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OBJECTIVE RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic β-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic β-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells. RESEARCH DESIGN AND METHODS DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1−/−, PD-1−/− or bone marrow chimeric mice. RESULTS Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1−/− or PD-1−/− mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic β-cells or of PD-1 in autoreactive CD8 T-cells induced EAD. CONCLUSIONS An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic β-cells can trigger pancreatic β-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the β-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.

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.

Only small populations of nonactivated, nonproliferating Foxp3+ CD4 regulatory T cell (TR) cells are found in the nonparenchymal cell compartment of the mouse liver while liver-draining celiac nodes contain expanded, activated TR cell populations (similar to other lymph nodes). Liver Foxp3+ CD4 TR cells suppress activation of T cell responses. Polyclonal, systemic T cell activation in vivo (via anti-CD3 antibody injection) is accompanied by intrahepatic accumulation of T blasts and a rapid but transient intrahepatic increase of activated, proliferating Foxp3+ CD4 TR cells. Following vaccination, the appearance of peripherally primed, specific CD8 T blasts in the liver is preceded by a transient rise of Foxp3+ CD4 TR cells in the liver. The adoptive transfer of immune CD8 T cells into congenic hosts that express the relevant antigen only in the liver leads to the accumulation of specific donor CD8 T cells and of host Foxp3+ CD4 TR cells in the liver. Conclusion: Although it contains only a small population of quiescent Foxp3+ CD4 TR cells, the liver can rapidly mobilize and/or recruit this T cell control in response to the intrahepatic appearance of peripherally or locally generated CD8 T blasts. (HEPATOLOGY 2008;48:1954-1963.)

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.

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.

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.

Background : The liver efficiently eliminates activated CD8 + T blasts. It is unknown if vaccine-primed CD8 + T blasts migrate to and establish functional CD8 + T cell immunity in the liver post-immunization. Aims : We tested, if functional CD8 + T cell populations can be detected in the liver post-vaccination. Methods : Murine CD8 + T cells with different epitope/restriction specificities were primed by intramuscular injection of protein- or DNA-based vaccines. The kinetics of appearance in the liver, as well as the surface phenotype and functional competence of intrahepatic, specific CD8 + T cell populations was tested. Results : High numbers of specific CD8 + T cells appear in the liver after vaccination that are activated (CD69 + CD44 + ), express effector functions (CD27 lo /CD28 lo phenotype, interferon γ secretion, specific cytolytic reactivity), but show no evidence of apoptosis (annexin V − , B220 lo , similar numbers/kinetics in primed, congenic lpr/lpr mice). Specific CD8 + T cells from the liver adoptively transferred into a naive, syngeneic host successfully reconstitute specific CD8 + T cell immunity. Conclusions : Specific, functionally competent CD8 + effector/memory T cell populations are established in the liver for months post-vaccination.

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.

Immune responses elicited by plasmid DNA vaccination can be enhanced and modulated by codelivery of cytokine-encoding plasmids. We studied whether priming of cytotoxic T lymphocyte (CTL) responses against hepatitis B surface antigen (HBsAg) by DNA vaccines injected either intramusculary or intradermally with the gene gun is enhanced by codelivery of cytokine-encoding plasmids. From a panel of tested cytokine plasmids only mouse IFNbeta, IL-15, and GM-CSF encoding plasmids showed an effect. Intradermal gene gun vaccination with 1 micro g plasmid DNA encoding intracellular HBsAg (large LS) showed enhanced CTL priming when IFNbeta, IL15, or GM-CSF encoding plasmids were codelivered; this was not observed when a DNA vaccine encoding secreted HBsAg (small S) was injected. Intramuscular injection of low (5 micro g) doses of a DNA vaccine encoding large HBsAg did not prime CTL when delivered without cytokines, with IFNbeta or IL15-encoding plasmids. However, codelivery with GM-CSF encoding plasmid DNA primed potent, specific CTL immunity detected either in a cytotoxic assay or by determining the frequency of L(d)-restricted CD8(+) T cells specifically inducible to IFNgamma production. The codelivery of GM-CSF encoding plasmids with the DNA vaccine furthermore enhanced CTL priming to a subdominant, D(d)-restricted epitope of HBsAg. The adjuvant effect of cytokine-encoding plasmids on CTL priming by DNA vaccines is thus complex and depends on: (a) the type of cytokine (or combination of cytokines) codelivered, (b) the type (intracellular vs. secreted) and dose (1-50 micro g) of the DNA vaccine, (c) the method of DNA vaccine delivery ("naked" vs. particle-coated DNA), and (d) the (intramuscular vs. intradermal) route of delivery of the DNA vaccine.

L(d)- and K(b)-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-gamma 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 NH(4)Cl (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 L(d)- and K(b)-binding epitopes from exogenous HBsAg particles by pulsed DC for presentation was TAP independent. Surface-associated 'empty' (presentation-deficient) 64(+) L(d) molecules (defined by the mAb 64-3-7), but not trimeric (presentation-competent) 30(+) L(d) molecules (defined by the mAb 30-5-7) had to be available during the pulse of DC with exogenous HBsAg particles to generate 30(+) L(d)molecules that present the antigenic S(28-39) peptide. Exogenous beta2-microglobulin present during the pulse of DC with HBsAg particles facilitated presentation of L(d)- and K(b)-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.

