In this paper, we have analyzed antigen presentation in influenza-infected mice using the induction of T cell responses in vivo to monitor viral antigen persistence after the resolution of an acute respiratory virus infection. We have found that processed viral antigen may persist for an extended time period, i.e., up to day 70 p.i., in the lung-draining MLN, as measured by the ability of the transferred antigen-specific TCR Tg T cells to proliferate in the MLN. This finding is in agreement with previously reported observations (Jelley-Gibbs et al., 2005
; Zammit et al., 2006
; Fazilleau et al., 2007a
), which implicated the secondary lymphoid organs (e.g., LNs draining the site of the initial infection) as sites of persistent antigen deposition after infection or vaccination. However, we have gone on to demonstrate that, in the case of respiratory virus infection with influenza, this small persistent reservoir of viral antigen (in the form of influenza viral RNA and protein) is localized to the site of infection, i.e., the lungs. Viral antigen (influenza NP) colocalized with both CD45−
and, to a lesser extent, CD45+
lung cells within residual small inflammatory loci. We were able to reconcile this apparent discrepancy, i.e., MLN for the site of T cell activation and lungs for antigen persistence, by demonstrating that RDCs capture the residual viral antigen deposited in the previously infected lung and then migrate to the MLN where they present the in vivo–processed antigen to T cells. Our results further suggest that the presentation of residual viral antigen by the migrant RDC in the MLN appears to have a previously unrecognized role in regulating the memory T cell pool in the draining MLN after infectious virus clearance and recovery from the infection.
There is accumulating evidence in several models of experimental respiratory tract viral infection, i.e., VSV, RSV, or influenza virus, that viral antigen in a form of processed peptide persists in the LN, draining the site of initial pathogen entry (Schwarze et al., 2004
; Jelley-Gibbs et al., 2005
; Turner et al., 2007
). It has been speculated that the viral antigen in the DLN may be retained in cells capable of presenting the antigen to antigen-specific memory T cells. In spite of efforts to identify the cell type that supports the proliferation of T cells in LNs draining sites of infection, the nature of antigen-presenting cells in the DLN has not been elucidated. Our analysis suggests that, in the case of acute viral infection of the respiratory tract, it is these two subsets of CD11chi
MHC Class IIhi
RDCs, i.e., CD103+
RDCs (Sung et al., 2006
; Kim and Braciale, 2009
), which carry out this APC activity in the DLN after their migration from the previously infected lungs. Two lines of evidence support this view: CD103+
RDC are the only DC subsets capable of supporting significant T cell proliferation when isolated from the lung-draining MLN and analyzed directly ex vivo, and elimination of these two RDC subsets from the previously infected respiratory tract abolishes the capacity of the draining MLN to support T cell activation/proliferation. In this regard, it is noteworthy that resident DCs within secondary lymphoid organs, for example, conventional DCs and pDCs, are thought to have relatively short life spans (from 3 to 7 d; Kamath et al., 2002
), and these blood-derived DCs are thus unlikely to retain a pool of antigen within the DLN because of their rapid turnover. RDCs display a basal level of migration out of the respiratory tract to the draining MLN (without an acute inflammatory stimulus; Jakubzick et al., 2008
; Kim and Braciale, 2009
). As we demonstrate in this paper, these RDCs continued to migrate from the lungs to the draining MLN well after nominal clearance of infectious virus, probably in response to the low level of residual inflammation in the infected lungs at late times after infection. This property makes RDCs ideal candidates to capture antigen from a reservoir of viral protein within the previously infected lungs and transport it to the DLN.
