The contributions of different subsets of memory CD8+ T cells to recall responses at mucosal sites of infection are poorly understood. Here, we analyzed the CD8+ T cell recall responses to respiratory virus infection in mice and demonstrate that activation markers, such as CD27 and CD43, define three distinct subpopulations of memory CD8+ T cells that differ in their capacities to mount recall responses. These subpopulations are distinct from effector– and central–memory subsets, coordinately express other markers associated with activation status, including CXCR3, CD127, and killer cell lectin-like receptor G1, and are superior to CD62L in predicting the capacity of memory T cells to mediate recall responses. Furthermore, the capacity of vaccines to elicit these memory T cell subpopulations predicted the efficacy of the recall response. These findings extend our understanding of how recall responses are generated and suggest that activation and migration markers define distinct, and unrelated, characteristics of memory T cells.

A defining feature of the adaptive immune system is the capacity to “remember” past encounters with a pathogen and subsequently mount superior responses (recall responses) to a secondary challenge with the same, or related, pathogen (1). In the case of influenza and parainfluenza virus infections, it has been established that antigen-specific CD8+ T cells elicited during a primary infection persist at high frequencies for years after clearance of the virus (24). These memory CD8+ T cells are able to mediate recall responses that result in accelerated viral clearance and survival after challenge with an otherwise lethal dose of virus (5, 6). The enhanced responsiveness of antigen-specific memory CD8+ T cells as compared with naive CD8+ T cells is due to increased cell numbers, higher activation status, reduced stimulatory requirements, more rapid acquisition of effector functions, and altered homing patterns (1, 7, 8).

Memory CD8+ T cells are heterogeneous in terms of phenotype, function, and anatomical distribution. This heterogeneity is thought to reflect the different functional characteristics and migratory properties of memory CD8+ T cell subsets and has led to the paradigm that memory cells can be divided into two major subsets: central– and effector–memory T cells (2, 3, 6, 915). Central–memory T cells express high levels of CD62L and CCR7, which directs them to enter high endothelial venules and thereby localize predominantly in secondary lymphoid tissues. Effector–memory T cells express low levels of CD62L and CCR7 and tend to localize predominantly in nonlymphoid and peripheral tissues (15, 16). The different characteristics of central– and effector–memory CD8+ T cells suggest that these cells play different roles in recall responses. However, the relative contributions of each subpopulation to the accumulation of fully activated effector cells during a recall response after secondary infection are unclear (1719). One possibility is that effector–memory T cells that accumulate in peripheral sites such as the lung mediate early and nonsustained responses to the pathogenic challenge, whereas central–memory cells in secondary lymphoid organs mediate a late but sustained proliferative response to the challenge (20). However, studies on the capacities of effector– and central–memory cell subpopulations to mediate recall responses in vivo have been contradictory. Several groups have shown that central–memory T cells dominate recall responses to pathogens that mediate systemic infections, although this does not necessarily correlate with protective efficacy (16, 2125). Conversely, other studies have shown that effector–memory CD8+ T cells can contribute equally to, or even dominate, recall responses (26, 27). We have used a dual adoptive transfer system in which the relative contributions of Sendai virus–specific effector– and central–memory cells to recall responses in the lung could be directly compared on a per-cell basis. These studies demonstrated that the relative contribution of effector– and central–memory cells to the recall response in the lung varied depending on how recently memory had been established (28, 29). Although effector–memory CD8+ T cells dominated the recall response relatively soon after infection (up to 6 mo after infection), central–memory CD8+ T cells dominated the recall response at later times (12 mo after infection). Furthermore, the data showed that on a per-cell basis, both central– and effector–memory T cells isolated from the spleens of mice 1 yr after infection mediated dramatically stronger recall responses to Sendai virus infection than the same subpopulations of memory T cells isolated 1 mo after infection (28). The overall conclusion from these and other studies is that there is no direct or generalized relationship between central– and effector–memory phenotype of the memory T cell pool and the ultimate composition of the recall response at the site of infection.

Here, we define three distinct subpopulations of memory CD8+ T cells that are superior to central– and effector–memory phenotype in predicting recall efficacy. The data resolve discrepancies regarding the contribution of central– and effector–memory T cells to recall responses and suggest an alternative or additional scheme to classify memory T cells. The findings extend our understanding of how recall responses are generated and indicate which types of memory T cells should be targeted by vaccines.

Results

Activation markers identify distinct memory CD8+ T cell subsets

Studies addressing the contribution of central– and effector–memory CD8+ T cell subpopulations to the recall response have been contradictory and inconclusive (16, 2124, 26, 28). Therefore, we speculated that recall efficacy correlated with other characteristics of memory cells that were independent of their central– or effector–memory status. To test this notion, we analyzed the phenotype of memory CD8+ T cells generated by intranasal Sendai virus infection (specific for the immunodominant NP324–332/Kb epitope) with a particular emphasis on the expression of markers that distinguish quiescent and semi-activated memory T cell subsets, such as CD27, CD43 (activated isoform), and CD127 (3033). As shown in Fig. 1, analyses of CD27 and CD43 (1B11) expression at 1 mo after Sendai virus infection divided NP324–332/Kb-specific T cells into three distinct subpopulations: CD27hi/CD43hi, CD27hi/CD43lo, and CD27lo/CD43lo.

