Infection with influenza A virus can lead to increased susceptibility to subsequent bacterial infection, often with Streptococcus pneumoniae. Given the substantial modification of the lung environment that occurs following pathogen infection, there is significant potential for modulation of immune responses. In this study, we show that infection of mice with influenza virus, followed by the noninvasive EF3030 strain of Streptococcus pneumoniae, leads to a significant decrease in the virus-specific CD8+ T cell response in the lung. Adoptive-transfer studies suggest that this reduction contributes to disease in coinfected animals. The reduced number of lung effector cells in coinfected animals was associated with increased death, as well as a reduction in cytokine production in surviving cells. Further, cells that retained the ability to produce IFN-γ exhibited a decreased potential for coproduction of TNF-α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node was minimal compared with that present in the lung, supporting a model of selective regulation in the tissue harboring high pathogen burden. These results show that entry of a coinfecting pathogen can have profound immunoregulatory effects on an ongoing immune response. Together, these findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.

Influenza A virus (IAV)-associated bacterial pneumonia is a significant cause of morbidity and mortality (13). During the 1918 “Spanish flu” pandemic, the vast majority (>95%) of fatal cases were complicated by bacterial pneumonias (4), with Streptococcus pneumoniae accounting for the majority of bacterial infections. Data from more recent pandemics in 1957, 1968, and 2009 reveal a similar phenomenon; for example, in 2009 as many as 56% of hospitalized patients tested positive for IAV-associated bacterial pneumonias (58).

Given the significant disease associated with influenza virus and pneumococcus infections, considerable effort has been directed toward understanding the mechanisms responsible for bacterial outgrowth under these circumstances. These studies revealed influenza-mediated alterations in the innate immune system that promote bacterial survival and growth, including decreased phagocytosis and loss of alveolar macrophages (1, 9, 10). Interestingly, in addition to increased bacterial burden, there is evidence that viral load in the lung is augmented following bacterial coinfection (11, 12), suggesting bacteria-mediated changes that promote virus infection and/or growth.

Pneumococcus in the lung is associated with a number of changes in the immune environment, including the entry of neutrophils and macrophages, as well as differentiation of T cells into Th17, Th2, and regulatory subsets, the last of which results in increased IL-10 (1, 1320). In addition, bacterial products have the ability to directly modulate inflammatory responses. For example, pneumococcal components can reduce asthma-associated inflammation by regulating effector function (2123). Along with the immune-modulatory effects on the lung environment that result from S. pneumoniae infection, there is evidence that pneumococcus can directly impact T cell survival. For example, peripheral blood T cells from patients with bacteremia and sepsis exhibit high amounts of death (2426). Further, in vitro studies show pneumolysin, the cholesterol-dependent cytolysin produced by S. pneumoniae, can induce T cell death (27). Based on these findings, we hypothesized that the entry and growth of S. pneumoniae in the lung may impact the ongoing T cell response to influenza virus.

Clearance of acute influenza virus infection is dependent on the presence of a potent adaptive immune response. In support of this, severe cases of influenza infection in humans have been associated with the lack of an effective CD8+ T cell response in the lung (28). CD8+ T cells were shown to mediate viral clearance through secretion of IFN-γ, as well as cytolytic granule release (29, 30). A study performed during the 2009–2010 H1N1 pandemic found a strong negative correlation between the severity of symptoms and the number of IFNγ+IL-2 CD8+ T cells (31), suggesting an important role for this cytokine in humans in the context of influenza.

We tested the hypothesis that S. pneumoniae negatively regulates the influenza-specific CD8+ T cell response. We found a marked decrease in the overall size and quality of the influenza-specific CD8+ T cell response in the lung. The decrease in number was due, at least in part, to increased lymphocyte death following coinfection. We also detected a decrease in the quality of influenza-specific CD8+ T cells, as evidenced by the reduced ability to coproduce IFN-γ and TNF-α in response to peptide stimulation. The decrease in cytokine-producing cells correlated with an increase in cells that exhibited cytolysis as their sole effector function. The selective inhibition of the production of cytokine correlated with marked decreases in IFN-γ mRNA. The altered influenza-specific T cell response appeared to contribute to disease in coinfected animals, because reconstitution of the response by adoptive transfer significantly increased survival. The negative regulation of the influenza-specific T cell response observed in our study was not associated with changes in lung dendritic cells (DCs), but it did correlate with an increase in regulatory T cells (Tregs). The changes in effector number and function were manifest predominantly in the lung, the primary site of bacterial and viral infection, suggesting that high pathogen burden is necessary for the negative regulation of the influenza-specific CD8+ T cell response.

All research performed on mice in this study complied with federal and institutional guidelines set forth by the Wake Forest University Animal Care and Use Committee. All studies were approved by the Wake Forest University Animal Care and Use Committee.

S. pneumoniae EF3030 is a serotype 19F clinical isolate noted for its ability to colonize the nasopharynx for ≥21 d, as well as its inability to cause invasive disease even when injected i.v. (32). S. pneumoniae was grown in Brain-Heart Infusion (Difco) broth supplemented with 10% heat-inactivated horse serum (Life Technologies) and 10% catalase (3 mg/ml) to an OD600 of 0.8, correlating to ∼1 × 108 CFU/ml. Broth cultures were mixed 1:1 with a 50% glycerol solution and frozen at −80°C for future use. For CFU enumeration, S. pneumoniae was grown on tryptic soy agar plates made with tryptic soy broth and 1.5% agar (both from Becton Dickinson) supplemented with 5% defibrinated sheep’s blood (Hemostat) and 4 μg/ ml gentamicin (Sigma-Aldrich).

Virus stocks were grown and titered in fertilized chicken eggs (median egg infectious dose [EID50]), essentially as described previously (33). Stocks were diluted in PBS, flash frozen, and stored at −80°C.

Ten- to twelve-week-old female BALB/c mice were purchased from The Jackson Laboratory. Mice were housed in a biosafety level 2 facility with access to food and water ad libitum.

Mice were anesthetized with Avertin (2,2,2-tribromoethanol) by i.p. injection. Virus (103 EID50) or PBS as a control was administered via the intranasal (i.n.) route in 50 μl PBS. Four days postinfection, mice were anesthetized with Avertin and bacteria (104 CFU) or Brain Heart Infusion broth administered i.n. in 20 μl. Disease was quantified using the following guidelines: 0, no disease; 1, ruffled fur; 2, ruffled fur, limited mobility, slight hunching; 3, ruffled fur, ataxia, hunched, hypoxia, dehydration; and 4, ruffled fur, ataxia, hunching, respiratory distress, hypoxia, and dehydration. Animals receiving a disease score of 4 were removed from the study and humanely euthanized.

Aliquots of lung homogenates were serially diluted and plated on tryptose blood agar plates. Bacterial colonies were quantified following a 20-h incubation at 37°C.

