Initial Antigen Encounter Programs CD8+ T Cells Competent to Develop into Memory Cells That Are Activated in an Antigen-Free, IL-7- and IL-15-Rich Environment

Roberto Carrio, Oliver F. Bathe and Thomas R. Malek
J Immunol June 15, 2004, 172 (12) 7315-7323; DOI: https://doi.org/10.4049/jimmunol.172.12.7315

Abstract

Although much is known concerning the immunobiology of CD8+ T memory cells, the initial events favoring the generation of CD8+ T memory cells remain poorly defined. Using a culture system that yields memory-like CD8+ T cells, we show that 1 day after Ag encounter, Ag-activated T cells developed into memory-like T cells, but this optimally occurred 3 days after Ag encounter. Key phenotypic, functional, and molecular properties that typify central memory T cells were expressed within 48 h when the activated CD8+ T cells were cultured with IL-7 or IL-15 in the absence of Ag or following transfer into normal mice. These data support a model whereby Ag activation of naive CD8+ T cells not only programs effector cell expansion and contraction but the potential to develop into a memory cell which ensues in an Ag-free environment containing IL-7 or IL-15.

Over the last several years there has been progress in defining features that typify the induction of a CD8+ T cell immune response, including the production of long-lived Ag-specific CD8+ T memory cells (1). Upon an initial encounter with Ag, naive CD8+ T cells undergo programmed expansion, followed by differentiation into effector CTL, and then programmed contraction through apoptosis (2, 3, 4, 5). Ag-activated CD8+ T cells that escape apoptosis go on to differentiate into memory cells that are detected in vivo long after the initial Ag encounter (6, 7, 8, 9, 10, 11, 12). The long-term persistence of memory CD8+ T cells depends upon constant slow turnover mediated by IL-7 and IL-15 (10, 11, 12, 13, 14, 15, 16, 17). However, very little is known concerning signals that favor memory cell development. Understanding the nature of such signals is fundamental toward improving the efficacy of vaccines.

Two types of memory CD8+ T cells have been identified. One is designated effector-memory cells as they exhibit phenotypic and functional properties similar to effector CTL, but persist after Ag clearance, predominately in nonlymphoid tissue. The other is called central memory cells, which are primarily found in secondary lymphoid tissue and are functionally and phenotypically distinct from effector CTL, effector-memory cells, and naive T cells (18, 19). Characteristic properties of central memory cells include expression of CD44high, CD62Lhigh, IL-2Rβ+, Ly-6Chigh, and CCR7+ and enhanced sensitivity to Ag that leads to a rapid reinduction of the effector program (20, 21, 22, 23, 24). Recent studies indicate that effector-memory cells eventually convert to central memory cells which have the greatest potential to persist in vivo (25).

Current data favor a linear differentiation model for memory cell development. The main tenet in this model is that memory CD8+ T cells are direct descendants of effector CTL. Thus, after Ag-activated naive CD8+ T cells expand and differentiate into CTL, some of these effector cells escape apoptosis and differentiate over several weeks to fully express properties of central memory cells. This process is complex, requiring the regulation of many genes (26) and optimally occurs after Ag clearance. However, it is still unclear at what point effector cells optimally acquire competency to develop into memory cells. This issue is difficult to study in vivo during an immune response, as the magnitude of the expansion of the effector CTL potentially obscures the initial emergence of memory cells. Typically, memory cell development in this setting has been assessed only after the contraction of effector cells, where the detection of memory-like T cells becomes practical.

The inherent complexity of investigating memory cell development solely in vivo as a consequence of Ag activation of naive Ag-specific precursor cells accentuates a need for other experimental systems, including in vitro models, that simplify and emulate one or more aspects of memory cell development. There is especially a need for more flexibility in investigating the impact of individual agents or conditions on the cellular and molecular basis of T cell memory. In this regard, we previously demonstrated that short-term (4–5 days) in vitro Ag-activated CD8+ T cells readily developed into persistent central memory cells upon adoptive transfer into normal syngeneic mice in the absence of Ag (24). This approach directly permits assessing the development of memory cells from effector cells without complication of an ongoing immune response. Furthermore, von Andrian and colleagues (17, 27) demonstrated that T cells with properties of central memory cells were obtained when Ag-activated CD8+ T cells were cultured for an extended time with IL-15, a cytokine already implicated in regulating the homeostasis of memory CD8+ T cells. These experiments illustrate the ability to model memory cell development solely in vitro.

In the present study, we have refined an in vitro system that leads to the generation of central memory-like CD8+ T cells. By using this system, we evaluated the competency of effector CTL to develop into memory T cells. Our data support the view that CTL optimally develop into central memory cells in the absence of Ag during a discrete time frame and that some changes toward the memory phenotype are induced within days. IL-7 was shown to be as efficient as IL-15 in promoting memory phenotypic cells. These data support a model whereby Ag activation of naive CD8+ T cells not only programs effector cell expansion and contraction but the potential to develop into memory cells, which ensues in an Ag-free environment containing IL-7 or IL-15. This latter finding raises the possibility that IL-7 and IL-15 not only promote the survival and homeostasis of CD8+ T memory cells but also instruct aspects of a developmental program that typifies these cells.

