Type I IFN Negatively Regulates CD8+ T Cell Responses through IL-10-Producing CD4+ T Regulatory Cells
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免疫学杂志 2005年第1期
Abstract
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 (TR) 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+ TR 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+ TR cells and CD4+IL-10+ TR1 cells, but only development of the latter TR cells depends on type I IFN.
Introduction
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 (TR1) cells are defined by their ability to produce high levels of IL-10 and TGF- (36). IL-10 promotes the differentiation of CD11clowCD45RBhigh DC that promote the development of TR1 cells both in vitro and in vivo (37), and IFN- enhances IL-10-induced differentiation of functional TR1 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+ TR 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+ TR1 cells in the course of specific CD8+ T cell responses.
Materials and Methods
Mice
C57BL/6J (B6) mice (H-2b), IFNAR-deficient (IFNAR–/–) B6 mice (39), IFN--deficient (IFN-–/–) B6 mice (40), Ab/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 Kb-restricted S2190–197 (VWLSVIWM) HBsAg peptide (42) was fused to the cationic HIV-tat50–57 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). HBsAgayw 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+ TR 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+ TR cells and CD11chigh 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 Ab-binding OVA323–339 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 Kb-binding peptide S2 (HBsAg190–197) VWLSVIWM, or the Kb-binding peptide S1 (HBsAg208–215) ILSPFLPL, or the peptide OVA257–264 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/Kb (peptide VWLSVIWM bound to Kb), 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 (Kb/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
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/Kb tet), or specifically inducing IFN- or TNF- expression by a 4-h peptide stimulation ex vivo (Fig. 2).
Antiviral CD8+ T cell responses to the Kb-restricted HBsAg epitope S2 were efficiently primed in normal and IFNAR-deficient mice by peptide (S2/Kb-tat)- or DNA (pCI/S)-based vaccination (42, 43). Both vaccination protocols primed CD8+ T cell responses in IFNAR–/–, IFN-–/–, and normal B6 mice that were readily detectable in the spleen. High frequencies (but lower numbers) of specific CD8+ T cells were also found in the liver, confirming our previous reports (45, 48) (Fig. 2A and Table II). Two to three times more specific CD8+ T cells were found postvaccination in liver and spleen from IFNAR- or IFN--deficient than normal mice, irrespective of the immunization protocol used (Fig. 2B). Although the adjuvant effect of CpG-containing ODNs or DNA vaccines has been proposed to be IFN-I dependent (8, 49, 50), the data indicate that the efficacy in CD8+ T cell priming of the ODN-dependent vaccines tested is enhanced in the absence of IFN-I. The frequencies of specific CD8+ T cells in spleen and liver were followed for 6 wk postvaccination (Fig. 3A). The data confirm that larger numbers of tetramer (S2/Kb tet)+ or IFN--expressing specific CD8+ T cells are found in IFNAR–/– than normal B6 mice throughout this observation period.
Primed CD8+ T cells specifically inducible to IFN- and/or TNF- expression were equally increased in number in vaccinated IFNAR–/– mice (Fig. 2B). IFN- (or its absence) thus does not modulate the expression profile of these two cytokines in vaccine-primed CD8+ T cells. This was apparent in specific CD8+ T cells primed by different vaccination protocols. The specific cytolytic activity of CD8+ T cells primed in IFNAR- and IFN--deficient mice was similar to that detected in normal mice (data not shown). Thus, IFN--independent priming generates functionally competent CD8+ T cell populations.
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, TR 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.
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.
CD25+CD4+ TR cells from IFNAR- and IFN--deficient mice down-modulate CD8+ T cell responses
CD25+CD4+ TR cells are a major (but not the only) TR cell subset that down-modulates CD8+ T cell responses (51, 52, 53, 54, 55). These TR 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+ TR cells in IFNAR–/– and IFN-–/– B6 mice. Both lines contained normal numbers of CD25+CD4+ TR 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+ TR 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+ TR cells are thus IFN- independent, and the down-modulation of CD8+ T cell responses by IFN-I is not mediated by these TR cells.
Reduced numbers of IL-10-producing CD4+ TR1 cells are generated in IFNAR-deficient mice
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 CD11chighB220– 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+ TR1 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+ TR1 cells that down-modulate CD8+ T cell responses.
Discussion
CD8+ T cell responses are primed more efficiently by different vaccination protocols in IFN--deficient than in normal mice. Regulation of the response by natural CD25+FoxP3+CD4+ TR cells, and the clonal expansion of primed CD8+ T cells were not affected by IFN-. In contrast, priming of CD8+ T cells was down-regulated by IFN-I, and the generation of adaptive, IL-10-producing TR1 cells was up-regulated in the presence of IFN-. Although MHC class I-restricted presentation of Ag to T cells is facilitated by IFN-I, the overall effect of these IFNs seems to be a down-modulation of CD8+ T cell responses.
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).
TR 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+ TR subsets have been implicated in the control of specific CD8+ T cell immunity: natural CD25+FoxP3+ TR cells and adaptive IL-10+ TR1 cells (65). The number of CD25+ TR 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 TR1 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+ TR cell and CD4+ TR1 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+ TR cells are required or facilitate the response of adaptive IL-10+ TR1 cells (65). This IFN-I-mediated copriming of suppressive TR1 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/Kb tetramers (peptide VWLSVIWM bound to Kb) 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@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; TR, regulatory T.
