Neem Leaf Glycoprotein Induces Perforin-mediated Tumor Cell Killing by T and NK Cells Through Differential Regulation of IFNγ Signaling

We have demonstrated augmentation of the CD3-CD56+ natural killer (NK) and CD8+CD56_ T-cell-mediated tumor cell cytotoxicity by neem leaf glycoprotein (NLGP). These NK and T cells were isolated from the peripheral blood of head and neck squamous cell carcinoma patients with a state of immunosuppression. NLGP induces TCRalphabeta-associated cytotoxic T lymphocyte (CTL) reaction to kill oral cancer (KB) cells. This CTL reaction is assisted by NLGP-mediated up-regulation of CD28 on T cells and HLA-ABC, CD80/86 on monocytes. CTL-mediated killing of KB cells and NK-cell-mediated killing of K562 (erythroleukemic) cells are associated with activation of these cells by NLGP. This activation is evidenced by increased expression of early activation marker CD69 with altered expression of CD45RO/CD45RA. NLGP is a strong inducer of IFNgamma from both T and NK cells; however, IFNgamma regulates the T-cell-mediated cytotoxicity only without affecting NK-cell-mediated one. Reason of this differential regulation may lie within up-regulated expression of IFNgamma-receptor on T-cell surface, not on NK cells. This NLGP-induced cytotoxicity is dependent on up-regulated perforin/granzyme B expression in killer cells, which is again IFNgamma dependent in T cells and independent in NK cells. Although, FasL expression is increased by NLGP, it may not be truly linked with the cytotoxic functions, as brefeldin A could not block such NLGP-mediated cytotoxicity, like, concanamycin A, a perforin inhibitor. On the basis of these results, we conclude that NLGP might be effective to recover the suppressed cytotoxic functions of NK and T cells from head and neck squamous cell carcinoma patients.