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The Journal of Immunology, 2007, 178, 7616-7622
Copyright © 2007 by The American Association of Immunologists, Inc.
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Disregulated Influenza A Virus-Specific CD8+ T Cell Homeostasis in the Absence of IFN-{gamma} Signaling1

Stephen J. Turner2,*,{dagger}, Elvia Olivas*, Astrid Gutierrez*, Gabriela Diaz* and Peter C. Doherty*,{dagger}

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent studies indicate that IFN-{gamma} may influence both the expansion and the trafficking of virus-specific CD8+ CTL, though the effects are not necessarily consistent for different models of viral and bacterial disease. Influenza A virus infection of mice deficient for IFN-{gamma} (IFN-{gamma}–/–) or deficient for the IFN-{gamma} receptor 1 (IFNGR1–/–) was, when compared with the wild-type (WT) B6 controls, associated with increased Ag-specific CD8+ T cell counts in the spleen and mediastinal lymph nodes. At the same time, fewer of these CTL effectors were found in the bronchoalveolar lavage population recovered from the IFN-{gamma}–/– mice. Comparable effects were observed for WT mice treated with a neutralizing IFN-{gamma}-specific mAb. Transfer of WT memory Thy1.1+ CD8+ populations into Thy1.2+ B6 IFN-{gamma}–/– or IFNGR1–/– mice followed by intranasal virus challenge demonstrated both that IFN-{gamma} produced by the host was important for the regulation of Ag-specific CTL numbers and that IFN-{gamma} was likely to act directly on the T cells themselves. In addition, the prevalence of CTLs undergoing apoptosis in spleen was lower when measured directly ex vivo for IFN-{gamma}–/– vs WT B6 mice. The present analysis is the first comprehensive demonstration that IFN-{gamma} signaling can differentially regulate both Ag-specific CTL homeostasis in secondary lymphoid organs and trafficking to a site of virus-induced pathology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Following virus challenge, CD8+ T cells specific for peptide plus MHC class I glycoprotein complexes (epitopes) proliferate and differentiate to become CTL effectors (1) while simultaneously acquiring the capacity to secrete cytokines, such as IFN-{gamma} and TNF-{alpha}, in an Ag-dependent manner (2). These expanded CTL populations then contract after virus clearance, leading to a stable pool of memory T cells that persist in both secondary lymphoid and nonlymphoid organs (3, 4). This homeostatic control of Ag-specific CTL pool size seems to be independent of both epitope specificity and tissue of isolation (4). The TH1 cytokine IFN-{gamma} is considered to influence the expression of molecules involved in MHC class I Ag presentation (5) and to play some part in the homeostatic control of Ag-specific CTL cell numbers (6, 7, 8, 9), though the mechanistic basis of the latter effect is far from clear.

After infection with Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV),3 or Mycobacterium bovis, IFN-{gamma}-deficient (IFN-{gamma}–/–) mice showed normal profiles of CTL expansion, but delayed contraction of effector T cell populations after pathogen clearance (7, 8). Such results imply that IFN-{gamma} has a role in modulating the post-Ag phase of T cell responsiveness. By contrast, other results with LCMV-infected IFN-{gamma} receptor 1-deficient (IFNGR1–/–) mice suggested that IFN-{gamma} plays a positive role in promoting Ag-driven T cell proliferation (9). Furthermore, IFN-{gamma} apparently mediates this effect by acting directly on the LCMV-specific effectors (9). In addition, the lack IFN-{gamma} has also been associated with diminished cellular infiltration into sites of inflammatory pathology in models of virus infection and autoimmunity (10, 11).

Given the lack of agreement concerning the role of IFN-{gamma} in the development and maintenance of pathogen-specific responses, we have revisited the issue for C57BL/6 (B6, H2b) mice infected intranasally (i.n.) with the A/HKx31 (H3N2) influenza A virus. Such challenge induces an acute, localized pneumonia (12) together with a diverse CTL response directed against at least six viral epitopes (13). Among the most prominent (14, 15, 16) are those derived from the viral nucleoprotein (NP)366–374 (DbNP366), polymerase A (PA)224–236 (DbPA224), and the basic polymerase B subunit 1 (PB1)703–711 (KbPB1703). Although some experiments have indicated that IFN-{gamma} plays a part in controlling influenza virus infection (14), IFN-{gamma}–/– mice have been found to generate normal CTL responses and to clear virus effectively (15). However, as these studies were completed before the availability of tetramer staining reagents (17, 18) no accurate measurements were made of T cell response profiles. The present analysis uses contemporary technology to demonstrate that a lack of IFN-{gamma} signaling results in the accumulation of Ag-specific CTLs in the spleen, with a resultant diminution in Ag-specific CTL numbers in the virus-infected lung. Importantly, this homeostatic control of CTL numbers and circulation profiles was dependent on extrinsically produced IFN-{gamma} acting directly on the Ag-specific T cells.


