The long-term objective of this proposal is to understand the mechanism of internalization of integrins and their utility for targeting drugs to specific cells. The short-term objective of this proposal is to study the mechanism of internalization of LFA-1 on T cells and to utilize this mechanism for targeting drugs to leukocytes. The central hypothesis is that an ICAM-1-derived peptide (cIBR) can bind to and be internalized by LFA-1. Therefore, a cytotoxic drug (methotrexate, MTX) will be linked with cIBR peptide to give MTX-cIBR conjugate; this conjugate will have dual functions: (a) to inhibit T-cell activation via inhibition of the cell adhesion signal and (b) to induce T-cell death by internalizing MTX. The MTX-cIBR conjugate has selective toxicity for LFA-1-expressing T cells but not LFA-1-deficient KB epithelial cells. MTXcIBR conjugate suppressed rheumatoid arthritis in the collagen-induced arthritis (CIA) mouse model better than MTX alone. Therefore, the first objective is to evaluate the mechanism of suppression of arthritis by MTX-cIBR conjugate (Specific Aim 1). The mechanism of MTX-cIBR conjugate in altering the activation of T cells from Th1 to Th2 response in the CIA mouse model will be elucidated. In addition, the long-term kinetic effects of MTX-cIBR on the T-cell commitment in the CIA mouse model will be determined. Secondly, the internalization of MTX-cIBR or FITC-cIBR conjugates will be released by LFA-1 in the cytoplasm; alternatively, it could (a) remain with the endosome, (b) be recycled to the cell surface, or (c) be targeted to other membrane compartments. Therefore, the internalization process and localization of these conjugates will be determined (Specific Aim 2). Thirdly, the internalization of MTX-cIBR may also be via reduced folate carrier (RFC) or membrane folate binding protein (mFBP).
In Specific Aim 3, MTX-cIBR will be compared using wild type and RFC-deficient mouse leukemia T cells. In addition, this conjugate will be compared with MTX in mFBP over-expressing leukemia mouse cells in the presence and absence of folic acid. Finally, the exact mechanism of the LFA-1 internalization and the identity of other proteins that are involved in this process are not well understood.
In Specific Aim 4, cIBR peptide will be modified and cross-linked to LFA-1 on activated T cells; the crosslink will be isolated along with proteins that interact with LFA-1. These isolated proteins will be identified.
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