Magnetic cell sorting
Interferon type I
The CD11c+ cell population in the non-parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c+ DC population from immunocompetent or immunodeficient [recombinase-activating gene-1 (RAG1)–/–] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220+ CD11cint subset of ‘plasmacytoid’ DC, and a B220– CD11c+ DC subset. The latter DC population could be subdivided into a major, immature (CD40lo CD80lo CD86lo MHC class IIlo) CD11cint subset, and a minor, mature (CD40hi CD80hi CD86hi MHC class IIhi) CD11chi subset. Stimulated B220+ but not B220– DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220– DC three- to fourfold within 18 h post-injection, and up-regulated their surface expression of activation marker, while it contracted the B220+ DC population. Early in virus infection, the hepatic B220+ DC subset expanded, and both, the B220+ as well as B220– DC populations in the liver matured. In vitro, B220– but not B220+ DC primed CD4+ or CD8+T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220+ and B220– subsets in CD11c+ DC populations freshly isolated from the mouse liver.
Hepatic dendritic cells (DC) are supposed to mediate tolerance induction, as well as the intrahepatic induction and restimulation of specific T cell immunity. The key role of the liver in tolerance induction has been reviewed 1–3. Intrahepatic DC activation and expansion can override hepatic tolerance induction, as liver allograft acceptance is abrogated by pretreating donors with the DC-expanding growth factor Flt3 ligand. It is of interest to define hepatic DC subsets and characterize their surface phenotype and function to test whether these contrasting effects of the liver on specific T cell immunity can be assigned to distinct liver DC subsets.
In this paper we identify B220+ and B220– DC subsets in the mouse liver. Particular care is taken to exclude from the analysis CD11c+ cells in hepatic non-parenchymal cell (NPC) populations that do not belong to DC lineages. We determine the surface phenotype of mouse hepatic B220+ and B220– DC subsets in the liver, and extend the analysis to subsets within the B220– DC subset. Interferon type I (IFN-I) release in response to virus infection is analyzed in vitro using cell sorter-purified hepatic DC subsets. The early changes in hepatic DC subsets and their surface phenotype after in vivo NKT cell activation or virus infection are followed. The data indicate that B220+ and B220– CD11c+ DC are distinct hepatic DC subsets in the mouse while the subset status of hepatic CD11b+ and CD8α+ DC remains uncertain.
2.1 CD11c+ non-parenchymal cells in the liver
We isolated 6×106–7×106 NPC from the liver of a normal, young adult C57BL/6 (B6) mouse (Table 1). Similar numbers of hepatic NPC were recovered from standard pathogen-free (SPF) and germ-free (GF) B6 mice, and from immunocompetent and immunodeficient [recombinase-activating gene-1 (RAG1)–/–] B6 mice. Approximately 20% of the cells within the hepatic NPC population were CD11c+ (Table 1). A two- to threefold increase in CD11c+ cells was found in hepatic NPC populations from immunodeficient RAG1–/– B6 mice (Fig. 1A). Surface expression of CD11c by liver NPC showed a broad spectrum including subsets with intermediate (CD11cint) and high (CD11chi) CD11c surface expression (Fig. 1A). Less than 10% of hepatic CD3+ T cells were CD11c+. In contrast, a large fraction (50–80%) of hepatic DX5+ NK cells were CD11cint; this subset was strikingly expanded in RAG1–/– B6 mice (Fig. 1A). Few CD11c+ CD19+ B cells were found in the mouse liver (data not shown).
Hence, in addition to DC, hepatic CD11c+ NPC populations contain NK cells, B cells and T cells. We therefore rigorously depleted (by negative selection in vitro, antibody treatment in vivo, and/or the use of RAG1–/– B6 mice) T cells, B cells and NK cells from hepatic CD11c+ NPC populations before analyzing DC populations of the liver. In addition, NK and T/B cells were gated out electronically in four-color flow cytometry (FCM) analyses.
