Plasmacytoid dendritic cells control T-cell response to chronic viral infection

Abstract

Infections with persistent viruses are a frequent cause of immunosuppression, autoimmune sequelae, and/or neoplastic disease. Plasmacytoid dendritic cells (pDCs) are innate immune cells that produce type I interferon (IFN-I) and other cytokines in response to virus-derived nucleic acids. Persistent viruses often cause depletion or functional impairment of pDCs, but the role of pDCs in the control of these viruses remains unclear. We used conditional targeting of pDC-specific transcription factor E2-2 to generate mice that constitutively lack pDCs in peripheral lymphoid organs and tissues. The profound impact of pDC deficiency on innate antiviral responses was revealed by the failure to control acute infection with the cytopathic mouse hepatitis virus. Furthermore, pDC-deficient animals failed to clear lymphocytic choriomeningitis virus (LCMV) from hematopoietic organs during persistent LCMV infection. This failure was associated with reduced numbers and functionality of LCMV-specific CD4⁺ helper T cells and impaired antiviral CD8⁺ T-cell responses. Adoptive transfer of LCMV-specific T cells revealed that both CD4⁺ and CD8⁺ T cells required IFN-I for expansion, but only CD4⁺ T cells required the presence of pDCs. In contrast, mice with pDC-specific loss of MHC class II expression supported normal CD4⁺ T-cell response to LCMV. These data suggest that pDCs facilitate CD4⁺ helper T-cell responses to persistent viruses independently of direct antigen presentation. Thus pDCs provide an essential link between innate and adaptive immunity to chronic viral infection, likely through the secretion of IFN-I and other cytokines.

Infections with persistent viruses such as the human hepatitis C virus (HCV) or HIV often result in immunodeficiency, tissue degeneration, or neoplastic disease. Persistent viruses typically undergo massive and rapid initial replication followed by chronic persistence in the target tissue. Lymphocytic choriomeningitis virus (LCMV) provides a useful murine model that recapitulates key features of human chronic infections (1). LCMV is a noncytopathic RNA virus that, depending on its strain, can cause acute (e.g., strains WE, Armstrong) or chronic (e.g., strains Clone 13, Docile) infection (2). For instance, infection with LCMV Docile leads to rapid virus spread followed by clearance from blood and lymphoid organs but persistence in nonlymphoid tissues such as the kidneys (3). Persistent LCMV strains sustain prolonged replication and reach higher peak titers than acute strains, in part because of their higher replication rate, enhanced binding to target cells such as dendritic cells (DCs), and active suppression of innate immune response (4–6). The clearance of viruses is mediated primarily by virus-specific CD8⁺ cytotoxic T cells, which receive obligate help from IFN-γ–producing CD4⁺ T cells (7, 8). Massive replication of the persistent virus not only outpaces T-cell differentiation but also may induce the T-cell exhaustion that prevents long-term virus clearance (9). The exhaustion of virus-specific T cells may reflect their clonal deletion (10) or hyporesponsiveness marked by the expression of inhibitory receptors such as programmed death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) (11).

Type I interferons (interferons α and β, IFN-I) are antiviral cytokines that restrict viral replication and are used to treat chronic infections such as HCV (12). In the case of LCMV infection, IFN-I is required to control the initial virus spread and thus prevent the exhaustive activation and loss of virus-specific T cells (13). Furthermore, T-cell–intrinsic IFN-I signaling was reported to facilitate effector differentiation of CD4⁺ and CD8⁺ T cells (14, 15) or to substitute for T-cell help to cytotoxic T cells (16). However, the cellular source of IFN-I and its role in adaptive immune responses during chronic viral infections are not well understood.