Viral RNA was extracted from lung homogenates using a QIAamp Viral RNA Mini Kit (QIAGEN). cDNA was synthesized from mRNA by reverse transcription using a Superscript III RT Kit and random primers (both from Invitrogen). RNA primer-probe sets specific for H1N1 were used (BEI Resources) for viral quantification. Quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems 7500 real-time PCR system.

At the designated days postinfluenza infection (day 8 or 11), perfused lungs and mediastinal lymph nodes (MLNs) were isolated. Lungs were homogenized and incubated for 1 h at 37°C with collagenase D (100 μg/ml; Roche). Mononuclear cells were isolated by passage over a Histopaque gradient. MLNs were mechanically disrupted, and RBCs were removed by lysis with ACK. Cells were stimulated ex vivo with 10−7 M NP147–155 peptide in the presence of monensin and brefeldin A (BD Biosciences). Following a 5-h incubation period, cells were stained for flow cytometric analysis. Fluorochrome-conjugated Ab detection reagents included anti-mouse CD8α, LFA-1, and CD107a (all from BioLegend). Allophycocyanin-conjugated NP147–155/Kd tetramer was used to identify IAV-specific CD8+ T cells (graciously supplied by the National Institutes of Health tetramer facility). For cells stimulated with peptide, tetramer was included during the stimulation. This allowed tetramer labeling that otherwise may have been hampered as a result of TCR downregulation. Unstimulated samples were stained with tetramer during the surface stain only to circumvent tetramer-induced cytokine production. Cells were then fixed and permeabilized (Cytofix/Cytoperm kit; BD Biosciences), followed by incubation with Abs specific for IFN-γ and, in some cases, TNF-α. When 7-aminoactinomycin D (7-AAD; BioLegend) was used to determine cell viability, cells were incubated with 7-AAD following Ab staining. Cells were then washed extensively. For the subsequent detection of active caspase-3, cells were fixed and permeabilized (BD Biosciences) following 7-AAD staining. For the detection of Tregs, the FoxP3 detection Fix/Perm kit (BioLegend) was used with PE-conjugated anti-FoxP3 Ab (BioLegend) and CD4+ and CD25+ staining. Data were acquired using a FACSCanto II flow cytometer and analyzed using FACSDiva software.

On day 7 postinfluenza infection, MLNs were harvested and stained with allophycocyanin-conjugated Thy1-specific Ab (BD Biosciences). Thy1.2+ cells were isolated using anti-allophycocyanin beads and MACS columns (both from Miltenyi Biotec), as per the manufacturer’s instructions. The purification was monitored by flow cytometric analysis. Populations were 90–95% Thy1.2+. Coinfected animals received either PBS or 2.6 × 106 isolated Thy1.2+ cells via tail vein injection 5.5 d postinfluenza virus infection. Animals were monitored through day 11 for disease.

On day 8 postvirus infection, perfused lungs were homogenized and incubated for 1 h at 37°C with collagenase D (100 μg/ml; Roche). Single-cell suspensions were passed over a Histopaque gradient to enrich for mononuclear cells. Recovered cells were stained with Abs specific for CD11c, CD11b, B220, MHC class II (all from BioLegend), and CD103 (BD Biosciences). Data were acquired using a FACSCanto II flow cytometer and analyzed using FACSDiva software.

Isolated lymphocytes were stained with CD8 and allophycocyanin-conjugated NP147–155/Kd tetramer. Tetramer+ and tetramer CD8+ lymphocytes were isolated using a FACSAria cell sorter. Following a 5-h peptide stimulation ex vivo, lymphocyte RNA was isolated by a standard TRIzol (Invitrogen) phenol/chloroform extraction. cDNA was synthesized from mRNA by reverse transcription using a SuperScript III Reverse Transcriptase Kit and random primers (all from Invitrogen). For IFN-γ, perforin, granzyme B, IL-15, IL-18, TGF-β, and GAPDH mRNA analysis, commercially available TaqMan primer-probe sets specific for the gene targets were used. qRT-PCR was performed using the Applied Biosystems 7500 real-time PCR system. Raw data values were normalized to GAPDH mRNA levels.

To assess the potential for coinfection with S. pneumoniae to regulate the ongoing anti-influenza virus adaptive immune response, we developed a model that allowed survival of most animals to day 11 postinfection with influenza virus. We used the well-characterized mouse-adapted influenza A/PR/8/34 [H1N1] (PR8), together with the pneumococcal strain EF3030. This strain is of the 19F serotype and is considered to lack the capacity to cause lethal invasive disease in mice (32, 34).

BALB/c mice received either PR8 (3.5 × 103 EID50) or PBS i.n. Four days following PR8 infection, mice were given either 3.5 × 104 CFU of EF3030 or Brain Heart Infusion media (mock) i.n. as a control (Fig. 1A). Animals were monitored daily for signs of disease. Animals receiving virus alone exhibited mild disease (disease score of 1–1.5) over the course of the infection (Fig 1B). In contrast, coinfected animals exhibited increased morbidity as early as day 6 post-PR8 infection (2 d post-EF3030), with severe disease (score ≥ 3) by day 7 (Fig. 1B). Coinfected animals also exhibited significantly increased mortality, with roughly 50% succumbing to the infection by day 11 post-PR8 infection (Fig. 1C). Animals receiving bacteria alone did not exhibit disease (Fig. 1B). Although mortality was increased in the presence of coinfection, a large percentage of animals survive, thereby allowing assessment of the potential for S. pneumoniae–mediated effects on the adaptive PR8-specific immune response.

FIGURE 1.

Coinfection with EF3030 + PR8 leads to enhanced disease and mortality. (A) Overview of experimental design. Animals infected as shown in (A) (singly infected with PR8 or EF3030 or coinfected) were monitored for disease (B) and mortality (C). Disease was quantified using an adapted version of Tate’s disease scoring criteria, which used the following parameters: coat ruffling, labored breathing, hypoxia, dehydration, ataxia and lethargy, conjunctivitis, and coat hygiene. Animals euthanized because of disease state were excluded from the disease score calculation after removal. Dotted line indicates day of bacterial infection. Data are derived from 26 (PR8), 44 (PR8 + EF3030), or 20 (EF3030) animals assessed across three experiments. ***p < 0.0002, ****p < 0.0001, Kruskal–Wallis nonparametric ANOVA with Dunn posttest (B) and Fischer exact test (C).

FIGURE 1.