|Cellsa)||×10–5 (± SEM)/mouseb)||%|
|CD11c+ DX5+ NK cells||2.2±0.3||3.4|
|CD11c+ DX5– CD3– DC||9.9±1.2||14.8|
|Subset A: B220– CD11chi DC||1.1±0.1||11.1±0.9c)|
|Subset B: B220– CD11cint DC||5.2±0.7||53.0±8.3c)|
|Subset C: B220+ CD11cint DC||3.5±0.6||35.6±6.9c)|
2.2 Hepatic CD11c+ DC subsets
We searched for markers that define subsets within hepatic CD11c+ (CD3– CD19– DX5–) DC populations from normal or RAG1–/– B6 mice. The CD45 isoform B220 defined a B220– subset (65–70%) that contained CD11chi cells (subset A, 10–15% of hepatic DC) and CD11cint cells (subset B, 45–50% of hepatic DC); and B220+ CD11cint cells (subset C, 30–35% of hepatic DC) (Fig. 1B). Similar DC subsets were found in the spleen of immunocompetent and immunodeficient B6 mice, but the relative and absolute numbers of DC within the subsets A–C differed between liver and spleen (Fig. 1B and data not shown). The B220– CD11chi DC subset A predominated in the spleen but not in the liver (55–60% versus 15–20%); in contrast, the B220+ or B220– CD11cint DC subsets B or C were strikingly smaller in the spleen than in the liver (15–20% versus 30–35%, and 10–15% versus 45–50%, respectively). Surface expression of the CD11c and B220 molecules thus identifies distinct DC subsets in spleen and liver.
Surface expression of the integrin CD11b and the CD8α coreceptor have been used to define ‘myeloid’ (CD11b+ CD8–) and ‘lymphoid’ (CD11b– CD8+) DC subsets. We found non-overlapping CD8α+ and CD11b+ subsets in hepatic CD11c+ DC populations (Fig. 1C). CD8α+ CD11b– DC were present in hepatic B220+ and B220– DC (Fig. 1C). CD11b+ CD8α– DC formed a major subset in B220– but not B220+ hepatic DC (Fig. 1C). Many splenic and hepatic DC were CD8α– CD11b– (Fig. 1C, data not shown). These marker profiles of splenic and hepatic DC were found in normal and immunodeficient RAG1–/– B6 mice, and in B6 mice raised under SPF or GF conditions (data not shown). Immunohistology studies of the liver showed that CD11c+ B220– DC were predominantly found in periportal fields and around central veins, while CD11c+ B220+ DC were present mainly in the lobular parenchyma but only rarely detected in periportal fields (data not shown). B220+ and B220– DC are thus present in spleen and liver of mice, but the population size of DC within each subset strikingly differs between the two organs. As the B220 marker most clearly identified hepatic DC subsets, we asked whether these subsets express a distinct functional marker.
2.3 Selective induction of IFN-I release in hepatic B220+ DC by virus infection
We tested whether the splenic and hepatic B220+ DC have the unique ability to produce large amounts of IFN-I in response to virus infection, as reported in mice 4, 5 and humans 6, 7. Freshly isolated, cell sorter-purified B220+ or B220– CD11c+ DC subpopulations from spleen or liver were infected with the murine cytomegalovirus (MCMV) in vitro. IFN-I release during a 24-h post-infection incubation period was determined using a sensitive bioassay. B220+ but not B220– DC infected with MCMV produced IFN (Fig. 2). An IFN-I-neutralizing antibody blocked 80–90% of the antiviral activity, indicating that the released IFN-I mediates the antiviral activity (data not shown). The preparative isolation of B220+ DC did not stimulate their ‘spontaneous’ IFN-I release. Virus infection or stimulation with CpG-containing oligonucleotides, but not stimulation with poly(I/C) induced IFN-I release (data not shown). Splenic B220+ DC reproducibly produced three- to sixfold more IFN-I per DC than hepatic DC (Fig. 2). These data support the notion that the B220 marker identifies a functionally distinct mouse DC subset.
2.4 The surface phenotype of hepatic DC within the B220+ and B220– subset
The minor CD11chi subset A in the hepatic B220– DC population was more mature than the major CD11cint subset B, because a major fraction of DC in subset A expressed the CD40, CD80 and CD86 costimulator molecules on the surface together with high levels of MHC class II molecules (Fig. 3). In contrast, only few DC from subset B expressed low levels of CD40, CD80 and CD86 costimulator and intermediate levels of MHC class II molecules on the surface (Fig. 3). A similar surface phenotype prevailed in hepatic B220+ CD11cint DC of subset C (Fig. 3). CD8+ cells were found in all three subsets of hepatic DC; B220+ CD11cint DC (subset C) contained a clearly defined CD8+ subset, while a broad range of CD8 surface expression made it difficult to delineate CD8+ subsets in hepatic B220– DC populations (Fig. 3). A ‘mature’ CD11chi B220– DC subset A predominates the splenic DC population while an ‘immature’ CD11cint B220– DC subset B predominates the hepatic DC population (Fig. 1B). Surface expression of CD11b, CD8α, CD40, CD80, CD86 and MHC class II by the splenic DC subsets A–C showed a profile similar to that of the respective hepatic DC subsets (data not shown).
