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Type I IFN Negatively Regulates CD8⁺ T Cell Responses through IL-10-Producing CD4⁺ T Regulatory 1 Cells¹

Nektarios Dikopoulos^2,*, Antonio Bertoletti, Andrea Kröger, Hansjörg Hauser, Reinhold Schirmbeck^* and Jörg Reimann^*

^* Department of Medical Microbiology and Immunology, University of Ulm, Ulm, Germany; Institute of Hepatology, University College of London, London, United Kingdom; and German Research Centre for Biotechnology, Braunschweig, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8⁺ T cell responses in IFN- (IFN-^–/–)- or type I IFN receptor (IFNAR^–/–)-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR^–/– or IFN-^–/– mice have normal numbers of CD4⁺ and CD8⁺ T cells, and CD25⁺FoxP3⁺ T regulatory (T_R) cells in liver and spleen. Twice as many CD8⁺ T cells specific for different class I-restricted epitopes develop in IFNAR^–/– or IFN-^–/– mice than in normal animals after peptide- or DNA-based vaccination. IFN- and TNF- production and clonal expansion of specific CD8⁺ T cells from normal and knockout mice are similar. CD25⁺FoxP3⁺ T_R cells down-modulate vaccine-primed CD8⁺ T cell responses in normal, IFNAR^–/–, or IFN-^–/– mice to a comparable extent. Low IFN- or IFN- doses (500–10³ U/mouse) down-modulate CD8⁺ T cells priming in vivo. IFNAR- and IFN--deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4⁺ T cells after polyclonal or specific stimulation in vitro or in vivo. CD8⁺ T cell responses are thus subjected to negative control by both CD25⁺FoxP3⁺ T_R cells and CD4⁺IL-10⁺ T_R1 cells, but only development of the latter T_R cells depends on type I IFN.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Priming, clonal expansion, memory generation, and the response to Ag challenge of CD8⁺ T cells are regulated by APCs, suppressor, and Th cells, and humoral mediators. Limited information is available on the regulation of vaccine-induced, specific CD8⁺ T cell immunity, although the choice of Ag delivery and coinjected adjuvants decisively influences the type of specific cellular immunity that is primed. Furthermore, treatment protocols for the control of chronic virus infections aim to re-establish specific CD8⁺ T cell immunity in an attempt to attenuate the clinical course of the associated disease. Preclinical studies in mice can define mediators that help or suppress vaccine-induced CD8⁺ T cell responses, thereby paving the way to optimize current prophylactic or therapeutic immunization strategies.

Type I IFN (IFN-I or IFN-)-deficient mice have a defective antiviral defense (1, 2). Immunomodulatory effects of IFN-I on CD8⁺ T cell responses are pleiotropic (3). IFN-I keeps activated CD8⁺ T blasts alive (4) and supports their IL-2-driven proliferative response (5), thereby promoting CD8⁺ T cell priming (6, 7, 8). IFN-I-stimulated IL-15 release is required for CD8⁺ T cell memory maintenance and turnover (6, 7, 9, 10), and for survival and proliferation of NK cells that facilitate CD8⁺ T cell priming (11). But IFNs are also antiproliferative and can induce CD8⁺ T cell memory attrition (12, 13, 14, 15), although recently activated T blasts may be partially refractory to this effect by restraining transcriptional responses to IFN-I (16). The IFN--induced and dsRNA-activated kinase (PKR)³ negatively regulates CD8⁺ T cell function (17). Although IFN- and/or IFN- enhance IFN- production of CD8⁺ T cells (18), possibly in synergy with IL-18 (19, 20), they suppress CD40 ligation (but not LPS)-triggered IL-12 production (21, 22, 23), inhibit development of protective Th1 immunity (24), and are used therapeutically to induce Th2 immunity (25, 26). A major source of IFN- are immature, plasmacytoid dendritic cells (DC) (i.e., IFN--producing cells), and IFN-I also mediates autocrine survival and differentiation of this DC subset (27, 28). IFN- selectively blocks development of myeloid DC from bone marrow precursors (29) and monocytes (30), but enhances their CD40L-mediated activation (31).

Knowledge about the potential impact of IFN-I on virus-specific CD8⁺ T cell responses is particularly important in chronic infections with hepatitis B (HBV) or hepatitis C virus (HCV) in which IFN- represents the first treatment of choice. Its potential immunomodulatory role can have a direct impact on the long-term efficacy of the therapy because a sustained and robust virus-specific CD8⁺ T cell response is associated with long-term virus control in both infections.

