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The Journal of Immunology, 2005, 175: 4686-4696.
Copyright © 2005 by The American Association of Immunologists

Functional Properties and Lineage Relationship of CD8+ T Cell Subsets Identified by Expression of IL-7 Receptor {alpha} and CD62L1

Martin F. Bachmann*, Petra Wolint{dagger}, Katrin Schwarz*, Petra Jäger* and Annette Oxenius2,{dagger}

* Cytos Biotechnology, Zurich-Schlieren, Switzerland; and {dagger} Swiss Federal Institute of Technology, Institute for Microbiology, Zurich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Three major subsets of Ag-experienced CD8+ T cells have been identified according to their expression of CD62L and CD127. These markers are associated with central memory T cells (CD62L+CD127+), effector memory T cells (CD162L–CD127+), and effector T cells (CD62L–CD127–). In this study we characterized the development of these three populations during acute and chronic viral infections and after immunization with virus-like particles and determined their lineage relation and functional and protective properties. We found that the balance between the three subsets was critically regulated by the availability of Ag and time. After initial down-regulation of CD127, the responding CD8+ T cell population down-regulated CD62L and re-expressed CD127. Dependent on Ag availability, the cells then further differentiated into CD62L–CD127– effector cells or, in the absence of Ag, re-expressed CD62L to become central memory T cells. Although all three populations efficiently produced effector cytokines such as IFN-{gamma}, CD62L–CD127– effector cells exhibited the highest ex vivo lytic potential. In contrast, CD62L+CD127+ central memory T cells most efficiently produced IL-2 and proliferated extensively in vitro and in vivo upon antigenic restimulation. Strikingly, only effector and effector memory, but not central memory, T cells were able to protect against peripheral infection with vaccinia virus, whereas central memory T cells were most potent at protecting against systemic infection with lymphocytic choriomeningitis virus, indicating that the antiviral protective capacities of specific CD8+ T cell subsets are closely related to the nature of the challenging pathogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naive CD8+ T cells are activated in secondary lymphoid organs, and the nature of the APC, the duration and strength of Ag encounter, the cytokine milieu, and the presence of Th cells have been implicated in the outcome of this activation process (reviewed in Refs.1, 2, 3, 4). Efficient activation of naive CD8+ T cells to become effector cells and to generate elevated frequencies of long-lived memory cells involves priming by mature, activated dendritic cells (5, 6), prolonged in vivo Ag encounter for at least 1 wk (7), and the presence of Th cells (8) for the induction of proliferation competent memory cells (9, 10, 11, 12, 13). Such a priming event induces a differentiation program in CD8+ T cells that involves clonal expansion and acquisition of effector functions, such as the ability to produce cytokines and cytolytic potential (14, 15). Concomitantly, cell surface receptors involved in migration and homing are modulated; activated CD8+ T cells down-regulate the lymph node (LN)3-homing receptors CD62L and CCR7 (16) as well as cell surface molecules involved in LN retention, such as sphingosine-1-P (17), and they up-regulate tissue-homing receptors, such as the integrins VLA-4 (18), VLA-1 (19), and cutaneous lymphocyte-associated Ag (20, 21). Moreover, precursors of long-lived memory CD8+ T cells up-regulate antiapoptotic molecules and surface expression of IL-15R and IL-7R{alpha}, which allows their homeostatic maintenance in the absence of further Ag challenge (13, 22).

In vitro studies have shown that a short Ag contact induces a synchronized full differentiation program in naive CD8+ T cells (14, 15). In contrast, in vivo Ag-specific effector and memory CD8+ T cells represent a heterogeneous population of Ag-experienced CD8+ T cells with respect to their phenotype, function, and localization (4, 23). Such heterogeneity seems to be partially inherent of a nonsynchronized in vivo activation process and may allow the identification of lineage relationships between subsets of memory cells (24, 25, 26). However, such heterogeneity is also crucially influenced by Ag persistence (23, 27).

Early studies divided memory T cells into activated and resting cells (28, 29). Sallusto et al. (16) subsequently proposed to divide memory T cells into central (TCM) and effector memory (TEM) T cells according to their expression of CCR7 or CD62L (16, 30). CD62L–CCR7– TEM and CD62L+CCR7+ TCM have different recirculation patterns and hence reside mainly in different anatomical compartments. TCM are prevalent in LNs, whereas TEM are mostly localized in peripheral tissues; both subsets are found in blood and spleen. Furthermore, it was shown that TCM and TEM may differ in their functional abilities, such as cytokine secretion and immediate cytolytic potential (16, 31, 32, 33). However, there is some discrepancy on this issue between different studies conducted in different experimental systems and between Ag-specific CD4+ and CD8+ T cells (24, 34, 35, 36). Although different cytokine secretion profiles, in particular for IFN-{gamma}, were observed between central and effector CD4+ T cells, there seems to be less difference in IFN-{gamma} secretion potential between TCM and TEM CD8+ T cells (24, 34, 35, 36). In contrast, a significant difference in immediate cytolytic potential between TCM and TEM CD8+ T cells has been observed, with TEM CD8+ T cells exhibiting increased levels of cytotoxicity (13, 32, 36, 37).

More recently it has been shown that CD127 (IL-7R {alpha}-chain) and/or CD8{alpha}{alpha} expression is a hallmark of primed CD8+ T cells that are able to develop into long-lived memory cells (13, 22, 38, 39). CD127 expression during the acute phase of an infection identified a subset of effector cells that were able to persist into the memory phase. Adoptively transferred CD127+CD8+ T but not CD127–CD8+ T cells survived in the absence of Ag by homeostatic proliferation and thus maintenance via CD127 (13, 22).

