[Show abstract][Hide abstract] ABSTRACT: Despite the strides that immune therapy has made in mediating tumor regression, the clinical effect is often transient, and more durable responses are still needed. The temporary nature of the immune response is attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, T regulatory cells (Treg). Although the depletion of Treg has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. Here, we found that PI3K-Akt pathway inhibitors selectively inhibit Treg with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Treg in comparison to Tconv cells when treated with different Akt and PI3K inhibitors. This effect was observed both in human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Treg both in naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors lead to a significant therapeutic anti-tumor effect, which was shown to be Treg dependent. In this work, we report using PI3K-Akt pathway inhibitors as potent selective agents for the depletion of suppressive Treg. These inhibitors are shown to enhance the anti-tumor immune response and are therefore promising potential Treg-depletion clinical reagents.
[Show abstract][Hide abstract] ABSTRACT: Studies have shown that a regulatory T cell (Treg) cell decrease accompanied complete regression of tumor growth induced by a Listeria monocytogenes (Lm)-based vaccine expressing a fusion protein consisting of truncated listeriolysin O (LLO) and human papilloma virus (HPV) E7 protein (Lm-LLO-E7). However, how Lm-based vaccine causes Treg decrease remains unclear yet. Using a highly attenuated Lm dal dat ∆actA strain (LmddA)-based vaccine, we report here that the vector LmddA itself was sufficient to induce a decrease in the proportion of Treg cells by preferentially expanding CD4+FoxP3- T cells and CD8+ T cells, by a mechanism dependent on and directly mediated by LLO. Episomal expression of a nonhemolytic truncated LLO in Lm (LmddA-LLO) significantly augmented the expansion, thus decreasing Treg frequency to a lower degree. While adoptive transfer of Tregs compromised the anti-tumor efficacy of LmddA-LLO-E7 vaccine, a combination of LmddA-LLO and an Lm-based vaccine expressing E7 protein (Lm-E7) induced complete regression against established TC-1 tumors. An Lm recombinant replacing LLO with perfringolysin O (PFO), allowing exit from the phagolysosome but without LLO, confirmed that the adjuvant effect was dependent on LLO itself. These results suggest that LLO may serve as a promising adjuvant by preferentially inducing CD4+FoxP3- T cell and CD8+ T cell expansion, thus improving the ratio of Tregs to CD4+FoxP3- T cells and to CD8+ T cells and favoring immune responses to eradicate tumor.
[Show abstract][Hide abstract] ABSTRACT: Programmed death receptor 1 (PD-1) is an important signaling molecule often involved in tumor-mediated suppression of activated immune cells. Binding of this receptor to its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), attenuates T cell activation, reduces IL-2 and IFN-γ secretion, decreases proliferation and cytotoxicity, and induces apoptosis. B7-DC-Ig is a recombinant protein that binds and targets PD-1. It is composed of an extracellular domain of murine B7-DC fused to the Fc portion of murine IgG2a. In this study, we demonstrate that B7-DC-Ig can enhance the therapeutic efficacy of vaccine when combined with cyclophosphamide. We show that this combination significantly enhances Ag-specific immune responses and leads to complete eradication of established tumors in 60% of mice and that this effect is CD8 dependent. We identified a novel mechanism by which B7-DC-Ig exerts its therapeutic effect that is distinctly different from direct blocking of the PD-L1-PD-1 interaction. In this study, we demonstrate that there are significant differences between levels and timing of surface PD-1 expression on different T cell subsets. We found that these differences play critical roles in anti-tumor immune effect exhibited by B7-DC-Ig through inhibiting proliferation of PD-1(high) CD4 T cells, leading to a significant decrease in the level of these cells, which are enriched for regulatory T cells, within the tumor. In addition, it also leads to a decrease in PD-1(high) CD8 T cells, tipping the balance toward nonexhausted functional PD-1(low) CD8 T cells. We believe that the PD-1 expression level on T cells is a crucial factor that needs to be considered when designing PD-1-targeting immune therapies.
Preview · Article · Jul 2012 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Western Blot Analysis of FPGT expression in SP and MP cells of SKOV3 and A224 cell lines (above). Immunoflourescence of ADAM19 in A224 SP and MP cells: The A224 cells were sorted for SP and MP and grown on the cover slip. The tissue culture media was removed and washed the wells with PBS twice. The cells were then fixed using 4% Para formaldehyde for 12 minutes and two quick washes were given with PBS. The cells were then permeabilized using 1% Triton in 0.02% BSA in PBS for 2 minutes. The cells were then incubated with blocking serum (20% heat inactivated serum with 2% BSA in PBS) for 20 minutes in room temperature. The cells were washed with PBS and control were incubated with Blocking Peptide (BP, ADAM19-P, SC-25989, Santa Cruz Biotechnology). The cells were stained with primary antibody (ADAM19 Goat plyclonal IgG, SC25989, Santa Cruz Biotechnology) for 2 hours at room temperature. The cells were washed thrice with PBS and stained for secondary antibody (Donkey anti-goat IgG-FITC, SC-2024, Santa Cruz Biotechnology) for 30 minutes at room temperature. The cells washed thrice with PBS and stained with DAPI for 5 minutes. SP and MP cells showed 64±4% and 33±5% positively stained cells for ADAM19. Representative figures showed above.
[Show abstract][Hide abstract] ABSTRACT: Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.
[Show abstract][Hide abstract] ABSTRACT: CA 125 staining for ascites samples: The CA 125 staining and analysis using FACS confirmed the ovarian origin of cells isolated from ascites and the ovarian cancer cell line SKOV3.