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The ₁₁ Integrin and TNF Receptor II Protect Airway CD8⁺ Effector T Cells from Apoptosis during Influenza Infection¹

Department of Microbiology and Immunology and the David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester, Rochester, NY 14642

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Primary viral infections of the lung induce potent effector CD8 T cell responses. To function in the influenza-infected airways, CD8 T cells must be able to resist cell death. The majority of the CD8 T cells in the airways and lung parenchyma expressed CD49a, the -chain of the type IV collagen receptor VLA-1, and these cells were highly activated, producing both IFN- and TNF-. In the airways, where type IV collagen is abundant, but not the spleen, the CD49a⁺ CD8 cells had reduced proportions of annexin V and caspase 8, and >80% expressed the TNF- receptor II, while Fas, TNFR-I, and CD27 expression were similar to CD49a^– cells. Furthermore, the CD49a⁺, but not CD49a^–, CD8 T cells from the airways were resistant to active induction of apoptosis in the presence of type IV collagen and TNF- in vitro. We propose that TNFR-II and the VLA-1 synergize to protect effector CD8 T cells in the infected airways from apoptosis during the acute infection.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Primary viral infections of the lung induce potent effector CD8 T cells. For influenza infection, these cells must migrate to and function within the infected airways. Elimination of infected cells and reduction of virus titers occurs through the release of antiviral cytokines IFN- and TNF-, the directed release of lytic proteins such as perforin and granzymes (1, 2, 3), and Fas/FasL-mediated induction of apoptosis (1). This has the potential to create an environment that is hostile for the T cells, and yet few specific mechanisms have been described to protect the effector T cells from apoptosis in the tissues.

T cells, once activated, can migrate into peripheral tissues, including the lung and infected airways regardless of whether they express an extralymphoid/effector (CD44^high/CD62L^low/CCR7^low) or central/lymphoid (CD44^high/CD62L^high/CCR7^high) phenotype (4, 5, 6). Therefore, the predominance of the extralymphoid/effector phenotype cells in peripheral sites during infection must be explained by a mechanism other than the ability to migrate to those sites. One possibility is that T cells with a lymphoid phenotype either lack the machinery to be retained, or are stimulated to migrate quickly out of peripheral tissues. This is supported by evidence that activated CCR7^high T cells that do not see Ag, fail to down-modulate CCR7, and are therefore responsive to chemokine gradients that direct them toward draining lymphatics (7). An alternative possibility is that different T cell subsets vary in the ability to survive in peripheral tissues. The latter is supported by evidence that virus-specific CD8 T cells in peripheral tissues are more resistant to apoptosis than those in lymphoid organs (8).

A short time ago, we identified a mechanism by which influenza-specific CD8 T cells were retained in the lung and airways during the memory phase of the response (9). The collagen adhesion molecule VLA-1 was shown to be essential for maintaining substantial numbers of flu-specific CD8 T cells in a variety of nonlymphoid tissues. In addition, we demonstrated that, during acute infection, VLA-1⁺ CD8 T cells from the airways had reduced TUNEL staining (9), though the mechanism to explain this observation was lacking.

Effector CD8 T cells in lymph nodes and spleen are protected from apoptosis by signals through CD27, the receptor for membrane-bound CD70 (10). However, it is not clear whether CD27 exerts the same function in peripheral tissues. The TNF/TNFR family members are regarded as the primary means by which the CD8 T cell response is regulated. For example, signals through TNFR-I, TNFR-II, and TRAIL can cause the death of activated CD8 T cells (11, 12), while CD27/CD70 and CD40/CD40L interactions can be both costimulatory and inhibit apoptosis (13). The outcome depends on the context in which the signals are delivered.

Ligation of integrins can also affect the survival of many cell types, including T cells (14). In fact, attachment to extracellular matrix is an essential survival signal for normal epithelial and endothelial cells (15). The reduced TUNEL staining of VLA-1⁺ CD8 T cells we observed during acute influenza infection led us to investigate whether this populations was unique in its ability to survive in the infected airways.

In the present study, we examined the relationship between the various markers of effector and lymphoid T cells, integrin expression, as well as several apoptotic markers and signals. We found that CD8 T cells that expressed CD49a, the -chain of VLA-1, and TNFR-II were less apoptotic in the lung, and resistant to active induction of apoptosis in vitro when in the presence of their natural ligands. From this, we propose a mechanism wherein VLA-1 and TNF- synergize to protect effector CD8 T cells in the lung and preserve immune function during infection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

Female C57BL/6 (B6) mice were obtained from the National Cancer Institute (Rockville, MD) or from The Jackson Laboratory at 5 wk of age. All animals were housed in the University of Rochester Vivarium facilities under specific pathogen-free conditions using microisolator technology. Inoculation with influenza virus was performed in animals 6–8 wk of age.

Viruses

The H3N2 A/Hong Kong/X31 (X31) influenza virus was grown and titered in embryonated chicken eggs and harvested as allantoic fluid preparations (16).

Influenza infection of mice

Mice were sedated with 5.4 mg/kg 2,2,2-tribromo-ethanol (Avertin) and positioned in a surgical plane. Thirty microliters of X31 (10⁵ 50% egg infectious dose in PBS) were instilled intranasally. Mice were returned to their cages and the appropriate incubation times were respected.