Materials and Methods

Mice

OT-I TCR-transgenic mice (28) were maintained by breeding heterozygous OT-I mice to wild-type C57BL/6J mice. B6.SJL-Ptprc/BoAiTac mice, congenic for CD45 and expressing the CD45.1 allele were purchased from Taconic Farms (Germantown, MD). C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed and bred under viral-Ag-free conditions.

Cell culture conditions

Spleen cells from OT-I mice (1 × 106/well) were cultured in 24-well plates in 1 ml of complete RPMI 1640 medium (CM)3 (29), with OVA257–264 peptide (0.1 nM; synthesized by Research Genetics, Huntsville, AL) and either IL-2, IL-7, or IL-15 (all cytokines (10 ng/ml) from PeproTech, Rocky Hill, NJ). After 1 or 3 days in culture, the cells were harvested, washed three times with RPMI 1640, and the CD8+ OT-I T cells were purified by positive selection using anti-CD8 magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). These cells were typically >98% CD8+. Positive selection was always used to study day 1 Ag-activated OT-I T cells for subsequent in vitro or in vivo experiments and the day 3 cultured cells for adoptive transfer experiments. Given the high proportion (>90%) of activated CD8+ T cells after the day 3 culture, positive selection of the CD8+ T cells was found not to be required and not performed for in vitro experiments. These activated OT-I T cells were recultured in T25 culture flasks at 105cells/ml in 10 ml of CM without OVA257–264, but with the indicated cytokines. After an additional 2 days in culture, the cells were harvested and recultured, if required, with the same cytokines for 2–3 more days. In some experiments to generate large number of cells, the day 3 cultures were expanded using T75 tissue culture flasks containing 30 ml of CM. These cultures were initiated at 3 × 105 T cells/ml when using IL-7 or IL-15. Viability was determined at various time points by trypan blue exclusion or flow cytometry as described below.

Flow cytometry

The following Abs were used for flow cytometry and purchased from BD PharMingen (San Diego, CA): FITC-CD8 (53.6.7), FITC-CD45.2 (104), PE-Vα2 (B20.1), Cyc-CD8 (53.6.7), biotin-CD44 (Pgp-1), biotin-CD62L (MEL-14), biotin-CD69 (H1.2F3), biotin-Ly-6C (AL-21), biotin-Vβ5.1,5.2 (MR9-4), and biotin-CD25 (IL-2Rβ) (7D4). The cells were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA) and CellQuest software (BD Biosciences) as previously described (24). Typically 1 × 105 cells/spleen or lymph node sample and 1 × 104 cultured cells/sample were analyzed. Dead cells were visualized with 7-aminoactinomycin D (BD PharMingen). Cultured cells were labeled using CFSE (Molecular Probes; Eugene, OR) as previously described (30). For cell cycle analysis, T cells were harvested, washed, and lysed in a buffer containing 0.1% sodium citrate, 0.01% Triton X-100, and 0.1 mg/ml propidium iodide and incubated overnight before FACS analysis.

CTL and cytokine assays

Cytotoxicity was measured by a standard 51Cr release assay as previously described (31) against EL4 targets alone or after a 1-h incubation at 37°C with 0.1 nM OVA257–264 (32). IFN-γ secretion was assessed for cytokine-stimulated OT-I T cells by reculture of washed cells (1 × 106 cells/well) in 1 ml of CM in 24-well plates previously coated with anti-CD3 (1 μg/ml) and anti-CD28 (3 μg/ml). Supernatant fluids were collected 48 h later and IFN-γ was measured by ELISA using Ab pairs from BD PharMingen according to the manufacturer’s instructions.

Proliferation assay

Ag-driven proliferation was determined essentially as previously described (33). In brief, to measure Ag-driven proliferation, naive OT-I spleen cells (1 × 105 cells/well), or cytokine-expanded OT-I T cells (2 × 104/well) with T-depleted mitomycin C-treated normal C57BL/6 spleen cells (8 × 104cells/well), as a source of APC, were cultured in 96-well plates with OVA257–264 (0.1 nM). [3H]Thymidine was added during the last 4 h of a 48-h culture.

Adoptive transfer

The indicated population of cultured OT-I T cells (10 × 106 cells) was injected via the tail vein in 0.5 ml of HBSS into nonirradiated mice.

mRNA analysis

Apoptosis and cell cycle gene expression array systems were purchased from SuperArray Bioscence (Frederick, MD). Total RNA was extracted from the indicated cell population with TRIzol (Life Technologies, Grand Island, NY). cDNA was prepared from this total RNA and hybridized to the arrayed filters according to the manufacturer’s instruction. The resulting hybridization signal was visualized by chemiluminescence. Data were subjected to densitometric analysis using Scion Image Software (Scion, Frederick, MD) (34). RNA levels were expressed as relative OD measurement after normalizing to the hybridization signals to β-actin.