Plasmacytoid dendritic cells (pDCs) sense virus-derived nucleic acids through Toll-like receptors (TLRs) 7 and 9 and respond with massive IFN-I production (17). Persistent viruses such as HIV (18) and LCMV (5) infect pDCs and elicit IFN-I production from these cells. Moreover, pDCs were shown to sense and respond directly to cells infected with persistent viruses such as HIV and HCV (19, 20). Indeed, many chronic viral infections are associated with profound depletion and/or functional impairment of pDCs (21). Notably, chronic LCMV infection is associated with reduced IFN-I production by pDCs; this reduced IFN-I production may increase susceptibility to opportunistic viral infections (22, 23). However, the role of pDCs in the adaptive immunity during chronic viral infections has not been examined directly. Indeed, the studies of pDC function have been limited to short-term ablation by antibodies (24) or transgenic toxin receptor-based systems (25). Apart from the issues of specificity and induced massive cell death, these approaches do not permit the long-term pDC ablation required for the study of chronic viral infections. Conversely, gene-targeted mice that lack pDCs, such as hypomorphic Ikaros mutants (26), have pleiotropic immune defects that complicate the interpretation of results. In this study, we developed a mouse model that constitutively lacks pDCs and used it to study their role in chronic LCMV infection.

Results

Conditional Targeting of E2-2 Causes Constitutive Loss of pDCs.

The development and maintenance of pDCs is controlled by transcription factor E2-2 (gene symbol Tcf4), whose global deletion leads to specific loss of pDCs (27, 28). We therefore undertook conditional targeting of E2-2 as a way to ablate the pDC lineage constitutively from birth. E2-2 is expressed in pDCs but is absent from classical dendritic cells (cDCs); however it also is expressed in certain lymphoid cell types such as B cells. To limit the deletion of E2-2 to the relevant cell type, we used the Itgax-Cre (CD11c-Cre) deleter strain specific for DC lineage (29). In the resulting conditional knockout (CKO) mice carrying one null and one LoxP-flanked E2-2 allele (Itgax-Cre⁺ Tcf4^flox/−), Cre recombination occurred primarily in cDCs and pDCs but was minimal in other E2-2–expressing cell types such as B cells (Fig. S1). The pDC population was reduced significantly in the bone marrow and was nearly absent in the secondary lymphoid organs and peripheral tissues of CKO mice (Fig. 1 A and C and Fig. S2). Consistent with the lack of E2-2 expression in cDCs, the cDC lineage in CKO mice was not affected (Fig. 1 B and C). The control and CKO animals were on a homogeneous (B6 × 129)F1 background and therefore were suitable for functional studies.

Fig. 1.

A mouse model of constitutive pDC ablation. CKO animals with DC-specific E2-2 deletion (Tcf4^flox/− Itgax-Cre⁺) were analyzed in parallel to Tcf4^flox/+ Itgax-Cre⁻ littermate controls (Ctrl). Statistically significant differences are indicated as follows: ***P < 0.001; **P < 0.01; *P < 0.05. (A) The pDC population in CKO mice. Shown are representative staining profiles of the bone marrow (BM) or splenic cells, with the frequencies of Bst2⁺ CD11c^low pDC population indicated (mean ± SD of three or four animals for the bone marrow and nine animals for the spleen). (B) The cDC population in CKO mice. Shown are staining profiles of total splenocytes with the CD11c^hi MHC class II⁺ (MHCII) cDC subset highlighted and the two cDC subsets within the gated cDC population. Subset frequencies represent mean ± SD of seven animals. The increase of the cDC fraction in CKO spleens is significant (P = 0.006). The B220⁺ CD8⁺ cDC-like cells arising from E2-2–deficient peripheral pDCs (28) could not be detected reliably in CKO mice. (C) Absolute numbers of pDCs and cDCs in lymphoid organs. Data shown are mean ± SD of four animals. (D) IFN-α production by total bone marrow cells or splenocytes cultured for 48 h with CpG DNA. Shown are IFN-α concentrations in the supernatant (mean ± SD of three to five animals). (E) IFN-α production after in vivo challenge with CpG DNA. Shown are serum IFN-α concentrations 6 h after CpG injection (mean ± SD of three animals).

