APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells.

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.

CpG oligodeoxynucleotides enhance monoclonal antibody therapy of a murine lymphoma.

Bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine dinucleotides known as cytosine phosphorothioate guanine oligodeoxynucleotides (CpG ODN) can activate various immune-cell subsets, including cells that participate in antibody-dependent cell-mediated cytotoxicity (ADCC). Studies have shown that CpG ODN enhance the efficacy of antitumor monoclonal antibody (MoAb) therapy in the 38C13 murine B-cell lymphoma. We performed a series of in vivo experiments using this tumor model to better characterize combination therapy with MoAb and CpG ODN. CpG ODN enhanced the efficacy of MoAb therapy of lymphoma in a dose-dependent manner. This effect was seen whether the CpG ODN was given before or after the MoAb therapy, but was decreased when CpG ODN was given more than 2 days after MoAb therapy. Three doses of CpG ODN and MoAb were more effective than single doses. There was no obvious toxicity with multiple dosing. These studies confirm that immunostimulatory CpG ODN enhance the efficacy of MoAb therapy, and that multiple courses of combination therapy with CpG ODN can serve as an effective therapy for lymphoma. Further exploration of this potentially potent combination of treatments, including clinical evaluation, is indicated.

Immunostimulatory CpG oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocyte-macrophage colony-stimulating factor.

Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor (GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13 murine lymphoma system to evaluate the immune response to a combination of these two adjuvants. Immunization using antigen, CpG ODN, and soluble GM-CSF enhanced production of antigen-specific antibody and shifted production towards the IgG2a isotype, suggesting an enhanced TH1 response. This effect was most pronounced after repeat immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days before tumor inoculation prevented tumor growth. CpG ODN enhanced the production of interleukin-12 by bone marrow-derived dendritic cells and increased expression of major histocompatibility complex class I and class II molecules, particularly when cells were pulsed with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN in combination with strategies involving GM-CSF enhances the immune response to antigen and shifts the response towards a TH1 response and that this approach deserves further evaluation in tumor immunization approaches and other conditions in which an antigen-specific TH1 response is desirable.

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization.

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund's adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund's adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund's adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

Isolating RNA from clinical samples with Catrimox-14 and lithium chloride.

RNA is a highly informative molecule that has great potential as a target for diagnostic studies. This potential can be reached only when reliable methods for isolating RNA are available in the clinical environment. Cationic surfactants lyse cells and precipitate nucleic acids. We have described a novel cationic surfactant (tetradecyltrimethylammonium oxalate, Catrimox-14), which is particularly effective in precipitating RNA from cells and which can be applied to clinical specimens. We examine the utility of a method of recovering RNA from the surfactant-nucleic acid precipitate, in which 2 M lithium chloride is used to extract the DNA and surfactant from the precipitate; RNA (being insoluble in lithium chloride solution) remains in the pellet. The yield of RNA from peripheral blood mononuclear cells by the Catrimox-LiCl method we describe was the same yield by a conventional method using guanidine thiocyanate, phenol, and chloroform (GPC). The quality of the RNA, judged by agarose gel electrophoresis, A260/280 ratio and its ability to serve as a target for reverse transcription and PCR, was the same. RNA was isolated and amplified from blood stored for at least 2 weeks in Catrimox solution at room temperature. RNA was also easily isolated with the Catrimox-LiCl method in good yield from frozen sections of mouse liver, spleen, kidney and brain, and from core biopsies of liver and kidney. RNA isolated from needle aspirates of liver, spleen, kidney, pancreas, and brain was easily amplified by RT-PCR. The Catrimox-LiCl method is simple and does not call for the use of corrosive reagents. The Catrimox-LiCl method removes 98% of the DNA. We conclude that the Catrimox-LiCl method is suitable for use in clinical applications of RNA-based diagnosis.

T-cell activation induced by anti-CD3 x anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand.

T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific antibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy.

Isolation of RNA from cells in culture using Catrimox-14 cationic surfactant.

