During viral infection of mice, a mixed type of Th response is generated (2
). As suggested by the antibody isotype profile (20
), the cytokine pattern elicited during the influenza virus response is likely to differ depending on viral replication and the location at which it is initiated. The dependence of IL-4 production on the expression of cellular NA (4
) suggests that influenza virus NA could polarize the T-cell response toward a Th2 type. We questioned whether infection of DC, the primary antigen-presenting cell type, with influenza virus would polarize an alloreactive T-cell response toward a type that produces IL-4.
Our previous work showed that the ability of DC to stimulate the proliferation of alloreactive T cells is changed by infection with influenza virus. At low doses of PR8, the stimulatory capacity of DC is enhanced by the activity of NA on the DC surface (23
). This effect, however, is dependent on the dose of PR8, so that at higher numbers of virus particles per cell, this increased response is not observed. This change is abrogated in the presence of antibodies that neutralize TGF-β1 (24
). In this report we show that the pattern of cytokines secreted in response to influenza virus-infected DC is also dose dependent. At low MOI, the response favors production of Th1-type cytokines IL-2 and IFN-γ, while at high MOI, Th2-type cytokines IL-4 and IL-10 are secreted (Fig. ).
The dose-dependent IL-2 and IFN-γ response reflects the proliferative response. Like proliferation, the increase observed in response to DC infected with low doses of PR8 is dependent on NA activity. This dependence is demonstrated by a lack of IL-2 and IFN-γ production when allogeneic T cells are stimulated with DC infected with NWS-Mvi, a mutant virus that lacks NA (Fig. ), and by inhibition of IL-2 and IFN-γ production in the presence of NA-specific antibodies (Fig. ) or zanamivir (Fig. ). To observe the increased Th1-type response, infection is not required, since UV-inactivated PR8 (Fig. ), PR8 that is neutralized by the addition of anti-HA (Fig. ), and purified NA (results not shown) generate similar increases. The possible mechanisms by which NA facilitates this response have been described in a previous publication (23
Since IL-12 supports the production of IFN-γ by T cells (9
), this cytokine, which is secreted by DC infected with low but not high doses of PR8 (Table ), is likely to support the NA-dependent Th1-type response. Other investigators also did not detect IL-12 in the supernatants of human macrophages that were infected with an apparently high dose of an H3N2 virus (a 1/10 dilution of allantoic fluid containing the virus was used to infect cells). However, under these conditions, the production of IFN-γ was retained and was supported by the production of IFN-α/β and IL-18 (27
). We have not included a complete analysis of cytokines present in our cultures; therefore, it is not possible to accurately predict which may be more important, especially since different pathways promote a Th1-type response (5
) and various outcomes are dependent on the mixture of cytokines present. For example, TGF-β inhibits the development of Th1 cells (28
) but in the presence of IL-4 supports IL-12-independent Th1 differentiation (16
). Our future experiments will therefore address mechanisms that may result in T-cell polarization in this in vitro system and will determine the relationship between NA and each of the relevant cytokines.
Since alloreactive T-cell proliferation is largely dependent on IL-2, it is not clear whether increased proliferation follows an increase in IL-2 secretion or whether the increased amount of IL-2 simply reflects the number of T cells in the culture. Clearly the reduced proliferation observed in cultures that were stimulated with DC infected with high doses of PR8 was not due to a lack of IL-2—supplementation with large amounts of this cytokine did not restore proliferation to levels obtained in cultures stimulated with either uninfected DC or DC infected at low MOI (results not shown).
