Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.

Characterization of N-linked oligosaccharides bearing sialyl lewis x moieties on an alternatively glycosylated form of soluble complement receptor type 1 (sCR1).

We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLe(x)) moiety. This modified glycoprotein could combine anti-complement activity with the ability to inhibit selectin-mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N-linked oligosaccharides in the alpha1-3 linkage, which is necessary for sLe(x) glycosylation. The glycoprotein purified from these cells was designated sCR1sLe(x), and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLe(x) was performed to confirm that the N-linked oligosaccharides possessed sLe(x) moieties and also to determine the extent of sLe(x) glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore-assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non-core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage-specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLe(x) moieties on the N-linked oligosaccharides of sCR1sLe(x). sCR1sLe(x) is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.

Six chains of the human T cell antigen receptor.CD3 complex are necessary and sufficient for processing the receptor heterodimer to the cell surface.

The T cell antigen receptor (TCR) plays a key role in the process of antigen recognition. It is a complex of at least seven peptide chains (alpha beta gamma delta epsilon zeta-zeta). It is found on the surface of mature T cells and functions in antigen binding in the presence of the major histocompatibility complex. It has been known for some time that physical associations between the CD3 proteins and the TCR chains are essential for efficient transport of either component to the surface of T cells. For example, T cells that lack either the alpha, beta, or delta chains synthesize partial complexes that are eventually degraded. cDNAs encoding the six chains of receptor have become available recently. We have used transfection techniques to generate a panel of Chinese hamster ovary cells that contain partial receptor complexes of known composition and also cells that express all six subunits of the TCR.CD3 complex. Cells in this panel were analyzed for the ability to form alpha-beta heterodimers and also an ability to transport the synthesized chains to the plasma membrane. These studies have allowed us to define the minimum requirements for TCR.CD3 expression on the cell surface.

Recognition for degradation in the endoplasmic reticulum and lysosomes prevents the transport of single TCR beta and CD3 delta subunits of the T-cell antigen receptor to the surface of cells.

The T cell antigen receptor is a multiple subunit membrane protein made from six different polypeptide chains (alpha beta gamma delta epsilon zeta). The subunits are transmembrane proteins but only receptors assembled from all six chains are transported efficiently to the plasma membrane. Partial receptors and single subunits fail to reach the Golgi apparatus. This study has used transfected fibroblasts to follow the intracellular fate of the TCR beta and CD3 delta subunits in detail. After a lag period of approximately 90 min both chains were degraded by a process which did not require their transport to the medial Golgi. Degradation was inhibited at temperatures below 18 degrees but was unaffected by agents that disrupted the ER to Golgi transport. Experiments using transfected Chinese hamster ovary Lec 1 cells suggested that CD3 delta was degraded without transport to the cis-Golgi. Lysosomotropic agents had no effect on the proteolysis of the beta chain but did prevent the degradation of approximately 25% of the delta subunit. In the presence of chloroquine the delta subunit could be detected in lysosomes. The experiments show that proteolysis in or close to the endoplasmic reticulum plays a major role in preventing the surface expression of single beta and delta subunits of the T cell antigen receptor; nevertheless, some of the delta chain is able to evade this process. Interestingly, there is a second check on the transport of delta to the plasma membrane, and the subunit is removed from the secretory pathway and delivered to lysosomes for degradation.

Induction of cell-mediated cytotoxicity by shark 19S IgM.

Plasma from unimmunized nurse sharks can mediate a reaction similar to antibody-dependent cell-mediated cytotoxicity (ADCC). Normal shark plasma contains numerous natural antibodies reactive with a variety of antigens, including the target employed. Adsorption of plasma with target cells removed a significant amount of activity, suggesting involvement of antibody. Purified 19s IgM was shown to be a component of shark plasma capable of inducing cytotoxicity. These cytotoxic reactions differ from observations in homeothermic vertebrates in that shark immunoglobulin appears to bind more avidly to the effector cells than to the targets. The effector leukocytes are glass adherent, but not susceptible to carbonyl iron treatment, which clearly separates them from the phagocytic effectors of spontaneous cytotoxicity. Thus, the shark possesses leukocytes with the capability of mediating an ADCC-like reaction. These leukocytes, in concert with those mediating spontaneous cytotoxicity, could provide the shark with an effective immunosurveillance system. These data also indicate that ADCC mechanisms, with IgM as the primary effector molecule, appeared early in evolution.

The T-cell receptor gamma chain-CD3 complex: implication in the cytotoxic activity of a CD3+ CD4- CD8- human natural killer clone.

