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The Journal of Immunology, 2008, 180, 5300 -5308
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Viral Infections

Derivation and Fluidity of Acutely Induced Dysfunctional CD8+ T Cells1

Gabriela Plesa*, Adam E. Snook{dagger}, Scott A. Waldman{dagger} and Laurence C. Eisenlohr2,*

* Department of Microbiology and Immunology and {dagger} Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dysfunctional CD8+ T (TCD8+) cells lacking cytokine production have been identified in many viral infections, but their genesis is not well understood. Established results indicate that such cells could be either high avidity that enter a refractory state due to overstimulation or low avidity that are only partially stimulated. Using an acute, resolving infection model that results in rapid production of dysfunctional cells, we show that this IL2 unresponsive phenotype emerges from the low end of the avidity spectrum and is characterized by broad TCR usage and a reduced proliferation rate. Furthermore, the dysfunctional population is extremely fluid, being sustained by high Ag dose but virtually eliminated following low dose boosting. Together, these results suggest that persistence of dysfunctional cells generated in this manner depends upon continual exposure to high Ag levels and that such cells may ultimately predominate if functional cells become exhausted.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The main goal of all vaccine strategies is to induce long-lasting protective immunological memory. Although traditional vaccines have sought to induce neutralizing Abs, many recent vaccines aim to elicit potent TCD8+ cells. This has remained challenging, because the mechanisms that regulate TCD8+ cell responses and memory are not well understood (1).

It has been demonstrated that the protective capacity of a TCD8+ cell response depends on the size and average avidity of memory pool. Thus, higher numbers of Ag-specific TCD8+ cells are more protective against viral infection than lower numbers (2) and high avidity TCD8+ cells protect against viral infections (3, 4) and tumor cells (5) better than low avidity cells. However, these two parameters are influenced in opposite ways by Ag dose. The magnitude of a TCD8+ cell response and the resulting memory population are directly proportional to virus dose (6) or dose of a single epitope (7, 8). In contrast, the average avidity of activated Ag-specific TCD8+ cells inversely correlates with the Ag level both in vitro (4) and in vivo (9, 10). T cell avidity impacts effector function, such as cytokine secretion in both TCD4+ (11) and TCD8+ cells (12, 13). These data suggest that Ag density is an important factor that affects the qualitative and quantitative aspects of immune responses and consequently their protective capacity.

Using a vaccinia-based expression system that allows for different levels of the same epitope with equivalent viral doses, we have previously demonstrated that acute expression of low/intermediate Ag doses induces a population that is uniformly capable of cytotoxicity and IFN-{gamma} release. In some cases, however, when excessively high levels of epitope are expressed, a fraction of the responding TCD8+ cells is neither cytotoxic nor IFN-{gamma} producing (14). Other viral systems have shown similar TCD8+ cell loss of function at high viral doses (15, 16). This dysfunctional phenotype has clinical significance. It has been identified in acute viral infections (8, 17), but mainly in chronic infections of humans (18, 19, 20, 21, 22, 23, 24, 25), rhesus macaque (26, 27) and murine models (16, 28, 29, 30, 31, 32). Furthermore, appearance of such cells has been associated with lack of immune protection (33, 34). Thus, for various reasons associated with the induction and control of TCD8+ cell responses, a better understanding of the genesis and evolution of the dysfunctional phenotype is essential.

The generally accepted model based on in vitro studies is that low levels of Ag activate high avidity T cells and high epitope densities activate T cells with the full range of avidities. Our previous results are in agreement with this model, because the dysfunctional phenotype develops only at high Ag levels. However, the basis for this phenotype remained unknown. We considered two models that seemed equally plausible. On the one hand, dysfunctional cells might be overstimulated high avidity T cells that enter a refractory state, in line with in vivo studies reporting that high avidity TCD8+ cells persist without mediating effector functions under conditions of high Ag doses (35, 36, 37, 38). Alternatively, dysfunctional cells might be low avidity T cells that are unresponsive to limiting Ag and partially stimulated under conditions of high Ag level, resulting in limited or absent effector function. Indeed, low avidity TCD8+ clones can be tolerized in vivo by high Ag expression (39) and low affinity pMHC-TCR interactions of TCD4+ and TCD8+ cells in vitro result in partial intracellular signaling (35, 40, 41, 42) and functional unresponsiveness (43, 44). The experiments reported here were designed to distinguish between these two possibilities as well as to gain insight into other aspects of the acutely dysfunctional phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and infections