Further analysis revealed that the chemokine receptor CXCR3 was coordinately expressed with CD27 (not depicted) and that the same three subpopulations of memory CD8+ T cells could also be defined by CXCR3/CD43 expression (Fig. 1 A). There was also a similar pattern of expression for CD127 in that all of the CD127+ cells were also CD27+. The correlation between CD127 and CD27 is illustrated in Fig. 1 B. Analysis of the three coordinated memory T cell subsets at various times after infection revealed that the CD27hi/CD43lo subpopulation progressively dominated the memory T cell pool, representing ∼90% of the antigen-specific T cells detected at 2 yr after infection (Fig. 1 A). In contrast, the CD27lo/CD43lo subpopulation progressively declines and disappeared by about 1 yr after infection. Thus, the representation of these distinct subpopulations within the total memory CD8+ T cell pool progressively changes over time.

The identification of three distinct memory populations expressing coordinated patterns of markers was of interest because very few markers are coordinately expressed on memory T cells. One question was whether these subpopulations corresponded to the central– (CD62Lhi) and effector– (CD62Llo) memory CD8+ T cells subsets. Consistent with previous reports (3, 28), ∼10% of Sendai virus NP324–332/Kb-specific memory CD8+ T cells expressed a central–memory (CD62Lhi) phenotype at 1 mo after infection, and this percentage rose to 90% at 12 mo after infection. Regardless of the time point, both central– and effector–memory CD8+ T cells were present in roughly equal frequencies in each of the subpopulations (CXCR3hi/CD43hi, CXCR3hi/CD43lo, and CXCR3lo/CD43lo; Fig. 1 C). Collectively, the data indicate that the division of memory CD8+ T cells into three major subpopulations is independent of central– or effector–memory status.

To determine whether these subpopulations of memory T cells expressed markers typically associated with T cell senescence or exhaustion, we analyzed the expression of killer cell lectin-like receptor G1 (KLRG1), a marker of senescent cells linked to poor in vitro proliferative capacity, and programmed death-1 (PD-1), a marker of T cell exhaustion (3436). As shown in Fig. 1 D, ∼40% of the CD27lo/CD43lo cells expressed KLRG1, whereas none of the cells expressed PD-1. The data suggest that the CD27lo/CD43lo population may include cells that have the characteristics of immune senescence.

The three memory cell subpopulations (CD27hi/CD43hi, CD27hi/CD43lo, and CD27lo/CD43lo) were not a unique feature of Sendai virus or a mucosal route of infection. As shown in Fig. 1 E, identical subpopulations were also identified in the pool of memory CD8+ T cells elicited by intranasal influenza virus infection (specific for both the influenza NP366–374/Db and PA224–233/Db epitopes). The same subpopulations of memory CD8+ T cells were also observed after intraperitoneal (systemic) Sendai and influenza virus infection (not depicted).

We next asked whether the rate of homeostatic turnover differed for each of the three memory cell subpopulations (CD27hi/CD43hi, CD27hi/CD43lo, and CD27lo/CD43lo; references 37 and 38). Mice were intranasally infected with Sendai virus and 1 mo later were administered BrdU for 14 d in the drinking water. Memory T cells in each of the sub populations became BrdU+ over this timeframe, indicating that all subpopulations were being maintained to some extent by homeostatic proliferation (Fig. 2).

However, the rate of BrdU accumulation was markedly different for each of the three subpopulations. For example, the CXCR3lo/CD43lo subpopulation accumulated BrdU at one quarter the rate of the CXCR3hi/CD43hi subpopulation. The slower turnover rate of the CXCR3lo/CD43lo subpopulation may account for the progressive disappearance of this population from the memory T cell pool over time. The same patterns of homeostatic proliferation were observed when the three subpopulations were identified on the basis of CD27 (consistent with the coordinate expression of this marker with CXCR3) and CD127 (Fig. 2).

We also analyzed the effector functions of the different memory T cell subpopulations. All of the tetramer+ cells in each subpopulation were functional in terms of the capacity to secrete IFN-γ in an intracellular cytokine assay, consistent with previous studies showing that the entire NP324–332/Kb-specific CD8+ memory T cell population is functional in terms of IFN-γ secretion (reference 3 and not depicted). In addition, the fraction of IFN-γ+ cells that produced TNF-α and IL-2 was similar for each subpopulation, although the frequency of IL-2+ cells was highest among the CD27hi/CD43lo subpopulation (Fig. 3 A).

In contrast, there was a notable difference in granzyme B expression among the three memory T cell subpopulations. Although the CD27hi/CD43hi and CD27hi/CD43lo cells were granzyme B, the CD27lo/CD43lo cells were granzyme B+ (Fig. 3 B). However, granzyme B was expressed by all three subpopulations in the context of a recall response to Sendai virus infection (Fig. 3 C).

Memory CD8+ T cell subpopulations differ in recall efficacy in the lung

CD8+ T cell recall responses to secondary respiratory virus infections are characterized by an increased accumulation of activated effector CD8+ T cells within the lungs and lung airways. To determine the individual contributions of the different memory T cell subpopulations to the accumulation of these cells in the lungs, we used a dual adoptive transfer approach in which we could directly compare the responses of two separate populations of memory T cells in the same animal under the same infection conditions (28, 29). Initial experiments were focused on memory T cells that were either positive or negative for CD43 expression. Spleen cells from C57BL/6 and B6-CD45.1 donors were enriched for CD8+ cells on a negative selection column and flow cytometrically sorted on the basis of CD44 and CD43 expression. The CD44hi/CD43hi and CD44hi/CD43lo subpopulation from each donor was then mixed together, such that the ratio of NP324–332/Kb cells was 1:1, and intravenously injected into B6-CD90.1 recipient mice. 1 d later, recipient mice were intranasally infected with Sendai virus, and NP324–332/Kb-specific T cells were analyzed 11 d later in the lung airways (bronchoalveolar lavage), lung parenchyma, pleural cavity lavage, mediastinal lymph nodes (MLNs), and spleens. Cells derived from each donor and from the host were distinguished on the basis of CD90 and CD45 expression, and the ratio of the response of the two donors was determined (39). An example of the sorting/transfer strategy and the data generated is presented in Fig. 4.