Coinfection with EF3030 + PR8 leads to enhanced disease and mortality. (A) Overview of experimental design. Animals infected as shown in (A) (singly infected with PR8 or EF3030 or coinfected) were monitored for disease (B) and mortality (C). Disease was quantified using an adapted version of Tate’s disease scoring criteria, which used the following parameters: coat ruffling, labored breathing, hypoxia, dehydration, ataxia and lethargy, conjunctivitis, and coat hygiene. Animals euthanized because of disease state were excluded from the disease score calculation after removal. Dotted line indicates day of bacterial infection. Data are derived from 26 (PR8), 44 (PR8 + EF3030), or 20 (EF3030) animals assessed across three experiments. ***p < 0.0002, ****p < 0.0001, Kruskal–Wallis nonparametric ANOVA with Dunn posttest (B) and Fischer exact test (C).

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One potential factor that could contribute to increased disease in coinfected animals was an increase in the level of bacteria and/or virus. To test this possibility, bacterial and viral load in the lung was determined on days 5, 8, and 11 post-PR8 infection (days 1, 4, and 7 post-EF3030). Viral burden was assessed by qRT-PCR following the protocol set forward by the Centers for Disease Control and Prevention (35). Total RNA from the supernatants of lung homogenates was reverse transcribed using random primers to generate a cDNA library. Viral RNA was quantified using a primer-probe set that targets hemagglutinin of H1N1 IAV. Using a DNA standard, we calculated the viral RNA copy number as a measure of total viral burden. As shown in Fig. 2A, a significant increase in viral RNA was detected at 24 h following delivery of the bacteria to PR8-infected animals compared with animals receiving PR8 alone. By days 8 and 11 post-PR8 infection, virus was comparable in both groups.

FIGURE 2.

Coinfection with PR8 and EF3030 leads to significantly increased viral RNA 24 h after coinfection and significantly increased bacterial burden in the lung through day 11. Animals were infected as described in Fig. 1. Lungs were harvested for viral and bacterial load analysis at the indicated times postinfluenza infection. (A) Viral RNA was extracted from lung homogenates using a QIAamp Viral RNA Mini Kit. cDNA was synthesized from mRNA by reverse transcription and subjected to qRT-PCR using a primer-probe set specific for PR8 hemagglutinin to enumerate viral RNA copies/lung. (B) A portion of the lung homogenate was serially diluted and plated on tryptose blood agar plates for bacterial quantification. Data are the average of 24–29 individually analyzed animals/infection condition assayed over three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-tailed Student t test.

FIGURE 2.

Coinfection with PR8 and EF3030 leads to significantly increased viral RNA 24 h after coinfection and significantly increased bacterial burden in the lung through day 11. Animals were infected as described in Fig. 1. Lungs were harvested for viral and bacterial load analysis at the indicated times postinfluenza infection. (A) Viral RNA was extracted from lung homogenates using a QIAamp Viral RNA Mini Kit. cDNA was synthesized from mRNA by reverse transcription and subjected to qRT-PCR using a primer-probe set specific for PR8 hemagglutinin to enumerate viral RNA copies/lung. (B) A portion of the lung homogenate was serially diluted and plated on tryptose blood agar plates for bacterial quantification. Data are the average of 24–29 individually analyzed animals/infection condition assayed over three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-tailed Student t test.

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As shown in Fig. 2B, on day 5 post-PR8 (day 1 post-EF3030), we observed an approximately 3-log increase in detectable bacteria in coinfected animals compared with animals receiving bacteria alone. Bacterial counts in coinfected animals remained high through day 11 post-PR8 infection. Animals receiving EF3030 alone had begun to clear bacteria by day 11, with a majority (5/9) having bacterial counts below the limit of detection in contrast with coinfected animals (0/12 had cleared bacteria). Thus, coinfection resulted in increases in both bacterial and viral load in the lungs.

CD8+ T cells play a critical role in the clearance of influenza virus. Given the high bacterial counts found in coinfected animals, we postulated that the presence of pneumococcus could alter the ongoing anti-influenza CD8+ T cell response. To assess the CD8+ T cell response elicited in these animals, we quantified the presence of IFN-γ–producing CD8+ T cells specific for the influenza virus immunodominant epitope NP147–155 in the lung and draining MLNs. IFN-γ was chosen because it is an important mediator of CD8+ T cell effector function in the context of influenza virus infection (30). By day 8 post-PR8 infection, a sizable virus-specific response was detected in the lungs of animals singly infected with PR8 (Fig. 3A). When lungs of animals coinfected with EF3030 were analyzed, a significant reduction (10.4-fold) in the total number of PR8-specific IFNγ+CD8+ T cells was detected (Fig. 3B). Assessment of the MLNs showed only a modest reduction (1.6-fold) in the PR8-specific cells in coinfected animals. The significant reduction in IFNγ+ cells in the lung remained at day 11, although the decrease compared with singly infected animals was not as pronounced as at day 8. The number of cells in the MLN was similar between the two groups at this time. These data suggest a strong negative regulation of the anti-influenza CD8+ T cell response at the site of active coinfection (i.e., the lung).

FIGURE 3.

Coinfection with PR8 and EF3030 results in a substantial reduction in the number of IFN-producing NP-specific CD8+ T cells in the lungs and a modest reduction in the MLN. Mice singly infected with influenza virus or coinfected were euthanized on days 8 and 11 postinfluenza virus infection. Perfused lungs and MLNs were harvested. Isolated cells were stimulated ex vivo with influenza NP147–155 peptide. Data shown are pregated on CD8+LFA-1hi cells. (A) Representative flow plots. (B) Averaged data from 17 or 18 (d8) or 8–11 (d11) individually analyzed animals assessed over three independent experiments. *p < 0.05, ****p < 0.0001, two-tailed Student t test.

FIGURE 3.

Coinfection with PR8 and EF3030 results in a substantial reduction in the number of IFN-producing NP-specific CD8+ T cells in the lungs and a modest reduction in the MLN. Mice singly infected with influenza virus or coinfected were euthanized on days 8 and 11 postinfluenza virus infection. Perfused lungs and MLNs were harvested. Isolated cells were stimulated ex vivo with influenza NP147–155 peptide. Data shown are pregated on CD8+LFA-1hi cells. (A) Representative flow plots. (B) Averaged data from 17 or 18 (d8) or 8–11 (d11) individually analyzed animals assessed over three independent experiments. *p < 0.05, ****p < 0.0001, two-tailed Student t test.

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We hypothesized that the dramatic reduction in IFN-γ–producing influenza-specific T cells in the lungs contributed to the overall disease process during coinfection. To test this possibility, Thy1.2+ cells were isolated from the MLN of influenza virus–infected animals. Isolated cells were adoptively transferred into coinfected animals on day 5.5 postinfluenza virus infection (1.5 d following S. pneumoniae infection). This time frame was chosen because it approximates the time at which effector cells begin to enter the lung and critically is a time at which mice remain healthy enough to undergo the transfer procedure. As above, mice were euthanized when they became moribund. As shown in Fig. 4, mice receiving influenza-specific T cells exhibited significantly increased survival. At day 11 postinfluenza virus infection (5.5 d following the adoptive transfer), 41% of animals that received cells were alive in contrast to 8% of animals that received PBS. These data show that increasing the number of influenza virus–specific T cells in coinfected animals promotes increased survival.

