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The Journal of Immunology, 2008, 181, 3818 -3822
Copyright © 2008 by The American Association of Immunologists, Inc.
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Cell Cycle-Related Acquisition of Cytotoxic Mediators Defines the Progressive Differentiation to Effector Status for Virus-Specific CD8+ T Cells1

Misty R. Jenkins2,*, Justine Mintern*, Nicole L. La Gruta*, Katherine Kedzierska*, Peter C. Doherty*,{dagger} and Stephen J. Turner3,*

* Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; and {dagger} Department of Immunology St. Jude Children’s Research Hospital, Memphis, TN 38105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although analysis of virus-specific CTL function at the peak of infection suggests that granzyme (grz) and perforin (pfp) gene expression is not coregulated, early differentiation events leading to acquisition of function are poorly understood. Using a combination of CFSE dilutions and single-cell RT-PCR, effector gene expression was determined early after CTL activation. There were low levels of pfp and grz expression at division 3, with increased expression by divisions 6–8. The increase in effector mRNA expression with division correlated with increasing ex vivo cytotoxicity. Of the mRNA transcripts detected at division 3, there was an increased frequency of grzB and grzK (compared with grzA or pfp), and this pattern was also observed at later divisions. The prevalence of OT-I CTL expressing grz/pfp mRNA was equivalent for the divided CD62Lhigh and CD62Llow sets, but the concentrations of grzB protein, levels of CTL activity, and the absolute amounts of grzB transcript were substantially greater for the CD62Llow population. Thus, while effector gene expression can be acquired early, maturation of cytotoxic capacity requires extended differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Following primary virus infection, naive, Ag-specific CD8+ CTLs undergo a program of proliferation and differentiation that results in the acquisition of CTL effector function (1, 2, 3). Virus-specific CTL-mediated lysis normally operates via the targeted secretion of perforin (pfp),4 a pore-forming protein, and various proteases known collectively as granzymes (grzs). With T cell activation, pfp and grz proteins are synthesized and sorted to cytoplasmic granules that polarize to the TCR/peptide MHC class I glycoprotein signaling domain and release their contents toward the virus-infected target cell (4). The pfp and grzs work synergistically to induce programmed cell death of virus-infected cells with rapid clearance of the resultant apoptotic debris limiting the extent of inflammatory damage to the host (5). The more familiar grzA and grzB are generally considered to be the most abundant within cytotoxic granules (5), but mRNA for the much less investigated grzK has also been detected at substantial frequency in both activated and memory T cells recovered from mice after influenza (6) and lymphocytic choriomeningitis virus infection (2).

Molecular profiling has established that a high frequency of influenza A virus-specific memory CTLs maintain expression of grzB and grzK mRNA in the long term (6), explaining the capacity of these T cells to mediate rapid cytotoxicity (7). Based on expression of the CD62L lymph node homing receptor (8), memory CTL can be divided into a less activated CD62Lhigh central (TCM) set that has the capacity to access draining lymph nodes directly from the blood (8, 9), and CD62Llow "effector memory" (TEM) T cells that distribute more to peripheral sites and show evidence of immediate CTL activity (9, 10). Recent analysis of TCR usage has established that these CD62Lhigh and CD62Llow memory CTLs can be derived from the same clonal expansions (11). However, while it is clear that individual Ag-specific T cell clones can contribute to both the TCM and TEM populations during the initial proliferative response to infection (12), the differential expression of effector mRNAs has not been analyzed for these two differentiating subsets.

Progressive cell division appears to be a key regulator enabling the induction of various T cell functions (13). Acquisition of CTL activity has been found to occur early after infection with the arming of activated CTL occurring before migration from the draining lymph node (14, 15) and dissemination to other tissues to mediate effector function (10, 16, 17). Although experiments with the influenza A virus mouse pneumonia model have shown that the expression of both grzB and IFN-{gamma} protein correlates with cell division (16) in lympoid tissue, this has not been extended to other effector molecules. Molecular profiling of pfp and grz mRNA expression in single CTLs specific for the influenza A virus DbNP366 and DbPA224 epitopes established that different T cells express varied spectra of "effector" mRNAs (6, 18, 19). However, there is no firm evidence that the expression of other grzs and pfp by recently activated CTL is also dependent on cell division. Furthermore, any direct link between cell division, induction of effector gene expression, and immediate cytotoxic function is yet to be established.

