FLOW CYTOMETRIC ANALYSIS AS SURROGATE FOR LYSIS

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Description: (provided by applicant) Cytotoxic T cell activity (CTL) has long been thought to be an important effector arm in mediating an anti-tumor immune response. However, the use of chromium release assays to delineate CTL specific responses has been fraught with problems including poor reproducibility, assay insensitivity, and lack of available biologically relevant targets for lysis in some human systems. Several methods to assess the CD8+ T cell response have been developed such as cytokine release and tetramer assays, but none have been directly compared to lysis assays to see if they are a surrogate for CTL activity. This pilot will propose to develop a more reproducible surrogate for the chromium release assay utilizing flow cytometric methods. Multiparameter flow cytometry will be used to investigate multiple cell surface, cytoplasmic, and nuclear markers that may, individually or collectively, correlate with CTL activity. Surface markers to be examined will include CD27, CD28, CD44, CD45RO/RA, CD62L, and CD95. These are all markers that can be used, singly or in combination, to distinguish naive from memory or effector cells. Cytoplasmic markers will include interferon gamma production in response to antigen stimulation, as well as perforin, granzyme B, and granulysin expression. The latter markers are all associated with cytoplasmic granule formation. We will assess what T cell marker patterns, after antigen stimulation, correlate with chromium release in both an infectious disease (CMV and flu) and tumor antigen model system. The goal of this pilot project will be to develop a rapid cell-based assay that is predictive of cytotoxic T lymphocyte (CTL) activity, as measured by standard 51Cr release assay after in vitro restimulation. Our specific aim will be to determine whether IFN -producing and/or proliferating CD8+ T cells, in combination with a phenotypic marker(s), correlate with CTL activity in the following systems: CMV and flu and at least one of the candidate cancer antigens to be evaluated by the consortium (MAGE3, HER2, or CEA). If the assays developed in this pilot correlate to lysis then consideration of their clinical development will be presented to the IMC investigators.