Antigenicity of human melanoma cells transfected to express the B7-1 co-stimulatory molecule (CD80) varies with the level of B7-1 expression.

The aim of this study was to compare the antigenicity of human melanoma cells molecularly modified by particle-mediated gene transfer to have transient or stable expression of the B7-1 co-stimulatory molecule (CD80). The unmodified melanoma cells (mel5, m21) had no constitutive expression of B7-1, but 22%-28% of cells had transient B7-1 expression 24 h following transfection with cDNA for B7-1 (mel5-B7, m21-B7). In addition, 85%-90% of cells had stable B7-1 expression following transfection with cDNA for B7-1 and in vitro culture under selection conditions (mel5-B7neo, m21-B7neo). Allogeneic HLA-unmatched normal donor peripheral blood mononuclear cells (PBMC) secreted greater amounts of granulocyte/macrophage-colony-stimulating factor (GM-CSF) when incubated for 3 days with m21-B7neo than did PBMC incubated with m21-B7, which, in turn, secreted greater amount of GM-CSF than PBMC incubated with m21. Similarly, cell-mediated cytotoxicity against unmodified melanoma cells by PBMC co-cultured for 5 days with the modified or unmodified melanoma cells was proportional to the level of B7-1 expression on the stimulating cells. This cytolytic activity had both an HLA-class-I-restricted and an HLA-class-I-unrestricted component. Following 5 days of co-culture, PBMC expression of CD28, the ligand for B7-1, was down-regulated in proportion to the level of B7-1 expression on the stimulating melanoma cells. Thus, particle-mediated gene delivery of cDNA for B7-1 into human melanoma cells increased expression of functional B7-1 and enhanced the antigenicity of the gene-modified cells in proportion to their level of B7-1 expression.

Dual expression of human leukocyte antigen molecules and the B7-1 costimulatory molecule (CD80) on human melanoma cells after particle-mediated gene transfer.

The aim of this study was to determine if human melanoma cells could be molecularly modified by particle-mediated gene transfer with a "gene gun", using genes for interferon-gamma (IFN-gamma), the B7-1 costimulatory molecule (CD80), and human leukocyte antigen (HLA)-A2, to augment expression of both HLA molecules and B7-1. Established and early passage melanoma cells transfected with human IFN-gamma complementary DNA (cDNA) produced IFN-gamma (50-5,000 pg/mL). The biological effect of this IFN-gamma transgene included an upregulation, or de novo appearance, of HLA expression. These melanoma cells had no detectable baseline surface expression of the B7-1 costimulatory molecule, but 8% to 31% of these cells became B7-1 positive with no selection procedure after gene transfer with human B7-1 cDNA. After combination gene transfer with cDNAs for both IFN-gamma and B7-1, 9% to 33% of these cells expressed both HLA-DR and B7-1. In combination gene transfer experiments with cDNAs for both HLA-A2 and B7-1, dual expression of HLA-A2 and B7-1 was achieved in 10% to 17% of the melanoma cells. Thus, the molecular modification of human melanoma cells to increase expression of both HLA and B7-1 can be achieved by particle-mediated gene delivery and presents a promising strategy to stimulate antimelanoma T-cell immunity. Key words: Melanoma; T cells; B7-1 costimulatory molecule (CD80); major histocompatibility complex.

Human genes for the immunoglobulin gamma-chain (Gm) allotypes and for the T cell receptor alpha-chain are distantly linked or unlinked.

Since the genes for two of the T cell receptor (TCR) chains (alpha and delta) and for the immunoglobulin (Ig) heavy chains (alpha, gamma, delta, epsilon and mu) are all on the long arm of human chromosome 14, it is of interest to determine if they are genetically linked and, if so, how strongly. We tested genetic polymorphisms of the TCR alpha-locus and Ig gamma-locus in 19 families comprising 107 members, and asssessed the frequency of recombination between the two loci. The results rule out strong linkage: all recombination fractions below 0.26 gave lod scores less than -2 (odds less than 1:100). Combining the data on maternal and paternal meioses, the most likely recombination fraction was 0.5 (nonlinkage).

HLA-DQA2 (DX alpha) polymorphism and insulin dependent diabetes.