C57BL/6 mice and B6.PL-Thy1 (no. 000406) mice were purchased from The Jackson Laboratory. OT1 Rag+/+ mice (45) were a gift from Dr. K. A. Hogquist (University of Minnesota, Twin Cities, MN) and OT1 Rag–/– (no. 004175-MM) mice were purchased from Taconic Laboratories. The animal protocols were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. For boosting experiments, C57BL/6 mice were infected i.p. with 107 PFU of vaccinia virus (Vac),3 boosted with 107 foci-forming units of rabies virus (RV) and 50 hemagglutinating units of influenza (Flu) at 28-day intervals. The adoptive transfer experiments were performed as follows: after RBC lysing and washing, 5 x 106 OT1Rag+/+ or OT1Rag–/– Thy1.2 splenocytes were injected in the tail vein of C57BL/6 Thy1.1 mice. The next day, mice (four per group) were immunized i.p. with 106 PFU Vac.

Viruses

Recombinant Vacs expressing the SIINFEKL epitope (Vac-SIIN, Vac-18SIIN, NP/SIIN) and the negative control virus expressing the influenza NP147–155 epitope have been described (14). Vac-16SIIN was generated as described (14). Engineered RVs are described elsewhere (46). Engineered Flu viruses (47) were a gift from Dr. D. J. Topham (University of Rochester, Rochester, NY). Recombinant adenoviruses (Adeno) were generated using the ViraPower adenoviral expression system (Invitrogen) according to the manufacturer’s instructions. In brief, the gene of interest was subcloned into the pENTR/D/TOPO entry vector (Invitrogen) and then recombined into the pAd/CMV/V5-DEST vector provided in the kit using Gateway cloning. Virus was produced by transfection of 293 cells, purified using the Adeno-X virus purification kit (BD Biosciences) and titered using the Adeno-X Rapid Titer kit (BD Biosciences) according to the manufacturer’s instructions.

Quantification of Kb/SIIN complexes

Quantification was done as described (14). In summary, L929 cells that stably express the H-2Kb MHC class I molecule, LKb, were infected with 5 PFU of various Vacs and the surface expression of Kb/SIIN complexes was measured by staining with culture supernatant of a Kb/SIIN-specific Ab (48) followed by FITC-labeled anti-mouse IgG (Vector Laboratories). The standard curve used to estimate the number of Kb/SIIN complexes was generated using Immuno-Brite fluorospheres with various FITC intensities (Beckman Coulter).

Flow cytometry, intracellular staining

Kb/SIIN-specific tetramer was purchased from Beckman Coulter. Anti-CD8{alpha}-FITC Ab was purchased from Caltag, and all the other Abs were purchased from BD Pharmingen. For intracellular staining (ICS), we used the Cytofix/Cytoperm Plus kit (BD Pharmingen) according to the manufacturer’s instructions. The background staining of cells stimulated with an irrelevant peptide was subtracted. The samples were run on a Becton Dickinson flow cytometer (Kimmel Cancer Center Flow Cytometry Facility, Thomas Jefferson University). The analysis was done using WinMDI software. For vaccinia-specific immune responses, the spleen cells were stimulated with LKb cells previously infected with control Vac and stained as above.

Isolation of Ag-specific IFN-{gamma}+ and IFN-{gamma}– T cell subsets

C57BL/6 mice were primed with 107 foci-forming units RV-SIIN, boosted with Vac-NP/SIIN after at least 30 days and 4–5 spleens were processed 5 days later. Cells were stained with Kb/SIIN tetramer at 24°C and on ice with anti-CD8{alpha} and IFN-{gamma}-catch bispecific Abs (mouse IFN-{gamma} secretion assay detection kit; Miltenyi Biotec). The cells were diluted in warm medium (37°C) for 1 h, washed with cold buffer, and stained with IFN-{gamma} detection Ab. A negative control sample kept on ice was used for proper gating on the IFN-{gamma}+ population. The CD8+Tet+IFN-{gamma}+ and CD8+Tet+IFN-{gamma}– populations were sorted with a MoFlo sorter (Wistar Institute Flow Cytometry Facility), rested overnight in the presence of IL2 cytokine (40 U/ml) and used in specific assays.

Tetramer dissociation assay

The sorted populations were re-stained with excess Kb/SIIN tetramer at 24°C, washed, resuspended in FACS buffer, together with a Fab preparation of Kb/SIIN-specific Ab and transferred to 4°C. At different time points, a sample of 100 µl was removed and fixed in 200 µl of 2% paraformaldehyde. The samples were run on a Becton Dickinson flow cytometer and analyzed with WinMDI software to determine the mean fluorescence intensity (MFI) of tetramer staining.