The pDC-deficient CKO mice were grossly normal, did not exhibit any outward signs of autoimmunity or inflammation, and remained pDC-deficient until 18 mo of age. The secretion of IFN-α in response to TLR9 ligand unmethylated CpG-containing DNA (CpG) is mediated primarily by pDCs (30). Indeed, IFN-α levels were severely reduced in the CpG-containing cultures of total CKO splenocytes and bone marrow cells (Fig. 1D) and in the sera of CpG-induced CKO mice (Fig. 1E). Thus, adult E2-2 CKO mice manifest phenotypic and functional loss of peripheral pDCs.

Loss of pDCs Abolishes Innate Control of Coronavirus Infection.

Mouse hepatitis virus (MHV) is a highly cytopathic coronavirus causing acute hepatitis with subsequent clearance in immunocompetent mice. The innate control of MHV requires TLR7, IFN-I signaling, and the presence of pDCs, which secrete IFN-I to protect cDCs and macrophages (31, 32). Two days after MHV infection, CKO mice showed reduced serum IFN-α levels (Fig. 2A), up to 1,000-fold elevated viral load in the liver and spleen, and spread of virus to additional tissues such as the lungs (Fig. 2B). The hepatitis in CKO mice was exacerbated severely as judged by elevated serum alanine transaminase (ALT) (Fig. 2C) and massive leukocytic infiltration (Fig. 2D). These data confirm the functional loss of pDCs in CKO mice and emphasize the essential role of pDCs in the control of MHV. Previously, pDCs were found to be important for innate protection against several experimental viral infections such as genital herpes simplex virus (33) and pulmonary respiratory syncytial virus (34). In contrast, relatively minor effects of pDC depletion on IFN-I response and/or virus replication were observed in other viral infections (25, 26). Thus, the precise role of pDCs in the early innate control of viral infections varies in different viruses and infection models. However, pDCs appear to be absolutely essential for IFN-I–mediated protection against MHV, a prototypical acute cytopathic coronavirus. Given the ubiquitous presence of coronavirus infections such as MHV in mice and human common cold virus HCoV 229E, these data suggest a common evolutionary pressure on pDC function across different species.

Fig. 2.

The loss of pDCs abolishes innate control of acute MHV infection. Groups of five control and five CKO mice were infected with 50 pfu MHV A59 and analyzed 48 h later. Statistical significance is indicated as in Fig. 1. (A) Serum IFN-α concentrations determined by ELISA (mean ± SD). (B) Virus titers in the indicated organs (mean ± SD). (C) Serum levels of ALT indicative of liver damage (mean ± SD). (D) Representative liver sections stained with H&E. Arrows indicate inflammatory foci in livers of CKO mice. (Scale bar, 100 μm.)

pDC-Deficient Mice Fail to Control Chronic LCMV Infection.

Studies with IFN-I reporter mice showed that pDCs produce IFN-I early upon LCMV infection (35). However, pDC depletion did not affect systemic IFN-I production in response to LCMV Armstrong (24), suggesting that this response is mediated primarily by other cell types. Indeed, control and CKO mice showed similar titers and tissue distribution of acute LCMV strain WE early after infection (Fig. S3). The same was observed for LCMV Armstrong, which was fully cleared from all organs by day 8 (Table S1). These results confirm that pDCs are dispensable for the control of acute LCMV infection. Next, mice were infected with a relatively high dose (10⁵ pfu) of the persistent LCMV strain Docile. We found that the virus was cleared from the blood of control mice between days 21–28, but it persisted at high levels as late as day 53 in CKO mice (Fig. 3). Indeed, CKO mice showed increased viral titers in both spleen and blood on days 5–7 and failed to clear the virus by day 30 (Table S1). Importantly, the virus persisted in the kidneys of control mice on day 30, confirming the chronic infection (Table S1). Thus, pDCs are required to prevent broad virus persistence at hematopoietic sites during chronic LCMV infection.

Fig. 3.

