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. Author manuscript; available in PMC: 2014 Nov 14.
Published in final edited form as: Immunity. 2013 Nov 14;39(5):10.1016/j.immuni.2013.09.013. doi: 10.1016/j.immuni.2013.09.013

Lung airway-surveilling CXCR3hi memory CD8+ T cells are critical for protection against Influenza A virus

Bram Slütter 1, Lecia L Pewe 1, Susan M Kaech 2,3, John T Harty 1,*
PMCID: PMC3872058  NIHMSID: NIHMS537237  PMID: 24238342

Summary

Inducing memory CD8+ T-cells specific for conserved antigens from Influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8 T-cells in the airways played an important role in early control of IAV. Expression of chemokine receptor CXCR3 was critical for memory CD8 T-cells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8 T-cells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8 T-cells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12, enhanced CXCR3 expression, sustained airway localization of memory CD8 T-cells and resulted in superior protection against IAV.

Introduction

Antibody inducing Influenza A virus (IAV) vaccines are available, however protection is suboptimal (Osterholm et al., 2012) and requires annual reformulation of the vaccine. IAV can escape neutralization by preexisting antibodies due to the high rate of mutagenesis in the primary targets of neutralization (hemagglutinin, HA and neuraminidase, NA) and due to its capacity to recombine in non-human hosts. In the absence of neutralizing antibodies, memory CD8+ T-cell specific for epitopes located in conserved regions of IAV proteins like the internal components nucleoprotein (NP), polymerase A and matrix protein may confer protection (Christensen et al., 2000; Heiny et al., 2007; Liang et al., 1994). However, the contribution of memory CD8+ T-cells to protection against IAV is still under debate, as vaccination or previous IAV exposure does not protect from subsequent heterosubtypic IAV infection (Steinhoff et al., 1993; Wilkinson et al., 2012). Boosting the numbers of broadly protective memory CD8+ T-cells may be a strategy to bolster their protective capacity (Slütter et al., 2013), however little is known about the functional requirements for robust CD8+ T-cell mediated protection against IAV. Memory CD8+ T-cells constitute a very heterogeneous population, in terms of their capacity to proliferate, generate cytokines/cytolytic mediators and the tissues in which they reside (Jameson and Masopust, 2009). For instance, multifunctional memory CD8+ T-cells that produce IFN-y, TNF-α and IL-2 after restimulation are generally associated with more robust proliferation and protection against viral infections (Nolz and Harty, 2011; Seder et al., 2008). Additionally, variation in the expression of selectins, integrins and chemokine receptors leads to differential migratory patterns and localization of memory CD8+ T-cells (Gebhardt and Mackay, 2012; Hikono et al., 2007; Sallusto et al., 1999), which can impact their protective capacity (Jiang et al., 2012). Determining the optimal memory CD8+ T-cell characteristics for protection against IAV and understanding how such memory could be generated will be crucial to future influenza vaccine development.

Here we report that the cytokine milieu evoked during primary or booster immunization can greatly influence the protective capacity of memory CD8+ T-cells against IAV, by altering CXCR3 expression and the capacity of memory CD8+ T-cells to survey the respiratory tract.

Results

The protective capacity of IAV specific memory CD8+ T-cells is shaped by the booster agent

We previously reported an accelerated prime-boost strategy to induce large numbers of memory CD8+ T-cells (Badovinac et al., 2005; Pham et al., 2010; Schmidt et al., 2008) and adopted this protocol to establish a large NP-specific memory CD8+ T-cell population. Priming BALB/c mice with mature dendritic cells coated with the H2-Kd restricted NP147-155 epitope (DC-NP147) and boosting with attenuated Listeria monocytogenes expressing IAV NP (LM-NP) resulted in a large memory CD8+ T-cell population (Figure S1a), which was sufficient to protect mice from a lethal A/PR/08/34 (PR8) infection (Figure S1b). Depletion of CD8+ T-cells prior to challenge rendered prime-boosted mice completely susceptible to lethal PR8, demonstrating that protection was CD8+ T-cell mediated (Figure S1b).

To assess whether the booster agent influences the protective capacity of memory NP-specific CD8+ T-cells, DC-NP147 primed mice were boosted with LM-NP or vaccinia virus expressing NP (VV-NP). LM-NP and VV-NP boosting resulted in similar high frequencies of NP147-specific memory CD8+ T-cells in blood (Figure 1a). However, although LM-NP and VV-NP boosted mice both survived lethal infection with PR8, the VV-NP boosted mice exhibited less morbidity and recovered more rapidly than their LM-NP boosted counterparts (Figure 1b). This suggested that VV-NP boosted mice more effectively controlled the IAV infection. Indeed, as early as 2–4 days post IAV infection, the viral titer in the lungs of VV-NP boosted mice was significantly lower than in the lungs of LM-NP boosted mice (p<0.05, Figure 1c). Thus despite inducing a similar frequency of IAV specific memory CD8+ T-cells in the blood, VV-NP boosting provided superior protection and accelerated viral clearance compared to LM-NP boosting.

Figure 1. Boosting with LM and VV results in NP-specific CD8+ T-cell memory populations of similar size but differential ability to protect against IAV infection.

Figure 1

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a) BALB/c mice were primed with DC coated with NP147 and boosted 1 week later with either LM-NP or VV-NP. Percentage of circulating NP147-specific CD8+ T cells was determined 45 days post boost. Each dot represents a single mouse. b) The prime-boosted mice and naïve mice were challenged with a lethal dose of PR8. Morbidity was assessed daily and the number of surviving/total challenged mice are indicated and c) viral titers in lung were determined at day 2, 4 and 6 post infection. n=5 +/−SEM, representative of 4 experiments * p<0.05 Also see Fig S1.