Proliferation assay

Mice were primed with RV-SIIN and challenged 30 days later with Vac-NP/SIIN and given BrdU (1 mg) every day by i.p. injection. Five days later, the splenocytes were stained and the double-positive CD8+Tet+ population was sorted. Cells were rested overnight in the presence of IL2 and stimulated for 6 h with SIINFEKL peptide-pulsed LKb cells in presence of brefeldin A. BrdU and IFN-{gamma} staining was performed using the BrdU flow kit (BD Pharmingen) as specified by the manufacturer.

Spectratyping analysis

RNA was extracted using Trizol reagent (Invitrogen). The primer sequences have been previously published: Cβ-long (49), Vβ1, 2, 3, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20 (50), Cβ, Vβ4, 5.1, 5.2, 5.3, 8.1, 8.2, 8.3, 14 (51) and were obtained from the Kimmel Cancer Center Nucleic Acid Facility (Thomas Jefferson University). The reverse transcription reaction used avian reverse transcriptase (Invitrogen). PCR was performed using the Vβ sense, Cβ-long antisense oligoprimers, and Ampli Gold Taq polymerase (Applied Biosystems) to amplify the specific Vβ sequences. Semi-nested PCR used a second fluorescently labeled Cβ antisense oligoprimer (Applied Biosystems) internal to the first Cβ-long and Taq polymerase (Fischer Scientific). The PCR products were run on a sequencing gel and analyzed by the Genotyper GeneScan software program (Kimmel Cancer Center Nucleic Acid Facility).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Variable epitope levels generated by minigene expressing viruses

Previously, we examined the impact of Ag dose on generation of immune responses by using recombinant Vacs expressing different levels of Ova257–264 peptide (SIINFEKL, SIIN), an H-2Kb-restricted immunodominant epitope of OVA. For those studies, the epitope was expressed either from a minigene or in the context of influenza virus nucleoprotein (14). The minigene-encoded epitope requires minimal intracellular processing and reaches exceedingly high levels at the cell surface whereas the same epitope embedded within a protein reaches substantially lower surface levels, reflecting the inefficiency of Ag processing (52) (Fig. 1A). One complication in interpreting results with the original constructs is that the epitope expressed in these two contexts (in isolation or within a full-length protein) is likely to have qualitatively different effects on various aspects of the host response, such as cross-presentation (53, 54) and such effects rather than epitope dose could be the basis for the differing phenotypes. To circumvent this, we focused on minigene-based Vac and modulated epitope expression through insertion of a hairpin between the promoter and the epitope (14). The hairpin length inversely correlates with the amount of epitope expressed at the cell surface. SIINFEKL epitope encoded by a synthetic minigene was cloned behind a hairpin of 18 bp (Vac-18SIIN), 16 bp (Vac-16SIIN), or no hairpin (Vac-SIIN).


Figure 1
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  		FIGURE 1. Dysfunctional TCD8+ cells are acutely generated by high Ag doses. A, Density of Kb/SIIN complexes on the surfaces of LKb cells infected with various recombinant Vacs. LKb cells were infected at 5 PFU/cell and stained with a Kb/SIIN-specific Ab at 6 h postinfection. Similar results were obtained in three additional independent experiments. B and C, Primary immune responses to Vac expressing different epitope levels. C57BL/6 mice were primed with Vac expressing various levels of SIINFEKL epitope or a negative control virus. Fourteen days later, the response was assessed by staining with CD8- and CD44-specific Abs, and either Kb/SIIN tetramer or intracellular staining for IFN-{gamma} after SIINFEKL peptide stimulation (10–6 M) in vitro. Cells were gated on the CD8 population. The bottom panel represents Vac-specific functional responses assessed by ICS for IFN-{gamma} as described in Materials and Methods. Similar results were obtained in three independent experiments. Error bars represent SD of four mice per group. C, Representative dot plots for B.

 
The surface level of SIINFEKL epitope expressed by various Vacs was determined by infecting LKb cells and staining with an Ab specific for the Kb/SIIN complex (48). We also included Vacs that express OVA (Ova) protein or the SIINFEKL epitope inserted into influenza nucleoprotein (NP/SIIN). Expression of Kb/SIIN complexes by minigene viruses require no Ag processing thus occurs faster than that of protein-expressing Vacs. Accordingly, peak levels of epitope expression from the minigene Vacs were measured at 6 h postinfection. As expected (Fig. 1A), the Vac-SIIN virus produces the highest level of expression. Infection with Vac-16SIIN leads to an epitope density three times lower than Vac-SIIN, comparable to epitope production from the NP/SIIN virus in the same period of time. The Vac-18SIIN reduced expression by 10-fold, comparable to that produced by the Ova virus. These data confirm that the hairpin system allows for high (Vac-SIIN), intermediate (Vac-16SIIN), and low (Vac-18SIIN) epitope densities from the minigene context.