The strength of this basic approach is that the response of two polyclonal (i.e., nontransgenic) populations of cells is compared in the same animal under identical infection conditions. In addition, it effectively integrates all of the factors involved in the accumulation of activated effector T cells at the site of infection, such as their capacity to migrate to lymph nodes before infection, the overall proliferative and death rates of the responding cells, and the capacity of the cells to migrate to inflammatory sites (27). As shown in Fig. 5, data obtained from several experiments established that CD43lo memory CD8+ T cells were two- to fivefold more efficient than CD43hi cells at mediating the recall response to Sendai virus infection in the airways, parenchyma, and pleural cavities of the lung (similar data were obtained in the MLN and spleen; not depicted).

This relative dominance of the CD43lo subpopulation was observed regardless of whether the memory T cells were isolated 1 or 6 mo after the primary infection (Fig. 5). These data support the finding that CD43lo memory CD8+ T cells mediate stronger recall responses than CD43hi cells in terms of their accumulation at the site of infection, and this dominance is maintained over the long term.

Using the same approach, we then compared the recall efficacy of CD27hi and CD27lo, as well as CXCR3hi and CXCR3lo, memory subpopulations. As shown in Fig. 5, data from several independent experiments showed that the CD27hi and CXCR3hi memory subpopulations were 2–10-fold more efficient than their counterparts at contributing to the recall responses in different compartments of the lung. In the case of CXCR3 sorted cells, we also analyzed the phenotype of effector CD8+ T cells that accumulated in the lungs of host mice on day 10 of infection. Donor memory cells retained their original CXCR3 phenotype in both the lung and spleen (not depicted), suggesting that expression of this chemokine receptor is not necessary for memory cell activation or migration into a site of inflammation. The original phenotypic pattern was also retained through day 35 after transfer (day 34 after infection), when the donor cells had returned to a resting memory phenotype (Fig. 6, A and B).

Furthermore, the CXCR3lo cells retained their original CD127lo and CD43lo phenotype when analyzed at this time point. However, retention of the original phenotype by effector– or secondary memory CD8+ T cells was not characteristic of cells sorted on the basis of CD43 expression inasmuch as each of the CXCR3hi/CD43hi and CXCR3hi/CD43lo subpopulations was capable of generating all three of the memory T cell subpopulations after reactivation (not depicted). Collectively, the data suggest that memory CD8+ T cells expressing either a CD27hi, CXCR3hi, or a CD43lo phenotype mediate the strongest recall response. Furthermore, cells expressing either a CXCR3hi or CXCR3lo phenotype retain this phenotype throughout the response and into the establishment of secondary memory cells. However, it is currently unclear whether this phenotype is retained over the long term.

CD27hi/CD43lo memory CD8+ T cells mediate the strongest recall response in the lung

The data thus far indicate that memory CD8+ T cells with either a CD27hi or a CD43lo phenotype mounted superior recall responses. Collectively, these data suggest that the CD27hi/CD43lo memory CD8+ T cell subpopulation mounted the strongest recall response in the lung. To test this idea, we sorted memory CD8+ T cells from the spleens of mice that had recovered from a prior Sendai virus infection (1 mo after infection) into the three memory T cells subpopulations and used dual adoptive transfer studies to compare the ability of each subpopulation to mount a recall response (the sorting strategy is illustrated in Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20070322/DC1). Because CXCR3 and CD27 are coordinately expressed (Fig. 1 and not depicted), the anti-CXCR3 antibody, rather than the anti-CD27 antibody, was used for sorting as it yielded superior separation of the memory cell subsets. These three-way sort/dual adoptive transfer experiments clearly demonstrate that the three CXCR3hi/CD43hi, CXCR3hi/CD43lo, and CXCR3lo/ CD43lo memory cell subpopulations differ in their capacity to mount recall responses in the lungs after secondary Sendai virus challenge (Fig. 7).

CXCR3hi/CD43lo cells (labeled as population A in Fig. 7) were superior to both CXCR3lo/CD43lo cells (labeled as population B) and CXCR3hi/CD43hi cells (labeled as population C). Furthermore, CXCR3lo/CD43lo cells (population B) mounted the weakest recall response compared with the other two memory T cell populations. These data, together with the fact that CXCR3 and CD27 are coordinately expressed, support a hierarchy of CD27hi/CD43lo > CD27hi/CD43hi > CD27lo/CD43lo memory cells in terms of mediating recall responses in the lung.

Vaccination elicits different subpopulations of memory CD8+ T cells

The differences in recall efficacy of memory CD8+ T cell subpopulations have substantial implications for the design of vaccines. Therefore, we compared the capacity of Sendai NP324–332 peptide-loaded dendritic cells or peptide administered in CFA to elicit memory CD8+ T cells. In the course of these analyses, we observed that Sendai NP324–332 peptide/CFA induced very high frequencies of CD27lo/CD43lo memory CD8+ T cells, predicted by our previous analysis to be poor responders to secondary Sendai virus challenge (Fig. 8 A).