Using different DNA vaccination techniques, we studied the IgG1/IgG2a antibody and MHC-I-restricted cytotoxic T lymphocyte (CTL) responses to the hepatitis B surface antigen (HBsAg) in mice. A single intramuscular injection of 100 µg HBsAg-encoding pCI/S plasmid DNA efficiently primed IgG2a antibody (IgG1/IgG2a ratio 80) but there was no CTL response (Th2 immunity). Injection of immune-stimulating CpG-containing oligodeoxy-nucleotides (ODN) into the skin area used for gene-gun-mediated pCI/S DNA delivery shifted the polarization of the response towards Th1 immunity. A similar shift from Th2 to Th1 immunity was observed when the skin area used for gene-gun-mediated DNA transfer was conditioned by injection of recombinant IL-12. DNA vaccination can thus prime polarized immunity to HBsAg. The polarization of immunity is determined by the technique of plasmid DNA delivery as well as by conditions of the tissue into which DNA is inoculated. Th1 immunity to HBsAg (primed by injection of naked pCI/S DNA) dominated Th2 immunity (primed by gene-gun-mediated pCI/S DNA). In contrast, an established HBsAg-specific Th2 immunity was readily shifted towards Th1 immunity (including specific CTL priming) by an intramuscular boost injection of pCI/S DNA. These data contribute to the rational design of DNA vaccination strategies to efficiently prime anti-viral Th1 immune effector specificities using the gene gun.

Dendritic cells (DC) derived from bone marrow precursors of BALB/c or C57BL/6 mice in low-serum cultures supplemented with granulocyte macrophage colony stimulating factor and Flt(3) ligand were pulsed in vitro with hepatitis B surface antigen (HBsAg) particles. DC processed exogenous HBsAg and presented its MHC class I-binding epitopes to cytotoxic T lymphocytes (CTL). This specific and restricted interaction of DC with CTL stimulated release of IFN-gamma and macrophage inflammatory protein (MIP)-1 alpha from the responding CTL. MIP-1 alpha enhanced the survival of DC in vitro but did not induce proliferation. Furthermore, co-delivery of MIP-1 alpha facilitated CTL priming in vivo to exogenous HBsAg in low responder C57BL/6 (H-2(b)) mice: a single injection of a low dose of HBsAg particles (without further adjuvants) successfully primed K(b)-restricted CTL responses to HBsAg only when the exogenous antigen was co-delivered with 100 ng MIP-1 alpha. These in vitro and in vivo data point to an important role of MIP-1 alpha in the DC-dependent priming of CTL to exogenous viral antigens.

The concept of distinct endogenous and exogenous pathways for generating peptides for MHC-I and MHC-II-restricted presentation to CD4+ or CD8+ T cells fits well with the bulk of experimental data. Nevertheless, evidence is emerging for alternative processing pathways that generate peptides for MHC-I-restricted presentation. Using a well characterized, particulate viral antigen of prominent medical importance (the hepatitis B surface antigen), we summarize our evidence that the efficient, endolysosomal processing of exogenous antigens can lead to peptide-loaded MHC-I molecules. In addition, we describe evidence for endolysosomal processing of mutant, stress protein-bound, endogenous antigens that liberate peptides binding to (and presented by) MHC-I molecules. The putative biological role of alternative processing of antigens generating cytotoxic T-lymphocyte-stimulating epitopes is discussed.

Intramuscular (i.m.) or s.c. injection of plasmid DNA encoding hepatitis B small surface antigen (HBsAg) primes potent MHC I-restricted cytotoxic T lymphocyte (CTL) responses in H-2(d) (BALB/c) and H-2(b) (C57BL/6) mice. In contrast, i.m. or s.c. injection of exogenous HBsAg particles without adjuvants primes CTL responses in 'high responder' H-2(d) but not 'low responder' H-2(b) mice. We have shown that processing of exogenous but not endogenous HBsAg generates the Kb-binding S208-215 peptide ILSPFLPL. This system allowed us to optimize conditions for stimulating murine CTL responses to exogenous antigen by identifying adjuvants that facilitate priming of Kb-restricted CTL by injecting recombinant HBsAg particles into 'low responder' H-2(b) mice. Synthetic oligodeoxynucleotides with immunostimulating sequences or the recombinant cytokine IL-12 efficiently enhanced priming of CTL to exogenous HBsAg. Hence, the adjuvanticity of DNA sequences that induce Th1 cytokines facilitate priming of MHC I-restricted T cell responses to exogenous antigen and are therefore of potential value in formulating vaccines designed to enhance CTL priming to exogenous antigen.

