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The Journal of Immunology, 2007, 179, 64 -70
Copyright © 2007 by The American Association of Immunologists, Inc.
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Heterogeneity of Effector Phenotype for Acute Phase and Memory Influenza A Virus-Specific CTL1

Misty R. Jenkins2,*, 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
 
Ag-specific, CD8+ CTLs clear influenza A viruses from the lung via granzyme (Gzm) and perforin-dependent mechanisms. Ex vivo analysis of perforin-Gzm mRNA profiles demonstrated substantial heterogeneity in patterns of effector mRNA transcription of CD8+ DbNP366- or DbPA224-specific CTL. The only difference between the two epitope-specific sets was apparent very early after infection with similar molecular profiles seen in peak primary and secondary responses and in long-term memory. Surprisingly, memory T cells also expressed a diverse pattern of effector mRNA profile with an emphasis on GzmB and, surprisingly, GzmK. This analysis thus defines how naive, effector, and memory T cells differ in cytotoxic potential and provides novel insight into the molecular signatures of effector molecules observed at various stages after infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The process of virus infection induces naive CD8+ T cells to embark on a program of Ag-driven differentiation and proliferation (1, 2) that culminates in the acquisition of various effector functions. During the course of the acute response, these expanded effector populations disseminate from the draining lymph nodes to other anatomical locations, particularly the site of infection (3, 4). The size of the various epitope-specific CTL sets then contracts after virus clearance to form stable memory T cell pools that persist for the life of a laboratory mouse (5, 6, 7) while retaining at least a measure of effector function (5, 8, 9). Much of the molecular detail in this sequence of activation, proliferation, majority CTL death, and minority memory T cell persistence is yet to be explored, although the broad picture is both established and generally agreed (5).

Activated CD8+ CTLs play a major part in the acute control of most virus infections (10). Prominent in the CTL cytoplasm are secretory granules that contain a spectrum of cytotoxic molecules including perforin (Pfp)4 and a group of serine proteases (11) called granzymes (granule enzymes; Gzm). After CTL engagement via TCR-mediated recognition of viral peptides (p) complexed with self-class I MHC glycoproteins (pMHCI), the granules move toward the CTL target interface where secretion of the cytotoxic contents results in apoptotic cell death. Although GzmA and GzmB are generally considered to be the most abundant Gzms (12), the cytotoxic granules also contain other Gzms with less well-defined functions. The pore-forming Pfp protein (13) facilitates the efficient delivery of GzmB to the target cell cytoplasm and then works synergistically to induce rapid apoptosis via primarily caspase-dependent mechanisms (14, 15). By contrast, GzmA has been shown to trigger cell death in a caspase-independent way (16). Despite that GzmA/B-deficient CTL are defective inducers of classical apoptosis, they still function as effective mediators of target cell death, suggesting that other Gzms may be operating via alternative pathways (17, 18). Another cytotoxic molecule, GzmK, is also found within the CTL granules, albeit at low levels in human lymphocytes (19). Less is known about the cell death pathways activated by GzmK, although purified GzmK can induce cell death independent of either GzmA or GzmB (20).

Influenza virus-specific CTL differentiate early to express GzmB protein (21) correlating with observations that, although effectors in the virus-infected lung may be much more potent (22, 23), differentiating CD8+ T cells can acquire CTL activity before exiting the draining lymph nodes (24, 25). After polyclonal in vitro stimulation of naive CD8+ T cells, early transcription of Pfp and GzmB mRNA is followed by GzmA mRNA expression 3–4 days later (26). The single-cell effector mRNA profiles were very heterogeneous, suggesting that Gzm and Pfp transcription may not be coregulated (26, 27). An indication that this functional diversity may also apply in vivo was found for influenza A virus-specific CTL isolated from both lung and mesenteric lymph nodes 7 days after primary challenge (27).