The effect of epitope level on functionality of TCD8+ cells

We primed C57BL/6 mice with equivalent doses of the hairpin-minigene Vac and analyzed the immune responses by Kb/SIIN tetramer staining (Tet+) and ICS for IFN-{gamma}. We also stained for the CD44 marker that is up-regulated on activated and memory TCD8+ cells. The resulting populations were clearly distinct. Upon priming with Vac-18SIIN and Vac-16SIIN (Fig. 1B), the number of Tet+ cells is similar to that of IFN-{gamma}-producing cells (Tet+IFN-{gamma}+). In contrast, Vac-SIIN induces a mixture of functional Tet+IFN-{gamma}+ and dysfunctional Tet+IFN-{gamma}– cells (Fig. 1, B and C) as previously reported (8). All cells down-modulate TCR in response to peptide stimulation (data not shown), indicating that the IFN-{gamma}– population is not the result of nonspecific bystander activation. As an internal control, we measured Vac-specific responses in all groups and observed them to be similar (Fig. 1B, bottom). These results emphasize the impact of Ag density on in vivo priming, regardless of the context, minigene or protein.

It has been reported that TCD8+ cells lacking effector functions can be induced in vitro to secrete IFN-{gamma} upon exposure to IL2 (17, 26). To determine whether this applied in our case, we treated a mixture of functional and dysfunctional cells from Vac-SIIN-immunized mice with IL2 (30 U/ml) in vitro and observed no increase in the proportion of IFN-{gamma}+ cells at different time points (3 and 6 days, data not shown) suggesting that the acutely induced dysfunctional phenotype is relatively refractory.

High epitope doses generate and maintain a dysfunctional TCD8+ cell phenotype

Having determined that regulated minigene expression could be used to explore the dysfunctional phenotype, and with the goal of understanding the association of this phenotype with chronic infections, we first investigated the durability of this subset. Specifically, we investigated the fate of this population following secondary immunization using a panel of viral vectors—RV, Flu, and Adeno—that express the SIINFEKL peptide from various contexts (Table I). None of these viruses is known to cause a persistent infection under the conditions that were used. Before using these viruses in vivo, we characterized them in terms of epitope output. LKb cells were infected with Vac, RV, or Flu and CMT93 cells were infected with Vac and Adeno. At different time points, the peak level of surface Kb/SIIN complex expression was measured by flow cytometry (48). Surface Kb/SIIN expression induced by Flu-SIIN was not above background levels. However, Vac, RV, and Adeno express similar levels of Kb/SIIN complexes (Fig. 2A) which are substantially higher than the levels produced by Flu (not shown).


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  		Table I. Viral vectors expressing SIINFEKL

 

Figure 2
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  		FIGURE 2. Dysfunctional TCD8+ cells are maintained by high Ag doses. A, Density of Kb/SIIN complexes expressed by Vac, rabies, and adenovirus vectors. LKb cells were infected with Vac or rabies viruses (top) and CMT93 cells were infected with Vac or adenovirus (bottom) expressing SIINFEKL epitope either as a protein (NP/SIIN) or minigene (SIIN). Densities of Kb/SIIN complexes were determined by staining the cells with a Kb/SIIN-specific Ab at different times: 6 h postinfection for Vac and 48 h for rabies and adenoviruses. Similar results were obtained in two additional independent experiments. B, The dysfunctional population is maintained during the secondary TCD8+ cell response to high expresser RV-NP/SIIN but not to low expresser Flu-SIIN. Mice were primed with Vac-SIIN and boosted with either RV-NP/SIIN or Flu-SIIN; the secondary response was assessed 28 days later as described in Fig. 1B. C, The dysfunctional population is maintained during the tertiary TCD8+ cell response to high expresser Adeno-NP/SIIN but not to low expresser Flu-SIIN. Mice were primed with Vac-SIIN, boosted with RV-NP/SIIN, and re-boosted with either Adeno-NP/SIIN or Flu-SIIN; the tertiary response was assessed 28 days later as described in Fig. 1B. Error bars represent SD of four mice per group.