In this study we used CD62L and CD127 expression to longitudinally characterize virus-specific CD8+ T cell populations induced by various immunization regimens that differed in the level and duration of Ag availability. Three major distinct populations of CD8+ T cells contributed to the CD8+ T cell memory pool and their relative proportions depended on the timing of analysis after infection, the nature of the priming Ag, and the level of Ag persistence. Importantly, the phenotypic differences in these memory populations were linked to distinct functional and antiviral protective capacities. Finally, we characterized the lineage relationship among these subpopulations and showed that CD62L–CD127+ T cells are the first emerging population of Ag-primed cells that, upon additional Ag contact, down-regulate CD127 to become CD62L–CD127– cells or, in the absence of additional Ag contact, slowly up-regulate CD62L to become CD62L+CD127+ cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, viruses, virus-like particles, and peptides

C57BL/6 and BALB/c mice were purchased from Janvier, Ly5.1+ 327 TCR transgenic (tg) mice (40) were maintained in a specific pathogen-free facility, and mice were immunized between 8 and 12 wk of age. Animal experiments were performed according to the regulations of the cantonal veterinary office.

The lymphocytic choriomeningitis virus (LCMV) isolates WE and Docile were provided by Dr. R. M. Zinkernagel (University Hospital, Zurich, Switzerland) and were propagated at low multiplicity of infection on L929 fibroblast cells or on Madin-Darby kidney cells, respectively. Mice were infected i.v. with 200 PFU of LCMV-WE or with 5 x 106 PFU of LCMV Docile. The LCMV glycoprotein peptide aa 33–41 (gp33 peptide, KAVYNFATM) was purchased from Neosystem.

Recombinant vaccinia virus expressing LCMV glycoprotein (VVG2) was originally obtained from Dr. D. H. L. Bishop (Oxford University, Oxford, U.K.) and grown on BSC cells at low multiplicity of infection, and quantification was performed as described previously (41).

Gp33-virus-like particles (gp-VLPs) based on peptide gp33 coupled to VLPs derived from the bacteriophage Q{beta} have been described previously (42). Packaging of CpG oligonucleotides (5'-GGGGTCAACGTTGAGGGGGG-3', thioester stabilized) into the gp33-VLPs was performed as described previously (42). Mice were immunized with 150 µg of gp33-VLPs.

Bacterial artificial chromosome-derived murine CMV (MCMV) MW97.01 was provided by Prof. U. H. Koszinowski (Max von Pettenkofer-Institut, Munich, Germany) and has previously been shown to be biologically equivalent to MCMV Smith strain ATCC VR-194 (recently re-accessioned as VR-1399; American Type Culture Collection) and is here referred to as MCMV (43). MCMV was grown on mouse embryonic fibroblasts and purified by sucrose cushion centrifugation according to established protocols (44). MCMV titers of virus stocks were determined by virus plaque assays on mouse embryonic fibroblasts as described using centrifugal enhancement of infectivity (44).

Viral challenge

Adoptively transfused female mice were infected i.p. with 5 x 105 PFU of VVG2. Four days later, ovaries were collected, and vaccinia titers were determined by plaque assay on BSC40 cells as previously described (41). Alternatively, adoptively transfused mice were challenged with 200 PFU of LCMV WE, and viral titers were determined in the spleen 4 days later by plaque assays of organ homogenates on MC57G cells (45).

Abs and peptide MHC class I tetramers

Allophycocyanin- or PE-conjugated peptide/MHC class I tetrameric complexes were generated as previously described (46). The following anti-mouse mAbs were purchased from BD Pharmingen: anti-CD45.1 (PE or biotin), anti-CD127 (PeCy5), anti-IFN-{gamma} (FITC, PE, or allophycocyanin), anti-IL-2 (allophycocyanin), anti-CD8 (PerCP or allophycocyanin), and anti-CD62L (FITC or allophycocyanin). Anti-CD127 (FITC) was purchased from eBioscience.

Cell stimulation, immunofluorescent staining, and analysis

For direct staining, whole blood or single-cell suspensions from spleens, LNs, or ovaries were used. Lymphocytes were isolated from lung and liver as described previously (47, 48). Cells were incubated for 20 min at 4°C with peptide/MHC tetramers or anti-CD45.1 Ab together with anti-CD8-, anti-CD127-, and anti-CD62L-specific Abs. For intracellular IFN-{gamma} or IL-2 staining, cells were stimulated with gp33 peptide for 6 h in the presence of monensin A, washed, surface stained at 4°C, and fixed/permeabilized using 500 µl of FIX/perm solution (FIX/perm solution (FACSLyse; BD Pharmingen) diluted to 2x concentration with H2O and 0.05% Tween 20 (Sigma-Aldrich)). Cells were washed once and incubated at room temperature with directly conjugated Abs specific for intracellular proteins. Cells were washed and resuspended in PBS containing 1% paraformaldehyde (Sigma-Aldrich). Four-color flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) with CellQuest software (BD Biosciences). List mode data were analyzed using WinList software (Verity Software House).

Isolation of T cell subsets

In most experiments 105 Ly5.1+ TCR tg spleen cells were adoptively transfused in naive C57BL/6 recipients, followed by infection with 200 PFU of LCMV WE. CD8+ T cells were purified first by magnetic cell sorting (Miltenyi Biotec) according to the manufacturer’s instructions. For further purification of CD62L/CD127-expressing cell subsets, Ly5.1+CD8+ cells were purified by FACS sorting (FACS Aria) according to the following expression profiles: CD127+CD62L+, CD127+CD62L–, and CD127–CD62L–.

Direct ex vivo cytotoxicity assay

51Cr release assays were used for the determination of LCMV-gp33-specific cytotoxicity ex vivo on EL4 target cells as described previously (49). In all cases, the starting E:T cell ratio was adjusted to obtain identical ratios of Db-gp33-specific CD8+ T cells to target cells. Spontaneous release was <25% in all 6-h assays and <40% in all 14-h assays.