Although CD8+ T cell responses to the nucleoprotein (NP366) and acid polymerase (PA224) peptides bound to H2-Db (28, 29) are approximately equivalent in magnitude following primary influenza A virus challenge of C57BL/6J (B6, H2b) mice, the DbPA224-specific T cells tend to emerge a little earlier and to show evidence of a more potent effector function (29, 30). Although both DbNP366- and DbPA224-specific CTL demonstrate potent cytotoxicity directly ex vivo (23), little is known about the patterns of CTL-specific effector gene transcription within these populations. How, and to what extent, these profiles of effector mRNA expression are maintained into memory is even less well understood. The present analysis pursues these questions for DbNP366- and DbPA224-specific CD8+ T cells recovered from both naive and previously primed mice after influenza virus challenge. The findings provide insight into both the development and character of the effector and memory phases of virus-specific CD8+ T cell-mediated immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and virus infection

Female C57BL (B6) mice were bred at the University of Melbourne (Melbourne, Austsralia) and were held under specific pathogen-free conditions. Naive mice (6–8 wk of age), or mice previously primed i.p. with 1.5 x 107 PFU of the PR8 influenza A virus at least 6 wk before challenge, were lightly anesthetized by inhalation of methoxyfluorane and infected intranasally (i.n.) with 104 PFU of the HKx31 influenza A virus. All experiments were approved by the University of Melbourne Animal Experimental Ethics committee.

Tissue sampling and cell preparation

Lymphocytes obtained by bronchoalveolar lavage (BAL) were incubated on plastic for 1 h at 37°C to remove adherent macrophages. Single-cell mesenteric lymph nodes and spleen suspensions were prepared and enriched for the CD8+ set using tissue culture plates precoated with 200 µg/ml anti-IgG and anti-IgM (Jackson ImmunoResearch Laboratories) mAbs for 1 h at 37°C, 5% CO2.

Tetramer and protein staining

Influenza-specific CD8+ T cells were identified using either the DbNP366 or DbPA224 tetrameric complexes conjugated to streptavidin-PE. Cells stained with tetramer were held at room temperature for 1 h, followed by two washes with sort buffer (PBS with 0.1% BSA), then incubated on ice for 20 min with anti-CD8{alpha}-FITC (BD Pharmingen). The cells were then washed, resuspended in sort buffer and transferred to polypropylene tubes for sorting. Cells stained for tetramer and CD8{alpha} were also fixed and permeabilized using a BD Cytofix/Cytoperm kit (BD Pharmingen); then intracellular GzmB was detected using anti-human GzmB-APC (clone GB12; Caltag Laboratories). The flow cytometry analysis utilized a BD FACSCalibur (BD Biosciences) and CellQuest software.

Single-cell sorting and RT-PCR

Lymphocytes were isolated using a FACS ARIA (BD Biosciences) fitted with a single-cell deposition unit (BD BioSciences). Single virus-specific (DbNP366 or DbPA224 tetramer binding) or naive (CD8+CD44high/low) cells were deposited directly into each well of a 96-well PCR plate (Eppendorf) containing 5 µl of a cDNA reaction mix (7). A total of 10 negative controls were included, and 80 single cells were sorted into each plate. Once sorted, the plates were capped and incubated at 37°C for 90 min to synthesize cDNA, before incubating at 95°C for 5 min to inactivate residual reverse transcriptase activity. The two rounds of nested PCR utilized 5 µl of cDNA in a 25-µl reaction volume. The first round of PCR amplified a larger region and used external primers for Pfp, GzmA, GzmB, GzmC, GzmK, and CD8{alpha} (as a positive control). For the second round of PCR, 2 µl of the first round were used as template to amplify a smaller (internal region) or each of the products separately. The PCR utilized 1 U of Taq polymerase (Invitrogen Life Technologies), 1.5 mM MgCl2, 0.2 mM dNTP, and 20 pmol each of the sense and antisense primers. The majority of the primers used to amplify each region have been described previously (26). Otherwise, the primers were: GzmK, EX sense 5'-GGCCATTTATGGCGTCCATCCAG-3', antisense 5'-TCACCTGGCATTTGGTCCCA-3' and IN sense 5'-GCAAGCATATTTGTGGAGGA-3', antisense 5'-GTCGTGAGAATGGGATGAAC-3'; CD8{alpha}, EX sense 5'-ATGGACGCCGAACTTGGTCA-3', antisense 5'-GTTCGCAGCACTGGCTTGGT-3', IN sense 5'-GTCAGAAGGTGGACCTGGTATG-3', antisense 5'-GAACTTGTTCAGGGT GAGAACG-3'.