Persistence of LCMV Docile in the blood of pDC-deficient mice. CKO mice or littermate controls were infected with 10⁵ pfu of LCMV Docile, and viral titers in blood were determined at the indicated time points postinfection. Data shown are mean ± SD of six mice per group.

Next, we examined the T-cell response to acute and chronic LCMV infection. Consistent with the efficient control of acute infection, normal CD8⁺ and CD4⁺ T-cell responses to LCMV Armstrong were observed in CKO mice after virus clearance (Fig. S4). Similarly, CKO mice mounted an efficient CD8⁺ T-cell response to the intracellular bacterium Listeria monocytogenes (Fig. S5), ruling out a general defect of T-cell priming in these animals. In contrast, infection with LCMV Docile was accompanied by a profound reduction of LCMV-specific MHC class I tetramer-binding CD8⁺ T cells in CKO mice on days 7 and 30 (Fig. 4 A and B). Similarly, IFN-γ production by CD8⁺ T cells in response to LCMV peptide restimulation was strongly reduced on day 7 and was nearly absent on day 30 (Fig. 4 A and C).

Fig. 4.

Impaired T-cell response to chronic LCMV infection in the absence of pDCs. CKO mice or littermate controls were infected with 10⁵ pfu of LCMV Docile, and LCMV-specific T-cell responses were analyzed on days 7 or 30. Total splenocytes were stained with LCMV peptide-MHC class I tetramers (tet) or were stimulated with LCMV peptides and stained for intracellular IFN-γ. Statistical significance is indicated as in Fig. 1. (A) Representative staining profiles of splenocytes highlighting gp33-specific CD8⁺ T cells and gp33-responsive IFN-γ–producing CD8⁺ T cells. (B and C) Frequencies of tetramer-positive CD8⁺ T cells (B) and of IFN-γ–producing CD8⁺ T cells (C) specific for the three indicated LCMV peptides. Data shown are mean ± SD of 8–11 mice pooled from three independent experiments. (D) Representative staining profiles of splenocytes highlighting gp61-responsive IFN-γ–producing CD4⁺ T cells. Values in the upper right quadrant indicate the percentage of IFN-γ–producing cells in the CD4⁺ T cell compartment. (E) The frequency of gp61-responsive IFN-γ–producing CD4⁺ T cells. Data shown are mean ± SD of seven mice from two independent experiments. p.i., postinfection.

Given the importance of CD4⁺ T-cell help in CD8⁺ T-cell responses, we examined the CD4⁺ T-cell response to LCMV Docile infection. Indeed, the frequency of CD4⁺ T cells producing IFN-γ in response to the MHC class II-restricted LCMV peptide gp61 was severely reduced on day 7 and remained low on day 30 postinfection (Fig. 4 D and E). Importantly, this reduction was not the result of increased regulatory T-cell expansion in CKO mice (Fig. S6). These results suggest that pDCs are required for optimal differentiation of IFN-γ–producing CD4⁺ T helper cells during chronic LCMV infection. Because day 7 postinfection is the earliest time point when LCMV-specific IFN-γ⁺ CD4⁺ T cells can be detected reliably, pDC deficiency appears to impair the early steps of CD4⁺ T-cell induction.

pDCs Are Required for Helper T-Cell Response to Persistent LCMV Infection.

To dissect further the role of pDCs in the activation of CD4⁺ T cells during chronic LCMV infection, we used transgenic CD4⁺ T cells expressing the T-cell receptor (TCR) specific for the LCMV peptide gp61 (SMARTA T cells) (36). SMARTA T cells were adoptively transferred into recipient mice that had been infected with LCMV Docile 1 d before the transfer, and their expansion was measured 7 d later. SMARTA T-cell expansion was impaired in Tlr7-deficient recipients (Fig. 5A), which show reduced IFN-I response and increased susceptibility to LCMV (35, 37). These data confirm that innate sensing of viral RNA facilitates the CD4⁺ T-cell response and suggest that Tlr7-expressing pDCs may be possible mediators of the process. Indeed, we found that the frequency of transferred T cells was reduced by more than fivefold in pDC-deficient CKO mice after LCMV Docile infection (Fig. 5B). As with endogenous CD4⁺ T cells, day 7 represented the earliest time point of detectable SMARTA T-cell expansion, suggesting that pDC deficiency impairs the induction of CD4⁺ T-cell response. In addition, the residual SMARTA T cells in CKO mice expressed higher levels of PD-1 and BTLA, the coinhibitory receptors associated with T-cell exhaustion (Fig. 5C).