In mixed cultures, the amount of IL-10 produced is dependent on the multiplicity of influenza virus particles (Fig. D) and IL-10 is produced in small amounts even when the virus is not infectious (Fig. D). It is noteworthy that IL-10 secretion is inhibited by the inclusion of zanamivir during the first 4 h of infection (Fig. D), suggesting that IL-10 production is facilitated by changes on the desialylated DC surface. Unlike IL-2 and IFN-γ production, which parallels the number of T cells present in mixed cultures, IL-10 secretion continues to increase when DC are infected with high doses of PR8 and T-cell proliferation is reduced. This suggests that the changes that occur in mixed cultures stimulated by DC that are infected at high MOI support the increased production of IL-10. TGF-β1 supports the production of IL-10 (19
). Since latent TGF-β1 can be activated by influenza virus NA (29
) and increased levels of TGF-β1 in culture supernatants are observed when DC are infected at high but not low MOI (Table ), the NA dependence of IL-10 production in our culture system may be due to the support of activated TGF-β1.
The production of IL-4 is clearly NA dependent. Following infection at high doses, IL-4 secretion by alloreactive T cells is almost completely inhibited by zanamivir when it is added throughout the culture of DC and T cells (Fig. ). Under these conditions, DC are infected since NA is not required for infection, but virus is not released from the host cell surface (7
). When zanamivir was added during the first 4 h of infection only, IL-4 was secreted by the responding allogeneic T cells (Fig. C), supporting the idea that removal of sialic acid from the surface of T cells and not of DC facilitates IL-4 production in response to influenza virus-infected DC. It is likely that the desialylation of T cells is required for the production of IL-4 (4
). When either DC or T cells are treated with an exogenous source of purified viral NA, the greatest levels of IL-4 are produced in the mixed lymphocyte cultures that contain desialylated T cells (Table ).
Since IL-4 is produced by infection with PR8 at high MOI only (Fig. C) and this is dependent on the activity of viral NA (Fig. C, C, and C), we propose that under these conditions, viral NA present in the supernatant cleaves terminal sialic acids from glycoconjugates on the T-cell surface. This idea is supported by the quantitation of sialic acid released from cells under these conditions. The amount of sialic acid in supernatants of infected DC was dose dependent (24
), and T cells treated with an amount of NA that was even smaller than that measured in supernatants of DC infected with high doses of PR8 released sialic acid into the medium. When T cells that had been stimulated with DC infected at high doses were washed and then treated with NA, the concentration of sialic acid measured in the supernatant was 0.312 ± 0.015 μg/ml, compared with 0.598 ± 0.019 μg/ml released by NA treatment from T cells that had been stimulated with uninfected DC. This suggests that some sialic acid was cleaved from T-cell surface glycoconjugates during culture with DC infected at high doses.
Electron microscopy, culture of the virus, and quantitation of NA activity show that virus particles that have active NA are released from DC infected with a high but not a low number of infectious particles per cell (24
), providing a rationale for these observations of dose dependence. The reason why virus particles are not formed by DC that are infected with a lower dose of PR8 is not clear. DC represent a cell type that, because it is essential for the activation of the adaptive immune response, may have mechanisms different from those of other cells to protect itself from the consequences of viral replication. Perhaps the inhibition of virus assembly by such a protective mechanism is overcome in the presence of an excessive number of virus particles or by defective interfering particles, which are likely to be present at higher MOI. Under these conditions the production of virions may be permitted, and consequently IL-4 production would be facilitated.
We predict that when NA is tethered to the surface of the antigen-presenting cell, a Th1-type response will predominate, but when NA cleaves both DC and T-cell surface substrates, a Th2-type response can be induced. Our in vitro studies therefore suggest that the types of cytokines produced during influenza virus infection may be determined in part by the location of NA activity.
Other DC surface molecules or cytokines produced by DC that can modulate the immune response probably also contribute to the polarization of the response, since Th1-Th2 development is the result of the strength of both T-cell receptor and cytokine signals (11
). These supporting factors may be NA independent (for example, influenza virus-infected DC have increased expression of ICAM-1 [23
]) or NA dependent (for example, the activation of TGF-β [24
]). Our future studies will therefore determine the mechanisms by which NA facilitates polarization of the T-cell response and will address the role of NA in polarizing T-cell responses in vivo.