DX5+ cells as well as all NK cell-mediated functions tested (cytolytic activity against YAC-1 targets; poly(I/C)- or IL-12-induced IFN-γ release) were eliminated when NK cells were depleted from hepatic NPC invivo (by repeated injections of anti-Asialo-GM1 antibody) or in vitro [by treatment with anti-Asialo-GM1 antibody plus C', or depletion of DX5+ cells by magnetic cell sorting (MACS)] (data not shown). NK1.1+ DC were detected within these NK cell-depleted CD11c+ liver NPC populations. These DC were exclusively found in hepatic B220– CD11cint DC populations (subset B) and comprised 60–70% of the cells in this subset; no NK1.1+ DC were found in the B220+ CD11cint subset C or B220– CD11chi subset A (Fig. 4A). NK1+ DC were not cytolytic (Fig. 4B), but primed CD4+ T cell responses in vitro (Fig. 4C). Hepatic NK1.1+ CD11c+ DC showed low CD40 and intermediate CD86 and MHC class II surface expression (data not shown).
2.5 Changes in hepatic DC subpopulations in response to NKT cell activation
Injection of the CD1d-binding glycolipid α-galactosylceramide (αGalCer) into mice activates hepatic NKT cells and DC 8, 9. All hepatic DC express CD1d on the surface (data not shown). Within 18 h post-injection, the number of hepatic DC increased moderately (Table 2). The CD11cint B220+ DC subset C strikingly decreased in absolute and relative numbers, while the CD11chi B220– DC subset A increased two- to threefold in number (Table 2, Fig. 5A). These changes in DC numbers and subset distribution in response to i.v. or i.p. αGalCer injection were more prominent in hepatic than splenic DC populations, presumably because the livers harbors a larger αGalCer-reactive NKT cell population than the spleen (Fig. 5A). Hence, within hours after αGalCer injection, the hepatic B220– CD11c+ DC subsets A and B are activated and expand while the B220+ CD11cint DC subset C (though also activated) contracts. Following αGalCer injection, the hepatic DC up-regulated CD40, CD86, CD86 and MHC class II surface expression (Fig. 5B). Similar changes were observed in splenic DC subsets from αGalCer-injected mice (data not shown). Control RAG1–/– or MHC class I-deficient (β2m–/–) B6 mice injected with αGalCer showed no changes in the hepatic DC subsets (data not shown).
|Cells||Non-injected (×10–5/mouse)||%||αGalCer-injected (×10–5/mouse)||%|
|Subset A: B220–CD11chi DC||1.0±0.1||11.1±0.9||3.2±1.0||27.0±5.0|
|Subset B: B220–CD11cint DC||4.9±0.7||53.0±4.3||6.8±2.2||54.0±9.0|
|Subset C: B220+CD11cint DC||3.1±0.5||35.6±2.8||0.9±0.3||8.5±1.5|
2.6 Changes in the hepatic DC population after virus infection
MCMV infection in vitro induces IFN-I release by B220+ CD11cint subset C DC from spleen and liver (Fig. 2). We tested whether MCMV infection in vivo induces changes in hepatic DC (sub)populations similar to NKT cell activation. MCMV infection of immunocompetent or immunodeficient (RAG1–/–) B6 mice leads to a moderate increase in the number of hepatic DC 36 h post-infection (Table 3). This resulted mainly from an increase in B220+ CD11cint subset C DC in the liver (Table 3). In infected mice, the hepatic subset C DC showed evidence of activation, i.e. up-regulation of CD40 and CD86, and to a lesser extent CD80 (Fig. 6). Hepatic DC of subset A up-regulated surface expression of CD40 and CD86, but not CD80 (Fig. 6). Unexpectedly, MHC class II surface expression by liver DC in subset C was down-regulated early after infection (Fig. 6).
