Levels of Art2+ cells but not soluble Art2 protein correlate with expression of autoimmune diabetes in the BB rat.

ART2a and ART2b are isoenzymes expressed on the surface of mature T cells and intraepithelial lymphocytes (IELs) in the rat. They exhibit both adenosine diphosphoribosyltransferase and nicotine adenine dinucleotide (NAD) glycohydrolase activities, and both can generate a transmembrane signal that modulates T cell activation. The presence or absence of ART2+ T cells modulates the expression of autoimmune diabetes in the BB rat. ART2 also circulates in a soluble form whose function is unknown. We tested the hypothesis that circulating ART2 protein regulates the expression of autoimmunity. We compared the kinetics, regulation, and source of soluble ART2 in normal rats and in rats with autoimmune diabetes. Basal levels of soluble ART2 varied greatly among strains of rats and were lowest in the diabetes-prone BB (BBDP/Wor) rat. In diabetes-resistant BB (BBDR/Wor) rats, administration of anti-ART2a antibody, which is known to induce diabetes, resulted in transient clearing of soluble ART2a that was followed rapidly by a rebound increase. Repeated treatment of BBDR/Wor rats with anti-ART2a antibody resulted in sustained supraphysiologic levels of soluble ART2a. Although the number of peripheral ART2a+ T cells is known to correlate with the expression of diabetes in BBDR/Wor rats, the level of soluble ART2a protein did not. The source of the soluble ART2 protein in the rat appeared to be the gut. The results suggest that ART2+ T cells and soluble ART2 protein may subserve different immunomodulatory functions.

Switch recombination in a transfected plasmid occurs preferentially in a B cell line that undergoes switch recombination of its chromosomal Ig heavy chain genes.

Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e. g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29 mu), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of S mu-S alpha recombination in the plasmid. Recombination of the plasmid in I.29 mu cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29 mu cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.

Ontogeny and immunohistochemical localization of thymus-dependent and thymus-independent RT6+ cells in the rat.

RT6 is a cell surface alloantigen that identifies a regulatory subset of peripheral T cells in the rat. Diabetes-prone BB rats are deficient in peripheral RT6+ T cells and develop spontaneous autoimmune insulin-dependent diabetes mellitus. Diabetes-resistant BB rats have normal numbers of RT6+ T cells, and insulin-dependent diabetes mellitus can be induced in these animals by in vivo depletion of peripheral RT6+ cells. Athymic rats are also severely deficient in peripheral RT6+ T cells. Although very different with respect to the peripheral RT6+ cell compartment, normal, athymic, and diabetes-prone BB rats all generate RT6+ intestinal epithelial lymphocytes (IELs). The goal of these studies was to analyze the ontogeny of RT6+ IELs and peripheral lymphoid cells by in situ immunohistochemistry. We observed the following. 1) RT6+ IELs appear before alpha(beta) T-cell-receptor- expressing IELs in diabetes-prone BB, diabetes-resistant BB, and athymic WAG rats. 2) In vivo depletion of peripheral RT6+ T cells in diabetes-resistant BB rats using a cytotoxic monoclonal antibody is not accompanied by depletion of RT6+ IELs. 3) A population of RT6+ T-cell-receptor-negative IELs is present in normal, euthymic diabetes-resistant BB rats, constitutes a larger percentage of the euthymic but lymphopenic diabetes-prone BB rat IEL population, and is the predominant IEL phenotype in athymic WAG rats. These results suggest that RT6+ cells are composed of both thymus-dependent and thymus-independent cell subsets that have different developmental characteristics and may differ in function.

The RT6 rat lymphocyte alloantigen circulates in soluble form.

RT6 is a rat maturational lymphocyte alloantigen that appears to subserve important immunoregulatory functions. The lymphopenic diabetes-prone BioBreeding (BB)/Worcester rat is severely deficient in RT6+ T cells and develops spontaneous autoimmune diabetes mellitus. Transfusion of RT6+ T cells prevents the disease. Conversely, in vivo immune elimination of RT6+ T cells from the diabetes-resistant line of BB rats induces diabetes and thyroiditis. RT6 protein is expressed in two allotypic forms, each linked to the cell surface by a phosphatidylinositol (PI) anchor. The mechanism by which RT6+ T cells exert their regulatory function is not known, nor is the function of the RT6 protein defined. In this study, we investigated the possibility that, like other PI-linked proteins, RT6 also exists in a soluble form in the circulation. Using standard biochemical procedures we observed: (i) Soluble RT6 circulates in readily detectable amounts in all rat strains studied. (ii) The diabetes-prone BB rat circulates less RT6.1 than does any other strain, including the coisogenic diabetes-resistant line. (iii) Injections of monoclonal anti-RT6.1 antibody rapidly eliminate soluble RT6 from the circulation of diabetes resistant BB rats. The existence of a soluble form of a protein associated with immunoregulatory T cells suggests the possibility that soluble RT6 itself might possess immunomodulatory properties.