 
Priming with Vac-SIIN and boosting with RV-NP/SIIN resulted in a mixed functional and dysfunctional population; however, boosting with Flu-SIIN induced a functional TCD8+ cell population only (Fig. 2B). Both RV-NP/SIIN and Flu-SIIN viruses boosted the SIINFEKL-specific TCD8+ cell population, because the overall magnitude of the secondary response was higher than that of primary response.

To further examine the fluidity of the dysfunctional TCD8+ cell population, we re-boosted the Vac-RV secondary response with Adeno-NP/SIIN and Flu-SIIN (Fig. 1C). The tertiary response induced by Adeno-NP/SIIN was a mixture of functional and dysfunctional cells. Tet+ cells outnumbered dysfunctional TCD8+ cells (Tet+IFN-{gamma}–) ~2- to 3-fold whether comparing the memory responses under the primary or boosting conditions (data not shown). As predicted by results from primary boosting, the population that persisted after Flu-SIIN boosting was of a functional phenotype only.

Together these results suggested that the Tet+IFN-{gamma}– phenotype develops acutely following expression of epitope at sufficiently high levels, is stable in the absence of additional restimulation, and depends upon high epitope levels during restimulation to be sustained. These characteristics suggested to us that the Tet+IFN-{gamma}– population expresses relatively low avidity TCRs; to be stimulated at all in the primary response, relatively high levels of epitope are required. To be sustained in a secondary or tertiary response, again high epitope doses are required. Under conditions of low epitope expression for the rechallenge, this population may not be able to compete with higher avidity epitope-specific cells. To test this model the relative avidities of the two populations were assessed as follows.

Sorting functional and dysfunctional TCD8+ cells

Because the dysfunctional subset displays no identified effector functions, assessment of avidity by methods such as peptide titration was not feasible. We therefore turned to tetramer dissociation in which the rate of tetramer elution from the T cell surface is used to assess relative avidity. To use this technique, however, separation of the functional and dysfunctional populations before analysis was necessary. This was a challenge because we could not use in vitro Ag stimulation to separate IFN-{gamma}+ and IFN-{gamma}– cells due the ensuing TCR down-regulation that would compromise tetramer staining (55). Furthermore, standard ICS was not an option because cell viability was necessary for subsequent analysis. Instead, we exploited the observation of Slifka et al. (56) that TCD8+ cells produce IFN-{gamma} while conjugated to APC, but cease IFN-{gamma} production when conjugates are disrupted.

To obtain in vivo activated SIINFEKL-specific TCD8+ cells, we primed mice with RV-SIIN, a protocol that also induces a mixture of functional and dysfunctional cells (Fig. 3A), and 28 days later boosted with Vac-NP/SIIN. Five days later, the SIINFEKL-specific TCD8+ cell response was composed of 16.4% Tet+ cells and 9.1% functional IFN-{gamma}+ cells (Fig. 3B). Spleens were disrupted in ice-cold buffer, a temperature that prevents IFN-{gamma} secretion by in vivo activated SIINFEKL-specific TCD8+ cells, and splenocytes were stained with tetramer and IFN-{gamma} capture Ab then warmed at 37°C to allow IFN-{gamma} secretion of functional cells. This technique detected 6.2% functional IFN-{gamma}+ at 37°C compared with cells that were kept on ice (Fig. 3C). Detection of more IFN-{gamma}+ cells following in vitro peptide stimulation than ex vivo analysis has also been reported in a different system (57). By careful gating of CD8+Tet+IFN-{gamma}+ and CD8+Tet+IFN-{gamma}– cells, we were able to sort these populations with an efficiency of at least 95% (Fig. 3D). It has been reported that in vivo production of IFN-{gamma} occurs in only a subset of responsive cells at any point in time (58). We therefore retested the IFN-{gamma} secretion of sorted populations after in vitro peptide stimulation using ELISA. The IFN-{gamma}+ population responded to peptide, at concentrations as low as 10–10 M. The IFN-{gamma}– population contained a subset of cells (15–20%) that responded with IFN-{gamma} secretion only at very high peptide concentrations (≥10–6 M), and therefore of low functional avidity (data not shown). Thus, effective separation of the two populations was achieved with this method. The distinctiveness of the sorted populations was also confirmed by spectratype analysis of TCR usage (Fig. 5).