Recruitment into the gut of CD4+ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4+ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4+ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-gamma and TNF-alpha but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-gamma- and TNF-alpha-producing effector CD4+ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4+ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4+ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-Lhi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-Lhi CD4+ T cells were enriched in IFN-gamma-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-Llo CD4+ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4+ T blasts that were almost exclusively P-Lhi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4+ T cells residing in inflamed and non-inflamed tissues during the development of colitis.

Induction of humoral and MHC (major histocompatibility complex)-I-restricted, cytotoxic T lymphocyte (CTL) responses of Balb/c mice to the small hepatitis B surface antigen (HBsAg) were studied with a protein antigen or a DNA vaccine. Different routes were used to deliver the HBsAg-encoding plasmid DNA or the recombinant HBsAg particles: different doses of expression plasmid DNA (10 micrograms or 100 micrograms per mouse) or of recombinant HBsAg lipoprotein particles were injected into different (normal or regenerating) muscles (m. tibialis anterior and m. quadriceps), into subcutaneous tissue (at the base of the tail), into the peritoneal cavity, or intravenously (into the tail vein). At different time points post-vaccination, the induction of HBsAg specific, MHC-I-restricted CD8+ T cells and of serum antibodies to HBsAg was monitored. The data show that the intramuscular and subcutaneous but not the intravenous and intraperitoneal injection of 'naked' DNA efficiently and reliably primes cellular and humoral immune responses. In contrast, recombinant HBsAg particles injected by all four routes (without adjuvants) efficiently primed specific humoral and CTL responses. These data demonstrate that the choice of routes to deliver 'naked' plasmid DNA for obtaining efficacious immunogenicity of the expressed antigen is restricted.

Proteinaceous and nonproteinaceous antigens from exogenous microorganisms can be processed by the host for MHC class I restricted presentation to T cells. Macrophages, B cells, mast cells and dendritic cells are antigen-presenting cells that process such exogenous antigens through multiple pathways before MHC-restricted epitope presentation. New conceptual frameworks are emerging regarding the processing and presentation to T cells of peptide or nonpeptide epitopes from bacteria in the context of conventional MHC class I molecules, nonconventional MHC class I molecules, or CD1 molecules. Animal experiments have demonstrated that these pathways are of central importance for generating protective antibacterial T cell responses. These findings form the basis for new vaccine designs that specifically target MHC class I restricted T cell reactivity.

Polyclonal, mucosa-seeking memory/effector CD4+ T cells containing a large fraction of blasts activated in situ accumulate in the gut lamina propria of severe-combined immunodeficient (SCID) mice developing colitis after CD4+ T cell transplantation. CD4+ T cells isolated from different repopulated lymphoid tissues of transplanted SCID mice proliferate in vitro in the presence of interleukin (IL)-2 + IL-7. CD3 ligation enhances this cytokine-supported proliferation in CD4+ T cells from the spleen and the mesenteric lymph node of transplanted SCID mice; CD3 ligation suppresses the cytokine-supported proliferation in CD4+ T cells from the gut lamina propria in a cell density- and dose-dependent manner. Almost all CD4+ T cells from repopulated lymphoid tissues of transplanted SCID mice express CD95 (Fas) on the cell surface, and a large fraction of CD4+ T cells from the gut lamina propria of transplanted SCID mice express the Fas ligand on the surface. Gut lamina propria CD4+ T cells show Fas-dependent cytotoxicity. A large fraction of gut lamina propria CD4+ T cells that infiltrate the inflamed colon in transplanted SCID mice are activated in situ and many CD4+ T cells are apoptotic. Hence, a large fraction of colitis-inducing CD4+ T cells undergo activation-induced cell death in situ and can damage other cells through Fas-dependent cytotoxicity.

H-2d mice generated a major histocompatibility complex (MHC) class I (Ld)-restricted T-cell response of defined restriction and epitope specificity to the hepatitis B virus small

Data from long-term non-progressing human immunodeficiency virus (HIV)-infected individuals and populations at high risk suggest that an early cytolytic T cell response rather than the humoral immune response might be involved in controlling disease progression. These observations may be used as a guide to the type of response that a vaccine should induce. To clarify the role of different arms of the immune system in conferring protection, the candidate vaccine should allow a regulated, selective induction of different immune responses. Based on a better understanding of the molecular mechanisms regulating the morphogenesis of HIV, we developed an autologous, non-replicating and safe antigen delivery system. This system takes advantage of molecular characteristics of the HIV group-specific antigens (gag) to self-assemble to highly immunogenic virus-like particles (VLP). The immunogenicity of the gag-derived VLP was expanded either by replacing defined domains by selected HIV-1 cytotoxic T lymphocyte (CTL) epitopes (type 1 VLP) or by stable anchoring derivatives of the HIV-1 envelope protein on the surface of the VLP (type 2 VLP). In complete absence of adjuvants, type 1 and type 2 VLP stimulated CD8+ CTL in BALB/c mice, which specifically recognised HIV sequences. In contrast to type 1 VLP, generating an HIV-specific CTL response in the absence of env-specific antibodies, type 2 VLP induced both arms of the immune system including reasonable levels of neutralising antibodies. Initial studies performed in rhesus macaques confirmed these results. Thus, depending on the type and formulation of the VLP, the proposed antigen delivery system allows either the induction of a CTL response (1) in the absence and (2) the presence of an envelope-specific antibody response. A comparison of these approaches in appropriate animal models might contribute to further define the correlates of protection.