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

The IFN-{gamma}–/–, B6 IFNGR1–/–, B6 CCR5-deficient (CCR5–/–) mice and wild-type (WT) C57BL/6J (B6) female mice were purchased from The Jackson Laboratory and housed under specific pathogen-free conditions in an American Association of Laboratory Animal Care-approved animal facility. All experiments were approved by the St. Jude Children’s Research Hospital Animal Ethics Committee. Naive mice were anesthetized by i.p. injection of Avertin (2,2,2 tribromoethanol), and challenged i.n. with 6.8 log10 egg ID50 (EID50) of the A/HKx31 (H3N2) influenza A virus.

Peptides and tetramers

NP366–374 (ASNENMETM), PA224–233 (SSLENFRAYV), and PB1703–711 (SSYRRPVGI) peptides were made at the Center for Biotechnology, St. Jude Children’s Research Hospital (Memphis, TN), using a PerkinElmer 433A peptide synthesizer and then purified by HPLC. The production and characterization of the DbNP366, DbPA224, and KbPB1703 tetramers have been previously described (16, 17, 18).

Flow cytometric analysis

At various times after infection, mice were anesthetized and exsanguinated via the axillary artery. Lymphocytes were isolated from lung by bronchoalveolar lavage (BAL) and CD8+ T cells were enriched from single-cell suspensions of the mediastinal lymph node (MLN) and spleen using mAbs to CD4 (GK1.5) and MHC class II (TIB120) followed by anti-rat and anti-mouse Ig-coated magnetic beads (Dynal Biotech). Inflammatory cell populations recovered by BAL were adsorbed on plastic for 1 h at 37°C to remove adherent cells. The lymphocytes were then stained with the PE for DbNP366, DbPA224, and DbPB1703 tetramers (1/100 in PBS/0.1% BSA/0.02% sodium azide as FACS buffer) for 1 h at room temperature. Lymphocytes were then washed and stained with anti-CD8 PerCP-Cy5.5 (BD Pharmingen) alone or in a mixture of anti-CD8 and mAbs specific for various cell surface markers. A minimum of 50,000 lymphocytes/CD8+-gated event/sample was collected on a FACSCalibur (BD Biosciences) and analyzed using CellQuest software.

Sorting and transfer of Thy1.1+ CD44+CD8+ T cells

Single-cell suspensions were prepared from the spleens of Thy1.1+ mice i.p. primed 8–12 wk earlier with 1 x 108 EID50 of the PR8 virus, erythrocytes were lysed and B cells were removed by panning in T175 cm2 flasks coated with goat anti-mouse IgG/IgM (Jackson ImmunoResearch Laboratories) for 1 h at 37°C, 5% CO2. The nonadherent cells were incubated with mAbs to CD4 (GK1.5) and MHC class II (TIB120) followed by sheep anti-mouse Ig and sheep anti-rat Ig-coated beads (Dynal Biotech), then enriched for the CD8+ subset by magnetic depletion. The predominantly CD8+ population was resuspended at 20 x 106 cells/ml in PBS/0.1% BSA (sort buffer), and stained with anti-CD8 FITC (BD Pharmingen) and anti-CD44 PE (BD Pharmingen) for 30 min at 4°C. After a further wash, the CD8+CD44+ set was separated using a MoFlo high-speed cell sorter (DakoCytomation). The cells were then washed twice in PBS, resuspended at 5 x 106 cells/ml in PBS and i.v. injected (200 µl) into B6, IFN-{gamma}–/–, and IFNGR1–/– Thy1.2+ recipients. Three days after transfer, mice were infected with the HKx31 virus and sampled subsequently as described.

Treatment of mice with XMG1.2

The depletion protocol has been previously described (10, 19). Briefly, mice were injected i.p with XMG1.2 anti-IFN-{gamma} mAb (2 mg/mouse) or rat Ig (Jackson ImmunoResearch Laboratories) on days –4, –2, 0, +2, and +4 in reference to i.n. infection with 6.8 log10 EID50 of the HKx31 virus. Evidence of IFN-{gamma} neutralization was confirmed by the absence of free IFN-{gamma} in the BAL fluid as previously described (20).