Although the ratio of the three CD27hi/CD43lo, CD27hi/CD43hi, and CD27lo/CD43lo memory CD8+ T cell subpopulations was ∼1:1:1 in mice that had recovered from a prior Sendai virus infection (1 mo after infection), the ratio of these subpopulations was ∼1:2:6 in peptide/CFA-vaccinated mice. Also, a substantial population of cells with a fourth phenotype (CD27lo/CD43hi) was observed in peptide CFA-vaccinated mice. In addition, the memory T cell pool elicited by peptide CFA vaccination was exclusively of the effector–memory phenotype, and ∼90% of the cells were CD127hi (Fig. 8 B). Consistent with data generated from mice that had recovered from a Sendai virus infection, KLRG1+ cells were highly enriched among the CD27lo subpopulations of memory T cells elicited by peptide CFA vaccination (Fig. 8 C). Because CD27lo cells represented a greater proportion of the memory pool generated by CFA vaccination (compared with viral infection), a much higher frequency of the total NP324–332/Kb-specific population was also KLRG1+ (Fig. 8 D).

Based on these findings, we hypothesized that the overall pool of NP324–332/Kb-specific memory T cells generated by NP324–332 peptide/CFA vaccination would mount weaker recall responses compared with the pool of NP324–332/Kb- specific memory T cells generated by a prior Sendai virus infection. Because peptide/CFA vaccination induced sufficient numbers of memory T cells available for dual transfer studies, we were able to test this hypothesis by directly comparing the recall efficacy of memory T cells isolated from vaccinated mice to those isolated from mice that had recovered from a prior Sendai virus infection. As shown in Fig. 9 A, data from several experiments demonstrated that memory CD8+ T cells from peptide/CFA-vaccinated mice were 10-fold less efficient than memory cells from Sendai virus– infected mice at contributing to a recall response to Sendai virus infection in the lung airways and parenchyma (similar data were obtained in the MLN and spleen; not depicted).

Thus, consistent with our phenotypic analyses and our hypothesis, peptide/CFA vaccination generated comparatively poor quality memory T cells.

To investigate the memory CD8+ T cells generated by peptide/CFA vaccination further, we isolated memory subpopulations on the basis of CD43hi/CD43lo or CXCR3hi/ CXCR3lo expression and compared their abilities to contribute to the recall response to intranasal Sendai virus infection. Consistent with memory cells generated by Sendai virus infection, CXCR3hi cells were superior to CXCR3lo cells in mounting recall responses (Fig. 9 B). Interestingly, CD43hi cells resulting from peptide/CFA-vaccinated mice were superior to CD43lo cells, a finding opposite to that seen with memory cells generated by Sendai virus infection. However, this difference is fully consistent with the fact that CD43lo cells isolated from peptide/CFA-vaccinated mice are dominated by poorly responsive CXCR3lo/CD43lo cells (Fig. 8 A). In summary, the distribution of distinct memory CD8+ T cell subpopulations generated by vaccination or infection can accurately forecast the recall potential of the memory T cell pool.

Discussion

Despite progress over the last few years in characterizing the properties of memory T cells, it is still not understood how different types of memory T cells actually contribute to recall responses or which memory T cells are the most appropriate for different pathogens. A key advance has been the realization that memory cells express distinct patterns of homing receptors. This in turn has led to the idea that effector–memory T cells in the periphery play a key role in mediating immediate responses to mucosal or peripheral infections, whereas central–memory T cells are more important for mediating proliferative responses to systemic infections. However, this idea has been difficult to reconcile with data showing that both central– and effector–memory T cells mount potent proliferative responses (16, 2126, 28). In this study, we have analyzed T cell memory subpopulations from the perspective of activation markers. The data indicate that patterns of CD27 and CD43 (1B11) expression define three distinct subpopulations of memory CD8+ T cells that differ significantly in their capacities to contribute to the accumulation of effector cells in the lungs during a respiratory virus challenge. These subpopulations coordinately express an array of other markers associated with activation status, including CXCR3, CD127, and KLRG1, and are independent of effector– and central–memory status (23, 31, 34, 40). In general, there appears to be an inverse correlation between activation status and proliferative potential, such that cells with the most “rested” phenotype have the capacity to mediate the strongest recall responses. For example, the subpopulation of memory T cells able to mediate the strongest recall response was shown to express a CD27hi/CD43lo phenotype (41). In contrast, the subpopulation of memory T cells that mediated the weakest recall responses expressed a CD27lo/CD43lo phenotype. Indeed many, but not all, of the cells in this latter subpopulation expressed characteristics of senescent T cells, including expression of KLRG1. Although all of the subpopulations of memory cells are based on markers typically associated with activation, the cells are nevertheless small, nonblasted, and do not express acute activation markers such as CD69 or CD25. Thus, our data support a new functional classification of memory CD8+ T cells that is based on activation phenotype rather than the widely accepted central– and effector–memory T cell classification (based on markers such as CD62L and CCR7). The most potent memory T cells, in terms of their proliferative capacity, express the most quiescent activation phenotype, and these cells tend to accumulate over time. This population also becomes increasingly dominated by cells with a central–memory phenotype (CD62Lhi) and ultimately comes to most resemble naive T cells. Overall, these findings suggest a pattern of memory T cell differentiation in which many markers and properties change over time, but at different rates.