The particulate form of the major envelope or surface (S) protein of hepatitis B virus (HBV) can be taken up by antigen-presenting cells and processed for class I presentation as an exogenous protein. We have used several DNA plasmid vectors expressing the HBV envelope proteins to determine whether these sequences are able to induce cytotoxic T lymphocyte (CTL) responses in BALB/c mice after intramuscular DNA injection. A potent and specific induction was obtained, which can be detected ex vivo using either specific or nonspecific (interleukin-2) stimulation in cell culture, and the DNA-primed CTL responses are stronger than those obtained with protein injection with either stimulation protocol. The CTL response induced by DNA-based immunization is both canonical and highly specific as indicated by the nature of the epitope presented (amino acids 28-39), the class I allele used (Ld), and the T lymphocytes involved (CD8+). The CTL response is initiated between 3 and 6 days after DNA injection. By 6-12 days after a single DNA injection, ex vivo cytolytic activity is nearly maximal, and similar high levels of activity can still be detected 4 months after injection. The possibility is discussed that the unusual mode of delivery of the antigen to the immune system provided by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation as an exogenous protein in addition to activating potentially the classical endogenous pathway.

We investigated the surface phenotype of CD3+CD4+ T cell receptor (TCR) alpha beta + T cells repopulating the intestinal lymphoid tissues of C.B-17 scid/scid (severe-combined immunodeficient; scid) (H-2d, Ld+) mice. CD4+ CD8- T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2d, Ld+) or semi-syngeneic dm2 (H-2d, Ld-) immunocompetent donor mice. After transfer of 10(5) cells into young scid mice, a mucosa-homing, memory CD44hi CD45RBlo CD4+ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3+ CD2+ CD28+ CD4+ CD8- T cells that expressed the alpha 4 integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type double-positive (DP) CD4+ CD8 alpha + CD8 beta - T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4+ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the alpha 4 integrin chain CD49d, but expressed high levels of the alpha IEL integrin chain CD103. The TCR-V beta repertoire of DP but not SP intraepithelial CD4+ T cells was biased towards usage of the V beta 6 and V beta 8 viable domains. Highly purified populations of SP and DP CD4+ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4+ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4+ T cells did not. Hence, a subset of CD3+ CD4+ TCR alpha beta + T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.

We investigated the major histocompatibility complex (MHC) class I-restricted presentation of an epitope of the hepatitis B virus small surface (S) antigen particle to cloned murine cytotoxic T lymphocytes (CTL). Efficient Ld-restricted presentation of the S28-39 epitope to CTL is observed in cells of different tissue origin pulsed in vitro, either with the antigenic S28-39 12-mer S-peptide, or with particulate S-antigen. The kinetics of epitope presentation differ in S-peptide-pulsed and in S-particle-pulsed cells: while a 15-min pulse with the antigenic peptide sensitizes targets for class I-restricted CTL lysis, presentation of S-particles requires 30-60 min to sensitize cells for CTL lysis. Uptake of antigenic material and active metabolism of the presenting cell are required for processing of S-particles, but not for sensitizing targets with S-peptides. Intracellular processing and presentation of S-particles is blocked in cells treated with chloroquine, NH4Cl, primaquine, or leupeptin, but not by treatment with cycloheximide or brefeldin A. This processing pathway operates efficiently in peptide-transporter-deficient, Ld-transfected T2 cells, revealing a novel endosomal/lysosomal processing pathway for class I-restricted presentation of peptides derived from exogenous S-particles.

We selected three peptides from the germ-line sequence of the V beta 8.2 and J beta 2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative Kd- and Ld-restricted epitopes. Immunization of BALB/c (H-2d) mice with the V beta 8.2(67-90) 23-mer peptide 1 as well as the 15-mer V beta 8.2(95-108)-peptide 2 efficiently primed specific CD8+ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-V beta 8.2 epitopes. V beta 8.2+ T cells were not deleted in TCR peptide-immunized mice because the fractions of V beta 8.2+ CD4+ and V beta 8.2+ CD8+ T cells in spleen and lymph nodes were not altered. The proliferative response of V beta 8.2+ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60-80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the J beta 2.3-derived peptide 3 stimulated a CD8+ CTL response against a class I-restricted epitope within this J beta segment that was also generated during natural "endogenous" processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8+ CTL recognize class I-restricted, natural V beta/J beta-TCR epitopes. Such anti-TCR CTL may, thus, operate in V beta-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast-derived surface antigen (S-antigen or S-protein) of hepatitis B virus (HBV). Native S-protein molecules self-assemble into stable 22-nm particles. BALB/c mice immunized with low doses of native S-particles without adjuvants efficiently generated an H-2 class I-restricted CD8+ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S-particle or linear epitopes of the denatured S-protein. Disruption of S-particles with sodium dodecyl sulfate and beta-2-mercaptoethanol generated p24 S-monomers. Injection of an equal dose of S-monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S-protein. In vivo priming of CTL by S-particles or S-monomers required "endogenous" processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S-derived 12-mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S-antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.

