Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (TCM). In this study, using relB−/− mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP366–374) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.

Influenza virus infects humans by gaining entry through the airway. Vaccination with formalin-inactivated, whole virus influenza vaccines protect against infection by inducing Abs that intercept the virus at the portal of entry (1). Abs against the virus hemagglutinin Ag are involved in protection against infection, whereas Abs against the neuraminidase Ag decrease the amount of virus released from infected cells. However, because of antigenic changes in the virus, vaccines need to be reformulated periodically to compensate for antigenic shift and drift (2). In contrast, protection against disease is mediated by MHC class I-restricted CD8 T lymphocytes that eliminate virus-infected cells. CTLs are directed against peptides of the core nucleoprotein (NP)3 or the matrix (M1) protein on the inner side of the virus envelope (3, 4, 5). These Ags are more resistant to antigenic shift and drift and are generally conserved among different virus types. There exists ample epidemiological evidence that T cell immunity is important in protection against disease in humans (6, 7) and that protection is lost by antigenic drift that causes virus escape (8). However, whereas Abs protect against infection and CTLs protect against disease, upon secondary virus challenge Abs can only neutralize viruses of the same type whereas CTLs may lyse cells infected with different virus types (9, 10).

In mice as in humans, influenza virus infection is an inflammatory disease of the airway (11). CTL responses are coordinately associated with attenuation of symptoms and protection from disease (12, 13). During natural infection the T cell response is initiated in the draining lymph nodes (LNs) of the airway tract (cervical and mediastinal) where dendritic cells (DCs) and macrophages traffic from inflamed lung tissue and present viral Ags to precursor T lymphocytes, causing their clonal expansion. This peaks at around days 7–10 (14) and then contracts while recovery from infection occurs and T cell memory is gradually generated and thereafter maintained. Memory T lymphocytes enable mice to mount a rapid and more potent response upon secondary virus challenge. The anamnestic response contains disease and prevents death. Presently, however, vaccination directed at inducing influenza virus-specific CD8 T cell responses is not an option for humans and is used experimentally to understand the pathodynamics of infection and the mechanisms of protection.

Vaccination with B lymphocytes genetically programmed to present a dominant CD8 T cell epitope of the influenza virus NP induces potent CD8-specific T cell responses in vivo (15) and provides complete protection against lethal challenge with influenza A virus (15) even if injected in low numbers (300/mouse) (16). Protection against lethal virus challenge in naive C57BL/6 mice is also obtained by the adoptive transfer of immune L-selectin (CD62Lhigh) CD8 T cells (16), suggesting that central memory CD8 T cells (TCM) are the correlate of protection in this disease. This is consistent with the notion that CD8 TCM cells possess greater Ag-driven proliferation potential (17) and greater ability to kill in vivo (18) compared with effector memory CD8 T cells and that TCM cells are the correlate of protection in a number of diseases (19).

In this study, to further understand the mechanism of protection against lethal influenza A virus infection by vaccine-induced CD8 TCM cells, we undertook studies on the role of bone marrow (BM)-derived DCs. Herein, we investigated the ability of memory CD8 T cells to confer protection against lethal influenza A virus infection in DC-compromised relB BM chimeras (BMC). We found that lack of functional BM-derived DCs in LNs draining the airway tract abrogates protection by memory CD8 T cells. We found that defective presentation of viral Ag by DCs to CD8 TCM cells in the draining LNs of the airway tract and poor homing of memory T cells have a negative effect on the protective memory T cell response. We conclude that re-presentation of Ag to memory T cells in draining LNs is a necessary event for protection against disease induced by influenza A virus to occur.

Eight- to 10-wk-old C57BL/6 (CD45.2+) mice were purchased from The Jackson Laboratory. Homozygous relB−/− mice were bred in the animal facility of the University of California San Diego (La Jolla, CA). BMCs were generated by injecting i.v. 5 × 106 BM cells from relB−/− mice into lethally irradiated (1100 rad) heterozygous relB+/− (−/− → +/−) or C57BL/6 (−/− → +/+) mice. As control, 5 × 106 BM cells from C57BL/6 mice were injected into lethally irradiated C57BL/6 (+/+ → +/+) mice. Mice were used 5–6 wk after BM transfer. OVA TCR OT-I CD45.1+RAG−/− transgenic mice were bred in the animal facility of the University of California San Diego. OVA TCR OT-II transgenic mice were a gift from Dr. M. Croft (La Jolla Institute for Allergy and Immunology, La Jolla, CA).

Plasmid γ1NV2NP3 was engineered as described (20). To allow for detection of transgenic cells by FACS, the gene for enhanced GFP (EGFP) was inserted at the C terminus of the γ1 constant region. Plasmid DNA were purified using a Qiagen MegaPrep kit (Qiagen) and stored at −20°C until use. OVA (grade VII) was purchased from Sigma-Aldrich. FITC-conjugated OVA was purchased from Molecular Probes. A/PR8/34 (A/Puerto Rico/8/34 strain) influenza virus (2 × 108 PFU/ml) was a gift from Dr. A. Garcia-Sastre (Mount Sinai School of Medicine, New York, NY). A/HKx31-OVA (H3N2) influenza virus (107 EID50/ml; EID50 is 50% egg infectious dose) and the A/HKx31 (H3N2) control influenza virus were gifts from Dr. P. G. Thomas (St. Jude Children’s Research Hospital, Memphis, TN).