CFSE-based proliferation assay

Sorted Ly5.1+ TCR tg populations (105) were mixed with 106 naive Ly5.2+ C57BL/6 spleen cells and were CFSE labeled (50). Cells were incubated in RPMI 1640/10% FCS for 3 days in the presence of 10–9 M gp33 peptide or PMA/ionomycin. Cells were stained for Ly5.1 and CD8, and proliferation was assessed by CFSE dilution.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotype of effector and memory CD8+ T cells

CD127 (IL-7R{alpha}) is expressed on naive CD8+ T cells and on long-lived Ag-independent memory CD8+ T cells (13, 22). In long-lived memory CD8+ T cells, IL-7R and IL-15R expression allow for their cytokine-driven homeostatic maintenance (51, 52, 53, 54). CD62L (L-selectin) expression and CCR7 expression on T cells are required for homing of T cells via high endothelial venules to T regions of secondary lymphoid organs (16, 55, 56). Effector and TEM T cell populations express low levels of CD62L and CCR7, whereas naive and central memory cells express high levels of CD62L and CCR7.

We used CD127 and CD62L surface expression to longitudinally characterize the phenotype of Ag-specific CD8+ T cells after different viral infections or after immunization with replication-incompetent VLP, which were covalently decorated with the LCMV gp33–41 epitope (gp33-VLP) (42). We used gp33-VLP immunization or viral infections that exhibited various degrees of persistence to vary the duration and extent of Ag exposure: acute/resolved (gp33-VLP, low dose LCMV strain WE), low level persistent (MCMV), or high level persistent (high dose LCMV strain Docile; Fig. 1A).



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  		FIGURE 1. A, Long-term in vivo evolution of CD62L and CD127 expression on Ag-specific CD8+ T cells. C57BL/6 mice were immunized s.c. with 150 µg of gp33-VLP or were infected with 200 PFU of LCMV WE or 106 PFU of LCMV Docile. BALB/c mice were infected i.v. with 106 PFU of MCMV. H-2 Db-restricted gp33-specific CD8+ T cells (for gp33-VLP and LCMV) and H-2Ld-restricted pp89-specific CD8+ T cells (for MCMV) were longitudinally followed in blood by tetramer staining. CD127 and CD62L expression on CD8+tetramer+ T cells is shown for the respective time points. Representative stainings are shown for three to five mice per group. <det., below detection; n.d., not determined (because LCMV viral load was controlled at this time point). The percentages of tetramer+ cells of CD8+ T cells were: gp33-VLP: 7.9% (day 8), 1.0% (day 22), 0.4% (day 34), 0.25% (day 50); LCMV WE: 13.6% (day 8), 10.8% (day 22), 12.9% (day 34), 7.3% (day 110), 6.2% (day 180); MCMV: 6.7% (day 8), 4.2% (day 22), 6.8% (day 50), 11.7% (day 110), 13.7 (day 180); LCMV Docile: 4.9% (day 8), 7.8% (day 22), 6.2% (day 50), 4.8% (day 110). B, Phenotype of LCMV- and MCMV-specific memory CD8+ T cells in various organs. Lymphocytes from blood, spleen, LN, ovary, lung, and liver from LCMV WE-infected (day 414) or MCMV-infected (day 341) mice were stained by tetramer for LCMV gp33-specific or MCMV pp89-specific CD8+ T cells, and CD62L and CD127 expression was analyzed. The percentages of tetramer+ cells of CD8+ T cells were: LCMV: 15.9% (blood), 14.7% (spleen), 12.0% (LN), 11.7% (ovary), 3.6% (liver), 16.7% (lung); MCMV: 12.7% (blood), 11.6% (spleen), 6.7% (LN), 9.4% (ovary), 55.4% (liver), 27.4% (lung). Representative stainings are shown for three mice per group.

 
Eight days after immunization or infection, the majority of Ag-specific CD8+ T cells were within the CD62L–CD127– subset, consistent with an effector phenotype. With time, this CD62L–CD127– effector subset vanished in VLP-immunized or low dose LCMV WE-infected mice; particularly after LCMV infection, a robust population of CD62L–CD127+ cells emerged up to 50 days after infection. At later time points (days 110–180), the majority of LCMV-specific CD8+ T cells were CD62L+CD127+, compatible with a central memory phenotype. Such a steady evolution toward a CD62L+CD127+ T cell population was not observed after low or high level persistent viral infections. Specifically, between 20 and 180 days after MCMV infection, a relatively stable composition of three major subsets of MCMV-specific CD8+ T cells was observed: CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+. The long-term maintenance of CD62L–CD127–CD8+ T cells was even more pronounced after high dose LCMV Docile infection; in fact, up to day 110 after infection, no significant population of CD127+CD8+ T cells was observed. These results indicate that the level of Ag persistence is an important determinant of the relative composition of memory CD8+ T cell subsets with respect to CD62L and CD127 expression. The presence of increased frequencies of CD62L–CD127– cells during acute and high level persistent infections suggests that these are effector cells, whereas the CD62L–CD127+ and CD62L+CD127+ subsets may relate to TEM and TCM cells, as suggested previously (13).

To assess whether the analysis of CD8+ T cell memory phenotypes in blood was representative for other tissues, we compared CD62L and CD127 expression on LCMV- or MCMV-specific memory CD8+ T cells from blood, spleen, LN, ovary, lung, and liver (Fig. 1B). In the case of LCMV gp33-specific memory CD8+ T cells, almost all cells were CD62L+CD127+ on day 414 after infection in all organs analyzed, with only slightly increased frequencies of CD62L–CD127+ cells in ovaries and liver. Thus, the expression of CD62L and CD127 may not necessarily correlate with tissue distribution. In contrast, the population of MCMV pp89-specific CD8+ T cells was still composed of all three populations (CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+) in blood and spleen on day 341 after infection. The relative contributions of these three populations, however, varied with the organ analyzed; although most of the cells were CD62L+CD127+ or CD62L–CD127+ in LNs, spleen and blood contained an additional population of CD62L–CD127– cells. In contrast, in the lung, ovaries, and particularly liver, the majority of cells were CD62L–CD127–. These differences in relative composition of the phenotype of MCMV-specific memory CD8+ T cells in various organs is most likely due to organ-specific MCMV reactivation events (57, 58).