The superior protection of VV-NP boosted mice was not mediated by CD4+ T-cells or NP-specific antibodies as CD4+ T-cell depletion of VV-NP boosted mice or transfer of serum contain NP-specific antibodies to LM-NP boosted mice prior to lethal infection did not affect protection against IAV (Fig S1c, d). In contrast depletion of CD8+ T-cells prior to IAV challenge rendered both LM-NP and VV-NP boosted mice completely vulnerable to lethal PR8 infection (Figure S1e). To determine whether the NP147-specific CD8+ T-cells from LM-NP and VV-NP boosted have a different protective capacity against IAV, CD8+ T-cell enriched splenocytes from both groups were transferred and recipient mice were challenged with a lethal dose of PR8. Although the CD8+ T cell recipient mice lost substantially more weight after challenge than those that were prime-boosted (Figure 1b), transfer of 3×105 NP147-specific memory CD8 T-cells from VV-NP boosted mice was sufficient to protect mice from lethal IAV infection. In contrast, transfer of the same number of LM-NP boosted memory CD8+ T-cells was not protective (Figure S1f, g). Therefore these data show VV-NP boosting resulted in memory CD8+ T-cells that were, on a per cell basis, more protective against IAV infection than those that were induced by LM-NP boosting.

Similar function but different localization of LM-NP and VV-NP boosted memory CD8+ T-cells

CD8+ T-cells can provide protection by killing infected cells and through production of cytokines. Variation in amounts of serine proteases like Granzyme B and/or a differential ability to produce cytokines could explain the superior protective capacity of VV-NP boosted IAV specific memory CD8+ T-cells. However after peptide stimulation similar frequencies of LM-NP and VV-NP boosted NP147-specific memory CD8+ T-cells produced IFN-γ, TNF-α and IL-2 (Figure S2a), exhibited no difference in Granzyme B expression (Figure S2b) and exhibited similar antigen sensitivity for cytokine production, when obtained from the spleen (Supplemental Figure 2c) as well as the lung (data not shown). This suggests that LM-NP and VV-NP boosting resulted in memory CD8 T-cells with a similar capacity to kill infected cells and produce cytokines.

Because we found no differences in circulating CD8+ T-cell numbers and effector functions we next evaluated the distribution of the memory populations. Along with similar frequencies in the blood, we observed equal numbers of NP147-specific CD8+ T-cells in the spleen (Figure 2a). Moreover, perfused lungs, which may still contain CD8+ T-cells in lung capillary beds (Anderson et al., 2012), had similar total numbers of NP147-specific CD8 T-cells in LM-NP and VV-NP boosted mice. In contrast, we found 7x more NP147-specific CD8+ T-cells in bronchio-alveolar lavage (BAL) fluid from VV-NP boosted mice compared to LM-NP boosted mice (p<0.05 Figure 2a). Thus, as the cellular contents of the BAL fluid correlates with the cellular contents of the airways (Papiris et al., 2005; Weiland et al., 1989), LM-NP and VV-NP boosting resulted in differential localization of memory CD8+ T-cells, with VV-NP boosting favoring the establishment of memory CD8+ T-cells lining the airways.

Figure 2. NP147-specific memory CD8+ T-cells in the airway mediate early control of IAV infection.

Figure 2

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a) Number of NP147-specific CD8+ T-cells in blood, spleen, lung and airways determined by tetramer analysis at day 50 after boosting with either LM-NP or VV-NP. b) VV-NP boosted memory mice received 5 μg Rat IgG or αCD8 intranasally. After 24 hours CD8 T-cells in BAL fluid and lungs were analyzed by staining for Thy1.2 and CD8β. c–d) LM-NP and VV-NP boosted memory mice received 5 μg Rat IgG or αCD8 intranasally. After 48 hours mice were challenged with a lethal dose of PR8. Morbidity was assessed daily (c) and viral titers were determined 4 days post infection (d). n=5 +/− SEM, representative of 3 experiments * p<0.05. Also see Fig S2.

Although we consistently observed significantly more NP147-specific memory CD8+ T cells in the BAL after VV-NP boosting, the recovered numbers were low. This was likely due, in part to inefficient recovery of respiratory CD8+ T cells during BAL lavage and thus, these numbers were likely a substantial underestimate of the actual populations. To assess whether the NP147-specific memory CD8+ T-cells lining the airways mediated the enhanced protection against IAV, we developed a local depletion strategy that drastically decreased the number CD8+ T-cells recovered by BAL, but did not affect the total number of CD8+ T-cells recovered from the lung (Figure 2b). LM-NP and VV-NP boosted memory mice were treated with control IgG or 5 μg αCD8 intranasally, followed 48 hours later by lethal infection with PR8. Strikingly, intranasal αCD8 treatment enhanced morbidity (Figure 2c) and increased viral titers (Figure 2d) in VV-NP boosted mice such that they resembled Ig-treated LM-NP boosted mice. However, morbidity and viral clearance in LM-NP boosted memory mice, which contained very few NP147-specific CD8+ T-cells in the airways at the time of challenge, was not affected by local CD8+ T-cell depletion. This is consistent with CD8+ T-cells in or lining the airways mediating the additional protection observed in VV-NP boosted mice.