The PCR conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 90 s, followed by 1 cycle of 95°C for 30 s, 55°C for 1 min, and 72°C for 7 min. Then 5 µl of each PCR product were resolved on a 2% agarose gel and visualized using ethidium bromide staining. Samples that were not CD8{alpha} positive were excluded from analysis, and bulk-sorted CD8+ spleen cDNA isolated from primary influenza virus-infected mice was used as a positive control for all primer products.

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

RNA was isolated from >1 x 105 bulk-sorted DbNP366- and DbPA224-tetramer-specific CD8+ T cells using TRIzol reagent (Invitrogen Life Technologies) followed by chloroform extraction and isopropanol precipitation. The RNA pellets were resuspended in 30 µl of RNA Storage Solution buffer (Ambion) and incubated at 60°C for 5 min. RNA was quantified by OD260 nm, and cDNA was synthesized using an Omniscript kit (Qiagen). The cDNA mix was added to the samples to give the following final concentrations: 0.2 U/µl Omniscript reverse transcriptase (Qiagen), 1x cDNA buffer, 0.5 mM each dNTP (Omniscript RT kit; Qiagen), 1 µM oligodeoxythymidylate primer (Promega) and 0.5 U/µl RNAsin (Invitrogen Life Technologies); 100 ng of total RNA were used in each reaction. Real-time PCR was performed using TaqMan Gene MGB primer/probes (FAM labeled; Applied Biosystems) specific for Pfp, GzmA, GzmB, GzmC, GzmK, and mitochondrial ribosomal protein L32 as an internal standard, in a 25-µl reaction volume. Real-time PCR was performed using an ABI PRISM 7700 thermocycler (Applied Biosystems) under the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Each cDNA sample was assayed in duplicate, and the results are reported as the mean of replicates, with results showing the mean fold difference of four individual mice per group ± SD. Real-time data were analyzed using Sequence detector software, and the relative fold differences were determined using the {Delta}CT method according to the manufacturer’s instructions (Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Single-cell analysis of DbNP366+ and DbPA224+ T cells

Naive (primary; Fig. 1) or PR8-immune (secondary; Fig. 2) B6 mice were challenged i.n. with the HKx31 influenza A virus, then CD8+ T cells that stain with the DbNP366 and DbPA224 tetramers were taken for single-cell FACS separation and mRNA profiling at intervals thereafter. The various patterns of effector mRNA expression within individual CTL are shown in Figs. 1 and 2. In general, the essentially random patterns of Pfp/Gzm mRNA expression observed after polyclonal stimulation of naive CD8+ CTL (26) were replicated for both primary (Fig. 1) and secondary (Fig. 2) DbNP366- and DbPA224-specific CTL responses. Little evidence was found for co-ordinated expression of effector mRNAs within and between the Ag-specific CTL populations. Moreover, heterogeneity of effector mRNA expression found in the acute phase of the primary response persisted into early (Fig. 1, day 18) and long-term (Fig. 2, days 60 and 365) memory CTL. Overall, there seemed to be little coregulation of effector mRNA acquisition, whereas Pfp/Gzm mRNA profiles are independent of whether the response is initiated from naive or memory CTL precursors.


Figure 1
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  		FIGURE 1. Pfp and Gzm mRNA profiles for the primary and early memory DbNP- and DbPA-specific CD8+ T cell responses. DbNP366- and DbPA224 tetramer+CD8+ T cells from naive B6 mice (primary; 1°) were challenged i.n. with 1 x 104 PFU of the A/HKx31 virus and the BAL (days (d) 5 and 7) and spleen cells (days 5, 7, and 18) were sampled at various times after infection. Single tetramer+CD8+ T cells were sorted directly into wells of a 96-well plate containing cDNA buffer and RT-PCR was performed to detect expression of CD8, GzmA, GzmB, GzmC, GzmK, 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 pooled DbNP366 and DbPA224-specific populations are listed for pooled mice (n = 3).