Similar changes were seen in immunocompetent B6 mice and congenic, immunodeficient RAG1–/– B6 mice, supporting the notion that the innate but not the specific immune system induces these changes in DC in the early stage of virus infection (Fig. 6). Similar changes were evident early after virus infection in splenic DC populations of subset A and C (data not shown). Hence, within 36 h after virus infection, the IFN-I-producing subset C (B220+ CD11cint) expands or is recruited to the liver, and is activated. This response differs from the early response to NKT cell activation, in which a depletion of subset C is observed in the liver DC population.
|Cell subset||Non-infected (×10–5/mouse)||%||MCMV-infected (×10–5/mouse)||%|
|Total NPC recovereda)||65±2.5b)||115±2.0|
|Subset A: B220–CD11chi DC||1.2±0.2||11.0±0.5||1.4±0.1||9.0±1.0|
|Subset B: B220–CD11cint DC||6.6±1.4||62.0±3.5||8.2±0.6||50±5.5|
|Subset C: B220+CD11cint DC||2.9±0.6||27.5±1.5||6.6±0.5||41±4.0|
2.7 In vitro priming of CD4+ and CD8+ T cells by hepatic B220– but not B220+ DC
Purified splenic CD4+ T cells (from OT-II mice) and CD8+ T cells (from OT-I mice) were co-cultured in vitro for 2–5 days with peptide-pulsed, splenic or hepatic B220– or B220+ CD11c+ DC. B220– but not B220+ DC from spleen or liver supported the specific in vitro priming of TCR-transgenic CD4+ or CD8+ T cells (Fig. 7). Proliferation as well as cytokine (IFN-γ, IL-2) release were induced by B220– DC, with the splenic DC being more potent APC than hepatic DC.
3.1 Identifying DC in mouse hepatic CD11c+ NPC populations
The αx-chain of the gp150/95 integrin (CD11c/CD18) is widely used to identify and preparatively isolate murine DC. Because CD11c is also expressed on the surface of NK cells, B cells and T cells, these CD11c+ cell subsets have to be distinguished from DC. CD3 or CD19 expression readily excludes T and B cells from the analysis of CD11c+ cells. The mAb DX5, which binds the α2-chain of the α2β1 integrin very-late antigen (VLA)-2 (CD49b/CD29), is expressed by most murine NK cells but not by DC. Only by depleting NK cells in vivo or in vitro, using T/B cell-deficient RAG1–/– B6 mice and/or depleting T and B cells in vitro, we could define and preparatively isolate hepatic CD11c+ DC.
In the first part of the study, we analyzed exclusively freshly isolated liver DC from normal mice. Animals were not pretreated with Flt3 ligand to expand hepatic DC, and the isolated DC were not exposed to cytokines in vitro or cultured before the analyses. Collagenase treatment, a well-established technique to isolate DC from tissues, was used to obtain NPC from the liver. The yield of NPC obtained from the liver without collagenase treatment was low, and the DC subset distribution was different. Control experiments indicated that collagenase treatment did not change the DC phenotype.
3.2 Plasmacytoid B220+ DC
Natural IFN-I-producing cells have been first discovered in human blood 7. Shortly thereafter, the mouse homologue of the IFN-I-producing cell was identified as the ‘plasmacytoid’ DC (PDC), also termed preDC or preDC2. PDC generated from precursors in mouse bone marrow or blood by Flt3 ligand 10–14 develop independently of the T cell lineage 15 in an IFN regulatory factor (IRF)-8-dependent way 16. PDC from the thymus 17, spleen and LN 18, 19 or the liver 20 produce IFN-I after stimulation 4, 5, 11, 21, 22. These DC are long-lived with a slow turnover 19 and express a heterogeneous surface phenotype profile 19. B220+ DC seem to be immature APC 12 or tolerogenic 5, 23. We could not prime CD4+ or CD8+ T cells in vitro with B220+ PDC. Following maturation, PDC may be able to prime Th1 and Th2 T cell responses 24.
3.3 Hepatic DC subsets
DC subset classifications are controversial. CD11b and CD8 are frequently used to identify ‘myeloid’ and ‘lymphoid’ DC characterized by their exclusive CD8 and CD11b surface expression 25–30. CD8+ CD11b– ‘lymphoid’ and CD8– CD11b+ ‘myeloid’ DC are found in the mouse liver (Fig. 1, 31). The origin of these two DC subsets in lymphoid organs and in the liver is unresolved. Some data indicate that the in vivo differentiation of these DC subpopulations may be unrelated to their experimental generation invitro32. In our attempt to define subsets in freshly isolated hepatic DC populations using these markers, we encountered two problems: (1) a substantial fraction of hepatic DC are CD8– CD11b– (Fig. 3); and (2) CD8 and CD11b surface expression is variable in hepatic CD11c+ DC, which makes it difficult to define a subset. These difficulties led to the search for an alternative subset definition for hepatic DC.
