CD8+ T cell population and numbers in the bottom panels indicate the percentage of TSKB20/Kb+ cells out of the total CD8+ T cell population. D, Proliferation and phenotype of donor cells following challenge infection. Dashed lines show the total CD8+ T cell donor population and solid lines show the TSKB20-specific donor population. The top and bottom numbers in each overlay represent the total CD8+ T cell donor population and the TSKB20-specific donor population, respectively, that incorporated BrdU (top panel) or that are CD127high (lower panel). Results are an average of three mice per group. Gating for the distribution of CD127 staining on donor cells was set using an unstained control and the staining pattern of CD127 on bulk naive and chronic CD8+ T cells. Gating for BrdU incorporation in donor cells was set based on an unstained negative control (FMO control). Data are representative of two independent experiments.

 
To determine the in vivo proliferative capacity of the transferred cell populations, the mice that received donor cells were infected with T. cruzi at 21 days after transfer and administered BrdU in drinking water. Examination of the CD8+ T cell population in the spleen 12 days after infection revealed that donor cells in the recipients of both CD127high and CD127low cells from chronically infected mice had expanded substantially as revealed by the frequency of donor cells (Fig. 4C) and by BrdU incorporation (Fig. 4D). The donor CD127high cells exhibited the highest frequency as expected based on their greater survival during the 21-day Ag-free period. Also, the majority of the initially CD127high and CD127low donor cells were CD127low following challenge, indicating the conversion to a TE/TEM phenotype (Fig. 4D). Naive CD127high donor cells had also expanded following challenge, although far fewer of these cells incorporated BrdU and converted to a CD127low phenotype compared with chronic donor cells (Fig. 4D).

TSKB20-specific CD8+ T cells also expanded following challenge infection in the CD127high and CD127low chronic donor populations but were undetectable in the naive CD127high population (Fig. 4C). Transferred TSBK20-specific and total CD8+ T cells from chronic CD127high and CD127low donors incorporated similar amounts of BrdU (Fig. 4D). These results indicate that CD127highCD8+ T cells from chronically infected mice are more efficiently maintained in naive mice than CD127low cells; however, donor cells in the recipients of both CD127high and CD127low cells expand robustly following antigenic challenge.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate that Ag-independent CD8+ T cell memory can be generated and maintained despite pathogen persistence in T. cruzi infection. A heterogeneous subset of Ag-specific CD8+ T cells was detected in chronically infected mice that expressed markers typically associated with long-term memory, including CD127, CD62L, and CCR7. These CD127high Ag-specific cells were capable of producing IFN-{gamma} in response to T. cruzi peptides, were present in CD8+ T cell populations with distinct T. cruzi Ag specificities and frequencies, and were stably maintained when transferred to an Ag-free environment. These data are in contrast to numerous studies (3, 11, 12, 20), suggesting that persistent infections in general compromise the maintenance of long-term, Ag-independent CD8+ T cell memory.

Although the characteristics of CD8+ T cell memory generated during acute resolving infections have been well documented, only recently has work focused on memory responses during chronic infections, revealing that memory T cells present in chronic infections are often phenotypically and functionally distinct from memory T cells in acute resolving infections (1). Specifically, the detrimental effects of persistent Ag on pathogen-specific CD8+ T cells often reported in viral models of infection include loss of cytokine production and cytolytic activity, impaired proliferation, and eventual deletion in the most extreme circumstances (11, 12, 21, 22). Maintenance of CD8+ T cell memory is also affected, as responsiveness to IL-7 and IL-15 is impaired in antiviral CD8+ T cell responses in persistent infections (3, 20, 23), suggesting that long-term immunity may be compromised following infection resolution. Although these findings highlight important characteristics of CD8+ T cell memory during high-load chronic infections, many factors, including the frequency and duration of Ag encounter as well as the cell tropism of the pathogen, may dictate the final outcome of Ag-specific CD8+ T cells during various persistent infections. T. cruzi infection differs dramatically from models of high-load persistent viral infections in that parasites persist at low levels and in muscle, adipose, and neuronal tissues. It is likely that these factors account at least partially for the generation of Ag-independent CD8+ T cell memory in this persistent infection.

CD8+ T cell memory during chronic infections is thought to be sustained primarily by the presence of Ag, resulting in a population of relatively short-lived TEM cells (3, 20). Consistent with this idea, we have previously shown that the majority of CD8+ T cells exhibit a CD44highCD62Llow TEM-like phenotype during chronic infection with T. cruzi (24). In the present study, we chose to focus on expression of CD127, a marker that identifies memory CD8+ T cell precursors capable of generating long-lived memory CD8+ T cells. CD127 is down-regulated on CD8+ T cells following recent TCR stimulation (25, 26) and, as a result, CD8+ T cells in chronic infections generally fail to express CD127 due to ongoing Ag stimulation (3, 11, 12, 20). Consistent with these findings, the majority of TSKB20-specific CD8+ T cells showed a CD127low phenotype over the entire course of T. cruzi infection. However, a subpopulation of CD127high TSKB20-specific CD8+ T cells was detected throughout the acute phase of infection which increased in frequency during the chronic phase, suggesting that memory precursors are generated during acute infection.