FIGURE 4.

Adoptive transfer (AT) of influenza-specific T cells into coinfected animals results in significantly increased survival. Thy1.2+ cells were isolated from the MLN of influenza–infected mice on day 7 postinfection. Isolated cells were transferred into coinfected recipients on day 5.5 following influenza virus infection (day 1.5 following S. pneumoniae infection). Animals were monitored, and those that reached a disease score of 4 were euthanized. Results represent data from two independent experiments that together assessed a total of 12 or 13 mice/group. *p < 0.05, Fisher exact test.

FIGURE 4.

Adoptive transfer (AT) of influenza-specific T cells into coinfected animals results in significantly increased survival. Thy1.2+ cells were isolated from the MLN of influenza–infected mice on day 7 postinfection. Isolated cells were transferred into coinfected recipients on day 5.5 following influenza virus infection (day 1.5 following S. pneumoniae infection). Animals were monitored, and those that reached a disease score of 4 were euthanized. Results represent data from two independent experiments that together assessed a total of 12 or 13 mice/group. *p < 0.05, Fisher exact test.

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The decrease in cytokine-producing cells could result from either a reduction in the absolute number of NP-specific CD8+ T cells or, alternatively, from negative regulation of function in this population. To test the former possibility, lung and MLN cells from infected animals were stained with NP147–155/Kd tetramer to quantify Ag-specific cells (Fig. 5A). Although the percentage of tetramer+ cells was similar in the CD8+ population on day 8 post-PR8 infection, the total number of tetramer+ cells was significantly reduced (8.8-fold) in the lungs of coinfected animals compared with PR8 singly infected animals (Fig. 5B). Thus, the reduced number of IFNγ+ cells can be accounted for, in part, by a reduction in the number of NP-specific cells in the lung. The number of NP147–155-specific CD8+ T cells in the MLN was not significantly different at this time, although there was a decrease on average. Analysis of the response at day 11 showed no difference in the number of NP-specific cells in either tissue (Fig. 5B). Given the observed reduction in the number of IFN-γ–producing cells in the lung on day 11 post-PR8 infection, there appears to be functional inactivation of lung cells at this time. These findings indicate that a decrease in the number of NP-specific CD8+ T cells makes a significant contribution to the reduced IFNγ+CD8+ T cell response observed in the lungs of coinfected animals.

FIGURE 5.

Coinfection with EF3030 results in a substantial reduction in the number of NP-specific CD8+ T cells in the lung but not the lung-draining MLNs. Lungs and MLNs from influenza virus–infected or coinfected mice were isolated on days 8 and 11 postinfluenza and processed as previously described. (A) Representative flow plots. Cells were pregated on CD8 and LFA-1hi expression. (B) Averaged data from 11–14 (d8) or 8–11 (d11) individually analyzed animals assessed over two independent experiments. ****p < 0.0001, two-tailed Student t test.

FIGURE 5.

Coinfection with EF3030 results in a substantial reduction in the number of NP-specific CD8+ T cells in the lung but not the lung-draining MLNs. Lungs and MLNs from influenza virus–infected or coinfected mice were isolated on days 8 and 11 postinfluenza and processed as previously described. (A) Representative flow plots. Cells were pregated on CD8 and LFA-1hi expression. (B) Averaged data from 11–14 (d8) or 8–11 (d11) individually analyzed animals assessed over two independent experiments. ****p < 0.0001, two-tailed Student t test.

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We hypothesized that the decrease in the overall size of the NP-specific CD8+ T cell population was due to increased death in the coinfected lung environment. To test this possibility, MLNs and lungs were harvested from PR8-infected or coinfected mice at days 8 and 11 post-PR8 infection. Isolated cells were stained for CD8 together with NP147–155/Kd tetramer. We used a modified staining approach that allowed concurrent analysis of annexin V, active caspase-3, and 7-AAD to assess CD8+ T cell viability (36). By day 8 post-PR8 infection, there was a significant increase in the percentage of tetramer+CD8+ T cells in the lungs of coinfected animals that were positive for 7-AAD (Fig. 6A, 6B). There was no significant increase in 7-AAD+ cells in the MLN (Fig. 6A, 6B). Despite the decreased viability of the NP-specific CD8+ T cell population, increases in 7-AAD positivity did not correlate with the increased presence of active caspase-3 (Fig. 6C). This finding suggests that death in coinfected animals is induced in a caspase-3–independent manner. These data support a model wherein the presence of EF3030 in the lungs of PR8-infected animals results in increased death of NP-specific CD8+ T cells.

FIGURE 6.

Decreases in the NP-specific CD8+ T cell response in the lungs of coinfected animals on day 8 postinfluenza infection correlate with increased cell death. Cells were isolated from the lungs and MLNs of animals infected with influenza virus or coinfected. Following staining for CD8 and tetramer, 7-AAD was added to identify cells that had lost membrane integrity as an indicator of cell death. (A) Representative flow plots. Cells were pregated on CD8 and tetramer. (B) Averaged data from two or three independent experiments assessing a total of 21 or 22 (d8) or 8–11 (d11) mice/infection condition. (C) Cells were isolated from the lung and MLNs of animals infected with influenza virus alone or coinfected. Following staining for CD8 and tetramer, cells were fixed, permeabilized, and stained with Abs specific for active caspase-3. The percentage of CD8+tetramer+ cells that stained positive for active caspase-3 is shown. Data are the average from four to six mice/condition. **p < 0.01, ****p < 0.0001, two-tailed Student t test.

FIGURE 6.

Decreases in the NP-specific CD8+ T cell response in the lungs of coinfected animals on day 8 postinfluenza infection correlate with increased cell death. Cells were isolated from the lungs and MLNs of animals infected with influenza virus or coinfected. Following staining for CD8 and tetramer, 7-AAD was added to identify cells that had lost membrane integrity as an indicator of cell death. (A) Representative flow plots. Cells were pregated on CD8 and tetramer. (B) Averaged data from two or three independent experiments assessing a total of 21 or 22 (d8) or 8–11 (d11) mice/infection condition. (C) Cells were isolated from the lung and MLNs of animals infected with influenza virus alone or coinfected. Following staining for CD8 and tetramer, cells were fixed, permeabilized, and stained with Abs specific for active caspase-3. The percentage of CD8+tetramer+ cells that stained positive for active caspase-3 is shown. Data are the average from four to six mice/condition. **p < 0.01, ****p < 0.0001, two-tailed Student t test.