IL-10 plays a key role in blocking proinflammatory cytokine production, costimulation, MHC class II expression, and chemokine secretion (32). Cross talk between IL-10 and IFN- has been revealed in vivo and in vitro. IL-10 reduces virus-induced IFN- responses of DC (33). In vitro, IFN- induces (in an IFN regulatory factor-1- and STAT3-dependent pathway) IL-10 production in human T cells cocultured with DC (21, 34). In vivo, IFN- induces IL-10 responses in virus-infected murine lungs (35). Type-1 CD4⁺ T regulatory (T_R1) cells are defined by their ability to produce high levels of IL-10 and TGF- (36). IL-10 promotes the differentiation of CD11c^lowCD45RB^high DC that promote the development of T_R1 cells both in vitro and in vivo (37), and IFN- enhances IL-10-induced differentiation of functional T_R1 cells (38). Type I IFNs could thus exert an IL-10-dependent, immunomodulatory effect on T cell responses.

We investigated the effect of type I IFNs on vaccine-induced CD8⁺ T cell immunity using mice lacking either the type I IFN receptor (IFNAR^–/–), or IFN- (IFN-^–/–). We selected this model because it allows us to study immunomodulatory effects of IFN-I without the concomitant effects of a virus infection. The observation of enhanced CD8⁺ T cell immunity with extended longevity prompted us to search for targets of the immunosuppressive effect of type I IFNs on CD8⁺ T cell responses. Although the suppressive effect of natural CD25⁺CD4⁺ T_R cells and the clonal expansion of T cells postpriming were normal in IFNAR- and IFN--deficient mice, these animals showed a deficiency of adaptive IL-10-producing CD4⁺ T_R1 cells in the course of specific CD8⁺ T cell responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6J (B6) mice (H-2^b), IFNAR-deficient (IFNAR^–/–) B6 mice (39), IFN--deficient (IFN-^–/–) B6 mice (40), A^b/OVA transgenic OT-II B6 mice, and RAG2^–/– B6 mice were bred and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). Male and female mice were used at 12–16 wk of age.

Vaccines and immunization protocols

DNA from the pCI/S expression vector (41) encoding the small hepatitis B surface Ag (HBsAg) under control of the HCMV IE promoter was prepared by PlasmidFactory. The K^b-restricted S2_190–197 (VWLSVIWM) HBsAg peptide (42) was fused to the cationic HIV-tat_50–57 KKRRQRRR domain (43). This peptide was dissolved in DMSO at a concentration of 10 mg/ml. To generate peptide/oligodeoxynucleotide (ODN) complexes, ODN were incubated for 30 min with peptides in PBS (pH 7.4) (43). HBsAg_ayw particles were obtained from K. Melber (Rhein-Biotech, Düsseldorf, Germany)

Mice were immunized by a single i.m. injection of either 100 µg of plasmid DNA (DNA vaccination), 50 µg of peptide complexed to 30 µgof ODN (peptide vaccination), or 10 µg of HBsAg particles with 30 µg of ODN (protein vaccination). We injected 50 µl into each tibialis anterior muscle. Three mice per group were used in all experiments. Representative data from one of at least three independent experiments are presented.

In vivo depletion of CD25⁺CD4⁺ T cells, IFN, and anti-CD3 mAb treatment of mice

CD25⁺ T_R cells were depleted using the mAb PC61 (or an isotype-matched control mAb), as described (44). Depletion of CD25⁺CD4⁺ cells was confirmed by flow cytometry. Mice were vaccinated 3 days after the Ab injection.

IFN- (catalog 12100-1; PBL Biomedical Laboratories) or IFN- (supernatants of a transfected Chinese hamster ovary (CHO) producer cell line) was injected s.c. Groups of mice were treated with (10², 5 x 10², or 10³ U/mouse) IFN- or IFN- 2 days before the immunization with pCI/S DNA. Alternatively, 10³ U/mouse IFN- was injected either 2 days before or 2 days after priming. Groups of mice were treated with supernatants from either CHO cells stably expressing the mouse IFN- gene, or nontransfected CHO cells of the identical subline. IFN- activity of the supernatant was tested in a bioassay, and 100, 500, or 1000 U was injected.

The anti-CD3 mAb 145-2C11 was injected i.p. once (10 µg/mouse in 200 µl of PBS) to polyclonally activated T cells in vivo.

Isolation of liver and spleen cells

Mice were sacrificed at different time points postvaccination. Spleens were removed and passed through a metal mesh to obtain a single cell suspension, and erythrocytes were lysed in NH₄Cl buffer (0.14 M NH₄Cl, 0.17 M Tris, pH 7.2). Liver nonparenchymal cells (NPC) were obtained, as described (45). CD4⁺ T cells were purified from spleen cell populations using MACS isolation kit (Miltenyi Biotec; CD4⁺ T cell isolation kit catalog no. 130-090-860). The purity of the isolated CD4⁺ T cells was >96%, as verified by flow cytometry (FCM). Splenic CD4⁺CD25⁺ T_R cells and CD11c^high B220^– DC were isolated from total spleen cells by electronic cell sorting to >98% purity.