IAV specific memory CD8+ T-cells situated in or in close proximity to the airways could be perfectly located to detect infection early and remove infected cells, thereby limiting viral replication. Additionally, the number of these cells may rapidly increase through local proliferation (Cuburu et al., 2012) or enhanced recruitment of additional memory CD8+ T cells from the systemic circulation (Schenkel et al., 2013). To assess whether protection in VV-NP boosted mice is mediated by the pre-existing memory CD8+ T-cells in the airways or through rapid proliferation or migration, the number of NP147 specific CD8+ T-cells in the BAL fluid of LM-NP and VV-NP boosted mice was determined during the first 4 days post IAV infection. Although the number of NP147 specific CD8+ T-cells in the BAL fluid was higher in VV-NP than LM-NP boosted mice throughout the evaluated time period (Figure S3a), no significant increase in either group was observed during this early period after challenge (p>0.05), suggesting limited proliferation or recruitment of CD8 T-cells during the first 4 days after IAV infection. However, NP147 specific CD8+ T-cells in the BAL rapidly expressed CD69 following IAV infection (Figure S3b), which suggested recent antigen encounter. Thus, this suggested that the increased protection by NP147 specific memory CD8+ T-cells in VV-NP boosted mice was mediated through the pre-existing local memory CD8+ T-cell population in the airways.

Memory CD8+ T-cells are recruited into the respiratory tract under steady state conditions

Having established that CD8+ T-cells in the airways mediated enhanced protection against IAV infection, we next investigated how VV-NP boosting resulted in a larger memory CD8+ T-cell population in the BAL fluid than LM-NP boosting. Similar numbers of NP147-specific CD8+ T-cells in the BAL fluid were observed at 8 days post boosting with LM-NP and VV-NP (Figure 3a). However this population contracted more sharply in LM-NP boosted mice, and resulted in more memory NP147-specific CD8+ T-cell in the respiratory tract of VV-NP boosted mice by day 20 and beyond (Figure 3a). These data suggested that either more VV-NP boosted memory CD8+ T-cells survived the contraction phase in the BAL fluid and progressed into memory, or more VV-NP boosted memory CD8+ T-cells continued to traffic into the airways over time. To address these possibilities, CD8+ T-cells in airways were locally depleted on day 7 post VV-NP boost, which effectively reduced the effector population in the BAL fluid on day 8. Despite this early depletion the NP147-specific memory CD8+ T-cell population at day 40 after depletion in the airways was similar to the memory populations found in control Ig treated VV-NP boosted animals (Figure 3b). This suggested that airway memory CD8+ T-cells were not resident memory (Jiang et al., 2012; Masopust et al., 2006) but were continually repopulating the airways under steady state conditions.

Figure 3. Memory CD8+ T-cells derived from VV-NP boosted mice traffic to the airways more efficiently than memory CD8+ T-cells from LM-NP boosted mice.

Figure 3

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a) Number of NP147-specific CD8+ T-cells in BAL fluid at the indicated days after LM-NP or VV-NP boost. b) VV-NP boosted mice received 5 μg Rat IgG or αCD8 intranasally 6 days post boost. Number of NP147-specfic CD8+ T-cells in the BAL fluid was determined at the indicated time points c) Splenocytes containing equal numbers of NP-specific memory CD8+ T cells (Thy1.2) from LM-NP or VV-NP boosted mice were transferred into Thy1.1 recipients. Total numbers of NP147-specific CD8+ T-cells in the BAL fluid and lungs were determined 5 days post transfer. Mean n=5 +/− SEM, representative of 2 experiments * p<0.05 Also see Fig S3.

To directly determine if memory CD8+ T-cells can traffic to the airways from the circulation during the steady state, T-cells from spleens of LM-NP and VV-NP boosted memory mice were transferred intravenously into naïve recipients. Within days after transfer, donor CD8+ T-cells were detected in the BAL fluid (Figure 3c), showing that memory CD8+ T-cells could traffic into the respiratory tract during the steady state. Consistent with the results in immunized mice, more transferred VV-NP boosted CD8+ T-cells were detected in BAL fluid than LM-NP boosted CD8+ T-cells, suggesting the VV-NP boosted memory CD8+ T-cells had a superior ability to traffic to the respiratory tract under steady state conditions.

CXCR3 mediates memory CD8+ T-cell trafficking into the airways under steady state conditions

The observation that memory CD8+ T-cells could traffic into the airways during the steady state prompted us to investigate the requirements for memory CD8+ T-cells to reach the BAL fluid. Analysis of a variety of surface molecules associated with T-cell migration such as CD44, core 2 O-glycosylated CD43 (1B11), CD62L, CD103, CXCR3 and CCR5, show that NP147-specific memory CD8+ T-cell in the BAL fluid were exclusively CD44hi, CD62Llo, CD103lo (Figure S4) and CXCR3hi (Figure 4a). Of note, the uniform CXCR3 expression on NP147-specific CD8+ T-cells in the BAL fluid was distinct from the fraction of cells expressing CXCR3 in the spleen (Figure 4a). Moreover, expression of CXCR3 was significantly (p>0.05) higher on VV-NP boosted than LM-NP boosted circulating memory CD8+ T-cells (Figure 4b) and the CXCR3hi cells were also VLA-1hi (Figure S4b) indicative of a lung tropism (Ray et al., 2004). These data strongly suggested a role for CXCR3 in the localization of memory CD8 T-cells to the respiratory tract and may potentially explain the superior numbers in the BAL of VV-NP boosted mice.