For protection experiments, mice were challenged intranasally (i.n.) with 50 μl of A/PR8/34 virus at 4 × 106 PFU/ml (lethal dose). Mice survival was monitored daily. Unprotected mice die between days 10 and 12. For experiments shown in Figs. 2 and 7, mice were challenged i.n. with 30 μl of A/HKx31-OVA virus at 3.33 × 106 EID50/ml (sublethal dose). Mice were sacrificed on days 1 and 3 after virus challenge.

Spontaneous transgenesis was performed as described (15). Briefly, spleen cells were harvested, washed with PBS, and incubated with 25 μg of plasmid DNA for 1 h at 37°C together with the pMACS Kk plasmid (Miltenyi Biotec) coding for a truncated mouse H-2 Kk molecule as a selectable cell surface marker. After overnight incubation, transgenic cells were magnetically sorted and analyzed by flow cytometry for enhanced GFP expression on a FACScalibur apparatus (BD Biosciences). Mice were immunized by single injection via the tail vein with a suspension of spleen lymphocytes containing 5,000 transgenic B lymphocytes (determined by FACS analysis). OVA and FITC-conjugated OVA were diluted in sterile PBS to a final concentration of 10 mg/ml. Mice were lightly anesthetized with isoflurane (Abbott Laboratories) and administered i.n. with 50 μl of Ag solution.

Airway and nonairway LNs, pooled from 2–6 mice per group depending on the experiment, were harvested 24 h after the administration of FITC-OVA or 48 h after the administration OVA. LNs were digested in collagenase D (1 mg/ml; Roche Molecular Biochemicals) and DNase (0.02 mg/ml, grade II bovine pancreatic; Boehringer) for 30 min at 37°C. LNs were then minced and passed through a cell strainer. Due to the photosensitivity of the FITC material, LNs from FITC-OVA-treated mice were protected from direct light throughout the manipulation. Positive selection of DCs from LNs was done by magnetic cell sorting after incubation with anti-CD11c magnetic beads (Miltenyi Biotech) according to the manufacturer’s instructions. Purified DCs from FITC-OVA-administered mice were stained with biotin-conjugated mAb against mouse CD11c (clone HL3) (BD Pharmingen). After 20–30 min at 4°C, cells were washed with PBS containing 0.5% BSA and 0.05% NaN3, and stained with CyChrome-streptavidin and PE-conjugated anti-IAb (clone AF6–120.1). Cells were analyzed by flow cytometry on a FACSCalibur apparatus (Becton Dickinson) and gates were set to select for viable DCs.

Airway and nonairway LN DCs pooled from 2–6 mice per group depending on the experiment were cultured (0–105/well) with 2 × 105 OVA-specific CD8 T cells from OT-I mice or with 2 × 105 OVA-specific CD4 T cells from OT-II mice. OT-I and OT-II splenocytes were depleted of APCs by using a mixture of mAbs plus rabbit complement. The mAbs M5114 anti-I-A, CA4 anti-class II, RA3.6.B2 anti-B220, PK136 anti-NK, M1/70 anti-CD11b, 3.155 anti-CD8, and GK1.5 anti-CD4 were added to OT-II and OT-I splenocytes for CD8 and CD4 depletion, respectively. [3H]Thymidine was added at 1 μCi/well and the cells were incubated for 16–18 h at 37°C. Cells were harvested onto glass fiber filter mats using a TomTec cell harvester and the radioactivity was measured in a liquid scintillation counter (Betaplate; Wallac). Results are expressed as means ± SD of the counts per minute of triplicate cultures. Supernatants for cytokine detection were harvested after 40 h and stored at −20°C. IL-2 and IFN-γ were measured using the Opt EIA mouse set (BD Pharmingen).

For protection experiments, C57BL/6 mice were immunized i.v. with 5 × 103 B lymphocytes transgenic for plasmid γ1NV2NP3. Three weeks later mice were sacrificed and single cell suspensions were prepared from the spleens. CD8 T cells were isolated by negative selection using a mixture of biotin-conjugated mAbs as the primary labeling reagent and anti-biotin mAbs conjugated to microbeads the as the secondary labeling reagent (CD8 T cell isolation kit; Miltenyi Biotec). CD62Lhigh (TCM) cells were sorted from the enriched CD8 T cell fraction using anti-CD62L magnetic beads (Miltenyi Biotec). Cells were then washed twice in PBS and 1 × 106 CD62Lhigh CD8 T cells were injected i.v. into relB BMC, C57BL/6 BMC, and C57BL/6 recipients. Mice were lethally challenged 48 h later. As a control, CD62Lhigh CD8 T cells were sorted from C57BL/6 naive mice and injected i.v. into C57BL/6 recipients. In DC reconstitution experiments (see Table III), DCs were prepared from the BM of C57BL/6 mice as described previously (21). Briefly, cells were cultured in Iscove’s complete medium supplemented with 10% FCS (HyClone), recombinant mouse GM-CSF (1000 U/ml; BD Pharmingen), and recombinant mouse IL-4 (4 ng/ml; R&D Systems). On day 7 of culture nonadherent cells were washed, resuspended at 5 × 106 cells/ml, and pulsed with the NP366 peptide (5 μg/ml) for 1 h at 37°C. Cells were then washed and injected i.v. (5 × 105 DCs per mouse) into relB BMC. Mice were subsequently challenged with a lethal dose of A/PR8/34 virus.

Table III.