Fig. 5.

The absence of pDCs impairs CD4⁺ T-cell response to chronic LCMV infection. The indicated mice were infected with 10⁵ pfu of LCMV Docile, received adoptive transfer of LCMV peptide gp61-specific SMARTA1 (SM1) TCR transgenic CD4⁺ T cells on day 1, and were analyzed on day 8. Statistical significance is indicated as in Fig. 1. (A) Expansion of the transferred SM1 TCR transgenic CD4⁺ T cells in Tlr7-deficient mice. Shown is the fraction of Thy1.1⁺ transferred T cells among the splenic CD4⁺ T-cell population in Tlr7^−/− or control C57BL/6 (B6) mice (mean ± SD; n = 5). (B) Expansion of SM1 T cells in pDC-deficient CKO or control (Ctrl) mice (mean ± SD; n = 8). (C) Surface levels of PD-1 and BTLA on the transferred T cells, with the mean fluorescence intensities indicated (mean ± SD; n = 8). (D) Expansion of IFNAR-deficient TCR transgenic CD4⁺ T cells. A 1:1 mixture of wild-type (Thy1.1⁺ Ly5.1⁻) and IFNAR-deficient (Thy1.1⁻ Ly5.1⁺) SM1 T cells was transferred into LCMV-infected control and CKO mice. Shown are fractions of transferred T cells among the splenic CD4⁺ T-cell population on day 8 (mean ± SD; n = 8). (E) Cytokine production by wild-type SM1 T cells transferred into LCMV-infected control or CKO recipients. Splenocytes were stimulated with gp61 peptide 8 d after the transfer. Shown is the frequency of cells producing indicated cytokines among the transferred Thy1.1⁺ SM1 T cells (mean ± SD; n = 6). (F) Expansion of SM1 cells in mice with pDC-specific deletion of MHC class II. The experiment was done as in D, with SM1 T cells transferred into LCMV-infected control (μMT:WT) or pDC-specific MHC class II knockout (μMT×PIII+IV-KO:WT) mice. Shown are fractions of transferred T cells among the splenic CD4⁺ T-cell population on day 8 (mean ± SD; n = 4). (G) Production of IFN-γ by wild-type SM1 T cells transferred into LCMV-infected control (μMT:WT) or pDC-specific MHC class II knockout (μMT×PIII+IV-KO:WT) mice. The results are shown as in E (mean ± SD; n = 4).

To explore further the role of IFN-I signaling in antiviral helper T-cell response (15), we used SMARTA CD4⁺ T cells that were deficient in the IFN-I receptor (IFNAR). A 1:1 mixture of wild-type and IFNAR-deficient SMARTA T cells was transferred into control or CKO mice 1 d after infection, and the relative expansion of donor T cells was measured 7 d later using allelic surface markers (Fig. S7). IFNAR-deficient T cells were barely detectable in both control and CKO mice, revealing the essential role of IFN-I in the expansion of CD4⁺ T cells (Fig. 5C). Importantly, the expansion of IFNAR-proficient SMARTA T cells was severely reduced in CKO mice (Fig. 5D), and the residual T cells were deficient in the production of cytokines IFN-γ, TNF-α and IL-2 (Fig. 5E). Thus, pDC-deficient animals show defective induction of an antiviral CD4⁺ T-cell response, whereas the few developing CD4⁺ T cells become functionally exhausted. This finding suggests that pDC deficiency causes a qualitative impairment of an LCMV-specific adaptive response rather than a mere delay in LCMV clearance.