Figure 4. CXCR3 expression mediates memory CD8+ T-cell migration to the BAL and enhances protection against IAV.

Figure 4

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a) CXCR3 expression on NP147-specific memory CD8+ T-cells in the BAL, lung and spleen of VV-NP boosted mice. b) CXCR3 expression on NP147-specific memory CD8 T-cells in the PBL. C) 5000 P14 or 5000 Cxcr3−/− P14 were transferred into naïve recipients. Mice were primed with DC-GP33 and boosted with VV-GP33. Numbers of P14 in the BAL fluid, lung and spleen were quantified at day 40 post boost. d) 106 Wildtype (Thy1.1/1.1) and Cxcr3−/− memory P14 (Thy1.1/1.2) were transferred in a 1:1 ratio to naïve recipients. After 5 days the ratio of WT:Cxcr3−/− P14 was determined in the spleen, lung and BAL. e) Wildtype and Cxcr3−/− memory P14 generated by priming with DC-GP33 and boosting with either LM-GP33 or VV-GP33 were transferred to naïve recipients and challenged with PR8-GP33. Viral titers in the lung were determined 3 days post infection. Bars represent mean n=5 +/− SEM, representative of 3 experiments. *p<0.01. Also see Fig S4.

Thus we next asked whether CXCR3 expression is required to establish a memory CD8+ T-cell population in the respiratory tract. LM and VV boosting in C57Bl/6 mice led to similar differences in CXCR3 expression on memory CD8+ T-cells as in BALB/c mice (data not shown). Therefore we switched to C57Bl/6 mice, enabling us to use P14 transgenic CD8 T-cells and IAV expressing the GP33 epitope. Wildtype (WT) and Cxcr3−/− P14 memory CD8 T-cells were generated through DC-GP33 prime and VV-GP33 boosting, ensuring that the WT P14 were CXCR3hi (data not shown). At a memory time point, similar numbers of WT and Cxcr3−/− P14 were detected in the spleen and lung, but significantly (p>0.05) more WT memory P14 were found in the BAL fluid compared to Cxcr3−/− memory P14 (Figure 4c). Thus the absence of CXCR3 impaired the formation of a memory CD8 T-cell population in the airways. Secondly, we asked whether CXCR3 plays a direct role in the trafficking of memory P14 to the BAL under steady state conditions. To address this we transferred a 1:1 mixture of WT and Cxcr3−/− memory P14 into the same recipients and analyzed the distribution of these populations in the spleen, lung and BAL fluid after 5 days (Figure 4d). WT memory P14 had a marked advantage for accumulating in the BAL fluid, whereas equal numbers of WT and Cxcr3−/− P14 were recovered from the spleen and perfused lung. As VLA-1 expression was pronounced on CXCR3hi cells (Figure S4b) and VLA-1 (α1β1 integrin) has been implicated in the localization of memory CD8 T-cells to airways (Ray et al., 2004) this integrin may provide an alternative explanation for the decreased accumulation of Cxcr3−/− memory P14. However, no difference in VLA-1 expression of WT and Cxcr3−/− memory P14 was observed (Figure S4c). Finally, Cxcr3−/− memory P14 were significantly less able to control IAV expressing GP33 then WT memory P14 (p<0.01, Figure 4e). Overall these data established that CXCR3 was directly involved in localization of memory CD8 T-cells to the airways under steady state conditions.

CXCR3 ligands CXCL9 and CXCL10 are IFN-γ inducible chemokines and are highly expressed in inflamed tissue. Whether these ligands are present in lung tissue during the steady state is unclear. Using IFN-γ deficient mice as a negative control, we showed a large fraction of wildtype C57Bl/6 mice contained detectable Cxcl9, Cxcl10 and Ifng mRNA in their lungs as detected by qPCR (Figure S4d), indicating that IFN-γ inducible chemokines, could play role in the accumulation of CD8 T-cells in the airways during the steady state.

IL-12 signaling diminished CXCR3 expression on memory CD8+ T-cells and localization to the airways

Differential CXCR3 expression between LM-NP and VV-NP boosted mice could play a critical role for trafficking of memory CD8+ T-cells to BAL and subsequent protection against IAV. A major difference between LM and VV is the inflammatory milieu they generate upon infection (Keppler et al., 2012; Wirth et al., 2011). Specifically LM infection drove substantially higher serum amounts of IL-12 and type I interferons than VV infection (Figure S5a). To address whether these cytokines modulated CXCR3 expression on memory CD8 T-cells WT, IL-12 receptor deficient (Il12rb1−/−) and type I interferon receptor deficient (Ifnαβr−/−) B6 mice were primed with DC-OVA257 and infected with attenuated LM expressing ovalbumin (LM-OVA). CXCR3 expression on OVA257-specific effector and memory CD8+ T-cells was unaltered in Ifnαβr−/− mice, but was increased in Il12rb1−/− mice when both were compared to WT mice (Figure 5a). These data suggested IL-12R signaling reduced CXCR3 expression on CD8+ T-cells. To assess whether this resulted from CD8+ T-cell intrinsic IL-12 signaling, WT and Il12rb1−/− OT-I T-cells were transferred into the same WT hosts, primed with DC-OVA and boosted with LM-OVA. A higher fraction of Il12rb1−/− OT-I expressed CXCR3 than WT OT-I at the effector as well as the memory stage (Figure 5b). This demonstrated that direct IL-12R signaling to CD8+ T-cell had a negative effect on the expression of CXCR3.

Figure 5. IL-12 receptor signaling reduced CXCR3 expression and localization to airways.