CD62L–CD127–CD8+ T cells have reduced surface TCR expression levels

Recently activated T cells show reduced TCR surface expression levels (59, 60). We therefore reasoned that TCR expression levels might differ in CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ CD8+ T cells early after immunization. TCR tg CD8+ T cells specific for the LCMV gp33–41 epitope were adoptively transferred into naive B6 hosts, followed by immunization with gp33-VLPs or infection with low dose LCMV WE (Fig. 2). Ten days later, gp33-specific CD8+ T cells were stained ex vivo with gp33-tetramers, and CD62L/CD127 expression was compared between CD8highTCRhigh and CD8lowTCRlow cells. After both immunizations, CD8lowTCRlow cells were predominantly CD62L–CD127–, whereas CD8highTCRhigh cells contained few CD62L–CD127– cells, but were enriched for CD127+ and CD62L+ cells. These results further strengthen the idea that CD62L–CD127– cells, at least early during T cell priming, are recently activated effector cells, whereas for CD127+ and CD62L+ CD8+ T cells, more time has elapsed since their last TCR engagement.



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  		FIGURE 2. Ag-specific CD8lowTCRlow cells predominantly contain CD62L–CD127– cells. Naive gp33–41-specific TCR tg CD8+ T cells (105) were adoptively transferred into naive B6 recipients and immunized s.c. with 150 µg of gp33-VLP or infected with 200 PFU of LCMV WE. Ten days later, splenocytes were stained with anti-CD8, gp33-tetramers, anti-CD62L, and anti-CD127. The upper plots show CD8-tetramer stainings. The lower plots are gated on the total population of CD8+tet+ cells (CD8+TCR+), on gp33-specific CD8highTCRhigh cells (R1), and on gp33-specific CD8lowTCRlow cells (R2). One of three experiments is shown.

 
Short-term lineage relationship between CD127+/– and CD62L+/– CD8+ T cells

We next studied in more detail the short-term in vivo kinetics of CD62L and CD127 expression level changes in naive Ag-specific CD8+ T cells and their relation to Ag load. Thus, naive Ly5.1+ TCR tg CD8+ T cells were purified, CFSE labeled, and adoptively transferred into Ly5.2+ recipient mice that had been immunized s.c. with gp33-VLPs 4 h, 3 days, or 6 days previously to vary the intensity and duration of Ag exposure. Three days after adoptive transfer, draining LNs were removed, and proliferation, phenotype, and function of the transferred cells were analyzed (Fig. 3, A and B). In all situations, transferred TCR tg CD8+ T cells had proliferated, although the most pronounced proliferation was observed in mice that were immunized on the day of cell transfer (Fig. 3A). However, significant CD62L down-regulation was only apparent in mice that were immunized at the time of transfer and only in cells that had undergone more than five divisions. In contrast, CD127 was rapidly down-regulated in cells that had a least undergone one cell division. Of note, re-expression of CD127 was only seen in mice that were immunized at the time of transfer and only in cells that had undergone more than five divisions. In fact, all CD62L– cells in these mice re-expressed CD127 (not shown).



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  		FIGURE 3. Short-term in vivo evolution of CD62L and CD127 expression on primed gp33-specific CD8+ T cells. Naive Ly5.1+ gp33–41-specific TCR tg CD8+ T cells (6 x 106) were CFSE labeled and adoptively transferred into naive Ly5.2+ B6 mice that had been immunized s.c. with 150 µg of gp33-VLP 6 days previously, 3 days previously, or on the day of transfer. Three days later, draining LNs were removed and stained for Ly5.1, CD62L, and CD127 expression (A) or restimulated in vitro with gp33 peptide and analyzed for intracellular IFN-{gamma} production (B). Plots are gated on Ly5.1+ TCR tg CD8+ T cells. The total numbers of Ly5.1+ TCR tg CD8+ T cells recovered from draining LNs were 4 x 105 ± 1.7 x 104 (day –6), 1.1 x 106 ± 1.8 x 105 (day –3), and 2.6 x 106 ± 8.5 x 105 (day 0). C, Secondary responses of TCR tg CD8+ T cells, transferred into recipients that had been gp33-VLP immunized 4 days (day –4) or 2 days (day –2) previously or at the time of transfer (day 0). On day 47, mice were challenged with 5 x 106 PFU of VVG2, and secondary expansion was analyzed in blood and ovaries. The middle graphs show a more detailed analysis of TCR tg CD8+ T cell frequencies in blood before and after VVG2 challenge, and the lower graph shows frequencies of TCR tg CD8+ T cells in ovaries 7 days after VVG2 challenge.

 
The early induction of activated CD62L+ T cells was confirmed in vitro; gp33-stimulated naive T cells exhibited a CD62L+ phenotype 48 h after stimulation. Interestingly, specific T cells down-regulated CD62L within hours after stimulation, presumably by proteolytic cleavage of the molecule. However, re-expression was gained within 24 h, confirming that sustained down-regulation of CD62L only occurred after a few days of stimulation (not shown).