To test whether the observed requirement for pDCs was based on their antigen-presentation capacity (38), we used mice that lack MHC class II expression selectively in pDCs. These mice harbor targeted mutation of the cell type-specific promoters III and IV of the MHC class II transactivator (CIITA PIII+IV) and are on the B-cell–deficient μMT background (39). SMARTA T cells were transferred into LCMV Docile-infected animals reconstituted with μMT×PIII+IV^−/− or control μMT bone marrow. No difference in the expansion (Fig. 5F) or function (Fig. 5G) of SMARTA T cells was observed, ruling out the role of MHC class II-dependent antigen presentation by pDCs. Thus, LCMV-specific CD4⁺ T cells depend on IFN-I signaling and on the presence of pDCs but not on their antigen-presentation capacity.

Next, we asked whether pDCs directly support the CD8⁺ T-cell response to LCMV Docile independently of CD4⁺ T-cell help. We used a similar adoptive transfer approach with P14 TCR transgenic CD8⁺ T cells specific for H2-D^b–restricted LCMV peptide gp33 (40). Importantly, the number of P14 T-cells used for adoptive transfer (5 × 10³) was much higher than the estimated number (100–200) of endogenous gp33-specific T cells (41). This number supported reproducible transfer and detection of T cells (42) and circumvented the need for endogenous CD4⁺ T-cell help in this setting. As shown in Fig. 6A, the expansion of LCMV-specific CD8⁺ T cells was reduced only marginally in CKO mice; furthermore, the expanded T cells showed normal expression levels of PD-1 and BTLA (Fig. 6B). Next, we performed competitive transfer of wild-type and IFNAR-deficient P14 CD8⁺ T cells (Fig. S7). IFNAR-deficient P14 T cells failed to expand in both control and CKO recipients (Fig. 6C), consistent with the role of IFN-I in LCMV-driven CD8⁺ T-cell expansion (14). However, the expansion of IFNAR-proficient CD8⁺ T cells was not reduced significantly in pDC-deficient CKO recipients and even was enhanced at higher T-cell numbers (Fig. S8). Thus, pDCs are not required directly for CD8⁺ T-cell expansion but appear to control T-cell response and virus clearance through CD4⁺ helper T-cell differentiation.

Fig. 6.

The absence of pDCs does not directly affect CD8⁺ T-cell response to chronic LCMV infection. CKO or control mice were infected with 10⁵ pfu of LCMV Docile, received adoptive transfer of LCMV peptide gp33-specific P14 TCR transgenic CD8⁺ T cells on day 1, and were analyzed on day 8. Data represent eight animals per group pooled from two independent experiments. Statistical significance is indicated as in Fig. 1. (A) Expansion of the transferred P14 TCR transgenic CD8⁺ T cells. Shown is the fraction of Ly5.1⁺ transferred T cells among the splenic CD8⁺ T-cell population in control or CKO mice (mean ± SD). (B) Surface levels of PD-1 and BTLA on the transferred T cells, with the mean fluorescence intensities indicated. (C) Expansion of IFNAR-deficient TCR transgenic CD8⁺ T cells. A 1:1 mixture of wild-type (Thy1.1⁻ Ly5.1⁺) and IFNAR-deficient (Thy1.1⁺ Ly5.1⁻) TCR transgenic CD8⁺ T cells was transferred into LCMV-infected control and CKO mice. Shown are fractions of transferred T cells among the splenic CD8⁺ T-cell population on day 8 (mean ± SD).