Cell culture, cytokine determination (ELISA), and real-time PCR analysis

Cells were cultured in 200 µl flat-bottom microwells in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine, and antibiotics. DC were pulsed for 4 h with the indicated doses of the A^b-binding OVA_323–339 peptide ISQAVHAAHAEINEAGR (recognized by the transgene-encoded TCR of OT-II mice). Pulsed DC were washed twice, and cocultured (at 1 x 10⁴ DC/well) with 1 x 10⁵ purified responder CD4⁺ (OT-II) T cells.

Cytokine release was detected in supernatants by double-sandwich ELISA. All purified capture and biotinylated Abs and all recombinant mouse cytokine standards were from BD Biosciences. Extinction was analyzed at 405/490 nm on a TECAN microplate ELISA reader (TECAN) using the EasyWin software (TECAN).

The expression of FoxP3 mRNA was determined by real-time RT-PCR and relative quantification using hypoxanthine phosphoribosyltransferase as a reference gene (46). A total of 3 x 10⁵ cell sorter-purified splenic CD25⁺ or CD25^– CD4⁺ T cells was washed in PBS and lysed in TRIzol reagent (catalog no. 15596-026; Invitrogen Life Technologies), their RNA was extracted and reverse transcribed, and the reaction product was submitted to real-time PCR using the iQ SYBR Green supermix (catalog no. 170-8880; Bio-Rad) (47). Primer sequences for FoxP3, 5'-CCCAGGAAAGACAGCAACCTT-3' and 5'-TTCTCACAACCAGGCCACTTG-3'; for hypoxanthine phosphoribosyltransferase, 5'-TGAAGAGCTACTGTAATGATCAGTCAAC-3' and 5'-AGCAAGCTTGCAACCTTAACCA-3' (46).

Determination of specific CD8⁺ and CD4⁺ T cell frequencies

For determination of CD8⁺ T cell frequencies, cells (1 x 10⁷/ml) were pulsed in the presence of 2.5 µg/ml brefeldin A (BFA) (catalog no. 15870; Sigma-Aldrich) for 4 h in RPMI 1640 medium with 1 µg/ml K^b-binding peptide S2 (HBsAg_190–197) VWLSVIWM, or the K^b-binding peptide S1 (HBsAg_208–215) ILSPFLPL, or the peptide OVA_257–264 SIINFEKL. Spleen cells from anti-CD3 mAb-treated mice were restimulated in vitro in the presence of BFA (2.5 µg/ml) for 4 h with 50 ng/ml PMA and 500 ng/ml ionomycin. For determination of CD4⁺ T cell frequencies, spleen cells from immunized mice were restimulated in vitro in the presence of BFA (2.5 µg/ml) for 4 h with rHBsAg particles (10 µg/ml). Cells were harvested, washed, and stained with PE-conjugated anti-CD8 mAb (catalog no. 553032; BD Biosciences) or anti-CD4 mAb (catalog no. 553730; BD Biosciences). Surface-stained cells were fixed (2% paraformaldehyde in PBS); resuspended in permeabilization buffer (HBSS, 0.5% BSA, 0.5% saponin, 0.05% sodium azide); and incubated with FITC-conjugated anti-IFN- (catalog no. 554411; BD Biosciences), anti-TNF- (catalog no. 554418; BD Biosciences), or anti-IL-10 mAb (catalog no. 554466; BD Biosciences) for 30 min at room temperature, and washed twice in permeabilization buffer. Stained cells were resuspended in PBS supplemented with 0.3% w/v BSA and 0.1% w/v sodium azide. The number of cytokine-expressing CD8⁺ or CD4⁺ T cells per 10⁵ splenic CD8⁺ or CD4⁺ T cells was determined by FCM analysis.

Alternatively, freshly isolated cells were washed twice in FACS buffer (PBS/0.3% w/v BSA supplemented with 0.1% w/v sodium azide). Nonspecific binding of Abs to Fc receptor was blocked by preincubating cells with mAb 2.4G2 (catalog 01241D; BD Biosciences) directed against the FcRIII/II CD16/CD32 (0.5 µg of mAb/10⁶ cells/100 µl). Cells were incubated with allophycocyanin-conjugated anti-CD8 mAb (catalog no. 553035; BD Biosciences) and PE-conjugated tetramer S2/K^b (peptide VWLSVIWM bound to K^b), kindly provided by the National Institute of Allergy and Infectious Diseases Tetramer Facility, for 30 min at 4°C. Cells were washed twice in FACS buffer and analyzed by FCM.