 

Figure 2
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  		FIGURE 2. Pfp and Gmz mRNA profiles for the long-term memory and secondary DbNP- and DbPA-specific CD8+ T cell responses. DbNP366- and DbPA224 tetramer+CD8+ T cells from naive B6 mice were challenged i.n. with 1 x 104 PFU of the A/HKx31 virus and the spleen cells were sampled at on days (d) 60 (n = 3) and 365 (n = 2) after primary (1°) infection. For secondary (2°) responses, mice primed i.p. at least 6 wk previously with 1 x 107 PFU of the A/PR8 virus, were challenged i.n. with 1 x 104 PFU of the A/HKx31 virus and sampled at various times after infection. Lymphocytes were isolated from BAL (day 5) and the spleen (days 5 and 28) and single tetramer+CD8+ T cells were sorted directly into wells of a 96-well plate containing cDNA buffer and RT-PCR was performed to detect expression of CD8, GzmA, GzmB, GzmC, GzmK, 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 pooled DbNP366 and DbPA224-specific populations are listed for pooled mice (n = 3).

 
The analysis in Figs. 1 and 2 represent pooled data. The results acquired from different individuals are summarized as mean ± SD in Figs. 3 and 4. Fig. 3 shows the frequency of individual CTL expressing a given effector mRNA, whereas Fig. 4 summarizes the frequency of individual CTL expressing multiple effector mRNAs. The situation for bulk, naive CD8+CD44low and "activated/memory" CD8+CD44high T cells is also described for mice that have never been exposed to any influenza A virus (Figs. 3A and 4A). The CD8+CD44low set was essentially negative for all effector mRNAs, whereas the (presumably) Ag-experienced CD8+CD44high T cells (Fig. 3A) showed profiles that were not identical with those found for the influenza-specific memory populations (Fig. 3, H–K). In particular, the CD8+CD44low T cells were more likely to be GzmA+ and GzmC+, and less likely to express GzmK than the CD8+DbNP366+ and CD8+DbPA224+ sets (Fig. 3, A and H–K). This comparison between influenza-experienced and naive mice suggests that, given the relative convergence in the results for the DbNP366- and DbPA224-specific CTLs, different infectious (or perhaps homeostatic) experiences may set up varied spectra of cytotoxic effector molecule mRNA heterogeneity in memory T cells.


Figure 3
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  		FIGURE 3. Frequency of single DbNP366 and DbPA224-specific CTL T cells expressing different effector mRNAs. Frequency analysis of effector mRNA expression by single Ag-specific CD8+ CTL is illustrated various times after infection. These are the same mice described in Figs. 1 and 2. A, Frequency of effector mRNA expression for splenic CD8+CD44low (naive; gray bars) and CD8+CD44high (memory; striped bars) T cells from naive mice that have never been exposed to an influenza A virus. B–G, Frequency of single-cell effector mRNA expression for acute primary (B, C, E, and F) and secondary (D and G) DbNP366- and DbPA224-specific responses. Shown are lymphocyte populations isolated from the spleen (B–D) or BAL (E–G). H–K, Results for splenic memory T cells established after primary (H and I) or secondary (J and K) infection. Each data point (n = 3) shows mean ± SD; significance was determined using a paired Student t test. *, p < 0.05; **, p < 0.002), except for day (d) 365 (I) where n = 2.

 

Figure 4
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  		FIGURE 4. Comparison of single DbNP366- and DbPA224-specific CTL T cells expressing multiple effector mRNAs. Proportion of single CD8+tetramer+ CTLs expressing various effector mRNAs (either Ppf, GmzA, GmzB, GmzC, or GmzK) at various times after infection. A, Frequency of effector mRNA expression for splenic CD8+CD44low (naive; gray bars) and CD8+CD44high (memory; striped bars). T cells from naive mice that have never been exposed to an influenza A virus. B–G, Frequency of single-cell effector mRNA expression for acute primary (B, C, E, and F) and secondary (D and G) DbNP366- and DbPA224-specific responses. Shown are lymphocyte populations isolated from the spleen (B–D) or BAL (E–G). H–K, Results for splenic memory T cells established after primary (H and I) or secondary (J and K) infection. The numerical values on the x-axis are: 0 = CD8+tetramer+ CTL not expressing other effector mRNAs; 1 = CD8+ CTL plus one other effector mRNA; 2 = CD8+ CTL plus two other effector mRNA; 3 = CD8+ CTL plus three other effector mRNA; 4 = CD8+ CTL plus four other effector mRNA; 5 = CD8+ CTL positive for all effector mRNAs tested; d, day.