Real-time PCR

Total RNA was isolated from 1 x 106 sorted DbNP366-specific CTL using TRIzol reagent (Invitrogen Life Technologies).A 50 ng of total RNA was used for amplification using a One-Step Real-Time PCR kit (Applied Biosystems) with primers specific for GAPDH, CCR1, CCR5, CCR8, CXCR3, CXCR4, and CXCR6 and using SYBR Green chemistry. The real-time PCR conditions were 48°C for 30 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 15 s using an ABI7700 (Applied Biosystems) real-time PCR product. Results were analyzed using sequence detector software, and relative fold differences were determined using the threshold cycle ({Delta}Ct) method as described by the manufacturer. Amplification of a specific product was confirmed by measuring the disassociation curve according to the manufacturer’s instructions.

Virus titration

Lungs from B6 or IFN-{gamma}–/– mice infected with the HKx31 virus were isolated on days 5, 7, and 10 after infection, frozen (–70°C) and later homogenized for virus isolation by allantoic inoculation in embryonated hen eggs (12). Virus titers are expressed as log10 EID50/lung.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Enrichment of influenza-specific CTL in the lymphoid tissue of IFN-{gamma}–/– mice

Given the conflicting roles attributed to IFN-{gamma} (7, 9, 21), we analyzed the Ag-specific response of naive B6 and IFN-{gamma}–/– mice to respiratory challenge with the HKx31 influenza A virus. The number of CD8+ T cells binding the DbNP366 (Fig. 1, A, D, and G), DbPA224 (Fig. 1, B, E, and H), and KbPB1703 (Fig. 1, C, F, and I) tetramers was determined for BAL (Fig. 1, A–C), MLN (Fig. 1, D–F), and spleen (Fig. 1, G–I) populations at various time points after infection. Comparison on day 11 showed that there were more Ag-specific T cells in the MLNs and spleens of the IFN-{gamma}–/– mice. The effect was greatest (four to five times) for the CD8+DbNP366+ sets, but the differences were still significant (p < 0.02) for the DbPA224- and KbPB1703-specific populations. This IFN-{gamma}–/– B6 profile was already apparent by day 8 in the spleen, but not in the draining lymph node (Fig. 1, D–F). The high Ag-specific T cell counts in the lymphoid tissue of the IFN-{gamma}–/– mice dropped dramatically by day 14, but the differences were still significant for the DbNP366 and DbPA224 populations from the MLN (p < 0.02 and p < 0.05, respectively) (Fig. 1, D and E) and spleen (p < 0.01 and p < 0.05, respectively) (Fig. 1, G and H) at this time point. Similar to a previous study (7), there was evidence of altered immunodominance hierarchies in the spleen and MLN of IFN-{gamma}–/– mice. The DbNP366- and DbPA224-specific responses are normally codominant during the primary response to infection (Fig. 1, D, E, G, and H) (17, 22). However, the DbNP366-specific response was immunodominant over both DbPA224- and KbPB1703-specific responses in IFN-{gamma}–/– mice at all time points measured (Fig. 1, D, E, G, and H).


Figure 1
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  		FIGURE 1. Enumeration of epitope-specific CD8+ T cells after influenza A virus infection of WT and IFN-{gamma}–/– mice. B6 WT (•) or IFN-{gamma}–/– ({square}) mice were i.n. challenged with the HKx31 virus. BAL (A–C), MLN (D–F), and spleen (G–I) populations (n = 5 each) were taken on days 8, 11, and 14 after infection and stained with the DbNP366 (A, D, and G), DbPA224 (B, E, and F), or KbPB1703 tetramers (C, F, and I) and a mAb to CD8{alpha}. The number of CD8+ tetramer-positive T cells (mean ± SD) shown is calculated from the total counts and the percentage of cells staining. Data are representative of three independent experiments. *, p < 0.01, **, p < 0.02, ***, p < 0.05) determined using a Student’s pairwise t test.

 
The converse situation was found for the BAL, with significantly fewer epitope specific CTL being detected in the IFN-{gamma}–/– vs B6 mice on day 8 after infection (Fig. 1, A–C). This divergence was less apparent on day 11, and not obvious at all by day 14. The impression is that the T cells are being held in the lymphoid tissue of the IFN-{gamma}–/– mice during the Ag-driven phase of the response, but that this had no major effect in the long term. Furthermore, despite the low counts in the BAL on day 8, there was no effect on virus clearance (Fig. 2).


Figure 2
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  		FIGURE 2. Comparison of virus clearance for WT and IFN-{gamma}–/– mice. Naive WT ({square}) or IFN-{gamma}–/– ({blacksquare}) mice (n = 5 each) were infected with the HKx31 virus and sampled 5, 7, or 10 days later. Lungs were homogenized for virus titration by allantoic inoculation in embryonated hen eggs. Titers are expressed as log10 EID50 per lung. The data are representative of two independent experiments.