A certain HLA-DQA2 locus TaqI fragment, DX alpha"U", has been reported to be associated with insulin-dependent diabetes mellitus (IDDM). Reports of various studies in this vein have ranged from stating that the association of DQA2"U" with IDDM exists even among subjects positive for HLA-DR3 and -DR4 to stating that the association of DQA2"U" with diabetes can be attributed to linkage disequilibrium between the DQA2"U" and some component(s) on the affected haplotypes. Using a synthetic 97-base probe corresponding to a portion of an intron of DQA2, in a Southern blot analysis of IDDM and control subjects from Wisconsin, we were able to confirm the association of DQA2"U" with diabetes. However, among DR3 subjects there was no significant association between DQA2"U" and diabetes (p = 0.26). Although there was a (nonsignificant) association of IDDM with DQA2"U" among DR4-positive subjects (p = 0.14), this can be completely attributed to linkage disequilibrium between DQA2"U" and DQw8. We also sequenced most of the second exon (corresponding to the alpha 1 domain of the DQA2 glycoprotein) from five individuals that were homozygous for either DQA2"U" or DQA2"L." The only polymorphisms observed were a "silent" mutation at position 36 and one example of a difference that would result in a change of amino acid at position 41.

Allelic T-cell receptor alpha complexes have little or no influence on susceptibility to type 1 diabetes.

We performed a multiple-affected-sib study to determine if T-cell receptor alpha-chain alleles affect susceptibility to insulin-dependent diabetes mellitus. Restriction fragment length polymorphisms were used to follow the segregation of allelic T-cell receptor alpha complexes within the families. The segregation of T-cell receptor alpha alleles in 29 multiplex families revealed no significant tendency for affected sibs to share T-cell receptor alpha-chain alleles more often than would be expected by chance alone (p greater than 0.2). In contrast, the same type of analysis for HLA alleles easily detected the well-known linkage of insulin-dependent diabetes mellitus susceptibility to the HLA complex (p = 0.003). We suggest that the importance of HLA alleles in insulin-dependent diabetes mellitus susceptibility and the lack of importance of T-cell receptor alpha alleles result from the different strategies by which HLA and T-cell receptor molecules achieve antigen-binding diversity: multiple loci and allelic diversity in the case of HLA; combinatorial, junctional, and N-region diversity in the case of the T-cell receptor. In this paper we also describe three new restriction fragment length polymorphisms of the T-cell receptor alpha complex and a new method for testing the significance of linkage in multiple-affected-sib studies.

Interaction of insulin-like growth factor II with rat chondrocytes: receptor binding, internalization, and degradation.

We have characterized the interaction of insulin-like growth factor II (IGF-II) with its plasma membrane receptor(s) on cultured rat chondrocytes. Our studies, paralleling those already reported for IGF-I, demonstrate that [125I]IGF-II binds to these receptors with a high degree of affinity and that this process is reversible, specific, and time, temperature, and concentration dependent. At 4 C, unlabeled IGF-II causes half-maximal displacement of the labeled ligand at a concentration of 22 ng/ml, whereas IGF-I is approximately 1/200th as potent, and insulin does not displace [125I]IGF-II even at a concentration of 10 micrograms/ml. Maximum binding to chondrocytes (44% of added radioactivity) occurred after 4-5 h of incubation at 15 C. Compared to [125I]IGF-I binding, this value is 7-fold higher and is consistent with an affinity constant (Ka = 3.8 X 10(8) M-1) approximately 1 order of magnitude greater. Photoaffinity labeling studies disclose that IGF-II binds primarily to the type II IGF receptor, which has an apparent mol wt of 220K when electrophoresed under nonreducing conditions and 270K under reducing conditions. Nanomolar concentrations of IGF-II stimulated the synthesis of DNA and RNA in a dose-related manner, and micromolar concentrations of insulin demonstrated an additive effect with respect to the incorporation of [3H]thymidine into DNA, but not [3H]uridine into RNA. Preincubation of rat chondrocytes with increasing concentrations of insulin caused a marked dose-related increase in [125I]IGF-II binding, a phenomenon previously reported in several other cell types. In addition to defining the binding characteristics of IGF-II, we used the lysosomotropic agents chloroquine and ammonium chloride to demonstrate that its ligand-receptor complex, like that of IGF-I, is internalized and degraded partially via the lysosomal pathway.

Nutritionally-induced changes in hepatic insulin-like growth factor I (IGF-I) gene expression in rats.