Results

Modeling the development of CD8+ central memory T cells in vitro

Previous studies have demonstrated that when Ag-activated CD8+ T cells were further cultured for 6 days with IL-15, the majority of the cells expressed a cell surface phenotype and the functional characteristics of central memory cells (17, 27). In the current study, we used OVA-specific MHC class I-restricted CD8+ T cells from the OT-I TCR-transgenic mouse to more precisely model the induction of memory-like T cells in vitro. Initially, we determined the kinetics by which CTL expressed a memory phenotype. The ability of IL-7 and IL-15 to induce such changes was also compared, as both cytokines function redundantly in vivo to promote the homeostasis of memory T cells.

Naive OT-I T cells were cultured with OVA257–264 and exogenous IL-2 for 3 days to generate effector cells. Since memory cell development in vivo is best visualized after Ag has been eliminated, these cells were washed to remove Ag. In some cases the CD8+ T cells were purified by positive selection to minimize the presence of APC and then recultured with IL-2, IL-7, or IL-15. We confirmed that the day 3 OT-I effector T cells were not contaminated with residual OVA257–264 and APC as the coculture of naive OT-I spleen cells with unfractionated or purified OT-I effector cells did not induce a proliferative response by the naive OT-I T cells (Table I⇓). After the initial 3-day culture, >90% of the viable recovered cells were CD8+ T cells that coexpressed Vα2 and Vβ5, characteristic of the OT-I TCR (data not shown). At that time, activated OT-I T cells expressed CD69, CD25, and high levels of CD44, while CD62L was down-regulated (Fig. 1⇓A). These cells exhibited potent CTL activity against OVA257–264-pulsed EL4 (H-2b) targets (Fig. 1⇓B). Thus, after 3 days in culture, these T cells typified effector CTL.

FIGURE 1.
FIGURE 1.

IL-7 or IL-15 rapidly induced CTL to express phenotypic and functional properties of memory cells in vitro. Splenocytes from naive OT-I mice were activated with OVA257–264 and IL-2 for 3 days and then the CD8+ OT-I T cells were purified and recultured in only the indicated cytokine for an additional 1–2 days. Cell size and surface phenotype (A), as assessed by FACS, or CTL activity using OVA257–264- pulsed EL4 cells (B) was determined for OT-I T cells at the indicated time point after culture. Data are representative of five experiments.

View this table:
Table I.

OT-I effector cells lack residual Aga

Upon subsequent culture in IL-2 for 2 days, the T cells expanded ∼10-fold and maintained phenotypic and the functional properties of CTL. In marked contrast, culture of the CTL in either IL-7 or IL-15 resulted in a time-dependent decrease in cell size and a shift in phenotype toward a central memory cell, i.e., CD69 and CD25 were substantially down-regulated while CD62L was up-regulated to a uniformly high level of expression. CTL activity was essentially undetectable (Fig. 1⇑B). High expression of Ly-6C represents another property of memory CD8+ T cells (22), and a higher percentage of OT-I T cells expressed Ly-6Chigh after culture with IL-7 or IL-15 (Fig. 1⇑A). This shift in phenotype required ∼48 h, as cells cultured for only 24 h in IL-7 and IL-15 expressed an intermediate phenotype. Furthermore, these phenotypic changes were accompanied by an ∼4- to 8-fold increase in the number of OT-I T cells, indicating that the cells which expressed these properties did not simply represent the survival of a rare subpopulation of T cells that was present within the initial priming culture. Thus, these data demonstrate that a population of effector cells is rather uniformly and rapidly converted to express several properties characteristic of central memory T cells.

Role for Ag, IL-7, and IL-15 in the development of memory-like T cells in vitro

We next evaluated to what extent Ag-activated OT-I T cells remained competent to develop into memory-like T cells when maintained for different periods of time as effector cells. For these experiments, OT-I T cells were primed with OVA257–264 and APC for 1 or 3 days and then recultured in the absence of Ag with either IL-2, IL-7, IL-15, or no exogenous cytokine for an additional 2 days (Fig. 2⇓, A and B). Alternatively, the 3-day OVA-primed OT-I T cells were further cultured without Ag, using IL-2 for an additional 2–4 days before switching to IL-7 and IL-15 (Fig. 2⇓, C and D). Optimal conversion into memory-like cells occurred after 3 days of priming with IL-2 and Ag as these cells were most polarized by IL-7 or IL-15 with respect to down-regulation of CD69 and CD25 and up-regulation of CD62L (Fig. 2⇓B). For CTL that were solely cultured in medium, cell expansion did not occur and viability was low. For the viable cells, some trends toward a memory phenotype were observed, e.g., down-regulation of CD69 and CD25, while others were not, e.g., up-regulation of CD62L (Fig. 2⇓B). At later time points there we too few viable cells to analyze.

FIGURE 2.
FIGURE 2.

