We explored the mechanism of RSV-induced immunosuppression by first simplifying the experimental system to MDDC and autologous CD4 T cells, thus eliminating interactions with CD8 T cells, B cells, and NK cells. Our approach was similar to that of Bartz and colleagues (5
), who used cord blood CD4 T cells, which are all naive, and cord blood CD34+
cells to derive dendritic cells (DC). In that report, treatment of DC with RSV suppressed IFN-γ secretion but not proliferation in response to superantigen. Preliminarily, we have seen no difference in suppression by RSV-infected MDDC of sorted naive versus memory CD4 T cells (B. Chi and R. L. Rabin, unpublished data), but the difference in the source of DC, age of donor, or both may easily account for the difference in functional suppression between the two reports.
Since RSV had no direct effect on CD4 T cells (Fig. ), we could concentrate on MDDC as a source of suppressive activity, and since suppressive activity could be transferred with supernatants from MDDC (Fig. ), we turned our attention toward soluble factors secreted by them. And since suppression of T-cell proliferation is induced only with infection of MDDC by live RSV (Fig. and ), we ruled out the possibility that live RSV directly suppresses CD4 T-cell proliferation by demonstrating, first, that CD4 T-cell suppression was unaffected by addition of palivizumab to supernatants from RSV-infected MDDC (
and, second, that direct addition of live (Fig. ) or UV-killed RSV (not shown) to CD4 T cells did not suppress proliferation in response to SEB. Furthermore, consistent with a previous report using a similar experimental system (5
), live RSV, and RSV-derived protein levels in harvested supernatants actually diminished. However, there are some obvious limitations to these experiments that must be acknowledged. First, it is not known if palivizumab blocks virus or F binding to cells or if palivizumab neutralization is mediated at a postbinding step. Because palivizumab specifically interacts with a single epitope within the F1 region, it would not be expected to block virus attachment mediated by the F2 portion of the fusion glycoprotein or inhibit interactions with the G glycoprotein. Second, these experiments do not rule out the possibility that RSV-secreted G glycoprotein may play a role in T-cell suppression. It is apparent, however, that type I and III IFN-mediated suppression is distinct from inhibition of NF-κB mediated by RSV-soluble G glycoprotein, the latter of which is not dependent on live virus (1
). Taken together, it appeared unlikely that RSV virions or proteins directly suppress T cells in our experiments; thus, we focused on MDDC-derived soluble factors.
One such factor that has been held responsible for RSV-induced immunosuppression is IL-1RA (32
). We found high levels of IL-1RA in the MDDC supernatants but very little IL-1α and IL-1β, suggesting that IL-1RA probably has no role in this context. Similarly, TGF-β1 and IL-10 are immunosuppressive cytokines that were ruled out because TGF-β1 was undetectable in the MDDC supernatants (not shown) and IL-10 levels were similar in all experimental conditions. Furthermore, neither addition of rhTGF-β1 or rhIL-10 to control MDDC supernatants nor exclusion of suppressor CD4 T cells (CD4+
lymphocytes), an important source of TGF-β1 and IL-10 blocked RSV-induced suppression (not shown). IL-12 enhances T-cell proliferation (61
) and was induced by RSV and UV-RSV. However, neither neutralization with anti-IL-12 MAb nor supplementation with rhIL-12 blocked RSV-induced immunosuppression (not shown). Similarly, IFN-γ, classically a product of T and NK cells, can be also expressed by DC (60
) and may be immunosuppressive (12
). Consistent with a previous report (39
), neutralizing MAb to IFN-γ enhanced, rather than diminished, suppression of proliferation (data not shown).