In liver NPC populations rigorously depleted of T/B cells and NK cells, we defined B220+ and a B220– DC subsets (Fig. 1, 3, 5). IFN-I production correlated with B220 surface expression, but not with CD8+ or CD4+ surface expression in B220+ DC of subset C, confirming recent reports 5, 20. The CD11cint B220+ DC subset C is prominent in the liver where it represents about a third of the total hepatic DC pool. Its identification is unambiguous. It contains a well-defined CD8α+ subset (20–30% of the DC in the B220+ subset C) but no CD11b+ DC. The hepatic B220– DC subsets A and B also contain CD8α+ cells. In vivo stimulation of hepatic DC, particularly by NKT cell activation, induced a depletion of CD8α+ DC (either by down-modulating marker expression, or by preferential loss of CD8α+ DC). Virus infection activates and expands hepatic B220+ DC in vivo but does not lead to up-regulation of CD8 surface expression.
About half of the B220– DC of subset A and B in the liver were CD11b+ and would be classified as ‘myeloid’ DC, while only a minority (10%) of CD11b– B220– DC were CD8+, confirming the heterogeneity of this subset 20. Tissue-specific variability is a problem in the definition of DC subsets because CD11b+ and CD8+ B220– DC subsets are readily identified in splenic but not hepatic DC populations. The surface phenotype and IFN-I release thus define B220+/B220– subsets more easily than CD11b+/CD8+ subsets in hepatic DC.
NK1 (CD161), a member of the NKR-P1 gene family, is a type II transmembrane C-type lectin expressed on the surface of T cells and NK cells as disulfide-linked homodimers. The NK1.1 allelic form to which the mAb PK136 binds is expressed in selected strains of mice, including B6 mice. We found NK1 expression by subset B (CD11cint B220–) DC with low/intermediate MHC class II and costimulator expression. More hepatic than splenic DC were NK1+. NK1 expression by hepatic CD11c+ NPC has been described 20, but the NK1+ CD11c+ NPC were considered non-DC and excluded from the study. This selectively excludes 70% of liver DC in subset B from the analysis and makes the DC population structure in the liver seeming more similar to that in the spleen. We demonstrate that the NK1+ CD11c+ DC mediate no NK cell functions but are APC. It remains to be elucidated whether NK1 molecules expressed by DC have a functional role.
3.4 Response of hepatic DC subsets to NKT cell activation and virus infection
The notion that B220+ and B220– DC populations in the liver are functionally distinct subsets is supported by their different early responses in vivo to stimuli that activate hepatic DC, i.e. NKT cell activation or MCMV infection. The expansion/contraction of the absolute and relative sizeof the two subsets and their changes in surface phenotype differed. In particular, the selective increase of IFN-I-producing hepatic B220+ DC shortly after virus infection suggests a role of these immature DC in the early response of this vulnerable organ to virus infection. Factors that regulate these responses in the liver are under investigation. Furthermore, the effects on specific T cell priming by the changes induced in the phenotype of hepatic DC subsets are the focus of ongoing studies.
4 Materials and methods
H-2b B6 mice, RAG1–/– B6 mice, and TCR transgenic OT-I and OT-II B6 mice were bred and kept under SPF conditions in the animal colony of the University of Ulm (Ulm, Germany).B6 mice raised under GF conditions were also used. Mice were used at 10–16 weeks of age.
4.2 Isolation of hepatic and splenic mononuclear cells
Hepatic NPC 8 and splenic DC 9 were isolated as described. Hepatic and splenic CD11chi/int B220– and CD11cint B220+ DCused for IFN-I release experiments were isolated by electronic cell sorting to >98% purity.
4.3 Bioassay to measure IFN-I release
The IFN concentration in the cell culture supernatants was determined by an antiviral assay using mouse L929 cells infected with vesicular stomatitis virus as described 33. To confirm the specificity of the antiviral activity, a neutralizing mAb directed against mouse IFN-I was added to the supernatant before addition to the test cells.