 
In the primary response, the great majority of the DbNP366- and DbPA224-specific T cells detected in spleen at the earliest time point (day 5) that can be analyzed after primary infection were either Gzm and Pfp negative, or expressed only one or two effector signatures (1°, day 5 spleen; Fig. 4B). The few virus-specific CD8+ T cells recovered on day 5 by BAL of the infected lung were, however, clearly more activated (1°, day 5 BAL, Figs. 3E and 4E), with three or more mRNAs being found in >40% of the DbPA224-specific set (Fig. 4E). By day 7, the greatly expanded spleen and BAL populations were showing further increase in the frequency (Fig. 3, C and F) and spectrum of effector mRNA expression (Fig. 4, C and F). Both the numbers of positive cells and the range of the mRNA expression profiles then fell substantially by day 18 (Figs. 3H and 4H), the stage when the numerical contraction of the CD8+ T cell response is very apparent in the BAL and the spleen. Thereafter, many of the CD8+DbNP366+ and CD8+DbPA224+ T cells continued to express at least one effector mRNA into long-term memory (day 365 spleen; Fig. 4I).

The recall from memory measured on day 5 after secondary i.n. challenge showed high levels of activation, with >70% of the CD8+DbNP366+ and CD8+DbPA224+ sets recovered in both the spleen and BAL populations expressing at least three mRNA signatures (2°, day 5; Fig. 4, D and G). By day 28, however, although T cell numbers are still substantially increased over the counts that would be found at this stage following primary infection (4), there was nothing in the mRNA phenotype to distinguish primary and secondary CD8+ T cell memory populations (compare 2° day 28 in Figs. 3J and 4J with 1° days 18 and 60 in Figs. 3, H and I, and 4, H and I). Similarly, there was no indication that secondary CD8+ memory T cells are likely to have any long term advantage (compare 1° day 365 with 2° day 540 in Figs. 2K and 3K). Despite evidence of greater TCR avidity for DbPA224-specific populations (30) and the much greater expansion of the CD8+DbNP366+ set that follows secondary exposure (29, 30, 31), the Pfp and Gzm mRNA expression profiles tended to be broadly comparable for both the peak of the primary and secondary effector phase, then into memory, for these two Ag-specific T cell subsets (Figs. 3 and 4). Only the Pfp/Gzm mRNA analysis for the day 5 primary BAL population gave the only substantial indication that the higher avidity CD8+DbPA224+ CTLs (30) are being activated more rapidly than the CD8+DbNP366+ effectors (Figs. 3E and 4E). Somewhat unexpectedly, the numbers of CTLs expressing GzmA seemed to fall off rapidly after the acute phase of the response, whereas GzmK continued to be prominent in the long–term (Fig. 3; compare B–G with H–K). The expression of GzmC mRNA was consistently low for both these CD8+ T cell populations (Fig. 3, B–K).

Quantitation of effector molecule mRNA expression

Although the single-cell multiplex RT-PCR analysis (Figs. 1–3) describes the prevalence of effector mRNA acquisition within a CTL population, it does not measure the amount of mRNA expressed. An analysis was thus done to compare mean mRNA levels for bulk-sorted CD8+DbNP366+ and CD8+DbPA224+ T cells sampled at intervals after primary or secondary challenge (Table I). Real–time PCR was performed using primer-probe sets specific for Pfp and GzmA, GzmB, GzmC, and GzmK. The minimalist profile was confirmed for GzmC, with no evidence of expression being found for day 6 secondary acute, or day 60 secondary memory T cells. In general, the results of the real–time PCR analysis supported the single-cell study, with the expected levels of Pfp and GzmB/K mRNA being detected within CTL populations. Also, as predicted from an earlier cDNA array analysis (23), greater levels of GzmA mRNA were present in splenic than in BAL CTL populations. The main difference from the single-cell results was, however, that the relative abundance of GzmA mRNA in memory T cells was much greater than expected from the low frequency of GzmA+ CTLs identified in the kinetic analysis (Figs. 3 and 4), especially in CTL isolated from secondary DbNP366 and DbPA224-specific populations. Further analysis by quantitative, single-cell RT PCR might well yield additional insights.