The adoptive transfer of CD8 TCM (CD62Lhigh) cells protects relB−/− BMC injected with peptide-pulsed DCsa

Transfer of CD62Lhigh T cells (1 × 106/mouse)DC ReconstitutionbVirus Challenge i.n. (4 × 106 PFU/ml)Survivalc
C57BL/6 BMC + − + 4/4 
relB BMC + + 5/5 
C57BL/6 + − + 0/4 
Transfer of CD62Lhigh T cells (1 × 106/mouse)DC ReconstitutionbVirus Challenge i.n. (4 × 106 PFU/ml)Survivalc
C57BL/6 BMC + − + 4/4 
relB BMC + + 5/5 
C57BL/6 + − + 0/4 
a

Mice were adoptively transferred with 106 CD62Lhigh CD8 T cells from immune C57Bl/6 mice.

b

BM-derived C57BL/6 DCs pulsed with NP366 peptide were injected 24 h after adoptive transfer of memory CD8 T cells and were allowed to park in vivo 10 days prior to virus challenge.

c

Survival was recorded over a period of 3 wk.

For adoptive transfer experiments, spleen and LN CD8 T cells were harvested from OT-I mice. Spleen and LN cell suspension was prepared and 107 cells per mouse were injected i.v. into C57BL/6 mice (7–10 mice were injected per experiment). Twenty-four hours later mice were immunized i.p. with the OVA peptide SIINFEKL (100 μg/mouse) together with the helper peptide TPPAYRPPNAPIL (60 μg/mouse) of the hepatitis B virus core (HBVc) Ag in IFA (Difco). Three weeks later, mice were sacrificed and a single cell suspension was prepared from the spleens and LNs. CD8 T cells were isolated by negative selection using a CD8 T cell isolation kit (Miltenyi Biotec) and EasySep CD8 T cell enrichment kit (StemCell Technologies). Enriched CD8 cells were stained using anti-CD8 (PerCP) (clone 53-6.7), anti-CD45.1 (FITC) (clone A20), anti-CD44 (PE) (clone IM7), anti-CD62L (PE) (clone MEL-14), and anti-CD25 (PE) (clone PC61) mAbs (BD Pharmingen). Enriched CD8 T cells were labeled with 10 μM CFSE (Molecular Probes) for 10 min at room temperature. Approximately 1.5 × 104 CD8/CD45.1 TCM cells were injected i.v. into relB BMC or C57BL/6 recipients. Mice were infected 24 h later with 30 μl of A/HKx31-OVA virus 3.33 × 106 EID50/ml.

Isolated airway LNs were harvested on days 1 and 3 after infection. Cells were stained with anti-CD8 PerCP, anti-CD45.1 allophycocyanin (clone A20), and anti-SIINFEKL/Kb Ab 25-D1.1b. DC number and phenotype were assessed by staining with mAb anti-CD11c (clone HL3), CD86 (clone GL-1), and MHC class II (clone AF6-120.1). For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-CD8 PerCP, anti-CD45.1 allophycocyanin, and anti-IFN-γ PE (clone XMG1.2).

Tissue was processed as previously described (22). Briefly, LNs and spleens were removed, embedded in Tissue-Tek (Sakura Finetek), snap frozen in precooled isopentane, and stored at −80°C. Cryostat sections (10 μm thick) were fixed in acetone for 10 min, rehydrated in PBS, and treated with avidin/biotin blocking reagent (Vector Laboratories) and anti-FcR (clone 2.4G2) to block nonspecific binding. Sections were processed in a humidified chamber using the Tyramide Signal Amplification (TSA) systems according to the manufacturer’s instructions (PerkinElmer) using biotin-labeled 25-D1.16, HRP-labeled streptavidin, biotin-labeled tyramide and streptavidin-Cy3, and FITC-labeled anti-B220 (eBioscience). The slides were then washed three times in PBS and mounted in Vectashield (Vector Laboratories). Digitized images were acquired using a Nikon imaging station and processed with Photoshop Software (Adobe Systems).

Homozygous relB−/− mice have an atrophic thymic medulla, possess no LNs, and lack BM-derived DCs (23), although they possess a population of CD8α+ lymphoid DCs in the spleen (24). BMCs generated by transferring homozygous (−/−) relB BM cells into lethally irradiated (1100 rad) hemizygous (+/−) relB recipients carry the same DC defect as relB−/− mice but have a longer life span (25). Notably, in the absence of the severe inflammation characteristic of homozygous mice, BM DCs from relB−/− mice are able to populate tissues when a normal radioresistant infrastructure already exists within the tissue (26). Although in relB−/− mice spleen DCs are unable to prime T cells (27, 28), we previously showed that relB BMCs immunized with transgenic B lymphocytes as APCs prime CD4 and CD8 T cells (15). Thus, this model is ideally suited to investigate the role of BM-derived DCs in the mechanism of protection against lethal influenza A virus infection.

In this study, we asked the question as to whether memory CD8 T cells induced by immunization with transgenic B lymphocytes expressing the dominant CTL epitope of the virus nucleoprotein (NP366) (4) could protect relB BMC from lethal virus challenge. C57BL/6 BMCs served as a control for the effects of irradiation and cell reconstitution. We found that while transgenic B lymphocyte-immunized C57BL/6 BMCs were protected, none of the relB BMC or naive C56BL/6 mice used as a control survived the lethal virus challenge (Table I). Notably, time to death in relB BMC mice was shorter than that in unprotected C57BL/6 mice used as controls (7 days vs 10 days). This prompted studies on the role of BM-derived DCs in protection mediated by memory CD8 T cells against the influenza A virus.