IFN-α levels were elevated in supernatants of MDDC exposed to live RSV (Fig. ). Similarly, RSV induces expression of IFN-λ in monocyte-derived macrophages (58
) and MDDC (Fig. ). IFN-λ1, IFN-λ2, and IFN-λ3 comprise the newly described type III IFN, which may be expressed by a variety of cells, including plasmacytoid DC, MDDC, and macrophages in response to a variety of stimuli, including viruses (13
). The receptor for the type III IFN consists of the ligand-binding chain, IL-28R, and the accessory receptor chain, IL-10R2 (27
). IL-28R is unique to the type III IFN, but IL-10R2 also dimerizes with IL-10R1, IL-22R, and IL-26R to signal in response to their respective cytokines (for a review, see reference 18
). In contrast to these three IL-10 family members, STAT1 and STAT2 primarily transduce signals through the IFN-λR complex, as they do for the IFN-α/β and -γ receptors (16
To determine whether IFN-α and IFN-λ contribute to RSV-induced immunosuppression, we used MAb to one of the two chains of IFN-α/β receptor, IFNAR2 (CD118), and to the two chains of the IFN-λR complex, IL-10R2 and IL-28R, to block receptor binding. In contrast to a previous report that IFN-α is solely responsible for RSV immunosuppression (42
), anti-IFNAR2 only modestly diminished immunosuppression (Fig. ). Similarly, anti-IL10R2 or anti-IL-28R was relatively ineffective. But when anti-IFNAR2 was combined with either anti-IL-10R2 or anti-IL-28R, the level of suppression was substantially decreased, thus demonstrating synergism between the two IFN. Since type I and III IFN both signal through STAT1 and STAT2, the two IFN might synergize by surpassing a threshold of signaling that neither could achieve alone. On the other hand, the two IFN may qualitatively signal such that only when present together are unique pathways activated or blocked. To our knowledge, this is the first report that type III IFN affect T-cell function.
The dependence of RSV-induced immunosuppression on type I and III IFN is paradoxical considering that (i) IFN are expressed in response to a variety of stimuli, including many other viruses that are not associated with T-cell suppression; (ii) plasmacytoid DC may express 100-fold as much IFN-α as MDDC and yet are critical to the ultimate antiviral adaptive response (56
); and (iii) type I IFN prevent activation-induced death of murine T cells in vitro after stimulation with superantigen in vivo (31
). The emerging story, however, is that the effects of type I IFN on CD4 T cells are dependent on the context in which they are exposed. For example, Dondi and colleagues showed that, in the context of TcR stimulation, exogenous IFN-α is antiapoptotic early and proapoptotic later (17
). Most relevant to our report, entry into the cell cycle was delayed when TcR stimulation followed IFN-α exposure (17
), apparently due to sequestration by the type I IFN receptor complex of CD45, Lck, and ZAP-70 (40
), all of which are necessary to initiate, but not to sustain, cellular responses to stimulation through the TcR (11
). Similarly, high concentrations of IFN-α cause STAT2 to associate with STAT6 and enhance signaling through the IL-4 receptor for the first 6 h of exposure; thereafter, IFN-α inhibits IL-4 responses (20
). Finally, IFN-α may activate STAT4 (36
), which while insufficient to induce Th1 differentiation (7
), may enhance it when induced by IL-12. It is likely that the contextual complexity of T-cell responses to IFN-α is only increased by the addition of type III IFN into the equation. The combination of type I and type III IFN could favor signaling through pathways that preferentially activate antiproliferative effects of IFN rather than those pathways that mediate the antiviral effects.
RSV has a number of strategies to minimize its vulnerability to IFN-α. The products of the NS1 and NS2 genes inhibit the upstream expression of IFN-α (8
) by suppressing activation and nuclear translocation of interferon regulatory factor 3 (8
) and block downstream responses by decreasing STAT2 levels (2
). Since cellular responses to IFN-α are complex and dependent upon context, it is reasonable to suspect that RSV may have achieved the fine balance of impairing antiviral properties of IFN-α while its suppressive activity may persist. Proliferation is not likely the only impaired T-cell response. For example, since IL-4 attenuates CD8 T-cell cytolytic function (3
), cytolysis may be further impaired when IFN-α enhances signaling in response to IL-4 (20
). This is but one potential mechanism by which RSV may deviate T-cell responses in ways that are highly relevant to infection with RSV and its sequelae, including childhood wheezing and asthma.
Conversely, suppression of T-cell responses during RSV infection may actually offer some benefit to the host. Our model system uses MDDC and autologous CD4 T cells derived from healthy, young adult donors, a population in which RSV lower respiratory tract disease is rare (22
). Since the host inflammatory response contributes significantly to lower respiratory disease (34
), diminished or absent suppressive activity in infants 2 to 6 months of age (22
) and the infirm elderly (21
) may account for their susceptibility to RSV pneumonia.