 
The effect in IFNGR1–/– mice

Naive B6 and IFNGR1–/– mice were i.n. infected with the HKx31 virus and the day 8 CD8+DbNP366+, CD8+DbPA224+, and CD8+KbPB1703+ responses were compared for the BAL (Fig. 3A) and spleen (Fig. 3B). Consistent with the earlier result in IFN-{gamma}–/– mice, there was a four to five time reduction in the number of epitope-specific CTLs recovered from the lungs of the IFNGR1–/– mice (p < 0.05, p < 0.02, p < 0.02 for DbNP366, DbPA224, and KbPB1703, respectively) (Fig. 3A). Again, as with the IFN-{gamma}–/– mice (Fig. 1), there were significantly more DbNP366- (p < 0.002), DbPA224- (p < 0.02), and KbPB1703-specific (p < 0.002) T cells in the spleens (Fig. 3B) of the IFNGR1–/– mice. The magnitude of the difference was comparable to the four to five time increase seen earlier.


Figure 3
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  		FIGURE 3. Enumeration of epitope-specific CD8+ T cells after influenza A virus infection of WT and IFNGR1–/– mice. Naive B6 ({square}) or IFNGR1–/– ({blacksquare}) mice were infected with the HKx31 virus 8 days before BAL (A) and spleen (B) populations (n = 5 each) were isolated and stained with the DbNP366, DbPA224, or KbPB1703 tetramers and anti-CD8. The number of CD8+ tetramer-positive T cells (mean ± SD) shown is calculated from the total counts and the percentage of cells staining. Data are representative of three independent experiments. *, p < 0.002; **, p < 0.02; ***, p < 0.05 determined using a Student’s pairwise t test.

 
IFN-{gamma} neutralization results in a similar pattern

Similar to the results in Figs. 1 and 3, treatment of influenza virus-infected BALB/c mice with a neutralizing Ab specific for IFN-{gamma} resulted in a decreased cellular infiltrate in the lung parenchyma that was maintained for up to 10 days after infection (10). To determine whether IFN-{gamma} neutralization also increased CTL number in the spleen, naive B6 mice were i.p. injected with either the anti-IFN-{gamma} mAb XMG1.2 or with a rat isotype control (20), and i.n. infected with the HKx31 virus. Analysis of day 8 BAL populations showed a significant decrease in the number of DbNP366- (p < 0.05), DbPA224- (p < 0.02), and KbPB1703-specific (p < 0.02) T cells (Fig. 4A) in the anti-IFN-{gamma}-treated mice. Conversely, dosing with anti-IFN-{gamma} resulted in significantly increased Ag-specific CTL counts in spleen (DbNP366, p < 0.001); DbPA224, p < 0.001; and KbPB1703, p < 0.02). Although these effects are similar to those observed in IFN-{gamma}–/– and IFNGR1–/– mice, the fold differences in BAL and spleen T cell counts were relatively less in the Ab-treated group (one and one-half to three times vs four to five times). Again, removing IFN-{gamma} throughout the course of the infectious process had no effect on virus clearance (data not shown).


Figure 4
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  		FIGURE 4. The effect of anti-IFN-{gamma} mAb treatment on virus-specific CD8+ T cell numbers after primary infection. Naive B6 mice were treated with either XMG1.2 (anti-IFN-{gamma} mAb ({blacksquare}) or rat Ig ({square}) at days –4, –2, 0, +2, and +4 relative to i.n. infection with the HKx31 virus. BAL (A) and spleen (B) populations (n = 5 each) were isolated 8 days after virus challenge and stained with the DbNP366, DbPA224, or KbPB1703 tetramers and anti-CD8. The number of CD8+ tetramer-positive T cells (mean ± SD) shown is calculated from the total counts and the percentage of cells staining. Data are representative of two independent experiments. *, p < 0.05; **, p < 0.02; ***, p < 0.001 determined using a Student’s pairwise t test.

 
Adhesion markers and chemokine receptors

The diminution in Ag-specific CTL recruitment to the site of virus-induced pathology in the lung may reflect diminished migration potential due to the disregulated expression of adhesion molecules. Lymphocyte populations were isolated from the BAL (Fig. 5, A–C) and spleen (Fig. 5, D–F) of B6, IFN-{gamma}–/–, or IFNGR1–/– mice infected with the HKx31 virus, and DbNP366-specific CTL were stained for the expression of various adhesion molecules. No differences were found in the levels of CD49b, CD54, {alpha}E integrin, and CD62L for any of these groups (data not shown). This was also true for CD43a (Fig. 5, A and D) but the CD8+DbNP366+ T cells from IFN-{gamma}–/– and IFNGR1–/– mice expressed more CD49d (Fig. 5, B and E) and less CD11a (Fig. 5, C and F) than the comparable sets from the B6 controls. Increased expression of CD49d, part of the VLA-4 cell adhesion complex, has been previously described for total lymphocyte populations from mice treated with the anti-IFN-{gamma} neutralizing Ab (10).