FCM analyses

Cells were washed twice in PBS/0.3% w/v BSA supplemented with 0.1% w/v sodium azide. Nonspecific binding of Abs to Fc receptor was blocked by preincubating cells with mAb 2.4G2 (catalog no. 01241D; BD Biosciences) directed against the FcRIII/II CD16/CD32 (0.5 µg of mAb/10⁶ cells/100 µl). Cells were washed and incubated with 0.5 µg/10⁶ cells of the relevant mAb for 30 min at 4°C, and washed again twice. In most experiments, cells were subsequently incubated with a second-step reagent for 10 min at 4°C. Four-color FCM analyses were performed by FACScan (BD Biosciences). The forward narrow angle light scatter was used as an additional parameter to facilitate exclusion of dead cells and aggregated cell clumps. Data were analyzed using the WinMDI software. The following reagents and mAb were obtained from BD Biosciences: FITC-conjugated and biotinylated anti-CD3 mAb 145-2C11 (catalog no. 553062 and 553060, respectively), FITC- and PE-conjugated anti-NK1.1 mAb PK136 (catalog no. 553165 and 553165, respectively), PE-conjugated anti-CD4 mAb GK1.5 (catalog no. 553730) and allophycocyanin-conjugated anti-CD4 mAb (catalog no. 553051), PE-conjugated and allophycocyanin-conjugated anti-CD8 mAb (catalog no. 553032 and 553035), biotinylated anti-CD44 (Pgp-1) mAb (catalog no. 553132), biotinylated anti-CD69 mAb H1.2F3 (catalog no. 553235), and biotinylated anti-CD25 mAb (catalog no. 553070). SA-Red 670 was obtained from Invitrogen Life Technologies (catalog no. 19543-024).

Adoptive transfer of T cells

For CD3⁺ T cell isolation, total spleen cells were first stained with PE-conjugated anti-H57 mAb recognizing the -chain of the TCR (BD Pharmingen; catalog no. 553172) and then selected by MACS using anti-PE microbeads (Miltenyi Biotec; catalog no. 130-048-801). The purity of the isolated CD3⁺ T cells was >96%, as verified by FCM. A total of 1 x 10⁷ T cells in 500 µl of PBS was injected i.p. into naive, syngeneic immunodeficient RAG2^–/– recipients. Two days posttransfer, transplanted mice were immunized with plasmid pCI/S DNA. The frequencies of splenic, specific CD8⁺ T cells (K^b/S2 tetramer⁺ CD8⁺ T cells and IFN-⁺ CD8⁺ T cells) were assessed 12 days postimmunization for individual mice.

Statistical analyses

Data were analyzed using the GraphPad prism software (version 4.0). Values are presented as means ± SEM. The statistical significance of differences in the measured mean T cell frequencies between groups was calculated using Student’s two-tailed t test for two groups, or the one-way ANOVA, followed by Kruskal-Wallis test for three groups. A p value <0.05 was considered significantly different.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8⁺ T cell responses are enhanced in IFNAR- and IFN--deficient mice

Numbers and percentages of both CD4⁺ and CD8⁺ T cells and the distribution of these subsets in liver and spleen of IFNAR^–/– or IFN-^–/– B6 mice are indistinguishable to those in normal B6 mice (Fig. 1 and Table I). T cells from both subsets display similar surface marker profiles (CD69, CD44, CD45RB, CD62L) in normal and IFN-I-deficient mice (data not shown). Furthermore, similar numbers of DX5⁺ NK cells were found in liver and spleen of normal and IFNAR^–/– or IFN-^–/– B6-deficient mice (data not shown). We used these IFN-I-deficient mice in vaccination experiments to test the immunomodulatory effect of type I IFN on CD8⁺ T cell responses. Specific CD8⁺ T cell responses were detected in spleen and liver by either identifying specific CD8⁺ T cells with tetramers (S2/K^b tet), or specifically inducing IFN- or TNF- expression by a 4-h peptide stimulation ex vivo (Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Hepatic and splenic T cell populations in normal, IFNAR–/–, and IFN-–/– mice. Freshly isolated spleen cells and hepatic NPC were stained for CD3, CD4, and CD8. The percentage of CD4+ or CD8+ T cells within the gated CD3high T cell population in representative, individual mice (of five mice per group analyzed) is shown.

View this table: [in this window] [in a new window]   Table I. Hepatic and splenic T cell populations in normal, IFNAR-, and IFN--deficient B6 mice

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Enhanced CD8+ T cell responses in IFNAR–/– and IFN-–/– mice. Normal, IFNAR–/–, or IFN-–/– B6 mice were immunized with either the pCI/S DNA vaccine (encoding HBsAg), or the S2/Kb-tat peptide (Kb-restricted HBsAg epitope S2 fused to the cationic HIV-tat50–57 peptide) complexed to immune-stimulating oligonucleotides. Twelve days postimmunization, liver NPC and spleen cells were isolated. A, Freshly isolated cells were stained with anti-CD8 mAb and S2/Kb tetramer (S2/Kb tet), or CD8+ T cells restimulated for 4 h ex vivo with the S2 peptide (in the presence of 2.5 µg/ml BFA) were stained for intracellular IFN-. The S2/Kb tet+CD8+ T cells, or IFN-+CD8+ T cells in the hepatic or splenic CD8+ T cell populations from naive or primed, normal, or IFNAR-deficient B6 mice are shown. Representative data of one (of three independent) experiment(s) with four mice per group are shown. B, The frequencies of specific IFN-+ CD8+ T cells or TNF-+CD8+ T cells in liver and spleen from naive or vaccinated normal, IFNAR–/–, and IFN-–/– B6 mice are shown as mean values (±SEM) of four mice per group of three independent experiments. The differences were considered statistically significant when p < 0.05 (*).

