The results presented herein suggest that generation of an effective influenza-specific CD8 T cell response requires activated CD8 T cells to interact with pulmonary pDCs, CD8α+ DCs, or iDCs in a MHC class I–, viral epitope–dependent manner once they enter the lungs. This secondary interaction is in addition to the initial DC–T cell interactions that occur in the LN during activation of naive CD8 T cells. To our knowledge, this is the first study detailing a critical role for peripheral DC–CD8 T cell interactions after initial programming of primary effector T cells in the LN. This suggests that the influenza-specific CD8 T cell response may be regulated by a “two-hit” model of development and that the magnitude, and possibly phenotype, of the peripheral CD8 T cells generated may be related to both the initial programming that occurs in the LN, but also by secondary contacts with DC subsets at the site of the infection. Of note, we have observed differential magnitudes of recruitment into the lungs of the various DC subsets described in this study during i.n. infections with various substrains of influenza A viruses, as well as during high and low dose infections with the same strain (unpublished data). These results suggest that the characteristics and subsets of the pulmonary DCs present in the lungs during respiratory challenges could potentially have a profound effect on the ensuing immune response and its outcome.
A two-hit model would allow the immune system to titrate, in an antigen-specific way, the magnitude and duration of the T cell response generated. This would be critically important in a vital organ such as the lungs, where T cell responses sufficient to control an infection are required, but, if overly robust, are known to induce harmful immunopathology (37
). Therein, a two-hit mechanism would allow the immune response to balance the benefit and cost of pulmonary CD8 T cell immunity.
Whether interactions of DCs with T cells in nonlymphoid tissues is a common or more global mechanism through which the immune system titrates the magnitude of the immune response in peripheral tissues is unclear at this time. However, a recent study has described a similar peripheral interaction of DCs and memory CD8 T cells in the dorsal root ganglia (38
). After reactivation of latent herpes simplex virus, a direct DC–memory T cell interaction in this nonlymphoid tissue allows the peripheral expansion of the T cells to reestablish control of the virus. Further, Smit et al. have recently shown that during respiratory syncytial virus (RSV) infections, increased numbers of pulmonary pDCs in the lungs enhance the magnitude to the RSV-specific CD8 T cell response (39
). Although this study did not directly show interaction of the CD8 T cells and pDCs in the lungs, their results would be consistent with those observed in this study where lung-resident pDCs increase the magnitude of the virus-specific pulmonary CD8 T cell response.
Interestingly, influenza virus infections lead to the recruitment of CD8α+
DCs and pDCs into the lungs. Both of these subsets have well-established roles in viral infection (33
). The results described herein show an as of yet unknown direct role for these DCs in amplification of influenza-specific CD8 T cell immune responses. Although many reports have described pDC recruitment into the lungs after respiratory challenge (40
), to our knowledge, this is the first study describing recruitment of CD8α+
DCs into the lungs. CD8α+
DCs are potent cross-presenters of viral antigens and are normally associated with lymphoid tissues. The mechanism controlling the recruitment of these cells and their locations within the infected lungs remains unknown. Given the affinity of CD8α+
DCs for lymphoid tissue, it is possible that these cells are being recruited into the recently described inducible bronchus-associated lymphoid tissue (42
), where naive T cell–DC interactions have been described (42
). Lung-recruited pDCs, on the other hand, are often associated with the alveolar interstitium (43
), suggesting that the pDCs and CD8α+
DCs could be mediating their effects in distinct locations in the lungs.
The source of the recruited pDCs and CD8α+
DCs currently remains unclear. Whereas aDCs and iDCs are thought to differentiate from blood-derived monocytes (44
) or precursors that reside in the lungs (48
), pDCs and CD8α+
DCs are found differentiated within the blood and/or other tissues before infection and can develop from a single precursor (49
). In a preliminary experiment, where liposome-clodronate was administered i.v. (38
) instead of i.n. at 48 h a.i., we observed a significant decrease in pulmonary aDCs, iDCs, pDCs, and CD8α+
DCs, but no change in aM
numbers (unpublished data). Because i.v. liposome-clodronate administration alters cell populations in the blood, bone marrow, spleen, and liver, but not in the lungs, this result suggests that the pDCs and CD8α+
DCs, or their precursors, most likely are recruited from one of these tissues.
The mechanism by which pDCs, CD8α+
DCs, and iDCs drive pulmonary influenza–specific T cell expansion is MHC I dependent and appears to be largely dependent on viral antigens. This increase in the magnitude of the T cell response could potentially occur through three processes: (a) increasing antigen-specific T cell migration into the lungs; (b) inducing subsequent T cell proliferation; and/or (c) protecting the cells from apoptosis. Because influenza-specific T cell numbers are similar in the blood and spleens of control and aDC-depleted mice (not depicted) and transfer of influenza B DCs only leads to a limited, at best, increase in antigen-specific CD8 T cell numbers in the lungs (), altered T cell migration to the lungs does not appear to be the dominant pathway involved in DC rescue of T cell responses. However, our results do show that an intermediate CFSE division phenotype exists in some aDC-depleted T cells found in the lungs on day 5 a.i. (), and in vitro co-culture of the aDC-depleted T cells with the DCs induces proliferation. These results suggest that a second round of DC-induced proliferation of the T cells may occur after the T cells enter the lungs (53
). Finally, recent studies have suggested that peripheral expression of costimulatory ligands such as OX40L and 4-1BBL may protect CD8 T cells from apoptosis (54
). To date, we have found no difference in expression of the classical costimulation molecules (i.e., CD40, CD80, and CD86; unpublished data) on those DCs, which increase the magnitude of the T cell response. However, the potential individual contributions of the various DCs expressed costimulatory molecules in the rescue of the T cell response awaits more detailed analysis.