Table I.

Immunized relB−/− mice are not protected against lethal challenge with A/PR8/34 influenza virusa

Virus Challenge i.n. (4 × 106 PFU/ml)SurvivalTime to Death (Days)
C57BL/6 BMC + 4/4  
relB BMC + 0/7 7 ± 0.7 
C57BL/6 + 4/4 10 ± 0.7 
Virus Challenge i.n. (4 × 106 PFU/ml)SurvivalTime to Death (Days)
C57BL/6 BMC + 4/4  
relB BMC + 0/7 7 ± 0.7 
C57BL/6 + 4/4 10 ± 0.7 
a

Mice were vaccinated with 5 × 103 transgenic B lymphocytes expressing the NP366 CTL epitope. Lethal challenge was given 21 days after priming.

Previously, we showed that in this murine model of infection CD8 TCM cells are the correlate of protection from disease and death (16). We also demonstrated that CD4 and CD8 T cell responses can be induced in relB BMCs by vaccination with Ag-presenting transgenic B lymphocytes (15). Therefore, we reasoned that lack of protection in immunized relB BMCs could result from a defective reactivation of memory CD8 T cells in the LNs draining the lung rather than an impaired T cell response. To test this hypothesis, CD62Lhigh CD8 memory T cells from immune C57BL/6 mice were adoptively transferred in relB BMCs or in C57BL/6 BMCs as a control (Fig. 1). Consistent with our previous data (16), C57BL/6 BMCs given immune CD62Lhigh CD8 T cells were fully protected (4/4) whereas none (0/6) of the relB BMC survived the virus challenge (Table II). To verify that protection was mediated only by adoptively transferred memory T cells and not by naive T cells cotransferred within the memory T cell pool, CD62Lhigh CD8 T cells from naive mice were injected into C57BL/6 recipients. After lethal virus challenge, none of the mice that had received CD62Lhigh CD8 naive T cells were protected (Table II). This is consistent with data from Cerwenka et al. (29) who showed that the transfer of as many as 107 naive hemagglutinin TCR Tg CD8 T cells failed to protect mice. Furthermore, it raised the possibility that adoptively transferred memory CD8 T cells failed to protect due to inadequate reactivation by relB−/− DCs in the draining LNs. Finally, to corroborate the idea that protection requires re-presentation of Ag by BM-derived DCs, we sought to rescue the inability of relB BMCs to resist lethal virus challenge upon adoptive transfer of TCM cells by injecting 5 × 105 C57BL/6 BM-derived DCs pulsed with the NP366 peptide before virus challenge. As indicated in Table III, relB BMCs given both TCM cells and NP366 peptide-pulsed competent DCs were protected from lethal A/PR8/34 virus challenge.

FIGURE 1.

Experimental design of the adoptive transfer of memory CD62Lhigh CD8 T cells in BMC mice. Memory CD62Lhigh CD8 T cells were purified from spleens of C57BL/6 mice immunized with 5 × 103 B lymphocytes transgenic for γ1NV2NP3. The purity of the sorted populations was determined by FACS analysis. Purified CD62Lhigh memory CD8 T cells were transferred into naive relB BMC and C57BL/6 BMC (1 × 106 cells/mouse). After 48 h mice were challenged with a lethal dose of A/PR8/34 virus and subsequently monitored daily for 3 wk after challenge. TgB cells, Transgenic B cells.

FIGURE 1.

Experimental design of the adoptive transfer of memory CD62Lhigh CD8 T cells in BMC mice. Memory CD62Lhigh CD8 T cells were purified from spleens of C57BL/6 mice immunized with 5 × 103 B lymphocytes transgenic for γ1NV2NP3. The purity of the sorted populations was determined by FACS analysis. Purified CD62Lhigh memory CD8 T cells were transferred into naive relB BMC and C57BL/6 BMC (1 × 106 cells/mouse). After 48 h mice were challenged with a lethal dose of A/PR8/34 virus and subsequently monitored daily for 3 wk after challenge. TgB cells, Transgenic B cells.

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Table II.

The adoptive transfer of CD8 TCM (CD62Lhigh) cells fails to protect relB−/− BMCa

Transfer of CD62Lhigh T CellsVirus Challenge i.n. (4 × 106 PFU/ml)aSurvivalb
Immune (1 × 106/mouse)Naive (1 × 106/mouse)
C57BL/6 − − 0/2 
C57BL/6 BMC − 4/4 
relB−/− BMC − 0/6 
C57BL/6 − 0/5 
Transfer of CD62Lhigh T CellsVirus Challenge i.n. (4 × 106 PFU/ml)aSurvivalb
Immune (1 × 106/mouse)Naive (1 × 106/mouse)
C57BL/6 − − 0/2 
C57BL/6 BMC − 4/4 
relB−/− BMC − 0/6 
C57BL/6 − 0/5 
a

Lethal challenge was given 48 h after the adoptive transfer of 106 CD62Lhigh CD8 T cells from immune or naive C57BL/6 mice.

b

Survival was recorded over a period of 3 wk.

