Development of neoplastic phenotype following transfection of HNF cells with sarcoma DNA

DNA isolated from chondrosarcoma cells effectively transformedNIH-3T3 cells and human foreskin fibroblasts. The transfectedNIH-3T3 cells, directly implanted three or four passages later,formed progressively growing tumors ( 2.0 cm in diameter) subcutaneouslyin nude mice. No metastasis was evident upon pathological examinationof the tumor bearing mice. Transfected human foreskin fibroblaststhat exhibited anchorage independent growth formed only smalltumors in nude mice (<0.6 cm in diameter). The transfectedhuman cells which exhibited anchorage independent growth reactedwith the monoclonal antibody 345.134S, specific for an epitopeexpressed by human sarcoma cells. The transfected NIH-3T3 cellsdid not exhibit reactivity with the same monoclonal antibody.Southern blot analysis of the DNA prepared from the transfectedNIH-3T3 cells, that developed as a progressively growing tumorin a nude mouse, revealed the presence of human repetitive DNAsequences.     

HLA-D restriction of antigen-specific proliferative T cell responses

The specificity of the inhibitory activity of anti HLA-DR antisera on the proliferative response of human T cells to soluble antigens has been recently challenged. Furthermore, there are conflicting reports about the effect of antisera to the stimulatory antigen. Therefore, we investigated the inhibitory activity of different antisera on the proliferative response of T lymphocytes from sensitized donors to the antigens HSV and PPD. Alloantisera to DR specificities shared between antigen presenting cells (APC) and T cells displayed a strong inhibitory activity: alloantisera to HLA-DR specificities expressed only by APC or the T cell donor displayed lower or no inhibitory activity. Monoclonal antibodies to monomorphic determinants of HLA-DR molecules were inhibitory, but only when used at a high concentration. Antisera to HLA-ABC molecules and to HSV displayed little if any inhibition. These findings provide further evidence that the HLA-DR molecules as such in the APC membrane, which were also present during initial sensitization, are restriction elements for T cells.     

Loss of LMP and TAP expression in human melanoma cells

The goal of this study was to determine the frequency of LMP2, LMP7, TAP1 and/or TAP2 loss in melanoma cell lines and in surgically removed melanoma lesions. Cell extracts from control and IFN-γ treated melanoma cell lines were tested in Western blotting with antisera elicited with LMP2, LMP7, TAP1 and TAP2-specific peptides. LMP2 and LMP7 were not detected in 7 out of 9 cell lines. Expression of both LMP subunits was induced in 5 of the 7 negative cell lines by treatment with IFN-γ. TAP1 and TAP2 were not detected in 3 out of 9 cell lines and were induced in one of them by IFN-γ. Surgically removed lesions were stained in the immunoperoxidase reaction with antibodies purified from the antisera by affinity chromatography on the immunizing peptides. LMP2 and LMP7 were not detected in 20% and 6%, respectively, of 32 primary lesions and in 40% and 12%, respectively, of 25 metastatic lesions. TAP1 and TAP2 were not detected in 15% and 18%, respectively, of 32 primary lesions and in 20% and 24%, respectively, of 25 metastatic lesions. Moreover, the frequency of TAP loss is increased in primary lesions from patients with recurrence of their disease, suggesting a potential role in the course of the disease and a negative impact on the outcome of T cell-based immunotherapy.     

Antibody-coated protein A-bearing Staphylococcus aureus: a versatile and stable immune reagent.

The human beta2-microglobulin antigen-antibody system was used as a model to illustrate the versatility of a microradioimmunoassay technique using protein A-bearing Staphylococcus aureus Cowan I strain (SACI) bacteria as a non-specific immunoadsorbent in place of a second antibody. Experimental conditions are described for a sensitive microassay which makes it possible to process large numbers of samples more rapidly and with minimum handling. Furthermore, SACI coated with specific antibodies by mixing with unfractionated antisera are a versatile reagent not only for radioimmunoassays but also for use as molecular probes to characterize cell surface antigens. Antibody-coated SACI could be lyophilized and proved extremely stable in storage thus providing a unique advantage for use in binding inhibition assays and as versatile reagent for clinical and investigative immunology.     