The TSKB20-specific population in chronically infected mice was heterogeneous, as approximately one-third of CD127hightetramer+ cells also expressed CD62L. The heterogeneity of the CD127high population could suggest that parasite-specific cells may have distinct histories of Ag encounter and/or may be at different points along a differentiation pathway, leading to the acquisition of a stable TCM phenotype. This is certainly a possibility, as studies by Kaech et al. (9) have demonstrated that acquisition of CD62L expression occurs over time and in the absence of antigenic stimulation. Expression of KLRG1, a receptor indicating repetitive Ag stimulation (27) or replicative senescence (19), was also examined on the CD127high TSKB20-specific population. Approximately 60% of CD127high cells were KLRG1low, thus resembling Ag-specific CD8+ T cells from acute resolving infections and indicating that this population of T. cruzi-specific CD8+ T cells has not been recently or repeatedly activated. This is perhaps not surprising given that T. cruzi persists at very low levels in only certain tissues, making the likelihood of encountering Ag lower than that during infections characterized by high pathogen load. Recruitment of naive CD8+ T cells into the memory pool during the chronic phase of infection also is likely to contribute to the heterogeneity of these TCM populations. Indeed, Vezys et al. (28) have shown that newly generated naive CD8+ T cells primed during low-level persistent polyoma virus infection contribute to the diversity of the antiviral memory pool, and preliminary data suggest that naive priming is also occurring during chronic T. cruzi infection (L. M. Bixby and R. L. Tarleton, unpublished data).

Memory phenotype CD127highCD8+ T cells from chronically infected mice were better maintained following transfer into naive hosts than their CD127low counterparts, presumably due to their ability to receive survival signals from IL-7 through expression of CD127. Nonetheless, a smaller fraction of CD127low sorted donor cells were also maintained following transfer to an Ag-free environment. Notably, nearly half of these sorted CD127low donors did not maintain their original phenotype and were CD127high 20 days following transfer to naive mice. These data could suggest that CD127low cells are capable of recovering a CD127high phenotype, allowing their long-term survival, following removal from Ag. However, we cannot rule out the possibility that a minor contaminating CD127high population survived and expanded in these recipients. These rested donor cells in recipients of both CD127high and CD127low cells also expanded following challenge infection and showed a CD127low phenotype, consistent with a TE or TEM phenotype expected after activation.

Collectively, our data support a model in which as Ag load is controlled during T. cruzi infection, a TCM-like population is observed that is composed of a pool of cells that rarely encounters Ag and likely includes newly recruited cells from the naive population. Although these TCM-like cells are presumably activated periodically by Ag as a consequence of parasite persistence, the TCM phenotype may restrict homing of these cells to lymphoid tissues, making their presence at sites of parasite persistence rare. TCM cells in this model can therefore serve several functions. TCM cells could provide a pool for the production of additional TE/TEM cells when Ag does reach a draining lymph node, either during persistent infection or following reinfection. Additionally, TCM cells likely represent a subset of cells that can preserve long-term T cell memory in the case that Ag is eventually cleared. Thus, the data presented here have significant implications for long-term immunological protection in individuals that have been successfully drug treated during T. cruzi infection. Benznidazole is the principle drug available for the treatment of T. cruzi infection, although this compound has serious potential side effects and a reported variable efficacy in clearing the infection. Nonetheless, we have recently documented complete parasite clearance in mice treated with benznidazole that resulted in the emergence of a protective T. cruzi-specific, predominantly CD127high TCM population (29). These parasite-specific TCM cells present in mice following drug cure were detected for >300 days after treatment and likely arise from the minor CD127high TCM-like population we have detected in chronically infected mice and have reported on here. These findings have implications for the majority of individuals with Chagas’ disease, who are not diagnosed and therefore do not receive drug treatment until symptoms appear decades following infection.

Similar evidence has recently been presented for TCM-like cells during persistent hepatitis C and {gamma}-herpesvirus infection (8, 25), both of which are also characterized by low Ag load. These studies and the data presented here demonstrate that not all chronic infections uniformly result in CD8+ T cell dysfunction, including the failure to generate Ag-independent memory, and emphasize that the presence of TEM/TCM cells is not determined by whether Ag is cleared, but individually at the T cell level based on how frequently Ag is encountered.


