Essential requirement of an invariant V alpha 14 T cell antigen receptor expression in the development of natural killer T cells.

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.

The 5' coding region of Paramecium surface antigen genes controls mutually exclusive transcription.

Paramecium tetraurelia stock 51 can express at least 11 different types of surface antigens, yet only a single type is expressed on the surface of an individual cell at any one time. The differential expression of stock 51 type A and B surface antigen genes (51A and 51B) is regulated at the level of transcription. Previously, we reported that nucleotide sequences upstream of position -26 (relative to the start of translation) in the 51A and 51B surface antigen genes are necessary for transcriptional activity but are not sufficient to direct differential transcriptional control. In this report we demonstrate that at least some of the critical elements necessary for differential transcription of the 51A and 51B genes lie within the 5' coding region. A hybrid gene that contains 51B upstream sequences (-475 to +1) attached to the ATG start codon of 51A is not cotranscribed with the 51B gene. In contrast, further substitution with 51B sequences (-1647 to +885) allows the chimeric gene to be coexpressed with 51B. A different hybrid gene containing a substitution of 51B sequence from -26 to +885 in the 51A gene is also coexpressed with 51B, revealing that the critical elements within the coding region of 51B do not require 51B upstream sequences for their effect. Coinjection of the 51A gene with the chimeric gene that contains 51B up to +885 showed that the same sequences that allow coexpression with 51B prevent cotranscription with 51A. Together, these results demonstrate that a region downstream of the transcriptional start site between nucleotide positions +1 and +885 (relative to translational start) is necessary to control differential transcriptional activity.

Major histocompatibility complex class I molecule serves as a ligand for presentation of the superantigen staphylococcal enterotoxin B to T cells.

Superantigens, such as staphylococcal enterotoxin B (SEB), elicit a strong proliferative response in T cells when presented in the context of major histocompatibility complex (MHC) class II molecules. We observed a similar T-cell response, when MHC class II-negative epidermal cell lines were employed as antigen-presenting cells. Immunoprecipitation studies indicated that the ligand to which SEB bound had a molecular mass of 46 kDa. Radiolabeled SEB could be immunoprecipitated from isolated membrane proteins on the SCC13 epidermal cell line with a monoclonal antibody directed against the MHC class I molecule, and transfection of the K-562 cell line with MHC class I molecules showed a 75% increased SEB-binding capacity compared with the nontransfected MHC class I- and class II-negative counterpart. In functional studies, antibodies to the MHC class I molecule inhibited T-cell proliferation by at least 50%. From these studies, we conclude that MHC class I molecules on malignant squamous cell carcinomas serve as ligands for SEB, which, given the appropriate costimulatory signals, is sufficient to allow for superantigen-induced T-cell proliferation.

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 10(9) different clones bearing a 30-residue peptide fused to gene III. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the alpha-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.

Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen.

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

The BCL2 major breakpoint region is a sequence- and cell-cycle-specific binding site of the Ku antigen.

The majority of translocations involving BCL2 are very narrowly targeted to three breakpoint clusters evenly spaced over a 100-bp region of the gene's terminal exon. We have recently shown that the immediate upstream boundary of this major breakpoint region (mbr) is a specific recognition site for single-strand DNA (ssDNA) binding proteins on the sense and antisense strands. The downstream flank of the mbr is a helicase binding site. In this report we demonstrate that the helicase and ssDNA binding proteins show reciprocal changes in binding activity over the cell cycle. The helicase is maximally active in G1 and early S phases; the ssDNA binding proteins are maximally active in late S and G2/M phases. An inhibitor of helicase binding appears in late S and G2/M. Finally, at least one component of the helicase binding complex is the Ku antigen. Thus, a protein with helicase activity implicated in repair of double-strand breaks, variable (diversity) joining recombination, and, potentially, cell-cycle regulation is targeted to the BCL2 mbr.

Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors. Preliminary data indicated that one of the major components of IBP-s was human estrogen-related receptor 1 (hERR1). We show here that several members of the steroid/thyroid hormone receptor superfamily, including testis receptor 2, thyroid receptor alpha 1 in combination with retinoid X receptor alpha, chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2), as well as hERR1, possess the properties of IBP-s. These receptors bind specifically to hormone receptor binding sites present in the SV40 major late promoter. Recombinant COUP-TF1 specifically represses transcription from the SV40 major late promoter in a cell-free transcription system. Expression of COUP-TF1, COUP-TF2, or hERR1 in monkey cells results in repression of the SV40 late promoter, but not the early promoter, in the absence of the virally encoded large tumor antigen. Overexpression of COUP-TF1 leads to a delay in the early-to-late switch in SV40 gene expression during the lytic cycle of infection. Thus, members of this superfamily can play major direct roles in regulating expression of SV40. Possibly, natural or synthetic ligands to these receptors can serve as antiviral drugs. Our findings also provide the basis for the development of assays to screen for the ligands to testis receptor 2 and hERR1.

Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.

Expression of endogenous peptide-major histocompatibility complex class II complexes derived from invariant chain-antigen fusion proteins.

CD4+ T cells recognize major histocompatibility complex (MHC) class II-bound peptides that are primarily obtained from extracellular sources. Endogenously synthesized proteins that readily enter the MHC class I presentation pathway are generally excluded from the MHC class II presentation pathway. We show here that endogenously synthesized ovalbumin or hen egg lysozyme can be efficiently presented as peptide-MHC class II complexes when they are expressed as fusion proteins with the invariant chain (Ii). Similar to the wild-type Ii, the Ii-antigen fusion proteins were associated intracellularly with MHC molecules. Most efficient expression of endogenous peptide-MHC complex was obtained with fusion proteins that contained the endosomal targeting signal within the N-terminal cytoplasmic Ii residues but did not require the luminal residues of Ii that are known to bind MHC molecules. These results suggest that signals within the Ii can allow endogenously synthesized proteins to efficiently enter the MHC class II presentation pathway. They also suggest a strategy for identifying unknown antigens presented by MHC class II molecules.