In light of the above results we decided to test the possibility that the reactivation of memory CD8 T cells by DCs in the draining LNs is necessary for protection. To test this hypothesis, new experiments were designed in which memory CD8 T cells generated in DC-competent mice were adoptively transferred into relB BMC. To enhance the resolution of the experiment, we used CD8 T cells TCR transgenic for a MHC class I-restricted epitope (SIINFEKL) of OVA and the recombinant A/HKx31-OVA influenza virus that codes for OVA (30) so that MHC-restricted presentation of SIINFEKL could be easily monitored in the context of viral infection. The experimental design is depicted in Fig. 2. A total of 8 × 106 CD8 T cells that contained ∼9.6 × 104 memory OT-I cells (0.12%; Fig. 2inset) as estimated from a phenotypic analysis of CD45.1+ cells (Fig. 2inset) were CFSE-labeled and immediately injected into three relB BMC or three C57BL/6 recipients (for a total of 1.5 × 104 memory T cells per mouse). The experiment was performed twice. Twenty-four hours after adoptive transfer, mice were infected i.n. with a sublethal dose (3.3 × 106 EID50/ml) of A/HKx31-OVA influenza virus. Three days after virus challenge mice were sacrificed and the draining LNs were removed and analyzed. Fig. 3 shows that adoptively transferred memory OT-1 cells accumulated within the draining LNs of C57BL/6 mice where they represented 0.06% of the total LN population. However, the percentage of memory OT-1 cells detected in relB BMC was substantially lowered (0.01%), indicating a limited migration and homing to the regional LNs (Fig. 3). These results raised the possibility that the homing of memory CD8 T cells may play a role in protection against a lethal influenza A virus infection.

FIGURE 2.

Experimental design for the generation and adoptive transfer of SIINFEKL-specific memory CD8 T cells in relB BMC and C57BL/6 mice. Spleens and LN cells (107) from OT-1 TCR transgenic CD45.1 Rag−/− mice were injected i.v. into 7–10 C57BL/6 recipients and allowed to reach homeostatic distribution in different tissues compartments for 24 h. Mice were then injected i.p. with OVA257–264 (SIINFEKL;100 μg/mouse) together with the hepatitis B virus core (HBVc) helper peptide in IFA. Three weeks later mice were sacrificed and a single cell suspension was prepared from the spleens and LNs. CD8 T cells were isolated by negative selection, CFSE-labeled and adoptively transferred (∼8 × 106 purified CD8 T cells/mouse) into relB BMC and C57BL/6 mice. After negative selection and before injection, the cell suspension was analyzed and found to contain a small (0.12%) number of CD45.1+ OT-I CD8 T cells (inset). A phenotypic analysis of cells harvested on day 21 showed that these cells are CD62LintCD44high/CD25low (where int is intermediate). Mice were then infected with 30 μl of A/HKx31-OVA virus (3.33 × 106 EID50/ml). Draining LNs and/or spleens were harvested on days 3 and 8 after infection.

FIGURE 2.

Experimental design for the generation and adoptive transfer of SIINFEKL-specific memory CD8 T cells in relB BMC and C57BL/6 mice. Spleens and LN cells (107) from OT-1 TCR transgenic CD45.1 Rag−/− mice were injected i.v. into 7–10 C57BL/6 recipients and allowed to reach homeostatic distribution in different tissues compartments for 24 h. Mice were then injected i.p. with OVA257–264 (SIINFEKL;100 μg/mouse) together with the hepatitis B virus core (HBVc) helper peptide in IFA. Three weeks later mice were sacrificed and a single cell suspension was prepared from the spleens and LNs. CD8 T cells were isolated by negative selection, CFSE-labeled and adoptively transferred (∼8 × 106 purified CD8 T cells/mouse) into relB BMC and C57BL/6 mice. After negative selection and before injection, the cell suspension was analyzed and found to contain a small (0.12%) number of CD45.1+ OT-I CD8 T cells (inset). A phenotypic analysis of cells harvested on day 21 showed that these cells are CD62LintCD44high/CD25low (where int is intermediate). Mice were then infected with 30 μl of A/HKx31-OVA virus (3.33 × 106 EID50/ml). Draining LNs and/or spleens were harvested on days 3 and 8 after infection.

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FIGURE 3.

Adoptively transferred memory CD8 T cells have impaired homing to secondary lymphoid organs in relB BMC. CFSE-labeled CD45.1+ CD8 memory T cells were adoptively transferred into relB BMC and C57BL/6 mice. Mice were infected with 30 μl of A/HKx31-OVA virus (3.33 × 106 EID50/ml). Draining LNs were harvested on day 3 after infection. Live cells were analyzed for CD45.1 and CD8 expression by flow cytometry. Numbers indicate the percentage of memory CD8 T cells that migrated and home the draining LNs. Data are representative of two experiments.

FIGURE 3.

Adoptively transferred memory CD8 T cells have impaired homing to secondary lymphoid organs in relB BMC. CFSE-labeled CD45.1+ CD8 memory T cells were adoptively transferred into relB BMC and C57BL/6 mice. Mice were infected with 30 μl of A/HKx31-OVA virus (3.33 × 106 EID50/ml). Draining LNs were harvested on day 3 after infection. Live cells were analyzed for CD45.1 and CD8 expression by flow cytometry. Numbers indicate the percentage of memory CD8 T cells that migrated and home the draining LNs. Data are representative of two experiments.