   Acknowledgments
 
We thank Julie Nelson of the Center for Tropical and Emerging Global Diseases Flow Cytometry Facility at the University of Georgia for technical assistance; Juan Bustamante, Matthew Collins, Charles Rosenberg, and Angel Padilla for technical support; Kimberly Klonowski for advice on this manuscript; and Diana Martin for critical reading of this manuscript.


   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 Research Grants AI-022070 and AI-033106 (to R.L.T.). Back

2 Address correspondence and reprint requests to Dr. Rick L. Tarleton, Center for Tropical and Emerging Global Diseases, 310 Coverdell Center, 500 D.W. Brooks Drive, University of Georgia, Athens, GA 30602. E-mail address: Tarleton{at}cb.uga.edu Back

3 Abbreviations used in this paper: TCM, T central memory; TE, T effector; TEM, T effector memory; TCT, tissue culture trypomastigote; SC, spleen cell; CRZP, cruzipain; GFT, β-galactofuranosyltransferase; FMO, fluorescence minus one. Back

Received for publication February 22, 2008. Accepted for publication June 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Shin, H., E. J. Wherry. 2007. CD8 T cell dysfunction during chronic viral infection. Curr. Opin. Immunol. 19: 408-415. [Medline]
  2. Murali-Krishna, K., L. L. Lau, S. Sambhara, F. Lemonnier, J. Altman, R. Ahmed. 1999. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science 286: 1377-1381. [Abstract/Free Full Text]
  3. Wherry, E. J., D. L. Barber, S. M. Kaech, J. N. Blattman, R. Ahmed. 2004. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc. Natl. Acad. Sci. USA 101: 16004-16009. [Abstract/Free Full Text]
  4. Schluns, K. S., K. Williams, A. Ma, X. X. Zheng, L. Lefrancois. 2002. Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells. J. Immunol. 168: 4827-4831. [Abstract/Free Full Text]
  5. Becker, T. C., E. J. Wherry, D. Boone, K. Murali-Krishna, R. Antia, A. Ma, R. Ahmed. 2002. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. J. Exp. Med. 195: 1541-1548. [Abstract/Free Full Text]
  6. Tan, J. T., B. Ernst, W. C. Kieper, E. LeRoy, J. Sprent, C. D. Surh. 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med. 195: 1523-1532. [Abstract/Free Full Text]
  7. Northrop, J. K., H. Shen. 2004. CD8+ T-cell memory: only the good ones last. Curr. Opin. Immunol. 16: 451-455. [Medline]
  8. Cush, S. S., K. M. Anderson, D. H. Ravneberg, J. L. Weslow-Schmidt, E. Flano. 2007. Memory generation and maintenance of CD8+ T cell function during viral persistence. J. Immunol. 179: 141-153. [Abstract/Free Full Text]
  9. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  10. Kaech, S. M., S. Hemby, E. Kersh, R. Ahmed. 2002. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111: 837-851. [Medline]
  11. Lang, K. S., M. Recher, A. A. Navarini, N. L. Harris, M. Lohning, T. Junt, H. C. Probst, H. Hengartner, R. M. Zinkernagel. 2005. Inverse correlation between IL-7 receptor expression and CD8 T cell exhaustion during persistent antigen stimulation. Eur. J. Immunol. 35: 738-745. [Medline]
  12. Fuller, M. J., D. A. Hildeman, S. Sabbaj, D. E. Gaddis, A. E. Tebo, L. Shang, P. A. Goepfert, A. J. Zajac. 2005. Cutting edge: emergence of CD127high functionally competent memory T cells is compromised by high viral loads and inadequate T cell help. J. Immunol. 174: 5926-5930. [Abstract/Free Full Text]
  13. Tewari, K., J. Sacha, X. Gao, M. Suresh. 2004. Effect of chronic viral infection on epitope selection, cytokine production, and surface phenotype of CD8 T cells and the role of IFN-{gamma} receptor in immune regulation. J. Immunol. 172: 1491-1500. [Abstract/Free Full Text]
  14. Sierro, S., R. Rothkopf, P. Klenerman. 2005. Evolution of diverse antiviral CD8+ T cell populations after murine cytomegalovirus infection. Eur. J. Immunol. 35: 1113-1123. [Medline]
  15. 2002. Control of Chagas disease. World Health Organization Technical Report Series 905: i-vi, 1–109, back cover.
  16. Zhang, L., R. L. Tarleton. 1999. Parasite persistence correlates with disease severity and localization in chronic Chagas’ disease. J. Infect. Dis. 180: 480-486. [Medline]
  17. Brandariz, S., A. Schijman, C. Vigliano, P. Arteman, R. Viotti, C. Beldjord, M. J. Levin. 1995. Detection of parasite DNA in Chagas’ heart disease. Lancet 346: 1370-1371. [Medline]