Organ isolations

Mice were subjected to deep anesthesia with 10 mg/kg avertin. The peritoneal cavity was exposed and the spleen was removed and placed in complete MEM (cMEM)³ on ice. An incision was made and skin was removed to expose the trachea. The trachea was cannulated, and three 1-ml (cMEM) bronchoalveolar lavages (BAL) were performed to collect cells present in the airways. The collected fluid was immediately placed on ice. Lungs were perfused with PBS by cardiac puncture and each lobe was removed and placed on ice in cMEM. The mediastinal lymph node was located, removed, and placed on ice in cMEM.

Cell isolations

Spleens and mediastinal lymph nodes were placed in Dounce homogenizers and single-cell suspensions were prepared. Homogenized samples were filtered through a 90-µm nylon mesh, centrifuged, and resuspended in 1 ml of cMEM and stored on ice. Spleen samples were depleted of RBC using a buffered ammonium chloride solution (Gey’s solution) for 4 min. Lung single-cell suspensions were obtained by pressing the organs through a 200-gauge wire mesh and filtered through a 90-µm nylon mesh. Lung lymphocytes were isolated at the interface of a 30 min, 1500 x g centrifugation step with Histopaque 1083 (Sigma Diagnostics). Airway cells obtained by BAL were washed with cMEM and centrifuged at 400 x g and 4°C for 5 min. All cell counts were obtained by trypan blue exclusion.

Staining for apoptotic markers

Aliquots of 1 x 10⁶ cells were stained with a fluorescently labeled inhibitor of caspases 8 labeled with FITC (BioVision). Active caspase 8 was identified using FITC-IETD-FMK (1:300) in a 45-min incubation in cMEM at 37°C. Cells were then washed and an FcR-blocking step was performed using unlabeled anti-CD16/32 (1:200; BD Pharmingen) for 20 min. Cells were then washed and labeled with primary Abs as described below. Following surface staining, cells were resuspended in a 1x annexin V buffer containing 10 mM HEPES/NaOH, 140 mM NaCl and 2.5 mM CaCl₂ and labeled with annexin V-PE-Cy5 (1:200; Abcam) for 15 min at room temperature. Live vs dead cells were discriminated by staining with Sytox blue (1:5000 in the 1x annexin V buffer) for 10 min immediately before analysis. The Sytox blue solution was left in the tubes but was diluted 2-fold.

Intracellular cytokine staining

Cells freshly isolated from organs were seeded in a 96-well plate at 1 x 10⁶ cells per 200 µl of medium containing brefeldin A (10 mg/µl) and IL-2 (50 U/ml) in the presence or absence of 1 µM NP_366–374 peptide. The cells were incubated for 6 h at 37°C and 5% CO₂. After incubation, cells were washed with PBS-BSA containing brefeldin A and stained for caspase 8, surface markers, and annexin V as described above. Cells were then thoroughly resuspended in 100 µl of CytoFix/CytoPerm (BD Biosciences) to which calcium chloride was added (2 mM final concentration) and incubated for 20 min at 4°C. A total of 100 µl of perm/wash buffer was added and cells were spun down and liquid was removed. An additional wash was performed with 200 µl of perm/wash buffer. Cells were than stained for intracellular IFN- (PE; 1:200) and TNF- (allophycocyanin; 1:200) for 30 min at 4°C. Cells were then washed twice with perm/wash buffer, resuspended in staining buffer and analyzed by FACS.

Polychromatic flow cytometric analysis

Lymphocyte populations were stained as aliquots of 1 x 10⁶ cells with various combinations of mAbs to CD4, CD8, CD44, CD62L, CD11a, CD120 (a and b), and CD49a conjugated to FITC, PE, PE-Cy5, PE-Cy5.5, PE-Cy7, allophycocyanin, allophycocyanin-Cy5.5, or allophycocyanin-Cy7. Conjugated mAbs were purchased from BD Pharmingen, Caltag Laboratories, eBioscience, or Serotec and are referenced in their current catalogs. CD49a Ab was also conjugated to Pacific Orange succinimidyl ester according to manufacturer’s protocol (Molecular Probes). Tetrameric complexes of H-2D^b/influenza NP_366–374 (D^bNP) were prepared by the Trudeau Institute Molecular Biology Core Facility and used as described previously (2, 17). All events collected were analyzed with FACSDiva software (BD Biosciences), using a BD Biosciences LSR II. All postacquisition analysis was performed using FlowJo (Tree Star).

Histology

On day 8, the peritoneal cavity was opened and spleens were removed, placed in OCT, and frozen using a mixture of dry ice and 2-methyl butane (–45°C). The rib cage was opened and a canula was placed in an incision made in the trachea and tightly fixed using suture. Warmed OCT (0.8 ml) was slowly injected using a 1-ml syringe to inflate the lungs, and held in place with suture. Lungs were then carefully excised and placed in OCT as described for spleens. Tissues were stored in a –80°C freezer. Sections (5–10 µm) were cut using a cryostat. For staining, sections were thawed and residual OCT was removed by incubating for 5 min with 1 ml of PBS. Sections were fixed in a mixture of methanol-acetone (1:1) for 5 min. Following fixation, sections were left to dry for 15 min and then rehydrated in a 5-min incubation with PBS-Tween 20 (0.05%). After this point, all incubation and washing steps were made using PBS-Tween 20. An FcR-blocking step was performed using unlabeled anti-CD16/32 (1:200; BD Pharmingen) for 20 min. Sections were washed twice for 5 min and stained with PE-labeled rat anti-mouse CD8a (1:100; eBioscience) and unlabeled rabbit anti-mouse type IV collagen (1:200; Chemicon International) for 60 min. Sections were washed and stained with a secondary donkey anti-rabbit Alexa 647 (1:100; Molecular Probes) for 45 min. The sections were then washed and mounted using a cover slip. Fluorescence microscopy was performed using a Nikon Eclipse E600 fluorescence microscope equipped with a 100 W mercury lamp (Chiu Technical) and a SPOT RT Color digital camera (Diagnostic Instruments).