CD8+ cytotoxic T lymphocytes (CTL) mediate protective immunity against many intracellular pathogens. New generation vaccines that exploit the potential of recombinant protein technology have to meet the challenge to identify immunodominant antigen systems of the pathogen of interest, to produce these antigens in recombinant form, and to find ways to selectively deliver them to the compartments of the specific immune system that mediate the protective responses. Immunization with soluble protein antigens usually primers CD4+ T cells but not CD8+ T cells because of the stringent requirements for 'endogenous processing' for major histocompatibility complex (MHC) class I-restricted epitope presentation to CD8+ CTL. This rule is not absolute because priming of class I-restricted CTL by soluble, recombinant viral protein antigens injected without adjuvants has been reported in various antigen systems. An understanding of the cell biology of alternative pathways of protein antigen processing for MHC class I-restricted epitope presentation is emerging. We describe subcellular sites, alternative peptide transport mechanisms, and alternative modes of MHC class I molecule recycling that allow different modes of peptide loading of class I molecules. Experimental evidence for some of these novel pathways of 'endogenous' processing for class I-restricted epitope presentation is discussed. Some pathways are still hypothetical. The issue of alternative modes of 'endogenous' processing for class I-restricted antigen presentation is of theoretical and practical relevance. Immunologists are interested in the question from which source protein antigens are derived that are potentially accessible to specific recognition by different T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunization of C57BL/6 (B6) mice (H-2b) with the "large tumor antigen" (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. CD8+ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure(s) required for a soluble protein to enter the "endogenous" class I-restricted antigen processing and presentation pathway for CD8+ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapeptides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the "endogenous" processing pathway and prime CD8+ CTL in vivo and in vitro.

Young (H-2d, Ld+) severe combined immunodeficiency (scid) mice were injected intravenously with 10(5) CD4+CD8- T cells purified from spleen, thymus or lymph nodes (LN) of dm2 (H-2d, Ld-) donor mice. In the immunodeficient recipients, the lymphoid compartment in the splenic white pulp was repopulated with donor-type T cells and cellularity in the red pulp was increased. In addition, donor-type CD4+ T cells repopulated the peritoneal cavity, mesenteric LN and the lamina propria of the small intestine of scid mice, but were undetectable in thymus and peripheral (inguinal, axillary) LN. Histological examination of repopulated mesenteric LN showed expanded subcapsular sinuses, repopulated cortical areas, but poorly developed high endothelial venules (HEV) indicating deficient blood-LN lymphocyte recirculation. The engrafted CD4+ T cell population had the surface phenotype of memory T cells (CD44/Pgp-1high CD45RB(low) and expressed the Peyer's patch HEV-specific homing receptor CD49d (LPAM-1), but not the LN HEV-specific homing receptor LECAM-1. The CD4+ T cell population in spleen and mesenteric LN of transplanted scid mice displayed a diverse T cell receptor-V beta repertoire. Transfer of titrated numbers (10(3), 10(4), 10(5) cells per mouse) of CD4+ T cells into scid mice established donor-type T cell populations with this unusual homing pattern in all recipients. Repeated serial transfers of dm2 CD4+ T cells through young scid mice revealed an extensive in vivo expansion potential of transferred cells for > 18 months. The experimental system described represents an in vivo model to study the functional competence and the differentiation potential of a murine memory CD4+ T cell subset.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.

Immunodominance hierarchies operating in immune responses to viral antigens limit the diversity of the elicited T cell responses. The L(d)/S(28-39)-restricted CD8 T cell response to the hepatitis B surface antigen (HBsAg or S) prevents copriming of D(d)- and K(b)-restricted CD8 T cell responses. We exchanged L to V at position S(39) of HBsAg to construct mutant S(L39V). Comparable levels of wild-type S and mutant S(L39V) were produced by transiently transfected cells, and mice immunized with the pCI/S and pCI/S(L39V) DNA vaccines showed comparable serum antibody responses to HBsAg. The pCI/S but not pCI/S(L39V) DNA vaccination induced L(d)/S(28-39)-specific CD8 T cell responses. However, the pCI/S(L39V) DNA vaccine efficiently primed CD8 T cell responses to the subdominant D(d)- and K(b)-restricted epitopes, confirming the immunosuppressive phenotype of the L(d)/S(28-39)-specific CD8 T cell response. A single point mutation within the HBsAg can hence completely silence a 'dominant' CD8 T cell response thereby facilitating priming of a multispecific repertoire of suppressed, 'subdominant' epitopes. The data have practical implications for understanding HBV-specific CD8 T cell responses and for the design of novel vaccination strategies.