Traditional RNA isolation methods use chaotropic agents and anionic detergents to lyse cells and solubilize nucleic acids. In contrast, the cationic surfactant, Catrimox-14, lyses cells and simultaneously precipitates RNA, thereby protecting it from RNases. We describe and compare four methods for extracting RNA from cultured cells that differ in the technique used to extract the RNA from the precipitate. The first uses a high-salt solution (guanidinium isothiocyanate). In the second, the RNA is extracted with a polar solvent (formamide). The third involves conversion of the RNA to the sodium salt by treatment of the precipitate in situ with sodium acetate in ethanol. The fourth uses 2 M lithium chloride to convert the RNA in the pellet to the lithium salt in situ. We applied these methods to human leukemia cells growing in culture and each method resulted in excellent yields of RNA (typically 23 micrograms/million K562 cells, 13 micrograms/million HL-60 cells) over a wide range of cell concentrations (1 x 10(5) - 3 x 10(7)/ml) and of good to excellent quality as judged by agarose electrophoresis and UV absorbance data (OD260/280 1.90-2.05). The advantages and limitations of each method are discussed.

Isolating RNA from whole blood--the dawn of RNA-based diagnosis?

A novel cationic surfactant solution is used to lyse blood cells and precipitate RNA and DNA. The RNA is recovered by extracting the pellet with a small volume of guanidinium isothiocyanate or formamide.

Identification and characterization of a new surface membrane antigen found predominantly on malignant B lymphocytes.

A monoclonal antibody, 1D10, was derived that identifies a new antigenic epitope on the surface of malignant B lymphocytes. Normal resting and stimulated lymphocytes do not express the antigen. The majority of individuals with acute Epstein-Barr virus infection express the antigen on their lymphocytes, and in these patients, the T lymphocyte may also be antigen positive. The antigen was found on B-lymphoid neoplasia from the early pre-B cell stage through terminally differentiated plasma cells, a characteristic not reported for other B cell-associated antigens. Studies on homozygous typing cells and cells from individuals with known HLA phenotypes indicate that the antigen does not segregate in a pattern characteristic for major histocompatibility antigens. The molecule is a heterodimeric polypeptide with the molecular weight and isoelectric points of the alpha and beta chains being 32,000 d/4 and 28,000 d/6, respectively. Evidence is presented that the 1D10 molecule is not HLA-DR, -DP, or -DQ. By extrapolation, we suggest that this novel molecule may represent HLA D-region gene expression of a gene(s) not normally expressed. Potential candidates are D-region pseudogenes. We conclude that the antigenic epitope identified by the 1D10 monoclonal antibody is unique among previously described B-lymphocyte antigens. Further studies of the factors controlling the expression of this molecule, as well as studies designed to look at the possible cellular function, may provide insights for understanding crucial events in the malignant transformation of lymphocytes.

Identification and characterization of an endothelial, cell-specific antigen with a monoclonal antibody.

The purpose of these studies was to use monoclonal antibodies to identify and characterize plasma membrane components unique to the vascular endothelium. Our assumption is that such components may perform some of the specialized functions of the endothelium and, by their identification with antibody probes, we may be able to study further their function and structure. Thus, primary cultures of human umbilical vein endothelium were used to immunize mice whose spleen cells were fused with the mouse myeloma cell NS-1. HEC-1 is a monoclonal antibody derived from such a fusion that appears to react with an antigen located only on endothelial cells. The antigen has been characterized by immunoprecipitation and polyacrylamide gel electrophoresis as a glycoprotein with a mol wt of 180,000 daltons under nonreducing conditions and 90,000 daltons under reducing conditions. Despite a close resemblance to a membrane component shown by others to be a receptor for transferrin, several lines of evidence reported in this paper indicate that this is not the function of the HEC-1 antigen. These data show that monoclonal antibodies can be used to identify and characterize membrane components of the vascular endothelium. Moreover, these probes can be used to inquire about the structure and function of the antigen with which they react.