4.4 Flow cytometry analyses
Hepatic NPC or spleen cells were depleted of T, B and NK cells by MACS (Miltenyi Biotec) using anti-CD5, anti-CD19 and anti-DX5 MicroBeads (Cat. No. 130–049–301, 130–052–201 and 130–052–501). Cells were washed twice in PBS/0.3% (w/v) BSA supplemented with 0.1% (w/v) sodium azide. Nonspecific binding of antibodies to Fc receptor was blocked by pre-incubating cells with mAb 2.4G2 (Cat. No. 01241D; BD Biosciences) directed against the FcγRIII/II CD16/CD32 (0.5 μg mAb/106 cells/100 μl). After washing, the cells were 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. Three- or four-color FCM analyses wereperformed by FACS (Becton & Dickinson, Mountain View, CA). The forward narrow angle light scatter was used as an additional parameter to facilitate exclusion of dead cells and aggregated cell clumps.
The following reagents and mAb were obtained from BD Biosciences: FITC-conjugated anti-CD3ϵ mAb 145-2C11 (Cat. No. 553062), PE-conjugated anti-β-TCR H57, (Cat. No. 03105A), FITC- and PE-conjugated CD49b/pan-NK-cells mAb DX5 (Cat. No. 09944D and 553858), PE- and allophycocyanin-conjugated anti-CD11c mAb HL3 (Cat. No. 553802 and 550261), FITC-conjugated anti-CD4 mAb GK1.5 (Cat. No. 553729), anti-CD8α mAb 53-6.7 (Cat. No. 553031), anti-CD11b mAb M1/70 (Cat. No. 557396), anti-CD80 mAb 16-10A1 (Cat. No. 553768), and anti-CD86 mAb GL1 (Cat. No. 553691), biotinylated anti-CD8α 53–6.7 (Cat. No. 553029), anti-CD11b mAb M1/70 (Cat. No. 01712D), anti-CD40 mAb 3/23 (Cat. No. 09662D) and anti-I-Ab mAb AF6–120.1 (Cat. No. 553550). Second-step reagents used were SA-PerCP (Cat. No. 554064; BD Biosciences) and SA-Red 670 (Cat. No. 19543–024; Gibco-BRL, Berlin, Germany).
4.5 Injection of α-galactosylceramide into mice
The glycolipid αGalCer, a kind gift of Dr. Y. Koezuka (Kirin Brewery Co Ltd, Pharmaceutical Research Laboratory, Gunma, Japan), was dissolved in 0.5 ml PBS and injected i.v. into mice (1 μg/mouse).
4.6 MCMV infection of mice
B6 or RAG1–/– mice were infected by i.p. injection of 3×104 PFU MCMV (a kind gift of Dr. D. Michel, Dept of Virology, University of Ulm). Mice were killed 36 h post-infection. DC (105/well) were infected in vitro by 3×105 PFU MCMV in medium for 1 h at 37°C, 5% CO2, washed and cultured in 200-μl wells in flat-bottom 96-wel plates in triplicates. Non-infected cells were used as a control. After 24 h the supernatants were collected for IFN-I detection.
4.7 T cell priming in vitro
Cells were cultured in 200 μl round-bottom wells in RPMI 1640 medium supplemented with 5% FCS (Cat. No. A15–649; PAA Laboratories, Linz, Austria), 2 mM L-glutamine and antibiotics. DC were pulsed with the indicated doses of either the Kb-binding OVA257–264 peptide SIINFEKL (recognized by the transgene-encoded TCR expressed by OT-I mice), or the Ab-binding OVA323–339 peptide ISQAVHAAHAEINEAGR (recognized by the transgene-encoded TCR expressed by OT-II mice). After 3 h of incubation DC were washed twice, and 1×104 DC/wellwere co-cultured with 1×105 purified responder CD8+ (OT-I) or CD4+ (OT-II) T cells. After a 48-h co-culture, IFN-γ and IL-2 were detected in supernatants by double-sandwich ELISA. All purified capture and biotinylated antibodies and all recombinant mouse cytokines standards were from BD Biosciences. Extinction was analyzed at 405/490 nm on a TECAN micro plate-ELISA reader (TECAN, Crailsheim, Germany) using the EasyWin software (TECAN).
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Re549/10–1) and the University of Ulm (IZKF-E3) to R.S. and J.R. We greatly appreciate the expert technical assistance of Tanja Güntert and Katja Ölberger, the help of Dr. P. Conradt with cell sorting, the MCMV from Dr. D. Michel (Dept. of Virology, Ulm), and Dr. Y. Koezuka(Kirin Brewery Co Ltd, Pharmaceutical Research Laboratory, Gunma, Japan) for the αGalCer. We gratefully acknowledge the helpful comments of Dr. M. J. Wick (Gothenberg, Sweden).