In vitro collagen binding and apoptotic marker analysis

Cells were isolated from mice 8 days postinfection, as described in Organ isolations and Cell isolations, and counted. Cells were plated at 2 x 10⁵ cells/well on plastic or collagen type IV-coated plates. Cells were left untreated or were treated with TNF- (10 ng/ml) or anti-Fas Jo2 Ab (10 µg/ml) or a combination of the two for 4 h. In some cases, cells were pretreated with anti-TNFR-II. Cells were removed from wells using a versene solution, Fc blocked, and stained for CD49a-Alexa 488, annexin V-PE, 7-aminoactinomycin D (7AAD), and CD8-allophycocyanin.

Statistical analysis

Data are presented as means ± SD or SEM (as indicated in the figure legends). Statistical significance was evaluated using the Student t test when comparing appropriate groups. A p value of <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Phenotypic analysis of CD49a^– and CD49a⁺ CD8 T cells isolated from the airways of influenza-infected mice

At 8 days after infection, 80% of CD8⁺ T cells in the airways express CD49a (9). CD49a⁺ CD8 cells presented a highly activated phenotype characterized by high expression levels of CD44 and CD11a (-chain of the LFA-1, _L2 integrin) (Fig. 1, A and B) and CD49d (the -chain of VLA-4; data not shown). Furthermore, the CD49a⁺ cells showed uniformly low surface levels of CD62L (Fig. 1C) and CCR7 (data not shown), consistent with decreased tropism for lymph nodes.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. VLA-1+ cells present a higher activation state than VLA-1– cells. Phenotypes of airway CD49a+ and CD49a– subsets of CD8 T cells were compared in mice 8 days postinfluenza infection. Cells were harvested by BAL and stained as described in Materials and Methods. Using a wide lymphocyte gate, CD8+ cells were identified in a CD8 vs CD4 scatter plot and CD8+ cells were subset using an anti-CD49a Ab to the -chain of VLA-1 into CD49a+ (A–C) and CD49a– (D–F) cells. Within these subpopulations, staining for CD62L (A and D), CD44 (B and E), and CD11a (C and F) was examined. Resting splenocytes were used as comparison controls in these experiments. Numbers in the upper right corners of each panel represent the mean fluorescence intensity of each marker. Plots shown are representative of five separate experiments.

Cytokine effector CD8 T cells are present in both the CD49a⁺ and CD49a^– subsets

Because surface analysis placed the CD49a⁺ CD8 T cells as potential effectors, we investigated the relationship between integrin expression and cytokine secretion. CD8 T cells retrieved from the airways were stimulated with NP_366–374 peptide in an intracellular cytokine assay, or stained with a D^bNP tetramer (2). D^bNP⁺ and cytokine-producing cells retrieved from influenza infected mice 8 days postinfection showed a predominantly CD62L^low profile (Fig. 2, A–C), and roughly 75% of the D^bNP⁺ and cytokine-producing cells were CD49a⁺ (Fig. 2, D–F). Furthermore, a much higher proportion of CD49a⁺ cells produced IFN-, TNF-, or both in response to NP peptide compared with the CD49a^– subset (19 vs 8% respectively; Fig. 2, G and H), suggesting Ag-specific cytokine-producing CD8 T cells are enriched within the CD49a⁺ subset. However, double TNF-/IFN- producers, considered the most activated effector type (18), were similar (49 vs 54% of the cytokine-producing cells) between CD49a⁺ and CD49a^– cells. Thus, effector CD8 T cells were present in both subsets. When cytokine production was examined in relation to the expression of the integrin LFA-1, IFN- and TNF--secreting CD8 T cells were present only among the LFA-1^high subset, likely because LFA-1-negative CD8 T cells have difficulty engaging target cells (19). Of the LFA-1^high cytokine⁺ CD8 T cells, most (>80%) were also CD49a⁺, with few CD49a^– LFA-1^low cells producing cytokine in the assay (data not shown). Together, the data show that the majority of functional effector CD8 T cells in the airways express both VLA-1 and LFA-1 integrins.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. VLA-1+ cells are enriched in Ag-specific effector cells and in cytokine producers. Eight days following influenza infection, Ag-specific and cytokine-producing populations were examined in cells harvested from airways of influenza-infected mice. After gating on CD8+ cells in a wide lymphocyte gate, Db-NP-tetramer+CD8+ cells were analyzed in relation to CD62L (A) and CD49a (VLA-1) (D) expression. Cytokine production was analyzed among CD8+ T cells responding to a 6-h in vitro NP366–374 peptide (1 µM) stimulation. Cells were stained as described in Materials and Methods. IFN- and TNF- producers were identified within the CD62L (B and C) and CD49a (E and F) populations of CD8+ lymphocytes. IFN- and TNF- single and double producers were identified within the CD49a– (G) and CD49a+ (H) subsets of CD8 cells. Numbers in quadrants represent the percentage of positive cells in that quadrant. Plots shown are representative of five (A) and two (B–H) separate experiments.

Because differences in apoptotic markers were observed between CD49a⁺ and CD49a^– CD8 T cells (9), the question arose as to whether cells that had started to undergo apoptosis were still functional and could still produce cytokines. Cells isolated from the airways and spleen that were positive for a fluorescent caspase 8 substrate (see insets in Fig. 3, A and B) that recognizes only the activated enzyme and not the proenzyme (20, 21, 22), and the membrane integrity indicator dye Sytox blue (23) (data not shown), produced very little to no TNF- or IFN- (Fig. 3, A and B) in the intracellular cytokine assays. However, a significant proportion of airway caspase 8-negative, annexin V⁺ cells produced IFN-, or both IFN- and TNF- (Fig. 3, C vs D) with an 2-fold greater proportion of IFN- single producers (17%) and IFN-/TNF- double producers (20%) (Fig. 3D). These differences were enhanced among splenic effectors (Fig. 3, E vs F) where there was an increased proportion of annexin V⁺ T cells, with 50% of these cells producing cytokine, particularly both TNF- and IFN-, in response to NP peptide (Fig. 3F). We conclude that in this assay, influenza-specific CD8 T cells that were in the early stages of apoptosis were still able to produce cytokines and were enriched within the annexin V⁺ subset. Though we could not distinguish whether the annexin V phenotype was acquired during the assay (8), it was clear that those T cells that have activated caspase 8 have lost effector capacity.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Influenza-specific CD8 T cells in the early stages of apoptosis are still able to produce cytokines. Eight days following influenza infection, cytokine-producing cells were examined in cells harvested from airways of influenza-infected mice. After gating on CD8+ cells in a wide lymphocyte gate, cytokine production was analyzed among CD8+ T cells responding to a 6-h in vitro NP366–374 peptide (1 µM) stimulation. Cells were stained for apoptotic markers and surface markers as described in Materials and Methods. IFN- and TNF- producers were identified within the annexin V and caspase 8-positive and -negative populations of airway (A, C, and D) and spleen (B, E, and F) cells. Cytokine producers were examined within caspase 8+ and annexin V+ cells of the airways (A) and spleen (B). Insets in A and B represent caspase 8 staining during the intracellular cytokine assay and gating strategy for caspase 8-positive and -negative cells. CD8 T cells were then gated on caspase 8– cells and further subset into annexin V-negative and -positive cells in the airways (C and D) and spleen (E and F), and cytokine production was examined. Numbers in quadrants represent the percentage of positive cells in that quadrant. Plots shown are representative of two separate experiments.

Our results so far showed that most CD8 T cells in the airways with an activated effector surface phenotype were also CD49a⁺, and cytokine⁺ cells were CD11a^high and caspase 8 negative. Given that CD49a⁺CD8⁺ T cells had also been shown to have a reduced proportion of TUNEL⁺ staining, we investigated the relationship between VLA-1, LFA-1 (CD11a), CD44, and CD62L expression and apoptotic markers.

Among the total CD8 T cells in the airways, the majority of annexin V-positive cells were CD62L^low, CD11a^low, and CD49a^low/– (Table I). CD44 expression did not distinguish the annexin V-positive and -negative subsets. In contrast, among the D^bNP⁺CD8⁺ T cells that were annexin V⁺, the majority were CD62L^high, CD11a^low, and CD49a^low/– (Table I), consistent with a more "lymphoid" profile (7). Similar results were obtained for caspase 8 and Sytox blue.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Ag-specific CD8 T cells in the airways and lungs of influenza infected mice show similar levels of annexin V during the acute phase of the response but annexin V and caspase 8 are reduced within the CD49a-positive and -negative subsets. Lungs and BALs were obtained 7, 9, and 10 days following A/HK/X31 infection of C57BL/6 mice. Pooled samples from three to five mice were stained for several surface markers and proapoptotic markers as described in Materials and Methods. Using a wide lymphocyte gate, cells were gated on CD8+ and Ag-specific cells were identified by gating on DbNP-tetramer+ cells. The proportion of these cells that stained positive for annexin V in the airways () and lung tissue () was evaluated (A). Cells from the airways were further subset into CD49a+ (B) and CD49a– (C) populations, and annexin V+ and caspase 8+ cells were identified within these two subsets on days 7 (), 9 (), and 10 (). Results shown are from a single experiment using pooled samples from three to five mice per time point to ensure that adequate Ag-specific cell numbers were obtained for subsetting and analysis, and that staining was uniform for all three time points for comparison but are representative of three independent experiments.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. For similar levels of Fas expression, infiltrating VLA-1+ airway CD8 T cells express high levels of TNFR-II and have reduced caspase 8 compared with VLA-1– cells and spleen cells. Cells were obtained from BAL and spleen 8 days postinfluenza infection. A, CD8+ cells were identified among lymphocytes in each sample with anti-CD8-PE and anti-CD49a-Alexa 488 staining was examined among these cells. B, TNFR-II (anti-CD120b-PE) and Fas (anti-CD95-allophycocyanin) expression was analyzed within the CD49a– (B; upper panels) and CD49a+ (B; lower panels) subsets of CD8+ lymphocytes found in airways (B; left panels) and spleens (B; right panels) of influenza-infected mice. C, Active caspase 8 levels in the CD49a– and CD49a+ subsets of airway and spleen DbNP tetramer+CD8+ lymphocytes were compared. This was performed following staining with anti-CD8-PE, anti-CD49a-Pacific Orange, and caspase 8 (FITC-IETD-FMK). Numbers in quadrants represent percentage of positive cells in that quadrant and those in parentheses represent the percentage of cells positive for active caspase 8 within the CD49a– and CD49a+ subsets. Plots shown represent four separate experiments.

Caspase 8 is downstream of both the TCR (20) and death receptor-mediated apoptotic signals (24, 25). One hypothesis to explain the reduced caspase 8 (and TUNEL or annexin V) activity is that expression of one or more of the TNF family of receptors was reduced or absent among the CD49a⁺ subset. A number of TNFR family members were investigated. Within the total CD8⁺ population in the airways, there was no difference in Fas (CD95) or TNFR-I expression between the CD49a⁺ and the CD49a^– subsets compared with resting spleen CD8⁺ T cells (Fig. 5, A and D, B and E). However, a much greater proportion of CD49a⁺ CD8 T cells expressed TNFR-II (>80%; see Fig. 6B), with roughly twice as much TNFR-II expressed by the CD49a⁺ population based on mean fluorescence intensity (Fig. 5, C and F). This suggests a mechanism by which caspase 8 and other apoptotic signals could be modified (26, 27).

View larger version (34K): [in this window] [in a new window]   FIGURE 5. For similar levels of FAS and TNFR-I, VLA-1+ cells present higher levels of TNFR-II than do VLA-1– cells. The phenotypes of CD49a+ (A–C) and CD49a– (D–F) subsets of CD8 T cells were compared in BALs of mice 8 days postinfluenza infection. Cells were stained for the following markers: anti-CD8-Alexa 405, anti-CD49a-Alexa 488, anti-CD120a (TNFR-I) or anti-CD120b (TNFR-II)-PE, anti-CD44-PE-Cy5.5, anti-CD11a-PE-Cy7, anti-CD95-allophycocyanin (Fas), anti-CD62L-allophycocyanin-Cy7. Using a wide lymphocyte gate, CD8+ cells were identified in a CD8 vs CD4 scatter plot and CD8+ cells were subset into CD49a+ (A–C) and CD49a– (D–F) cells. Within these subpopulations, staining for Fas (A and D), TNFR-I (B and E), and TNFR-II (C and F) was examined. Resting splenocytes were used as comparison controls in these experiments. Numbers in upper right corners of each panel represent mean fluorescence intensity. Plots shown are representative of four experiments.

The differences in apoptotic markers and TNFRs between T cell populations could be explained by selection within the tissue. Infiltrating CD49a-positive and -negative CD8 T cells were identified among airway and splenic populations of CD8⁺ T cells (Fig. 6A). Although a similar proportion of CD49a⁺ cells isolated from the airways (42.3%) and the spleen (42.6%) expressed Fas on their surface (Fig. 6B, lower panels), a smaller proportion of spleen cells expressed TNFR-II, regardless of CD49a expression. In fact, only 18% of the CD49a^– subset and 34% of the CD49a⁺CD8⁺ cells in the spleen expressed the receptor (Fig. 6B, right panels). This was in sharp contrast to the observations made in the airways where 42% of the CD49a^– cells and 81% of the CD49a⁺ cells expressed TNFR-II (Fig. 6B, left panels). Because the spleen contains many naive and resting T cells, the CD44^highCD62L^low subsets in both tissues were examined. Although a high proportion of the activated CD44^highCD62L^low CD8 T cells in the airways expressed TNFR-II (70% of the CD49a^– cells and 80% of the CD49a⁺ cells), the proportions were again reduced in the spleen with little difference between the CD49a⁺ and CD49a^– subsets. There is little evidence that VLA-1 is required for TNFR-II expression, because about half of CD49a-deficient CD8 T cells from the airways of influenza-infected mice expressed TNFR-II (data not shown). The airway CD8 T cells are also distinguished from splenic cells in that CD49a⁺ influenza-specific cells had reduced caspase 8 compared with CD49a^– cells in the airways, but no difference was observed between these populations in the spleen (Fig. 6C). These observations suggest that specific subpopulations of CD8 T cells selectively persist in the airways, possibly due to differences in the presence of their ligands.

VLA-1⁺TNFR-II⁺ cells show reduced proapoptotic markers in the airways of influenza-infected mice

Access to collagen by VLA-1 and a high level of TNFR-II expression may be important factors in determining the dynamics and fate of cell subpopulations infiltrating the airways during virus infection. We sought to determine whether the expression of VLA-1 and TNFR-II was segregated or coincident with expression of apoptotic markers on CD8 T cells within the same extralymphoid tissue. Total CD8⁺ T cells (Fig. 7, A and B) or Ag-specific (D^b-NP-tetramer⁺) CD8⁺ T cells in the airways (Fig. 7C) were further subdivided into populations based on TNFR-II and CD49a (CD49a⁺, TNFR-II⁺ vs CD49a^–, TNFR-II^–). The highest and lowest proportions of cells exhibiting annexin V and caspase 8 activity occurred, respectively, among the CD49a^–, TNFR-II^– and CD49a⁺, TNFR-II⁺ subsets (36.2 ± 1.95% vs 23.56 ± 3.72%; p = 0.039), while cells expressing alternate combinations of VLA-1 and TNFR-II showed intermediate proportions of apoptotic markers (Fig. 7B). In particular, the proportion of caspase 8⁺ cells was higher in the CD49a^–, TNFR-II^– subset compared to CD49a⁺ TNFR-II⁺ cells (20.73 ± 3.35% compared with 4.48 ± 1.38%; p = 0.011) (Fig. 7B). This was even more striking among the Ag-specific population where the proportions of total annexin V⁺ and caspase 8⁺ cells were, respectively, almost 3-fold (p < 0.01) and 13-fold (p < 0.001) lower in the CD49a⁺, TNFR-II⁺ cells compared to cells lacking both receptors (Fig. 7C). These data suggest that coexpression of VLA-1 and TNFR-II may act in concert to protect CD8 T cells from induction of apoptosis in the infected airways.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Reduced caspase 8 and annexin V among VLA-1 and TNFR-II-positive T cells in the airways. Among the airway cells, expression of proapoptotic markers annexin V and caspase 8 was determined in relation to VLA-1 and TNFR-II (A). Plots shown are representative of three separate experiments. The bar graph (A) represents the percentage of annexin V+caspase 8+ cells in each quadrant of (A) corresponding to the alternate combinations of VLA-1 and TNFR-II; x-axis values indicate each quadrant in a clockwise manner starting in the upper left. Annexin V and active caspase 8 staining within the VLA-1–, TNFR-II– (), and VLA-1+, TNFR-II+ () subsets of total CD8+ (B) and Db-NP-tetramer+CD8+ (C) cells (n = 3). Numbers in quadrants represent percentage of positive cells in that quadrant. Results are means ± SEM; *, p < 0.05; **, p < 0.01.

Although we have shown that CD8 T cells in the lung are preferentially in close proximity to collagen IV (9, 28), their proximity to collagen in the spleen is unknown. Lungs and spleens from acutely infected animals were removed, and frozen sections (5–10 µm thick) were stained with fluorescence-labeled rat anti-mouse CD8a and rabbit anti-mouse type IV collagen. Although the lung tissue contained abundant type IV collagen around the airways, blood vessels, and alveoli (Fig. 8A), in the spleen, type IV collagen could be identified only in the structural portion of the red pulp and white pulp, most likely corresponding to the basement membranes of the marginal sinuses and central arterioles (Fig. 8B) (29, 30). CD8⁺ cells in the lungs were found in close proximity to type IV collagen, in particular around the blood vessels and the infected airways (Fig. 8A), while in the spleen, most CD8⁺ cells were found in large clusters near periarteriolar lymphocyte sheaths in which very little type IV collagen could be found, with a few CD8⁺ cells scattered in the structural portion of the tissue where type IV collagen was more abundant (Fig. 8B). Taken together, these findings show differences in access to type IV collagen between the lung and spleen and may explain the observed differences in apoptosis among CD8 T cell subsets.

View larger version (55K): [in this window] [in a new window]   FIGURE 8. Histological examination of the distribution of CD8+ cells in lungs (A) and spleen (B) of mice 8 days postintranasal influenza infection. Five-micrometer sections were obtained from tissues and stained with rat anti-mouse CD8-PE (red) and unlabeled rabbit anti-mouse type IV collagen followed by an Alexa 647-labeled donkey anti-rabbit secondary Ab labeling (pseudo-colored green). Images were overlaid in Adobe PhotoShop and color brightness was adjusted. Images were taken at x400 and are representative of several individual sections. The calibration mark at the bottom left of each image represents 100 µm. A, alveoli; AW, airway; BV, blood vessel; CA, central arteriole; MZ, marginal zones; PALS, periarteriolar lymphocyte sheaths.

VLA-1-expressing cells resist apoptosis in the presence of type IV collagen and TNF- in vitro