Temporal requirements for the induction of memory-like T cells by IL-7 or IL-15 in vitro. Splenocytes from naive OT-I mice were activated with OVA257–264 and IL-2 for 1 (A) or 3 (B–D) days and further cultured with IL-2 for in the absence of Ag for 2 (C) or 4 (D) days. After these cultures, the OT-I T cells were recultured for 2 additional days with either IL-2, IL-7, or IL-15 as indicated. On the day indicated, cell viability and surface phenotype for the indicated markers were assessed by FACS for the OT-I T cells. The FACS gating for CD69+ or CD62Lhigh is shown in Fig. 1⇑A, day 0. The expression for CD25 is represented as the mean fluorescent intensity (MFI) for all cells. Data represent the mean ± SD of three experiments. B, Splenocytes activated with OVA257–264 and IL-2 for 1 day were recultured in the indicated cytokine for 1–2 days (day 2 or day 3, respectively), and CTL activity was assessed using OVA257–264-pulsed EL4 cells. Data are representative of two experiments.

When the Ag-activated cells were expanded with IL-2 for an additional 2–4 days, IL-7 and IL-15 were less apt to encourage memory-like T cell development. This was apparent based on the phenotype, as progressively fewer CD62Lhigh cells were detected, and on the cell recoveries, as OT-I T cell viability progressively decreased (Fig. 2⇑, C and D). Interestingly, IL-7 and IL-15 also favored the development of memory-like cells when OT-I T cells were primed for as little as 24 h with Ag and APC, as the majority of cells 2 days later were CD62Lhigh and exhibited progressively lower CTL activity when further cultured with IL-7 or IL-15 (Fig. 2⇑, A and E). During these cultures, there was at least a 9-fold cell expansion using either IL-2, IL-7, or IL-15. Therefore, even after the initial encounter with Ag, when cells are programmed for expansion and differentiation into CTL, IL-7 and IL-15 influenced this program to promote properties characteristic of memory cells. However, as apparent by OT-I T cells that were primed for 3 days with OVA257–264, this is most efficient after several days as an effector CTL.

OT-I T cells always exhibited the best growth when cultured with IL-2 (Fig. 3⇓). For the first week in culture, IL-2 generated an approximate 1000-fold increase, or at least 10 cell doublings, of OT-I T cells. As shown in Fig. 2⇑D, after 7 days in culture with IL-2, OT-I CTL were inefficiently converted to memory-like cells when placed in IL-7 or IL-15. At this point OT-I T cells were somewhat responsive to IL-2, but cell recoveries in IL-7 or IL-15 were <50% of the input (Fig. 3⇓D). Furthermore, after a 1 (Fig. 3⇓A)- or 3 (Fig. 3⇓B)-day priming with IL-2 and OVA257–264, subsequent culture of the primed cells with either IL-7 or IL-15 always resulted in a lower growth rate, with cell expansion of ∼40-fold over this period. After this time, there was minimal expansion by IL-7 or IL-15 (Fig. 3⇓, C and D). Collectively, our data indicate that optimal generation of memory T cells by IL-7 and IL-15, i.e., between days 3 and 5 of cell culture, occurs after at least five to six cell divisions, based on these cell yields.

FIGURE 3.
FIGURE 3.

Proliferative properties of cytokine-stimulated OT-I T cells. Naive OT-I splenocytes were cultured exactly as described in Fig. 2⇑. On the day indicated (A–D), cell numbers were assessed by trypan blue exclusion. Data represent the mean ± SD of two to three experiments.

All of the preceding experiments tested the capacity of IL-7 and IL-15 to promote memory cell development after removal of Ag. To investigate whether Ag removal was in fact necessary, we examined the phenotype and CTL activity of OT-I T cells that were primed for 3 days with OVA257–264 in the continuous presence of IL-2, IL-7, or IL-15. Under these conditions, all cytokines supported similar cell recoveries (data not shown). Each condition also generated activated OT-I T cells with a cell surface phenotype characteristic of effector T cells, with strong CTL activity (Fig. 4⇓, day 3) that was best in the cultures containing exogenous IL-2. Therefore, in the presence of Ag, IL-7 and IL-15 were largely ineffective in promoting memory-like T cells. However, as before, if Ag was removed by washing, and the cells were then further cultured in the cytokines used during priming, 2 days later, the OT-I T cells cultured in IL-7 or IL-15 resembled central memory cells (Fig. 4⇓A, day 5), with minimal CTL activity (Fig. 4⇓B, day 5). In contrast, those cells cultured with IL-2 behaved as effector CTL. For the most part, extending the culture period for 7 days resulted in a cell surface phenotype that was somewhat more uniformly characteristic of activated effector cells in IL-2 or memory cells in IL-7 or IL-15 (Fig. 4⇓A, day 7) with relatively high cell viability in all cultures. Furthermore, OT-I T cells primed in the presence of OVA257–264 and IL-7 or IL-15 for 3 days and maintained with these cytokines for an additional 6 days (data not shown) exhibited growth rates similar to those in Fig. 3⇑ for cells primed in the presence of IL-2 and then shifted to IL-7 or IL-15.

FIGURE 4.
FIGURE 4.