We could detect mRNA encoding the influenza NP gene product, as well as mRNA for the alternatively spliced NS gene product NEP and spliced M gene product M2, in the lungs for up to 30 d p.i, which is well after infectious virus clearance from lungs (). Moreover, immunofluorescent staining of lung tissue revealed that the viral NP could likewise be detected in the previously infected lungs for up to 30 d p.i., largely within CD45−
cells located in sites of focal inflammation reminiscent of iBALT. The presence of viral transcripts and viral protein in the lungs at these later times after infection in the absence of detectable viral gene expression in the draining MLN makes this depot of viral antigen the likely ultimate source of the viral antigen presented by migrant RDC to T cells in the draining nodes. It is also noteworthy that viral gene expression persists in the face of a vigorous adaptive immune response, which results in the elimination of both infectious virus and the vast majority of virally infected cells in the respiratory tract. We cannot formally determine whether infectious virions are assembled and released from this viral antigen reservoir, as high levels of circulating neutralizing antibodies in these animals preclude detection of infectious virions in the lung tissue at these later times p.i. The cell types expressing the influenza viral genes and the mechanism by which these cells escape elimination by the immune system remain to be determined. However, beyond 30 d p.i., although viral RNA and/or protein is not demonstrable, viral antigen is present and retained in some recognizable form by influenza-specific CD8+
and, more notably, by CD4+
T cells because T cell proliferation is demonstrable in the draining MLN for >70 d p.i. (Jelley-Gibbs et al., 2005
; unpublished data). The nature of this reservoir of the viral antigen is open to speculation. One potential cellular reservoir of the antigen, which is long lived, irradiation resistant, and CD45−
, is FDC (Mandels et al., 1980
). Although these cells are normally not found in the lungs of naive mice after influenza infection and the formation of iBALT, FDCs can localize in follicles formed within the iBALT (Moyron-Quiroz et al., 2004
). Our findings using in vivo depletion of FDC by LTβR blockade (Fig. S5) support the idea that FDC present in the previously infected lungs may, like CD11chi
RDC, play an important role in controlling the retention of memory T cells within the DLNs. Whether these cells serve as the ultimate, or even most effective, reservoir for residual viral antigen remains to be determined. Nevertheless, although not formally explored in this paper, we would speculate that iBALT-like structures within the previously infected lungs may be important sites of influenza viral antigen deposition where CD45−
cells, such as FDC, may interact with CD45+
RDC to regulate memory T cell populations within the DLNs.
The preferential localization of memory CD8+
T cells to the LNs draining the site of infection, observed by us in this paper and by others (Hogan et al., 2001
; Marshall et al., 2001
; Jelley-Gibbs et al., 2005
; Zammit et al., 2006
), is a potentially useful adaptation for the adaptive immune system to deal with repeat infections by a given pathogen. This strategy allows for rapid mobilization of the memory response through a concentration of central memory cells in the LNs draining the likely site of subsequent infection. Using the DTR mouse model, we demonstrate that elimination of the critical CD103+
RDC from the influenza-infected respiratory tract before the RDC migration to the DLN substantially decreases the numbers of virus-specific endogenous (wild type) memory CD8+
T cells present in the DLN within days of RDC elimination. Furthermore, adoptive transfer of DC (but not alveolar macrophages) into the respiratory tract of DTx-treated DTR mice restores memory T cell numbers within the DLN. Although strong inflammatory stimuli can alter the architecture of LNs draining the site of the inflammatory stimulus, resulting in preferential accumulation of T cells within the DLN (Soderberg et al., 2005
), our findings, in particular the rapid loss of memory T cells from the DLN after RDC depletion from the lungs, are more consistent with an active role for these migrant viral antigen-bearing RDCs in retaining memory cell numbers within the DLN. The nature of the interaction between the memory T cells and the RDC leading to T cell retention in the DLN remains to be elucidated. Our analysis (Fig. S7) does, however, indicate that the accumulation of the memory T cells in the draining MLN appears not to be a result of ongoing proliferation of the memory T cells. Rather, the elevated expression of CD69 would favor a model where the interaction of memory T cells with the migrant RDC results in the CD69-dependent (Shiow et al., 2006
) selective retention of the memory T cells within the draining nodes.
In summary, our study provides insight into the process by which viral antigen persists after acute respiratory virus infection and the mechanism through which the prolonged in vivo antigen presentation occurs. Our results suggest that RDCs found at the site of primary infection (i.e., the lungs) play a pivotal role in sampling the residual viral antigen and, upon migration to the DLN, directly present the in vivo–processed peptides to the antigen-specific T cells. As a consequence, this ongoing residual antigen recognition by memory T cells may be an important event resulting in the selective enrichment of virus-specific memory T cells in the DLN after the clearance of infectious agent. This enriched population of antigen-specific T cells maintained in the antigen DLN would likely provide a more rapid and robust recall T cell response to a reinfection. These findings suggest that immunization strategies using long-lasting vaccines deposited at body surfaces where the target pathogen enters the body (e.g., the respiratory tract or GI tract) may be the most effective way to sustain and recall a memory adaptive immune response.