The observation that no CD62L down-regulation and CD127 re-expression occurred in recipients that were immunized 3 or 6 days before cell transfer despite the presence of cells that had undergone five divisions suggests that a strong stimulus is required for the generation of CD62L–CD127+ cells. Furthermore, based on these results, we propose that the CD62L–CD127+ TEM cell is the first population of Ag-experienced cells that emerges 3 days after priming and is likely to be the source of the expanded CD8+ T cells observed later in the periphery (Fig. 3C). Re-expression of CD127 and down-modulation of CD62L correlated with effector function, because only cells that had undergone more than six divisions were able to produce high amounts of IFN-{gamma} (Fig. 3B).

We further analyzed the long-term consequences of these different priming regimens on generation of peripheral effector and memory CD8+ T cells. Thus, we longitudinally followed the frequencies of transferred CD8+ T cells in the blood up to 48 days after transfer (Fig. 3C). Interestingly, only cells that were transferred on the day of priming showed considerable expansion 7 days later before frequencies started to decline over the next month. Thus, optimal expansion of transferred cells was dependent on the availability of sufficient Ag during the priming period and was associated with the initial in vivo differentiation of CD62L–CD127+ cells. Strikingly, after challenge infection with a recombinant vaccinia virus expressing the LCMV glycoprotein (VVG2), secondary expansion of gp33-specific CD8+ T cells was exclusively observed in mice in which initial priming and adoptive transfer of naive TCR tg CD8+ T cells were performed on the same day (Fig. 3C). Together with the observation that virtually all T cells showed an activated phenotype 3 days after priming, these results indicate that brief antigenic exposure may ultimately result in an abortive T cell response and even a state of unresponsiveness. Thus, a very short-term Ag encounter in vivo is not sufficient to drive the full differentiation program in naive CD8+ T cells, including the development of memory T cells.

Functional characteristics of different memory CD8+ T cell subpopulations

We also addressed the question of whether the different memory populations, defined by CD62L and CD127 expression, differed in their functional capacities. We transferred naive Ly5.1+ TCR tg CD8+ T cells to Ly5.2 recipients and infected them with 200 PFU of LCMV WE. Seven or 21 days after infection, Ly5.1+ TCR tg CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ populations. Sorted cells were analyzed subsequently for their in vitro proliferative capacity, their cytokine secretion capacity, and their direct cytotoxic activity.

Proliferative capacity. Sorted Ly5.1+ cells from days 7 and 21 after infection were CFSE labeled together with Ly5.2+ splenic APCs from naive mice and stimulated with 10–9 M gp33 peptide or PMA/ionomycin for 3 days. Proliferation was analyzed by CFSE dilution (Fig. 4). All gp33-specific populations that were sorted 7 days after LCMV infection were refractory to gp33 stimulation. In contrast, PMA/ionomycin stimulation induced significant proliferation of CD62L+CD127+ cells, modest proliferation of CD62L–CD127+ cells, and almost no proliferation of CD62L–CD127– cells. The gp33-specific populations that were sorted 21 days after LCMV infection were no longer refractory to gp33 stimulation; in fact, all populations proliferated well after peptide stimulation. However, although CFSElow cells accumulated in high numbers with both CD127+ populations, CFSElow cells did not accumulate in the CD62L–CD127– population (compare size of CFSElow peaks), suggesting that proliferating CD62L–CD127– cells were unable to survive.



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  		FIGURE 4. In vitro proliferation of the three CD8+ T cell subsets. Naive Ly5.1+ gp33–41-specific TCR tg CD8+ T cells (1 x 105) were adoptively transferred into naive Ly5.2+ B6 mice, followed by infection with 200 PFU of LCMV WE. Seven or 21 days after infection, CD8+ T cells were purified from the spleen and Ly5.1+CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, or CD62L+CD127+. FACS-sorted cells were mixed with Ly5.2+ splenocytes (serving as APCs), CFSE labeled, and stimulated with PMA/ionomycin or 10–9 M gp33 peptide. CFSE dilution was analyzed after 3-day culture. Dotted lines, naive Ly5.2+ splenocytes; solid line, FACS-sorted Ly5.1+ TCR tg CD8+ T cells. Note that naive Ly5.2+ splenic APCs proliferate upon stimulation with PNA/ionomycin.

 
Cytokine production. Next, the cytokine secretion profiles of the three subpopulations were analyzed. Sorted cells were stimulated with gp33 peptide, and 6 h later, IFN-{gamma} and IL-2 production was assessed (Fig. 5). The CD62L+CD127+ cells contained the highest percentage of IFN-{gamma}-producing cells and the highest intensity of IFN-{gamma} staining, followed by CD62L–CD127+ and CD62L–CD127– cells. The ability of cells to respond to antigenic stimulation by cytokine production depends on their history of recent Ag encounter; cells that have recently encountered Ag become more refractory to further antigenic stimulation (61, 62, 63, 64). The same ranking as that for IFN-{gamma} was observed for specific IL-2 production, with the CD62L–CD127– cells being completely incapable of IL-2 production.



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  		FIGURE 5. IFN-{gamma} and IL-2 production of the three CD8+ T cell subsets. Naive Ly5.1+ gp33–41-specific TCR tg CD8+ T cells (1 x 105) were adoptively transferred into naive Ly5.2+ B6 mice, followed by infection with 200 PFU of LCMV WE. Twenty-three days after infection, CD8+ T cells were purified from the spleen, and Ly5.1+ CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, or CD62L+CD127+; restimulated in vitro with gp33-loaded naive Ly5.2+ splenocytes; and analyzed for intracellular IFN-{gamma} and IL-2 production. Cells are gated on CD8+Ly5.1+. Numbers indicate the percentage of IFN-{gamma}- or IL-2-expressing T cells.