Discussion

We found that constitutive absence of pDCs is detrimental and could not be compensated by other cell types in two virus infection models, acute MHV and chronic LCMV Docile. The latter result reveals a specific function of pDCs in the adaptive immune response against chronic viral infection. In response to massive virus replication characteristic of persisting viruses such as LCMV Docile, pDCs facilitated optimal CD4⁺ T-cell activation and prevented their early exhaustion. A modest ∼twofold decrease in virus-specific CD8⁺ T cells was reported in pDC-depleted mice shortly after vesicular stomatitis virus infection, although the mechanism and consequences for virus control remained unclear (25). In contrast, our results demonstrate the absolute requirement for pDCs in the sustained CD8⁺ T-cell response to a persistent virus and in the resulting control of virus spread and viremia. These results imply that the profound impairment of pDC function during chronic LCMV infection (22, 23) is an essential adaptation by the virus that blunts T-cell responses and maintains virus persistence. In contrast, pDCs appear to be less important for T-cell responses to acute LCMV and other viral infections (25) that exhibit lower initial replication rates. Thus, our results reveal two essential functions of pDCs in antiviral responses: innate protection against acute cytopathic viruses such as MHV and facilitation of adaptive immune responses to rapidly replicating, noncytopathic viruses such as LCMV Docile.

The absence of pDCs caused an early and prolonged impairment of both helper and cytotoxic T-cell responses to persistent LCMV infection. It is likely that pDCs facilitate antiviral T-cell responses through a direct effect on CD4⁺ T cells, because only CD4⁺ but not CD8⁺ LCMV-specific T cells were dependent on the presence of pDCs in adoptive recipients. Importantly, pDCs were required to induce the clonal expansion of virus-specific CD4⁺ T cells as well as to prevent functional exhaustion of the resulting T cells. However, MHC class II-restricted antigen presentation by pDCs was dispensable, suggesting the cytokine secretion by pDCs as a primary mechanism. Given the absolute dependence of LCMV-specific T cells on IFN-I, the unique capacity of pDCs for IFN-I secretion is likely to play a major role. A formal proof of this notion would require an experimental model in which the IFN-I capacity of pDCs is selectively abolished. In the absence of such model and of pDC-specific gene targeting in general, the role of other pDC-derived cytokines cannot be ruled out. Interestingly, systemic levels of IFN after infection by LCMV Armstrong (24) are not affected by the absence of pDCs. Thus, any effect of pDC-derived IFN-I on CD4⁺ T-cell expansion would be likely mediated locally in a paracrine fashion.

The observations in the LCMV system are relevant for persistent human viruses that exhibit high initial replication rates and thereby can exhaust virus-specific T cells. Indeed, human viruses such as HIV induce pDC depletion and/or block pDC function through inhibitory pDC receptors, likely facilitating virus persistence (18, 21). Our data imply an important role of pDCs in the activation of antiviral T cells in chronic human virus infections such as HIV and HCV. Moreover, the therapeutic efficacy of IFN-I against persistent viruses such as HCV may result in part from its enhancement of T-cell responses in lieu of defective pDC function (43). Altogether, these results establish IFN-I secretion by pDCs as a critical event in the control of chronic infections and an attractive target for specific therapy.

Materials and Methods

Animals.

Mice carrying the conditional Tcf4^flox allele with dendritic cell-specific Itgax-Cre deleter transgene (C57BL/6 background) and mice carrying Tcf4 germline null allele (129SvEvTac background) have been described (27). They were intercrossed to generate CKO (Tcf4^flox/− Itgax-Cre⁺) and sex-matched littermate control (Tcf4^flox/+ Itgax-Cre⁻) mice on a homogeneous (B6 × 129)F1 background. These animals were generated at the Columbia University animal facility and were shipped to the Kantonal Hospital St. Gallen for analysis. The Tcf4^flox/− Itgax-Cre⁺ mice carrying the Cre-inducible fluorescent reporter allele (Gt(ROSA)26Sor-EYFP) have been described (28). Tlr7-deficient mice on a C57BL/6 background have been described (44). Bone marrow chimeric mice lacking MHC class II expression specifically on pDCs (μMTxpIII+IV^−/−:WT) and the respective controls (mMT:WT) were generated as described previously (39).