Figure 3
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  		FIGURE 3. Sorting functional and dysfunctional TCD8+ cells. A, Generation of the dysfunctional population during primary TCD8+ cell response to high expresser RV-SIIN. Mice were primed with RV-SIIN or a negative control virus and the primary responses were assessed 14 days later as described in Fig. 1B. B, The dysfunctional population is maintained in mice primed with RV-SIIN and boosted with Vac-NP/SIIN. Splenocytes were analyzed 5 days after the boost by Kb/SIIN tetramer staining (left panel) and intracellular staining for IFN-{gamma} after SIINFEKL peptide stimulation (10–6 M) in vitro (right panel). C, Surface staining with Kb/SIIN tetramer, CD8, IFN-{gamma}-specific capture and detection Abs of splenocytes from mice primed and boosted as above. The test sample was incubated at 37°C (right panel) and the control sample was kept at 0°C (left panel). Cells are gated on the CD8 population. Numbers represent percentages of the CD8 population. D, Sorted cells were reanalyzed by flow cytometry. The numbers represent percentages of CD8+Tet+IFN-{gamma}– (left) and CD8+Tet+IFN-{gamma}+ (right) cells of the total sorted populations.

 

Figure 5
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  		FIGURE 5. TCR-Vβ usage of dysfunctional cells is broader than that of functional cells. The RNA of sorted CD8+Tet+IFN-{gamma}– (IFN-{gamma}–) and CD8+Tet+IFN-{gamma}+ (IFN-{gamma}+) cells was extracted and used for spectratype analysis. White box, no Vβ detected; gray box, Vβ detected.

 
Avidities of functional and dysfunctional TCD8+ cells

To analyze relative avidities, we compared the tetramer dissociation from sorted IFN-{gamma}+ and IFN-{gamma}– cells. Sorted cells were re-stained with excess Kb/SIIN tetramer, an Fab preparation of Kb/SIIN-specific Ab was added to prevent reassociation of eluted tetramer, and the intensity of tetramer bound on cell surface was measured at different time points by flow cytometry. Tetramer dissociation was clearly and reproducibly faster for the IFN-{gamma}– cells (Fig. 4A), indicating that the functional population is derived from the high avidity precursors and dysfunctional TCD8+ cells are derived from the lower end of the avidity spectrum. TCR levels on the sorted populations were similar (Fig. 3D).


Figure 4
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  		FIGURE 4. Dysfunctional TCD8+ cells have lower avidity than their functional counterparts. A, Tetramer dissociation analyses of sorted cells re-stained with excess Kb/SIIN tetramer and analyzed by flow cytometry at different time points. The MFI for tetramer staining is expressed as percentage of the maximum MFI at time 0. Data are representative of three experiments. B, Monoclonal transgenic TCD8+ cells of high avidity generate functional cells only. Naive spleen cells from OT1 mice (Thy1.2) were adoptively transferred into naive C57BL/6 (Thy1.1) mice. On the following day, mice were primed and 20 days later, the splenocytes were stained with Thy1.2-, CD8-, CD44-specific Abs and either Kb/SIIN tetramer (white bars) or IFN-{gamma}-specific Abs (ICS) after SIINFEKL peptide stimulation (10–10 M) in vitro (black bars). Cells are gated on Thy1.2+CD8+ donor cells. The percentages of CD44highTet+ and CD44highIFN-{gamma}+ are shown. The experiment was repeated three times. Error bars represent SD of three mice per group. The bottom panel depicts Vac-specific functional responses assessed by ICS for IFN-{gamma} as described in Materials and Methods.

 
To bolster the notion that dysfunctional cells are not derived from high avidity clones, we tested the effect of Ag dose on a monoclonal population of relative high avidity. OT1 mice have a TCR transgenic TCD8+ cell population specific for Kb/SIIN complexes (45, 59). We adoptively transferred naive splenocytes from OT1 mice into naive C57BL/6 mice, primed with high and low expresser Vacs and analyzed the phenotype of donor cells. Fig. 4B indicates that low Ag doses induce functional TCD8+ cells and that high doses induce a small fraction of dysfunctional cells that we speculated to arise from the minor endogenous T cells present in Rag+/+ mice. We repeated the experiment using OT1 Rag–/– mice, and found that high Ag densities induce an entirely functional population. As expected the Vac-specific responses were similar in all groups (Fig. 4B, bottom). Tetramers remained associated with the OT1 population at a level of 90–95%, confirming the high avidity of these transgenic cells (data not shown). These data further support the concept that dysfunctional TCD8+ cells emerge from the low avidity pool.