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Kinetics of CD8+ T cell responses in liver and spleen of normal, IFNAR–/–, and IFN- –/– B6 (H-2b) mice. Mice were immunized i.m. by the pCI/S DNA vaccine (100 µg/mouse) encoding HBsAg. At the indicated time points postimmunization, liver NPC and spleen cells were isolated and restimulated for 4 h in vitro with the Kb-binding S2 peptide (1 µg/ml) in the presence of BFA (2.5 µg/ml). Cells were labeled for surface CD8 and intracellular IFN-. Alternatively, freshly isolated cells were labeled with anti-CD8 mAb and the S2/Kb tetramer (tet) without prior in vitro restimulation. A, Frequencies of specific IFN-+CD8+ T cells or S2/Kb tet+CD8+ T cells in CD8+ T cell populations from liver or spleen of vaccinated, normal, or IFNAR-deficient B6 mice at different time points postimmunization. Mean values (±SEM) of four mice per group and per time point (of two independent) experiment(s) are shown. The differences were considered statistically significant when p < 0.05 (*). B, Frequencies of specific IFN-+CD8+ T cells in CD8+ T cell populations from liver or spleen of vaccinated, normal, or IFN-–/– B6 mice at different time points postimmunization. Mean values (±SEM) of four mice per group and per time point (of two independent experiments) are shown. The differences were considered statistically significant when p < 0.05 (*).

In addition to IFNAR^–/– B6 mice, we used B6 mice with a disrupted IFN--encoding gene in the next vaccination experiments. Similar to IFNAR^–/– B6 mice, IFN-^–/– B6 mice generated larger populations of specific CD8⁺ T cells after DNA- or peptide-based vaccinations (Fig. 2B). This was confirmed when we followed the kinetics of appearance and decline of specific CD8⁺ T cells in liver and spleen of IFN-^–/– mice after vaccination (Fig. 3B). Similar to the data described for IFNAR^–/– mice, the specifically triggered cytokine expression profile and cytolytic activity of CD8⁺ T cell primed in the absence of IFN- were indistinguishable from those primed in normal B6 mice (Fig. 2B, data not shown). Similar to IFNAR deficiency, IFN- deficiency thus leads to enhanced, specific CD8⁺ T cell responses, indicating that neither IFN- nor IFN-I signaling plays a critical role in CD8⁺ T cell priming.

IFN-I down-modulates CD8⁺ T cell priming

Enhanced CD8⁺ T cell responses in IFNAR- and IFN--deficient mice may result from type I IFNs: 1) impairing priming of CD8⁺ T cell precursors; 2) restricting clonal expansion of primed CD8⁺ T cells; and/or 3) activating a suppressive, T_R subset. The following experiments were designed to test each of these hypotheses.

Larger populations of specific CD8⁺ T cells developing in the absence of IFN- may result from more efficient T cell priming in the absence of IFN-I. We therefore tested whether IFN-I down-modulates CD8⁺ T cell priming. The s.c. injections of different doses of rIFN- 2 days before immunization (Fig. 4A) or a single injection (2 days before or 2 days after vaccination) of 10³ U of IFN- (Fig. 4B) into B6 mice down-modulated priming of CD8⁺ T cell responses apparent in the lower numbers of specific CD8⁺ IFN-⁺ T cells in liver and spleen. Similarly, CD8⁺ T cell responses were down-modulated in mice injected with 100, 500, or 1000 U of IFN- (supernatants with titrated IFN activity from CHO cells stably expressing a mouse IFN- gene), but not in mice injected with supernatants from nontransfected CHO cells (data not shown). Thus, IFN-I down-regulates CD8⁺ T cell priming in vivo.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. IFN- or IFN- down-modulates CD8+ T cell priming in vivo and in vitro. A, B6 mice were injected s.c. with the indicated doses of IFN-. Two days after the IFN injection, mice were immunized with pCI/S DNA. B, Alternatively, mice received a single injection of 103 U of IFN- either 2 days before or 2 days after priming with the pCI/S DNA vaccine. Liver NPC and spleen cells were isolated 12 days postimmunization and restimulated in vitro for 4 h with the Kb-restricted S2 peptide (1 µg/ml) in the presence of BFA (2.5 µg/ml). Mean values (±SEM) of IFN-+ CD8+ T cells/105 CD8+ T cells of four mice per group (of three independent experiments) are shown. The differences compared with the untreated control group were considered statistically significant when p < 0.05 (*).