The present analysis uses a combination of adoptive transfer, single-cell sorting, and RT-PCR-based approaches to ask whether the progressive acquisition of CTL-mediated lytic activity and the transcription of pfp and the various grzs follows predictable patterns that are in turn dependent on cell division. The findings also raise questions concerning which of these various differentiation stages can give rise to memory T cells, and should cause us to ask what exactly do we mean when we categorize a CTL as an effector (1)?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

The Ly5.2+ C57BL/6J (B6) and congenic OT-I-Ly5.1 (OT-I) (20) mice were bred at the University of Melbourne, and experiments were conducted according to approval obtained from the institutional ethics committee.

Tissue sampling and cell preparation

Spleen and mediastinal lymph node (MLN) samples were recovered from mice at various time points after infection and OT-1 T cell transfer, then enriched for CD8+ T cells by panning on plates coated with goat anti-mouse IgG and IgM Abs to remove B cells for 1 h at 37°C (Jackson ImmunoResearch Laboratories).

Adoptive transfer and analysis of CFSE-labeled OT-I cells

Naive OT-I splenocytes were resuspended at 1 x 107 per ml and stained with 5 µM CFSE (Sigma-Aldrich) at 37°C for 10 min. CFSE-labeled cells were washed with PBS and 2 x 106 cells transferred in 200 µl i.v. into B6 Ly5.2+ recipients. Given the CD8+ population is 20% of the total splenocyte population, this represents the transfer of 1 x 105 OT-I T cells. After 24 h, mice were anesthetized and infected intranasally (i.n.) with 104 PFU of the HKx31 influenza virus, engineered to express the OVA257–264 peptide in the neuraminidase stalk (HK-OVA) (21). Spleens and MLNs were sampled at d3.5 for analysis. Cells were gated on the CD8+Ly5.1+ set for FACS analysis and sorted on the basis of their CFSE profile into tubes for either bulk analysis (51Cr-release assay) or into 96-well plates for single-cell RT-PCR (6, 19).

grzB and CD62L staining

Cells were stained with anti-CD8{alpha}-PerCPCy5.5 and anti-CD62L-PE, then fixed and permeabilized using a BD cytofix/cytoperm kit (BD Pharmingen). Intracellular grzB was detected using anti-human grzB-allophycocyanin (clone GB12; Caltag Laboratories), and analyzed using a BD FACSCalibur (BD Immunocytometry Systems) and CellQuest software.

51Cr-release assay

The standard Na51Cr-release assay used EL4 (H-2b) target cells that had been pulsed with the SIINFEKL peptide for 60 min at 37°C, before 3x washing and plating at 10,000 targets per well. They were then incubated with 10,000 sorted effector cells per well for 5 h at 37°C before harvesting the supernatants for gamma counting and calculation of specific 51Cr release.

RNA extraction, cDNA synthesis, and real-time PCR analysis

After bulk sorting Ly5.1+ OT-I cells based on CFSE dilution, RNA was isolated using TRIzol reagent (Invitrogen) followed by chloroform extraction and isopropanol precipitation. Then cDNA was synthesized using an Omniscript kit (Qiagen), according to the manufacturer’s instructions. A total of 100 ng of total RNA was used in each reaction and analyzed using TaqMan Gene MGB primer/probes (FAM labeled) (Applied Biosystems) specific for grzB, and mitochondrial ribosomal protein L32 as an internal standard and an ABI PRISM 7700 thermocycler (Applied Biosystems). Each cDNA sample was assayed in duplicate and results are reported as the mean of replicates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Progressive acquisition of pfp/grz mRNA and CTL effector function correlates with cell division