A subset of human T cells has recently been described. These cells express the CD3 complex but they do not carry the classical T-cell receptor (TCR)-alpha/-beta heterodimer on their surface (WT31- CD3+). Instead, they express a TCR-gamma chain associated with another type of polypeptide termed TCR-delta. We report here that a T-cell clone with natural killer (NK)-like activity, WM-14, had a disulfide bridged TCR-gamma homodimer associated with CD3 on its surface. The TCR-gamma chains of WM-14 cells were present in three different glycosylation forms of 43, 40, and 38 kDa, but they appeared to contain the same polypeptide backbone. Since cytotoxicity by WM-14 could be inhibited by anti-CD3 antibodies, we concluded that the TCR-gamma-CD3 complex was involved in the NK-like unrestricted killer activity. Although normal CD3-gamma, CD3-delta, and CD3-epsilon chains were present in this clone, the association with the TCR-gamma homodimer may be the cause of a complete processing of the N-linked oligosaccharides attached to the CD3-delta chain.

T3-p28 is a protein associated with the delta and epsilon chains of the T cell receptor-T3 antigen complex during biosynthesis.

The human T cell receptor-T3 antigen complex is composed of at least five polypeptide chains. In addition to the 45-kDa/50-kDa heterodimer (alpha and beta chains) of the T cell receptor, the complex includes 25-kDa (T3-gamma) and 20-kDa (T3-delta) glycoproteins and a nonglycosylated 20-kDa (T3-epsilon) protein. Here we report that in pulse-chase biosynthetic labeling experiments we detect a new polypeptide chain (T3-p28) which is associated with the T3-delta and T3-epsilon chains during biosynthesis but not on the cell surface. T3-p28, which is not recognized by anti-T3 antibodies, can be chemically distinguished from the previously described T3-gamma chain. The carboxylic ionophore monensin blocks the apparent dissociation of T3-p28 from the T3-delta and T3-epsilon chains. Peripheral blood lymphocytes as well as all T cell leukemic lines tested contain T3-p28, except one HPB-ALL subline. Since the T3-p28 protein is only observed early in biosynthesis of T3-delta and T3-epsilon, it may function in intracellular transport or assembly of the T cell receptor-T3 complex.

Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex.

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.

A T3-like protein complex associated with the antigen receptor on murine T cells.

Antigen recognition by human T lymphocytes and initiation of T-cell activation are mediated by a group of integral membrane proteins, the T-cell antigen receptor (TCR) and the T3 complex. The polypeptides which comprise T3 (a gamma-chain of relative molecular mass (Mr) 25,000 (25K), and delta and epsilon chains of 20K each) are physically associated with the TCR chains. Surface expression of the complex requires the presence of all the component T3 and TCR proteins. In contrast to the human system, murine T3 has not been identified using antibodies. Here we describe a murine T3-like protein complex. It appears to be more complicated than human T3, containing three monomeric glycoproteins (21-28K), two of which have N-linked carbohydrate side chains and a novel family of TCR-associated homo- and heterodimers. The 28K protein is identified as the murine T3 delta-chain. The 21K protein is phosphorylated on cell activation with concanavalin A (Con A).

Immunotherapy of murine leukemia. XI. differential susceptibility of spleen cells from serum-protected mice to the in vitro immunosuppressive effects of Friend leukemia virus-infected splenocytes.

It has previously been shown that DBA/2 mice protected against the development of Friend leukemia virus (FLV)4-induced disease by the passive administration of heterologous antisera directed against disrupted virions or the major viral envelope glycoprotein (gp71) fail to undergo the generalized immunosuppression which characterizes FLV leukemogenesis. In the present studies, the susceptibility of spleen cells from serum-protected mice to the immunosuppressive effects of FLV-infected spleen cells has been examined by means of in vitro assays of antibody production and of natural killing (NK). In contrast to the parallel suppression of both functions in FLV-infected mice and their lack of suppression in serum-protected animals, a dichotomy was observed in the in vitro susceptibility of these activities of spleen cells from serum-protected mice to the suppressive effects of virus-infected splenocytes, implying that more than one mechanism of suppression is operative. Thus, the antibody-producing capability of serum-protected splenocytes was not suppressed by FLV-infected spleen cells, this suppression reflecting the activity of infectious virus, while the NK effector function of serum-protected spleen cells was as susceptible as that of normal splenocytes to suppression by virus-infected spleen cells. This suppression of NK activity is mediated by virus-induced suppressor cells and does not involve free infectious virus, in contrast to suppression of antibody production. These results thus indicate that, while serum therapy inhibits the development of the virus-induced NK suppressor cells found in the spleens of progressively infected mice, splenic NK effectors, which are present, demonstrate full susceptibility to the suppressive effects of such independently generated virus-induced suppressor cells. Preliminary attempts at characterizing these NK suppressors in the spleens of FLV-infected mice indicate that they are not eliminated by treatment with antisera directed against T cells, B cells, macrophages or FLV gp71. However, Percoll fractionation of spleen cells from infected mice has revealed the presence of an expanded (as compared to normal spleen cells) population of cells of density 1.077 which demonstrates high in vitro NK suppressor activity, presumably representing the virus-induced suppressor cells measured in this assay. At the present time, the mechanism of the resistance of serum-protected spleen cells' antibody production to suppression by FLV-infected splenocytes (i.e., by FLV itself) remains undefined.