 
Immediate cytotoxicity. Immediate cytotoxicity was determined by 51Cr release assays; sorted cells were incubated with gp33-labeled target cells, and specific target cell lysis was determined after 6 and 14 h (Fig. 6). CD62L–CD127– cells showed the highest level of direct killing after 6 and 14 h of incubation, consistent with an effector phenotype. Similar, but low, levels of cytotoxicity were observed for the other two populations. Direct cytotoxicity requires prestored lytic effector molecules in cytotoxic granules; such intracellular stores of lytic effector molecules are regulated by Ag and are most abundant in cells that have most recently encountered Ag (36). These findings indicate that the CD62L–CD127– population is the most differentiated effector cell subset with respect to direct cytotoxicity, suggesting that the cells within this population might have received a particular strong activation signal or might have encountered Ag more recently than the other subsets.



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  		FIGURE 6. Immediate cytotoxicity of the three CD8+ T cell subsets. Naive Ly5.1+ gp33–41-specific TCR tg CD8+ T cells (1 x 105) were adoptively transferred into naive Ly5.2+ B6 mice, followed by infection with 200 PFU of LCMV WE. Twenty-one days after infection, CD8+ T cells were purified from the spleen, and Ly5.1+ CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, or CD62L+CD127+. Sorted cells were used as effectors in 6- and 14-h 51Cr release assays with gp33-loaded or unloaded EL4 cells as targets. The E:T ratio indicates the ratio between purified TCR tg CD8+ T cells and target cells.

 
Antiviral protective capacity. The question of whether CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ gp33-specific CD8+ T cells exhibited different protective capacities against challenge infections with either LCMV or VVG2 was addressed next. To this end, naive Ly5.1+ gp33-specific TCR tg CD8+ T cells were transferred into Ly5.2+ B6 recipients and infected with low dose LCMV WE. Ten days later, Ly5.1+ CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ cells, and these cells were transferred into naive Ly5.2+ B6 recipients. The following day, mice were challenged either i.p. with 5 x 105 PFU VVG2 (Fig. 7A) or i.v. with 200 PFU of LCMV WE (Fig. 7B). The peripheral expansion of the transferred T cells was monitored in the blood 4 days after viral challenge. After both viral challenges, CD62L+CD127+ T cells proliferated vigorously; however, expansion was more pronounced after VVG2 challenge than after LCMV challenge, most likely due to the difference in Ag load. In contrast, CD62L–CD127+ T cells proliferated to much lower levels, and CD62L–CD127– cells showed virtually no expansion. This pattern was also observed in the spleen 4 days after LCMV challenge (the main site of LCMV replication). In the ovaries (the main site of VV replication) of VVG2-challenged mice, the descendants of CD62L+CD127+ T cells were also 5-fold more abundant than the descendants of CD62L– cells. However, in the case of VVG2 challenge, the proliferative potential of CD62L+CD127+ T cells did not correlate with their protective capacity. In fact, there was an inverse correlation between the proliferative potential of gp33-specific CD8+ T cells and their protective capacity against VVG2 challenge; CD62L–CD127– cells showed the highest degree of protection, CD62L–CD127+ cells showed an intermediate degree of protection, and CD62L+CD127+ cells showed no protection. Taking into account that all three populations are able to produce IFN-{gamma} and TNF-{alpha} (not shown), the critical cytokines for control of vaccinia virus (41, 65), the absent protective potential of CD62L+CD127+ cells was not due to inappropriate effector function. Rather, the distinct anatomical distribution of the three cell populations may be responsible for the difference. In such a scenario, CD62L– cells would be able to enter peripheral tissues such as the ovaries, where they would be present in sufficient numbers at the time of VVG2 challenge and thus confer protection. CD62L+CD127+ cells, in contrast, would primarily home to secondary lymphoid tissues and would have to be reactivated by drained Ag before they would be able to migrate to peripheral sites of viral replication (72).



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  		FIGURE 7. In vivo protective capacity of the three CD8+ T cell subsets. Naive Ly5.1+ gp33–41-specific TCR tg CD8+ T cells (1 x 105) were adoptively transferred into naive Ly5.2+ B6 mice, followed by infection with 200 PFU of LCMV WE. Fifteen days after infection, CD8+ T cells were purified from the spleen, and Ly5.1+ CD8+ T cells were FACS sorted into CD62L–CD127– (62L-127–), CD62L–CD127+ (62L-127+), or CD62L+CD127+ (62L+127+) populations. Sorted cells (7 x 105) were adoptively transferred into naive Ly5.2+ recipient mice, and the frequency of transferred cells was measured 1 day later in blood. Mice were then challenged either i.p. with 5 x 105 PFU of VVG2 (A) or i.v. with 200 PFU of LCMV (B). Peripheral expansion of transferred cells was analyzed in blood, spleen, and ovaries. Virus titers were determined 4 days after challenge in ovaries (VVG2) or spleen (LCMV). Symbols represent the average of at least three mice per group. One of three similar experiments is shown.

 
An almost reciprocal picture was observed regarding the protective capacity of the three subpopulations against LCMV challenge (Fig. 7B). Proliferation-competent CD127+ cells were able to confer complete protection whereas proliferation-incompetent CD127– cells were not able to confer complete protection, although they were able to significantly reduce LCMV replication compared with naive controls. Apparently, the proliferative capacity, combined with the acquisition of cytolytic effector function is key for protection against a virus infection that predominantly takes place in secondary lymphoid organs and is controlled by cell-contact dependent cytotoxicity. The proliferation and thus expansion of effector cell numbers are likely to be important in such a situation where an almost stoichometric effector-target cell contact is required for antiviral effector function.

