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Immunodominance limits the TCR diversity of specific antiviral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain Ags (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV) surface (S), core (C), and polymerase (Pol) proteins and/or the OVA Ag as stress protein-capturing fusion proteins. Priming of mono- or multispecific, HLA-A*0201- or Kb-restricted CD8 T cell responses by these DNA vaccines differed. Kb/OVA257–264- and Kb/S190–197-specific CD8 T cell responses did not allow priming of a Kb/C93–100-specific CD8 T cell response in mice immunized with multidomain vaccines. Tolerance to the S- Ag in transgenic Alb/HBs mice (that express large amounts of transgene-encoded S- Ag in the liver) facilitated priming of subdominant, Kb/C93–100-specific CD8 T cell immunity by multidomain Ags. The “weak” (i.e., easily suppressed) Kb/C93–100-specific CD8 T cell response was efficiently elicited by a HBV core Ag-encoding vector in 1.4HBV-Smut tg mice (that harbor a replicating HBV genome that produces HBV surface, core, and precore Ag in the liver). Kb/C93–100-specific CD8 T cells accumulated in the liver of vaccinated 1.4HBV-Smut transgenic mice where they suppressed HBV replication. Subdominant epitopes in vaccines can hence prime specific CD8 T cell immunity in a tolerogenic milieu that delivers specific antiviral effects to HBV-expressing hepatocytes.

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8+ conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d−/− mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-γ, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-γ but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.

We tested in B6 mice whether the local expansion of CD4 T cells producing proinflammatory cytokines including IL-17 (Th17 cells) in the colonic lamina propria (cLP) depends on the commensal microflora. High numbers of CD4 Th17 cells were found in the lamina propria of the ileum and colon but not the duodenum, jejunum, mesenteric lymph nodes, spleen, or liver of specific pathogen-free (SPF) mice. The microflora is required for the accumulation of cytokine (IL-17, IFN-γ, TNF-α, IL-10)-producing CD4 T cells in the cLP because only low numbers of cytokine-producing cLP CD4 T cells were found in syngeneic (age- and sex-matched) germfree mice. The fraction of cLP Th17 cells was higher in (type I and type II) IFN- but not IL-4- or IL-12p40-deficient SPF congenics. cLP CD4 Th17 cells produce IL-17 but not IFN-γ, TNF-α, IL-4, or IL-10. cLP CD4 Th17 cells accumulate locally in colitis induced by adoptive transfer of IFN-γ+/+ or IFN-γ−/− CD4 T cells into congenic SPF (but not germfree) RAG−/− hosts. In this colitis model, cLP CD4 T cells that “spontaneously” produce IL-17 progressively increase in number in the inflamed cLP, and increasing serum IL-17 levels appear as the disease progresses. Commensal bacteria-driven, local expansion of cLP CD4 Th17 cells may contribute to the pathogenesis of this inflammatory bowel disease.

Upon entering the liver CD8 T cells encounter large numbers of NKT cells patrolling the hepatocyte (HC) surface facing the perisinusoidal space. We asked whether hepatic NKT cells modulate the priming of CD8 T cells by HC. Hepatic (α-galactosyl-ceramide-loaded CD1d dimer binding) NKT cells produce predominantly IL-4 when stimulated with glycolipid-presenting HC but predominantly IFN-γ when stimulated with glycolipid-presenting dendritic cells. These NKT cells prime naive CD8 T cells to a (Kb-presented) peptide ligand if they simultaneously recognize a CD1d-binding glycolipid presented to them on the surface of the responding CD8 T cells that they prime. No IL-10-producing CD8 T cells are detected if these T cells are primed by either HC or NKT cells. In contrast, IL-10 is produced by HC-primed CD8 T cells if IFN-β-producing NKT cells are coactivated by the same HC presenting a glycolipid (in the context of CD1d) and an antigenic peptide (in the context of Kb). Hence, IL-10-producing CD8 T cells are generated in a type I IFN-dependent manner if the three cell types (CD8 T cells, NKT cells, and ligand-presenting HC) specifically and closely interact. IL-10-producing CD8 T cells generated under these conditions down-modulate IL-2 (and proliferative) responses of naive CD4 or CD8 T cells primed by DC. If in close proximity, NKT cells can thus locally modulate the phenotype of CD8 T cells during their priming by HC thereby limiting the local activation of proinflammatory immune effector cells and protecting the liver against immune injury.