We have used [32P]-labeled human and mouse IGF-I cDNA probes to identify three hepatic IGF-I transcripts (8.0, 1.8 and 1.1 kilobase) in rats and to quantify nutritionally-induced changes. During fasting, the major (8.0 kilobase) transcript decreased progressively after 6 h, and at 30 h was only 39% as abundant as in the nonfasted control. Refeeding animals fasted 24 h caused a marked rise in the 8.0-kb mRNA by 6 h, and an 18-fold increase at 30 h. The two minor transcripts also appeared to behave in a similar manner. Serum IGF-I concentrations paralleled levels of the major hepatic transcript during periods of both fasting and refeeding, verifying the central role of nutrition in growth regulation.

Interaction of insulin-like growth factor I/somatomedin-C with cultured rat chondrocytes: receptor binding and internalization.

We have characterized the interaction of insulin-like growth factor I/somatomedin C (IGF-I/Sm-C) with its plasma membrane receptors on cultured rat chondrocytes. Our studies have demonstrated that [125I]IGF-I/Sm-C binding to these receptors is a relatively specific, reversible, and time-, temperature-, pH-, and concentration-dependent process. Insulin displaces [125I]IGF-I/Sm-C from its receptors on rat chondrocytes, but with a potency only 10(-4) that of IGF (I and II). Using the known lysosomotropic agents chloroquine and ammonium chloride as well as the substituted diamine monodansylcadaverine, we have shown that this 125I-labeled Sm is internalized and partially degraded via the lysosomal pathway. These conclusions have been further supported by photoaffinity labeling studies which, surprisingly, demonstrate that the predominant IGF receptor on chondrocytes is the type II receptor, and that [125I]IGF-I/Sm-C is bound primarily in this ligand-receptor complex which is internalized and degraded, in part, by lysosomes.

Calcium-activated, phospholipid-dependent protein kinase activity and protein phosphorylation in HL60 cells induced to differentiate by retinoic acid.

Treatment of human promyelocytic (HL60) cells with retinoic acid for at least 48 h causes differentiation to more mature myeloid forms. Prior to commitment of cells to the myeloid pathway there is a marked increase in cytosolic calcium-activated, phospholipid-dependent protein kinase activity. This increase does not result from an intracellular redistribution of the enzyme. Concomitant with the increased enzyme activity there is enhanced phospholipid-dependent phosphorylation of proteins of 29, 49, 52, 58, 68, 69, 120, 170, 200 and 245 kDa.

Decreased insulin binding and degradation associated with depressed intracellular ATP content.

The effect of metabolic inhibitors, 2,4-dinitrophenol (DNP) and NaF, on insulin binding and degradation has been studied in cultured Buffalo rat liver (BRL) cells. In control studies, 1.8 fmol of 125I-insulin binds to 1.2 x 10(6) cells, possessing approximately 40,000 receptor sites per cell with binding affinity of 5.52 x 10(-8) M. When the cells were preincubated with increasing concentrations of either DNP or NaF, a dose- and time-dependent decrease in both insulin binding and degradation was observed. The total amount of 125I-insulin bound to BRL cells preincubated with metabolic inhibitors was reduced to 1.2 fmol per 1.2 x 10(6) cells. The point of 1/2 B max was achieved in the presence of 50 ng/ml of native insulin, 1.7 times that of the control level. The number of receptor sites was unaffected by either DNP or NaF, but an average affinity profile revealed a decrease in the affinity of the ATP-depleted cells for insulin (KD: 7.31 x 10(-8) M and 7.06 x 10(-8) M in DNP- and NaF-treated cells, respectively). The decrease in insulin binding and degradation following the exposure of the BRL cells to the metabolic inhibitors was associated with a 20% reduction in intracellular ATP and adenylate energy charge. DNP and NaF did not affect the equilibrium constant for the myokinase catalyzed reaction and the intracellular concentration of hypoxanthine was stable, confirming the integrity of the cells during the experiments. It is suggested that ATP levels must remain intact to maintain normal insulin receptor affinity. Furthermore, the rate of insulin degradation by ATP-depleted cells is slower than that of intact cells. It is conceivable that the depression of insulin degradation by partially ATP-depleted cells results from either diminished binding or decreased endocytosis and lysosomal activity, all of which appear to be energy dependent.