In light of these findings, we sought to investigate whether the phenotype observed directly ex vivo could be reproduced in a controlled in vitro environment. To do so, cells were isolated from the airways of acutely infected mice 8 days postinfection and cultured in presence or absence of type IV collagen in 96-well plates, and in the presence or absence of the caspase 8-triggering, apoptosis-inducing anti-Fas Ab Jo2, and/or TNF-. Apoptotic CD8⁺ cells were identified using annexin V and 7AAD and examined in relation to CD49a. After 4 h in culture, the CD49a⁺ CD8 T cells in medium alone had the lowest annexin V/7AAD⁺ profile (33.43 ± 0.90%) (Fig. 9A, lane 1) and this was set as the baseline and results were calculated as percentages above this baseline. In the presence of Jo2 Ab, significant apoptosis was induced above baseline in both CD49a⁺ (43.28 ± 4.01%; p < 0.01) and CD49a^– (68.56 ± 4.16%; p < 0.01) cells (Fig. 9, A and B, lane 2). In the presence of type IV collagen alone, Jo2-induced apoptosis was significantly inhibited in CD49a⁺ cells compared with CD49a^– cells (Fig. 9, A and B, lane 3), suggesting that binding to type IV collagen through VLA-1 can partially inhibit apoptosis induction in VLA-1⁺ cells. In the presence of TNF- alone, Jo2-induced apoptosis was inhibited by 27% (p < 0.01) in the CD49a⁺ subset but no effect was observed in the CD49a^– subset (Fig. 9, A and B, lane 4), suggesting that TNF- signaling can contribute to the inhibition of apoptosis in the CD49a⁺ subset that expresses high levels of TNFR-II compared with CD49a^– cells that express TNFR-II to a much lesser extent in proportion and level. Strikingly, when the cells were cultured in the presence of type IV collagen and TNF-, Jo2-induced apoptosis was blocked by 88.7% (p < 0.01) in the CD49a⁺ subset with no significant effect under these conditions in the CD49a^– group (Fig. 9, A and B, lane 5), suggesting that type IV collagen binding through VLA-1 and TNF- signaling can synergize to potently inhibit apoptosis in cells that express both the VLA-1 integrin and TNF- receptors. In a separate set of experiments, the inclusion of a TNFR-II-blocking Ab abrogated the protective effect of TNF- for the CD49a⁺ CD8 T cells, but had no effect on the CD49a^– subset (Fig. 9, C and D, lanes 2 vs 3), confirming that the TNF- effect signaled through TNFR-II which is highly expressed by VLA-1⁺ cells. In the experiments, neither TNF- nor the blocking Ab alone had an effect on the baseline. Taken together, these data suggest that signals through VLA-1 and TNFR-II can inhibit apoptotic signals among highly activated effector CD8 T cells in the airways.

View larger version (26K): [in this window] [in a new window]   FIGURE 9. Type IV collagen and TNF- act in synergy to protect VLA-1+ cells form apoptosis in vitro. Cells were harvested from airways of mice intranasally infected with influenza 8 days postinfection and pooled. Cells were then plated in presence or absence of type IV collagen and/or TNF- (10 ng/ml). Apoptosis was induced using the anti-Fas Jo2 Ab (10 µg/ml) in a 4h incubation. Cells were removed from plates and stained with anti-CD8-allophycocyanin, anti-CD49a-Alexa 488, annexin V-PE, and 7AAD. Apoptotic cells were identified as annexin V+, 7AAD+. The percentage of apoptotic cells was compared in CD49a+ (A and C) and CD49a– (B and D) cells in each well. In a separate experiment to determine that the effect of TNF- was through TNFR-II, a TNFR-II-blocking Ab (10 µg/ml) was added before TNF- and Jo2 in and apoptosis was evaluated in CD49a+ and CD49a– cells. Results in A and B are expressed as percentage above baseline apoptosis for CD49a+ cells which was 33.43 ± 0.90% annexin V+, 7AAD+ cells in a 4-h incubation without treatment. For C and D, the baseline was 26.90 ± 0.90% (data not shown). Results are means ± SD (n = 5 in two separate experiments); *, p < 0.01 compared with induced control.

	Discussion

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In addition to trafficking, one of the mechanisms that regulate the number of T cells present in a tissue at any given time is the ability of the T cells to be retained. Retention of T cells in turn can reflect both survival and failure to migrate out via efferent lymphatics. One prevailing view is that resolution of acute antiviral responses is mediated by apoptotic elimination of activated T cells after virus has been cleared. However, we found that the proportion of T cells expressing apoptotic markers was relatively similar at the expansion, peak, and resolution phases of the response. This suggested that apoptosis is a continual process and not just a feature of the resolution phase.

TNF- receptor signaling is regarded as the primary means by which the CD8 T cell response is controlled (11). However, depending on the model and the culture system used, conflicting results have been observed. Initial reports suggested that normal T cell blasts were sensitive to TNF- cytotoxicity and that this effect was mediated primarily through TNFR-II (11). Based on the latter, a recent report has suggested that TNFR-II-mediated editing of the influenza-specific CD8 T cell response functions to limit the number of effectors that have localized to site of infection in the lung (but not the spleen) (31). However, the limited TNF--mediated editing process described was dependent on the dose of peptide used and could not be a major factor involved in the return to lower CD8 T cell numbers during the resolution phase (31). In contrast, it has been suggested that TNFR-II (p75) functions as a costimulator for Ag-driven T cell responses in vivo (32). In a Listeria monocytogenes model, for example, TNFR-II regulated the threshold for clonal expansion of Ag-specific CD4 and CD8 T cells, and the CD8 T cells depended on TNFR-II for survival during the early phase of the response (32). In addition, the resulting memory pools were diminished in TNFR-II^–/– mice. Similarly, TNFR-associated factor 2 (TRAF2) adaptor protein dominant-negative mice show profound defects in T cell responses and poor generation of CD8 T cell memory (33). In humans, naive and memory subsets of CD8 T cells exhibit differential sensitivity to TNF--induced apoptosis such that effector/memory cells were resistant to TNF--induced apoptosis compared with naive and central memory CD8 T cells (34).

Our present results may help explain some of the discrepancies and identify a subpopulation of acutely activated T cells that are not only resistant to TNF--mediated apoptosis, but also signal through TNFR-II to make the cells resistant to other proapoptotic signals. The ability of TNF- to promote survival or apoptosis depends on both the dominant receptor expressed on the cell surface, as well as the intracellular context through which the signals are interpreted (20, 35, 36, 37, 38, 39). In our experiments, exposure to TNF- made the CD49a⁺TNFR-II⁺ CD8 T cells resistant to Fas-induced apoptosis, while it had little effect on the CD49a^– subset, despite the fact that a proportion of these cells also expressed TNFR-II. This can be explained possibly by the lower expression levels of TNFR-II on the CD49a^– cells, but it could also suggest modified signaling pathways for TNF- among the CD49a⁺ cells.