The fact that the aDC population, depleted during the initial liposome-clodronate treatment, did not restore pulmonary T cell responses to control levels was unexpected, given that these DCs are one of the primary populations that traffic from the lungs to the LNs during an influenza virus infection and participate in the initial activation of naive CD8 T cells in the regional LNs (1
). This, and the fact that aDCs can stimulate control T cells to proliferate during in vitro cultures (), suggests that the aDCs should have the necessary factors needed to stimulate the aDC-depleted T cells. Interestingly, Belz et al. recently showed a similar memory T cell ignorance of aDCs in the LNs. In this study, although the aDCs and CD8α+
DCs in the LNs could activate naive CD8 T cells, only the CD8α+
DC subset could drive secondary expansion of the memory T cells (4
). Further, although we have demonstrated that MHC I and influenza viral antigens are necessary to confer a full CD8 T cell response, it is likely that these factors alone are not sufficient, given that virally infected MHC I+
epithelial cells are not limiting during the course of an influenza infection, yet these cells are unable to provide the necessary signals to influenza-specific CD8 T cells in the lungs. Moreover, adoptive transfer of pDCs purified from the spleens of influenza virus–infected animals, unlike transfer of the aforementioned pulmonary pDCs, does not drive rescue of influenza-specific CD8 T cell responses (Fig. S6, available at http://www.jem.org/cgi/content/full/jem.20080314/DC1
). Together, these studies suggest pulmonary pDC-, CD8α+
DC–, and iDC-mediated rescue of T cells may require additional molecules, such as CD70 (4
), in addition to viral antigen and MHC class I.
The results presented herein largely suggest that the reduced influenza-specific CD8 T cells numbers in the lungs are responsible for the increased disease severity. Consistent with this idea, reconstitution of the lungs with pDCs, CD8α+
DCs, or iDCs leads to an increased T cell response, and therein reduced pulmonary virus titers (unpublished data). However, because the i.n. liposome-clodronate treatment eliminates pulmonary APC populations (i.e., aM
and aDC) and alters DC recruitment (i.e., pDCs and CD8α+
DCs), we cannot completely rule out that loss of these cells alters some form of APC direct control of the virus infection (55
) or at least decreases clearance of virus-related debris, thereby increasing mortality. When we have reconstituted the depleted lungs with aM
or aDCs (), we have not seen an abatement of influenza-associated morbidity or mortality (not depicted), suggesting that these cells, independent of full CD8 T cell responses, cannot alleviate the increased disease severity. It is of interest, however, that recent studies have identified an early role for aM
in immunity to RSV infections (56
) and a highly pathogenic 1918 HA/NA–containing influenza virus (55
). In these studies, differences in the production of cytokines and chemokines (TNF-α, IL-6, CCL3, IFN-α, and CCL5), as well as NK cell activation, after RSV infection and control of virus titers/morbidity during 1918 HA/NA–containing influenza virus infections of aM
-depleted animals were limited to day 1 a.i. (i.e., RSV) or required the depletion of aM
before infection (i.e., 1918 HA/NA–containing influenza virus). In this study, the aM
were not depleted until 48 h a.i. (i.e., after this first 24-h a.i. window), therefore it remains to be seen if changes in chemokine and cytokine production or NK activation have occurred. Regardless, the aM
, unlike pDCs, CD8α+
DCs, and iDCs, appear to have no direct role in amplifying the influenza-specific CD8 T cell response, and therein allow the elimination of the virus from the lungs.
Herein, we have shown that influenza virus infections induce the recruitment or expansion of CD8α+ pDCs and iDCs in the lungs. In the absence of these DC subsets, the influenza-specific CD8 T cell response is dramatically inhibited. This inhibition of T cell immunity is not caused by altered activation of naive T cells in the LN, as CD25, CD69, and T cell division (), as well as DC subset cell numbers and ability of day 3 and 5 a.i. purified LN DCs to activate naive CD8 T cells, appears identical in the aDC-depleted and control mice. Furthermore, after activation in the LN, the influenza-specific T cells are found in the blood and spleen, but not the lungs, at normal levels. Reconstitution with pDCs, CD8α+ DCs, and iDCs increases the numbers of influenza-specific CD8 T cells present in a DC–T cell contact–, MHC I–, and viral epitope–dependent manner. Collectively, our results suggest that pulmonary influenza–specific CD8 T cells require at least two antigen-specific interactions with DCs, one as naive cells in the LN, and a second as activated cells after arriving in the lungs.