Received for publication July 19, 2004. Accepted for publication September 16, 2004.
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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 (TR) 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+ TR 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+ TR cells and CD4+IL-10+ TR1 cells, but only development of the latter TR cells depends on type I IFN.
Introduction
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 (TR1) cells are defined by their ability to produce high levels of IL-10 and TGF- (36). IL-10 promotes the differentiation of CD11clowCD45RBhigh DC that promote the development of TR1 cells both in vitro and in vivo (37), and IFN- enhances IL-10-induced differentiation of functional TR1 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+ TR 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+ TR1 cells in the course of specific CD8+ T cell responses.
Materials and Methods
Mice
C57BL/6J (B6) mice (H-2b), IFNAR-deficient (IFNAR–/–) B6 mice (39), IFN--deficient (IFN-–/–) B6 mice (40), Ab/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 Kb-restricted S2190–197 (VWLSVIWM) HBsAg peptide (42) was fused to the cationic HIV-tat50–57 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). HBsAgayw 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+ TR 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+ TR cells and CD11chigh 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 Ab-binding OVA323–339 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 Kb-binding peptide S2 (HBsAg190–197) VWLSVIWM, or the Kb-binding peptide S1 (HBsAg208–215) ILSPFLPL, or the peptide OVA257–264 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/Kb (peptide VWLSVIWM bound to Kb), 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 (Kb/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
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/Kb tet), or specifically inducing IFN- or TNF- expression by a 4-h peptide stimulation ex vivo (Fig. 2).
Antiviral CD8+ T cell responses to the Kb-restricted HBsAg epitope S2 were efficiently primed in normal and IFNAR-deficient mice by peptide (S2/Kb-tat)- or DNA (pCI/S)-based vaccination (42, 43). Both vaccination protocols primed CD8+ T cell responses in IFNAR–/–, IFN-–/–, and normal B6 mice that were readily detectable in the spleen. High frequencies (but lower numbers) of specific CD8+ T cells were also found in the liver, confirming our previous reports (45, 48) (Fig. 2A and Table II). Two to three times more specific CD8+ T cells were found postvaccination in liver and spleen from IFNAR- or IFN--deficient than normal mice, irrespective of the immunization protocol used (Fig. 2B). Although the adjuvant effect of CpG-containing ODNs or DNA vaccines has been proposed to be IFN-I dependent (8, 49, 50), the data indicate that the efficacy in CD8+ T cell priming of the ODN-dependent vaccines tested is enhanced in the absence of IFN-I. The frequencies of specific CD8+ T cells in spleen and liver were followed for 6 wk postvaccination (Fig. 3A). The data confirm that larger numbers of tetramer (S2/Kb tet)+ or IFN--expressing specific CD8+ T cells are found in IFNAR–/– than normal B6 mice throughout this observation period.
Primed CD8+ T cells specifically inducible to IFN- and/or TNF- expression were equally increased in number in vaccinated IFNAR–/– mice (Fig. 2B). IFN- (or its absence) thus does not modulate the expression profile of these two cytokines in vaccine-primed CD8+ T cells. This was apparent in specific CD8+ T cells primed by different vaccination protocols. The specific cytolytic activity of CD8+ T cells primed in IFNAR- and IFN--deficient mice was similar to that detected in normal mice (data not shown). Thus, IFN--independent priming generates functionally competent CD8+ T cell populations.
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, TR 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.
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.
CD25+CD4+ TR cells from IFNAR- and IFN--deficient mice down-modulate CD8+ T cell responses
CD25+CD4+ TR cells are a major (but not the only) TR cell subset that down-modulates CD8+ T cell responses (51, 52, 53, 54, 55). These TR 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+ TR cells in IFNAR–/– and IFN-–/– B6 mice. Both lines contained normal numbers of CD25+CD4+ TR 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+ TR 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+ TR cells are thus IFN- independent, and the down-modulation of CD8+ T cell responses by IFN-I is not mediated by these TR cells.
Reduced numbers of IL-10-producing CD4+ TR1 cells are generated in IFNAR-deficient mice
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 CD11chighB220– 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+ TR1 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+ TR1 cells that down-modulate CD8+ T cell responses.
Discussion
CD8+ T cell responses are primed more efficiently by different vaccination protocols in IFN--deficient than in normal mice. Regulation of the response by natural CD25+FoxP3+CD4+ TR cells, and the clonal expansion of primed CD8+ T cells were not affected by IFN-. In contrast, priming of CD8+ T cells was down-regulated by IFN-I, and the generation of adaptive, IL-10-producing TR1 cells was up-regulated in the presence of IFN-. Although MHC class I-restricted presentation of Ag to T cells is facilitated by IFN-I, the overall effect of these IFNs seems to be a down-modulation of CD8+ T cell responses.
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).
TR 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+ TR subsets have been implicated in the control of specific CD8+ T cell immunity: natural CD25+FoxP3+ TR cells and adaptive IL-10+ TR1 cells (65). The number of CD25+ TR 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 TR1 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+ TR cell and CD4+ TR1 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+ TR cells are required or facilitate the response of adaptive IL-10+ TR1 cells (65). This IFN-I-mediated copriming of suppressive TR1 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/Kb tetramers (peptide VWLSVIWM bound to Kb) 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@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; TR, regulatory T.
Received for publication July 19, 2004. Accepted for publication September 16, 2004.
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