Virus-specific CD8+ T cells differentiate in the draining lymph node to develop CTL activity within days after infection (15). To determine whether the acquisition of immediate lytic capacity was related to cellular division, B6-Ly5.2+ recipients were given 2 x 106 CFSE-labeled B6-OT-I- Ly5.1+ T cells and rested for 24 h before i.n. challenge with 104 PFU of the HK-OVA influenza A virus (21). At the time of transfer, 95% of the OT-I T cells showed the naive, CD44lowCD69lowCD62Lhigh phenotype (data not shown). Spleen and MLN populations were then taken 3.5 days after infection and sorted on the basis of CFSE-loss as a measure of cell division for the subsequent analysis of CTL activity (spleen and MLN) and single-cell PCR to detect grz and pfp mRNA (MLN). The great majority of the OT-I T cells had divided more than once by day 3.5 (Fig. 1A), with the development of CTL effector function being clearly correlated with progressive cellular division (Fig. 1B). The highest level of CTL activity was found for spleen and MLN OT-I T cells that had undergone seven or eight divisions, with the values decreasing for three to five divisions and coming back to <5% for those that remained undivided (Fig. 1B). The acquisition of lytic function within the draining lymph node thus requires extended cellular division.


Figure 1
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  		FIGURE 1. Progressive cell division correlates with increased CTL cytotoxic function. A, Naive B6 Ly5.2+ mice were injected i.v. with 1 x 106 CFSE-labeled B6-OT-I-Ly5.1 T cells then rested for 24 h before i.n. infection with 104 PFU of the HK-OVA influenza A virus (21 ). The draining lymph node (MLN) and spleen were sampled 3.5 days after infection and the number of divisions measured by loss of CFSE staining. B, Lymphocytes from MLN and spleen were bulk sorted on the basis of cell division number and assayed at a 1:1 E:T ratio in a 51Cr-release assay using SIINFEKL-pulsed EL.4 target cells. Shown is the percent specific lysis ± SD (n = 3).

 
Heterogeneity of effector gene expression by recently activated CTL

Earlier experiments showed a marked heterogeneity of grz/pfp expression within individual, influenza virus-specific CTL effectors (6, 18, 19). Given that the expression of multiple effector molecules correlates with increased cytotoxic capacity (22), the next step was to determine the profiles of grz/pfp expression for single OT-I T cells at early and late divisions as the cells differentiate. Individual OT-I CTLs showed evidence of heterogeneous grz/pfp mRNA expression after three or six divisions. A representative example is shown in Fig. 2. Less than half of activated OT-1 T cells that had undergone three divisions expressed different effector mRNAs with the pattern of expression varying considerably from mouse to mouse (Figs. 2 and 3A). It was invariably the case that more cells were mRNA+ after six divisions (Figs. 2 and 3A). Even so, though pfp and grzA mRNAs were detected in significant numbers of T cells at the later divisions (Fig. 3A), those expressing message for one (or both) of these effector molecules were still at relatively low frequency when compared with message for grzB (~80%) or grzK (~50%). Overall, the single cell mRNA expression hierarchy was grzB>grzK>pfp>grzA (Figs. 2 and 3A). In agreement with earlier reports, there was minimal grzC up-regulation at any stage in these Ag-specific OT-I CTLs (6, 18, 19). Despite this heterogeneity of grz/pfp mRNA expression at both early and late divisions, there was a clear, progressive increase in the frequency of CTLs expressing multiple effector mRNA transcripts (Fig. 3B). Overall, the acquisition of enhanced cytotoxic capacity by recently activated virus-specific CTL correlates with progressive cellular division and increased frequency of effector mRNA transcription.


Figure 2
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  		FIGURE 2. Heterogeneity of grz/pfp expression is independent of cell division number. Naive B6 Ly5.2 mice were injected i.v. with 1 x 106 CFSE-labeled B6-OT-I-Ly5.1 T cells then rested for 24 h before i.n. infection with 104 PFU of the HK-OVA influenza A virus. The draining lymph node (MLN) was sampled 3.5 days after infection and the number of divisions measured by loss of CFSE staining. Single OT-I CD8+ CTL that had undergone either three or six divisions were sorted directly into wells of a 96-well plate containing cDNA buffer and RT-PCR was performed to detect expression of CD8, grzA, B, C, K, and pfp mRNA. The observed patterns of effector mRNA expression are represented by the shaded boxes with black representing expression of an effector mRNA. The frequency (%) of individual patterns within sorted OT-I CTL are listed for an individual mouse to the right. The values at the bottom of the figure represent the percent of individual effector mRNA expression within OT-I that divided either three or six times. Results are representative of three independent experiments.