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In addition to the production of IFN-γ, effector T cells contribute to viral clearance through the production of TNF-α, as well as cytolysis. Thus, we determined whether the cells from coinfected mice that were incapable of producing IFN-γ retain the ability to lyse or produce TNF-α. Lymphocytes were isolated from the lung and MLN of singly or coinfected animals on days 8 and 11 following PR8 infection. CD8+ T cells were stimulated with peptide in the presence of tetramer to facilitate optimal detection by tetramer in subsequent analyses. IFN-γ and TNF-α production, as well as CD107a at the cell surface, a surrogate for cytolysis, was assessed. This analysis revealed a significant decrease in the proportion of NP-specific cells in the lungs of coinfected animals that were polyfunctional (IFNγ+TNFα+CD107+) compared with cells isolated from singly infected animals (Fig. 7A). This decrease corresponded to a significant increase in the proportion of cells that exhibited cytolysis as their only effector function. The results in the MLN diverged from the lung, where there was no evidence of a shift toward cells that were only cytolytic (Fig. 7A). These data indicate selective regulation in the ability of NP-specific CD8+ T cells from coinfected lungs to produce cytokine. By day 11, responses in singly and coinfected animals were similar in both tissues.

FIGURE 7.

Coinfection results in qualitatively diminished CD8+ T cell function as measured by reduced polyfunctionality and coproduction of IFN-γ and TNF-α. Cells isolated from the lungs and MLNs of animals infected with influenza virus or coinfected were stimulated with peptide as described previously. Anti-CD107a Ab was included in the stimulation phase to identify cells releasing lytic granules in response to peptide. Following the stimulation period, cells were stained for CD8, tetramer, LFA-1, IFN-γ, and TNF-α. (A) Averaged data evaluating the distribution of the effector function of the influenza-specific response. (B) Percentage of NP-specific IFN-γ–producing cells that coproduced TNF-α. Data are the average from two or three independent experiments assessing a total of 12–18 (d8) or 10 or 11 (d11) mice/infection condition. *p < 0.05, **p < 0.01, ****p < 0.0001, two-tailed Student t test.

FIGURE 7.

Coinfection results in qualitatively diminished CD8+ T cell function as measured by reduced polyfunctionality and coproduction of IFN-γ and TNF-α. Cells isolated from the lungs and MLNs of animals infected with influenza virus or coinfected were stimulated with peptide as described previously. Anti-CD107a Ab was included in the stimulation phase to identify cells releasing lytic granules in response to peptide. Following the stimulation period, cells were stained for CD8, tetramer, LFA-1, IFN-γ, and TNF-α. (A) Averaged data evaluating the distribution of the effector function of the influenza-specific response. (B) Percentage of NP-specific IFN-γ–producing cells that coproduced TNF-α. Data are the average from two or three independent experiments assessing a total of 12–18 (d8) or 10 or 11 (d11) mice/infection condition. *p < 0.05, **p < 0.01, ****p < 0.0001, two-tailed Student t test.

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We also determined the extent to which cells that were capable of producing IFN-γ coproduced TNF-α. We found a significant decrease in the percentage of IFN-γ–producing cells in the lungs of coinfected animals that coproduced TNF-α on day 8 post-PR8 infection (Fig. 7B). Thus, TNF-α production appeared to be most susceptible to the negative regulatory effects of EF3030 in the lung, followed by IFN-γ. These data show that cytokine production is more susceptible to negative regulation by the presence of EF3030 compared with cytolytic function.

To begin to understand the regulation of cytokine production at a mechanistic level, we determined whether the failure to produce cytokine was associated with a decrease in cytokine message. On day 8 post-PR8 infection, NP147–155/Kd tetramer+ lung cells from singly or coinfected mice were purified by sorting. This approach allowed isolation of peptide-specific cells, regardless of function. As such, the cells from coinfected animals represented a heterogeneous population of IFN-γ–producing and nonproducing cells. Sorted cells were stimulated with NP peptide for 5 h, RNA was isolated, and qRT-PCR was performed with primer-probe sets specific for IFN-γ, perforin, granzyme B, and GAPDH. As shown in Fig. 8, a significantly reduced level of IFN-γ mRNA was detected in CD8+NP+ cells isolated from coinfected animals compared with singly infected animals. In contrast, no decrease was observed in the level of perforin or granzyme B mRNA, two of the primary components of cytolytic granules. These results mirrored the functional data showing that a population of cells from coinfected animals exhibit cytolytic potential in the absence of cytokine-producing capability. These data suggest that the lack of IFN-γ production by NP-specific CD8+ T cells is regulated at a step prior to transcription.

FIGURE 8.

The reduction in IFN-γ production in NP-specific cells in the lungs of coinfected animals is correlated with decreased IFN-γ mRNA. On day 8 postinfluenza virus infection, CD8+tetramer+ and CD8+tetramer lung cells from singly or coinfected animals were isolated by FACS sorting. The sorted populations were cultured in the presence of NP147–155 peptide for 5 h to induce cytokine production. Following stimulation, mRNA was isolated and IFN-γ, perforin, and granzyme B message was quantified by qRT-PCR. Message levels were normalized to GAPDH. In all cases, the fold increase compared with the level of each mRNA present in tetramerCD8+ T cells from PR8-infected animals was calculated. Data are the average from two independent experiments assessing a total of six influenza-infected or seven coinfected mice. *p < 0.05, **p < 0.01, two-tailed Student t test.

FIGURE 8.

The reduction in IFN-γ production in NP-specific cells in the lungs of coinfected animals is correlated with decreased IFN-γ mRNA. On day 8 postinfluenza virus infection, CD8+tetramer+ and CD8+tetramer lung cells from singly or coinfected animals were isolated by FACS sorting. The sorted populations were cultured in the presence of NP147–155 peptide for 5 h to induce cytokine production. Following stimulation, mRNA was isolated and IFN-γ, perforin, and granzyme B message was quantified by qRT-PCR. Message levels were normalized to GAPDH. In all cases, the fold increase compared with the level of each mRNA present in tetramerCD8+ T cells from PR8-infected animals was calculated. Data are the average from two independent experiments assessing a total of six influenza-infected or seven coinfected mice. *p < 0.05, **p < 0.01, two-tailed Student t test.

Close modal

CD8+ T cell responses are initiated upon engagement of the TCR and CD8 with cognate peptide Ag presented by MHC. TCR/coreceptor engagement induces signaling through Src-family kinase pathways that eventually lead to transcription factors that drive cytokine production (37). One possibility to explain the lack of cytokine production in effectors from coinfected animals is alteration of the TCR-signaling cascade, such that cells could release granules but not produce cytokine. We tested whether these cells were able to appropriately initiate TCR signaling by assessing effector function following addition of PMA and ionomycin (ION). These agents bypass the TCR by directly inducing PKC activation and calcium flux, respectively. Mice were singly infected or coinfected, and lung cells were stimulated with peptide or PMA/ION on day 8 post-PR8 infection. As shown in Fig. 9, PMA/ION stimulation did not increase IFN-γ production in the NP-specific cells isolated from the lungs of coinfected animals compared with that seen with peptide stimulation. These data exclude membrane-proximal defects in TCR signaling as a mechanism to account for the failure of cells from coinfected animals to produce cytokine.