IFN- does not down-modulate clonal expansion of CD8⁺ T cells

IFN- could limit clonal expansion of primed CD8⁺ T cells through its well-known antiproliferative effect. To test this, we transferred equal numbers of purified splenic T cells from normal or IFNAR^–/– B6 mice into congenic RAG^–/– B6 hosts. The mice were vaccinated 2 days posttransfer, and the absolute numbers of tetramer⁺CD8⁺ T cells and the frequencies of specific CD8⁺IFN-⁺ T cells were measured 12 days postpriming by flow cytometry. Specific CD8⁺ T cells from normal (IFN--responsive) or IFNAR^–/– (IFN--nonresponsive) mice expanded equally well in the adoptive host (Fig. 5). The suppressive effect of type I IFNs on CD8⁺ T cell responses is thus unlikely to be mediated by an antiproliferative effect on clonal expansion.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. In vivo priming of normal or IFNAR-deficient CD8+ T cells in syngeneic, adoptive RAG2–/– hosts. T cells (1 x 107/mouse in 500 µl of PBS) from normal or IFNAR-deficient B6 mice (isolated by MACS) were injected into syngeneic, immune-deficient RAG2–/– mice. Two days posttransfer, the mice were immunized i.m. with 100 µg of pCI/S DNA vaccine. Spleen cells obtained 12 days postimmunization were restimulated in vitro for 4 h with the Kb-binding S2190–197 peptide (1 µg/ml) in the presence of BFA (2.5 µg/ml) and labeled. Freshly isolated cells were costained with anti-CD8 mAb and S2/Kb tetramer (S2/Kb-tet). The frequencies of IFN-+CD8+ T cells or tet+CD8+ T cells/105 CD8+ T cells were determined. Mean values (±SEM) of three mice per group are shown.

CD25⁺CD4⁺ T_R cells from IFNAR- and IFN--deficient mice down-modulate CD8⁺ T cell responses

CD25⁺CD4⁺ T_R cells are a major (but not the only) T_R cell subset that down-modulates CD8⁺ T cell responses (51, 52, 53, 54, 55). These T_R cells are defective in STAT1^–/– knockout mice (56), an important signal transducer of the IFNAR (57). We therefore evaluated the numbers and the function of CD25⁺CD4⁺ T_R cells in IFNAR^–/– and IFN-^–/– B6 mice. Both lines contained normal numbers of CD25⁺CD4⁺ T_R cells expressing a surface profile indistinguishable from that of normal mice (Fig. 6A, data not shown). Comparable levels of expression of the transcription factor FoxP3, the most reliable marker of this T cell subset (46), were detectable in CD25⁺CD4⁺ T_R cells from naive or immunized mice by quantitative RT-PCR (Fig. 6B, data not shown). Most importantly, in vivo depletion of CD25⁺ T cells by treatment with the mAb PC61 before vaccination enhanced the vaccine-primed CD8⁺ T cell response in normal, and IFN-^–/– or IFNAR^–/– B6 mice (Fig. 6C, data not shown). Development and function of CD25⁺ T_R cells are thus IFN- independent, and the down-modulation of CD8⁺ T cell responses by IFN-I is not mediated by these T_R cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. CD4+CD25+ TR cells efficiently control CD8+ T cell responses in IFNAR-deficient mice. A, Freshly isolated spleen cells from normal and IFNAR-deficient B6 mice were stained for CD3, CD4, and CD25. The CD3+CD4+ T cell population was gated and analyzed for expression of CD25. The numbers in the dot plots represent the percentage of CD4+CD25+ T cells within the CD4+ T cell population. B, The CD4+CD25+ TR cell population and the control CD4+CD25– T cell population from naive or DNA-immunized mice were electronically sorted (purity >98%). Total RNA was prepared from sorted cells, and their mRNA expression was quantitatively determined by real-time PCR analysis. The mean values (±SEM) of mRNA expression from four different mice per group are shown. C, Mice were immunized with either the pCI/S DNA vaccine or the S2/Kb-tat peptide vaccine. In some groups, CD25+ T cells were depleted by i.p. injection of 1 mg of anti-CD25 mAb PC61 (three injections in the week before immunization). Twelve days postimmunization, liver NPC and spleen cells were isolated and restimulated in vitro either with the Kb-restricted S2190–197 or S1208–215 peptide (1 µg/ml) for 4 h in the presence of BFA (2.5 µg/ml). Mean values (±SEM) of IFN-+CD8+ T cells/105 CD8+ T cells of five mice per group (of two independent experiments) are shown. The differences were considered statistically significant when p < 0.05 (*).