 

Figure 3
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  		FIGURE 3. Effector mRNA expression by OT-I CTL increases with division number. CFSE-labeled OT-I CTL were recovered from the MLN of mice as described for Fig. 1. OT-I CTL that had undergone either three ({square}) or six to eight divisions ({blacksquare}) were single cell sorted and subjected to multiplex PCR for detection of grzA, B, C, K, and pfp mRNA as previously described (6 ). A, The frequency of individual grz or pfp expression by single OT-I CTL is shown for three individual mice. B, The frequency of single OT-I expressing either 0, 1–2, 3–4, or 5 effector mRNAs per cell determined from the data in A.

 
Effector molecule expression and function for CD62Lhigh and CD62Llow T cells

Following virus challenge, most (though not all) of the Ag-specific CTLs down-regulate cell surface CD62L expression and up-regulate cytoplasmic grzB levels (16). We have previously shown here that both CD62Lhigh and CD62Llow CTL are present at early (division 3) and late divisions (divisions 4–6) at day 3.5 after infection and that memory T cells can be generated within the lymph node early after infection (12). Given that TCM and TEM CTL subsets (8, 9) can be characterized by differences in CD62L expression and function (TCM: CD62Lhigh, low lytic activity; TEM: CD62Llow, high lytic activity), it was important to determine whether cytotoxic capacity would segregate based on CD62L expression at this early time point. Recently activated OT-I CTL from the draining lymph node were stained for CD62L expression (Fig. 4A). OT-I T cells that were either CD62Lhigh or CD62low populations were then directly sorted onto OVA257-loaded, 51Cr-labeled EL.4 target cells. The CD62Llow OT-I CTLs recovered on day 3.5 were more potent CTL effectors than the CD62Lhigh T cells that had undergone the same number of divisions (Fig. 4B), despite the fact that the frequency of grz/pfp mRNA expression was comparable at a single cell level for the two CD62L phenotypes (Fig. 4B). Thus, while the CD62Lhigh and CD62Llow OT-I CTLs both expressed similar molecular profiles indicative of an effector state, the CD62Llow OT-I CTLs were the more potent mediators of CTL lysis.


Figure 4
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  		FIGURE 4. The frequency of effector mRNA expression within OT-I CTL does not correlate with cytotoxic capacity. A, Gating strategy for isolation of recently activated OT-I CTL based on CD62L staining. CFSE-labeled OT-I CTL were recovered from the MLN of mice 3.5 days after infection as described for Fig. 1. Shown is the gating strategy for sorting of CD62Lhigh (upper box) and CD62Llow (lower box) populations. B, CFSE labeled OT-I were recovered from the MLN of mice as described for Fig. 1. CD62Lhigh or CD62Llow OT-I CTL that had undergone at least two divisions were sorted and ex vivo cytotoxic capacity assayed at a 1:1 E:T ratio in a 51Cr-release assay using SIINFEKL-pulsed EL.4 target cells. Shown is the percent specific lysis ± SD (n = 3). The percent specific lysis of CD62Lhigh and CD62Llow populations on unpulsed EL.4 cells was 0%. C, The frequency of grz/pfp expression by either CD62Lhigh or CD62Llow CTL that had undergone six to eight divisions was determined by single cell RT-PCR as described in Fig. 2 for 75–80 cells from pooled mice (n = 3).

 
Real-time PCR was then used to measure grzB mRNA levels within bulk-sorted, divided CD62Lhigh/low OT-I T cells. The OT-I populations recovered from spleen and MLN at day 3.5 after infection were found to express comparable levels of grzB mRNA, with the amounts being ~5 times greater for the CD62Llow sets (Fig. 5A). Analysis of grzB staining (Fig. 5B) demonstrated that the difference in mRNA levels between CD62Lhigh and CD62Llow OT-I correlated with an increased proportion of grzB+ OT-I CTLs (Fig. 5B) and enhanced levels of grzB expression (Fig. 5C), particularly at later divisions. Therefore, increased cytotoxic capacity of TEM most likely reflects an increase in either transcriptional activity, or RNA stability ensuring potent cytotoxic function.