FIGURE 9.

Stimulation with PMA/ION does not promote increased IFN-γ production in influenza-specific CD8+ T cells isolated from the lungs of coinfected animals. On day 8 postvirus infection, cells were isolated from the lungs of animals infected with influenza virus or coinfected and cultured in the presence of NP147–155 peptide or PMA/ION. IFN-γ production in the CD8+LFA-1hitetramer+ population was determined. Data are the average from two independent experiments in which a total of five influenza-infected or eight coinfected mice was individually assessed. **p < 0.01.

FIGURE 9.

Stimulation with PMA/ION does not promote increased IFN-γ production in influenza-specific CD8+ T cells isolated from the lungs of coinfected animals. On day 8 postvirus infection, cells were isolated from the lungs of animals infected with influenza virus or coinfected and cultured in the presence of NP147–155 peptide or PMA/ION. IFN-γ production in the CD8+LFA-1hitetramer+ population was determined. Data are the average from two independent experiments in which a total of five influenza-infected or eight coinfected mice was individually assessed. **p < 0.01.

Close modal

Transpresentation of IL-15 by lung DCs was shown to be an important signal for survival of effector cells (38). Thus, a possible contributor to the loss of cells in our model is a reduction in lung DCs capable of mediating this signal in coinfected animals. To determine whether this was the case, cells were isolated from the lungs of influenza virus–infected or coinfected animals on day 8 following PR8 infection. DC and macrophage subsets in the lung were identified as follows: airway macrophages, high side scatter (SSC) CD11c+CD11blo/−; interstitial macrophages, high SSC CD11c+CD11bint; recruited inflammatory macrophages, high SSC CD11c+CD11bhi; airway DCs, CD11c+CD11bCD103+MHCIIhi; parenchymal DCs, CD11c+CD11b+CD103MHCIIhi; monocyte-derived respiratory DCs, CD11c+CD11b+CD103MHCIIlo/int; and plasmacytoid DCs, CD11cloB220+MHCIIint. This strategy for subset identification is based on previously published results (9, 39). This analysis showed that there was no significant difference in the number of any of the DC subsets (Fig. 10A). Not surprisingly, we observed a significant reduction in the number of airway macrophages in the lungs of coinfected animals (Fig. 10A), consistent with a previous report (9).

FIGURE 10.

Differences in the number of lung DCs or the cytokines IL-15, IL-18, and TGF-β cannot account for the negative regulation of effector cells in coinfected animals. (A) Lung DCs and macrophages were quantified in virus-infected or coinfected animals on day 8 postinfluenza virus infection. DC and macrophage subsets were identified as follows. Live cells were gated on CD11c. Macrophages were identified based on forward/side scatter profile and CD11b staining, with recruited inflammatory macrophages defined as high expressers of CD11b and interstitial macrophages defined as intermediate expressers; alveolar macrophages expressed little or no CD11b. For DC subsets, plasmacytoid DCs were identified by intermediate levels of MHC class II and B220 positivity. Airway DCs were defined by CD103 and MHC class II positivity. Inflammatory monocyte-derived respiratory DCs (MoRDC) were defined by low to intermediate expression of MHC class II and positive staining for CD11b. Parenchymal DCs were defined as MHCIIhiCD11b+CD103. Data shown are the average of nine influenza-infected and seven coinfected mice assessed across two experiments. (BD) Cellular RNA was extracted from lung homogenates on day 8 following influenza virus infection. cDNA was synthesized from mRNA by reverse transcription and subjected to qRT-PCR using primer-probe sets specific for IL-15 (B), IL-18 (C), TGF-β (D), or GAPDH. Fold changes for each animal were calculated based on comparison with the average level of each mRNA detected in PR8-infected animals. Data shown are the average of 15 coinfected and 15 influenza virus–infected animals assayed across two independent experiments. *p < 0.05, two-tailed Student t test.

FIGURE 10.

Differences in the number of lung DCs or the cytokines IL-15, IL-18, and TGF-β cannot account for the negative regulation of effector cells in coinfected animals. (A) Lung DCs and macrophages were quantified in virus-infected or coinfected animals on day 8 postinfluenza virus infection. DC and macrophage subsets were identified as follows. Live cells were gated on CD11c. Macrophages were identified based on forward/side scatter profile and CD11b staining, with recruited inflammatory macrophages defined as high expressers of CD11b and interstitial macrophages defined as intermediate expressers; alveolar macrophages expressed little or no CD11b. For DC subsets, plasmacytoid DCs were identified by intermediate levels of MHC class II and B220 positivity. Airway DCs were defined by CD103 and MHC class II positivity. Inflammatory monocyte-derived respiratory DCs (MoRDC) were defined by low to intermediate expression of MHC class II and positive staining for CD11b. Parenchymal DCs were defined as MHCIIhiCD11b+CD103. Data shown are the average of nine influenza-infected and seven coinfected mice assessed across two experiments. (BD) Cellular RNA was extracted from lung homogenates on day 8 following influenza virus infection. cDNA was synthesized from mRNA by reverse transcription and subjected to qRT-PCR using primer-probe sets specific for IL-15 (B), IL-18 (C), TGF-β (D), or GAPDH. Fold changes for each animal were calculated based on comparison with the average level of each mRNA detected in PR8-infected animals. Data shown are the average of 15 coinfected and 15 influenza virus–infected animals assayed across two independent experiments. *p < 0.05, two-tailed Student t test.

Close modal

As noted above, the ability of lung DCs to provide survival signals is dependent on transpresentation of IL-15 (38). Thus, it was possible that DCs were present but did not produce IL-15 for presentation to CD8+ effector cells. To address this, IL-15 mRNA was measured in the lungs of coinfected or virus-infected animals on day 8 postinfluenza virus infection. No decrease was observed in coinfected lungs; surprisingly, however, there was an ∼2.5-fold increase in the amount of IL-15 mRNA in the lungs of coinfected animals (Fig. 10B). Together, these findings suggest that the increased death in effector cells from the lungs of coinfected animals is not the result of DC loss or the absence of IL-15–mediated survival signals.