  18. Martin, D. L., D. B. Weatherly, S. A. Laucella, M. A. Cabinian, M. T. Crim, S. Sullivan, M. Heiges, S. H. Craven, C. S. Rosenberg, M. H. Collins, et alCD8+ T-cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes. PLoS Pathog. 2: e77
  19. Voehringer, D., C. Blaser, P. Brawand, D. H. Raulet, T. Hanke, H. Pircher. 2001. Viral infections induce abundant numbers of senescent CD8 T cells. J. Immunol. 167: 4838-4843. [Abstract/Free Full Text]
  20. Shin, H., S. D. Blackburn, J. N. Blattman, E. J. Wherry. 2007. Viral antigen and extensive division maintain virus-specific CD8 T cells during chronic infection. J. Exp. Med. 204: 941-949. [Abstract/Free Full Text]
  21. Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, R. Ahmed. 2003. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77: 4911-4927. [Abstract/Free Full Text]
  22. Fuller, M. J., A. J. Zajac. 2003. Ablation of CD8 and CD4 T cell responses by high viral loads. J. Immunol. 170: 477-486. [Abstract/Free Full Text]
  23. Obar, J. J., S. G. Crist, E. K. Leung, E. J. Usherwood. 2004. IL-15-independent proliferative renewal of memory CD8+ T cells in latent {gamma}herpesvirus infection. J. Immunol. 173: 2705-2714. [Abstract/Free Full Text]
  24. Martin, D. L., R. L. Tarleton. 2005. Antigen-specific T cells maintain an effector memory phenotype during persistent Trypanosoma cruzi infection. J. Immunol. 174: 1594-1601. [Abstract/Free Full Text]
  25. Bengsch, B., H. C. Spangenberg, N. Kersting, C. Neumann-Haefelin, E. Panther, F. von Weizsacker, H. E. Blum, H. Pircher, R. Thimme. 2007. Analysis of CD127 and KLRG1 expression on hepatitis C virus-specific CD8+ T cells reveals the existence of different memory T-cell subsets in the peripheral blood and liver. J. Virol. 81: 945-953. [Abstract/Free Full Text]
  26. Franchimont, D., J. Galon, M. S. Vacchio, S. Fan, R. Visconti, D. M. Frucht, V. Geenen, G. P. Chrousos, J. D. Ashwell, J. J. O'Shea. 2002. Positive effects of glucocorticoids on T cell function by up-regulation of IL-7 receptor {alpha}. J. Immunol. 168: 2212-2218. [Abstract/Free Full Text]
  27. Thimme, R., V. Appay, M. Koschella, E. Panther, E. Roth, A. D. Hislop, A. B. Rickinson, S. L. Rowland-Jones, H. E. Blum, H. Pircher. 2005. Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation. J. Virol. 79: 12112-12116. [Abstract/Free Full Text]
  28. Vezys, V., D. Masopust, C. C. Kemball, D. L. Barber, L. A. O'Mara, C. P. Larsen, T. C. Pearson, R. Ahmed, A. E. Lukacher. 2006. Continuous recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells during persistent infection. J. Exp. Med. 203: 2263-2269. [Abstract/Free Full Text]
  29. Bustamante, J. M., L. M. Bixby, R. L. Tarleton. 2008. Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease. Nat. Med. 14: 542-550. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. C. Albareda, G. C. Olivera, S. A. Laucella, M. G. Alvarez, E. R. Fernandez, B. Lococo, R. Viotti, R. L. Tarleton, and M. Postan
Chronic Human Infection with Trypanosoma cruzi Drives CD4+ T Cells to Immune Senescence
J. Immunol., September 15, 2009; 183(6): 4103 - 4108.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
K. A. Jordan, E. H. Wilson, E. D. Tait, B. A. Fox, D. S. Roos, D. J. Bzik, F. Dzierszinski, and C. A. Hunter
Kinetics and Phenotype of Vaccine-Induced CD8+ T-Cell Responses to Toxoplasma gondii
Infect. Immun., September 1, 2009; 77(9): 3894 - 3901.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. L. Colpitts, N. M. Dalton, and P. Scott
IL-7 Receptor Expression Provides the Potential for Long-Term Survival of Both CD62Lhigh Central Memory T Cells and Th1 Effector Cells during Leishmania major Infection
J. Immunol., May 1, 2009; 182(9): 5702 - 5711.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. S. Cush and E. Flano
Protective Antigen-Independent CD8 T Cell Memory Is Maintained during {gamma}-Herpesvirus Persistence
J. Immunol., April 1, 2009; 182(7): 3995 - 4004.
[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 Bixby, L. M.
Right arrow Articles by Tarleton, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bixby, L. M.
Right arrow Articles by Tarleton, R. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS