Apoptosis During Influenza Infection Effector T Cells from + Protect

The α1β1 Integrin and TNF Receptor II Protect Airway CD8 + Effector T Cells from Apoptosis during Influenza Infection

This information is current as Martin V. Richter and David J. Topham of September 25, 2021. J Immunol 2007; 179:5054-5063; ; doi: 10.4049/jimmunol.179.8.5054 http://www.jimmunol.org/content/179/8/5054 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

؉ ␤ ␣ The 1 1 Integrin and TNF Receptor II Protect Airway CD8 Effector T Cells from Apoptosis during Inﬂuenza Infection1

Martin V. Richter and David J. Topham2

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. The Journal of Immunology, 2007, 179: 5054–5063. Downloaded from

rimary viral infections of the lung induce potent effector A short time ago, we identified a mechanism by which influen- CD8 T cells. For influenza infection, these cells must mi- za-specific CD8 T cells were retained in the lung and airways P grate to and function within the infected airways. Elimi- during the memory phase of the response (9). The collagen adhe- nation of infected cells and reduction of virus titers occurs through sion molecule VLA-1 was shown to be essential for maintaining the release of antiviral cytokines IFN-␥ and TNF-␣, the directed substantial numbers of flu-specific CD8 T cells in a variety of http://www.jimmunol.org/ release of lytic proteins such as perforin and granzymes (1–3), and nonlymphoid tissues. In addition, we demonstrated that, during Fas/FasL-mediated induction of apoptosis (1). This has the poten- acute infection, VLA-1ϩ CD8 T cells from the airways had re- tial to create an environment that is hostile for the T cells, and yet duced TUNEL staining (9), though the mechanism to explain this few specific mechanisms have been described to protect the effec- observation was lacking. tor T cells from apoptosis in the tissues. Effector CD8 T cells in lymph nodes and spleen are protected T cells, once activated, can migrate into peripheral tissues, from apoptosis by signals through CD27, the receptor for mem- including the lung and infected airways regardless of whether brane-bound CD70 (10). However, it is not clear whether CD27 they express an extralymphoid/effector (CD44high/CD62Llow/ exerts the same function in peripheral tissues. The TNF/TNFR low high high high CCR7 ) or central/lymphoid (CD44 /CD62L /CCR7 ) family members are regarded as the primary means by which the by guest on September 25, 2021 phenotype (4–6). Therefore, the predominance of the extralym- CD8 T cell response is regulated. For example, signals through phoid/effector phenotype cells in peripheral sites during infection TNFR-I, TNFR-II, and TRAIL can cause the death of activated must be explained by a mechanism other than the ability to migrate CD8 T cells (11, 12), while CD27/CD70 and CD40/CD40L inter- to those sites. One possibility is that T cells with a lymphoid phe- actions can be both costimulatory and inhibit apoptosis (13). The notype either lack the machinery to be retained, or are stimulated outcome depends on the context in which the signals are delivered. to migrate quickly out of peripheral tissues. This is supported by Ligation of integrins can also affect the survival of many cell evidence that activated CCR7high T cells that do not see Ag, fail to types, including T cells (14). In fact, attachment to extracellular down-modulate CCR7, and are therefore responsive to chemokine matrix is an essential survival signal for normal epithelial and en- gradients that direct them toward draining lymphatics (7). An al- dothelial cells (15). The reduced TUNEL staining of VLA-1ϩ CD8 ternative possibility is that different T cell subsets vary in the abil- T cells we observed during acute influenza infection led us to ity to survive in peripheral tissues. The latter is supported by ev- investigate whether this populations was unique in its ability to idence that virus-specific CD8 T cells in peripheral tissues are survive in the infected airways. more resistant to apoptosis than those in lymphoid organs (8). 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 Department of Microbiology and Immunology and the David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University TNFR-II were less apoptotic in the lung, and resistant to active of Rochester, Rochester, NY 14642 induction of apoptosis in vitro when in the presence of their natural Received for publication March 29, 2007. Accepted for publication July 31, 2007. ligands. From this, we propose a mechanism wherein VLA-1 and The costs of publication of this article were defrayed in part by the payment of page TNF-␣ synergize to protect effector CD8 T cells in the lung and charges. This article must therefore be hereby marked advertisement in accordance preserve immune function during infection. 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. Materials and Methods Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Animals Sciences, University of Rochester Medical Center, 601 Elmwood Avenue, Box 609, Rochester, NY 14642. E-mail address: [email protected] Female C57BL/6 (B6) mice were obtained from the National Cancer In- stitute (Rockville, MD) or from The Jackson Laboratory at 5 wk of age. All Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 animals were housed in the University of Rochester Vivarium facilities www.jimmunol.org The Journal of Immunology 5055

under specific pathogen-free conditions using microisolator technology. CD120 (a and b), and CD49a conjugated to FITC, PE, PE-Cy5, PE-Cy5.5, Inoculation with influenza virus was performed in animals 6–8 wk of age. PE-Cy7, allophycocyanin, allophycocyanin-Cy5.5, or allophycocyanin- Cy7. Conjugated mAbs were purchased from BD Pharmingen, Caltag Lab- Viruses oratories, eBioscience, or Serotec and are referenced in their current cat- The H3N2 A/Hong Kong/X31 (X31) influenza virus was grown and alogs. CD49a Ab was also conjugated to Pacific Orange succinimidyl ester according to manufacturer’s protocol (Molecular Probes). Tetrameric com- titered in embryonated chicken eggs and harvested as allantoic fluid b b preparations (16). plexes of H-2D /influenza NP366–374 (D NP) were prepared by the Trudeau Institute Molecular Biology Core Facility and used as described previously Influenza infection of mice (2, 17). All events collected were analyzed with FACSDiva software (BD Biosciences), using a BD Biosciences LSR II. All postacquisition analysis Mice were sedated with 5.4 mg/kg 2,2,2-tribromo-ethanol (Avertin) and was performed using FlowJo (Tree Star). positioned in a surgical plane. Thirty microliters of X31 (105 50% egg infectious dose in PBS) were instilled intranasally. Mice were returned to Histology their cages and the appropriate incubation times were respected. On day 8, the peritoneal cavity was opened and spleens were removed, Organ isolations placed in OCT, and frozen using a mixture of dry ice and 2-methyl butane (Ϫ45°C). The rib cage was opened and a canula was placed in an incision Mice were subjected to deep anesthesia with 10 mg/kg avertin. The peri- made in the trachea and tightly fixed using suture. Warmed OCT (0.8 ml) toneal cavity was exposed and the spleen was removed and placed in com- 3 was slowly injected using a 1-ml syringe to inflate the lungs, and held in plete MEM (cMEM) on ice. An incision was made and skin was removed place with suture. Lungs were then carefully excised and placed in OCT as to expose the trachea. The trachea was cannulated, and three 1-ml (cMEM) described for spleens. Tissues were stored in a Ϫ80°C freezer. Sections bronchoalveolar lavages (BAL) were performed to collect cells present in (5–10 ␮m) were cut using a cryostat. For staining, sections were thawed the airways. The collected fluid was immediately placed on ice. Lungs and residual OCT was removed by incubating for 5 min with ϳ1mlof Downloaded from were perfused with PBS by cardiac puncture and each lobe was removed PBS. Sections were fixed in a mixture of methanol-acetone (1:1) for 5 min. and placed on ice in cMEM. The mediastinal lymph node was located, Following fixation, sections were left to dry for 15 min and then rehydrated removed, and placed on ice in cMEM. in a 5-min incubation with PBS-Tween 20 (0.05%). After this point, all Cell isolations incubation and washing steps were made using PBS-Tween 20. An FcR- blocking step was performed using unlabeled anti-CD16/32 (1:200; BD Spleens and mediastinal lymph nodes were placed in Dounce homogeniz- Pharmingen) for 20 min. Sections were washed twice for 5 min and stained ers and single-cell suspensions were prepared. Homogenized samples were with PE-labeled rat anti-mouse CD8a (1:100; eBioscience) and unlabeled

filtered through a 90-␮m nylon mesh, centrifuged, and resuspended in 1 ml rabbit anti-mouse type IV collagen (1:200; Chemicon International) for 60 http://www.jimmunol.org/ of cMEM and stored on ice. Spleen samples were depleted of RBC using min. Sections were washed and stained with a secondary donkey anti-rabbit a buffered ammonium chloride solution (Gey’s solution) for 4 min. Lung Alexa 647 (1:100; Molecular Probes) for 45 min. The sections were then single-cell suspensions were obtained by pressing the organs through a washed and mounted using a cover slip. Fluorescence microscopy was 200-gauge wire mesh and filtered through a 90-␮m nylon mesh. Lung performed using a Nikon Eclipse E600 fluorescence microscope equipped lymphocytes were isolated at the interface of a 30 min, 1500 ϫ g centrif- with a 100 W mercury lamp (Chiu Technical) and a SPOT RT Color digital ugation step with Histopaque 1083 (Sigma Diagnostics). Airway cells ob- camera (Diagnostic Instruments). tained by BAL were washed with cMEM and centrifuged at 400 ϫ g and 4°C for 5 min. All cell counts were obtained by trypan blue exclusion. In vitro collagen binding and apoptotic marker analysis Staining for apoptotic markers Cells were isolated from mice 8 days postinfection, as described in Organ 5 isolations and Cell isolations, and counted. Cells were plated at 2 ϫ 10 by guest on September 25, 2021 Aliquots of 1 ϫ 106 cells were stained with a fluorescently labeled inhibitor cells/well on plastic or collagen type IV-coated plates. Cells were left un- of caspases 8 labeled with FITC (BioVision). Active caspase 8 was iden- treated or were treated with TNF-␣ (10 ng/ml) or anti-Fas Jo2 Ab (10 tified using FITC-IETD-FMK (1:300) in a 45-min incubation in cMEM at ␮g/ml) or a combination of the two for 4 h. In some cases, cells were 37°C. Cells were then washed and an FcR-blocking step was performed pretreated with anti-TNFR-II. Cells were removed from wells using a using unlabeled anti-CD16/32 (1:200; BD Pharmingen) for 20 min. Cells versene solution, Fc blocked, and stained for CD49a-Alexa 488, annexin were then washed and labeled with primary Abs as described below. Fol- V-PE, 7-aminoactinomycin D (7AAD), and CD8-allophycocyanin. lowing surface staining, cells were resuspended in a 1ϫ annexin V buffer

containing 10 mM HEPES/NaOH, 140 mM NaCl and 2.5 mM CaCl2 and Statistical analysis labeled with annexin V-PE-Cy5 (1:200; Abcam) for 15 min at room tem- Ϯ perature. Live vs dead cells were discriminated by staining with Sytox blue Data are presented as means SD or SEM (as indicated in the figure ϫ legends). Statistical significance was evaluated using the Student t test (1:5000 in the 1 annexin V buffer) for 10 min immediately before anal- Ͻ ysis. The Sytox blue solution was left in the tubes but was diluted 2-fold. when comparing appropriate groups. A p value of 0.05 was considered statistically significant. Intracellular cytokine staining

6 Results Cells freshly isolated from organs were seeded in a 96-well plate at 1 ϫ 10 Ϫ ϩ cells per 200 ␮l of medium containing brefeldin A (10 mg/␮l) and IL-2 (50 Phenotypic analysis of CD49a and CD49a CD8 T cells ␮ U/ml) in the presence or absence of 1 MNP366–374 peptide. The cells isolated from the airways of influenza-infected mice were incubated for 6 h at 37°C and 5% CO . After incubation, cells were ϩ 2 At 8 days after infection, ϳ80% of CD8 T cells in the airways washed with PBS-BSA containing brefeldin A and stained for caspase 8, ϩ surface markers, and annexin V as described above. Cells were then thor- express CD49a (9). CD49a CD8 cells presented a highly acti- oughly resuspended in 100 ␮l of CytoFix/CytoPerm (BD Biosciences) to vated phenotype characterized by high expression levels of CD44 which calcium chloride was added (2 mM final concentration) and incu- and CD11a (␣-chain of the LFA-1, ␣ ␤ integrin) (Fig. 1, A and B) ␮ L 2 bated for 20 min at 4°C. A total of 100 l of perm/wash buffer was added and CD49d (the ␣-chain of VLA-4; data not shown). Furthermore, and cells were spun down and liquid was removed. An additional wash was ϩ performed with 200 ␮l of perm/wash buffer. Cells were than stained for the CD49a cells showed uniformly low surface levels of CD62L intracellular IFN-␥ (PE; 1:200) and TNF-␣ (allophycocyanin; 1:200) for 30 (Fig. 1C) and CCR7 (data not shown), consistent with decreased min at 4°C. Cells were then washed twice with perm/wash buffer, resus- tropism for lymph nodes. pended in staining buffer and analyzed by FACS. CD49a-negative CD8 T cells, in turn, were much lower in pro- Polychromatic flow cytometric analysis portion, and more heterogeneous in their activation phenotypes. The CD49a-negative CD8 T cells had lower (though still positive) Lymphocyte populations were stained as aliquots of 1 ϫ 106 cells with various combinations of mAbs to CD4, CD8␣, CD44, CD62L, CD11a, levels of CD44 and LFA-1 (Fig. 1, D and E), and expressed CD49d. They also presented with a mixed CD62L profile with both CD62Lhigh and CD62Llow cells within the population (Fig. 3 Abbreviations used in this paper: cMEM, complete MEM; BAL, bronchoalveolar lavage; 7AAD, 7-aminoactinomycin D; NP, nucleoprotein; TRAF, TNFR-associated 1F), in line with the fact that lung infiltration is not restricted to the factor; DD, death domain; DISC, death-inducing signal complex. CD62Llow subset (5). 5056 VLA-1 AND TNF PROTECT FLU-SPECIFIC CD8ϩ T CELLS

FIGURE 1. VLA-1ϩ cells present a higher activation state than VLA-1Ϫ cells. Phenotypes of airway CD49aϩ and CD49aϪ 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. Downloaded from Using a wide lymphocyte gate, CD8ϩ cells were identified in a CD8 vs CD4 scatter plot and CD8ϩ cells were subset using an anti-CD49a Ab to the ␣-chain of VLA-1 into CD49aϩ (A–C) and CD49aϪ (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 cor- http://www.jimmunol.org/ ners 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 CD49aϩ and CD49aϪ subsets Because surface analysis placed the CD49aϩ CD8 T cells as po- tential effectors, we investigated the relationship between integrin expression and cytokine secretion. CD8 T cells retrieved from the by guest on September 25, 2021 airways were stimulated with NP366–374 peptide in an intracellular cytokine assay, or stained with a DbNP tetramer (2). DbNPϩ and cytokine-producing cells retrieved from influenza infected mice 8 days postinfection showed a predominantly CD62Llow profile (Fig. 2, A–C), and roughly 75% of the DbNPϩ and cytokine-producing cells were CD49aϩ (Fig. 2, D–F). Furthermore, a much higher proportion of CD49aϩ cells produced IFN-␥, TNF-␣, or both in response to NP peptide compared with the CD49aϪ subset (19 vs 8% respectively; Fig. 2, G and H), suggesting Ag-specific cytokine-producing CD8 T cells are enriched within the CD49aϩ sub- ϩ set. However, double TNF-␣/IFN-␥ producers, considered the FIGURE 2. VLA-1 cells are enriched in Ag-specific effector cells and most activated effector type (18), were similar (49 vs 54% of the in cytokine producers. Eight days following influenza infection, Ag-spe- ϩ Ϫ cific and cytokine-producing populations were examined in cells harvested cytokine-producing cells) between CD49a and CD49a cells. ϩ from airways of influenza-infected mice. After gating on CD8 cells in a Thus, effector CD8 T cells were present in both subsets. When wide lymphocyte gate, Db-NP-tetramerϩCD8ϩ cells were analyzed in re- cytokine production was examined in relation to the expression of lation to CD62L (A) and CD49a (VLA-1) (D) expression. Cytokine pro- the integrin LFA-1, IFN-␥ and TNF-␣-secreting CD8 T cells were duction was analyzed among CD8ϩ T cells responding to a 6-h in vitro high ␮ present only among the LFA-1 subset, likely because LFA-1- NP366–374 peptide (1 M) stimulation. Cells were stained as described in negative CD8 T cells have difficulty engaging target cells (19). Of Materials and Methods. IFN-␥ and TNF-␣ producers were identified the LFA-1high cytokineϩ CD8 T cells, most (Ͼ80%) were also within the CD62L (B and C) and CD49a (E and F) populations of CD8ϩ CD49aϩ, with few CD49aϪ LFA-1low cells producing cytokine in lymphocytes. IFN-␥ and TNF-␣ single and double producers were identi- Ϫ ϩ the assay (data not shown). Together, the data show that the ma- fied within the CD49a (G) and CD49a (H) subsets of CD8 cells. Num- jority of functional effector CD8 T cells in the airways express bers in quadrants represent the percentage of positive cells in that quadrant. Plots shown are representative of five (A) and two (B–H) separate both VLA-1 and LFA-1 integrins. experiments. Cytokine secretion and apoptotic phenotype Because differences in apoptotic markers were observed between substrate (see insets in Fig. 3, A and B) that recognizes only the CD49aϩ and CD49aϪ CD8 T cells (9), the question arose as to activated enzyme and not the proenzyme (20–22), and the mem- whether cells that had started to undergo apoptosis were still func- brane integrity indicator dye Sytox blue (23) (data not shown), tional and could still produce cytokines. Cells isolated from the produced very little to no TNF-␣ or IFN-␥ (Fig. 3, A and B)inthe airways and spleen that were positive for a fluorescent caspase 8 intracellular cytokine assays. However, a significant proportion of The Journal of Immunology 5057

Table I. Phenotypic analysis of total CD8 and DbNP-tetramerϩ CD8 T cell subsetsa

% Annexin Vϩ

Total CD8ϩ % NP-TetramerϩCD8ϩ

CD62Lhigh 2.55 55.60 CD62Llow 11.20 23.20 CD11ahigh 6.67 13.30 CD11alow 11.60 39.50 CD44high 8.30 20.00 CD44low 10.90 29.10 CD49ahigh 7.19 15.60 CD49alow 13.20 34.70

a BALs were obtained 10 days following A/HK/X31 infection of C57BL/6 mice. Pooled samples from five mice were stained for several surface markers and proapoptotic markers as described in Materials and Methods. Using a wide lymphocyte gate, CD8ϩ cells were identified in a CD8 vs CD4 scatter plot. Ag-specific cells were identified by gating on DbNP-tetramerϩ cells. These groups were subset into CD62L, CD44, CD11a, and CD49a high- and low-expressing cells. Within these subsets, the percentage of cells staining positive for annexin V was analyzed. Results are repre-

sentative of several independent experiments and were similar in the acute phase on Downloaded from days 7, 9, and 10 postinfection.

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. http://www.jimmunol.org/

CD49aϩLFA-1highCD62LlowCD44highCD8ϩ T cells have reduced caspase 8 and annexin V in the airways but not the lymphoid organs Our results so far showed that most CD8 T cells in the airways with an activated effector surface phenotype were also CD49aϩ, and cytokineϩ cells were CD11ahigh and caspase 8 negative. Given that CD49aϩCD8ϩ T cells had also been shown to have a reduced proportion of TUNELϩ staining, we investigated the relationship by guest on September 25, 2021 between VLA-1, LFA-1 (CD11a), CD44, and CD62L expression FIGURE 3. Influenza-specific CD8 T cells in the early stages of apo- and apoptotic markers. ptosis are still able to produce cytokines. Eight days following influenza Among the total CD8 T cells in the airways, the majority of infection, cytokine-producing cells were examined in cells harvested from low low ϩ annexin V-positive cells were CD62L , CD11a , and airways of influenza-infected mice. After gating on CD8 cells in a wide low/Ϫ lymphocyte gate, cytokine production was analyzed among CD8ϩ T cells CD49a (Table I). CD44 expression did not distinguish the responding to a 6-h in vitro NP peptide (1 ␮M) stimulation. Cells annexin V-positive and -negative subsets. In contrast, among the 366–374 b ϩ ϩ ϩ were stained for apoptotic markers and surface markers as described in D NP CD8 T cells that were annexin V , the majority were Ϫ Materials and Methods. IFN-␥ and TNF-␣ producers were identified CD62Lhigh, CD11alow, and CD49alow/ (Table I), consistent with within the annexin V and caspase 8-positive and -negative populations of a more “lymphoid” profile (7). Similar results were obtained for airway (A, C, and D) and spleen (B, E, and F) cells. Cytokine producers caspase 8 and Sytox blue. ϩ ϩ were examined within caspase 8 and annexin V cells of the airways (A) The majority of the influenza-specific CD8 T cells that were and spleen (B). Insets in A and B represent caspase 8 staining during the annexin V-negative had a CD44ϩCD62LlowCD11ahighCD49aϩ intracellular cytokine assay and gating strategy for caspase 8-positive and phenotype, placing them into a highly activated subset. Because -negative cells. CD8 T cells were then gated on caspase 8Ϫ cells and further animals were sampled at day 10, near the end of the acute cellular subset into annexin V-negative and -positive cells in the airways (C and D) response, it was not clear whether the relative resistance of the and spleen (E and F), and cytokine production was examined. Numbers in ϩ quadrants represent the percentage of positive cells in that quadrant. Plots CD49a subset reflected a process occurring continually or just shown are representative of two separate experiments. during the resolution of the acute response. CD8 T cells were examined for apoptotic markers at time points immediately before (day 7) and immediately after (days 9 and 10) the peak (day 8) of airway caspase 8-negative, annexin Vϩ cells produced IFN-␥,or cellular infiltration. Although it was expected that apoptotic indi- both IFN-␥ and TNF-␣ (Fig. 3, C vs D) with an ϳ2-fold greater cators would increase toward the end of the acute response, an- proportion of IFN-␥ single producers (ϳ17%) and IFN-␥/TNF-␣ nexin V and Sytox blue percentages remained relatively stable double producers (ϳ20%) (Fig. 3D). These differences were en- from days 7 through 10 at 15–20% of the nucleoprotein (NP)- hanced among splenic effectors (Fig. 3, E vs F) where there was an specific CD8 T cells (Fig. 4A). On day 7, a somewhat lower pro- increased proportion of annexin Vϩ T cells, with ϳ50% of these portion of CD49aϩ cells stained with annexin V or Sytox blue, and cells producing cytokine, particularly both TNF-␣ and IFN-␥,in caspase 8 was clearly reduced among CD49aϩ cells compared response to NP peptide (Fig. 3F). We conclude that in this assay, with CD49aϪ cells (13 vs 23%, respectively) (Fig. 4, B and C). At influenza-specific CD8 T cells that were in the early stages of days 9 and 10, the CD49aϩDbNPϩCD8ϩ T cells had markedly apoptosis were still able to produce cytokines and were enriched reduced levels of all three apoptotic markers, particularly caspase within the annexin Vϩ subset. Though we could not distinguish 8 (Fig. 4, B and C). These differences between the CD49aϩ and 5058 VLA-1 AND TNF PROTECT FLU-SPECIFIC CD8ϩ T CELLS

FIGURE 5. For similar levels of FAS and TNFR-I, VLA-1ϩ cells present higher levels of TNFR-II than do VLA-1Ϫ cells. The phenotypes of CD49aϩ (A–C) and CD49aϪ (D–F) subsets of CD8 T cells were compared in BALs of Downloaded from 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, CD8ϩ cells were identiﬁed in a CD8 vs CD4 scatter plot and CD8ϩ cells were subset into CD49aϩ (A–C) and CD49aϪ (D–F)

cells. Within these subpopulations, staining for Fas (A and D), TNFR-I (B and http://www.jimmunol.org/ 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.

phoid organs. This is consistent with the hypothesis that the VLA-1ϩ CD8 T cells are more resistant to apoptosis when they are

within the peripheral tissues. by guest on September 25, 2021

Airway CD49aϩ CD8 T cells express high levels of TNFR-II 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 CD49aϩ subset. A number of TNFR family members were investigated. Within the total CD8ϩ population in the airways, there was no difference in Fas (CD95) or TNFR-I expression between the CD49aϩ and the CD49aϪ subsets FIGURE 4. Ag-specific CD8 T cells in the airways and lungs of influenza compared with resting spleen CD8ϩ T cells (Fig. 5, A and D, B and infected mice show similar levels of annexin V during the acute phase of the E). However, a much greater proportion of CD49aϩ CD8 T cells response but annexin V and caspase 8 are reduced within the CD49a-positive expressed TNFR-II (Ͼ80%; see Fig. 6B), with roughly twice as and -negative subsets. Lungs and BALs were obtained 7, 9, and 10 days fol- ϩ much TNFR-II expressed by the CD49a population based on lowing 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 mean fluorescence intensity (Fig. 5, C and F). This suggests a described in Materials and Methods. Using a wide lymphocyte gate, cells were mechanism by which caspase 8 and other apoptotic signals could gated on CD8ϩ and Ag-specific cells were identified by gating on DbNP- be modified (26, 27). tetramerϩ cells. The proportion of these cells that stained positive for annexin V in the airways (f) and lung tissue (Ⅺ) was evaluated (A). Cells from the High proportions of infiltrating CD8 T cells express TNFR-II in airways were further subset into CD49aϩ (B) and CD49aϪ (C) populations, the lung airways compared with the spleen ϩ ϩ and annexin V and caspase 8 cells were identified within these two subsets The differences in apoptotic markers and TNFRs between T cell Ⅺ f u on days 7 ( ),9( ), and 10 ( ). Results shown are from a single experiment populations could be explained by selection within the tissue. In- using pooled samples from three to five mice per time point to ensure that filtrating CD49a-positive and -negative CD8 T cells were identi- adequate Ag-specific cell numbers were obtained for subsetting and analysis, fied among airway and splenic populations of CD8ϩ T cells (Fig. and that staining was uniform for all three time points for comparison but are ϩ representative of three independent experiments. 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 CD49aϪ subsets were not observed in the spleen or lymph nodes expressed TNFR-II, regardless of CD49a expression. In fact, only (see Fig. 6C), reinforcing an earlier (unpublished) observation that ϳ18% of the CD49aϪ subset and 34% of the CD49aϩCD8ϩ cells TUNELϩ cells were not reduced among CD49aϩ cells in the lym- in the spleen expressed the receptor (Fig. 6B, right panels). This The Journal of Immunology 5059

was in sharp contrast to the observations made in the airways where 42% of the CD49aϪ cells and 81% of the CD49aϩ cells expressed TNFR-II (Fig. 6B, left panels). Because the spleen contains many naive and resting T cells, the CD44highCD62Llow subsets in both tissues were examined. Although a high proportion of the activated CD44highCD62Llow CD8 T cells in the airways expressed TNFR-II (70% of the CD49aϪ cells and 80% of the CD49aϩ cells), the proportions were again reduced in the spleen with little difference between the CD49aϩ and CD49aϪ 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 CD49aϩ influenza-specific cells had reduced caspase 8 compared with CD49aϪ 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. Downloaded from VLA-1ϩTNFR-IIϩ 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 http://www.jimmunol.org/ 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 CD8ϩ T cells (Fig. 7, A and B) or Ag-specific (Db-NP-tetramerϩ) CD8ϩ T cells in the airways (Fig. 7C) were further subdivided into populations based on TNFR-II and CD49a (CD49aϩ, TNFR-IIϩ vs CD49aϪ, TNFR-IIϪ). The highest and lowest proportions of cells exhibiting annexin V and caspase 8 activity occurred, respectively, among the CD49aϪ, TNFR-IIϪ by guest on September 25, 2021 and CD49aϩ, TNFR-IIϩ subsets (36.2 Ϯ 1.95% vs 23.56 Ϯ 3.72%; p ϭ 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 8ϩ cells was higher in the CD49aϪ, TNFR-IIϪ subset compared to CD49aϩ TNFR-IIϩ cells (20.73 Ϯ 3.35% compared with 4.48 Ϯ 1.38%; p ϭ 0.011) (Fig. 7B). This was even more striking among the Ag-specific population where the proportions of total annexin Vϩ and caspase 8ϩ cells were, respectively, almost 3-fold ( p Ͻ 0.01) and 13-fold ( p Ͻ 0.001) lower in the CD49aϩ, TNFR-IIϩ 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 FIGURE 6. For similar levels of Fas expression, infiltrating VLA-1ϩ CD8 T cells from induction of apoptosis in the infected airways.

airway CD8 T cells express high levels of TNFR-II and have reduced ϩ caspase 8 compared with VLA-1Ϫ cells and spleen cells. Cells were ob- VLA-1 CD8 T cells have enhanced access to type IV collagen tained from BAL and spleen 8 days postinfluenza infection. A, CD8ϩ cells in the airways were identified among lymphocytes in each sample with anti-CD8-PE and Although we have shown that CD8 T cells in the lung are prefer- anti-CD49a-Alexa 488 staining was examined among these cells. B, entially in close proximity to collagen IV (9, 28), their proximity TNFR-II (anti-CD120b-PE) and Fas (anti-CD95-allophycocyanin) expres- ϩ to collagen in the spleen is unknown. Lungs and spleens from sion was analyzed within the CD49a– (B; upper panels) and CD49a (B; lower panels) subsets of CD8ϩ lymphocytes found in airways (B; left pan- acutely infected animals were removed, and frozen sections (5–10 ␮ els) and spleens (B; right panels) of influenza-infected mice. C, Active m thick) were stained with fluorescence-labeled rat anti-mouse caspase 8 levels in the CD49aϪ and CD49aϩ subsets of airway and CD8a and rabbit anti-mouse type IV collagen. Although the lung spleen DbNP tetramerϩCD8ϩ lymphocytes were compared. This was tissue contained abundant type IV collagen around the airways, performed following staining with anti-CD8-PE, anti-CD49a-Pacific blood vessels, and alveoli (Fig. 8A), in the spleen, type IV collagen Orange, and caspase 8 (FITC-IETD-FMK). Numbers in quadrants rep- could be identified only in the structural portion of the red pulp and resent percentage of positive cells in that quadrant and those in paren- white pulp, most likely corresponding to the basement membranes theses represent the percentage of cells positive for active caspase 8 Ϫ ϩ of the marginal sinuses and central arterioles (Fig. 8B) (29, 30). within the CD49a and CD49a subsets. Plots shown represent four CD8ϩ cells in the lungs were found in close proximity to type IV separate experiments. collagen, in particular around the blood vessels and the infected airways (Fig. 8A), while in the spleen, most CD8ϩ cells were 5060 VLA-1 AND TNF PROTECT FLU-SPECIFIC CD8ϩ T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 8. Histological examination of the distribution of CD8ϩ 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 Photo- Shop and color brightness was adjusted. Images were taken at ϫ400 and

are representative of several individual sections. The calibration mark at by guest on September 25, 2021 the bottom left of each image represents 100 ␮m. A, alveoli; AW, airway; BV, blood vessel; CA, central arteriole; MZ, marginal zones; PALS, peri- FIGURE 7. Reduced caspase 8 and annexin V among VLA-1 and arteriolar lymphocyte sheaths. 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 an- in the presence or absence of the caspase 8-triggering, apoptosis- ϩ nexin Vϩcaspase 8ϩ cells in each quadrant of (A) corresponding to the inducing anti-Fas Ab Jo2, and/or TNF-␣. Apoptotic CD8 cells alternate combinations of VLA-1 and TNFR-II; x-axis values indicate each were identified using annexin V and 7AAD and examined in re- quadrant in a clockwise manner starting in the upper left. Annexin V and lation to CD49a. After4hinculture, the CD49aϩ CD8 T cells in Ϫ Ϫ active caspase 8 staining within the VLA-1 , TNFR-II (Ⅺ), and VLA- medium alone had the lowest annexin V/7AADϩ profile (33.43 Ϯ ϩ ϩ f ϩ b ϩ ϩ 1 , TNFR-II ( ) subsets of total CD8 (B) and D -NP-tetramer CD8 0.90%) (Fig. 9A, lane 1) and this was set as the baseline and results ϭ (C) cells (n 3). Numbers in quadrants represent percentage of positive were calculated as percentages above this baseline. In the presence .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء ;cells in that quadrant. Results are means Ϯ SEM of Jo2 Ab, significant apoptosis was induced above baseline in both CD49aϩ (43.28 Ϯ 4.01%; p Ͻ 0.01) and CD49aϪ (68.56 Ϯ 4.16%; p Ͻ 0.01) cells (Fig. 9, A and B, lane 2). In the presence found in large clusters near periarteriolar lymphocyte sheaths in of type IV collagen alone, Jo2-induced apoptosis was significantly which very little type IV collagen could be found, with a few inhibited in CD49aϩ cells compared with CD49aϪ cells (Fig. 9, A ϩ CD8 cells scattered in the structural portion of the tissue where and B, lane 3), suggesting that binding to type IV collagen through type IV collagen was more abundant (Fig. 8B). Taken together, VLA-1 can partially inhibit apoptosis induction in VLA-1ϩ cells. these findings show differences in access to type IV collagen be- In the presence of TNF-␣ alone, Jo2-induced apoptosis was inhib- tween the lung and spleen and may explain the observed differ- ited by 27% ( p Ͻ 0.01) in the CD49aϩ subset but no effect was ences in apoptosis among CD8 T cell subsets. observed in the CD49aϪ subset (Fig. 9, A and B, lane 4), suggesting that TNF-␣ signaling can contribute to the inhibition of apo- VLA-1-expressing cells resist apoptosis in the presence of type ptosis in the CD49aϩ subset that expresses high levels of TNFR-II ␣ IV collagen and TNF- in vitro compared with CD49aϪ cells that express TNFR-II to a much In light of these findings, we sought to investigate whether the lesser extent in proportion and level. Strikingly, when the cells phenotype observed directly ex vivo could be reproduced in a con- were cultured in the presence of type IV collagen and TNF-␣, trolled in vitro environment. To do so, cells were isolated from the Jo2-induced apoptosis was blocked by 88.7% ( p Ͻ 0.01) in the airways of acutely infected mice 8 days postinfection and cultured CD49aϩ subset with no significant effect under these conditions in in presence or absence of type IV collagen in 96-well plates, and the CD49aϪ group (Fig. 9, A and B, lane 5), suggesting that type The Journal of Immunology 5061 Downloaded from http://www.jimmunol.org/

FIGURE 9. Type IV collagen and TNF-␣ act in synergy to protect VLA-1ϩ 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 ␮g/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 Vϩ, 7AADϩ. The percentage of apoptotic cells was compared in CD49aϩ (A and C) and CD49aϪ (B and D) cells in each well. In a separate experiment to determine that the effect of TNF-␣ was ␮ ␣ ϩ Ϫ

through TNFR-II, a TNFR-II-blocking Ab (10 g/ml) was added before TNF- and Jo2 in and apoptosis was evaluated in CD49a and CD49a cells. by guest on September 25, 2021 Results in A and B are expressed as percentage above baseline apoptosis for CD49aϩ cells which was 33.43 Ϯ 0.90% annexin Vϩ, 7AADϩ cells in a 4-h incubation without treatment. For C and D, the baseline was 26.90 Ϯ 0.90% (data not shown). Results are means Ϯ SD (n ϭ 5 in two separate experiments); .p Ͻ 0.01 compared with induced control ,ء

IV collagen binding through VLA-1 and TNF-␣ signaling can syn- In addition to trafficking, one of the mechanisms that regulate ergize to potently inhibit apoptosis in cells that express both the the number of T cells present in a tissue at any given time is the VLA-1 integrin and TNF-␣ receptors. In a separate set of exper- ability of the T cells to be retained. Retention of T cells in turn can iments, the inclusion of a TNFR-II-blocking Ab abrogated the pro- reflect both survival and failure to migrate out via efferent lym- tective effect of TNF-␣ for the CD49aϩ CD8 T cells, but had no phatics. One prevailing view is that resolution of acute antiviral effect on the CD49aϪ subset (Fig. 9, C and D, lanes 2 vs 3), responses is mediated by apoptotic elimination of activated T cells confirming that the TNF-␣ effect signaled through TNFR-II which after virus has been cleared. However, we found that the propor- is highly expressed by VLA-1ϩ cells. In the experiments, neither tion of T cells expressing apoptotic markers was relatively similar TNF-␣ nor the blocking Ab alone had an effect on the baseline. at the expansion, peak, and resolution phases of the response. This Taken together, these data suggest that signals through VLA-1 and suggested that apoptosis is a continual process and not just a fea- TNFR-II can inhibit apoptotic signals among highly activated ef- ture of the resolution phase. fector CD8 T cells in the airways. TNF-␣ receptor signaling is regarded as the primary means by which the CD8 T cell response is controlled (11). However, de- Discussion pending on the model and the culture system used, conflicting Despite our knowledge of memory CD8 T cell characteristics, less results have been observed. Initial reports suggested that normal T is known about CD8 T cell subpopulation dynamics and regulation cell blasts were sensitive to TNF-␣ cytotoxicity and that this effect ␣ ␤ during the acute immune response. The 1 1 collagen-binding in- was mediated primarily through TNFR-II (11). Based on the latter, tegrin VLA-1 is an important feature of memory CD8 T cells that a recent report has suggested that TNFR-II-mediated editing of the allows them to be maintained in extralymphoid sites (9) and reg- influenza-specific CD8 T cell response functions to limit the num- ulates the precise localization of CD4 and CD8 T cells within the ber of effectors that have localized to site of infection in the lung collagen-rich structure of the lung (28). In this present study, we (but not the spleen) (31). However, the limited TNF-␣-mediated focused on the function of VLA-1 during the acute phase of the editing process described was dependent on the dose of peptide infection. Although it had been shown that VLA-1ϩCD8ϩ T cells used and could not be a major factor involved in the return to lower increase in the lung over the course of the acute infection, it was CD8 T cell numbers during the resolution phase (31). In contrast, not clear whether this reflected preferential recruitment or it has been suggested that TNFR-II (p75) functions as a costimu- retention. lator for Ag-driven T cell responses in vivo (32). In a Listeria 5062 VLA-1 AND TNF PROTECT FLU-SPECIFIC CD8ϩ T CELLS monocytogenes model, for example, TNFR-II regulated the thresh- could be shared among lymphocytes and other cell types, it re- old for clonal expansion of Ag-specific CD4 and CD8 T cells, and mains to be determined which, if any, of the pathways are shared. the CD8 T cells depended on TNFR-II for survival during the early A precedent exists for T cells in that VLA-2-mediated ligation of phase of the response (32). In addition, the resulting memory pools collagen I was shown to inhibit activation-induced death of Jurkat were diminished in TNFR-IIϪ/Ϫ mice. Similarly, TNFR-associ- T cells (14), though in that study VLA-1 was not observed to have ated factor 2 (TRAF2) adaptor protein dominant-negative mice an effect. The interaction of VLA-1 and type IV collagen may show profound defects in T cell responses and poor generation of initiate signaling through focal adhesion kinase, which then acti- CD8 T cell memory (33). In humans, naive and memory subsets of vates the PI3K/AKT pathway leading to activation of ERK/MAPK CD8 T cells exhibit differential sensitivity to TNF-␣-induced ap- and NF-␬B (14, 44). MAPK/ERK signaling, in turn, can override optosis such that effector/memory cells were resistant to TNF-␣- apoptotic signals from Fas, TNF, and TRAIL receptors (45, 46) induced apoptosis compared with naive and central memory CD8 reinforcing the hypothesis that signals provided by integrins mod- T cells (34). ify TNFR signals. Our present results may help explain some of the discrepancies Why did we observe these antiapoptotic effects for T cells in the and identify a subpopulation of acutely activated T cells that are airways and not the spleen? VLA-1 is the major receptor for type not only resistant to TNF-␣-mediated apoptosis, but also signal IV collagen, which is abundant in the lung and found in the base- through TNFR-II to make the cells resistant to other proapoptotic ment membranes of the endothelium and epithelium (28, 47). In signals. The ability of TNF-␣ to promote survival or apoptosis lymphoid tissues, fibroblastic reticular cells envelop collagen depends on both the dominant receptor expressed on the cell sur- fibrils, and that these collagen fibrils are located in the interfol- face, as well as the intracellular context through which the signals licular zones (48, 49). Thus, the splenic tissue is much less abun- Downloaded from are interpreted (20, 35–39). In our experiments, exposure to dant in type IV collagen, with most of the collagen located in the TNF-␣ made the CD49aϩTNFR-IIϩ CD8 T cells resistant to Fas- basement membranes of the marginal sinuses and the central induced apoptosis, while it had little effect on the CD49aϪ subset, arterioles. despite the fact that a proportion of these cells also expressed We therefore propose a mechanism to protect activated effector TNFR-II. This can be explained possibly by the lower expression CD8 T cells in the lung during acute infection. Although both Ϫ ϩ Ϫ low high levels of TNFR-II on the CD49a cells, but it could also suggest CD49a and CD49a (and CD62L and CD62L ) CD8 T http://www.jimmunol.org/ modified signaling pathways for TNF-␣ among the CD49aϩ cells. cells can reach the airways; the CD49aϩ cells have a survival TNFR-I contains death domains (DD) that trimerize upon liga- advantage conferred by the ability to bind collagen and signal tion of TNF-␣ resulting in adaptor proteins TNFR-associated DD through VLA-1. The exertion of effector function in the form of and Fas-associated DD (24). This complex further interacts with TNF-␣ secretion potentiates this effect by promoting antiapoptotic procaspase 8 to form the death-inducing signal complex (DISC) signals through NF-␬B. This protects the effector CD8 T cells at and rapidly produces active caspase 8 resulting in the initiation of the site of infection, where they are likely to express and secrete the apoptotic pathway leading to the activation of downstream ef- both TNF-␣ and FasL. This mechanism results in a selection pro- fector caspases. This pathway is kept in check by several gene cess that is restricted to anatomical areas where collagen IV is products such as FLIP that inhibits the association of procaspase 8 easily accessible, explaining some of the differences observed by guest on September 25, 2021 to the DISC (40, 41) and can also activate the NF-␬B and ERK between the lung and the spleen. It could also function to limit pathways through adaptor proteins such as TRAF-1,-2, and -3, serine/ the accumulation of effector T cells in noninfected tissues threonine kinase receptor-interacting protein (RIP), and Raf (42). where TNF-␣ production would not be triggered. In addition to TNFR-II, which lacks death domains, stimulates the activation of the acute phase, this mechanism may have implications for the MAPKs and NF-␬B through TRAF2 and RIP leading to the inhibition formation of memory given the relationship of TRAF2 in sec- of apoptosis in mice and humans (43) through induction and ex- ondary influenza infections (33) and our observations that pression of several potent antiapoptotic genes including FLIP, VLA-1 deficiency impairs secondary influenza immunity (9). TRAF-1, and TRAF-2 (35–38). Thus, the intracellular balance of adaptor proteins such as TRAF1 and TRAF2 can determine Acknowledgments whether TNFR ligation leads to recruitment of the DISC to the We thank Dr. Deborah Fowell for use of fluorescence microscopy receptor, or to activation of NF-␬B (25). In this regard, the sec- equipment. ondary response to influenza was vastly impaired in TRAF2 dominant-negative mice compared with wild-type mice (33). 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