Figure 5

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a) WT, Il12rb1−/− and Ifnαβr−/− B6 mice were infected with 106 LM-OVA and CXCR3 expression on OVA257-specific CD8+ T-cells was tracked over time. b) 500 WT OT-I (Thy1.2/1.2, CD45.2) and 500 Il12rb1−/− deficient OT-1 (Thy1.1/1.2, CD45.2) were co-transferred and CD45.1 recipients were primed with DC-OVA and infected on day 7 with 106 LM-OVA. Expression of CXCR3 on OT-1 was determined at effector (day 7) and memory (day 45) time points after boosting. c) WT (Thy1.1/1.2) and Stat4−/− (Thy1.2/1.2) BALB/c bone marrow were introduced (in a 1:1 ratio) in irradiated BALB/c mice (Thy1.1/1.1) and after 2 months, chimeric recipients were primed with DC-NP147 and boosted with LM-NP. Panels shown were gated on KdNP147 tetramer positive CD8+ T-cells. CXCR3 expression on NP147-specific CD8+ T-cells was determined on day 7 and 41 post boost. d) At day 35 post boost the ratios of Stat4−/− and WT NP147-specific CD8+ T-cells were determined in blood, spleen, lung and BAL fluid. e) WT (Tbx21+/+) and Tbx21+/− P14 were transferred and prime-boosted using DC-GP33 and LM-GP33. Expression of CXCR3 was assessed at day 7 and day 41 time points. f) At the memory time point numbers of P14 in the spleen and BAL were determined. Bars represent mean n=5 +/− SEM, representative of 3 experiments. *p<0.001. Also see Fig S5.

To evaluate how signaling downstream of IL-12R leads to reduction of CXCR3 surface expression, we assessed the role of STAT4. WT (Thy1.1/1.2) and Stat4−/− (Thy1.2/1.2) BALB/c bone marrow was introduced into irradiated BALB/c recipients (Thy1.1/1.1) to generate chimaeras. After reconstitution mice were primed with DC-NP147 and boosted with LM-NP. The circulating NP147-specific CD8+ T-cell populations at day 7 and day 32 post boost were numerically larger for WT-derived CD8+ T-cells (Figure 5c). However less than 30% of the WT NP147-specifc CD8+ T-cells were CXCR3hi at these time points, whereas more than 70% of the Stat4−/− NP147-specific CD8+ T-cells were CXCR3hi (Figure 5c). Additionally, although the overall size of the WT NP147-specific memory population in the spleen and lung was larger, Stat4−/− NP147-specific memory CD8+ T-cell accumulated more effectively in the airways (Figure 5d).

Chipseq experiments have not identified the CXCR3 promoter as a potential binding site for STAT4 (Good et al., 2009), suggesting the CXCR3 repressing effect may be further downstream of this transcription factor. IL-12 and STAT4 signaling are reported to sustain levels of the T-box transcription factor T-bet in activated CD8+ T-cells (Takemoto et al., 2006; Yang et al., 2007). Correspondingly, T-bet expression in LM-NP boosted NP147-specific memory CD8 T-cells was higher than in VV-NP boosted memory CD8+ T-cells (Figure S5b). To evaluate the role T-bet on localization of memory CD8+ T-cells in the airways, heterozygous (Tbx21+/−) and WT (Tbx21+/+) P14 CD8+ T-cells were transferred into WT recipients and prime-boosted with DC-GP33 and LM-GP33. As reported previously (Joshi et al., 2011) CXCR3 expression in Tbx21+/− P14 was greatly increased compared to WT P14, both at an effector as well as a memory timepoint (Figure 5e), demonstrating that high levels of T-bet correlated with reduced CXCR3 expression on CD8+ T-cells. In line with the differences in CXCR3 expression, Tbx21+/− P14 established a larger memory population in the BAL fluid compared to Tbx21+/+ P14 (Figure 5f). Importantly, reduction of T-bet did not decrease the total number of systemic memory CD8 T-cells, as similar numbers of memory Tbx21+/+ and Tbx21+/− P14 were retrieved from spleens >40 days post boost (Figure 5f). Thus a reduction in T-bet expression did not affect the size of the systemic memory P14 population, but reduced the expression of CXCR3 and localization of memory CD8+ T-cells in the airways.

Neutralization of IL-12 increases protective capacity of IAV memory CD8+ T-cells

We next assessed whether manipulation of IL-12 signaling during boosting enhanced the localization of memory CD8+ T-cells and improved their protective capacity against IAV. Mice were treated with αIL-12 or control IgG on day 0 and 1 after LM-NP boost, when serum levels of IL-12 peaked (Figure S5a). Neutralization of IL-12 resulted in no discernable difference in the NP147-specific CD8+ T-cell frequency in the blood (Figure 6a,b). However, neutralization of IL-12 significantly increased (p<0.01) the frequency of CXCR3hi NP147-specific CD8+ T-cell both at effector and memory time points (Figure 6a,b). Consistent with this, αIL-12 treatment during LM-NP boost increased the number of NP147-specific memory CD8+ T-cells in BAL fluid, but not spleen or lung (Figure 6c). Finally, mice treated with αIL-12 during boosting with LM-NP exhibited significantly lower viral titers (p<0.001) after challenge with PR8 (Figure 6d). This showed that neutralization of IL-12 during booster immunization enhanced localization of the resulting memory CD8+ T-cell population to the airways and increased protection against IAV.