Intraperitoneal injection of 2 x 10(7) nonfractionated spleen cells (SC) from C57BL/6 (B6, H-2b) mice into completely allogeneic immunodeficient H-2d scid mice induced clinical and histological signs of acute graft-vs.-host disease (GVHD), with all transplanted severe combined immunodeficiency (scid) mice dying in the 3rd week post-transfer. In contrast four out of five scid mice survived for greater than 7 weeks after intravenous (i.v.) injections of equal numbers of B6 SC. Intravenously allotransplanted scid mice analyzed in the 8th week post-transfer had engrafted donor-type CD4+ and CD8+ T cells in the spleens but showed no clinical or histological evidence of GVHD. i.v. injection of 10(7) or 10(6)O B6 SC engrafted allogeneic T cells in spleens of scid recipients; in contrast, i.v. injection of 10(5) nonfractionated B6 SC or 3 x 10(5) cell sorter-purified, naive or anti-H-2d-primed splenic CD4+ or CD8+ B6 T cells led to rejection by young scid recipient mice. B6 T cells engrafted into spleens of scid mice after i.v. injection showed proliferative anti-host alloreactivity in vitro. No cytotoxic reactivity against host-type alloantigens was found in standard 4-h 51Cr-release assays. These data demonstrate that allogeneic T cells injected i.v. into immunodeficient scid mice are partially tolerized against host-type alloantigens.

Heat shock proteins (Hsp) of the Hsp70/90 families facilitate cellular immune responses to antigenic peptides or proteins bound to them and have therefore been used as vaccine vehicles. We developed an expression system in which chimeric proteins with an Hsp-capturing, viral J domain fused to diverse antigen-encoding sequences form stable complexes with eukaryotic (Hsp70, Hsp73) or bacterial (DnaK) stress proteins and accumulate to high steady-state levels. J domains from different species (viruses/SV40, bacteria/Chlamydia trachomatis or plants/Arabidopsis thaliana) efficiently capture murine or human stress proteins in this system, thus making different J domains available for vaccine production. A novel expression and purification method was developed to produce native Hsp/antigen complexes in transfectants. These purified Hsp/antigen complexes efficiently elicited antigen-specific CD8 T cell responses in mice when delivered as vaccines without adjuvants. In situ complex formation of antigen with Hsp was critical for CD8 T cell priming. Because the described expression system supports the flexible design of multivalent vaccines, it is an attractive strategy to elicit CD8 T cell responses either to recombinant proteins or to selected antigenic domains of these molecules.

Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product–specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.

CD4 Foxp3 regulatory T (TR) cells are well-defined regulator T cells known to develop in the thymus through positive selection by medium-to-high affinity TCR-MHC interactions. We asked whether Foxp3 TR cells can be generated in the complete absence of MHC class II molecules. CD4 Foxp3 TR cells are found in secondary lymphoid tissues (spleen and lymph nodes) and peripheral tissues (liver) but not the thymus of severely MHC class II-deficient (Aα−/− B6) mice. These TR cells preferentially express CD103 (but not CD25) but up-regulate CD25 surface expression to high levels in response to TCR-mediated activation. MHC class II-independent Foxp3 TR cells down modulate vaccine-induced, specific antiviral CD8 T cell responses of Aα−/− B6 mice in vivo. Furthermore, these TR cells suppress IL-2 release and proliferative responses in vitro of naive CD25− (CD4 or CD8) T cells from normal B6 mice primed by bead-coupled anti-CD3/anti-CD28 Ab as efficiently as CD4CD25high TR cells from congenic, normal B6 mice. MHC class II-independent CD4 Foxp3+ TR cells thus preferentially express the (TGF-β-induced) integrin molecule αE (CD103), are generated mainly in the periphery and efficiently mediate immunosuppressive effects.

A priority in current vaccine research is the development of multivalent vaccines that support the efficient priming of long-lasting CD8(+) T-cell immunity. We developed a novel vaccination strategy that used synthetic, cationic (positively charged), and antigenic peptides complexed to negatively charged nucleic acids: antigenic, major histocompatibility complex-class I-binding epitopes fused with a cationic sequence derived from the HIV tat protein (tat50-57: KKRRQRRR) were mixed with nucleic acids (e.g., CpG-containing oligonucleotides) to quantitatively form peptide/nucleic acid complexes. The injection of these complexes efficiently primed long-lasting, specific CD8(+) T-cell immunity of high magnitude. This chapter describes a novel strategy to codeliver complexes of cationic/antigenic peptides bound to antigen-encoding plasmid DNA vaccines in a way that enhances the immunogenicity of both components for T cells.