The TCR transgenic mice specific for the LCMV peptide gp61 (SMARTA1) Ly5.1⁺ (36) and SMARTA1⁺ Thy1.1⁺ on the Ifnar1^−/− background (45) were bred in the animal facilities of the Kantonal Hospital St. Gallen. TCR transgenic mice specific for the LCMV peptide gp33-41 (P14) Thy1.1⁺ (40) and P14⁺ IFNAR^−/− Ly5.1⁺ were kindly provided by M. F. Bachmann (Cytos AG, Schlieren, Switzerland) and A. Oxenius (Eidgenössische Technische Hochschule Zurich, Zurich). All animal work at the Kantonal Hospital St. Gallen was done in accordance with the Swiss Federal legislation on animal protection and was approved by the Veterinary Office of the Canton of St. Gallen. All animal work at Columbia University was done according to the protocol approved by the Institutional Animal Care and Use Committee.

Cells and Viruses.

MHV strain A59 (31) was propagated on 17Cl1 cells and titrated by plaque assay on L929 cells. Mice were injected i.p. with 50 pfu of MHV A59. LCMV strains Armstrong and Docile were propagated on L929 or MDCK cells, respectively, and titrated by focus-forming assay on MC57 cells. Mice were infected i.v. with 10³ pfu LCMV Armstrong or 10⁵ pfu LCMV Docile.

IFN Measurement.

The induction with CpG A (ODN 2216; Invivogen) was done as described (27). IFN-α concentration in culture supernatants and serum was measured by ELISA (PBL Biomedical Laboratories).

Histology and ALT Determination.

For histology, livers were fixed in 4% formalin and embedded in paraffin. Sections were stained with H&E. ALT was measured in serum using an Hitachi 747 autoanalyzer.

Antibodies and Peptide MHC Class I Tetramers and in Vitro Restimulation.

PE-conjugated tetramers against LCMV gp33-41 and LCMV np396-404 were generated as previously described (46). The PE-conjugated tetramer for the LCMV gp34-41 was purchased from Sanquin. Directly conjugated antibodies against the indicated surface markers were purchased from BD Biosciences, eBiosciences, or Biolegend. 7-amino-actinomycin D (Calbiochem) was used to discriminate dead cells in flow cytometric analysis.

For peptide-specific cytokine production, 10⁶ splenocytes were restimulated with gp33-41, gp34-41, np396-404, or gp61-80 peptides in the presence of Brefeldin A (5 μg/mL) for 5 h at 37 °C. Cells were stimulated with phorbol myristate acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) (both purchased from Sigma) as positive control or were left untreated as a negative control. For intracellular staining, restimulated cells were surface-stained and fixed with cytofix-cytoperm (BD Biosciences) for 20 min. Fixed cells were incubated at 4 °C for 40 min with permeabilization buffer [2% FCS and 0.5% (vol/vol) Saponin in PBS] containing anti–IFN-γ mAb. Samples were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson), and data were analyzed using CellQuest and FlowJo software (Tree Star, Inc.). Peptides gp33 (KAVYNFATC), gp34 (AVYNFATC), np396 (FQPQNGQFI), and gp61 (GLNGPDIYKGVYQFKSVEFD) were purchased from Neosystem.

Adoptive Transfer of TCR Transgenic Splenocytes.

Single-cell suspensions were obtained from spleens of SMARTA1⁺ Ly5.1⁺, SMARTA1⁺ IFNAR^−/− Thy1.1⁺, P14 Thy1.1⁺, and P14 IFNAR^−/− Ly5.1⁺ mice, and splenocytes were injected i.v. into the indicated recipient mice. For cotransfer experiments, cells were counted and mixed in a 1:1 ratio as confirmed by flow cytometry before injection.

Statistical Analysis.

All statistical analyses were performed with Prism 4.0 (Graphpad Software Inc.). Data were analyzed with the nonpaired Student's t test assuming that the values followed a Gaussian distribution.