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To gain further insights, we probed DCs lining the airway mucosa in relB BMC for their ability to capture Ag and migrate to draining LNs by using soluble OVA as the Ag (28). Briefly, OVA-FITC was administered (500 μg/mouse) to relB BMC, C57BL/6 BMC, or C57BL/6 mice by inhalation, hence mimicking the natural route of infection by the influenza virus. Draining LNs were harvested 24 h later and the percentage of CD11c+FITC+ cells was assessed by FACS analysis. Fig. 4 shows that whereas OVA-FITC uptake in C57BL/6 or C57BL/6 BMC varied between 30 and 40%, the percentage of CD11c+ FITC+ cells in relB BMC was lower (19%). This cannot be explained on the basis of defective repopulation, because the average number of CD11c+ cells in the airway LN pools was ∼80,000 for relB BMC (n = 12), ∼140,000 for C57BL/6 BMC (n = 10), and ∼93,000 for C57BL/6 mice (n = 7), suggesting no obvious repopulation defect in relB BMC, a finding in agreement with Crowley and Lo (26).

FIGURE 4.

Ag uptake by residual lung DCs in relB BMCs. Mice were injected i.n. with 50 μl of FITC-conjugated OVA (10 mg/ml). DCs were isolated 24 h later from pools of airway (AW) and nonairway (NAW) LNs, stained with an anti-CD11c mAb, and analyzed by flow cytometry. The percentage of FITC-positive DCs is shown in each panel.

FIGURE 4.

Ag uptake by residual lung DCs in relB BMCs. Mice were injected i.n. with 50 μl of FITC-conjugated OVA (10 mg/ml). DCs were isolated 24 h later from pools of airway (AW) and nonairway (NAW) LNs, stained with an anti-CD11c mAb, and analyzed by flow cytometry. The percentage of FITC-positive DCs is shown in each panel.

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Next, we probed the ability of DCs in LNs draining the airway tract of relB BMC to process and present OVA. OVA was administered (500 μg/mouse) by inhalation and the draining LNs were harvested 48 h later. In this experimental design, we allowed Ag uptake and Ag processing to occur in vivo. After 48 h we assessed the priming of OVA-specific CD8 (OT-I) and CD4 (OT-II) T lymphocytes in vitro. Fig. 5 A shows that only DCs isolated from the draining LNs of C57BL/6 BMCs induced a specific proliferation of OT-I (right panel) or OT-II (left panel) lymphocytes. In contrast, upon stimulation with DCs isolated from the draining LNs of relB BMCs no proliferation occurred in either OT-I or OT-II lymphocytes, irrespective of the DC:T cell ratio studied.

FIGURE 5.

relB−/− DCs in LNs draining the airway tract fail to prime CD4 and CD8 T cell responses against OVA. A, relB BMC (open and filled circles) and C57BL/6 BMC (open and filled squares) were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled circles and squares) and nonairway (open circles and squares) lymph nodes and cultured at different concentrations (0–105 cells/well) with 2 × 105 OVA specific OT-II (left panel) or OT-I (right panel) T cells. B, relB BMC (−/− → +/−) or (−/− → +/+) and C57BL/6 BMC were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled bars) and nonairway lymph nodes (open bars) and cultured at 5 × 104 cells/well with 2 × 105 OVA specific OT-II (left panel) or OT-I (right panel) T cells. [3H]Thymidine incorporation was measured after 72 h. Results are expressed as means ± SD of counts per minute of triplicate cultures.

FIGURE 5.

relB−/− DCs in LNs draining the airway tract fail to prime CD4 and CD8 T cell responses against OVA. A, relB BMC (open and filled circles) and C57BL/6 BMC (open and filled squares) were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled circles and squares) and nonairway (open circles and squares) lymph nodes and cultured at different concentrations (0–105 cells/well) with 2 × 105 OVA specific OT-II (left panel) or OT-I (right panel) T cells. B, relB BMC (−/− → +/−) or (−/− → +/+) and C57BL/6 BMC were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled bars) and nonairway lymph nodes (open bars) and cultured at 5 × 104 cells/well with 2 × 105 OVA specific OT-II (left panel) or OT-I (right panel) T cells. [3H]Thymidine incorporation was measured after 72 h. Results are expressed as means ± SD of counts per minute of triplicate cultures.

Close modal

We reasoned that lack of T cell activation could be due to an intrinsic defect of DC function in the LNs of relB BMC. To examine this issue, studies were repeated using in addition relB BMCs generated by transferring homozygous relB−/− BM cells into irradiated C57BL/6 recipients (−/− → +/+), considering that in these mice the defect intrinsic to relB−/− DCs would be the only defect present and that abnormalities in the stromal architecture of the LNs would be minimal. Fig. 5,B shows that neither activation of OT-I (left panel) nor that of OT-II (right panel) lymphocytes was rescued in relB (−/−) → +/+) BMCs. These results suggest that the LN environment does not contribute in a demonstrable way to the functional defect of relB−/− DCs in the draining LNs of the lung. To corroborate these findings, we measured the production of cytokines in the supernatant of T cell cultures seeded with DCs isolated from the draining LNs. Fig. 6,A shows that only OT-I lymphocytes seeded with DCs isolated from the draining LNs of C57BL/6 BMCs produced IL-2 (left panel) and IFN-γ (right panel). Similarly, Fig. 6 B shows IL-2 production only in cultures of OT-II lymphocytes seeded with DCs isolated from the draining LNs of C57BL/6 BMC. Taken together, these results demonstrate that relB−/− DCs in the draining LNs are intrinsically defective in Ag presentation to T cells.

