After s.c. delivery of infectious virions, the most rapid antigen presentation occurs via direct priming. Within seconds, lymphatics convey virions to LNs, where they can infect macrophages and DCs in the LN parenchyma (Hickman et al., 2008
). Because viral peptides are generated primarily from DRiPs (Dolan et al., 2011
), the onset of viral protein synthesis is immediately accompanied by the generation of class I peptide complexes, which can rapidly initiate CD8+
T cell priming. For VV, the initial phase of direct priming likely persists for 12–24 h (Hickman et al., 2008
), after which infected DCs and macrophages succumb to viral cytopathogenesis or NK-mediated lysis. Because the first 12 hpi are critical for activating antiviral T cells after s.c. VV infection (Hickman et al., 2008
), it is imperative that T cells rapidly contact the appropriate infected APC.
The second wave of antigen presentation likely involves transfer or acquisition of antigen by LN-resident APCs. CD8α+
DCs are particularly adept at cross-priming antigens acquired from dead or dying cells (den Haan et al., 2000
; Iyoda et al., 2002
). For viruses that infect dermal epithelial cells, like herpes simplex virus-1, these cells provide an antigen source for cross-priming by migratory dermal DCs (Bedoui et al., 2009
). To date, antiviral cross-priming has been investigated largely by ex vivo analyses of cells recovered from dissociated tissues (Allan et al., 2003
; Belz et al., 2004
; Bedoui et al., 2009
). It will be important in future studies to confirm the central role of CD8α+
DCs in antiviral cross-priming using MPM, although it will be challenging to unambiguously identify APCs actively engaged in cross-priming and not direct priming after synthesis of undetectable levels of viral antigens.
The LN CR, a reticular stromal structure at the border of the T/B zones, likely serves as a staging ground for both direct and cross-priming T cell–APC interactions (Katakai et al., 2004
). Migratory, antigen-bearing DCs are thought to accumulate in the CR, positioning themselves to maximize cross-priming interactions with nearby T cells (Bajénoff et al., 2003
; Lindquist et al., 2004
). We show here that direct priming interactions independent of DC migration also occur in the outermost edges of the CR as virions drain to the LN and are captured by resident APCs. After the first wave of virus-infected cells die or are eliminated in the LN, it will be interesting to determine the location of virus-specific CD8+
T cell–APC interactions in relation to the macrophage-rich and dendritic regions of the CR.
Controversy swirls around the definition of macrophages versus DCs and their putative differences in priming capacity. Hume (2008)
has eloquently argued that DCs are simply mononuclear phagocytes that do not comprise a unique cellular subset, and are not especially adept at priming. A key element of Hume’s argument is that the heightened priming ability of DCs relative to macrophages ex vivo is caused by removing suppressive macrophages during the process of isolating DCs. Here, we show that in vivo, T cells scan DCs and macrophages at different rates in infected LNs. Additionally, LN depletion of CD11c+
cells, which eliminates nearly all DCs and SCS macrophages, has profound effects on antiviral priming, despite our direct observations that CD8+
T cells now form contacts with macrophages that are indistinguishable by IVM from their contacts with DCs under nonablative conditions. This argues strongly that DCs have a unique role in direct priming that macrophages cannot replace.
What is the role for the large numbers of infected LN macrophages if not for priming naive CD8+
T cells? Recently, Asano et al. (2011)
showed that these macrophages serve as facile APCs via cross-presentation of tumor associated antigens, making their failure to drive antiviral T cell responses even more enigmatic. Several imaging studies have now shown antigen acquisition by SCS sinus macrophages and subsequent antigen donation to LN B cells (Batista and Harwood, 2009
). Perhaps virally infected nodal macrophages specialize in presentation to B cells and are largely ignored by T cells (much like CD8α+
DCs preferentially present to CD8+
and not CD4+
T cells; Dudziak et al., 2007
). Additionally, these macrophages express the molecule sialoadhesin, a newly identified participant in regulatory T cell function and expansion, raising the possibility that macrophage infection activates suppressive rather than effector T cells (Wu et al., 2009
We show that macrophages prime CD8+ T cells that are sub-optimally activated by the standard criteria. It remains to be determined, however, whether there is method to this madness and the “partially” activated cells are fully activated for a specific alternative function in primary or memory responses. Alternatively, macrophages may serve to dampen initial CD8+ T cell responses generated via direct priming, favoring instead cross-priming at later time points. In any event, it is a safe assumption that macrophages perform multiple functions in the infected LNs.
In clearly demonstrating the essential role DCs play in direct antiviral priming, our findings emphasize the importance of understanding the basis for CD8+
T cell attraction to infected PIR DCs. This knowledge is likely to be useful in maximizing the ability of vaccines to elicit effective CD8+
T cell responses. We provide the initial evidence that CCR5 and R1-signaling chemokines, known to guide CD8+
T cells to cross-priming DCs in noninfectious models (Castellino et al., 2006
; Hugues et al., 2007
), also play a critical role in direct priming during viral infection. We show, first, that viruses rapidly induce chemokines in the LN, and second, that an Ab cocktail that neutralizes several of the known CCR5 ligands inhibits both DC attraction of CD8+
T cells and CD8+
T cell IFN-γ responses. Additionally, these experiments likely underestimate the importance of CCR5 agonist chemokines in direct antiviral CD8+
T cell priming, since it is unlikely that the Ab cocktail we used covers all CCR5 agonists or completely neutralizes the agonists covered. Likewise, experiments using CCR5KO OT-I cells will similarly undervalue the effect of these chemokines on CD8+
T cell responses as the cells retain signaling through CCR1–CCL3 and –CCL5 interactions.
Because of its relevance for rational vaccine design, there is tremendous interest in identifying APC types that most effectively prime functional CD8+ T cell responses. We have identified a factor that optimizes CD8+ T cell targeting to appropriate APCs during viral infection. This raises the exciting possibility of engineering vaccines to express optimal antigens as well as a guidance system to attract CD8+ T cells to DCs for maximal priming of effector cells with optimal effector profiles.