Studies have shown that the ability of memory CD8 T cells to confer heterosubtypic immunity against influenza virus declines within a few months (1
). This decline was later found to result from a gradual loss of virus-specific memory CD8 T cells in the airways (4
). Because it is difficult to obtain a sufficient number of memory cells from the airways following a polyclonal CD8 T cell response, the mechanisms underlying the loss of airway memory CD8 T cells have not been properly examined. Indeed, current knowledge about memory T cell homeostasis has been largely derived from the analysis of cells from lymphoid organs (7
), in which cytokines IL-7 and IL-15 are identified as key regulatory factors. Whether the same or different mechanisms regulate memory T cell persistence in nonlymphoid locations, such as in the airways, however, are unknown.
In this report, we examined this issue by following a cohort of TCR transgenic T cells following influenza virus infection. The use of TCR transgenic T cells made it possible to recover sufficient numbers of memory CD8 T cells from the airways for analysis as well as compare memory T cell persistence, phenotype, and function in different organs without the confounding effects of the TCR heterogeneity of a polyclonal T cell response. We found similar levels of IL-7 protein in bronchial lavage and spleen supernatant and similar levels of IL-7 and IL-15 transcripts in the lung and the spleen. However, IL-7R and IL-15R expression was gradually lost from persisting memory CD8 T cells in the airways but not in the spleen. The observed selective retention of memory T cells with low IL-7R and IL-15R in the airways could result from either gradual down-regulation of the cytokine receptor expression (a gradual change in phenotype over time) or selection for memory T cells that do not express the cytokine receptors (selection of cells with a particular phenotype over time). Although our results do not unequivocally distinguish the two possibilities, the existing data are more consistent with the former possibility. The loss of receptor expression is specific for IL-7R and IL-15R because TCR, CD8, Thy-1, and CD44 expression on the same cells is stably maintained. When memory 2C cells from the spleen were adoptively transferred into the airways, expression of the IL-7R and IL-15R was gradually down-regulated. Consistent with our results, a recent study showed that when purified CD44high
memory CD8 T cells from spleen of Sendai virus-infected mice were transferred into the airways of naive mice, they also down-regulated the surface level of IL-7Rα as early as 18 h after the transfer (25
). These findings suggest that the down-regulation is induced by the airway environment, not by some intrinsic differences between airway and splenic memory CD8 T cells.
How does the airway environment induce the down-regulation of IL-7R and IL-15R on memory T cells? One possibility is that IL-7R and IL-15R molecules are shed from memory CD8 T cells in the airways (26
), perhaps via the action of a protease (27
). Based on the results that the levels of IL-7R and IL-15R were high on 2C cells in the airways 14 dpi () and that normal levels of TCR, CD8, CD44, and Thy-1 were detected on airway memory 2C cells 30 dpi, this possibility seems unlikely. Furthermore, the levels of IL-7R and IL-15R transcripts in memory 2C cells were lower in the airways than in the spleen, suggesting that the down-regulation is at the transcriptional level. Cytokines, such as IL-2, IL-4, IL-6, IL-15, and even IL-7 itself, and signaling through TCR, have been shown to down-regulate IL-7R expression at the transcriptional level (28
). The presence of these cytokines and other yet-to-beidentified molecules in the airway environment may have induced the down-regulation of IL-7R and IL-15R expression. Consistent with this possibility, IL-7 was readily detected in bronchial lavage and IL-15 transcripts were detected in the lung.
One consequence of IL-7R and IL-15R down-regulation is the loss of response of airway memory CD8 T cells to the corresponding cytokines. Stimulation of airway memory 2C cells with IL-7, IL-15, or both failed to induce Stat5 phosphorylation, survival, or proliferation, whereas upon the same stimulation, splenic memory 2C cells immediately underwent Stat5 phosphorylation, followed by proliferation and survival. Because airway and splenic memory 2C cells proliferated equally well to anti-CD3 stimulation and produced similar levels of IFN-γ in response to SIY peptide, the non-responsiveness of airway memory CD8 T cells to IL-7 and/or IL-15 stimulation is specific, not due to a general defect, because these cells are dying in the airways.
