The search for improved cytotoxic agents continues to be an important line of modern anticancer drug discovery and a promising mechanistic approach towards this goal is the functional inhibition of cellular microtubules. Tubulin inhibitors are compounds which either stabilize or destabilize microtubules in vitro, leading to G2/M cell cycle arrest and apoptosis in cancer cells. While destabilizing agents, such as vinca alkaloids inhibit the assembly of ??-tubulin heterodimers, stabilizing compounds like taxol induce the de novo formation of stable microtubules in vitro. In this study we have investigated a number of plant-derived compounds that have recently been reported to interact with the tubulin/microtubule system and to induce taxol-like effects. This includes the sesquiterpene lactones parthenolide and costunolide, the coumarin derivative ferulenol, and the jatrophane ester JTE1. In addition, we have screened a small natural product library (84 cytotoxic compounds) and 107 cytotoxic plant extracts in an assay sys- tem that allows the detection of both microtubule-stabilizing and -destabilizing agents in a 96-well setup within the same experimental format. None of the plant extracts inhibited or induced tubulin polymerization in vitro. From the compound library only the known plant-derived tubulin inhibitors vinblastine, colchicine, podophyllotoxin, chelidonine, rotenone, and taxol were identified as hits. Curcumin, which was recently reported to destabilize cellular microtubules, was inactive in our assay. Interestingly, rotenone, which is widely used as a mitochondrial respiration chain I inhibitor, potently inhibited microtubule assembly in vitro and showed higher affinity to ??-tubulin than vinblastine, although it was significantly less cytotoxic. None of the plant-derived natural products that were recently reported to be microtubule-stabilizing agents were found to be active in our assay system. In conclusion, plant-derived natural products clearly represent an interesting and productive source for microtubule-destabilizing agents. In contrast, apart from taxol and related structures, no plant-derived natural product with potent in vitro microtubule-stabilizing properties has yet been identified.
Abstract CD8+ T cells are primarily regarded as cytotoxic cells. We have identified a substantial subset of CD40L expressing non-cytotoxic T-cells among primary human CD8+ T cells. CD40L+ CD8+ T cells exert diverse characteristic Th-cell functions such as activation of B cells, induction of DC maturation and secretion of cytokines such as IL-2, IFNγ, TNFα or IL-4. Up to 25% of total human central and effector memory CD8+ T cells express CD40L including cells specific for pathogens such as influenza, CMV and EBV, as well as yellow fever virus specific cells after primary vaccination. At next we assessed the functional relevance of CD40L expression on CD8+ T cells during a anti-tumor response. We challenged Rag1-/- mice with SV40 Tag expressing tumor cells and injected in parallel wt or CD40L-/- CD8+ T cells. Only application of CD40L competent wt CD8+ T cells prevented the establishment of tumors, whereas injection of CD40L-/- CD8+ T cells resulted in non-controlled tumor progression similar to non-treated tumors, although tumor-specific T cells were primed in these mice. Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Especially, in situations of reduced CD4+ T-cell help and MHC-II antigen presentation and/or limited danger signals CD40L+ CD8+ T cells may exert essential helper responsibilities for immunity and thus are potent candidate T cells to execute or support effective anti-tumor or anti-pathogen immune therapies.
Recently, new methods have been introduced describing assessment of antigen-specific CD4+ T-cell immunity according to the induction of CD154 (CD40L) on CD4+ T cells during short-term activation. In our study, we have evaluated the influence of different stimulation conditions on the flow cytometric analysis of CD154 expression after antigenic in vitro activation. We used different cell preparation methods, antigen sources, and time periods of in vitro stimulation and analyzed their impact on intra and extracellular detection of antigen-induced CD154 expression on CD4+ T cells. We could demonstrate that analysis of CD4+ T-cell immunity according to CD154 expression displayed low intra-assay variability and was robust with respect to its induction in the course of a variety of stimulation conditions. For a basic quantitative evaluation of antigen-specific CD4+ T cells, surface CD154 analysis could be employed, enabling the fast analysis of live antigen-specific CD4+ T cells. Intracellular analysis of CD154 in combination with cytokines such as IL-2 and IFNgamma allowed quantitative and qualitative assessment of antigen-specific CD4+ T cells. The cytometric analysis of antigen-specific CD4+ T-cell immunity according to CD154 expression is characterized by robustness, high sensitivity, and low intra-assay variability.
Background. Polyomavirus BK virus (BKV) infection represents a serious complication leading to BKV-associated nephropathy (BKVAN) and subsequent kidney graft loss in up to 10% of transplant patients. Cellular immunity is known to play a crucial role in the control of BKV replication. However, the knowledge on the BKV-T-cell response is limited: only two (VP1 and large T antigen) of six known BKV proteins were evaluated for their antigenicity so far. Methods. By using 10-color flow cytometry and newly created overlapping peptide pools of five BKV antigens (VP1, VP2, VP3, large T antigen, and small t antigen), we performed cross-sectional phenotypic and functional analysis of BKV-specific T cells in kidney transplant patients with a history of BKVAN. Patients with clinically unapparent BKV infection (history of transient/no BKV reactivation) were used as control group. Results. Our data demonstrate for the first time the antigenic properties of all five evaluated proteins with VP3 as a new important target of cellular immunity. Further, we found a correlation between the severity of the previous BKV infection and the magnitude of memory CD4+ T-cell response. Thus, compared with the control group, patients with a history of BKVAN demonstrated significantly higher frequencies of interferon-γ- and interleukin-2-producing effector memory CD4+ T cells. In the control group, more patients with detectable interferon-γ+/interleukin-2+/tumor necrosis factor+ triple producers were found, suggesting possibly a protective function of these multifunctional T cells. Conclusions. In conclusion, our study results suggest an implementation of new targets for monitoring of BKV immunity. Further studies are required to evaluate the protective function of the found BKV-specific T-cell subsets.