IL-18 was shown to provide positive signals for CD8+ T cell cytokine production and survival (40, 41). In addition to provision of positive signals, cytokines can inhibit function. TGF-β is one such well-characterized inhibitory cytokine (42). Previous studies showed that TGF-β is induced following infection with S. pneumoniae (20, 43), making it an appealing candidate for negative regulation in the coinfected lung. IL-18 and TGF-β expression in the lung was assessed in singly and coinfected animals at day 8 postinfluenza virus infection. As shown in Fig. 10C and 10D, no difference in the level of mRNA for these cytokines was detected. These data suggest that the loss of influenza-specific CD8+ T cells in our model is not due to the loss of the supportive cytokine IL-18 or an increase in the inhibitory cytokine TGF-β.

Tregs are another potent mediator of negative regulation. Previous studies indicated that infection with S. pneumoniae or exposure to pneumococcal components can increase Tregs in the lungs of mice (21, 23). To determine whether there was differential expansion/recruitment of Tregs in coinfected versus influenza virus–infected animals, lung cells were analyzed for the presence of CD4+CD25+FoxP3+ cells (Fig. 11). In stark contrast to what was observed for CD8+ effector cells, a significant increase (4.2-fold) in the number of Tregs was observed in the lungs of coinfected animals.

FIGURE 11.

Coinfected animals have a significantly increased number of FoxP3+ Tregs. Cells were isolated from the lungs of singly or coinfected mice on day 8 postinfluenza virus infection. Tregs were detected based on positivity for CD4, CD25, and FoxP3. Data are the average of nine influenza-infected or seven coinfected mice assessed across two independent experiments. *p < 0.05, two-tailed Student t test.

FIGURE 11.

Coinfected animals have a significantly increased number of FoxP3+ Tregs. Cells were isolated from the lungs of singly or coinfected mice on day 8 postinfluenza virus infection. Tregs were detected based on positivity for CD4, CD25, and FoxP3. Data are the average of nine influenza-infected or seven coinfected mice assessed across two independent experiments. *p < 0.05, two-tailed Student t test.

Close modal

Infectious processes have the potential to significantly alter the lung environment. This is the product of signals resulting from tissue damage, cytokines/regulatory factors produced by tissue-resident cells, and innate immune cells that enter as a consequence of the presence of pathogen. Influenza virus is known to extensively modulate the lung environment. For example, infection results in the production of numerous inflammatory cytokines (including type I IFN, IL-6, MCP-1) (44), decreases in the ability of monocytes and neutrophils to phagocytose pathogens, and reduced mucosal ciliary action (1, 9, 10, 45).

Infection with pneumococcus also induces numerous changes to the lung environment. A robust neutrophilic infiltrate is one hallmark of S. pneumoniae infection (46). Other immune-modulatory signals present early postinfection include the production of the proinflammatory cytokines TNF-α, IFN-α/β, IL-1β, and IL-6 (47). In addition, the presence of S. pneumoniae components is associated with the activation of Tregs in the lungs (48). The complex array of immune signals present in this tissue, as well as the direct action of bacterial components, has the potential to regulate adaptive immune responses. Although much attention has been focused on the influenza virus–mediated changes that promote increased bacterial outgrowth (912), to our knowledge, the studies presented in this article are the first to address the impact of S. pneumoniae infection on the ongoing influenza-specific adaptive immune response. Investigation of this question led to the novel finding that the presence of pneumococcus negatively regulates the ongoing antiviral CD8+ T cell response in the lung. Our data show that there is a marked decrease in the total number of CD8+ NP-specific T cells, as well as a change in the distribution of effector function in these cells, with a pronounced shift away from cytokine-producing cells to those that are exclusively cytolytic. Our findings suggest that these changes in the effector population contribute to disease in coinfected animals, because adoptive transfer of influenza-specific cells resulted in increased survival. These data support previously published work demonstrating a strong correlation between the absolute number of IFNγ+CD8+ T cells and disease severity (31). Interestingly, the reduction in effector cell number was not associated with a prolonged period of increased viral load. Although higher levels of virus were detected in coinfected animals 1 d following S. pneumoniae infection, virus was similar in influenza virus–infected and coinfected animals by 4 d postinfection. Thus, there was not a direct relationship between virus load and disease. Previous studies reported that, under some circumstances, damaging inflammation is not correlated with influenza virus load (49, 50). The increase in survival observed following adoptive transfer leads to the intriguing hypothesis that the reduced virus-specific effector cell number/function alters the balance of cytokines or inflammatory cells in the lungs of coinfected animals and this contributes to disease. Altered production of IFN-γ by CD8+ effector cells was shown to affect the inflammatory milieu present following influenza virus infection (51). In addition, TNF-α has effects on multiple inflammatory cell types (52). Although not yet evaluated, it is also possible that chemokine production is dysregulated in influenza-specific effector cells. CD8+ T cells can produce a number of chemokines that regulate the recruitment and function of a broad array of cells (53, 54). In disease processes in which individuals survive past the initial phase of bacterial infection (24–48 h), as is the case in our model, a reduction in cytokines/chemokines has the potential to significantly impact ongoing inflammation and/or the response to tissue damage. Additional studies are warranted to gain a fuller understanding of the role of influenza-specific effector cell regulation in the disease observed in our model.

The reduction in effector cell number in coinfected animals is the result, in part, of increased death in cells residing in the coinfected lung. The defect in cytokine production is correlated with a decrease in cytokine mRNA. The failure of PMA/ION to induce cytokine production suggests that alteration of the membrane-proximal TCR signal-transduction pathway is not responsible for the lack of cytokine production. This finding suggests that these cells may be inherently incapable of producing cytokine. One possibility is that cells that are exclusively cytolytic preferentially survive in the coinfected lung. Given the death in our model, this is an attractive possibility that warrants further investigation. Alternatively, epigenetic changes induced as a consequence of signals present in the coinfected lung environment may have resulted in shut-off of cytokine production. Epigenetic regulation is a well-described mechanism for the control of cytokine gene expression in T cells (5557).

The question arises of whether the negative regulation of the adaptive immune response is due to changes in the immune environment or a direct effect of bacterial components. Immune signals in the form of cytokines have been shown to regulate the CD8+ T cell antiviral response (38, 40, 41). For example, IL-15 production by lung DCs was identified as a critical signal for the survival of influenza-specific CD8+ effector cells in the lung (38). However, our findings suggest that this is not the case in our model, because IL-15 expression in the lungs of coinfected animals was higher than that in animals infected only with influenza virus. IL-18 also was shown to promote the generation and sustained presence of functional effector T cells. Cells with an “exhausted” phenotype downregulate IL-18R, fail to produce cytokine, and have been implicated in the susceptibility to secondary bacterial infections (40). However, as was the case for IL-15, IL-18 expression was maintained in the lungs of coinfected animals and thus is also unlikely to be involved in the death of effector cells or the decrease in cytokine production observed in our model. An opposing factor to the positive signals delivered by cytokines is the presence of negative regulators (i.e., TGF-β). This immunomodulatory cytokine is known to be increased following S. pneumoniae infection (20, 43). Interestingly, we did not observe any differences in the amount of detectable TGF-β in the coinfected versus influenza virus–infected lung.

