The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets.

We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright yellow-green, with maximal excitation at approximately 360 nm and maximal emission at approximately approximately 530 nm. The ELF substrate was used with streptavidin-alkaline phosphatase, to fluorescently label site-specific probes bound to their targets, including cell-surface sites, cytoplasmic organelles, nuclear antigens and cytoskeletal networks. All targets were labelled successfully with both the ELF substrate and fluoresceinated probes or protein conjugates. However, the ELF method was frequently more sensitive, with lower background fluorescence, allowing detection of more lysosomes, actin filaments, microtubules and nuclear targets than were visible with corresponding fluoresceinated probes. The ELF substrate was also used with antifluorescein-alkaline phosphatase to amplify fluorescein signals. We found that the ELF signal was in all cases brighter and more photostable than fluorescein signals, permitting shorter film exposures and allowing more time for examining samples. Surprisingly, relative brightness and photostability depended on the target, rather than being a general phenomenon related to the choice of dye alone.

CD4(+)CD25(+) T cells facilitate the induction of T cell anergy.

T cell anergy is characterized by the inability of the T cell to produce IL-2 and proliferate. It is reversible by the addition of exogenous IL-2. A similar state of unresponsiveness is observed when the proliferative response of murine CD4(+)CD25(-) T cells is suppressed in vitro by coactivated CD4(+)CD25(+) T cells. We have developed a suppression system that uses beads coated with anti-CD3 and anti-CD28 Abs as surrogate APCs to study the interaction of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in vitro. CD4(+)CD25(+) T cell-induced suppression, in this model, was not abrogated by blocking the B7-CTLA-4 pathway. When the CD4(+)CD25(-) T cells were separated from the CD4(+)CD25(+) suppressor cells after 24 h of coactivation by the Ab-coated beads, the CD4(+)CD25(-) T cells were unable to proliferate or to produce IL-2 upon restimulation. The induction of this anergic phenotype in the CD4(+)CD25(-) T cells correlated with the up-regulated expression of the gene related to anergy in lymphocytes (GRAIL), a novel anergy-related gene that acts as a negative regulator of IL-2 transcription. This system constitutes a novel mechanism of anergy induction in the presence of costimulation.

Immunization with DNA encoding an immunodominant peptide of insulin prevents diabetes in NOD mice.

DNA vaccination is an effective means of protecting experimental animals against infectious pathogens and cancer and has more recently been used to prevent autoimmune disease. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T-cell-mediated destruction of the insulin-secreting beta cells in the pancreas. The NOD mouse is an animal model of IDDM in which several autoantigens, including insulin, have been identified. In this study we demonstrate that vaccination of NOD mice with DNA encoding an immunodominant peptide of insulin (residues 9-23 of the B chain) protects the animals from developing diabetes. Animals injected intramuscularly with a bacterial plasmid encoding the insulin B chain peptide show significantly lower disease incidence and delayed onset of disease when compared to controls. Protection appears to be mediated by insulin B (9-23)-specific down-regulation of IFN-gamma. Our results confirm that DNA vaccination has a protective effect on autoimmunity, the understanding of which will reveal new insights into the immune system and open doors for novel therapies.

The ELF-97 alkaline phosphatase substrate provides a bright, photostable, fluorescent signal amplification method for FISH.

We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at approximately 360 nm, with emission centered at approximately 530 nm. This large Stokes shift allows ELF-97 signals to be easily distinguished from sample autofluorescence and signals arising from counterstains or other fluorophores. The ELF-97 precipitate was extremely photostable compared to fluorescein, allowing multiple photographic exposures of samples without significant signal intensity loss. For RNA in situ hybridization, labeling was specific and localized well to targets in cultured cells, tissue sections, and whole-mount zebrafish embryos. ELF-97 signals developed in seconds to minutes and were easily distinguished from pigmented tissues or cells, unlike those obtained using colorimetric substrates. We used the substrate with singly biotinylated short oligonucleotides to detect actin mRNA in MDCK cells and actin and beta-galactosidase mRNA in LacZ+ mouse fibroblasts. We also used a biotinylated cDNA, complementary to the mRNA encoded by the constant region of the T-cell receptor beta-chain, to specifically identify T-cells in mouse lymph node tissue sections. With digoxigenin-labeled probes, we detected several developmentally expressed mRNAs in whole-mount zebrafish embryos. Hybridization to centromere repeat regions in human metaphase and interphase chromosomes was also detected; ELF-97 signals were manyfold brighter than signals obtained with fluorescein conjugates. Finally, Southern blot hybridization using singly labeled oligonucleotide probes yielded a sensitivity similar to that obtained with radioactivity.

