Detlef Stober

IL-12/IL-18-Dependent IFN-γ Release by Murine Dendritic Cells

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c+ myeloid DC from 7-day cultures stimulated with TNF-α, IFN-α, IFN-γ, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low “spontaneous” release of IL-18, and did not release IFN-γ. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-γ expression and release in 15–20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-γ release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8α+ and CD8α− DC (from immunocompetent and immunodeficient H-2d and H-2b mice) cultured with IL-12 and IL-18 released IFN-γ. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-γ release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-γ release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-γ generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.

NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires “help”. We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4+ T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4+ T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4+ T cell-independent priming of CTL. DC copresenting a Kb-binding antigenic peptide and the CD1d-binding glycolipid α-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.

Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5–20 ng RNA/μg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-γ release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20–100 μg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 μg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 μg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 on Murine Dendritic Cells Is a Potent Regulator of T Cell Stimulation

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1α, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4+ T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.

Activating Immunity in the Liver. II. IFN-β Attenuates NK Cell-Dependent Liver Injury Triggered by Liver NKT Cell Activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10–100 ng/mouse) of α-galactosyl ceramide (αGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following αGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to αGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-γ response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG−/− or HBs-tg/RAG−/−) hosts via the portal vein elicited IFN-γ responses of liver NK cells in situ. IFN-β down-regulates the pathogenic IL-12/IFN-γ cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-β suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (αGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-γ response of the specifically activated liver NKT cells. In vivo, IFN-β attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.

The immunodominant, Ld-restricted T cell response to hepatitis B surface antigen (HBsAg) efficiently suppresses T cell priming to multiple Dd-, Kd-, and Kb-restricted HBsAg epitopes.

MHC-I-restricted CTL responses of H-2d (Ld+ or Ld−) and F1 H-2dxb mice to hepatitis B surface Ag (HBsAg) are primed by either DNA vaccines or HBsAg particles. The Dd/S201–209 and Kd/S199–208 epitopes are generated by processing endogenous HBsAg; the Kb/S208–215 epitope is generated by processing exogenous HBsAg; and the Ld/S28–39 epitope is generated by exogenous as well as endogenous processing of HBsAg. DNA vaccination primed high numbers of CTL specific for the Ld/S28–39 HBsAg epitope, low numbers of CTL specific for the Dd/S201–209 or Kd/S199–208 HBsAg epitopes in BALB/c mice, and high numbers of Dd/S201–209- and Kd/S199–208-specific CTL in congenic H-2d/Ld− dm2 mice. In F1dxb mice, the Kd-, Dd-, and Kb-restricted CTL responses to HBsAg were strikingly suppressed in the presence but efficiently elicited in the absence of Ld/S28–39-specific CTL. Once primed, the Kd- and Dd-restricted CTL responses to HBsAg were resistant to suppression by immunodominant Ld/S28–39-specific CTL. The Ld-restricted immunodominant CTL reactivity to HBsAg can thus suppress priming to multiple alternative epitopes of HBsAg, independent of the processing pathway that generates the epitope, of the background of the mouse strain used, and of the presence/absence of different allelic variants of the K and D MHC class I molecules.

Dendritic cells pulsed with exogenous hepatitis B surface antigen particles efficiently present epitopes to MHC class I-restricted cytotoxic T cells

Ld‐ and Kb‐binding epitopes processed by murine dendritic cells (DC) pulsed with exogenous, particulate hepatitis B surface antigen (HBsAg) are presented to cytotoxic T lymphocytes (CTL). The specific and dose‐dependent induction of IFN‐γ release and cytotoxicity in CTL by metabolically active DC did not depend on antigenic peptides contaminating the particles, was cytochalasin D resistant, independent of the maturation state of DC, and blocked by primaquine, amiloride and NH4Cl (indicating involvement of acid proteolysis). The specific immunostimulatory phenotype of pulsed DC was maintained for about 3 h after the end of the pulse but rapidly decayed thereafter. Processing of Ld‐ and Kb‐binding epitopes from exogenous HBsAg particles by pulsed DC for presentation was TAP independent. Surface‐associated ‘empty’ (presentation‐deficient) 64+ Ld molecules (defined by the mAb 64‐3‐7), but not trimeric (presentation‐competent) 30+ Ld molecules (defined by the mAb 30‐5‐7) had to be available during the pulse of DC with exogenous HBsAg particles to generate 30+ Ldmolecules that present the antigenic S28–39 peptide. Exogenous β2‐microglobulin present during the pulse of DC with HBsAg particles facilitated presentation of Ld‐ and Kb‐restricted epitopes. DC generated from bone marrow progenitors in vitro, as well as splenic and liver DC (generated in vivo) presented epitopes to specific CTL. HBsAg particles thus efficiently enter an alternative processing pathway in DC that leads to presentation of epitopes to MHC class I‐restricted CTL.

Ongoing Murine T1 or T2 Immune Responses to the Hepatitis B Surface Antigen Are Excluded from the Liver that Expresses Transgene-Encoded Hepatitis B Surface Antigen

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.

Noncovalent Association with Stress Protein Facilitates Cross-Priming of CD8+ T Cells to Tumor Cell Antigens by Dendritic Cells

A viral oncogene carrying well-defined Kb/Db-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the Kb/Db-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-γ-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, Db/Kb-restricted IFN-γ-producing CD8+ T cells were primed during the growth of transfected H-2d Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F1(b × d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.