Finally, we evaluated the presence of FoxP3+ Tregs. Despite the decrease in influenza-specific effectors, there was a significant increase in Tregs in the lungs of coinfected mice. This was somewhat surprising given the similar levels of TGF-β detected. One possibility is that cytokine production is impaired in these cells, similar to the reduction in cytokine in virus-specific effector cells. Alternatively, other sources of TGF-β may mask the contribution of Tregs when overall levels in the lung are assessed. Tregs use a number of mechanisms to negatively regulate T cells, including production of adenosine, direct killing through the release of perforin and granzymes, and increases in intracellular cAMP (reviewed in Ref. 58). Whether these cells are directly involved in effector cell death or altered function requires further study.

In addition to the possibility for Treg-mediated regulation effectors, there is evidence that bacterial components can directly impact T cell function (22). In vitro studies suggest that the pneumococcal cholesterol–dependent cytotoxin pneumolysin can lead to lymphocyte death in a Fas-dependent manner when monocytes are present (27). Although this is an appealing model given the death observed in our system, Fas-mediated death would be expected to result in activation of caspase-3 (59), which was not detected in lung cells from coinfected animals. However, in the same study, a necrotic death pathway could be triggered when monocytes were not present. This latter possibility is most consistent with the absence of active caspase-3 in dying cells observed in our model. The potential role of pneumolysin warrants further investigation.

It is important to note that, despite sustained levels of bacteria at day 11, the IAV-specific CD8+ T cell response has become more similar in both size and quality to that of animals singly infected with PR8. This could be explained, in part, by the fact that levels of virus are at their lowest by day 11. It is feasible that the increased death and alterations of CD8+ T cell effector function in our model are dependent on signals from the presence of both pathogens. Alternatively, S. pneumoniae is known to form biofilms as part of its infectious life cycle (60). One consequence of biofilm formation is the reduction of autolysis that S. pneumoniae uses to release virulence factors, such as pneumolysin, to the surrounding environment (60). It is possible that EF3030 exists in a biofilm state by day 11 compared with day 8 post-IAV infection. If this were the case, bacteria may no longer be releasing components that could modulate the IAV-specific CD8+ T cell response. Alternatively, this could indicate that bronchus-associated lymphoid tissue (BALT) formation is impaired early by the presence of the bacteria. BALT was shown to be a critical early component for the generation and maintenance of adaptive immune responses in the lung (61). If inducible BALT formation was inhibited by EF3030, this could lead to fewer IAV-specific CD8+ T cells in the lung at day 8 post-IAV infection. By day 11, IAV-specific CD8+ T cells infiltrating the lung from the MLN could be reconstituting the IAV-specific CD8+ T cell response in the lung, which would be consistent with our data.

The consequences of S. pneumoniae coinfection on the influenza-specific response may extend beyond the acute CD8+ T cell response assessed in this study. For example, the reduced number of effector cells may impact the number of tissue-resident CD8+ memory T cells generated at the conclusion of the response. This could be exacerbated if the presence of S. pneumoniae drove differentiation toward short-lived effector cells, which are unlikely to survive long term, further decreasing the memory pool. S. pneumoniae–mediated increases in inflammatory cytokines, which are known to direct differentiation along the short-lived effector cell pathway (62), is an attractive mechanism for potential altered regulation of effector cell differentiation. Specifically, S. pneumoniae infection subsequent to influenza was shown to synergistically increase the level of type I IFN (63). In addition, it is possible that regulation of the acute effector pool impacts the quality of the memory cells (e.g., their cytokine-producing potential). A reduced or impaired tissue-resident memory pool in individuals who experienced coinfection would increase their susceptibility to reinfection with IAV. This is currently an area of study. Finally, our finding of increased cell death in coinfected animals raises the possibility that established tissue-resident memory CD8+ T cells specific to other respiratory pathogens that are present in the lung during coinfection may be negatively impacted. Our results raise the interesting possibility that coinfection with IAV and pneumococcus could potentiate the deletion of established cellular immunity in the lung environment.

In summary, our studies provide exciting new insights into polymicrobial disease states. Specifically, we show that coinfection with S. pneumoniae results in a marked decrease in the number of NP-specific CD8+ T cells in the lung. Further, surviving influenza-specific effector cells exhibited altered effector function (i.e., the reduced production of cytokine) at the population level. Importantly, our data support a role for these changes in the high mortality observed following coinfection. The loss of IFN-γ production in a subset of cells was associated with a decrease in the level of IFN-γ mRNA. The negative effects on the influenza-specific effector cell population were independent of changes in lung DC populations and were not associated with decreased IL-15 or IL-18 or increased TGF-β expression. However, there was an association with increased Tregs in the lungs of coinfected animals. These studies establish the ability of S. pneumoniae to modulate the ongoing adaptive immune response to an existing pathogen. It is likely that coinfecting pathogens are common in the population; thus, there is significant opportunity for cross-regulation of immune responses, similar to that reported in this article. Such cross-regulation is a potential contributor to pathogenesis and immunity in vivo.

We thank Drs. Jason Grayson and Karen Haas (Wake Forest University School of Medicine, Winston-Salem, NC) for discussions regarding this project. Swine Influenza A (H1N1) Real-Time RT-PCR Assay, NR-15577 was obtained through the National Institutes of Health Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Manassas, VA). We acknowledge the National Institutes of Health Tetramer Core Facility (Contract HHSN272201300006C) for provision of allophycocyanin-conjugated NP147–155/Kd tetramers. We acknowledge services provided by the Cell and Viral Vector Core and Flow Cytometry Core Laboratories of the Wake Forest University School of Medicine Comprehensive Cancer Center (Winston-Salem, NC), which is supported in part by National Cancer Institute P30 CA121291-37.

This work was supported by National Institutes of Health Grant 5-T32AI007401-22 (to L.K.B.), National Institutes of Health-National Institute on Deafness and Other Communication Disorders Grant R01DC10051 (to J.T.W.), and the Louis Argenta Physicians Scientist Scholarship (to J.T.W.).

Abbreviations used in this article:

     
  • 7-AAD

    7-aminoactinomycin D

  •  
  • BALT

    bronchus-associated lymphoid tissue

  •  
  • DC

    dendritic cell

  •  
  • EID50

    median egg infectious dose

  •  
  • IAV

    influenza A virus

  •  
  • i.n.

    intranasal(ly)

  •  
  • ION

    ionomycin

  •  
  • MLN

    mediastinal lymph node

  •  
  • PR8

    influenza A/PR/8/34

  •  
  • qRT-PCR

    quantitative real-time PCR

  •  
  • SSC

    side scatter

  •  
  • Treg

    regulatory T cell.

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The authors have no financial conflicts of interest.