TNFR-I contains death domains (DD) that trimerize upon ligation of TNF- resulting in adaptor proteins TNFR-associated DD and Fas-associated DD (24). This complex further interacts with procaspase 8 to form the death-inducing signal complex (DISC) and rapidly produces active caspase 8 resulting in the initiation of the apoptotic pathway leading to the activation of downstream effector caspases. This pathway is kept in check by several gene products such as FLIP that inhibits the association of procaspase 8 to the DISC (40, 41) and can also activate the NF-B and ERK pathways through adaptor proteins such as TRAF-1,-2, and -3, serine/threonine kinase receptor-interacting protein (RIP), and Raf (42). TNFR-II, which lacks death domains, stimulates the activation of MAPKs and NF-B through TRAF2 and RIP leading to the inhibition of apoptosis in mice and humans (43) through induction and expression of several potent antiapoptotic genes including FLIP, TRAF-1, and TRAF-2 (35, 36, 37, 38). Thus, the intracellular balance of adaptor proteins such as TRAF1 and TRAF2 can determine whether TNFR ligation leads to recruitment of the DISC to the receptor, or to activation of NF-B (25). In this regard, the secondary response to influenza was vastly impaired in TRAF2 dominant-negative mice compared with wild-type mice (33). Although the latter model was different from the one used in this study, it clearly shows a link between TRAF2, an adaptor protein involved in the signaling of many receptors of the TNF family, and the response to influenza. This is important because TRAF2 is the main adaptor protein that associated with TNFR-II and can also compete for TNFR-I to induce the expression of several potent antiapoptotic genes. However, the high expression of TNFR-II on the VLA-1⁺ CD8 T cells in the lung is more consistent with the hypothesis that this is sufficient for the delivery of a prosurvival, antiapoptotic signal.

The role that collagen IV ligation through VLA-1 plays in this process also remains to be fully explained. Integrin-mediated signals via extracellular matrix interactions are essential antiapoptotic, prosurvival signals for a variety of cell types, particularly endothelial and epithelial cell types (15). However, these are cells that are strictly contact dependent, whereas T cells are highly migratory. So while some of the antiapoptotic signaling pathways could be shared among lymphocytes and other cell types, it remains to be determined which, if any, of the pathways are shared. A precedent exists for T cells in that VLA-2-mediated ligation of collagen I was shown to inhibit activation-induced death of Jurkat T cells (14), though in that study VLA-1 was not observed to have an effect. The interaction of VLA-1 and type IV collagen may initiate signaling through focal adhesion kinase, which then activates the PI3K/AKT pathway leading to activation of ERK/MAPK and NF-B (14, 44). MAPK/ERK signaling, in turn, can override apoptotic signals from Fas, TNF, and TRAIL receptors (45, 46) reinforcing the hypothesis that signals provided by integrins modify TNFR signals.

Why did we observe these antiapoptotic effects for T cells in the airways and not the spleen? VLA-1 is the major receptor for type IV collagen, which is abundant in the lung and found in the basement membranes of the endothelium and epithelium (28, 47). In lymphoid tissues, fibroblastic reticular cells envelop collagen fibrils, and that these collagen fibrils are located in the interfollicular zones (48, 49). Thus, the splenic tissue is much less abundant in type IV collagen, with most of the collagen located in the basement membranes of the marginal sinuses and the central arterioles.

We therefore propose a mechanism to protect activated effector CD8 T cells in the lung during acute infection. Although both CD49a⁺ and CD49a^– (and CD62L^low and CD62L^high) CD8 T cells can reach the airways; the CD49a⁺ cells have a survival advantage conferred by the ability to bind collagen and signal through VLA-1. The exertion of effector function in the form of TNF- secretion potentiates this effect by promoting antiapoptotic signals through NF-B. This protects the effector CD8 T cells at the site of infection, where they are likely to express and secrete both TNF- and FasL. This mechanism results in a selection process that is restricted to anatomical areas where collagen IV is easily accessible, explaining some of the differences observed between the lung and the spleen. It could also function to limit the accumulation of effector T cells in noninfected tissues where TNF- production would not be triggered. In addition to the acute phase, this mechanism may have implications for the formation of memory given the relationship of TRAF2 in secondary influenza infections (33) and our observations that VLA-1 deficiency impairs secondary influenza immunity (9).

	Acknowledgments

 
We thank Dr. Deborah Fowell for use of fluorescence microscopy equipment.

	Disclosures

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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 Grants AG021970 and AI050020.

² Address correspondence and reprint requests to Dr. David J. Topham, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester Medical Center, 601 Elmwood Avenue, Box 609, Rochester, NY 14642. E-mail address: david_topham{at}urmc.rochester.edu

³ Abbreviations used in this paper: cMEM, complete MEM; BAL, bronchoalveolar lavage; 7AAD, 7-aminoactinomycin D; NP, nucleoprotein; TRAF, TNFR-associated factor; DD, death domain; DISC, death-inducing signal complex.

Received for publication March 29, 2007. Accepted for publication July 31, 2007.

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