Figure 5
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  		FIGURE 5. Cell surface expression of CD43a, CD49d, and CD11a on DbNP366-specific CTL isolated from BAL and spleen of wt, IFN-{gamma}–/– and IFN-{gamma}R1–/– mice. Naive WT, IFN-{gamma}–/–, or IFNGR1–/– mice were i.n. infected with the HKx31 virus. Lymphocyte populations were isolated from BAL (A–C) and spleen (D–F) on day 8 and stained with the DbNP PE tetramer, CD8 PerCP-Cy5.5, and FITC-conjugated Abs against a range of surface molecules. The samples were run on a FACSCalibur, and at least 50,000 CD8 lymphocyte events collected. Staining profiles for CD43a (A and D), CD49d (B and E), and CD11a (C and F) from WT (filled histogram), IFN-{gamma}–/– (open histogram), and IFNGR1–/– (dashed line histogram) mice are shown. Profiles presented are representative of five individual profiles.

 
Trafficking and adhesion of Ag-specific CTL involves a multistep process that requires chemokine binding and triggering to enable integrin adhesion and migration into tissues (23). Initial determination of mRNA levels of chemokine receptor mRNA, measured by real-time PCR for primary DbNP366-specific BAL and populations from B6 and IFN-{gamma}–/– mice demonstrated little difference in mRNA levels for CCR1, CCR8, CXCR4, CXCR6, and CXCR3 (data not shown). There was a 2-fold decrease in the levels of CCR5 mRNA expression in CTL from the BAL of IFN-{gamma}–/– mice compared with B6 controls (data not shown). Although the level of chemokine receptor expression did not seem greatly aberrant in the IFN-{gamma}–/– mice compared with the B6 controls, even a 2-fold difference may have biological significance. Previous reports have shown that blocking CCR5 decreases the migration of T cells into tissues (24) and that CCR5–/– mice are more susceptible to influenza virus infection (25). The finding that levels of CCR5 mRNA were two times higher for BAL (but not spleen) T cells from WT B6 vs IFN-{gamma}–/– mice (data not shown) suggested that diminished CCR5 expression could influence T cell recruitment profiles (Fig. 1). However, we found no difference in the number of influenza-specific T cells in BAL or spleen of HKx31-infected CCR5–/– or B6 mice (Fig. 6).


Figure 6
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  		FIGURE 6. Comparison of BAL and spleen epitope-specific CTL numbers from WT B6 and CCR5–/– mice. Naive B6 ({square}) or CCR5–/– ({blacksquare}) mice were infected with the HKx31 virus. BAL (A) and spleen (B) populations (n = 5 each) were isolated 8 days later and PE stained with the DbNP, DbPA, or KbPB1 tetramers and anti-CD8. The number of CD8+ tetramer-positive T cells (mean ± SD) is calculated from the total counts and the percentage of cells staining.

 
Regulation of Ag-specific CTL requires extrinsic IFN-{gamma}

Virus-specific CTLs up-regulate IFN-{gamma}R1 (9) and acquire the capacity to produce IFN-{gamma} following Ag recognition (2, 16, 17, 22), raising the possibility that the effects shown in this study (Figs. 1, 3, and 4) on T cell homeostasis could be an intrinsic property of the lymphocytes. The alternative is, of course, that IFN-{gamma} produced by some other cell type exerts an extrinsic influence on the responding T cells. To differentiate between these two possible mechanisms, influenza-immune CD8+CD44+ T cells were purified from B6 Thy1.1+ mice that had been i.p. primed with the PR8 virus and transferred into Thy1.2+ recipient B6, IFN-{gamma}–/– or IFNGR1–/– mice. As the DbNP-specific CTL dominate secondary responses (16, 17), this population was analyzed after i.n. HKx31 virus challenge of Thy1.2+ recipient mice (Fig. 7). Response profiles in the BAL and spleen were then measured for the recipient Thy1.2+ (Fig. 7A) and donor Thy1.1+ (IFN-{gamma}+IFNGR1+) DbNP366 (Fig. 7B). As previously found (Figs. 1, 3, and 4), there were significantly fewer host Thy1.2+ DbNP366-specific CTLs (p < 0.05) in the BAL and significantly more (p < 0.02) in the spleen populations from both IFN-{gamma}–/– and IFNGR–/– vs WT B6 mice.


