The objective of this proposal is to investigate and define the mononuclear cells which comprise benign and malignant ocular adnexal lymphoid neoplasms and the ocular inflammations. First, we shall test the hypothesis that histopathologically polymorphous and immunophenotypically polyclonal ocular pseudolymphomas often contain clonal B or T cells by analyzing the lymphoid cells isolated from these lesions 1) on a fluorescent activated cell sorter to determine clonal B cell excess not detectable by fluorescent microscopy and 2) by Southern blot hybridization to determine the presence of rearrangements of the genes which code for immunoglobulin and the T cell receptor in order to demonstrate clonality at the molecular DNA level. This will allow us to evaluate the diagnostic and prognostic value of these methodologies, and to clarify the pathogenetic relationship between ocular pseudolymphomas and B cell lymphomas. Second, we shall seek to identify cell surface antigens unique to ocular adnexal B cells and investigate the possibility that they differentiate along a distinctive B cell developmental pathway. We will accomplish this by 1) preparing hybridoma monoclonal antibodies with specificity for B cell associated differentiation antigens preferentially expressed on ocular adnexal B cells and by 2) inducing ocular adnexal and systemic monoclonal B cell proliferations to differentiate in vitro under the influence of phorbol ester (TPA) and evaluating the induced B cells for their similar or dissimilar expression of B cell associated differentiation antigens. Third, we shall employ monoclonal antibodies which define lineage, differentiation and subset-specific antigens in immunoperoxidase cryostat tissue section techniques to delineate the topographic distribution of the mononuclear cells and their constituent subsets which comprise the immunoarchitecture of the normal ocular adnexa. Fourth, we shall delineate the phenotypic and functional cellular immune abnormalities in ocular immune diseases. Mononuclear cells and their subsets will be investigated in microscopic and macroscopic ocular inflammations by immunoperoxidase examination of cryostat tissue sections with monoclonal antibodies. In addition, we shall isolate the T cells from the macroscopic ocular inflammations and investigate their ability to generate proliferative responses and help and suppress immunoglobulin production in vitro. These studies will allow us to determine qualitative and quantitative cellular immune abnormalities and evaluate the functional integrity of the immune system in patients with ocular immune diseases.