Effect of IL-7 and IL-15 during the initial Ag priming and subsequent development of memory-like T cells in vitro. A, Splenocytes from naive OT-I mice were activated with OVA257–264 and the indicated cytokine for 3 days and then recultured in only the indicated cytokine for an additional 2–4 days. On the indicated day, viability and cell surface phenotype for the indicated markers were assessed by FACS for the OT-I T cells. FACS gating is as described in the legend to Fig. 2⇑. Data represent the mean ± SD of three experiments. B, On days 3 and 5, CTL activity was assessed using OVA257–264-pulsed EL4 cells. Data are representative of three experiments. MFI, Mean fluorescence intensity.

Further analysis of cells that were cultured for 5 days indicated that, upon restimulation, each group secreted IFN-γ (Fig. 5⇓A), although much higher levels were noted for OT-I T cells cultured with exogenous IL-2. Continued exposure of 3-day OVA-primed OT-I T cells to IL-7 or IL-15 for 2 additional days resulted in strong proliferative responses when rechallenged with OVA257–264 (Fig. 5⇓B). By contrast, the OT-I effector cells maintained with IL-2 not only failed to proliferate to OVA257–264, but upon examining the cultures, most T cells appeared to have undergone activation-induced cell death. Thus, CTL exposed to IL-7 or IL-15, but not IL-2, exhibited intensified responsiveness to Ag, a property characteristic of central memory cells.

FIGURE 5.
FIGURE 5.

Functional properties of cytokine-stimulated OT-I T cells. Naive OT-I splenocytes were activated and cultured as described in Fig. 4⇑. On day 5, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 or OVA257–264 and APC. IFN-γ secretion (A) and proliferation (B), respectively, were assessed 48 h latter. Data are representative of three experiments.

Properties of in vitro effector and memory-like T cells upon adoptive transfer to normal syngeneic mice

The preceding experiments indicated that Ag-activated CD8+ T cells developed into memory-like T cells in the absence of Ag, but in the presence of IL-7 or IL-15, and this differentiation optimally occurred during a discrete time frame after initial activation. To validate some of these issues in vivo, we next examined the capacity of effector OT-I T cells to engraft and persist in vivo when generated for various periods of time in vitro. Effector cells were generated by priming with OVA257–264 and Ag for 1 or 3 days and then further expanded in IL-2 as necessary.

Regardless of when the CTL were adoptively transferred into syngeneic CD45.1-congenic C57BL/6 mice (Fig. 6⇓A), donor-derived OT-I T cells were detected when assayed 4 wk later. Optimal persistence was noted for those effector cells that were generated during a 3-day in vitro culture (Fig. 6⇓B). Importantly, the persistent cells uniformly expressed a phenotype, i.e., CD69neg, CD25neg, CD62Lhigh, and Ly-6Chigh, that typifies central memory cells when assayed in either the spleen (Fig. 6⇓D) or lymph nodes (data not shown) of recipient mice. Therefore, this time course for memory cell development in vivo largely parallels that for the in vitro conversion of IL-2-generated effector cells into memory-like cells.

FIGURE 6.
FIGURE 6.

Temporal requirements for the engraftment of in vitro-generated effector and memory-like OT-I T cells upon adoptive transfer to normal mice. Naive OT-I splenocytes were activated for 1 and 3 days with OVA257–264 and IL-2 and after washing were further expanded in IL-2 without Ag 2–5 additional days. The activated effector OT-I T cells were adoptively transferred into normal CD45.1 C57BL/6-congenic mice. A and B, After 4 wk, the number of donor cells in the spleen was determined by FACS by coexpression CD8 and CD45.2. Representative FACS dot plots (A) and summary of donor engraftment (B) for all mice that received OT-I T cells cultured in vitro for the indicted number of days. Data in B are the mean ± SD of six to seven mice per group from three experiments. C, Naive OT-I splenocytes were activated and cultured for 5 and 8 days as described in the legend to Fig. 4⇑ using exogenous IL-2, IL-7, or IL-15, as indicated, to prepare effector vs memory-like OT-I T cells. Four weeks after adoptive transfer of these cultured OT-I cells to CD45.1 C57BL/6-congenic mice, engraftment of OT-I T cells in the spleen was determined by FACS, as in A, for coexpression of CD8 and CD45.2. Data are the mean ± SD of seven mice per group from three experiments. D, The cell surface phenotype of the donor cells was determined for the indicated markers by FACS analysis after gating on CD8+CD45.2+ cells. For simplicity, shown are only the persistent OT-I T cells derived from day 3 effector CTL. Regardless of the level of engraftment and the type of cells transferred, the phenotype was the same as shown for both the spleen and lymph node.

We also compared the ability of in vitro-maintained effector vs memory-like T cells to persist upon adoptive transfer. Somewhat surprisingly, after 5 days in culture, there was no measurable difference in the capacity of OT-I T cells cultured in IL-2, IL-7, or IL-15 to persist as memory phenotypic T cells (Fig. 6⇑C, left), even though the latter two cell populations already expressed properties of memory cells. Therefore, at this juncture, OT-I CTL exhibited a high potential to develop and persist as memory cell in vivo. However, after 8 days in culture, only the transferred IL-7 or IL-15 memory-like T cells were readily detected in vivo 28 days after the transfer (Fig. 6⇑D, right). Therefore, the poor persistence of day 8 in vitro effector cells suggests that these OT-I T cells are less capable of developing into memory cells the longer they expand as effector cells.