Fluorescence-based signal amplification technology.

The ELF alkaline phosphate substrate can be used to fluorescently label a wide variety of biological targets. This substrate yields a bright, photostable yellow-green fluorescent precipitate at the site of enzymatic activity. ELF labeling can be as much as 40 times as bright and hundreds of times as photostable as labeling with conventional fluorophores and yields signals capable of very fine submicroscopic resolution. Signal development is also extremely rapid, making the signal amplification technology well suited for applications such as RNA in situ hybridization.

Simultaneous visualization of G- and F-actin in endothelial cells.

We developed site-specific fluorescent probes that permit simultaneous microscopic observation of G- and F-actin in bovine endothelial cells. G-actin distribution was visualized with fluorescein-deoxyribonuclease I (DNAse I). F-actin was labeled with phalloidin conjugated to the new long-wavelength fluorophore BODIPY 581/591 (581-nm excitation, 591-nm emission), which is spectrally similar to Texas Red. The G-actin appeared as pervasive green fluorescence that was more intense in the nuclear region, where cell thickness is greater and stress fibers are less frequent. In addition, we observed a punctate fluorescein pattern around the nuclei and in other parts of the cells, suggesting that some G-actin is localized to small discrete sites. F-actin was observed as red fluorescent filaments. Unlabeled DNAse I effectively prevented staining of G-actin by the fluorescent DNAse I conjugates. The specificity of DNAse I for G-actin was confirmed by the presence of a single labeled band with molecular weight corresponding to actin in a Western blot of total cytoplasmic endothelial proteins reacted with biotin-DNAse I-streptavidin-alkaline phosphatase. Anti-actin antibody, which associates with both G- and F-actin, in conjunction with fluorescent secondary antibody produced a pattern similar to that obtained by simultaneous visualization with fluorescein-DNAse I and BODIPY 581/591- or rhodamine-phalloidin.

Selection for amino acid sequence and J beta element usage in the beta chain of DBA/2V beta b- and DBA/2V beta a-derived myoglobin-specific T cell clones.

Previous experiments in our laboratory have demonstrated that there is a marked restriction on the TCR beta-chain usage in DBA/2 mice in response to the sperm whale myoglobin (SpWMb) determinant 110-121, predominated by the use of the V beta 8.2 gene element. We analyzed the response of mice that had been genetically depleted of V beta 8+ T cells by generating a DBA/2 line that carries the V beta a TCR haplotype. Despite the very limited TCR repertoire expressed by DBA/2V beta a mice, they made an excellent response after immunization with the SpWMb 110-121 peptide. Data presented in this manuscript demonstrate that there is an equally restricted TCR V beta-chain utilization in the T-cell response to the determinant SpWMb 110-121 in DBA/2V beta a mice. Unexpectedly, there was a shift of MHC restriction of this determinant to T cells in the V beta a strain when compared with the V beta b strain of DBA/2 mice. We had previously demonstrated that DBA/2 mice utilized both the hybrid E alpha dA beta d MHC molecule as well as the conventional A alpha dA beta d molecule as presenting elements in response to SpWMb 110-121. Data presented in this manuscript demonstrate that the T-cell response in DBA/2V beta a mice is entirely restricted by the A alpha dA beta d MHC class II molecule. By analyzing a panel of SpWMb 110-121-specific T-cell clones from DBA/2V beta a mice, we were able to study the TCR repertoire expressed on T cells from mice that lack the V beta 8.2 gene. The V beta usage by the panel of clones analyzed was remarkably homogeneous. Thirteen of the 17 clones analyzed used the V beta 1 gene segment. Perhaps more striking was the junctional region nucleotide and amino acid sequences that were shared among these clones and that were similar to the V beta 8.2 clones analyzed previously. All clones assayed used the J beta 2.6 element, as did the great majority of the V beta 8.2 clones analyzed from DBA/2 (and B10.D2) V beta b mice. Importantly, in each strain of mice, irrespective of the V beta utilized, each TCR appeared to have selected an acidic amino acid in the beta-chain at position 100.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ.