DNA vaccines encoding heat shock protein (hsp)-capturing, chimeric peptides containing antigenic determinants of the tumor-associated Ag (TAA) gp70 (an envelope protein of endogenous retrovirus) primed stable, specific, and tumor-protective CD8 T cell immunity. Expression of gp70 transcripts was detectable in most normal tissues but was particularly striking in some (but not all) tumor cell lines tested (including the adenocarcinoma cell line CT26). An ∼200 residue gp70 fragment or its Ld-binding antigenic AH1 peptide cloned in-frame behind an hsp-capturing (cT272) or noncapturing (T60) N-terminal large SV40 tumor Ag sequence was expressed as either hsp-binding or -nonbinding chimeric Ags. Only hsp-capturing, chimeric fusion proteins were expressed efficiently in transfected cell lines and primed TAA-specific CD8 T cell immunity. This immunity mediated protection in the CT26 and mKSA models. A vaccination strategy based on delivering antigenic, hsp-associated TAA fragments can thus prime protective CD8 T cell immunity even if these TAA are of low intrinsic immunogenicity.

We investigated the specific and cross-reactive CD8 T cell immunity to three natural variants (of different geno/serotype) of the small hepatitis B surface Ag (or S protein). The Dd-binding variants of the S201–209 epitope showed different immunogenicity. The loss of the consensus C-terminal (P9) anchor abrogated its immunogenicity. In contrast, a conservative (serine vs asparagine) exchange at P7 primed cross-reactive CD8 T cells that preferentially recognized the priming variant. Cross-reactive CD8 T cell responses to a variant could be primed in mice tolerant to an alternative variant of the Dd-binding S201–209 peptide. Loss of the C-terminal (P10) anchor in S185–194 eliminated its immunogenicity in HLA-A*0201(A2)-transgenic mice but two conservative exchanges (leucine vs valine in P2, and leucine vs isoleucine in P6) in S208–216 generated cross-reactive CD8 T cell responses with strong preference for the priming variant. Similar cross-reactive recognition of variant envelope epitopes were also found in S208–216-specific CD8 T cells from hepatitis B virus (HBV)-infected patients. Distinct CD8 T cell populations cross-reactive to natural variants of class I-restricted HBV epitopes can be primed by vaccination (of mice) or natural infection (of humans), and they may play a role in the “spontaneous remission” or the specific immunotherapy of chronic HBV infection.

To test whether simple expression units used in DNA vaccines can generate immunogenic, MHC class I-binding epitopes by translating other than the primary open reading frame (ORF), we constructed a vector (pCI/SX) that encodes the small hepatitis B surface Ag in the primary ORF, and a C-terminal fragment (residue 344–832) of the polymerase (Pol) in an alternative (out-of-frame) reading frame. pCI/SX efficiently primed multispecific, HLA-A2-restricted CD8+ T cell responses to epitopes of hepatitis B surface Ag and of Pol (Pol3, Pol803–811). Pol3-containing products generated from pCI/SX were detected only by T cell assays, but not by biochemical assays. Priming Pol-specific T cell responses to epitopes generated from alternative ORFs depended on promoter sequences that drive transcription in the DNA vaccine (human CMV-derived promoter sequences being more efficient than SV40-derived promoter sequences). Human CMV promoter-driven Pol constructs encoding different Pol fragments in primary or alternative reading frames elicited comparable levels of Pol3-specific T cell responses. We confirmed efficient T cell priming to epitopes from alternative ORFs by constructing DNA vaccines that encode an SV40-derived cT1–272 protein fused either in frame or out of frame with an immunogenic OVA fragment (OVA18–385). Similar OVA-specific CD8+ T cell responses were primed by both alternative vaccine constructs. Hence, DNA vaccine-stimulated T cell responses to epitopes generated from alternative ORFs seem to be a regular event, although its biological role and risks are largely unexplored.