FIGURE 6.

relB−/− DCs in LNs draining the airway tract fail to prime CD4 and CD8 T cell responses against OVA. A, relB BMC (−/− → +/−) or (−/− → +/+) and C57BL/6 BMC were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled bars) and nonairway lymph nodes (open bars) and cultured at 5 × 104 cells/well with 2 × 105 OVA-specific CD8 T cells. The supernatants for IL-2 and IFN-γ detection were collected after 40 h. B, relB−/− BMC (−/− → +/−) or (−/− → +/+) and C57BL/6 BMCs were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled bars) and nonairway (open bars) LNs and cultured at 5 × 104 cells/well with 2 × 105 OVA-specific CD4 T cells. The supernatants for IL-2 detection were collected after 40 h. The insets refer to cytokine production in cultures where OVA-specific T cells were seeded with different concentrations (0–105 cells/well) of airway LN DCs from relB BMC (open circle) or C57BL/6 BMC (filled circle).

FIGURE 6.

relB−/− DCs in LNs draining the airway tract fail to prime CD4 and CD8 T cell responses against OVA. A, relB BMC (−/− → +/−) or (−/− → +/+) and C57BL/6 BMC were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled bars) and nonairway lymph nodes (open bars) and cultured at 5 × 104 cells/well with 2 × 105 OVA-specific CD8 T cells. The supernatants for IL-2 and IFN-γ detection were collected after 40 h. B, relB−/− BMC (−/− → +/−) or (−/− → +/+) and C57BL/6 BMCs were injected i.n. with 50 μl of OVA (10 mg/ml). Forty-eight hours after injection, DC were purified from a pool of airway (filled bars) and nonairway (open bars) LNs and cultured at 5 × 104 cells/well with 2 × 105 OVA-specific CD4 T cells. The supernatants for IL-2 detection were collected after 40 h. The insets refer to cytokine production in cultures where OVA-specific T cells were seeded with different concentrations (0–105 cells/well) of airway LN DCs from relB BMC (open circle) or C57BL/6 BMC (filled circle).

Close modal

Based on the above results, it became important to monitor Ag presentation by relB−/− DCs in LNs draining the airway tract after intranasal virus challenge because the inflammatory component of virus infection could not be mimicked by inhalation of OVA-FITC. Briefly, using an experimental design similar to that shown in Fig. 2, C57BL/6 mice and relB BMC were challenged i.n. with a sublethal (3.3 × 106 EID50/ml) dose of A/HKx31-OVA influenza virus. DCs were isolated from the draining LNs 24 h after virus infection and stained using mAb 25.D-1.16, which recognizes the SIINFEKL peptide complexed with the Kb molecule (31). FACS analysis showed marked Ag presentation in DCs from C57BL/6 BMCs but not from relB BMCs (Fig. 7,A). We also sought SIINFEKL/Kb complex staining in frozen sections of LNs harvested 24 h after virus infection (Fig. 7,B). In the draining LNs of C57BL/6 mice, Ab 25.D-1.16 produced a diffuse and intense staining in the T cell area (red), suggesting active Ag presentation by BM-derived DCs. Minimal SIINFEKL/Kb complex staining was observed in LN sections of relB BMC (Fig. 7 B).

FIGURE 7.

Ag presentation by DCs in LNs draining the airway tract. Memory CD8 TCR transgenic OT-I cells were adoptively transferred into C57BL/6 mice or relB BMCs and mice were challenged 24 h later with a sublethal dose of A/HKx31-OVA influenza virus. Twenty-four hours after infection, draining LNs and spleens were removed, digested, and stained for DCs (CD11c+) and presentation of SIINFEKL/Kb (clone 25.D-1.16). A, CD11c+ cells presenting the SIINFEKL peptide complex with the Kb molecule are shown. Samples from C57BL/6 mice that were left uninfected or infected with A/HKx31virus were used as negative controls (data not shown). B, Immunofluorescence analysis on frozen tissue sections from airway-draining LN sections were performed using biotin-labeled 25-D1.16 followed by sequential incubation in HRP-labeled streptavidin, biotin-labeled tyramide, and streptavidin-Cy3. Costaining with FITC-conjugated anti-B220 Ab was used to identify B cell areas. Digitized images were acquired using a Nikon imaging station and processed using Photoshop software.

FIGURE 7.

Ag presentation by DCs in LNs draining the airway tract. Memory CD8 TCR transgenic OT-I cells were adoptively transferred into C57BL/6 mice or relB BMCs and mice were challenged 24 h later with a sublethal dose of A/HKx31-OVA influenza virus. Twenty-four hours after infection, draining LNs and spleens were removed, digested, and stained for DCs (CD11c+) and presentation of SIINFEKL/Kb (clone 25.D-1.16). A, CD11c+ cells presenting the SIINFEKL peptide complex with the Kb molecule are shown. Samples from C57BL/6 mice that were left uninfected or infected with A/HKx31virus were used as negative controls (data not shown). B, Immunofluorescence analysis on frozen tissue sections from airway-draining LN sections were performed using biotin-labeled 25-D1.16 followed by sequential incubation in HRP-labeled streptavidin, biotin-labeled tyramide, and streptavidin-Cy3. Costaining with FITC-conjugated anti-B220 Ab was used to identify B cell areas. Digitized images were acquired using a Nikon imaging station and processed using Photoshop software.