To determine the rapidity by which some of these changes toward memory cells occurred in vivo, the cell surface phenotype was examined 2 and 5 days after effector OT-I CTL were adoptively transferred to normal mice (Fig. 7⇓). The expression of CD69 and CD25 was both substantially down-regulated 2 days after adoptive transfer in a manner essentially identical to that of effector cells cultured with IL-7 or IL-15. Somewhat surprisingly, Ly-6C was expressed at a near uniform high level that characterizes CD8+ memory cells. This level of conversion was much greater than that seen for in vitro-cultured cells. The expression of CD62L was somewhat intermediate with only a subpopulation of transferred cells that expressed this molecule at high levels. Therefore, similar to in vitro, some changes occurred rapidly while other required additional time.

FIGURE 7.
FIGURE 7.

In vivo surface phenotype of adoptively transferred OT-I effector T cells. After 5 days in culture, IL-2-induced CTL cells were washed, labeled with CFSE to follow the donor cells, and adoptively transferred into normal C57BL/6 mice. Two and 5 days after adoptive transfer, FACS analysis was performed on splenocytes for the indicated markers after gating on the CFSE+CD8+ cells. Data are derived from six mice per group from two experiments.

Molecular profiles of in vitro-generated effector vs memory cells

To begin to assess some of the initial molecular changes during the development of memory CD8+ T cells, the expression of mRNAs that primarily regulate apoptosis (Fig. 8⇓A) and the cell cycle (Fig. 8⇓B) were compared for day 3 effector OT-I T cells vs cells cultured for 2 additional days with IL-2 or IL-15. For the mRNA of the 96 genes on the apoptosis array, 26 mRNAs were not detected, whereas 30 of the 70 remaining mRNAs were differentially expressed by at least 3-fold. Sixteen of the differentially expressed mRNA were coordinately up- (12) or down-regulated (4) by the day 5 effector and memory-like OT-I T cells (data not shown). However, 14 of the remaining differentially expressed mRNAs appeared to distinguish effector from memory cells (Fig. 8⇓A). When compared with day 3 effector cells, seven mRNAs (Bcl-x, Survivin, Bnip3, TNF-β, CD30, 4-1BB, and Rpa) were increased in the IL-2-expanded CTL but decreased in the IL-15 memory-like T cells. Three other mRNAs were either selectively increased (CD27 and TNFR1) or decreased (TNFR-associated factor 6) in the memory-like IL-15-cultured OT-I T cells. Thus, these 10 mRNAs may be directly and rapidly regulated by IL-15 as effector CTL develop into memory T cells. The remaining four mRNAs (Bak, caspase-3, caspase-8, and TNF-α) were highly increased after continued culture of CTL in IL-2. This pattern of mRNA expression is consistent with a cell that is a polarized effector cell poised for apoptosis. The IL-2-dependent CTL are likely protected from apoptosis through expression of heightened levels of Bcl-x.

FIGURE 8.
FIGURE 8.

Differential mRNA expression of cytokine-induced effector and memory-like OT-I T cells. Splenocytes from naive OT-I mice were activated with OVA257–264 and IL-2 for 3 days (D3) and then recultured with the indicated cytokine for an additional 2 days (D5). Total RNA was prepared from these cells and the reverse-transcribed cDNA was hybridized to the apoptosis (A) or cell cycle (B) GE array. Shown is the relative expression of those mRNAs that varied by >3-fold after densitometric analysis of the hybridized arrays and were uniquely characteristic of effector or memory-like OT-I T cells. Data represent the mean ± SD from RNA isolated from four distinct cultures for the day 5 cultured cells or mean of 1 (B) or 2 (A) distinct cultures for the day 3 CTL. These day 3 data are shown as a reference and were internally controlled by parallel analysis to day 5 cultured cells. C, Cell cycle analysis for day 5 cultured cells. Data are representative of three experiments.

On the cell cycle array, 42 mRNAs were not detected whereas 23 of the 54 remaining mRNAs were differentially expressed by at least 3-fold. Two of the differentially expressed mRNAs were coordinately up-regulated by the day 5 effector and memory-like OT-I T cells (data not shown). However, 21 of the remaining differentially expressed mRNAs appeared to distinguish effector from memory cells (Fig. 8⇑B). When compared with the day 3 or day 5 effector cells, 15 mRNAs (cyclin G2 and B, Mcm family, Dp1, Pcna, Csk1, Cdk1 and 6, Cdc20, and Prc1), which primarily promote cell cycle progression, were highly decreased in the IL-15 memory-like T cells. Four other mRNAs showed the highest expression in the IL-15-induced memory-like cells (E2F2, Cdk8, p27, and p15). The two most highly and differentially expressed mRNA were p15 and p27, which both promote cell cycle arrest. When compared with the IL-2-cultured OT-I T cells, cell cycle analysis revealed fewer cells in S-G2-M for the IL-15 memory-like cells (Fig. 8⇑C). Collectively, these molecular changes support the notion that the day 3 CTL consist of effector cells that have decreased proliferative and enhanced survival potential after exposure to IL-15. These characteristics typify slowing dividing, long-lived memory cells.