The division of memory cells into subpopulations based on the expression of activation markers may help explain certain features of CD8+ T cell memory. First, we have previously shown that the recall efficacy of memory CD8+ T cells progressively improves over time (28). This observation could not be explained by changes in the distribution of effector– and central–memory T cells over time, as the improvement occurred on a per-cell basis and within both of these subpopulations. However, the data here show that there is a progressive loss of memory cell subpopulations that express a weak (CD27lo/CD43lo) or intermediate (CD27hi/CD43hi) proliferative capacity from the total memory T cell pool. Although these less responsive phenotypes of cells represented ∼70% of the total memory T cell pool at 1 mo after infection, they only represented 5–10% of the population at 2 yr after infection (with the very poorly responding CD27lo/CD43lo subpopulation essentially gone by about 1 yr after infection). Thus, a progressive loss of poorly responding memory T cells may explain the finding that that efficacy of the total memory T cell pool increases over time. Second, we have previously shown that the relative contributions of effector– and central–memory T cell subpopulations to recall responses differ dramatically between recently generated and long-term pools of memory T cells (28, 29). Changes in the distribution of memory cells expressing activation markers within these subpopulations over time potentially explain these differences. For example, the strongly responsive CD27hi/CD43lo cells become increasingly represented in central–memory cells over time (for example, compare the distribution of CD43hi and CD43lo cells in the central– and effector–memory T cell pools at 1 and 12 mo after infection in Fig. 1). Third, our unpublished studies had suggested that although peptide/CFA vaccination was effective at establishing large numbers of memory T cells, it was relatively poor at eliciting recall responses. Our current observation that peptide/CFA vaccination generated very high frequencies of poorly responsive CD27lo/CD43lo memory CD8+ T cells likely explains the overall weak recall response elicited by this vaccine strategy. Thus, enumeration of these newly described memory CD8+ T cell subpopulations allows one to predict the overall efficacy of a recall response.

The significance of the CD27 and CD43 activation markers on memory T cells is poorly understood. CD27 is a TNF receptor family member that functions as a T and B cell costimulatory molecule (32, 42, 43). This marker has previously been shown to correlate with effector functions in humans (44). CD43 occurs as two distinct glycoforms (115 and 130 kD) of which the 130 kD glycoform is associated with T cell activation and is specifically detected by the 1B11 antibody (45). It is involved in cell adhesion and may also regulate apoptosis during acute and memory phases of an immune response (33, 4648). Although these markers clearly regulate different aspects of the immune response, it is unlikely that they actually mediate the differences in recall response observed in the current studies. Rather, they are probably surrogate markers of intrinsic programmed states of the memory T cell that affects recall efficacy. One possibility is that this phenotype is associated with optimal access to sites of antigen presentation within lymphoid tissues, although this is not entirely consistent with patterns of CD62L expression (27). Another possibility is that these cells may have undergone less expansion during the acute response and therefore retain more proliferative potential (49). We are currently investigating these possibilities.

The lack of any relationships between activation marker status and central/effector–memory phenotypes suggests that these two characteristics of memory T cells are completely independent of each other. Several studies have addressed the relative recall efficacy of central– and effector–memory T cells, and results from these studies have been either contradictory or inconclusive, probably reflecting the use of different infection models (16, 2128). Also, these studies did not account for the presence of KLRG1-expressing senescent cells within the populations of memory T cells examined (as discussed above; reference 34). Other issues to be considered are the migration and resulting distribution of the cells in adoptive transfer experiments. Cells separated on the basis of CD62L tend to distribute differently in recipients after adoptive transfer with CD62Lhi cells tending to localize preferentially in the lymph nodes (28, 29). An ability to localize the lymph nodes is likely important for responses that depend on dendritic cell and antigen trafficking to the lymph nodes (27). In these current studies, we found no evidence that memory CD8+ T cells expressing different activation markers had different patterns of migration to the lymph nodes (unpublished data). These data further suggest that activation markers (CD27/CD43) and migration markers (CD62L/CCR7) define distinct, and unrelated, characteristics of memory cells. Thus, activation marker expression may influence the general capacity of memory T cells to mediate a recall response, whereas migration markers may dictate the capacity of memory T cells to migrate to sites of antigen deposition.

In summary, the data presented here suggest that the expression of certain activation markers on memory CD8+ T cells is a better predictor of recall efficacy than central– and effector–memory phenotype. Separation of memory cell subpopulations by these phenotypes helps resolve prior discrepancies regarding the contribution of central– and effector–memory T cells to recall responses, and also supports the use of these phenotypes as an alternative or additional scheme to classify memory T cells. Although central– and effector–memory phenotypes are markers of memory T cell–trafficking patterns, activation marker expression likely indicates different functional qualities of memory T cells. Clearly, both factors need to be taken into consideration during vaccine development.

Materials And Methods

Virus, mice, infection, and vaccination.

Sendai virus (Enders strain) and influenza virus A/HK-x31 were grown, stored, and titered as described previously (50). 8–12-wk-old female C57BL/6J, B6.SJL-Ptprca Pep3/BoyJ (CD45.1), and B6.Pl-Thy1a/Cy (CD90.1) mice (The Jackson Laboratory) were anesthetized and intranasally infected with 250 50% egg infectious doses (EID50) of Sendai virus or 600 EID50 of x31 influenza virus. Vaccination was performed with 50 μg of Sendai virus nucleoprotein peptide (NP324–332) emulsified in 100 μl CFA by subcutaneous injection in the base of the tail. All animal studies were approved by the Trudeau Institute Animal Care and Use Committee.

Flow cytometric analyses.

Splenocytes were panned on goat anti–mouse IgG H+L (Jackson ImmunoResearch Laboratories) -coated flasks and stained with APC-conjugated tetramers (Sendai NP324–332/Kb, Flu NP366-374/Db, or Flu PA224-233/Db) and antibodies to cell surface markers (CD8, CD44, CD43 [1B11], CD62L, CD27, CXCR3, CD127, KLRG1, or PD-1). Samples were run on FACSCalibur (BD Biosciences) or CyAn ADP (DakoCytomation) flow cytometers. Data were analyzed using FlowJo software (Tree Star).

Intracellular BrdU staining.