Use of adoptive transfer and Winn assay procedures in the further analysis of antiviral acquired immunity in mice protected against Friend leukemia virus-induced disease by passive serum therapy.

Previous studies have demonstrated that spleen cells from DBA/2 mice protected against challenge with a leukemogenic dose of Friend leukemia virus (FLV) by passive administration of xenogeneic antiviral or anti-FLV Mr 71,000 viral envelope glycoprotein antisera can adoptively transfer antiviral resistance to unimmunized irradiated syngeneic recipients. In addition, elimination of T-cells by treatment with anti-Thy 1.2 antibodies plus complement had no effect on the ability of spleen cells from serum-protected mice to adoptively transfer antiviral resistance. We now show that similar depletion of B-cells with rabbit anti-mouse immunoglobulin G plus complement or macrophages by adherence to Sephadex G-10 columns also leaves intact the protective capacity of spleen cells from serum-protected mice. That these results reflect the ability of more than one spleen cell population to transfer antiviral resistance rather than the activity of a non-T, non-B, nonmacrophage cell compartment is supported by the finding that purified splenic T- or B-cells alone from serum-protected DBA/2 mice can adoptively transfer antiviral resistance. Given the previously reported effects of sublethal irradiation on FLV leukemogenesis which could potentially complicate the interpretation of adoptive transfer experiments carried out in this system, analogous studies were performed using a Winn-type assay in which putative effector cells were preincubated with virus before inoculation of the mixture in unirradiated mice. These Winn assay experiments yielded identical results in that serum-protected spleen cells again prevented viral leukemogenesis, and the separate elimination of T-cells, B-cells, or macrophages had no effect on their protective activity. In addition, mixed transfer of serum-protected and normal spleen cells also protected irradiated mice against FLV challenge, providing further evidence that this adoptive protection truly reflects the presence of virus-specific effector cells in the spleens of serum-protected mice and not an inability of these spleen cells to replace radiation-sensitive viral target cells in recipient animals, since these should be supplied by the normal spleen cells in the transferred mixture.

Temperature and cellular regulation of spontaneous cytotoxicity in the shark.

Spontaneous cytolytic activity of shark peripheral blood leukocytes is observed only during periods of decreased environmental temperature (less than 23 degrees C). The effector cell is adherent to glass and is phagocytic. Leukocytes tested during warmer periods (26-31 degrees C) exhibit no spontaneous activity; however, glass-adherent cells isolated from those fish are cytotoxic in vitro, indicating that the effector cell is present at all temperatures. During warmer temperatures, nonadherent cells added to adherent cells were shown to inhibit spontaneous cytotoxicity. This inhibition requires viable cells in contact with the spontaneous cytotoxic population. Thus decreased environmental temperature correlated with spontaneous cytotoxicity, and appears to affect a regulatory cell that is glass nonadherent. In addition, the cytotoxic effector cell is more active at 23 degrees C than 30 degrees C in vitro. These data show that by the time of emergence of the nurse shark, a temperature-dependent mechanism had evolved for cellular regulation of at least one immune function, spontaneous cytotoxicity.

Cell types in peripheral blood of the nurse shark: an approach to structure and function.

Ultrastructural and functional studies were carried out on nurse shark (Ginglymostoma cirratum) peripheral blood cells in order ot identify cells of definitive morphology and specific function. Along with erythrocytes and thrombocytes, four morphologically distinct leucocytes are recognized in peripheral blood: two types of granulocytes, the 'eosiniphil' and the 'granulocyte', and two mononuclear agranulocytic cells, one resembling mammalian macrophage and monocyte, the other resembling mammalian lymphocyte. Also present in peripheral circulation are blast-like cells and mitotic cells. In vitro phagocytosis was demonstrated by the monocyte-macrophage and the granulocyte while thrombocytes, eosinophils and lymphocytes showed no phagocytic activity in the system studied. It is stressed that care must be used in drawing functional analogies between blood cells of a mammal and an elasmobranch on the basis of morphological similarity alone.