Figure 6. Neutralization of IL-12 during boost improves the protective capacity of memory CD8 T-cells.

Figure 6

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DC-NP primed BALB/c mice were boosted with 106 LM-NP. On the day of boosting and the day after mice received αIL-12 (clone C17.8) or control antibody. a) Representative dot plots of NP147-specific CD8+ T-cells quantified using KdNP147 tetramers and CXCR3 expression on the KdNP147 positive cells. b) The percentage of NP147-specific CD8+ T-cells and their CXCR3 expression in the blood. c) On day 44 post-boost numbers of NP147-specific CD8+ T-cells in the BAL was determined. d) Peak viral titers (day 4) post infection with a lethal dose of PR8. n=5 +/− SEM, representative of 3 experiments *p<0.05.

From a vaccination perspective it may be more feasible to avoid using vaccine vectors or adjuvants that induce high systemic IL-12 levels, rather than neutralizing IL-12 after the immunization. Whereas the TLR9 agonist CpG oligonucleotides triggered a potent IL-12 response, the TLR3 agonist PolyIC primarily induced type I IFN (Figure 7a). To assess whether these 2 adjuvants would induce memory CD8+ T-cells with differential CXCR3 expression and protective capacity against IAV, mice were vaccinated with DC-OVA adjuvanted with CpG or PolyIC. Although adjuvanting with CpG resulted in enhanced numbers of OVA257-specific CD8+ T-cells at an effector time point compared to adjuvanting with PolyIC, an equal frequency of circulating CD8 T-cells was observed at a memory time point (Figure 7b). Of note, OVA257 specific CD8+ T-cells that resulted from immunization in the presence of CpG, expressed significantly less CXCR3 at an effector (p<0.001, Figure 7c) and a memory time point (Figure 7d). Concordantly, the BAL fluid of PolyIC-adjuvanted mice contained a larger number of OVA257-specific memory CD8+ T-cells than CpG adjuvanted mice (Figure 7e). When challenged with a lethal dose of PR8 expressing OVA257 (PR8-OVA), mice that were immunized with DC-OVA + PolyIC controlled the virus significantly (p<0.05) better than naïve or DC-OVA + CpG mice (Figure 7e). Overall these data demonstrated that the choice of adjuvant impacted CXCR3 expression, airway localization and protective capacity against IAV of the ensuing memory CD8+ T cells.

Figure 7. CXCR3 expression, airway localization and protection by memory CD8+ T cells can be manipulated by adjuvant choice.

Figure 7

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a) Serum levels of IL-12 and IFN-α were determined after DC-OVA vaccination adjuvanted with CpG or PolyIC. b) OVA257-specific CD8 T-cell response in the blood were tracked over time by tetramer analysis. c) CXCR3 expression on OVA257-specific CD8+ T-cells was determined by flow cytometry on day 7 post vaccination. d) At a memory time point (day 60 post vaccination) CXCR3 expression was reassessed and e) the number of OVA257-specific CD8+ T-cells in the BAL fluid was assessed. f) Viral titers 4 day post infection with PR8-OVA at a memory time point. Mean n=5 +/−SEM, representative of 2 experiments *p<0.05

Discussion

Memory CD8+ T-cells could be an important part of heterosubtypic protection against IAV as they can recognize conserved epitopes in viral proteins, which have not been subjected to immunological pressure (Yewdell et al., 1985; Zweerink et al., 1977). Although IAV specific memory CD8+ T-cells can be detected in humans with a history of IAV infection, immunity provided by these cells appears to be limited (Wilkinson et al., 2012). Increasing the number of IAV specific memory CD8+ T-cells through boosting can improve protection (Christensen et al., 2000; Slütter et al., 2013), however it is also clear that the boosting can have a profound effect on the phenotype of the resulting memory CD8 T-cell population (Wirth et al., 2011). Here we show that memory CD8+ T-cells with a superior protective capacity against IAV could be generated by manipulating the cytokine milieu via selection of vaccine vectors or adjuvants. Therefore this work may provide a rationale for the development of future influenza vaccines to induce protective CD8+ T-cells.

Increasing the number of IAV specific memory CD8+ T-cells may be achieved through the use of prime-boost strategies (Slütter et al., 2013) and several potent booster agents capable of inducing strong CD8+ T-cells responses, including adenoviruses, poxviruses and attenuated LM strains are currently under clinical investigation (Meyer et al., 2012; Sheehy et al., 2012; Sinnathamby et al., 2009). Although the focus is generally on maximizing the number memory CD8+ T-cells, our work argues that the inflammatory milieu induced by the vaccine vector can also greatly influence specific properties of memory CD8+ T-cells, including their localization. In turn, our data showed that the localization of IAV specific memory CD8+ T-cells in the airways improved their protective capacity. A correlation between local CD8+ T-cell memory in the lung and immunity against IAV infection has long been established (Liang et al., 1994), but direct evidence for a protective role of T-cells in the respiratory tract has only been reported for CD4+ T-cells (Hogan et al., 2001). Here we showed, through airway specific depletion of CD8+ T-cells, that IAV specific memory CD8+ T-cells in the airways substantially lowered the viral burden during the first days of IAV infection. The generation of a memory CD8+ T-cell population capable of populating the respiratory tract could therefore be an important goal for next generation IAV vaccines. In accordance with the findings of Ely et al. (Ely et al., 2006) establishing an airway memory CD8+ T-cell population does not seem to require local immunization or pulmonary inflammation, as memory CD8+ T-cells of splenic origin could be detected in the BAL fluid of recipient mice after intravenous transfer. Nasal or pulmonary immunization is therefore not a prerequisite for induction of respiratory memory CD8+ T-cells, but also parenteral immunization may lead to an airway localized memory population. Of note, the memory CD8+ T-cells in the BAL we described were not classical tissue resident memory (Trm) as found in the skin (Gebhardt et al., 2011), intestine (Masopust et al., 2010) or lung parenchyma (Wakim et al., 2013) where effector CD8+ T-cells are “educated” by the local microenvironment to form resident memory (Jiang et al., 2012) and provide rapid protection against re-infection (Jiang et al., 2012). Instead the lung, being a highly vascularized organ subjected to recurrent environmental exposures, may be more accessible to circulating CD8+ T-cells and allow memory CD8+ T-cells subtypes to migrate into the airways under steady state conditions.