BrdU staining was performed as described previously (51). Mice were administered 0.8 mg/ml BrdU in the drinking water for 14 d. Splenocytes were then stained as described above and fixed overnight at 4°C in 1% paraformaldehyde/PBS containing 0.05% Nonidet P-40. After washing, cellular DNA was denatured with DNase I (bovine pancreas; Sigma-Aldrich). Cells were subsequently stained with anti– BrdU-FITC.

Intracellular cytokine and granzyme B staining.

Sorted memory CD8+ T cell subsets were co-cultured at 37°C for 5 h with naive CD45.1+ splenocytes pulsed with 1 μg of Sendai NP324–332 peptide in 250 μl of CTM containing 10 μg/ml brefeldin A (Epicenter Biotechnologies) and 10 U/ml IL-2 (R&D Systems). After culture, the cells were stained with anti-CD8 and anti-CD45.2 in PBS/brefeldin A. The cells were then fixed in 2% formaldehyde, permeabilized with buffer containing 0.5% saponin, and stained with either anti–IFN-γ, anti–TNF-α, or anti–IL-2 monoclonal antibodies conjugated to APC (BD Biosciences). For granzyme B staining, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) after tetramer and surface marker staining. Cells were stained with anti–granzyme B or isotype control antibody conjugated to PE (Caltag Laboratories).

Isolation of memory CD8+ T cell subpopulations by FACS.

Lymphocytes were isolated from spleens and further enriched for CD8+ cells using negative selection T cell enrichment columns (R&D Systems). In two-way sorting, enriched CD8+ cells were stained with anti–CD44-FITC and either anti–CD43 (1B11)-PE, anti–CD27-PE, or anti–CXCR3-PE. CD44hi cells were sorted into two subsets based on the expression of the other cell surface marker; e.g., CD44hiCD43hi and CD44hiCD43lo. In three-way sorting, enriched CD8+ cells were stained with anti–CD44-APC-AF750 and anti–CD43 (1B11)-AF488 in combination with either anti–CXCR3-PE or anti–CD27-PE. CD44hi cells were sorted into three subsets based on the expression of CD43 and the other cell surface marker; e.g., CD44hiCD43loCXCR3hi, CD44hiCD43loCXCR3lo, and CD44hiCD43hi CXCR3hi (Fig. S1). Sorting was performed on a FACSVantage cell sorter with DIVA enhancement software (BD Biosciences).

Dual adoptive transfer of memory subsets.

Dual adoptive transfer experiments were performed as described previously (28, 29). In brief, memory subsets from the donors were combined such that the number of Sendai NP324–332/Kb-specific T cells in each donor population was equal. The combined cell subsets were intravenously transferred into naive recipients and then intranasally challenged with 250 EID50 of Sendai virus 1 d later. On day 11 after infection, lymphocytes were isolated from various tissues, stained with Sendai NP324–332/Kb tetramer-APC, anti–CD8-PerCP, anti–CD45.1-FITC, and anti–CD90.2-PE, and then analyzed by flow cytometry. The relative response in each tissue was calculated from the numbers of Sendai NP324–332/Kb-specific T cells derived from each donor. Control experiments demonstrated that neither anti-CD43 (1B11), anti-CD27, nor anti-CXCR3 monoclonal antibody staining negatively affected the ability of donor cells to mount a recall response in vivo.

Online supplemental material.

An example of the sorting strategy used to isolate memory CD8+ T cell subsets for functional studies is shown in Fig. S1, which is available at http://www.jem.org/cgi/content/full/jem.20070322/DC1.

Acknowledgments

We thank Simon Monard and Brandon Sells for assistance with flow cytometry, the Molecular Biology Core for producing tetramers, and Drs. Marcy Blackman and William Reiley for critically reviewing the manuscript.

This work was supported by National Institutes of Health grants AG021600, AI055500, HL63925, and AI58668, and funds from the Trudeau Institute.

The authors have no conflicting financial interests.

