Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-08-08
2004-03-30
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007220, C435S007230, C435S007240, C435S007250, C435S007310, C435S007320, C435S007330, C435S007920, C435S007940
Reexamination Certificate
active
06713250
ABSTRACT:
FIELD OF THE INVENTION
The subject invention is directed generally to immunology, and more particularly to a method for screening a test compound for the ability of the test compound to induce a response from human naive T-cells, and to a method for primary in vitro sensitization of human naive T-cells.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
There is a great need for in vitro methods for testing allergenicity of compounds. This need is driven by socioeconomic factors related to concerns over animal testing. In particular, a method is needed to screen for potential allergens in products intended for topical application, such as cosmetics, and toiletry products. Currently, testing of potential allergens is largely performed on human subjects at great expense (1). Unfortunately, even the most extensive clinical trials employing over 100 subjects are unlikely to detect severe allergic reactions that may occur in 1/1,000 persons. Yet these rare severe reactions may present a significant risk to the population, as well as a large legal liability to the manufacturer.
Significant progress has been made on the development of in vitro assays for irritancy (2). However, in vitro assays that can distinguish irritants from allergens have not yet been developed, despite an extensive effort funded by the Center for Alternatives to Animal Testing @ Johns Hopkins University (3). Most allergens are also either clinical or sub-clinical irritants which further complicates the development of an assay for allergenicity. Generally, both allergens and irritants evoke a similar cytokine profile (4).
Allergenicity of topically applied compounds can be manifest as allergic contact dermatitis, photoallergic dermatitis, or contact urticaria (5,6,7). Allergic contact dermatitis is mediated by T-lymphocytes, and is a variant of delayed hypersensitivity in which the primary antigen presenting cell is the Langerhans dendritic cell (8). Allergens thus function as antigens to induce a T-lymphocyte response.
Primary in vitro sensitization is the sensitization of naive T-lymphocytes to antigens which the donor has never encountered. Other investigators have been unable to achieve primary in vitro sensitization without the use of dendritic cells (9,10,11,12,13,14). The use of peripheral blood human dendritic cells to distinguish allergens from irritants has been previously suggested (15). Dendritic cells are difficult to isolate in significant numbers (16), which greatly limits their application to a commercial assay.
The ability of dendritic antigen presenting cells to induce a primary immune response to a novel antigen is probably a function of the high expression of co-stimulatory molecules by these cells. Presentation of antigen to T-lymphocytes involves the interactions of multiple molecules, and T-cell receptor occupancy is not sufficient to induce T-cell proliferation (17). The interaction of CD28 and B7-1 (CD80) or B7-2 (CD86) has been shown to deliver such an essential co-stimulatory signal to human (18,19) CD4+ and CD8+ (20) T-cells. However, B7/CD28 signalling alone is inadequate to explain the unique capabilities of dendritic cells (21). Other molecule pairs with a role in co-stimulation or signaling of T-cells include ICAM-1/LFA-1 (22), LFA-3/CD2 (23), CD40/CD40-ligand (24), and possibly Heat Stable antigen (25). These co-stimulatory molecules are expressed at high levels on dendritic antigen presenting cells, and probably explain the ability of these cells to induce a primary immune response.
Since compounds which are unable to induce a T-lymphocyte response would be unable to induce allergic contact dermatitis, an in vitro method which detects the ability of novel compounds to induce a T-lymphocyte response may thus function as a screening assay for contact allergens. Due to the difficulty in isolating significant numbers of dendritic cells, the use of such cells as antigen presenting cells for primary in vitro sensitization has limited practical applications. A need continues to exist for a practical in vitro method for testing allergenicity and for primary in vitro sensitization.
SUMMARY OF THE INVENTION
The subject invention addresses this need by providing for the addition of an immortalized B-cell line to a sensitization culture to enable a primary in vitro response to novel antigens. If the B-cell line is not derived from the same donor as the responding lymphocytes, there is a response against the foreign transplantation antigens of the B-cell line. Such an alloantigen response precludes detection of primary in vitro sensitization. Therefore, this response to transplantation antigens is avoided by using a B-cell line which lacks both the class I (HLA-DR) and class II (HLA-A,B,C) major histocompatibility antigens (MHC). T2 cells are a line of EBV transformed B-cells which have deleted the gene for class II major histocompatibility locus transplantation antigens, and also have very low expression of class I MHC transplantation antigens (26). Addition of mitomycin C treated T2 cells to primary in vitro sensitization cultures permitted the detection of T-lymphocyte responses to novel antigens (allergens). This culture system has the potential to detect the immunogenic potential of weak allergens such as Balsam of Peru, and does not react to irritants, such as sodium dodecyl sulfate (SDS). This technology thus provides for primary in vitro sensitization without the use of dendritic cells. This technology can be used to identify human allergens and haptens. It can also identify peptides, or epitopes recognized by human T-lymphocytes, which is useful for vaccine development.
More particularly, the subject invention provides a method for screening a test compound for the ability of the test compound to induce a response from human naive T-cells. The method comprises admixing human naive T cells, macrophages/monocytes, immortalized B cells lacking class I and class II major histocompatibility antigens, and a test compound; and determining whether the test compound induces a response from the human naive T cells.
The invention further provides a method for primary in vitro sensitization of human naive T-cells. The method comprises admixing human naive T cells, macrophages/monocytes, immortalized B cells lacking class I and class II major histocompatibility antigens, and an antigen.
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Degwert et al., in “Dendritic Cells in Fundamental and Clinical Immunology”, Banchereau and Schmitt, Eds., vol. 2, pp. 187-189, Plenum Press, New York (1995).
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Rogalsky & Weyand, LLP
Saunders David
The Research Foundation of State University of New York
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