Close modal

In this study we show that protection against lethal influenza A virus infection by memory CD8 T cells requires that DCs in the LNs draining the site of infection re-present viral Ag to memory CD8 T cells. Because lack of the relB gene severely impairs the Ag-presenting function of BM-derived DCs (28), our findings suggest that DCs play a key role in the mechanism of protection against influenza A virus infection. Upon infection, the path to presentation of viral Ags can be divided into two steps: 1) Ag uptake by DCs; and 2) Ag processing and presentation to T cells in the T cell zone. Our data suggest that relB−/− DCs in the LNs draining the airway tract are defective at both levels. This implies that protective memory CD8 T cells require reactivation in the LNs draining the airway tract by BM-derived DCs.

The key finding of this study is that memory CD8 T cells fail to protect if the Ag-presenting function of DCs in the draining LNs of the airway is impaired. Several lines of evidence support this conclusion. First, lung DCs isolated from the draining LNs of relB BMCs, unlike DCs from C57BL/6 BMC mice, showed a markedly decreased uptake of soluble Ag (Fig. 4). Second, Ag presentation by relB DCs in the draining LNs harvested within 24 h after virus infection was reduced compared with DCs from C57BL/6 mice. This was true whether DCs were analyzed as cell suspensions or as frozen tissue by direct immunofluorescence (Fig. 7, A and B). Third, relB−/− DCs in the draining LNs of the airway tract failed to prime CD8 or CD4 T cells in vitro (Fig. 5, A and B), a finding consistent with a previous report from this laboratory that residual splenic DCs in relB BMC are unable to prime CD4 T cells against soluble Ag whether Ag uptake occurs in vivo or in vitro (28). Fourth, analysis of the LNs draining the airway tract showed poor homing of memory CD8 T cells in relB BMCs and, hence, insufficient reactivation of memory CD8 T cells compared with C57BL/6 mice (Fig. 3). Of note, reconstitution experiments with C57BL/6 BM-derived DCs pulsed with the NP366 peptide restored protection in relB BMC (Table III). Collectively, the present data point to a critical role by BM-derived DCs in reactivating memory CD8 T cells in protection against lethal virus challenge. Because an abnormal stromal environment postirradiation and reconstitution did not apparently play a role (Fig. 5 B), a tempting hypothesis would then be that the defect of relB−/− DCs is linked to a defect in signaling through the lymphotoxin-β receptor, which was found to regulate the homeostasis of myeloid DCs (32).

The present results stress the importance of local immunity in protection against influenza A virus. Failure of adoptively transferred CD8 TCM cells to protect relB−/− BMCs clearly suggests that vaccine-induced, T cell-mediated protection against disease rests on at least two prerequisites. One is the induction of systemic T cell immunity with the generation of CD8 TCM cells (19). The other is the necessity for memory T cells to undergo Ag reactivation in the LNs draining the site of infection. Previous studies established that memory CD8 T cells localize to the lung (33) and mediate protection by accelerating the clearance of the influenza virus in the lung (34). Zammit et al. (35) demonstrated that the interaction with DCs is a major mechanism for reactivation of memory CD8 T cells in airway LNs following influenza virus infection. Similarly, Belz et al. (36) showed that T cell memory responses against influenza virus depend on BM-derived DCs for expansion in the spleen and in LNs 10 days after viral challenge. Interestingly, presentation of viral Ag in the draining LNs is also required for the maintenance of virus-specific CD8 T cells (37). Similarly, our data suggest that reactivation of memory CD8 T cells by BM-derived DCs in the draining LNs is critically necessary for protection against lethal influenza virus infection. Due to the fact that activation of memory CD8 T cells may require a longer time than one would anticipate (36) and that lethality by the virus occurs within 7–10 days, we suggest that the DC insufficiency in relB BMCs renders the host unable to take advantage of protective memory T cells. Notwithstanding this interpretation, we cannot rule out the possibility that relB BMCs may also have a defect in innate immunity that is known to be at play against influenza A virus infection (38). For instance, innate immunity could aid in the early control of viral load and this would facilitate the antiviral effect of memory CTL.

Current vaccines against influenza A virus infection induce Abs that block infection by intercepting the virus at the portal of entry but are subject to antigenic shift and antigenic drift. Vaccines that induce CD8 T cell responses against conserved viral Ags are candidates for protection against disease. Based on this study and recently published work (33, 34, 35, 36, 37), vaccine-induced, T cell-mediated protection against influenza A virus requires the induction of protective memory CD8 T cells (16) and their migration to the draining LNs to be sampled and reactivated by virus laden DCs (39). In this sequential program, qualitative events (i.e., the preferential induction of CD8 TCM cells) and geographical considerations (i.e., migration to draining LNs and local restimulation by DCs) need to occur coordinately. Our analysis in vivo using mice with defective DC function places emphasis on the interplay between these various phases and suggests that the characteristics of both systemic and local immunity play an important role. This understanding may facilitate the design of T cell protective vaccines against influenza A virus infection.

We thank Dr. Adolfo Garcia-Sastre (Mount Sinai School of Medicine, New York, NY) for the gift of the A/PR8/34 virus and Dr. Paul Thomas (St. Jude Children’s Research Hospital, Memphis, TN) for the gift of the A/HKx31 and A/HKx31-OVA viruses.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant RO1AI062894.

3

Abbreviations used in this paper: NP, nucleoprotein; BM, bone marrow; BMC, BM chimera; DC, dendritic cell; EID50, 50% egg infectious dose; LN, lymph node; TCM, central memory T cell.

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