Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Identification and characterization of engrafted human cells in human/goat xenogeneic transplantation chimerism

We have injected human CD34+lin- cells derived from cord blood (CB) into the goat fetuses via in utero at 45-55 days gestation under guidance of B-scan ultrasonograph. Sixty out of 68 fetuses injected survived to full term. The long-term survival of the human cells in transplant goat has been tested by various experimental methods, including FACS analysis, real-time PCR, RT-PCR, Southern-blot hybridization, FISH, as well as immunohistochemical assays. All the 60 transplant goats demonstrated engrafted human cells, including myeloid, B-lymphoid, and erythroid lineages. The yield of the human CD34+ cells varied, but was not linked with sex and age. High numbers of human cells could be detected for at least 16 months after birth. Immunohistochemical analyses revealed that the human cells were present not only in blood but also in other tissues, such as liver, of the transplant goats. In addition, a human-specific serum albumin and the hepatocyte nuclear factor (hHNF-3beta) mRNAs specific to human hepato-antigen could be readily detected in the livers of the transplant goats. Our results demonstrate that this in utero xenograft model should be useful for expansion of human HSC and possibly for the evaluating the effectiveness of prenatal treatment of human genetic diseases.     

Colony formation by human fibroblasts: Inhibition by multitransfusion and multiparous antisera

Human isoantisera from various sources were evaluated as potential selective agents for human diploid fibroblasts. Antisera inhibiting colony formation of some fibroblast strains were obtained from among subjects receiving multiple transfusions and also from multiparous women. Of 18 multitransfusion sera tested, 6 inhibited colony formation by some strains of a test panel of fibroblast strains. Thirty sera from multiparous women were tested for colony-inhibiting antibody against fibroblast strains from their offspring; two were positive. Antiserum fractionated by density gradient centrifugation contained colony-inhibiting activity in both the IgG and the IgM fractions. Multiparous antiserum reacted with a smaller proportion of tests strains than any of the multi-transfusion sera, suggesting that it had fewer antibody specificities. From studies of survival of mixtures of cells from an antiserum-sensitive and an antiserum-resistant strain in the presence of antiserum, it appeared that human isoantisera would be effective selective agents for human diploid fibroblasts.Supported by a grant (5R01 GM15883) and a Research Career Development Award (1K3 HD31, 721) from the National Institutes of Health.     

Isolation and characterization of murine Ia antigens

The isolation and characterization of Ia antigens from both lymphoid and nonlymphoid cells was attempted by SDS-polyacrylamide gel electrophoresis of radiolabeled, NP-40 solubilized, and anti-Ia precipitated lysates. The profiles obtained indicate that membrane proteins with a molecular weight of approximately 30,000 can be isolated from peripheral B but not from peripheral T cells. Ia antigens cannot be immunoprecipitated from cortisone-resistant thymocytes, total thymocytes, allogeneically activated T cells, Con A stimulated T cells, and anti-Ig immunoadsorbent purified T cells. Ia antigens seem to comprise only 1%–2% of labeled splenic intracellular and membrane-associated proteins. They differ from H-2 antigens and immunoglobulin H and L chains with respect to size and serological reactivity. Ia antigens cannot be found to be secreted from lymph node cells or splenocytes into the extracellular incubation media. Tissue distribution studies indicate that Ia antigens are present on macrophages, fetal liver cells, epidermal cells, and bone marrow cells. They have not been found on such tumor cells as myelomas, teratomas, and lymphocytic leukemias.     

Idiotype-specific inactivation by xenogeneic antisera of T killers but not of T-MIF producers immune to H-2 complex antigens

Following appropriate absorption rabbit serum to CBA anti-C57BL/6 lymph node cells (rabbit ISS) and rat serum to enriched CBA anti-C57BL/6 T killers (rat ISS) selectively inhibited the activity of K-anti-b T killers but did not affect T killers of other specificities (K-anti-d, d-anti-b). Rabbit ISS did not suppress the capacity of CBA anti-C57BL/6 (K-anti-b) T cells to produce MIF. Under the same conditions, anti-Thy-1.2 serum inactivated killers and K-anti-b MIF-producers. The findings indicate that the killers and MIF-producers immune to the antigens of H-2 complex constitute different subpopulations of T cells, which appear to carry nonidentical sets of idiotypes.     

Envelope proteins of human T cell leukemia virus type I: characterization by antisera to synthetic peptides and identification of a natural epitope

Three peptides corresponding to selected regions of the env gene products of human T cell leukemia virus type I were synthesized by solid-phase Merrifield techniques. The sequence of peptide designated SP-65 was identical to the predicted C-terminal 12 residues of the transmembrane protein p21env, and peptide SP-74 was inferred from a region shown to be highly conserved among mammalian retroviruses. The third peptide, SP-70, was derived from a C-terminal region of the surface glycoprotein gp46. Antibodies to each peptide were raised in rabbits and were used to identify and further characterize the proteins coded by the env gene. Despite being present at very low levels in purified viral preparations, these proteins were chromatographed by reverse-phase high pressure liquid chromatography and were located by Western blot analysis of the column fractions. Anti-SP-70 recognized the surface glycoprotein (gp46) and also its C-terminal cleavage fragment (gp16). Anti-SP-65 and anti-SP-74 both reacted with the hydrophobic transmembrane protein (p21) and provided evidence that this protein does not undergo apparent C-terminal processing during viral maturation, unlike the trans-membrane protein of murine leukemia virus. As expected, anti-SP-74 also reacted with homologous proteins from other Type C and Type D viruses, confirming that peptide SP-74 corresponds to a broadly conserved region of retroviral transmembrane proteins. SP-70, which is predicted to be quite near the C terminus of the major surface glycoprotein, was also reactive with sera of HTLV-I-positive patients, indicating that this peptide corresponds to, or is part of, a native epitope recognized by the natural host.     