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  		Table I. Relative levels of CTL effector mRNA expressed by influenza A virus-specific CTLa

 
Profiles of GzmB protein staining

The DbNP366- and DbPA224-specific CTLs were sampled at different time points and stained with a cross-reactive mAb to human GzmB (Fig. 5). As with the real-time PCR analysis, GzmB+ CTL were present at comparable frequency in the CD8+DbNP366+ and CD8+DbPA224+ populations recovered after primary (Fig. 5, A–F) or secondary (Fig. 5, G–L) challenge. During the acute phase, GzmB+ CTL were less prevalent in the epitope-specific sets recovered from the spleen (Fig. 5, A–C and G–I) vs the BAL (Fig. 5, D–F and J–L). The analysis was then continued through to long-term memory, with the results being expressed as both the percent CD8+tetramer+GzmB+ cells (Fig. 6, A–D) and the mean fluorescence intensity (MFI) of GzmB staining (Fig. 6, E–H). The scales for the MFI values differ for the various panels (Fig. 6, E–H). Throughout the analysis, there is nothing that discriminates between the CD8+DbNP366+ and CD8+DbPA224+ T cells, no matter what the time of sampling or site of recovery (Fig. 6). This convergence of GzmB expression levels for different pMHC-specific responses is intriguing.


Figure 5
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  		FIGURE 5. Typical GzmB staining profiles for CD8+DbNP366+ and CD8+DbPA224+ T cells. Shown are epitope-specific cells that were recovered from spleen (A–C and G–I) and BAL (D–F and J–L) of B6 mice at 10 days (d) after 1 (0) (A–F) or 8 days after 2° i.n. challenge (G–L). These FACS plots are representative from four experiments. The tetramer+ cells were stained with anti-CD8{alpha}-PerCPCy5.5 and then fixed, permeabilized, and stained for intracellular GzmB-FITC. The flow cytometric analysis used a FACSCalibur and CellQuest software. A minimum of 50,000 CD8+ lymphoctye events were collected.

 

Figure 6
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  		FIGURE 6. Kinetic and quantitative analysis of GzmB staining for DbNP366+ and DbPA224+ T cells from the acute phase through to long-term memory. Tetramer+ cells were isolated from the BAL and spleens of mice after both primary and secondary influenza challenge, stained with anti-CD8{alpha}-PerCPCy5.5, and then fixed, permeablized, and stained for intracellular GzmB-FITC. Results are expressed as the (mean ± SD, n = 5) percent of GzmB+ (A–D) within each tetramer+ set or as MFI for the positive cells (E–H). The analysis examined at the spleen (A, C, E, and G) over a long time course following 1° (A and E) or 2° (C and G) challenge, and the BAL at the acute phase of the 1° (B and F) or 2° (D and H) response. For the 1° response, the day (d) 6 BAL (B and F) and day 7 spleen (A and B) samples were the earliest obtained in sufficient numbers for adequate GzmB phenotyping.

 
After primary influenza virus infection of B6 mice, CD8+tetramer+ T cells first become apparent on days 5 and 6 after infection and peak in magnitude on day 10, whereas the peak of the secondary response occurs on day 8 (31). Interestingly, the proportion of GzmB+tetramer+ CTL peaked on days 7–8 of the primary (Fig. 6, A, B, E, and F) and day 6 of the secondary (Fig. 4, C, D, G, and H) responses. Both time points precede the usual peak in CTL number. The difference between the BAL and spleen populations reflects a spectrum of previous evidence that the T cells recovered from the virus-infected lung are much more in "effector" mode (23), with the GzmB MFI values being as much as 3 to 5 times above those for the concurrently sampled spleen sets (Fig. 6, compare E and F with G and H). However, compared with day 6 after infection, less GzmB protein is found within Ag-specific CTL at the peak of the acute primary (day 10, Fig. 6, B and F) and secondary (day 8, Fig. 6, D and H) responses in terms of both proportion of GzmB+ CTL and abundance of GzmB protein. This rapid drop in GzmB+ CTL numbers and GzmB MFI staining values for the BAL population could be thought to reflect the degranulation of effector T cells operating in the site of virus-induced pathology.

