Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones

We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.     

Decrease of Atrial Natriuretic Peptide Content in Rat Superior Cervical Sympathetic Ganglion After Denervation and Axotomy

We found atrial natriuretic peptide (ANP), known as a humoral factor in regulating body fluid volume and blood pressure, in considerable quantities in rat superior cervical sympathetic ganglion (SCG) by radioimmunoassay after separation with reverse-phase HPLC. Although the ANP content of the immature rat 1 week after birth was low, it doubled at 2 weeks and then increased gradually, until it reached the adult level. Denervation caused a rapid decrease in the ANP content to half of the intact SCG level after 3 h, which then fell to 10% of the control value on day 2 after operation. The time course of ANP content reduction after denervation was similar but rather faster than that of activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, an observation suggesting that ANP may partly contribute to cholinergic synaptic transmission. On the other hand, axotomy produced a rather slower decrease in the ANP content than did denervation. Enucleation and sialoadenectomy also caused a considerable reduction of the ANP content. Thus, part of the ANP found in the ganglion is apparently transported from sympathetically innervated extraganglionic organs via retrograde axoplasmic flow.     

Covalent structure of a low-molecular-mass protein, meleagrin, present in a turkey (Meleagris gallopavo) ovomucoid preparation

A low-molecular-mass protein, tentatively named meleagrin, was isolated from a commercial preparation of turkey (Meleagris gallopavo) ovomucoid. This 40-amino acid protein contains 3 disulfide bonds and high concentrations of aromatic residues (2 tryptophans and 3 tyrosines). It lacks threonine, methionine, phenylalanine, and arginine residues. The complete amino acid sequence was determined to be the following: less than Glu-Val-Leu-Lys-Tyr-Cys-Pro-Lys-Ile-Gly-Tyr-Cys-Ser-Ser-Lys-Cys-Ser-Lys- Ala- Glu-Val-Trp-Ala-Tyr-Ser-Pro-Asp-Cys-Lys-Val-His-Cys-Cys-Val-Pro-Ala-Asn- Gln-Lys - Trp. One of the three disulfide bonds exists between Cys12 and Cys28, and the two others links Cys32-Cys33 with Cys6 and Cys16. The amino acid sequence of meleagrin shows a strong homology to a similar basic protein, cygnin (Simpson, G.R. & Morgan, F.J. [1983] Int. J. Pep. Protein Res. 22, 476-481), of a rather distantly related aves, black swan (Cygnus atratus), suggesting some vital role of this protein in avian eggs. Similarity to a part (exon 9) of transferrins was also recognized.     

Infertility induced in rats by immunization with synthetic peptide segments of a sperm protein.

Three peptide segments (YAL-198, YAL-201 and YAL-212) corresponding to the extracellular domain of a human sperm protein designated as YWK-II antigen were synthesized as multiple antigen peptide (MAP). Male and female rats were immunized with the YWK-II-MAPs and fertility determined. In a group of 12 female rats immunized with YAL-198, seven animals were infertile and two animals were subfertile. When immunized with YAL-201 and YAL-212, 4 and 2 animals were infertile, respectively. In a group of 15 males immunized with YAL-198, 2 animals were infertile and 6 were subfertile. Two animals immunized with YAL-201 were subfertile. All control male and female rats immunized with bovine serum albumin and adjuvant were fertile. Sera obtained from infertile rats immunized with YAL-198 contained higher titers of antibodies compared to those obtained from fertile animals. The present study shows that immunization with synthetic peptide segments of a sperm protein can effectively reduce fertility.     

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.     

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.