References

References
1
Seder, R.A., and R. Ahmed.
2003
. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation.
Nat. Immunol.
4
:
835
–842.
2
Marshall, D.R., S.J. Turner, G.T. Belz, S. Wingo, S. Andreansky, M.Y. Sangster, J.M. Riberdy, T. Liu, M. Tan, and P.C. Doherty.
2001
. Measuring the diaspora for virus-specific CD8+ T cells.
Proc. Natl. Acad. Sci. USA.
98
:
6313
–6318.
3
Hogan, R.J., E.J. Usherwood, W. Zhong, A.D. Roberts, R.W. Dutton, A.G. Harmsen, and D.L. Woodland.
2001
. Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections.
J. Immunol.
166
:
1813
–1822.
4
Wiley, J.A., R.J. Hogan, D.L. Woodland, and A.G. Harmsen.
2001
. Antigen-specific CD8+ T cells persist in the upper respiratory tract following influenza virus infection.
J. Immunol.
167
:
3293
–3299.
5
Hogan, R.J., W. Zhong, E.J. Usherwood, T. Cookenham, A.D. Roberts, and D.L. Woodland.
2001
. Protection from respiratory virus infections can be mediated by antigen-specific CD4+ T cells that persist in the lungs.
J. Exp. Med.
193
:
981
–986.
6
Flynn, K.J., G.T. Belz, J.D. Altman, R. Ahmed, D.L. Woodland, and P.C. Doherty.
1998
. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia.
Immunity.
8
:
683
–691.
7
Dutton, R.W., L.M. Bradley, and S.L. Swain.
1998
. T cell memory.
Annu. Rev. Immunol.
16
:
201
–223.
8
Hammarlund, E., M.W. Lewis, S.G. Hansen, L.I. Strelow, J.A. Nelson, G.J. Sexton, J.M. Hanifin, and M.K. Slifka.
2003
. Duration of antiviral immunity after smallpox vaccination.
Nat. Med.
9
:
1131
–1137.
9
Usherwood, E.J., R.J. Hogan, G. Crowther, S.L. Surman, T.L. Hogg, J.D. Altman, and D.L. Woodland.
1999
. Functionally heterogeneous CD8+ T-cell memory is induced by Sendai virus infection of mice.
J. Virol.
73
:
7278
–7286.
10
Murali-Krishna, K., J.D. Altman, M. Suresh, D.J. Sourdive, A.J. Zajac, J.D. Miller, J. Slansky, and R. Ahmed.
1998
. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.
Immunity.
8
:
177
–187.
11
Masopust, D., V. Vezys, A.L. Marzo, and L. Lefrancois.
2001
. Preferential localization of effector memory cells in nonlymphoid tissue.
Science.
291
:
2413
–2417.
12
Reinhardt, R.L., A. Khoruts, R. Merica, T. Zell, and M.K. Jenkins.
2001
. Visualizing the generation of memory CD4 T cells in the whole body.
Nature.
410
:
101
–105.
13
Cauley, L.S., T. Cookenham, T.B. Miller, P.S. Adams, K.M. Vignali, D.A. Vignali, and D.L. Woodland.
2002
. Cutting edge: virus-specific CD4+ memory T cells in nonlymphoid tissues express a highly activated phenotype.
J. Immunol.
169
:
6655
–6658.
14
Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia.
1999
. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.
Nature.
401
:
708
–712.
15
Lefrancois, L., and D. Masopust.
2002
. T cell immunity in lymphoid and non-lymphoid tissues.
Curr. Opin. Immunol.
14
:
503
–508.
16
Wherry, E.J., V. Teichgraber, T.C. Becker, D. Masopust, S.M. Kaech, R. Antia, U.H. Von Andrian, and R. Ahmed.
2003
. Lineage relationship and protective immunity of memory CD8 T cell subsets.
Nat. Immunol.
3
:
225
–234.
17
Kapasi, Z.F., K. Murali-Krishna, M.L. McRae, and R. Ahmed.
2002
. Defective generation but normal maintenance of memory T cells in old mice.
Eur. J. Immunol.
32
:
1567
–1573.
18
Unsoeld, H., S. Krautwald, D. Voehringer, U. Kunzendorf, and H. Pircher.
2002
. Cutting edge: CCR7+ and CCR7- memory T cells do not differ in immediate effector cell function.
J. Immunol.
169
:
638
–641.
19
Bjorkdahl, O., K.A. Barber, S.J. Brett, M.G. Daly, C. Plumpton, N.A. Elshourbagy, J.P. Tite, and L.L. Thomsen.
2003
. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues.
Immunology.
110
:
170
–179.
20
Hikono, H., J.E. Kohlmeier, K.H. Ely, I. Scott, A.D. Roberts, M.A. Blackman, and D.L. Woodland.
2006
. T-cell memory and recall responses to respiratory virus infections.
Immunol. Rev.
211
:
119
–132.
21
Zanetti, M., and G. Franchini.
2006
. T cell memory and protective immunity by vaccination: is more better?
Trends Immunol.
27
:
511
–517.
22
Vaccari, M., C.J. Trindade, D. Venzon, M. Zanetti, and G. Franchini.
2005
. Vaccine-induced CD8+ central memory T cells in protection from simian AIDS.
J. Immunol.
175
:
3502
–3507.
23
Bachmann, M.F., P. Wolint, K. Schwarz, P. Jager, and A. Oxenius.
2005
. Functional properties and lineage relationship of CD8+ T cell subsets identified by expression of IL-7 receptor alpha and CD62L.
J. Immunol.
175
:
4686
–4696.
24
Marzo, A.L., K.D. Klonowski, A. Le Bon, P. Borrow, D.F. Tough, and L. Lefrancois.
2005
. Initial T cell frequency dictates memory CD8+ T cell lineage commitment.
Nat. Immunol.
6
:
793
–799.
25
Stock, A.T., C.M. Jones, W.R. Heath, and F.R. Carbone.
2006
. Cutting edge: central memory T cells do not show accelerated proliferation or tissue infiltration in response to localized herpes simplex virus-1 infection.
J. Immunol.
177
:
1411
–1415.
26
Berenzon, D., R.J. Schwenk, L. Letellier, M. Guebre-Xabier, J. Williams, and U. Krzych.
2003
. Protracted protection to Plasmodium berghei malaria is linked to functionally and phenotypically heterogeneous liver memory CD8+ T cells.
J. Immunol.
171
:
2024
–2034.
27
Klonowski, K.D., A.L. Marzo, K.J. Williams, S.J. Lee, Q.M. Pham, and L. Lefrancois.
2006
. CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen.
J. Immunol.
177
:
6738
–6746.
28
Roberts, A.D., K.H. Ely, and D.L. Woodland.
2005
. Differential contributions of central and effector memory T cells to recall responses.
J. Exp. Med.
202
:
123
–133.
29
Roberts, A.D., and D.L. Woodland.
2004
. Cutting edge: effector memory CD8+ T cells play a prominent role in recall responses to secondary viral infection in the lung.
J. Immunol.
172
:
6533
–6537.
30
Jones, A.T., B. Federsppiel, L.G. Ellies, M.J. Williams, R. Burgener, V. Duronio, C.A. Smith, F. Takei, and H.J. Ziltener.
1994
. Characterization of the activation-associated isoform of CD43 on murine T lymphocytes.
J. Immunol.
153
:
3426
–3439.
31
Kaech, S.M., J.T. Tan, E.J. Wherry, B.T. Konieczny, C.D. Surh, and R. Ahmed.
2003
. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells.
Nat. Immunol.
4
:
1191
–1198.
32
Croft, M.
2003
. Costimulation of T cells by OX40, 4-1BB, and CD27.
Cytokine Growth Factor Rev.
14
:
265
–273.
33
Onami, T.M., L.E. Harrington, M.A. Williams, M. Galvan, C.P. Larsen, T.C. Pearson, N. Manjunath, L.G. Baum, B.D. Pearce, and R. Ahmed.
2002
. Dynamic regulation of T cell immunity by CD43.
J. Immunol.
168
:
6022
–6031.
34
Voehringer, D., C. Blaser, P. Brawand, D.H. Raulet, T. Hanke, and H. Pircher.
2001
. Viral infections induce abundant numbers of senescent CD8 T cells.
J. Immunol.
167
:
4838
–4843.
35
Barber, D.L., E.J. Wherry, D. Masopust, B. Zhu, J.P. Allison, A.H. Sharpe, G.J. Freeman, and R. Ahmed.
2006
. Restoring function in exhausted CD8 T cells during chronic viral infection.
Nature.
439
:
682
–687.
36
Zajac, A.J., J.N. Blattman, K. Murali-Krishna, D.J. Sourdive, M. Suresh, J.D. Altman, and R. Ahmed.
1998
. Viral immune evasion due to persistence of activated T cells without effector function.
J. Exp. Med.
188
:
2205
–2213.
37
Tan, J.T., B. Ernst, W.C. Kieper, E. LeRoy, J. Sprent, and C.D. Surh.
2002
. Interleukin IL-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells.
J. Exp. Med.
195
:
1523
–1532.
38
Zhang, X., S. Sun, I. Hwang, D.F. Tough, and J. Sprent.
1998
. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15.
Immunity.
8
:
591
–599.
39
Badovinac, V.P., K.A. Messingham, S.E. Hamilton, and J.T. Harty.
2003
. Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host.
J. Immunol.
170
:
4933
–4942.
40
Agostini, C., M. Facco, M. Siviero, D. Carollo, S. Galvan, A.M. Cattelan, R. Zambello, L. Trentin, and G. Semenzato.
2000
. CXC chemokines IP-10 and mig expression and direct migration of pulmonary CD8+/CXCR3+ T cells in the lungs of patients with HIV infection and T-cell alveolitis.
Am. J. Respir. Crit. Care Med.
162
:
1466
–1473.
41
Appay, V., P.R. Dunbar, M. Callan, P. Klenerman, G.M. Gillespie, L. Papagno, G.S. Ogg, A. King, F. Lechner, C.A. Spina, et al.
2002
. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections.
Nat. Med.
8
:
379
–385.
42
Hendriks, J., Y. Xiao, J.W. Rossen, K.F. van der Sluijs, K. Sugamura, N. Ishii, and J. Borst.
2005
. During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively determine formation of CD8+ memory T cells and their capacity for secondary expansion.
J. Immunol.
175
:
1665
–1676.
43
Borst, J., J. Hendriks, and Y. Xiao.
2005
. CD27 and CD70 in T cell and B cell activation.
Curr. Opin. Immunol.
17
:
275
–281.
44
Hamann, D., P.A. Baars, M.H. Rep, B. Hooibrink, S.R. Kerkhof-Garde, M.R. Klein, and R.A. van Lier.
1997
. Phenotypic and functional separation of memory and effector human CD8+ T cells.
J. Exp. Med.
186
:
1407
–1418.
45
Ellies, L.G., A.T. Jones, M.J. Williams, and H.J. Ziltener.
1994
. Differential regulation of CD43 glycoforms on CD4+ and CD8+ T lymphocytes in graft-versus-host disease.
Glycobiology.
4
:
885
–893.
46
He, Y.W., and M.J. Bevan.
1999
. High level expression of CD43 inhibits T cell receptor/CD3-mediated apoptosis.
J. Exp. Med.
190
:
1903
–1908.
47
Matsumoto, M., K. Atarashi, E. Umemoto, Y. Furukawa, A. Shigeta, M. Miyasaka, and T. Hirata.
2005
. CD43 functions as a ligand for E-Selectin on activated T cells.
J. Immunol.
175
:
8042
–8050.
48
Stockton, B.M., G. Cheng, N. Manjunath, B. Ardman, and U.H. von Andrian.
1998
. Negative regulation of T cell homing by CD43.
Immunity.
8
:
373
–381.
49
D'Souza, W.N., and S.M. Hedrick.
2006
. Cutting edge: latecomer CD8 T cells are imprinted with a unique differentiation program.
J. Immunol.
177
:
777
–781.
50
Cole, G.A., T.L. Hogg, and D.L. Woodland.
1994
. The MHC class I-restricted T cell response to Sendai virus infection in C57BL/6 mice: a single immunodominant epitope elicits an extremely diverse repertoire of T cells.
Int. Immunol.
6
:
1767
–1775.
51
Cauley, L., T. Cookenham, R. Hogan, S. Crowe, and D. Woodland.
2003
. Renewal of peripheral CD8 memory T cells during secondary viral infection of antibody sufficient mice.
J. Immunol.
170
:
5597
–5606.

Abbreviations used: EID, egg infectious dose; KLRG1, killer cell lectin-like receptor G1; MLN, mediastinal lymph node; PD-1, programmed death-1.

H. Hikono and J.E. Kohlmeier contributed equally to this work.

H. Hikono's present address is National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan.

Supplementary data