The relative prevalence of splenic GzmB+ T cells declined fairly rapidly with the resolution of the acute phase of both the primary and secondary virus-specific CD8+ T cell responses (Fig. 6, A–C), although some cells expressing very low levels of GzmB protein could still be detected in the very long term. Comparison with the mRNA profiles (Fig. 3, H–K), however, suggests that many fewer memory T cells are making detectable GzmB protein (Fig. 6, A–E and C–G). Even so, it was only from the GzmB-staining profiles that we found a hint that there may be differences between primary and secondary CD8+ memory T cells. The percent GzmB+ within the CD8+tetramer+ sets was a little higher at the late time points after secondary (days 28 and 180 in Fig. 6C) rather than primary (days 28–180 in Fig. 6A) challenge. There is also an indication that upon recall, memory CTL are capable of expressing higher levels of GzmB protein when compared with naive CTL sampled early after challenge (Fig. 6, compare E and G).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Naive CTL develop the potential for cytolytic activity before disseminating to other tissues in virus-infected mice (25). Despite sharing similar functions that contribute to the development of the cytotoxic phenotype, the transcription of at least some of these effector mRNAs must be independently regulated. It also seems that the mechanisms controlling Pfp and Gzm mRNA expression are an inherent characteristic of CTL, as the diverse mRNA profiles for effector and memory T cells were similar for CTL of different specificities. Although this supports the idea that naive precursors embark on programmed proliferation leading to the development of both effector and memory CTLs (1, 32), the process evidently plays out differentially when it comes to Pfp and Gzm expression within individual, Ag-specific clonotypes. The results fit, in fact, with a random, stochastic model of effector T cell differentiation (33).

The expression of GzmK by Ag-specific CTL was striking, and confirmed observations that GzmK transcription is characteristic of both effector and memory CD8+ T cells (S. J. Turner and P. C. Doherty, unpublished data, and Ref. 5). The little that is known about GzmK indicates that it is somewhat like GzmA in function and substrate specificity (20) although, in the current experiments, the expression profile for GzmK was more like that of GzmB. Might GzmA and GzmK be essentially interchangeable when it comes to CTL effector function? It is known thatGzmAB-deficient CTL, which show no defect in GzmK expression, are still able to mediate rapid target cell death by a nonclassical pathway (17, 18).

Differences in the molecular profiles of effector mRNA expression were observed for endogenous CD8+DbNP366+ and CD8+DbPA224+ CTL only in the early phase after infection. Under inflammatory conditions in, for example, the virus-infected lung sampled by BAL, these CTL have clearly differentiated further to become fully activated effectors (22, 23). This activation-related acquisition of cytotoxic capacity may explain why, at the very earliest stage, the DbPA224-specific CTL recovered by BAL expressed more effector mRNAs than the corresponding CD8+DbNP366+ set. Perhaps the greater numbers of DbPA224-specific precursors within the naive repertoire (30, 34) facilitate more rapid proliferation and recruitment to the infected lung. By day 7, however, no differences in Pfp/Gzm mRNA profiles were apparent for the CD8+DbNP366+ and CD8+DbPA224+ sets recovered from lung or spleen. This is in direct contrast to the situation for increased IFN-{gamma}, TNF-{alpha} and IL-2 production by DbPA224-specific effector and memory CTL that in effect correlates with in vitro measurement of slower TCR disassociation (30).