Vaccines for the prophylactic and/or therapeutic immunization against hepatotropic pathogens (e.g., hepatitis B and hepatitis C virus) should establish long-lasting, specific antiviral effector/memory CD8+ T cell immunity in the liver. We describe a novel peptide-based vaccine in which antigenic major histocompatibility complex Class I-binding peptides are fused to a cationic (e.g., human immunodeficiency virus tat-derived) domain and complexed to immune-stimulating oligonucleotides. This vaccine formulation efficiently primes liver-homing, Class I-restricted CD8+ effector/memory T cell responses. In different antigen systems, this formulation was more potent in priming liver-homing CD8+ T cell responses than DNA-based vaccines delivering the same epitopes. CD8+ T cell priming was independent of CD4+ T cell "help" but submitted to regulatory control by CD25+ CD4+ T cells. The vaccine efficiently primed memory/effector CD8+ T cells detectable in the liver for more than 3 months after a single injection. With increasing time after priming, the phenotype of these specific memory CD8+ T cells shifted from an effector memory to a central memory type. The vaccine could override T cell tolerance in mice expressing the relevant antigen from a transgene in the liver. The CD8+ T cell immunity in the liver primed by this peptide formulation could be boosted by challenge injections. In conclusion, we describe a simple and potent vaccine formulation that has the potential to generate or reconstitute specific CD8+ T cell immunity to hepatotropic pathogens in the liver.

Large number of T cells traffic through the liver. In order to examine the effects of such traffic on the phenotype of hepatocytes, we vaccinated mice using DNA vaccines encoding antigens with MHC class I-binding epitopes. Small numbers of activated CD8(+) T blasts (10(5)-10(6)/liver) changed the surface phenotype and cytokine expression profile of hepatocytes (HCs). HCs upregulate surface expression of major histocompatibility complex (MHC) class I molecules and CD1d but not MHC class II molecules Qa-1, CD80, CD86, CD54, or CD95; in addition, they expressed/secreted interleukin (IL)-10 and IL-4 but not IL-1, IL-6, IL-13, interferon (IFN)-gamma, tumor necrosis factor (TNF), IL-4, or IL-27 (i.e., they acquire the HC* phenotype). HCs* (but not HCs) induced specific activation, proliferation, and IFN-gamma, TNF, and IL-13 release of cocultured naïve CD8(+) T cells. In contrast to the specific activation of naïve CD8(+) T cells by dendritic cells (DCs), specific CD8(+) T cell activation by HC* was not down-modulated by IFN-alphabeta. Only recently activated CD8(+) T blasts (but not recently activated CD4(+) T blasts or activated cells of the innate immune system, including natural killer T [NKT] cells) induced the HC* phenotype that is prominent from day 10 to day 20 postvaccination (i.e., time points at which peak numbers of recently primed CD8(+) T blasts are found in the liver). In conclusion, recently activated CD8(+) T blasts that enter the liver postimmunization in small numbers can transiently modulate the phenotype of HC, allowing them to activate naïve CD8(+) T cells with unrelated specificities.

DNA-based vaccination efficiently primes MHC-restricted T-cell responses. This technique specifically stimulates MHC-II-restricted CD4(+) T-cell responses and MHC-I-restricted CD8(+) T-cell responses against "strong" (immunodominant) or "weak" (subdominant or cryptic) epitopes of intracellular, secreted or membrane-associated protein antigens. In many experimental systems, T-cell-mediated effector functions have the potential to control tumor growth. In particular MHC-I-restricted cytotoxic T lymphocytes (CTL) can reject tumors. This has been shown using either defined tumor-associated antigens (TAA), or viral antigens containing well-defined, MHC-binding and CTL-stimulating epitopes that are expressed by transfected tumor cells.

A priority in current vaccine research is the development of adjuvants that support the efficient priming of long-lasting, CD4+ T cell help-independent CD8+ T cell immunity. Oligodeoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I-restricted CD8+ T cell responses to proteins or peptides. We show that the adjuvant effect of ISS+ ODN on CD8+ T cell priming to large, recombinant Ag is enhanced by binding them to short, cationic (arginine-rich) peptides that themselves have no adjuvant activity in CD8+ T cell priming. Fusing antigenic epitopes to cationic (8- to 10-mer) peptides bound to immune-stimulating ISS+ ODN or nonstimulating NSS+ ODN (without CpG-containing sequences) generated immunogens that efficiently primed long-lasting, specific CD8+ T cell immunity of high magnitude. Different MHC class I-binding epitopes fused to short cationic peptides of different origins showed this adjuvant activity. Quantitative ODN binding to cationic peptides strikingly reduced the toxicity of the latter, suggesting that it improves the safety profile of the adjuvant. CD8+ T cell priming supported by this adjuvant was Toll-like receptor 9 dependent, but required no CD4+ T cell help. ODN (with or without CpG-containing sequences) are thus potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of Ag delivery prevailing in many viral nucleocapsids.

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.