Long-term lineage relationship between CD127+/– and CD62L+/– CD8+ T cells

The long-term lineage relationship between CD127+/– and CD62L+/– CD8+ T cells was analyzed by adoptive transfer experiments of purified CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ CD8+ T cells. Naive Ly5.1+ gp33-specific TCR tg CD8+ T cells were transferred into Ly5.2+ B6 recipients and infected with low dose LCMV WE. Seven days later, Ly5.1+ CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ cells, and these were transferred into naive Ly5.2+ B6 recipients. CD127 and CD62L expression on the transferred cells was followed over a period of 60 days (Fig. 8). Starting 3 wk after transfer, a reversion of the surface phenotype was apparent; CD62L–CD127– cells up-regulated CD127 expression, and CD62L–CD127+ cells up-regulated CD62L expression. Specifically, CD62L–CD127– T cells first up-regulated CD127 expression, followed by an up-regulation of CD62L. By day 60 after transfer, the vast majority of cells were CD62L+CD127+ regardless of their phenotype at the time of transfer. Although we cannot completely rule out minor contaminations, our results nevertheless strongly suggest that CD62L–CD127– cells can at least partially revert to CD62L–CD127+ cells, and these, in turn, can revert to CD62L+CD127+ cells in the absence of Ag. If the number of surviving cells was assessed, the populations differed markedly, because CD62L+CD127+ T cells survived almost quantitatively, whereas the bulk of CD62L–CD127– cells disappeared over time (not shown) (13, 22). Note that the relatively large number of CD62L– cells in the CD62L+CD127+ sorted population is due to the fact that expression levels of CD62L+ and CD62L– populations are overlapping. However, because all sorted populations eventually reverted to a CD62L+CD127+ phenotype, we set the sorting gates in such a way that CD62L– cells would rather contaminate the CD62L+ population than the other way around.



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  		FIGURE 8. Long-term lineage relationship between CD127+/– and CD62L+/– CD8+ T cells. Naive Ly5.1+ gp33-specific TCR tg CD8+ T cells (105) were transferred into Ly5.2+ B6 recipients and infected with low dose LCMV WE. Seven days later, Ly5.1+ CD8+ T cells were FACS sorted into CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+ cells, and 1 x 106 sorted cells were transferred into naive Ly5.2+ B6 recipients. The purity of CD62L+CD127+ cells was 60%; the purity of CD62L–CD127– and CD62L–CD127+ cells was 80%. CD127 and CD62L expression on the transferred cells was followed over a period of 60 days. Graphs show the percentages of Ly5.1+ CD8+ T cells that are CD62L–CD127–, CD62L–CD127+, or CD62L+CD127+.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown that the nature of the priming Ag and the level of Ag persistence shape the composition of the memory CD8+ T cell population with respect to subpopulations defined by surface expression of CD62L and CD127. Upon acute infection, a majority of Ag-specific CD8+ T cells were CD62L–CD127–; however, with time and in the absence of measurable Ag persistence, an almost complete reversion to a CD62L+CD127+ phenotype occurred. Low level Ag persistence was associated with long-term maintenance of a heterogeneous memory CD8+ T cell population (CD62L–CD127–, CD62L–CD127+, and CD62L+CD127+), whereas a CD62L–CD127– population was maintained in a situation of high level Ag persistence. In a previous report, listeriolysin O91–99-specific CD8+ T cells were characterized by CD62L and CD127 expression during Listeria monocytogenes infection (13). Although the long-term evolution of these subsets was not analyzed, distinct functional properties were assigned to these subsets with respect to cytokine secretion and proliferative potential. IL-2 production was mainly confined to CD62L+CD127+ CD8+ T cells; CD62L–CD8+ T cells predominantly produced IFN-{gamma} and TNF-{alpha}, and CD62L–CD127– cells exhibited the highest degree of cytolytic potential (13). We confirmed for LCMV gp33-specific CD8+ T cells that IL-2 production is mainly restricted to CD62L+CD127+ T cells, whereas immediate cytotoxicity is largely restricted to CD62L–CD127– cells. Significant IFN-{gamma} production, however, was observed in all subpopulations, with the CD62L+CD127+ T cells producing the highest amounts of this cytokine. Reduced IFN-{gamma} production capacity has been shown for CD8+ T cells having had recent Ag encounter (61, 62, 63, 64); thus, our results showing that CD62L–CD127– cells produce less IFN-{gamma} than CD62L+CD127+ cells in addition to the observation that CD62L–CD127– cells have lower TCR expression levels suggest that the CD62L–CD127– population observed early after priming contains cells with relatively recent Ag encounter.

The in vivo protective potential against viral challenge infections of purified populations of CD62L–CD127–, CD62L–CD127+, or CD62L+CD127+ gp33-specific CD8+ T cells differed between systemic LCMV challenge (protection being mediated by contact-dependent perforin-mediated cytotoxicity) (66) and peripheral vaccinia virus challenge (protection being mediated by T cell produced IFN-{gamma} and TNF-{alpha}) (41, 65). Although proliferation-competent CD62L+CD127+ gp33-specific CD8+ T cells completely eliminated LCMV within 4 days after systemic viral challenge, the same cells were unable to mediate protection against peripheral vaccinia virus challenge. In contrast, poorly proliferating CD62L–CD127–CD8+ T cells were crucial for protection against vaccinia virus replication in the ovaries, i.e., in a peripheral solid organ. Because CD62L+CD127+ gp33-specific CD8+ T cells were efficiently producing IFN-{gamma} and TNF-{alpha} (not shown), the lack of protection was not due to functional incompetence, but was probably due to the secondary lymphoid organ homing properties of CD62L+CD127+ CD8+ T cells. Despite efficient reactivation and expansion of CD62L+CD127+ CD8+ T cells in secondary lymphoid organs, the time delay until the reactivated cells reached the ovaries where vaccinia virus replicated was too long for antiviral protection (72) (67, 68). In agreement with previous reports (24), proliferation-competent CD62L+CD127+ CD8+ T cells were most effective in protection against systemic LCMV challenge despite that fact that CD62L–CD127–CD8+ T cells exhibited the highest degree of immediate cytotoxicity. This suggests that clonal expansion and reacquisition of immediate cytolytic potential are the key for a protective mechanism that depends on direct cytotoxicity and hence on large numbers of effector cells required for an almost 1:1 stoichometric interaction with virally infected target cells. With respect to vaccinia virus protection, our results are in disagreement with a previous report that showed that proliferation-competent CD127+ cells are important for protection against vaccinia virus challenge (24). This discrepancy might be due to transfer of lower numbers of CD127– gp33-sepcific CD8+ T cells in the previous report, which would not home in large enough numbers to peripheral organs for mediating immediate and complete protection as we have seen in our experiments. In such a scenario, the expansion of proliferation-competent CD8+ T cells in secondary lymphoid organs might be relevant for the 10-fold reduction in viral titers seen 5 days after vaccinia virus challenge (24). However, in line with our results, immediate T cell-mediated protection against peripheral viral challenge was also dependent on the presence of tissue-resident T cells (TEM or effector cells) in other experimental systems, such as in intranasal challenge with influenza virus or Sendai virus (67, 68, 69). Thus, immediate protection against a fast replicating peripheral viral infection seems to critically depend on the presence of a sufficient number of specific T cells at the peripheral site of viral challenge (67, 68, 69), whereas protection against systemic infections or peripheral infections with slow replicating pathogens can afford restimulation and reactivation of TCM cells (24, 70).