It has been shown that CTL can acquire cytotoxic arming as soon as 24 h after infection (35) and certainly by the time activated CTL leave the draining lymph node and emigrate to the spleen (25). It is possible that, at this very early stage of CTL activation, effector mRNA expression within single Ag-specific CTL are in fact coordinated and are differentially regulated upon exit from the lymph node. However, naive CTL up-regulate GzmB mRNA more rapidly than Pfp mRNA after in vitro activation, suggesting that differential effector gene regulation is apparent even in the early stages of naive CTL activation. However, such results need to be confirmed directly ex vivo and experiments are currently under way to examine this issue.

As these CD8+DbNP366+ and CD8+DbPA224+ T cells proliferate during the acute, effector phase, then become long-term memory cells, the Pfp/Gzm mRNA expression profiles transit from heterogeneity to relative homogeneity, returning to a profile of greater heterogeneity with the contraction of the response that occurs after Ag elimination. Even so, many memory T cells continue to express at least one Gzm mRNA in the very long term, with GzmB and GzmK being the most common. The loss of the less diverse T cells may reflect selective death of the most potent effectors, although it is also conceivable that there could be a measure of dedifferentiation. Another possibility is that a stable pool of memory T cell precursors is established very early in the course of the response (36, 37).

Importantly, the maintenance of Pfp/Gzm mRNA in long-term memory is in direct contrast to the situation for cytokines like IFN-{gamma}, TNF-{alpha}, and IL-2 that require further Ag exposure to induce mRNA and protein expression (30). Thus, the regulatory mechanisms used to maintain different effector function within persisting memory CTL populations are clearly diverse. Also, despite the high frequency of GzmB mRNA, the level of protein staining within long-lived memory CTLs was generally very low, indicating that the regulation of GzmB expression is more at the level of translation than of transcription. Both IL-2 and IL-15 have been demonstrated to enhance transcription of Pfp and GrzB mRNA (38, 39). Given that IL-15 is important for maintenance of memory CTL homeostasis (40, 41), it is tempting to speculate that the actions of IL-15 may also play a role in maintaining effector mRNA transcription in long-term influenza-specific memory CTL. Such a mechanism may contribute to the enhanced induction of CTL effector function that is a hallmark of memory T cell responses (8, 9).

Previous experiments have shown that TCR diversity/avidity profiles, peptide-induced patterns of IFN-{gamma} and TNF-{alpha} production, estimates of in vivo Ag load and the spectrum of potential APCs all differ for the CD8+DbNP366+ and CD8+DbPA224+ responses (7, 30, 42, 43, 44). Despite that, both the frequency and intensity of GzmB protein staining and the extent of Pfp/Gzm mRNA heterogeneity were essentially comparable for these two Ag-specific sets from the peak of the acute response through to persistent memory. Perhaps what we are seeing here is that the character of Pfp/Gzm induction is a function of the overall response milieu associated with a particular pathogen. Would, for example, the long-term emphasis on GzmK found here with the influenza A viruses be apparent for other viruses? This heterogeneity in Pfp/Gzm profiles make biological sense, given that different infectious agents are known to have evolved various molecular evasion strategies for subverting cytotoxic effector pathways (45, 46). As such, it would be of interest to determine whether the molecular signatures observed in this model of infection were found in CTL induced after infection with other pathogens.


   Acknowledgments
 
We thank Drs. Nicole La Gruta, John Stambas, and Paul Thomas for critical review and discussion; Ken Field for cell sorting; and Dina Stockwell for excellent 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 (to M.R.J.), a National Health and Medical Research Council Burnet Award (to P.C.D.), a Victorian Government Science, Technology and Innovation Grant (to P.C.D.), an National Health and Medical Research Council Peter Doherty Postdoctoral Fellowship (to K.K.), an National Health and Medical Research Council R. D. Wright Fellowship (to S.J.T.), and a Melbourne University Early Career Researcher Grant (to S.J.T.). Back

2 Current address: Sir William Dunn School of Pathology, University of Oxford, Oxford, U.K. Back

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

4 Abbreviations used in this paper: Pfp, perforin; Gzm, granzyme; NP, nucleoprotein; PA, acid polymerase; i.n., intranasally; BAL, bronchoalveolar lavage; MFI, mean fluorescence intensity. Back

Received for publication February 8, 2007. Accepted for publication April 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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