Preparation and characterization of antisera to the myelin-associated glycoprotein

Rabbits were immunized with the myelin-associated glycoprotein (MAG) that had been purified from isolated rat brain myelin by selective extraction with lithium diiodosacicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. Antibodies to MAG were detected qualitatively by immunodiffusion and quantitatively by a double antibody assay utilizing [³H]fucose-labeled MAG as antigen. The antisera were capable of precipitating between 300 and 500 g of MAG/ml of serum under the conditions of the assay. Preincubation of the anti-MAG serum with other glycoproteins or glycolipids did not inhibit the precipitation of labeled MAG. Similarly, preincubation of the antiserum with LIS-phenol extracts of non-neural tissues did not inhibit the immune precipitation of MAG. The specificity of the antiserum was also indicated by the selective double antibody precipitation of MAG from solubilized whole myelin that contained a heterogeneous mixture of [³H]fucose-labeled glycoproteins. The antibodies to MAG were not effectively absorbed by whole brain homogenate or purified myelin, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification. Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Early recognition of a discordant xenogeneic organ by human circulating lymphocytes.

Cell-mediated immune mechanisms underlying discordant xenograft rejection are poorly characterized. In our study, using a human to rat xenogeneic ex vivo model, we show that a fraction of human lymphocytes, when perfused through the coronary system of a rat heart, rapidly and specifically adheres to the vascular endothelium and infiltrates the myocardium. Lymphocyte phenotypic analysis before and after perfusion, as well as the use of purified cell subpopulations, demonstrate preferential adhesion of CD3- CD16+ NK cells. NK cell adhesion occurs via xenoreactive antibody-dependent and -independent pathways, because the selective removal of human IgG from the perfusion buffer markedly reduces but does not completely abrogate NK cell sequestration. However, T lymphocytes are retained in the xenoorgan via an antibody-independent pathway, as assessed by the lack of influence of IgG removal. Leukocyte integrins appear to play a crucial role in mediating adhesion of both lymphocyte subsets, because the pretreatment of lymphocytes with anti-CD11a, anti-CD11b, and anti-CD18 antibodies markedly reduces their retention into the xenogeneic organ. Retained human lymphocytes mediate rapid and direct damage of the xenoorgan, as demonstrated by histologic and functional alterations of the endothelium, impaired vascular resistance and in vitro lysis of rat endothelial cells by human NK cells. Taken together, these findings suggest a role for cell-mediated mechanisms in the rapid recognition and rejection of vascularized xenografts.     

Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Immune cytolysis of human tumor cells mediated by xenogeneic "immune" RNA

Normal, nonimmune, human peripheral blood lymphocytes, when incubated with RNA extracted from lymphoid organs of guinea pigs or sheep immunized with human tumor cells, mediated the immune cytolysis of those tumor cells in vitro. Lymphocytes incubated without RNA, or with control RNA preparations, failed to evidence cytotoxic activity. Treatment of the active RNA preparations with ribonuclease abrogated the cytotoxic activity, but treatment with deoxyribonuclease or pronase did not effect activity.     

Specific antisera and immunological procedures for characterization of methanogenic bacteria.

Specific antisera were raised in rabbits to 19 methanogenic bacteria representing the species available in pure culture at the present time. The antisera were characterized, labeled, and organized in a bank to serve as a source of material for preparation of antibody probes and thus provide standardized reagents for immunological analysis of methanogens. An indirect immunofluorescence procedure was standardized for optimal staining of homologous and heterologous bacterial strains. Two immunoenzymatic assays were developed: (i) a simple slide assay, useful for rapid antibody detection in small samples, antibody titrations, and disclosure of cross-reactions among methanogens, and (ii) a quantitative method. The latter is useful for quantification of antigenic relatedness. Procedural details were developed to obtain optimal bacterial preparations for use as immunogens to raise antibodies in vivo, and as antigens for antibody assay in vitro.     

A novel xenogeneic co-culture system to examine neuronal differentiation capability of various adult human stem cells

Background

Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions.

Methods and Findings

This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal differentiation in stem cells could be found in the media supernatants of the co-cultures.

Conclusions

The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications.     

Protection of xenogeneic cells from human complement-mediated lysis by the expression of human DAF, CD59 and MCP

CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.     

The induction of TNP-altered, self-reactive human cytotoxic T cells by soluble factors: the role of Ia antigens.

This report demonstrates that human peripheral blood T lymphocytes, triggered by allogeneic cells or soluble antigens, elaborate helper factor(s) that promotes the in vitro differentiation of TNP altered-self reactive human CTL. Helper factor(s) alone is not sufficient for the generation of these killer cells, but requires the presence of TNP-derivatized autologous stimulators during sensitization. Additional experiments were performed with antisera to a human Ia-like antigen, p23, 30. These studies indicate that human Ia-like antigens play an important role in both the induction of helper factor(s) and in the functional activity of preformed helper factor(s) molecules.