Figure 5
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  		FIGURE 5. Correlation of cytolytic effector mRNA and protein expression with cell division and CD62L phenotype. A, CFSE-labeled CD8+ OT-I T cells were transferred into B6 naive mice, infected i.n. with the HK-OVA virus, and MLN harvested 3.5 days later. CD62Lhigh or CD62Llow CTL that had divided at least two times were bulk sorted and total RNA was extracted and cDNA synthesized. The cDNA was used as template in a real-time PCR using a FAM-labeled MGB-primer/probe set specific for grzB. A primer/probe set for L32 was used as a normalizer. Real-time PCRs were run on an Applied Biosystems ABI7700 Real Time PCR machine and data analyzed using Sequence detector software. Relative levels of grzB mRNA were determined using the {Delta}Ct method according to manufacturers instructions. B, CFSE-labeled CD8+ OT-I T cells were transferred into B6 naive mice, infected i.n. with the HK-OVA virus and MLN harvested 3.5 days later. OT-I CTL were stained with anti-CD8a-PerCPCy5.5, anti-CD62L, and anti-intracellular grzB. Shown is the grzB staining vs CFSE fluorescence for either CD62Lhigh (right panel) or CD62Llow (left panel) populations. C, The proportion of GrzB+ CD62Lhigh ({square}) or CD62Llow ({blacksquare}) OT-1 CTL that have undergone limited (division 1–3) vs extended (divisions 4–6) division is shown (n = 3). D, CFSE labeled cells were prepared as described for A. Shown is the mean fluorescence intensity of grzB staining (n = 3) for CD62Lhigh ({square}) and CD62Llow ({blacksquare}) OT-I CTL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Upon activation, naive CTL undergo a program of proliferation and acquisition of various effector functions (1, 2). Previous reports have established the relationship between gene expression and cell division for grzB and IFN-{gamma} protein (16) by naive, transgenic T cells differentiating in the regional lymph nodes of mice infected with an influenza A virus. Although other studies have demonstrated that naive CTL develop cytotoxic capacity early after infection before leaving the lymph node (14, 15), a direct correlation between effector molecule expression, cell division, and the acquisition of effector function has not been formally demonstrated. The present analysis establishes that the link between acquisition of effector function and cell division extends to pfp, grzA, and grzK mRNA. Moreover, this progressive acquisition of "effector" message correlates directly with the development of immediate CTL activity.

The divergence in prevalence for pfp and grzB mRNA suggests that independent regulatory pathways may operate to determine their expression. In fact, the results presented here indicate that differential regulation (18, 23) operates for all four of the CTL "effector" molecules analyzed here. Though there is a tendency for grzB and grzK to emerge earlier, the prevalence rates are not identical, each mRNA can be expressed independent of the other in single responding T cells and they are in no sense in "lock step." Furthermore, while our earlier experiments with polyclonal, endogenous DbNP366- and DbPA224-specific CD8+ CTL responses in this influenza A virus model (6) could be interpreted as suggesting that these diverse "effector" mRNA profiles are a reflection of varying TCR/peptide plus MHC class I glycoprotein avidities for TCR-different clonotypes, this explanation cannot be applied in the current situation where the response profiles are for homogeneous, TCR-transgenic OT-I T cells. The heterogeneity of both timing and frequency for "cytotoxic effector" mRNA expression is, therefore, an intrinsic property of responding CTLs that can be independent of TCR usage and Ag specificity. This in contrast to the situation for cytokine production where qualitative differences in IFN-{gamma}, TNF-{alpha}, and IL-2 production by virus-specific CTL have been correlated with differences in CTL avidity (24, 25, 26).

Recent studies have demonstrated that transfer of high numbers of TCR transgenic T cells results in phenotypic and functional outcomes that do not mimic activation of the endogenous repertoire (27, 28, 29). Given that there appears to be an upper limit of expansion regardless of precursor frequency (30), a higher precursor frequency means less expansion overall is required to reach that ceiling. As a consequence, expanded T cell populations peak earlier and have altered phenotypic and functional characteristics (27). Importantly, our study is a direct comparison of activated T cells at stages of cellular division and, therefore, at different stages of differentiation. As such, the issue of high precursor frequency does not impact the interpretation and biological relevance of our data. In fact, we demonstrate that acquisition of function occurs early after activation and is tightly regulated with the extent of division, therefore, supporting the notion that a lower level of expansion results in diminished functional capacity (27).

