The role of B7-1 and B7-2 costimulation for the generation of CTL responses in vivo

The role of B7-1 and B7-2 costimulatory molecules in the generation of Ag-specific CD8+ CTLs is not well understood. In this paper, we analyze the role of both B7-1 and B7-2 in the generation of CTLs to nonliving, exogenous Ag and to live virus. To analyze the role of B7 costimulation in the induction of CTLs, we blocked B7-1 and/or B7-2 in vivo by injecting C57BL/6 mice with anti-B7-1 and/or anti-B7-2 mAbs; the mice were subsequently immunized with either chicken OVA that had been cross-linked to beads as a model of exogenous Ags or with wild-type and recombinant vaccinia virus expressing different forms of chicken OVA as models of viral Ags. Our results indicate that B7 costimulation is necessary in the generation of CTLs for all of these Ags. Since the B7 molecules could be costimulating CD8+ and/or CD4+ T cells in wild-type animals, we also examined the role of costimulation in the generation of CTLs to exogenous and viral Ag in MHC class II-deficient mice lacking most CD4+ T cells. In these animals, a combination of both mAbs also blocked all CTL responses, indicating that the Th cell-independent activation of CTLs is dependent upon the B7-costimulatory signals supplied to the CD8+ cell. These findings contribute to the understanding of the role of costimulation for the generation of CTLs. We also discuss the implications of these findings on the role of professional APCs in the initiation of CTL responses.     

IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.     

Retrovirally transduced bone marrow-derived dendritic cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity

It has been extensively documented that murine dendritic cells loaded with tumor-associated Ag (TAA)-derived peptides or protein can prime Ag-specific CD8+ cytotoxic T cells in vivo and can elicit Ag-specific immunity. Optimal presentation of TAA might be achieved by retroviral transduction of DCs allowing long term and stable expression of the TAA-peptides as well as the presentation of multiple epitopes in the context of MHC class I and/or class II molecules. Here we show that retroviral transduction of bone marrow-derived dendritic cells (DCs) with chicken OVA cDNA or the reporter gene green fluorescent protein retained their potent stimulatory capacity and that the transduced DCs could process and present the endogenously expressed OVA protein. The DCs transduced with cDNA encoding native OVA protein presented OVA-derived peptides in the context of MHC class I as well as MHC class II and induced a strong Ag-specific CTL response. DCs expressing a cytosolic form of OVA presented OVA peptides only in the context of MHC class I and failed to induce an OVA-specific CTL response in vivo when they had been cultured in the absence of exogenous protein. Immunization with retrovirally transduced DCs resulted in an Ag-specific immunity and rejection of a tumor cell challenge and a significant survival advantage in tumor-bearing mice. These results obtained in this rapidly lethal tumor model suggest that DCs transduced with TAA may be useful for tumor immunotherapy and underscore the importance of the simultaneous delivery of T cell help in the development of Ag-specific cytotoxic T-cells.     

Gene vaccination with naked plasmid DNA: mechanism of CTL priming

The injection of naked plasmid DNA directly into the muscle cells of mice has been shown to induce potent humoral and cellular immune responses. The generation of a cytotoxic T lymphocyte (CTL) response after plasmid DNA injection may involve the presentation of the expressed antigen in the context of the injected myocytes' endogenous major histocompatibility (MHC)-encoded class I molecules or may use the MHC molecules of bone marrow-derived antigen presenting cells (APC) which are capable of providing co-stimulation as well. To resolve which cell type provides the specific restricting element for this method of vaccination we generated parent-->F1 bone marrow chimeras in which H-2bxd recipient mice received bone marrow that expressed only H-2b or H-2d MHC molecules. These mice were injected intramuscularly with naked plasmid DNA that encoded the nucleoprotein from the A/PR/8/34 influenza strain, which as a single antigen has epitopes for both H-2Db and H-2Kd. The resulting CTL responses were restricted to the MHC haplotype of the bone marrow alone and not to the second haplotype expressed by the recipient's myocytes. The role of somatic tissues that express protein from injected plasmids may be to serve as a reservoir for that antigen which is then transferred to the APC. Consequently, our data show that the mechanism of priming in this novel method for vaccination uses the MHC from bone marrow-derived APC, which are efficient at providing all of the necessary signals for priming the T cell.     