Figure 7
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  		FIGURE 7. Transfer of PR8-primed Thy1.1 CD44+CD8+ lymphocytes to Thy1.2+ B6 WT, IFN-{gamma}–/–, and IFNGR1–/– mice. Sorted CD8+CD44+ T cells were obtained from Thy1.1 congenic mice previously primed 8–12 wk earlier with the PR8 (H1N1) virus. The CD8+CD44+ T cells (1 x 106 cells/mouse) were transferred into naive WT B6 ({square}), IFN-{gamma}–/– ({blacksquare}), or IFNGR1–/– (Figure 7) mice. Three days after transfer, mice were i.n. infected with the HKx31 (H3N2) virus and sampled 8 days later. BAL (n = 5) and spleen (n = 5) populations were isolated and stained with the PE DbNP366 tetramer, anti-CD8, and anti-Thy1.1. The number of Thy1.2+CD8+DbNP366+ (A) or Thy1.1+CD8+DbNP366+ (B) T cells (mean ± SD) calculated from the total counts and the percentage of cells staining. *, p < 0.05; **, p < 0.02; ***, p < 0.001 determined using a Student’s pairwise t test.

 
The number of transferred CD8+DbNP366+Thy1.1+ T cells in the BAL and spleens of IFNGR1–/– and B6 mice were essentially equivalent (Fig. 7B). By contrast, significantly fewer DbNP366+Thy1.1+ CTL (Fig. 7B) were found in the BAL of the IFN-{gamma}–/– recipients (p < 0.02), although there were more in the spleen (p < 0.001). Despite the fact that the transferred WT CTL can make IFN-{gamma} following Ag recognition, the familiar (Fig. 1, 3, and 4) pattern of disrupted homeostasis and diminished recruitment to the site of pathology was maintained for the IFN-{gamma}–/– mice. These results imply that IFN-{gamma} produced by host cells other than the responding CTLs regulates T cell trafficking. Furthermore, given that the WT CD8+DbNP366+Thy1.1+ T cells showed similar distribution profiles in both B6 and IFNGR1–/– mice, it is apparent that IFN-{gamma} produced by other cell types acts directly on the Ag-specific effectors (9).

Impact of IFN-{gamma} on cell viability in the spleen but not the BAL

Previous reports have demonstrated that IFN-{gamma} can induce apoptosis of Ag-specific CD4+ T cells (6, 8), suggesting that the pattern of CTL accumulation in spleens shown in this study for IFN-{gamma}–/– and IFNGR1–/– mice (Figs. 1, 3, and 4) may reflect decreased apoptosis. When the CD8+DbNP366+ responders from B6 and IFN-{gamma}–/– mice were stained with Annexin V (Fig. 8), the increased number of splenic CTL in IFN-{gamma}–/– mice correlated with a decrease (relative to B6) in the proportion of Annexin V+ DbNP366-specific T cells (p < 0.03). However, despite the lower number of DbNP366-specific CTL in the BAL of IFN-{gamma}–/– mice (data not shown), Annexin V+ T cells were present at equivalent prevalence in the B6 and IFN-{gamma}–/– mice (Fig. 8). Perhaps the large numbers of activated macrophages in the influenza virus-infected lung mediate the rapid removal of Annexin V+ cells.


Figure 8
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  		FIGURE 8. Distribution of Annexin V+ DbNP366-specific CTL in spleens of WT and IFN-{gamma}–/– mice. Naive WT ({square}) or IFN-{gamma}–/– ({blacksquare}) mice were i.n. infected with the HKx31 virus and lymphocyte populations from the BAL and spleen were isolated on day 8 and stained with PE DbNP tetramer, CD8 PerCP-Cy5.5, propidium iodide (PI), and Annexin V-FITC. The samples were run on a FACSCalibur and at least 50,000 CD8 lymphocyte events were noted. Necrotic cells (Annexin V+ PI+) were excluded from the analysis. Data shown are the percentage (mean ± SD) of DbNP366+Annexin-V+PI– cells used to measure the extent of apoptosis in the population (n = 5). This is representative of three independent experiments. *, p < 0.03 compared using Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The conclusion that IFN-{gamma} deficiency does not compromise either virus clearance or the clinical outcome of influenza virus infection is in accord with earlier findings (15). Dissection of the CD8+ T cell response demonstrated that the lack of IFN-{gamma} signaling in IFN-{gamma}–/– and IFNGR1–/– mice led to an accumulation of tetramer-positive T cells in the lymphoid tissue at the peak of the influenza-specific response, a profile that was maintained at the initial contraction phase. By contrast, the inflammatory cells obtained by BAL of the influenza virus-infected lung contained fewer Ag-specific CTL in IFN-{gamma}–/– and IFNGR1–/– mice compared with the WT B6 controls. The number of Ag-specific CTL isolated from BAL reflects recruitment to the lung tissue during the acute stage of infection (4, 22). Given a previous report has demonstrated that inhibition of IFN-{gamma} results in diminished cellularity within infected lung tissue during the acute response (10), it is assumed that the decreased number of CTL in the BAL of infected IFN-{gamma}–/– and IFNGR1–/– mice will also be reflected in the lung tissue. The impression overall is that the lack of IFN-{gamma} signaling leads to the retention of responding T cells in the secondary lymphoid tissue, an effect that impacts negatively on efficient cellular recruitment to sites of pathology (10, 11). Importantly, extrinsic IFN-{gamma} acted directly on the Ag-specific T cells.