The differentiation pathways and lineage relationship among naive, effector, TEM, and TCM cells is still a matter of debate. In a first model, naive T cells differentiate either into effector cells (TE) upon strong and prolonged Ag encounter or into an intermediate activation stage (Tint) upon weak and short Ag stimulation. TE cells can then develop into TEM, Tint develop into TCM, and, at least in vitro, TCM can develop into TEM in the presence of IL-7 and IL-15 even in the absence of Ag (26). In a second model, a linear differentiation from naive to TE, TEM, and TCM is proposed on the basis of gene expression analysis of Ag-specific CD8+ T cells at various time points after infection (24, 71). Partly supporting the latter model it was recently shown that a majority of Ag-specific TCM and TEM cells are derived from one original T cell clone and that, upon secondary encounter with Ag, a fraction of TCM cells differentiated into TEM cells, whereas in the absence of secondary Ag challenge, some TEM cells re-expressed CD62L (25). We observed that naive CD8+ T cells adopted a TEM phenotype (CD62L–CD127+) early upon Ag stimulation. Prolonged Ag exposure induced CD127 down-regulation (TE phenotype), whereas the absence of further Ag contact allowed slow, but continuous, re-expression of CD62L (TCM phenotype). We therefore propose a model (Fig. 9) in which naive CD8+ T cells differentiate upon first Ag encounter into a transient intermediate stage (CD62L+CD127–). Arrest at this stage (due to short Ag encounter) is associated with lack of peripherally measurable clonal expansion and hence represents an abortive T cell activation process. With sufficient Ag exposure, this intermediate stage cell differentiates further to a CD62L–CD127+ cell (TEM phenotype) with effector function and with the potential of significant clonal expansion. Further Ag contact induces CD127 down-regulation (TE phenotype), and a majority of these cells will not develop into long-lived memory cells but will eventually die due to activation-induced cell death or to lack of response to homeostatic signals such as IL-7. However, we propose that in the absence of Ag, at least some TE cells can re-express CD127 to adopt a TEM phenotype, and later on, in the absence of Ag, some TEM cells can re-express CD62L to become cells with a TCM phenotype. Such dynamic lineage relationships allow the development of CD8+ TEM cells with different anatomical localization and different recall capabilities. Repeated or prolonged Ag exposure assures the distribution of T cells throughout lymphoid and nonlymphoid organs, whereas a short-term, limited Ag exposure favors the development of cells with excellent recall potential, but with limited long-term presence in extralymphatic tissues. Such considerations are relevant for the development of T cell-mediated vaccines with respect to the nature of the challenging pathogen and its central or peripheral protective requirements.



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  		FIGURE 9. Lineage relationship model. Naive CD62L+CD127+ CD8+ T cells first down-regulate CD127 (intermediate, one to four cell divisions) and subsequently down-regulate CD62L and re-express CD127 (effector memory phenotype, more than five cell divisions). Additional Ag contact induces CD62L down-regulation (effector), and a majority of those cells do not enter the long-lived memory pool, either due to antigenic overstimulation or lack of response to homeostatic cytokine signals. Some effector cells, however, might re-express CD127 and again adopt an effector memory phenotype. The prolonged absence of Ag allows re-expression of CD62L and thus development of secondary lymphoid organ-resident central memory cells with excellent recall potential.

 

   Acknowledgments
 
We thank Urs Karrer for critical reading of the manuscript, and Eva Niederer for excellent technical assistance in cell sorting.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
M. F. Bachmann, K. Schwarz, and P. Jaeger are employees of, and own stock or stock options in, Cytos Biotechnology AG.


   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 the Swiss National Science Foundation, the Vontobel Stiftung, and the Roche Research Fund for Biology. Back

2 Address correspondence and reprint requests to Dr. Annette Oxenius, Institute for Microbiology, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule Hönggerberg, Wolfgang Pauli Strasse 10, HCI G401, 8093 Zurich, Switzerland. E-mail address: oxenius{at}micro.biol.ethz.ch Back

3 Abbreviations used in this paper: LN, lymph node; LCMV, lymphocytic choriomeningitis virus; MCMV, murine CMV; TCM, central memory cell; TE, effector T cell; TEM, effector memory T cell; tg, transgenic; Tint, intermediate activation stage T cell; VLP, virus-like particle; VVG2, recombinant vaccinia virus expressing LCMV glycoprotein. Back

Received for publication May 25, 2005. Accepted for publication July 26, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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