Discussion

Immunological memory is operationally defined as an extremely rapid and efficient immune response upon a second encounter with an immunogen. Some of the key principles bearing on the cellular basis of immunological memory have emerged by analysis of the properties of Ag-specific lymphocytes that persist after a successful primary immune response in vivo (1). A critical aspect of our study is that the in vitro system utilized closely recapitulates key phenotypic and functional properties of CD8+ T memory cells. Therefore, conclusions derived from this culture system should directly relate to the mechanism of memory cell development in vivo. In this regard, most of our key findings were verified when the phenotype and persistence of in vitro-derived effector cells were analyzed following adoptive transfer into normal unmanipulated recipient mice.

One of the most difficult issues to study in vivo is the initial events that control the development of memory T cells from Ag-activated lymphocytes. For this reason, many of our experiments relied on a system where memory-like T cells were generated in vitro. Several important conclusions are evident from such experiments. First, there appears to be a window of time in which Ag-activated T cells are poised to develop into memory cells. Activation of naive CD8+ T cells by Ag for as little as 24 h was sufficient to endow the capability to develop into memory-like T cells. Optimal transition into memory-like CD8+ T cells required 3 days of activation in the presence of Ag. However, more extensive IL-2-driven expansion and polarization into a CTL rendered the cells less capable of expressing properties of memory T cells. Second, most, if not all, Ag-activated CD8+ T cells were competent to develop into memory cells. This was especially evident when naive OT-I T cells were cultured for 3 days with OVA257–264 and IL-2, leading to a population of effector CTL, which were essentially uniformly converted to memory-like cells within 48 h by IL-7 or IL-15. This conversion was accompanied by a 4- to 8-fold expansion of T cells, which was nearly comparable to expansion driven by IL-2. Thus, the expression of memory cell properties cannot be ascribed to a minor subset of Ag-activated T cells. Lastly, memory T cell development did not occur unless the Ag-activated T cells were in the correct environment, i.e., with IL-7 or IL-15, but in the absence of Ag. Importantly, in the presence of Ag, during the initial priming, IL-7 or IL-15 was ineffective in generating memory-like T cells. Although both cytokines are broadly and constitutively produced in an Ag-independent manner by nonlymphoid cells, this finding suggests that IL-7 or IL-15 cannot subvert the effector phase of an immune response until Ag is eliminated or the effector cells are within a niche that is Ag free.

Recently, Wherry et al. (25) demonstrated that, after lymphocytic choriomeningitis virus infection, central memory CD8+ T cells persisted much longer than effector-memory T cells. These investigators further demonstrated that, upon clearance of Ag, effector-memory T cells converted to central memory cells. We believe it is noteworthy that in both our in vitro and in vivo model systems, T cells with an effector-memory phenotype were essentially not detected. This result indicates that production of effector-memory T cells is not a prerequisite for the development of long-lived central memory cells and suggests that Ag removal is pivotal to produce central memory T cells.

Both IL-7 and IL-15 are critical cytokines for the survival and homeostasis of memory CD8+ T cells (10, 11, 12, 13, 14, 15, 16, 17, 35, 36). Our data suggest a much more active role for each of these cytokines in promoting the development of memory CD8+ T cells. In the presence of either cytokine, but in the absence of nominal Ag, cell surface phenotypic, functional, and molecular changes occurred that were characteristic of central memory cells, including down-regulation of CTL activity and reacquisition of Ag responsiveness. These data are consistent with a direct role for IL-7 and IL-15 in regulating these changes. However, it is highly unlikely that IL-7 or IL-15 are the only extrinsic factors responsible for the development of memory T cells. Notably, Ly-6C expression was highly, rapidly, and uniformly induced upon adoptive transfer of CTL to normal mice while this characteristic of CD8+ memory T cells was markedly less efficient in vitro with either IL-7 or IL-15. Furthermore, some properties of memory T cells, such as the down-regulation of CD69 and CD25, might simply represent the absence of IL-2 or Ag signals, as these changes were also favored when effector CTL were simply cultured in medium. Given the array of cellular and molecular modifications that occur as CTL transit to memory cells, it seems likely that both passive and active cytokine-dependent mechanisms are operative.

The ability of both IL-7 or IL-15 to redundantly favor memory-like activities in vitro may be related to the fact that receptors for both cytokines induce overlapping signaling pathways, notably Janus kinase 1, Janus kinase 3, Stat3, and Stat5 (37, 38, 39, 40, 41). The mere induction of these pathways, however, cannot explain their efficacy in promoting memory cell development. The IL-2 signaling pathway essentially entirely overlaps with that induced by IL-15, as both the IL-2 and IL-15 receptors share identical β and common γ-chain subunits (42), yet continued culture with IL-2 favors effector rather than memory T cells. This dichotomy between IL-2 and IL-15 signaling has been noted previously (43, 44). One explanation for the distinct outcomes of IL-2 vs IL-15 or IL-7 for CD8+ T cells may simply be related to the strength of signal, as other studies have shown that the culture of Ag-activated CD8+ T cells with a suboptimal dose of IL-2 in vitro primarily yielded memory phenotypic T cells (27). The alternative possibility, that cannot yet be entirely excluded, is that there is a unique signaling element, shared between IL-7 and IL-15, that favors the production of memory CD8+ T cells.