Bacterial DNA sequences containing unmethylated CpG motifs have recently been proposed to exhibit immunostimulatory effects on B-, T- and NK cells, leading to the induction of humoral and cell-mediated immune responses. In the present study we investigated the immunomodulatory effects of a CpG-containing oligodeoxynucleotide (CpG ODN) to the HIV-1 gp160 envelope (Env) protein in the BALB/c mouse model. Priming and boosting of mice with gp160 adsorbed to aluminium hydroxide (Alum) induced a typical T helper-2 (Th2)-dominated immune response with high titers of gp160-specific immunoglobulin (Ig)G1 isotypes but a weak IgG2a response. Specifically re-stimulated splenocytes from these mice predominantly secreted interleukin (IL)-5 but only minute amounts of interferon-γ (IFN-γ) upon specific re-stimulation. In contrast, a boost immunisation of gp160/Alum primed mice with a gp160/Alum/CpG combination resulted in a seven times higher production of IgG2a antibodies, without affecting the titers of IgG1 isotypes. Furthermore, approximately 10-fold increased levels of IFN-γ, but significantly reduced amounts of IL-5, were secreted from gp160-restimulated splenic cells. A further greater than 30-fold increase in the levels of specific IgG2a responses and a substantially elevated secretion of IFN-γ were observed when the mice received gp160/Alum/CpG combinations for priming and boost injections. Thus, CpG ODNs are useful as an adjuvant to induce a typical Th0/Th1 response to HIV gp160 proteins. However, despite the induction of a more Th1-like immune response, gp160/Alum/CpG combinations were not sufficient to prime an Env-specific cytotoxic T-cell (CTL) response.

Murine MHC class I-restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL-defined epitopes of this viral envelope protein are the Ld-binding 12-mer S28-39 peptide IPQSLDSWWTSL in H-2d mice, and the Kb-binding 8-mer S208-215 peptide ILSPFLPL in H-2b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H-2 class I-binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the Ld-binding peptide S28-39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the Kb-binding peptide S208-215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I-binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.

Scid mice transplanted with low numbers of syngeneic CD4+ T cells, develop a chronic and lethal inflammatory bowel disease (IBD) within 4-6 months. We have used in vivo 5-bromo2-deoxy-uridine (BrdU) labeling to assess the proliferation of lamina propria-derived CD4+ T cells in diseased scid mice. The hourly rate of renewal of colonic lamina propria CD4+ T cells in diseased mice was 7% compared with 1.5% in normal BALB/c control mice. Transplantation of scid mice with in vitro activated CD4+ T cells accelerated the disease onset and development in a cell dose-dependent fashion when compared with non-activated CD4+ T cells. In pulse-chase experiments it was shown that BrdU-labeled cells disappeared rapidly from the lamina propria of diseased mice. DNA analysis revealed that this was due to the presence of nearly four times as many apoptotic CD4+ T cells in diseased than in control mice. Further analyses showed that the apoptotic lamina propria CD4+ T cells were derived from cells having entered the cell cycle within the previous 8 h. These data clearly demonstrate that vigorous CD4+ T cell proliferation and death are involved throughout the course of IBD.

Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the constitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two Db-binding peptides and one Kb-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two Db-binding epitopes, but not the Kb-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi Db molecules is available for 2-3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.

The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared. The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols). Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions. CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide. The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality. Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg. The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure. Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes. The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.

Peptides recognized by CD8+ cytotoxic T lymphocytes in the context of major histocompatibility complex (MHC) class I molecules are usually derived from endogenous proteins synthesized within the cell. Exogenous 22-nm hepatitis B surface antigen (HBsAg) particles are taken up by many cells, and are processed in a novel peptide-transporter-independent, endosomal or lysosomal pathway for class I (Ld)-restricted epitope presentation. Here, we present evidence that 'empty' Ld molecules derived from the cell surface are involved in presenting antigenic peptides from endocytosed HBsAg particles. Intracellular assembly of presentation-competent, trimeric Ld molecules required endocytosis of the exogenous antigen and 'empty' Ld molecules. These data assign a functional role to surface-associated, 'empty' MHC class I molecules.

We tested the efficiency of induction of immune responses to the small hepatitis B surface antigen (HBsAg) in mice by intramuscular DNA immunization using different vector constructs that allow high levels of HBsAg expression in mouse cells. The HBsAg-specific responses of class I-restricted cytotoxic T lymphocytes (CTL) and of B cells (serum antibody titers) were measured. Following the intramuscular inoculation of 'naked' DNA, five different vector constructs of 4-8 kb, that contained or did not contain an intron and/or the neo gene, in which HBsAg expression was driven by promoter sequences derived from the immediate early region of HCMV, the SV40 enhancer/promoter region, or a retroviral 3' LTR efficiently primed responses of class I-restricted CD8+ CTL precursors. In contrast, the constructs in which HBsAg expression was driven by HCMV-derived promoter sequences stimulated significantly higher levels of HBsAg-specific serum antibody titers after intramuscular DNA injection than the SV40 or MPSV vector constructs. Large (15 kb) episomal vector constructs did not stimulate CTL or antibody responses. The data demonstrate that: (i) intramuscular DNA immunization represents an efficient technique for priming CTL and antibody responses to HBsAg; (ii) many vectors can be constructed that express an immunogenic product after intramuscular inoculation of 'naked' DNA; (iii) the efficiency of the tested vector constructs to prime after DNA immunization, either a CTL response, or an antibody response, differs.