A polytope DNA vaccine (pCI/pt10) was used that encodes within a 106-residue sequence 10-well characterized epitopes binding MHC class I molecules encoded by the K, D, or L locus (of H-2d, H-2b, and H-2k haplotype mice). The pCI/pt10 DNA vaccine efficiently primed all four Kb/Db-restricted CD8+ T cell responses in H-2b mice, but was deficient in stimulating most CD8+ T cell responses in H-2d mice. Comparing CD8+ T cell responses elicited with the pCI/pt10 DNA vaccine in Ld+ BALB/c and Ld− BALB/cdm2 (dm2) mice revealed that Ld-restricted CD8+ T cell responses down-regulated copriming of CD8+ T cell responses to other epitopes regardless of their restriction or epitope specificity. Although the pt10 vaccine could thus efficiently co prime multispecific CD8+ T cell responses, this priming was impaired by copriming Ld-restricted CD8+ T cell responses. When the pt10 sequence was fused to a 77-residue DnaJ-homologous, heat shock protein 73-binding domain (to generate a 183-residue cT77-pt10 fusion protein), expression and immunogenicity (for CD8+ T cells) of the chimeric Ag were greatly enhanced. Furthermore, priming of multispecific CD8+ T cell responses was readily elicited even under conditions in which the suppressive, Ld-dependent immunodominance operated. The expression of polytope vaccines as chimeric peptides that endogenously capture stress proteins during in situ production thus facilitates copriming of CD8+ T cell populations with a diverse repertoire.

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.

The immunodominant, conformational “a” determinant of hepatitis B surface Ag (HBsAg) elicits Ab responses. We selectively expressed the Ab-binding, glycosylated, native a determinant (residue 120–147) of HBsAg in a fusion protein containing C-terminally the HBsAg fragment SII (residue 80–180) fused to a SV40 T-Ag-derived hsp73-binding 77 aa (T77) or non-hsp-binding 60 aa (T60) N terminus. A DNA vaccine encoding non-hsp-binding secreted T60-SII fusion protein-stimulated murine Ab responses with a similar efficacy as a DNA vaccine encoding the secreted, native, small HBsAg. A DNA vaccine encoding hsp73-binding, intracellular T77-SII fusion protein-stimulated murine Ab responses less efficiently but comparable to a DNA vaccine encoding the intracellular, native, large HBsAg. HBsAg-specific Abs elicited by either the T60-SII-expressing or the T77-SII-expressing DNA vaccine suppressed HBsAg antigenemia in transgenic mice that produce HBsAg from a transgene in the liver; hence, a biologically active B cell response cross-reacting with the native, viral envelope epitope was primed by both DNA vaccine constructs. HBsAg-specific Ab and CTL responses were coprimed when an S20–50 fragment (containing the immunodominant, Ld-binding epitope S28–39) of HBsAg was fused C-terminally to the pCI/T77-SII sequence (pCI/T77-SII-Ld DNA vaccine). Chimeric, polyepitope DNA vaccines encoding conformational, Ab-binding epitopes and MHC class I-binding epitopes can thus efficiently deliver antigenic information to different compartments of the immune system in an immunogenic way.

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.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.

By exchanging sequences from the middle-surface (MS) and small-surface (S) Ag of hepatitis B virus (HBV) with corresponding sequences of the MS Ag of woodchuck hepatitis virus, we constructed chimeric MS variants. Using these constructs as DNA vaccines in mice, we selectively primed highly specific (non-cross-reactive) Ab responses to pre-S2 of the HBV MS Ag and the “a” determinant of the HBV S Ag, as well as Ld- or Kb-restricted CTL responses to HBV S epitopes. In transgenic mice that constitutively express large amounts of HBV surface Ag in the liver we could successfully suppress serum antigenemia (but not Ag production in the liver) by adoptive transfer of anti-pre-S2 or anti-“a” immunity but not CTL immunity. DNA vaccines greatly facilitate construction of chimeric fusion Ags that efficiently prime specific, high-affinity Ab and CTL responses. Such vaccines, in which sequences of an Ag of interest are exchanged between different but related viruses, are interesting tools for focusing humoral or cellular immunity on selected antigenic determinants and elucidating their biological role.