TCR usage of functional and dysfunctional T cell populations

Based on a previous publication by Villacres et al. (60), we predicted that the functional high avidity subset would be characterized by focused TCR usage whereas the dysfunctional, low avidity population would be characterized by broad, degenerate TCR usage. To test this prediction, we identified Vβ families in sorted cells by spectratype analysis, the numbers of sorted cells being too low for Vβ-specific Ab staining. As seen in Fig. 5, the Vβ14 family was repeatedly detected in the functional population, indicating that high avidity receptors belong to a small pool of T cells. In contrast, multiple Vβ families were identified in the dysfunctional population.

Relative proliferation rates of functional and dysfunctional TCD8+ cells

In aggregate, results thus far suggest that the dysfunctional and functional populations emerge from distinct clones and do not transition from one state to the other. Thus, in the low Ag dose boosting experiments described above (Fig. 2), it is likely that the dysfunctional clones disappear rather than convert to functional clones. To test the idea that dysfunctional, low avidity T cells compete poorly against functional high avidity T cells when Ag is limiting, we assessed the proliferative potentials of functional and dysfunctional population during a secondary response. According to conventional protocols (61), primed and boosted mice were dosed with BrdU for 5 days following boosting. The CD8+Tet+ population was sorted, stimulated in vitro with SIINFEKL peptide pulsed LKb cells and stained with IFN-{gamma} and BrdU-specific Abs (Fig. 6B). BrdU incorporation was significantly lower for CD8+Tet+IFN-{gamma}– than CD8+Tet+IFN-{gamma}+ cells suggesting that the high avidity and functional cells proliferate faster than the dysfunctional counterparts and replace them under conditions of low Ag boost.


Figure 6
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  		FIGURE 6. Dysfunctional TCD8+ cells proliferate more slowly than functional cells. Mice were primed with RV-SIIN, boosted with Vac-NP/SIIN and injected with BrdU every 24 h for 6 days. The splenic CD8+Tet+ population was sorted, rested overnight in the presence of IL2, restimulated with LKb cells pulsed with 10–6 M SIINFEKL peptide, and stained with IFN-{gamma}- and BrdU-specific Abs or isotype controls. The MFI of BrdU staining for CD8+Tet+IFN-{gamma}– (IFN-{gamma}–) and CD8+Tet+IFN-{gamma}+ (IFN-{gamma}+) cells is shown. Error bars represent SE of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Using a prime-boost system of acute transient viral infection, we generated and characterized the dysfunctional TCD8+ cell phenotype that is considered a hallmark of many chronic infections. Our results support a model in which limiting Ag activates only high avidity cells from a restricted Vβ repertoire to acquire effector functions. As availability of Ag increases, TCD8+ cells of progressively lower avidity and increased Vβ representation participate in the response. At a critical level of Ag, cells of very low avidity participate in the response albeit with reduced proliferation and without acquisition of effector function. With no further boosting the dysfunctional population is quite stable; however, persistence following a secondary response is critically dependent upon high Ag levels. As previously reported in other systems (7), we observed that there is a direct relationship between the Ag dose and the magnitude of TCD8+ cell primary responses in vivo (Fig. 1B). However, the increased size of the response to higher Ag doses does not necessarily enhance functionality, as dysfunctional T cells with lower avidity are eventually recruited and may, in fact, compromise protection (33, 34).

Stimulation with high epitope doses may result in deletion (62, 63) or incapacitation (64, 65) of high avidity cells. Our experiments (Fig. 4A) showing survival of two Ag-specific populations with different avidities do not rule out deletion of the highest avidity cells (and persistence of functional cells with intermediate avidity and dysfunctional cells of low avidity) even though we did not detect dysfunctional high avidity cells. It is possible that overstimulated dysfunctional cells with high avidity undergo relatively rapid apoptosis. In line with this notion, a reduction of Ag-specific TCD8+ cells in secondary responses was observed when the highest epitope expressing viruses, Vac-SIIN and RV-SIIN, were used for boosting (data not shown).

Persistence of low avidity dysfunctional populations after repetitive boosting depended upon Ag availability rather than a stochastic selection mechanism. Survival of high avidity functional memory cells (Fig. 6A) may be the direct result of their efficient competition for access to pMHC complexes (66) or the indirect effect of pMHC down-modulation from the APC surface (67), which would discourage lower avidity responses. The failure of lower avidity dysfunctional cells to persist under limiting boosting could be due to their inability to compete for survival niches, as previously suggested to explain memory attrition subsequent to heterologous virus infections (68). Evidently the delayed onset/slower proliferation rate of dysfunctional cells (Fig. 6) does not impede their persistence upon restimulation with high epitope levels. Despite the loss of many TCR specificities, elimination of dysfunctional cells under conditions of lower Ag levels could be advantageous to the host, as the memory pool would be populated by functional, protective TCD8+ cells only, unencumbered by potentially suppressive dysfunctional cells. We note, however, that the deficit in cytokine secretion of these cells was not restricted to IFN-{gamma}, but included all other cytokines tested including IL2, 4, 5, 6, 12, and TNF (data not shown). Thus, an active suppression mechanism (versus simply competition for space) is not implicated at this point.