We tested whether CD4⁺ T cell priming is modulated by IFN-I in vitro or in vivo. Purified, naive (IFN-I-responsive) CD4⁺ T cells from OT-II RAG1^–/– B6 mice were cocultured with peptide-pulsed, splenic CD11c^highB220^– DC from normal, IFN-^–/–, or IFNAR^–/– B6 mice (Fig. 7, data not shown). CD4⁺ T cells specifically stimulated by IFNAR- or IFN--deficient DC produced more IFN-, but less IL-10, over a 3-day incubation period. We tested whether lower numbers of suppressive, IL-10-producing CD4⁺ T_R1 cells are generated during polyclonal or specific T cell responses in vivo. B6 mice were injected i.p. with 10 µg of anti-CD3 mAb 145-2C11, and the inducible IFN- or IL-10 expression splenic CD4⁺ T blasts were assayed 12 h postinjection (Fig. 8A). The number of IFN--producing CD4⁺ T blasts was increased, while the number of IL-10-producing CD4⁺ T cells was reduced 3-fold in IFNAR-deficient mice. Similar data were obtained when we tested the specifically inducible IFN- or IL-10 expression profile of CD4⁺ T cells from normal or IFNAR- and IFN--deficient mice primed by vaccination with pCI/S plasmid DNA or HBsAg particles (Fig. 8B, data not shown). In IFNAR-deficient mice, the number of primed CD4⁺ T cells specifically inducible to IFN- expression was increased >2-fold, but the number of CD4⁺ T cells specifically inducible to IL-10 expression was reproducibly decreased 2-fold. No IL-10-producing, specific CD8⁺ T cells were detected in vaccinated normal or IFNAR-deficient B6 mice (data not shown). IFN-I thus facilitates generation of suppressive CD4⁺ T_R1 cells that down-modulate CD8⁺ T cell responses.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. In vitro priming of naive CD4+ OT-II T cells by normal or IFNAR-deficient DC. Splenic CD11c+ B220– DC (1 x 104 cells/well) from normal () or IFNAR–/– () mice were pulsed for 4 h with the Ab-binding OVA323–339 peptide ISQAVHAAHAEINEAGR (recognized by the transgene-encoded TCR of CD4+ OT-II T cells), washed twice, and cocultured with naive CD4+ OT-II T cells (1 x 105 cells/well). A, Release of IFN- and IL-10 by CD4+ OT-II T cells in a 48-h coculture with DC pulsed with increasing doses of peptide. B, Time kinetics of IFN- and IL-10 release by CD4+ OT-II T cells during a 72-h coculture. Mean values of triplicates (±SEM) of three independent experiments are shown. The differences were considered statistically significant when p < 0.05 (*).

View larger version (22K): [in this window] [in a new window]   FIGURE 8. CD4+ T cell responses in normal and IFNAR-deficient mice to polyclonal or specific activation in vivo. A, Spleen cells from normal or IFNAR-deficient B6 mice injected i.p. with 10 µg of anti-CD3 mAb 145-2C11 were obtained 12 h postinjection and restimulated in vitro for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of BFA (2.5 µg/ml). Cells were stained for surface CD4 and intracellular IFN- or IL-10. Frequencies of cytokine-expressing CD4+ T cells/105 CD4+ T cells were determined in FCM analysis. B, Normal or IFNAR-deficient B6 mice were immunized i.m. with the pCI/S DNA vaccine (100 µg/mouse). Twelve days postvaccination, cells were harvested and restimulated in vitro with rHBsAg particles (10 µg/ml) in the presence of BFA (2.5 µg/ml). Frequencies of IFN-- and/or IL-10-expressing CD4+ T cells/105 CD4+ T cells were determined. Mean values (± SEM) of four mice per group (of three independent) experiment(s) are shown. The differences were considered statistically significant when p < 0.05 (*).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Enhanced, vaccine-primed CD8⁺ T cell response in mice deficient in IFN- or IFNAR was similar. The origin of the IFN- required for this effect is unknown. Although all cell types can produce IFN-, the major producer cells in the immune system are plasmacytoid DCs. What triggers cells to induce IFN- gene activation? Because viruses or LPS can be excluded in our vaccination approach, few known triggers have to be considered, including receptor activation of NF-B ligand (RANKL) or TLR9. RANKL-mediated signals induce IFN- that blocks osteoclast precursor differentiation (58). A RANKL/RANK-induced IFN-I signal may impair the ability of DC to prime T cells. IFN-^–/– mice have been reported to show an altered splenic architecture, a reduction in some myeloid precursor subsets in the bone marrow, and decreased numbers of resident and circulating macrophages and granulocytes (59). Development and function of APC subsets may be altered in IFN--deficient mice.