We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.

Limited T cell receptor beta-chain usage in the sperm whale myoglobin 110-121/E alpha dA beta d response by H-2d congenic mouse strains.

The specificity and TCR gene usage of a panel of sperm whale myoglobin (SpWMb)-reactive T cell clones from DBA/2 mice have previously been characterized, to study structure-function relationships between components of the ternary complex consisting of Ag, TCR, and MHC class II molecules, whose interaction leads to Th cell activation. These DBA/2 clones were specific for epitopes within the residue 110 to 121 region of SpWMb, in the context of the mixed isotype molecule E alpha dA beta d, and expressed the TCR V beta 8.2 gene element. SpWMb-specific T cell hybridomas from the H-2d-congenic B10.D2 mouse strain, which differs from the DBA/2 strain only in the non-MHC background, were generated and compared with the T cell hybridomas from DBA/2 mice, in order to investigate the influence of non-MHC genes on the specificity of the T cell response to the 110-121 epitope. V beta usage by these hybridomas was very homogeneous; three of three DBA/2 and eight of nine B10.D2 hybridomas specific for the 110-121 epitope, in the context of the mixed isotype molecule E alpha dA beta d, expressed the V beta 8.2 gene product. Nucleotide and amino acid sequences of D beta, J beta, and N regions were also similar. One 110-121/E alpha dA beta d-specific B10.D2 hybridoma used V beta 7, a V beta that is clonally deleted in DBA/2 mice. These experiments suggest that a similar set of TCR beta genes are used to respond to a given epitope, regardless of non-MHC background, and they support the hypothesis that, despite great variability between individuals in their non-MHC background genes, human HLA-associated diseases might result from the formation of a particular ternary complex consisting of a shared MHC molecule, a common "disease-associated" epitope, and a shared TCR.

The presumptive CDR3 regions of both T cell receptor alpha and beta chains determine T cell specificity for myoglobin peptides.

The T cell receptor alpha/beta (TCR-alpha/beta) is encoded by variable (V), diversity (D), joining (J), and constant (C) segments assembled by recombination during thymocyte maturation to produce a heterodimer that imparts antigenic specificity to the T cell. Unlike immunoglobulins (Igs), which bind free antigen, the ligands of TCR-alpha/beta are cell surface complexes of intracellularly degraded antigens (i.e., peptides) bound to and presented by polymorphic products of the major histocompatibility complex (MHC). Therefore, antigen recognition by T cells is defined as MHC restricted. A model has been formulated based upon the similarity between TCR-alpha/beta V region and Ig Fab amino acid sequences, and the crystal structure of the MHC class I and Ig molecules. This model predicts that the complementarity determining regions (CDR) 1 and 2, composed of TCR V alpha and V beta segments, primarily contact residues of the MHC alpha helices, whereas V/J alpha and V/D/J beta junctional regions (the CDR3 equivalent) contact the peptide in the MHC binding groove. Because polymorphism in MHC proteins is limited relative to the enormous diversity of antigenic peptides, the TCR may have evolved to position the highly diverse junctional residues (CDR3), where they have maximal contact with antigen bound in the MHC peptide groove. Here, we demonstrate a definitive association between CDR3 sequences in both TCR alpha and beta chains, and differences in recognition of antigen fine specificity using a panel of I-Ed-restricted, myoglobin-reactive T cell clones. Acquisition of these data relied in part upon a modification of the polymerase chain reaction that uses a degenerate, consensus primer to amplify TCR alpha chains without foreknowledge of the V alpha segments they utilize.