Avidity maturation of T cell responses (7, 69) was observed in our system when comparing functional avidities of secondary and primary responses; however, we did not observe an additional increase upon tertiary stimulation (data not shown), suggesting that the effect is limited. Thus, elimination of dysfunctional cells may be seen as a new parameter defining maturation of the response.

The repertoire analysis of functional and dysfunctional cells showed markedly different TCR usages (Fig. 5). This analysis confirms and extends earlier findings of a broad Ova-specific TCD8+ cell repertoire (59). The TCR transgene of the OT1 mouse is composed of Vβ5.2 and V{alpha}2 chains and was selected based on its high avidity among the Vβ5.2 clones that paired with different V{alpha}-chains (59). However, it has not been confirmed that the highest avidity Ova-specific clones in a natural response belong to the Vβ5.2 family (45, 70, 71). We did not detect this subset in the functional population, possibly due to deletion of the highest avidity cells, although one of three experiments identified Vβ5.2 among the dysfunctional cells. Interestingly, we detected the previously identified Vβ14 family in both functional and dysfunctional populations. Additional analysis (G. Plesa, unpublished observations) revealed a Vβ14 clonotype unique to the dysfunctional cells and another that was present in both populations. This latter group might contain subfamilies that are either functional or nonfunctional. The indirect relationship between T cell avidity and broadness of the specific repertoire is in agreement with other studies (60) and may be explained by TCR degeneracy that has been proposed to be more pronounced for lower avidity T cells (72).

The presence of dysfunctional T cells is considered a hallmark of persistent immune activation such as seen in chronic viral infections and tumor microenvironments. Characterization and deciphering the molecular signature of this phenotype is therefore of increasing importance for the goal of restoring the function of these cells. It was reported that increased expression of PD-1 on CD4 and TCD8+ cell is associated with cell dysfunction and persistent viral infection in human and mice (73, 74). IL10 production by APC (75) and more specifically CD8{alpha}-DCs (76) at the early stages of infection also results in T cell dysfunction and viral persistence. However, the cause-and-effect relationship of IL10/IL10R and PD-1/PD-1L pathways in mediating Ag-specific unresponsiveness is still to be investigated. It is possible that higher Ag doses skew the early innate immune response toward an IL10 predominant environment that dampens the functionality of immune responses to limit immunopathology. Indeed, suppression of Ag-specific CD4 T cell proliferation has been observed under conditions of high viremia (77).

It remains to be seen whether the dysfunctional cells that become apparent in the later stages of many chronic infections are similar to those developed under acute conditions. We speculate that during the course of a chronic infection, low avidity cells specific for certain epitopes will expand as soon as Ag levels reach a critical point. In the cases where high epitope levels are achieved in the short term, this will be relatively soon after infection. In other cases, extensive and prolonged viral replication will be necessary. These dysfunctional cells will persist as long as Ag levels do not recede and will ultimately predominate when higher avidity, functional cells senesce (78) due to their higher rates of division.


   Acknowledgments
 
We thank Jeffrey Faust and Lester Acosta from Wistar Institute Flow Cytometry Facility for excellent help with sorting cells. We acknowledge Matt Loza for helpful discussions and also John Wherry and Joseph Comber for critical reading and helpful suggestions regarding this manuscript. We also thank the personnel of Kimmel Cancer Institute Nucleic Acid Facility and Flow Cytometry Facility 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 National Institutes of Health Grant AI046511. Back

2 Address correspondence and reprint requests to Dr. Laurence C. Eisenlohr, Thomas Jefferson University, 233 South 10th Street, Bluemie Life Sciences Building 730, Philadelphia, PA 19107. E-mail address: Laurence.Eisenlohr{at}jefferson.edu Back

3 Abbreviations used in this paper: Vac, vaccinia virus; RV, rabies virus; Flu, influenza; Adeno, adenovirus; MFI, mean fluorescence intensity; ICS, intracellular staining. Back

Received for publication November 16, 2007. Accepted for publication February 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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