Increased threshold for TCR-mediated signaling controls self reactivity of intraepithelial lymphocytes

To examine the effect of self Ag on activation requirements of TCR-alphabeta intestinal intraepithelial lymphocytes (IELs), we utilized the 2C transgenic (Tg) mouse model specific for a peptide self Ag presented by class I MHC, H-2Ld. CD8alpha alpha and CD4-CD8- IELs from syngeneic (H-2b, self Ag-) and self Ag-bearing (H-2b/d, self Ag+) strains were examined for their ability to respond in vitro to P815 (H-2d) cell lines expressing the endogenous antigenic peptide, p2Ca. Proliferation, cytokine production, and CTL activity were elicited in IEL T cells isolated from self Ag- H-2b mice when stimulated with P815 cells expressing basal levels of self Ag. These responses were enhanced following the addition of exogenous p2Ca peptide and ectopic expression of the costimulatory molecule, B7-1. By comparison, IEL from self Ag-bearing mice failed to respond to basal levels of self Ag presented by P815 cells even in the presence of B7-1-mediated costimulation. However, the addition of increasing amounts of exogenous p2Ca peptide induced a response from the in vivo "tolerized" T cells. These results suggest that exposure to self Ag in vivo increased the threshold of TCR activation of Ag-exposed self-reactive IELs. The dependence of increased signal 1 to activate self-reactive IELs suggests a defect in TCR signaling that may maintain self tolerance in vivo. These data suggest that conditions that overcome signal 1 IEL defects may initiate autoreactive responses in the intestine.     

Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro: enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-alpha

DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-alpha consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.     

CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge

CD40/CD40 ligand interactions have a central role in the induction of both humoral and cellular immunity. In this study, we examined whether a plasmid expressing CD40 ligand/trimer (CD40LT) could enhance immune responses in vivo. BALB/c mice were injected with plasmid expressing beta-galactosidase DNA with or without CD40LT DNA or IL-12 DNA, and immune responses were assessed. Mice vaccinated with beta-gal DNA plus CD40LT DNA or IL-12 DNA had a striking increase in Ag-specific production of IFN-gamma, cytolytic T cell activity, and IgG2a Ab. The mechanism by which CD40LT DNA enhanced these responses was further assessed by treating vaccinated mice with anti-IL-12 mAb or CTLA-4 Ig (CTLA4Ig). Production of IFN-gamma and CTL activity was abrogated by these treatments, suggesting that CD40LT DNA was mediating its effects on IFN-gamma and CTL activity through induction of IL-12 and enhancement of B7 expression, respectively. Physiologic relevance for the ability of CD40LT DNA to enhance immune responses by the aforementioned pathways was shown in two in vivo models. First, with regard to CTL activity, mice vaccinated with CD40LT DNA did not develop metastatic tumor following challenge with lethal dose of tumor. Moreover, in a mouse model requiring IL-12-dependent production of IFN-gamma, mice vaccinated with soluble Leishmania Ag and CD40LT DNA were able to control infection with Leishmania major. These data suggest that CD40LT DNA could be a useful vaccine adjuvant for diseases requiring cellular and/or humoral immunity.     

Virus-like particles induce MHC class I-restricted T-cell responses. Lessons learned from the hepatitis B small surface antigen

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 surface antigen (HBsAg). Here, we compare different vaccination techniques that prime in vivo class I-restricted, murine cytotoxic T lymphocyte (CTL) precursors and specific serum antibody responses. CTL were efficiently primed by the injection of low doses of recombinant native HBsAg particles without adjuvants, by the injection of low doses of denatured HBsAg monomers without adjuvants, by infection with recombinant vaccinia virus carrying a HBsAg-encoding gene, or by intramuscular transfer of plasmid DNA encoding HBsAg under appropriate promoter control. The observation that the injection of 100 ng to 1 microgram of native HBsAg "virus-like particles' (VLP) without adjuvants is an exogenous antigen preparation that efficiently primes class I-restricted CTL responses was unexpected. It reveals a novel aspect of the immunogenicity of VLP for T cells.     