One very striking aspect of this study is the rapidity, i.e., days, by which both IL-7 or IL-15 supported features of memory CD8+ T cells in vitro. Rapid changes toward memory phenotype were not just limited to in vitro culture, as several features of memory T cells were also noted 2–5 days after effector CTL were adoptively transferred in vivo to normal mice. This quick conversion in vivo may at least partially explain why the transfer of either CTL or memory-like cells to normal mice, after a 5-day culture, persisted to essentially identical levels when assayed 4 wk later (Fig. 6⇑C). The short time frame for these changes appears to conflict with several recent studies that favor a mechanism whereby the properties of memory cells are gradually acquired over several weeks after the induction of an effector CTL response (2, 20, 26). Our data, however, also indicate a somewhat extended time for full conversion to memory CD8+ T cells. Even after 5–8 days in culture under memory cell conditions or 5 days after adoptive transfer in vivo, the phenotype of these OT-I T cells was not identical to the persistent memory cells characterized 4 wk after adoptive transfer to normal mice (Figs. 1⇑ and 7⇑ vs Fig. 6⇑D) or after development in vivo after virus infection (21, 26, 45). Therefore, our findings support the notion that the cell fate decision whereby an effector CTL differentiates into a memory T cell is critically dependent upon a 48-h time period, at which point a number of the key properties of a memory CD8+ T cell are apparent. An extended time, however, is still necessary for full development into a long-term persistent memory cell.

A gene expression profile has been described for naive, effector, and memory cells by isolating the appropriate cell populations after infection (26, 45). This type of analysis provides an important snapshot of gene expression by the CD8+ T cells as a consequence of an immune response, but it does not ascribe particular changes in profile to a specific signal. A distinct feature of in vitro-generated memory-like CD8+ T cells is that large numbers of T cells are available for cellular and molecular analysis to assay the initial events as memory T cells develop from effector CTL. During this effector to memory transition, our molecular analysis indicates a large number of changes occurred during this 48-h culture period. Some of these changes, e.g., the relatively high mRNA expression for TNF-α, TNF-β, 4-1BB, CD30, and OX40 by effector CTL and CD27 by memory-liked T cells, has been noted by others (46, 47, 48, 49, 50). More importantly, IL-15 down-regulated a number of genes important for cell cycle progression and apoptosis that was accompanied by a decrease in cell size. Correspondingly, cell cycle analysis revealed fewer cells in the S-G2-M phase of the cell cycle for these IL-15-stimulated memory-like T cells. Thus, IL-15 supported alteration in the molecular profile of effector CTL to one with lower potential for cell growth and apoptosis, properties of memory CD8+ T cells.

Two models have been proposed to explain the development of memory CD8+ T cells. In one, effector CTL and memory T cells are derived from separate lineages (9, 27). In the other the development of effector CTL is a prerequisite for production of memory cells (25, 26, 51, 52). Our data support tenets inherent to both of these models. We favor the view that, during the initial encounter with Ag, a naive CD8+ T cell is programmed for expansion, contraction, and the potential to develop into an effector or memory cells. The potential for a CTL to develop into memory cells exists for a discrete window of time. These fate outcomes of these two cells are dictated by environmental factors, with the presence or absence of Ag as one factor favoring effector vs memory cells, respectively. However, the mere absence of Ag may not in itself be sufficient to promote memory cell development. Other factors, including but not limited to IL-7 or IL-15, are necessary to either rescue the CTL from apoptosis and/or to induce properties of memory cells. Our view predicts that, once activated with Ag, a CD8+ T cell has the potential to travel down the memory cell pathway without becoming a CTL. This was readily visualized by removing Ag and then either culturing the Ag-activated T cells in IL-7 or IL-15 or by adoptively transferring the cells into normal mice. In the physiological situation of an immune response to an infectious agent, removal of Ag requires a substantial immune response, including effector CTL. Upon removing the bulk of Ag, CTL that escape apoptosis then develop into memory cells. In practical terms, memory cells do not develop until after a vigorous effector response which is responsible for decreasing Ag levels to a permissive level. Therefore, the linear differentiation model of naive to effector to memory CD8+ T cells describes most immune responses in vivo.

Acknowledgments

We thank Lin Kong and Aixin Yu for technical assistance.

Footnotes

  • ↵1 This research was supported by National Institutes of Health Grant R01 AI40114.

  • ↵2 Address correspondence and reprint requests to Dr. Thomas R. Malek, Department of Microbiology and Immunology (R138), University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101. E-mail address: tmalek{at}med.miami.edu

  • ↵3 Abbreviation used in this paper: CM, complete medium.

  • Received November 19, 2003.
  • Accepted April 9, 2004.

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