Based on these results there may in fact be several levels of IFN-{gamma} control of the CTL response after influenza virus challenge. In contrast to infection with either L. monocytogenes or LCMV, the effect on spleen cell numbers in the influenza model seems to be transient: very obvious in the acute phase of the response and less associated with a late phenotype (7). Such divergent profiles for different pathogens may reflect the localized nature of influenza virus replication, which is substantially restricted to lung epithelium. The resultant limited spectrum of Ag presentation after influenza A virus challenge (26) is at variance with the heavy Ag loads induced by the systemic L. monocytogenes or LCMV infections. Perhaps deficiencies in IFN-{gamma} signaling are more apparent when less Ag is available. Providing a bigger stimulus may tend to minimize any effect of IFN-{gamma} on the acute phase of the response. An analysis over a longer time course is warranted to determine whether the influenza A virus-specific CTL number contracts to levels observed in WT mice overtime.

Infection of IFN-{gamma}–/– mice with L. monocytogenes resulted in an aberrant immunodominance hierarchy in which there was equal expansion of what is considered to be the normal subdominant p60217–225-specific and dominant LLO91–99 responses. The explanation advanced for this result was that IFN-{gamma} signaling changes immunoproteosome activity by promoting the incorporations of the induced low molecular mass polypeptide subunits LMP2 and LMP7, an effect that influences immunodominance hierarchies after infection in an epitope specific manner (7). There was alteration to the characteristic influenza A virus-specific immunodominance hierarchy. Rather than codominance of both DbNP366- and DbPA224-specific responses typically observed after primary infection, the DbNP366-specific response was immunodominant in IFN-{gamma}–/– mice. This response may reflect a need for IFN-{gamma}–/– signaling to increase the Ag presentation of the PA224 epitope. This interpretation is supported by earlier findings that presentation of the PA224 epitope is limited to professional APCs (26). In contrast, the cellular spectrum of NP366 epitope presentation is broader and therefore may be less dependent on IFN-{gamma}-dependent induction of the immunoproteosome.

The increased influenza-specific CTL counts in the secondary lymphoid tissue of both IFN-{gamma}–/– and IFNGR1–/– mice may, at least partially, reflect defective Ag-induced cell death, as the proportion of Annexin V+ DbNP366-specific CTL was increased in the spleens of WT B6 compared with the IFN-{gamma}–/– hosts. In support of this notion, IFN-{gamma} has been shown to play a part in limiting the number of Ag-specific CD4+ T cells (6, 8) via an apoptotic mechanism. Furthermore, IFN-{gamma} can up-regulate a number of Ag-induced cell death-related mediators, such as caspases 3 and 8, in activated CD4+ T cells (6). Interestingly, the proportion of Annexin V+ CTL in the BAL was equivalent for both WT and IFN-{gamma}–/– mice. One interpretation is that the homeostatic mechanisms that control the number of Ag-specific CTL are modulated by the particular tissue environment. For example, TNFR2 seems to be involved in eliminating influenza A virus-specific CTLs from the BAL (27). Other possibilities are that fewer epitope-specific T cells transit to the infected lung in the IFN-{gamma}–/– mice, or that any excess Annexin V+ T cells are rapidly removed by the large numbers of activated macrophages in this site (28).