A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increases protection conferred by a malaria DNA vaccine

Using the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-gamma responses of CD8+ T cells to the PyCSP 280-288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.     

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.     

Cutting edge: induction of enhanced CTL-dependent protective immunity in vivo by N-end rule targeting of a model tumor antigen

There is much interest in vaccines that will enhance the induction of CTL. One mechanism to enhance Ag-specific CTL responses involves targeting Ag to undergo rapid cytoplasmic degradation by the N-end rule pathway. We have analyzed the ability of N-end rule targeting to confer protection in an immunization-challenge setting. Using the HIV-1 nef protein as a model tumor Ag, we found that in mice immunized with a vaccinia vector expressing a form of nef that is targeted for rapid cytoplasmic degradation, there was enhanced induction of nef-specific CTL and protection from a lethal challenge with the syngeneic CT26 tumor cells that had been transfected with nef. Protection from tumor challenge correlated with the magnitude of the CTL response. Thus, the targeting of tumor or viral Ags for rapid cytoplasmic degradation by the N-end rule pathway may represent a strategy for the induction of protective Ag-specific CTL responses in vivo.     

A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.     

Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients

We have shown previously that granulocyte-macrophage colony-stimulating factor-stimulated mouse bone marrow-derived MHC class II+ dendritic cell (DC) progenitors that are deficient in cell surface expression of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) can induce alloantigen-specific T-cell anergy in vitro. To test the in vivo relevance of these findings, 2 x 10(6) B10 (H2b) mouse bone marrow-derived DC progenitors (NLDC 145+, MHC class II+, B7-1dim, B7-2-/dim) that induced T-cell hyporesponsiveness in vitro were injected systemically into normal C3H (H2k) recipients. Seven days later, the mice received heterotopic heart transplants from B10 donors. No immunosuppressive treatment was given. Median graft survival time was prolonged significantly from 9.5 to 22 days. Median graft survival time was also increased, although to a lesser extent (16.5 days), in mice that received third-party (BALB/c; H2d) DC progenitors. Ex vivo analysis of host T-cell responses to donor and third-party alloantigens 7 days after the injection of DC progenitors (the time of heart transplant) revealed minimal anti-donor mixed leukocyte reaction and cytotoxic T lymphocyte reactivity. These responses were reduced substantially compared with those of spleen cells from animals pretreated with "mature" granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DC (MHC class IIbright, B7-1+, B7-2bright), many of which rejected their heart grafts in an accelerated fashion. Among the injected donor MHC class II+ DC progenitors that migrated to recipient secondary lymphoid tissue were cells that appeared to have up-regulated cell surface B7-1 and B7-2 molecule expression. This observation may explain, at least in part, the temporary or unstable nature of the hyporesponsiveness induced by the DC progenitors in nonimmunosuppressed recipients.     

Early postoperative gadolinium-DTPA-enhanced MR imaging after successful lumbar discectomy

Cytotoxic T lymphocytes (CTLs) recognise antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules on virus-infected cells. The formation and transportation of antigenic peptides to class I MHC in the cells are multi-step reactions known as antigen processing. In order to design a good DNA vaccine, it is important to dissect the specificity of antigen processing. Here we describe the construction of an epitope-based plasmid vector as a device to investigate antigen processing in transfected cells. The epitope-based plasmid vector was constructed by insertion of an epitope-encoding minigene into the lacZ gene. We used a CTL epitope on influenza A virus nucleoprotein (NP366-374 epitope) as a model. Upon transfection, the epitope-based plasmid vector induced the expression of NP epitope antigenically as well as immunogenically. Immunization of mice with plasmid-transfected cells was able to induce NP epitope-specific CTLs in vivo. Moreover, the plasmid vector functioned as a gene vaccine; NP epitope-specific CTLs were primed in vivo upon transfection of the vector into dermis by electroporation. The results suggest that this epitope-based DNA delivery system may provide a new strategy for in vivo induction of epitope-specific CTLs to investigate antigen processing and presentation.     