We showed that CXCR3 was an important chemokine receptor that regulated access into the respiratory tract under steady state conditions, whereas Kohlmeier et al. (Kohlmeier et al., 2008) show that CXCR3 expression is dispensable for memory CD8+ T-cells to reach the respiratory tract, under inflammatory condition. Interestingly, Kohlmeier et al show that surface expression of CCR5 on memory CD8 T-cells is low during the steady state, but is quickly upregulated after infection (Kohlmeier et al., 2008). Together, these data indicate an active role for CCR5 in recruiting memory CD8+ T-cells to the airways during an episode of inflammation whereas during the steady state its role may be limited. Consistent with this hypothesis, we observed many CXCR3lo CD8+ T-cells in BAL fluid 7 days after boost with LM-NP (Figure 3a, and data not shown), suggesting CXCR3 independent recruitment. After a few weeks however CD8+ T-cells in the BAL fluid were exclusively CXCR3hi and recruitment to BAL was CXCR3 dependent (Figure 4). As the respiratory tract is exposed to frequent environmental stimuli, low levels of IFN-γ can be detected in BAL fluid of healthy humans (Paats et al., 2012), potentially allowing CXCR3 ligands to be continuously produced. Indeed, we were able to detect elevated levels of IFN-γ, CXCL9 and CXCL10 transcripts in lung tissues from a large subset of mice in our animal facility. This may provide a gradient sufficient for CXCR3hi CD8 T-cells to migrate continuously into the respiratory tract.

The identification of CXCR3 as a pivotal molecule for memory CD8+ T-cells to reach the respiratory tract could help to distinguish between high and low quality memory CD8 T-cells in terms of protection against IAV. We showed that the expression of CXCR3 on CD8+ T-cells was tuned by the inflammatory cytokines induced by the vaccine vector or adjuvant. In the case of CD4+ T-cells CXCR3 is a signature marker associated with a T-helper1 (Th1) phenotype (Sallusto et al., 1998). As such, IL-12 signaling, STAT4 activation and T-bet activity are key events for Th1 polarization and promote CXCR3 expression (Mullen et al., 2001; Thieu et al., 2008). Conversely we showed that, CXCR3 expression in memory CD8+ T-cells was negatively correlated with IL-12R signaling and T-bet expression. Such differential regulation of an important chemokine receptor fits with previous reports of differential migration of memory CD4+ and CD8+ T-cell to the epidermis (Gebhardt et al., 2011) and lung airways (Ely et al., 2006). Potentially T-bet directly suppresses CXCR3 expression in CD8+ T-cells as interaction of T-bet with the promoter region of CXCR3 in human CD4+ T-cells has been shown (Beima et al., 2006; Lewis et al., 2007). However, in CD4+ T-cells T-bet’s interaction with the CXCR3 promoter region is indisputably positively correlated with CXCR3 expression (Lewis et al., 2007; Matsuda et al., 2007), suggesting the regulation of CXCR3 by T-bet in CD8+ T cells may be indirect. For instance, T-bet has been reported to promote the expression IL-12Rβ2 (Afkarian et al., 2002) and as such enhances sensitivity to IL-12.

Although we showed that direct IL-12 signaling to CD8+ T-cells was detrimental to CXCR3 expression, IL-12 is also an important “signal 3” cytokine that promotes optimal accumulation of effector CD8+ T-cells (Curtsinger et al., 2003; Keppler et al., 2012). In this respect, it is interesting that another major signal 3 cytokine, type I IFN, did not suppress CXCR3 on effector and memory CD8+ T-cells. Therefore type I IFN could still serve as the important signal 3 cytokine, to allow maximal accumulation and memory formation of CD8+ T-cells (Curtsinger et al., 2003; Keppler et al., 2012), without reducing their protective capacity by limiting CXCR3 expression. For instance, the TLR3 agonist PolyIC induces high serum IFN-α levels, but little IL-12, which may be due to MyD88/IRAK independent signaling after TLR3 activation (Jiang et al., 2003). We showed as a proof of concept that the use of PolyIC as an adjuvant during DC vaccination led to a similar number of memory CD8+ T-cells as adjuvanting with TLR9 agonist CpG, but these cells expressed more CXCR3, localized more effectively to the airways and had superior protective capacity against IAV. Selection of adjuvants that induce little IL-12, but do provide type I IFN may therefore be a relevant approach to elicit high quality (CXCR3hi) CD8+ T-cells responses without the drawback of reducing the overall number memory CD8 T-cells.