What is the relationship between the down-regulation of IL-7R and IL-15R and gradual disappearance of memory T cells from the airways? One possibility is that the down-regulation is a consequence of memory T cells dying in the airways. Lung airways are exposed to the external environment and lined with surfactants, which inhibit T cell activation and survival (29
). Molecules such as NO produced by alveolar macrophages in the airways are also known to inhibit Ag- or mitogen-induced T cell proliferation (30
). Despite these unfavorable conditions, some of the transferred splenic memory 2C cells were able to persist in the airways for at least 2 wk (), suggesting that the airway environment can support significant memory cell survival, consistent with the presence of IL-7 in the bronchial lavage (). Therefore, an active suppression mechanism such as down-regulation of the cytokine receptors is likely required to limit their survival in the airways.
Our findings suggest that the down-regulation of IL-7R and IL-15R expression is a significant contributing factor to the poor persistence of memory CD8 T cells in the airways. The gradual down-regulation of cytokine receptors is temporally correlated with the gradual decline of memory T cells in the airways following influenza virus infection. When splenic memory T cells were adoptively transferred into the airways, the receptor down-regulation was also associated with the cessation of homeostatic proliferation. Cytokines IL-7 and IL-15 are key factors that promote the survival of memory T cells in the lymphoid organs (1
). The down-regulation of IL-7R and IL-15R obviously prevents airway memory T cells from responding to these cytokines. As the levels of IL-7 and IL-15 transcripts in the lung were generally higher than those in the spleen (), airway memory T cells likely have access to both cytokines as memory T cells do in the spleen. However, even in the presence of excess amount of IL-7 or IL-15, such as in vitro, airway memory 2C cells did not survive or proliferate, whereas splenic memory 2C cells responded readily to the cytokines. These findings demonstrate the essential role of IL-7R and IL-15R expression in determining the cellular responses to these cytokines. However, they also raise the question what, if any, factors help to keep the persisting memory T cell alive in the airways. Although further studies are needed to answer this question, controlling memory T cell survival by regulating IL-7R and IL-15R expression likely provides a more stringent control than that by cytokines. The unresponsiveness of airway memory CD8 T cells to IL-7 and IL-15 not only has a critical effect on their persistence, but also likely blocks their `bystander' proliferation (TCR independent) in response to subsequent unrelated infection in the airways (32
The difference in IL-7R and IL-15R expression by memory 2C cells in the airways and the spleen are unlikely due to the use of TCR transgenic T cells, which significantly increases the frequency of responding CD8 T cells (34
). First, identical with the endogenous polyclonal SIY-specific memory CD8 T cells (), memory 2C cells from the airways 30 dpi were all CD62Llow
, suggesting that the frequency of responding CD8 T cells does not alter memory T cell development in the airways. Second, the ratios of CD62Lhigh
memory CD8 T cells in the spleen were similar between the transferred 2C cells and the endogenous SIY-specific CD8 T cells, indicating that transferred 2C cells are not biased toward central memory cell differentiation. Third, both the memory 2C cells and the endogenous SIY-specific CD8 T cells exhibit similar surface phenotypic changes: high levels of IL-7Rα, IL-15Rβ, and γc
in the spleen and low levels in the airways. Fourth, transferring splenic memory 2C cells into the airways of naive recipient mice led to the down-regulation of cytokine receptors, indicating that down-regulation in the airways is not a matter of initial frequencies of responding T cells, but an active local process. Together, these observations suggest that the use of TCR transgenic T cells are unlikely to have affected the outcome of memory T cell persistence in the airways.
In summary, the down-regulation of IL-7R and IL-15R expression is likely a contributing factor for the poor survival of memory CD8 T cells in the airways. These findings provide a molecular explanation for the gradual loss of heterosubtypic immunity following influenza virus infection. Because the protective immunity mediated by memory CD8 T cells also wanes rapidly following other respiratory infection, such as respiratory syncytial virus (35
), the findings presented in this study may represent a general characteristic of airway memory CD8 T cells. As the lung is a major portal of infection, our findings also highlight the challenge to vaccine strategies aimed at inducing long-lasting cellular immunity against respiratory infections, such as influenza virus.