The number and surface phenotype of splenic and hepatic CD4⁺ or CD8⁺ T cells and NK cells in the two IFN-I-deficient B6 lines used in these studies were indistinguishable from that in normal B6 mice. It is thus unlikely that the enhanced CD8⁺ T cell responses in IFNAR- and IFN--deficient mice result from expanded populations of responding T cells. We found a reduction in NKT cells and plasmacytoid DC in liver and spleen of IFNAR-deficient mice (data not shown). The numbers of vaccine-primed, specific (tetramer⁺) CD8⁺ T cells were increased 2-fold in IFN-I-deficient mice. Enhanced clonal expansion of primed CD8⁺ T cells in the absence of type I IFN responsiveness does not explain the larger numbers of specific T cells detected postvaccination, as demonstrated by adoptive transfer experiments. No functional defects were found in CD8⁺ T cells developing in a type I IFN-deficient environment. The specific cytolytic activity of these T cells was not impaired (as assayed in the semiquantitative ⁵¹Cr release test using titrated E/T ratios; data not shown). Increased IFN- and TNF- expression of T cells in diseased IFN-^–/– mice has been reported (40), but IFN-^–/– mice also show reduced (constitutive and induced) expression of TNF- in macrophages (59). The fraction of CD8⁺ T cells inducible to IFN- and/or TNF- expression after a specific 4-h ex vivo restimulation was similar in IFNAR-, IFN--deficient, and normal B6 mice. Our data resemble the described negative regulation of CD8⁺ T cell responses by IFN--induced and dsRNA-activated kinase PKR (17). IFN-I is thus not required to prime functional CD8⁺ T cell response, but it down-modulates magnitude and longevity of the response.

The ability of type I IFNs to inhibit CD8⁺ T cell expansion might explain the apparent different impact of IFN- on HCV-specific CD4⁺ and CD8⁺ T cell response. IFN- treatment has been shown to increase HCV-specific CD4⁺ T cell responses (60, 61), but has little effect on HCV-specific CD8⁺ response (60). Furthermore, the potential ability of IFN- to inhibit T cell responses might partially explain the conflicting results about recovery of T cell response in HBV-infected patients treated with lamivudine only, or with a combination of lamivudine and IFN-. Although, in the former group, inhibition of HBV replication is followed by a recovery of HBV-specific CD4⁺ and CD8⁺ T cells (62, 63), patients treated with an IFN- plus lamivudine combination therapy did not show any recovery of T cell response despite identical inhibition of HBV replication (64).

T_R cells have a major (direct or indirect) effect on priming, clonal expansion, functional competence, and memory generation of specific CD8⁺ T cells. Two distinct CD4⁺ T_R subsets have been implicated in the control of specific CD8⁺ T cell immunity: natural CD25⁺FoxP3⁺ T_R cells and adaptive IL-10⁺ T_R1 cells (65). The number of CD25⁺ T_R cells, their surface phenotype, their FoxP3 expression, and, most importantly, their suppressive effect of ongoing CD8⁺ T cell responses were intact in IFNAR- and IFN--deficient mice. In contrast, in vitro and in vivo priming of IL-10-producing CD4⁺ T cell responses to two unrelated Ags was defective when the epitope was presented by DC in the absence of IFN-. These data confirm the essential role of IFN-I in triggering an IL-10 response of DC that supports the differentiation of T_R1 cells (34, 38). Negative feedback by IL-10 may down-regulate IFN-I production (33). The novel finding is that CD8⁺ T cell responses are under the dual, suppressive control of CD25⁺CD4⁺ T_R cell and CD4⁺ T_R1 cells. This may explain why some vaccine formulations prime CD8⁺ T cell responses more efficiently in CD4⁺ T cell-deficient than normal mice (48). It is unresolved whether natural CD25⁺ T_R cells are required or facilitate the response of adaptive IL-10⁺ T_R1 cells (65). This IFN-I-mediated copriming of suppressive T_R1 cells limits the magnitude of antiviral CD8⁺ T cell responses that tend to dramatically expand, but reach peak numbers late in acute virus infections when the virus titers have already been reduced substantially by effector mechanisms of the innate immune system.

	Acknowledgments

 
We greatly appreciate the expert technical assistance of Tanja Guentert, Daniela Schey, und Katrin Oelberger. We thank Dr. J. Demengeot (Lisbon, Portugal) for the generous gift of B6 IFNAR^–/– mice. The S2/K^b tetramers (peptide VWLSVIWM bound to K^b) were kindly provided by the National Institute of Allergy and Infectious Diseases Tetramer Facility.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Re549/10-1), the Wilhelm-Sander-Foundation (088.3), and the European Commission (QLK2-CT-2002-00700) to J.R.

² Address correspondence and reprint requests to Dr. Nektarios Dikopoulos, Department of Medical Microbiology and Immunology, University of Ulm, Helmholtzstr. 8/1, D-89081 Ulm, Germany. E-mail address: nektarios.dikopoulos{at}medizin.uni-ulm.de

³ Abbreviations used in this paper: PKR, IFN-I-induced dsRNA-activated kinase; BFA, brefeldin A; CHO, Chinese hamster ovary; DC, dendritic cell; FCM, flow cytometry analysis; HBsAg, hepatitis B virus surface Ag; HBV, hepatitis B virus; HCV, hepatitis C virus; IFNAR, IFN-I receptor; NPC, nonparenchymal cell; ODN, oligodeoxynucleotide; RANKL, receptor activation of NF-B ligand; T_R, regulatory T.

Received for publication July 19, 2004. Accepted for publication September 16, 2004.

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