Recent data has demonstrated that rather than CTL memory being established upon resolution of infection (2), memory precursors can be generated in the early stages of the immune response (12, 31). We demonstrated that functional characteristics of TCM (lower cytotoxic potential) and TEM (high cytotoxic potential) (9) are established early after infection. The difference in cytotoxic effector function for the CD62Lhigh/low subsets may reflect the capacity to transcribe high enough levels of mRNA for protein synthesis rather than a simple ± expression of transcript. Alternatively, there may be differences in effector mRNA stability. Furthermore, CD62Lhigh (TCM) CTL were capable of expressing a similar profile of cytotoxic gene transcripts to TEM, raising the question "at what stage should we describe these differentiating mRNA+ cells as effectors?" Rather than debating the proposal that memory T cells can be derived from "effector" precursors (1), we might more usefully ask: "when, if ever, are "effector" T cells terminally differentiated?"

Intriguingly, the level of specific killing was lower (for an E:T ratio of 1:1) than would have been expected for fully activated CTLs (32). Given the requirement for pfp to mediate potent CTL activity (33), this is likely due to the relatively low frequency pfp expression at the later divisions. As such, the profile of limited expression for pfp we observed suggests that the high level of cytotoxicity found after eight divisions in these experiments is mediated by, at the most, 30% or so of the activated T cells. At least in the influenza model, where no infectious virus is produced in the lymphoid tissue, the generation of full effector function may require further differentiation after the CTL leave the draining lymph node and localize to the site of virus-induced pathology in the respiratory tract. This possibility is supported by the observation that influenza-specific CTL recovered at the peak of the response by bronchoalveolar lavage of the infected lung exhibit focused (narrowed) gene expression and more potent cytotoxic capacity when compared with comparable T cells in the regional lymph nodes and spleen (10, 32). As it is possible that the lysis of Ag-presenting dendritic cells limits the expansion of Ag-specific CTLs (34), such delayed expression of pfp in lymph node T cells may serve to ensure that the initiation of a primary response is not aborted prematurely, before sufficient CTL numbers are generated. A similar scenario may play out for re-activation of Ag-specific TCM memory populations. A low level of effector mRNA and cytotoxic function within CD62Lhigh TCM may limit killing of dendritic cells within the draining lymph node, thus ensuring that Ag presentation persists for long enough to ensure a robust memory response.


   Acknowledgments
 
We thank Dr. John Stambas for critical review and discussion, Ken Field for cell sorting, and Dina Stockwell for technical assistance.


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


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

1 This work was supported by an Australian Postgraduate Scholarship awarded to M.R.J.; an National Health and Medical Research Council Burnet Award and a Victorian Government Science, Technology and Innovation grant awarded to P.C.D.; an National Health and Medical Research Council R.D. Wright Fellowship awarded to K.K. and N.L.G.; a Pfizer Senior Research Fellowship awarded to S.J.T.; a University of Melbourne C.R. Fellowship awarded to J.M.; and a Melbourne University Early Career Researcher grant awarded to S.J.T. and J.M. Back

2 Current address: University of Cambridge, Cambridge Institute for Medical Research, Wellcome/Medical Research Council Building, Hills Rd, Cambridge, CB2 0XY, United Kingdom. Back

3 Address correspondence and reprint requests to Dr. Stephen Turner, University of Melbourne, Department of Microbiology and Immunology, Parkville, Victoria, Australia 3010. E-mail address: sjturn{at}unimelb.edu.au Back

4 Abbreviations used in this paper: pfp, perforin; grz, granzyme; MLN, mediastinal lymph node; TEM, T effector memory cell; TCM, T central memory cell; i.n., intranasally. Back

Received for publication May 20, 2008. Accepted for publication July 15, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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O. Bannard, M. Kraman, and D. T. Fearon
Secondary Replicative Function of CD8+ T Cells That Had Developed an Effector Phenotype
Science, January 23, 2009; 323(5913): 505 - 509.
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