Synergistic regulation of inducible nitric oxide synthase gene by CCAAT/enhancer-binding protein beta and nuclear factor-kappaB in hepatocytes

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response.     

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen

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.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway

By conjugation of proteins to beads, Ags can be selectively targeted into the MHC class I pathway of phagocytes in vivo and can stimulate CTL responses. Because phagocytes also present particulate Ag on MHC class II molecules, we examined whether these Ags stimulated concomitant CD4 T cell immunity. Although the priming of CD4 T cells with soluble OVA required adjuvants, particulate Ag was stimulatory when injected in saline. We next examined whether CD4 T cell responses played a role in the generation of CTL to particulate Ag. At low concentrations of Ag, OVA primed CTLs in wild-type mice but not in MHC class II-deficient animals, indicating that MHC class II presentation of Ag was essential for CTL generation. These data both support a model where CD4 T cells collaborate with CTLs as part of a three-cell interaction and identify a phagocyte as the third cell in this reaction. Interestingly, injection of higher concentrations of the same Ag primed equivalent CTL responses in both wild-type and MHC class II-deficient mice. These results indicate that a key variable in determining whether CTL generation is helper cell dependent or independent is the dose of immunogen. This may explain in part why CTL responses to abundant Ags, such as viruses, tend to be helper independent, while responses to less abundant Ags, such as minor histocompatibility Ags, require T helper cells. In addition, these results also point to the potential of using particulate Ags to prime or boost responses in settings with CD4 immunodeficiency.     

Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocyte and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization

We examined the role of the cellular localization of antigen on the immune response after DNA immunization of mice with three forms of ovalbumin (OVA). DNA encoding OVA which was secreted (sOVA) generated 10- to 100-fold higher IgG responses with 50-and 100-fold higher levels of IgG1 than the cytoplasmic (cOVA) or membrane bound (mOVA) forms. An IgG2a predominance was seen only in cOVA and mOVA immunized mice. Although the antibody response was CD4+ T cell dependent, the differences in the antibody response could not be compensated for by provision of excess CD4+ T cell help in TCR transgenic mice. Together with our hapten-carrier studies, this would indicate that membrane or intracellular localization limits the availability of antigen for B cell priming which affects the magnitude and form of the antibody response. Surprisingly, stronger cytotoxic T lymphocyte (CTL) responses were generated for sOVA or mOVA than for cOVA via intramuscular (i.m.) injection. Since a cytoplasmic antigen should have best access to the canonical class I pathway for antigen presentation, our results indicate that priming of CTL responses after i.m. DNA immunization is probably by cross-presentation of antigen by non-transfected professional antigen-presenting cells. In contrast, intradermal immunization with cOVA produced optimal CTL responses but, as with mOVA, suboptimal antibody responses. This, together with our ex vivo RT-PCR analysis showing similar mRNA levels from all three constructs 7 days post-immunization, argues against the differential CTL response for i.m. injection to be due to dose.     

Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization

Interaction of CD40 with its ligand (CD154) can induce CD40-bearing APCs to express immune stimulatory accessory molecules that facilitate immune recognition. We evaluated whether a plasmid vector encoding CD154 (pCD40L) could influence the immune response to a transgene protein encoded by coinjected plasmid DNA. We found that coinjection of pCD40L in BALB/c mice enhanced the Ab response to beta-galactosidase induced by i.m. or intradermal injection of placZ, a plasmid DNA vector encoding beta-galactosidase. Furthermore, i.m. or intradermal coinjection of pCD40L with placZ enhanced the generation of CTL specific for P815 cells transfected with placZ. This study indicates that pCD40L can serve as a genetic adjuvant capable of augmenting humoral and cellular immune responses to Ags encoded by plasmid DNA expression vectors.