Structural studies of murine I-E and human DR antigens.

The structures of murine I-E antigens from two strains of mice were compared to each other and to human DR antigens. Murine and human antigens were isolated by using allo- and xenoantiserum, respectively, and purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The murine I-E and human DR antigens consist of two polypeptide chains designated α and β. The E_α and DR_α chains display a high degree of amino acid sequence homology as do the E_β and DR_β chains, provided a gap is inserted at position 1 of the DR_β chain. Comparison of N-terminal sequences reveals several differences between the β chains of I-E antigens from the two strains of mice. In contrast no sequence differences between the two α chains are observed. In addition, comparison of tryptic peptides examined by isoelectrofocusing reveals several differences between the two E_β chains, but not between the two E_α chains. Thus, the polymorphism of murine I-E antigens and by analogy human DR antigens, may result from structural differences in the smaller (β) chain.     

The monoclonal antibody AC1.59 defines a new polymorphic determinant on HLA-DR molecules

2-dimensional gel electrophoresis analysis and sequential immunoprecipitation studies have shown that the monoclonal antibody (MoAb) AC1.59 recognizes a gene product of the HLA-DR locus. The determinant defined by the MoAb AC-1.59 has an unusual distribution on HLA-DR allospecificities, since it is expressed by HLA-DR1, DRw8, DRw9 antigens and by subtypes of HLA-DRw6 and HLA-DR4 antigens. In the latter allospecificity the expression of the MoAb AC1.59 defined determinant correlates with the cellularly defined HLA-Dw4 alloantigen. The present investigation provides evidence of serological heterogeneity within HLA-DR alloantigens. Furthermore these results suggest that monoclonal antibodies can sharpen the discriminatory power of serological assays to dissect the heterogeneity and complexity of HLA-DR alloantigens.     

Inhibition by anti-HLA class II monoclonal antibodies of monocyte-dependent T cell proliferation induced by monoclonal antibody OKT3

The inhibitory effect of monoclonal antibodies (mAb) to monomorphic (locus-restricted and locus-shared) and polymorphic determinants of HLA class II antigens on the monocyte-dependent proliferation of T cells stimulated with mAb OKT3 has been studied. The effect appears to be specific, dose dependent, is not mediated by the Fc portion of mAb and reflects their interaction with the corresponding determinants. The anti-HLA class II mAb do not have to be present in the culture throughout the incubation period, but are essential in early phases of mAb OKT3 T cell activation. Both monocytes and T cells are the targets of the inhibition exerted by the anti-HLA class II mAb. Their inhibitory effect involves several steps in the sequence of events which leads to T cell proliferation, including interleukin (IL) 1 and 2 secretion, and IL2 receptor expression.     

Syngeneic antiidiotypic monoclonal antibodies to the murine anti-HLA-DR,DP monoclonal antibody CR11-462

Two out of 625 hybridomas constructed with splenocytes from a Balblc mouse immunized with the murine anti-HLA-DR, DP monoclonal antibody CR11-462 secrete antiidiotypic antibodies. Binding assays with a panel of 12 anti-HLA class I, 14 anti-HLA class II, and 9 anti-human melanoma-associated antigen MoAbs showed that the idiotopes recognized by MoAb F3-C25 and F3-B6 are not expressed by any of the tested antibodies, even those which inhibit the binding of MoAb CR11-462 to cultured B lymphoid cells. Crossblocking experiments showed that the two idiotypes are distinct and spatially distant. Both idiotopes require the association of heavy and light chain of MoAb CR11-462 for their expression. The idiotope recognized by MoAb F3-B6 is an idiotope, since it is located outside the antigen combining site of MoAb CR11-462. The idiotope recognized by MoAb F3-C25 is a γ idiotope, since it is antigen inhibitable but does not posses an internal image of the antigen. Interestingly the MoAb F3-C25 displays a differential inhibitory effect on the binding of MoAb CR11-462 to lymphoid cells that express different HLA phenotypes or share the HLA-DRw6 allospecificity. These results suggest that antiidiotypic antibodies may sharpen our ability to dissect the heterogeneity of HLA class II antigens.