Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200.

BACKGROUND AND OBJECTIVE: Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro. METHODS AND RESULTS: CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses. CONCLUSION: These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.

Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Positive control of yeast meiotic genes by the negative regulator UME6.

The yeast meiotic activator IME1 stimulates transcription of many early meiotic genes. These genes share a 5' sequence called URS1. URS1 sites function as repression sites in cells that lack IME1; we show here that URS1 sites are weak activation sequences in cells that express IME1. Repression through URS1 sites is known to depend upon the URS1-binding protein UME6. We have identified a UME6 allele (previously called rim16-12) that causes a defect in IME1-dependent activation of meiotic genes but not in repression through URS1 sites. In contrast, a ume6 null mutation causes defects in both IME1-dependent activation and in repression through URS1 sites. A LexA-UME6 fusion protein is an IME1-dependent transcriptional activator, whereas a LexA-UME6 fusion carrying the rim16-12 substitution cannot activate transcription. These findings argue that IME1 activates meiotic genes by converting UME6 from a negative regulator to a positive regulator; the rim16-12 mutant protein is defective in conversion to a positive regulator.

Analysis of RIM11, a yeast protein kinase that phosphorylates the meiotic activator IME1.

Many yeast genes that are essential for meiosis are expressed only in meiotic cells. Known regulators of early meiotic genes include IME1, which is required for their expression, and SIN3 and UME6, which prevent their expression in nonmeiotic cells. We report here the molecular characterization of the RIM11 gene, which we find is required for expression of several early meiotic genes. A close functional relationship between RIM11 and IME1 is supported by two observations. First, sin3 and ume6 mutations are epistatic to rim11 mutations; prior studies have demonstrated their epistasis to ime1 mutations. Second, overexpression of RIM11 can suppress an ime1 missense mutation (ime1-L321F) but not an ime1 deletion. Sequence analysis indicates that RIM11 specifies a protein kinase related to rat glycogen synthase kinase 3 and the Drosophila shaggy/zw3 gene product. Three partially defective rim11 mutations alter residues involved in ATP binding or catalysis, and a completely defective rim11 mutation alters a tyrosine residue that corresponds to the site of an essential phosphorylation for glycogen synthase kinase 3. Immune complexes containing a hemagglutinin (HA) epitope-tagged RIM11 derivative, HA-RIM11, phosphorylate two proteins, p58 and p60, whose biological function is undetermined. In addition, HA-RIM11 immune complexes phosphorylate a functional IME1 derivative but not the corresponding ime1-L321F derivative. We propose that RIM11 stimulates meiotic gene expression through phosphorylation of IME1.

Isolation and characterization of monoclonal antibodies to a recombinant human topoisomerase II polypeptide.

We have produced two murine monoclonal antibodies (SWT3D1 and SWR1C2) to a recombinant polypeptide corresponding to the carboxyl-terminal one-third (amino acid 854-amino acid 1447) of human topoisomerase II alpha. Each antibody is able to recognize intact human topoisomerase II using immunoblotting and enzyme-linked immunosorbent assay (ELISA) techniques. Data is presented demonstrating that the antibodies bind specifically to topoisomerase II alpha but do not interact with topoisomerase II beta. The monoclonal antibodies do not recognize murine or calf thymus topoisomerase II indicating that each may bind exclusively to the human enzyme. The topoisomerase II binding sites for each monoclonal antibody have been compared in a competition ELISA. The SWT3D1 antibody had no significant effect on the binding efficiency of biotinylated SWR1C2 antibody. Although SWR1C2 was capable of inhibiting the binding of biotinylated SWT3D1, this only occurred at concentrations approximately 1000-fold higher than those required of SWT3D1 to block binding of itself. These results suggest that SWT3D1 and SWR1C2 do not recognize identical epitopes on topoisomerase II.

Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae.

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.

Selection for early meiotic mutants in yeast.

In the yeast Saccharomyces cerevisiae, only a/alpha cells can enter meiosis; a and alpha cells cannot. Because a/alpha cells are typically diploid and a and alpha cells are typically haploid, this cell type restriction ensures that only diploid cells enter meiosis. Entry into meiosis is accompanied by an increase in expression of the IME1 gene; the IME1 product (IME1) then activates IME2 and other meiotic genes. We have found that IME1 expression is toxic to starved haploid cells, presumably because IME1 directs them into meiosis. IME1 toxicity is greater in rad52 mutants, in which meiotic recombination causes lethal damage. Suppressors of IME1 toxicity include recessive mutations in two genes, RIM11 and RIM16 (Regulator of Inducer of Meiosis), that are required for IME1 to activate IME2 expression. RIM11 maps near CIN4 on chromosome XIII.

Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms.

To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.

Yeast expression of a catalytic antibody with chorismate mutase activity.

The catalytic antibody 1F7 promotes the rearrangement of chorismate into prephenate. We cloned and sequenced the genes encoding this catalyst to determine the origin of the observed rates and specificity. The antibody cDNAs were modified and inserted into inducible expression vectors. Simultaneous intracellular expression of the light and truncated heavy chains in strains of the yeast Saccharomyces cerevisiae lacking natural chorismate mutase resulted in the production of properly folded and assembled Fab antibody. Assembly of the light and heavy immunoglobulin chains is roughly 60-70% efficient in our in vivo system, lagging behind light chain synthesis throughout log and stationary phase. Nevertheless, high intracellular levels of functional Fab antibody (0.1% of total cellular protein) were obtained with an ultra-high copy number plasmid. As yeast-derived 1F7(Fab) catalyzes the chorismate mutase reaction with the same specific activity as antibody isolated from the hybridoma, our expression system now makes possible the application of classical and "reverse" genetics to the study and improvement of this first-generation abzyme.

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations.

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.

Production of antibodies in transgenic plants.

Complementary DNAs derived from a mouse hybridoma messenger RNA were used to transform tobacco leaf segments followed by regeneration of mature plants. Plants expressing single gamma or kappa immunoglobulin chains were crossed to yield progeny in which both chains were expressed simultaneously. A functional antibody accumulated to 1.3% of total leaf protein in plants expressing full-length cDNAs containing leader sequences. Specific binding of the antigen recognized by these antibodies was similar to the hybridoma-derived antibody. Transformants having gamma- or kappa-chain cDNAs without leader sequences gave poor expression of the proteins. The increased abundance of both gamma- and kappa-chains in transformants expressing assembled gamma-kappa complexes was not reflected in increased mRNA levels. The results demonstrate that production of immunoglobulins and assembly of functional antibodies occurs very efficiently in tobacco. Assembly of subunits by sexual cross might be a generally applicable method for expression of heterologous multimers in plants.