Material and Methods

Mice

BALB/c Stat4−/−, C57Bl/6 Il12rb1−/−, and C57Bl/6 Cxcr3−/− were acquired from Jackson Laboratories (Bar Harbor, ME). C57Bl/6 Tbx21−/− P14 mice have been described previously (Kaech et al., 2003). Cxcr3−/− P14 and Il12rb1−/− OT-I mice were generated through crossing of Cxcr3−/− or Il12rb1−/− with the respectively transgenic mouse strain. C57Bl/6 Ifnαβr−/− mice were a gift from Dr. Mescher (U. of Minnesota, Minneapolis, MN). BALB/c and C57Bl/6 mice strains were originally derived from the National Cancer Institute (Fredericksburg, MD) and a colony is maintained in house. All animal studies and procedures were approved by the University of Iowa Animal Care and Use Committee, under PHS assurance, Office of Laboratory Animal Welfare guidelines.

Infections and vaccine vectors, adjuvants

actA/inlB double deficient L. monocytogenes expressing IAV NP were provided by Dr. P. Lauer, (Aduro Biotech, Berkely, CA), actA deficient LM expressing LCMV GP33 or OVA were provided by Dr. H. Shen (UPenn, Philadelphia, PA) and were propagated as described (Corbin and Harty, 2004). Vaccinia virus expressing LCMV GP33 and IAV NP were provided by Dr. J. Bennink (NIH, Bethesda MD) and were propagated using standard protocols. For immunization, LM and VV were diluted in phosphate buffered saline and injected intravenously. A/PR/08/34 virus was grown in chicken eggs as previously reported (Legge and Braciale, 2005). While lightly anesthetized, mice were challenged with a 10 fold LD50 (2×105 TCID50) in 50 μl PBS. All infected mice were monitored daily for morbidity, and mice losing 30% or more of initial body weight were euthanized per IACUC guidelines and considered to have succumbed to infection. Viral titers were defined as tissue culture infective dose 50 (TCID50) by titration on MDCK cells (Slütter et al., 2013). CpG ODN (IDT, Coralville, IA) and PolyIC (InVivogen, San Diego, CA) were injected intraperitoneally at 100 μg.

Depletion and neutralization

For systemic depletion of CD4+ T-cells two doses of 400 μg αCD4 (clone GK1.5) were injected intraperitonial (i.p.). Systemic depletion of CD8+ T-cells required one ip injection of 100 μg αCD8 (clone 2.43). Local depletion of CD8+ T-cells from the BAL was achieved through intranasal administration of 5 μg αCD8. IL-12 was neutralized through 2 ip injections of 400 μg of αIL-12 (clone C17.8). Control mice received an equivalent amount of rat IgG in each experiment. All hybridoma cell lines were acquired from ATCC (Manassas, VA) and purified antibodies were prepared using standard methods.

Adoptive transfer

For generation of CD8+ T-cell memory, naïve OT-I or P14 transgenic T-cells (Thy1.1+) were obtained from donor blood and were transferred (500 and 5000 respectively) into naïve (Thy1.2+) hosts. Dendritic cell vaccination was performed with LPS matured dendritic cells prepared as described (Schmidt et al., 2009) and coated with NP147-155, GP33-41 or OVA257-264 peptide. For priming 5×105 DC were injected intravenously. CpG ODN 1826 (IDT, Coralville, IA) and Poly(I:C) (Sigma, St. Louis, MO) were injected intraperitonally (100 μg). For transfer of memory CD8+ T-cells, memory P14 were isolated from donor spleens though positive selection on Th1.1 with magnetic beads (Mylteni, Auburn, CA). NP147-specific memory CD8 T-cells were enriched by depletion of CD4+ and CD19+ cells using magnetic beads (Mylteni).

T-cell analysis

Bronchio alveolar lavage was performed post mortem by inflating the lung with 1 ml of RPMI 3× through the trachea. Lungs were isolated after systemic perfusion with saline and collegenase/DNAse treated for 1 hour prior to homogenization.

Blood, BAL and homogenized lung, lymph nodes, and spleens were depleted of erythrocytes by treatment with ammonium chloride buffer and stained with the appropriate antibodies or in-house prepared Kd-NP147 tetramer complexes. All antibodies (αCXCR3 clone CXCR3-173, αCD8α clone 53-6.7, αCD8b clone YTS156.7.7, αThy1.2 clone 53-2.1 and αThy1.1 clone OX-7 and, were acquired from Biolegend (San Diego, CA). Flow cytometry was performed using a Fortessa flowcytometer (BD biosciences) and analyzed using Flowjo (Treestar, Ashland, OR) for MacIntosh.

Statistics

Unless indicated otherwise significance was calculated by one way ANOVA with Bonferroni’s post test using Graphpad Prism 4 for Macintosh. P-values lower 0.05 were considered significant.

Supplementary Material

01

Highlights.

  • Memory CD8+ T-cells in airways provide rapid protection against influenza A virus

  • CXCR3+ memory CD8+ T-cells accumulate in the airways during steady state conditions

  • IL-12 signaling reduces CXCR3 and airway localization of memory CD8+ T-cells

  • Limiting IL-12 enhances memory CD8+ T-cells in airways and immunity to IAV

Acknowledgments

We thank Jeff Nolz, Vladimir Badovinac and Stanley Perlman for critically comments on the manuscript, Peter Lauer for recombinant Listeria and Jack Bennink for recombinant vaccinia viruses. We also thank Lisa Hancox and Cathryn Varga for research support. This work was supported by a NWO Rubicon Fellowship, (BS), the Howard Hughes Medical Institute (SMK) and NIH grants AI42676, AI100527, AI106776 to JTH.

Footnotes

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