An RT6a gene is transcribed and translated in lymphopenic diabetes-prone BB rats.

T-cells expressing the RT6 surface alloantigen appear to perform important immunoregulatory functions in the rat. Diabetes-prone BB rats lack circulating RT6+ T-cells and spontaneously develop autoimmune diabetes mellitus and thyroiditis. The coisogenic diabetes-resistant BB rat does circulate RT6+ T-cells and is free of disease. Transfusions leading to engraftment of RT6+ T-cells prevent both diabetes and thyroiditis in the diabetes-prone rat. To investigate the absence of this subset in the lymphopenic BB rat, we used both molecular and biochemical procedures and made the following observations: 1) an mRNA encoding RT6 protein is present in diabetes-prone spleen cells; 2) nucleotide sequencing of this transcript reveals an intact coding sequence for the RT6.1 alloantigen; 3) sensitive chemiluminescent assay of diabetes-prone lymph node cell detergent extracts shows that diabetes-prone RT6 mRNA is translated in vivo; 4) quantitatively, diabetes-prone lymph node cells express < or = 10% of the RT6.1 protein found on similar numbers of diabetes-resistant BB cells; and 5) finally, we obtained evidence of an intact phosphatidylinositol linkage of the molecule to the cell surface and successfully immunoprecipitated the phosphatidylinositol-linked protein with DS4.23 monoclonal antibody, indicating that the RT6.1 antigen is correctly processed and folded in diabetes-prone lymph node cells. We conclude that the near total absence of RT6+ T-cells in the diabetes-prone BB rat is unlikely to be because of a defect in RT6 gene expression per se. Defects in RT6 gene regulation or other cellular defects leading to premature cell death in the T-cell lineage, alone or in combination, may instead be responsible.

Neonatal tolerance induction to Mlsa. II. T cells induce tolerance to Mlsa.

We have investigated the ability of murine T cell lines to induce neonatal tolerance to Mlsa (minor lymphocyte stimulating). Mlsb mice were injected within 24 hr of birth with MHC (major histocompatibility complex) identical T cell lines generated by culturing responders from Mlsa strains with stimulators from Mlsb strains. Injected mice were tested at 6 to 8 weeks of age for responses in either primary mixed leukocyte reaction or IL-2 limiting dilution analysis. Mlsa specific responses by injected tolerant mice relative to noninjected controls were reduced by 92-98% in MLR and by 2- to 10-fold in IL-2 LDA. In contrast, responses against third-party MHC antigens by either the injected or the noninjected mice were identical. Fifty percent of all mice injected with the T cell lines were tolerant to Mlsa. These results strongly suggest that murine T cells express the Mlsa gene product.

Neonatal tolerance induction to Mlsa. I. Tolerance to Mlsa is restricted by shared MHC determinants.

In this paper we have examined the influence of MHC (major histocompatibility complex) on neonatal tolerance to Mlsa (minor lymphocyte stimulating). By employing a novel approach we have shown that tolerance to Mlsa is restricted by shared MHC determinants. Thus, neonatal Mlsb mice, injected at birth with spleen cells from Mlsa mice, were tested as adults for Mlsa specific responses by interleukin-2 limiting dilution analysis, a technique which allows us to discriminate between responses to MHC + Mlsa and to MHC alone. Tolerance to Mlsa was in the context of any MHC type examined--donor, host, and third-party MHC products. These results show that tolerance to Mlsa is restricted by shared MHC determinants and extend previous studies indicating that activation of Mlsa responses is similarly restricted.

Quantitation of beta-lipoprotein in cord serum by rate nephelometric immunoassay: a potential screening test for familial hypercholesterolemia.

We measured beta-lipoprotein in 232 cord sera by rate nephelometry and compared the results with those obtained by electroimmunoassay. The mean (mean +/- two standard deviations) was 544 (293-1009) mg/L by rate nephelometry and 503 (265-955) mg/L by electroimmunoassay. The between-assay correlation coefficient was 0.891. Between-run coefficients of variation were 3.5-4.4% for nephelometry and 11-14% for electroimmunoassay. Nephelometry was faster, more precise, and less labor intensive than electroimmunoassay. For these reasons, rate nephelometry appears to be a more suitable assay system for mass screening, should detection of familial hypercholesterolemia prove feasible and desirable.