Non-Hodgkin's lymphoma is an AIDS-defining event in a significant percent of U.S. patients infected with the Human Immunodeficiency Virus (HIV). Advances in anti-retroviral treatment and management of opportunistic infection have witnessed an increase in the incidence of these lymphomas. Evidence of polyclonal B cell hyperactivity during the early phases of HIV-infection argues that chronic B cell stimulation may be the major process predisposing B cells in the HlV-infected individual to malignant transformation. The mechanism of this stimulation of normal B cells and its relationship to AIDS-associated lymphomas remain unexplored and represent the goals of the proposed studies. We hypothesize that the HlV- associated lymphomas represent transformed B cells selected from HIV stimulated B cells. To test this idea, we will determine the relationship between the subset and antigen specificity of those B cells from non- infected individuals stimulated by HIV and that of the HIV-associated lymphomas. Specifically, we will: 1) Determine the frequency of HIV-responsive B cells from non-infected individuals. The frequency of B cells responding in vitro to whole virus as well as recombinant viral proteins will be determined by flow cytometic analysis of acridine orange stained cells. We will determine if antigen receptor-dependent and -independent mechanisms exist for HIV-mediated stimulation. 2) Determine the molecular mechanism of HIV-induced activation. The biochemistry of HIV-mediated B cell stimulation will be determined. Depending upon results obtained in Aim l, we will explore both antigen receptor dependent and independent signalling pathways. 3) Determine to what extent AIDS-associated lymphoma cells are restricted with respect to surface phenotype and immunoglobulin (Ig) gene expression. FACS analysis will be used to determine the stage of B-cell differentiation, the involvement of B-cell subsets (e.g. CD5) and the expression of certain variable region heavy (VH) or light (VL) chain restricted idiotypes. Ig gene expression will be carried by nucleotide sequence analysis of expressed VH and VL genes. The Ig sequence analysis will also include an assessment for non-random nucleotide substitutions indicative of clonal selection by antigen. 4) Determine to what extent the Ig expressed by AIDS-associated lymphomas are directed to HIV related antigens. The lymphoma related VH and VL genes will be cloned into phagemid vectors which allow for the creation of phage displaying the relevant Fab fragments on their surfaces and the subsequent bacterial production of soluble Fab fragments. The specificity of these Fab fragments/phage displaying Fab will be assessed by ELISA using plates coated with multiple recombinant proteins and by Western blotting to evaluate for reactivity to particular HIV gene products.
| Przybylski, G K; Goldman, J; Ng, V L et al. (1996) Evidence for early B-cell activation preceding the development of Epstein-Barr virus-negative acquired immunodeficiency syndrome-related lymphoma. Blood 88:4620-9 |