When memory B6 Thy1.1+ CTL were transferred into naive Thy1.2+ B6 or IFNGR1–/– hosts that were then challenged i.n. with the HKx31 influenza A virus, a comparable number of donor CD8+DbNP366+Thy1.1+ T cells was found in the spleen and BAL populations from both sets of recipients, whereas the endogenous CD8+DbNP366+Thy1.2+ showed the divergent profiles characteristic of WT and IFNGR1–/– mice. Given that IFNGR1–/– mice are capable of producing IFN-{gamma}, and the host IFNGR1–/– CTL demonstrated the usual pattern of altered homeostasis, the findings fit the notion that IFN-{gamma} acting directly on Ag-specific IFNGR1+/+ CTLs promotes normal homeostasis and trafficking. By contrast, when WT Thy1.1+ memory populations were transferred into IFN-{gamma}–/– (Thy1.2+) hosts, the usual disruption in T cell localization profiles was observed for the Ag-specific Thy1.1+ IFNGR1+/+ T cells. These T cells were, of course, capable of producing IFN-{gamma}. The obvious conclusion is that extrinsic IFN-{gamma} acts to regulate CD8+ T cell homeostasis and trafficking (9).

The inability of the CTLs themselves to compensate for the lack of extrinsic IFN-{gamma} production by producing IFN-{gamma} themselves might be explained by the dependence on specific Ag-recognition for cytokine production (29). Single-cell RT-PCR analysis indicates that both DbNP366- and DbPA224-specific T cells are negative for IFN-{gamma} mRNA at the peak of the CTL response when Ag is effectively cleared (S. J. Turner and P. C. Doherty, unpublished data). Given the postulated role of IFN-{gamma} in the T cell contraction phase (7) following Ag clearance, it is unlikely that the influenza A virus-specific CTLs are capable of self-regulation via any IFN-{gamma}-mediated pathway.

IFN-{gamma} signaling has been shown to modulate both Ag-specific CD8+ and CD4+ T cell responses to pathogens (7, 9, 30). Therefore, the regulatory role of IFN-{gamma} correlates with regulated expression of IFNGR1 on activated T cells (9, 30, 31). Therefore, the lack of compensation may also be indicative of the levels of IFN-{gamma}R1 expression on DbNP366- and DbPA224-specific T cells during the effector phase of the primary CTL response. Importantly, the responsiveness of Ag-specific CTL to IFN-{gamma} has been shown to be dependent on IFNGR2 expression by activated T cells (32). Therefore, the levels of both IFNGR1 and IFNGR2 expression on influenza A virus-specific CTL should be examined in the future to better understand the of impact of IFN-{gamma} signaling on CTL homeostasis.

Ag-specific T cells from both IFN-{gamma}–/– and IFNGR1–/– mice express more CD49d ({alpha}4 integrin) and less CD11a (LFA-1). CD49d binds to the VCAM-1, fibronectin, and MadCAM molecules that are involved in lymphocyte adhesion, signaling, and dissemination (23, 33). It is tempting to speculate that the increased expression of CD49d by IFN-{gamma}–/– and IFNGR1–/– virus-specific CTLs is perhaps one mechanism that leads to preferential localization and/or retention in the spleen (34, 35). The adhesion molecule VLA-1 has also been implicated in T cell trafficking, with high levels being found on influenza-specific CTL from the lungs of virus infected mice (36). However, blocking VLA-1 did not impact on recruitment to the infected lung though it did reduce CTL retention in this site and it was associated with T cell accumulation in the spleen (33).

Analysis of chemokine receptor expression on Ag-specific CTLs from IFN-{gamma}–/– mice did not provide a clear explanation for the observed T cell localization phenotype. Some evidence was found for altered CCR5 levels on these IFN-{gamma}–/– T cells (24, 25), but experiments with CCR5–/– mice showed no effect on either resistance to influenza A virus infection or on the homeostatic control of Ag-specific CTL numbers. Although chemokine receptor expression profiles were not otherwise modified in this limited study of IFN-{gamma}–/– mice, many chemokines are inducible by IFN-{gamma} and it remains possible that our analysis was not sufficiently detailed to reveal effects that might contribute to dysregulated CTL homeostasis.


   Acknowledgments
 
We thank Drs. Nicole La Gruta, Katherine Kedzierska, John Stambas, Barbara Coulson, and Paul Thomas for helpful discussion and critical review of this manuscript.


   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 a National Health and Medical Research Council of Australia Burnet Award (to P.C.D.) and a R. D. Wright Fellowship (to S.J.T.), by Science, Technology, and Innovation funds from the Government of Victoria, Australia, by Grant AI29579 from the U.S. Public Health Service, National Institutes of Health, and by ALSAC, St. Jude Children’s Research Hospital. Back

2 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

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; PA, polymerase A; BAL, bronchoalveolar lavage; MLN, mediastinal lymph node; i.n., intranasally; EID50, egg ID50; NP, nucleoprotein; WT, wild type. Back

Received for publication December 21, 2006. Accepted for publication April 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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