Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice - PubMed (original) (raw)

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice

Lukas W Pfannenstiel et al. Cell Immunol. 2010.

Abstract

Subclinical doses of Paclitaxel (PTX) given 1day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu(+) tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8(+) T cells with enhanced lytic activity against neu(+) tumors. PTX treatment also enhances maturation marker expression on CD11c(+) DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8(+) T cells with greater lytic capability than DC's from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-gamma-secreting CD8(+) T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.

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Conflict of interest statement

Conflict of Interest Statement. Under a licensing agreement between Cell Genesys and the Johns Hopkins University, Johns Hopkins University is entitled to milestone payments and royalties on sales of the human vaccine product that is similar to the mouse vaccine described in this report. The terms of these arrangements are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

Figures

Figure 1. CY and PTX have synergistic antitumor effects when combined with a HER-neu specific whole cell vaccine

A. Tumor-bearing _neu-_N mice received 20mg/kg PTX, 100mg/kg CY, or both via intraperitoneal injection followed by vaccination with 3T3neuGM or 3T3GM mock vaccine cells. Spleens and vaccine-draining lymph nodes were collected at the indicated time points post vaccination. CD4 and Foxp3 expression was measured by flow cytometry and the percent of CD4+ cells positive for Foxp3 was determined. Data were analyzed each day with P values < .05 for both ANOVA and Kruskal Wallace. Individual groups were then compared by Unpaired Student’s T-test with * P < 0.001 for CY vs PTX treated splenocytes; ** P < 0.001 for CY vs PTX treated lymph node cells. B. The combination of CY and PTX delays the growth of NT2.5 tumors compared to CY or PTX alone. Mice were tumor challenged and treated as described in the Methods. Mice were monitored every 5 to 7 days. Weekly time points are shown to simplify the graph. Groups from day 29 to 48 were analyzed by Kruskal-Wallace and ANOVA p <.002 and .0001 respectively. Individual time points were analyzed by Unpaired Students t-Test. * P < 0.01 for Cy + PTX + Vac versus CY + Vac and PTX + Vac; ** P < 0.001 for CY + PTX + Vac combination versus CY + Vac, PTX + Vac, and combination + mock vaccine. Vac = vaccine. Data not shown: Mock: overlays Vac, CY + Mock: Overlays PTX + Mock, and CY + PTX + Mock: overlays CY + Vac.

Figure 1. CY and PTX have synergistic antitumor effects when combined with a HER-neu specific whole cell vaccine

Figure 2. PTX affects DC phenotype at an early differentiation stage

Maturation marker expression levels were analyzed by flow cytometry in DCs cultured for 6 days in either GM-CSF (dark columns) or 10 nM PTX + GM-CSF (white columns) w/o maturation with LPS (None), DCs cultured with PTX + GM-CSF and matured with LPS (LPS), and DCs cultured with PTX + GM-CSF and matured with various concentrations of PTX (column sets 3–5) At least 10,000 DCs were analyzed for expression of CD86, CD40, MHC Class II, and IL-12. Data are shown as the mean plus standard deviation and are representative of 3 individual experiments. P values from Student’s t-Test compare DCs cultured with GM and matured with LPS and DCs cultured in GM + PTX and matured with LPS.

Figure 3. PTX matures DCs with enhanced ability to induce high avidity CD8+ T cells with higher lytic activity

A. FVB/N-derived bone marrow DCs were cultured in 20ng/ml GM-CSF with or without 10nM PTX and matured with LPS as described in the Methods. At maturation, DCs were pulsed with 20ug of RNEU(420–429) and cultured with Clone100 transgenic T cells for five days. C100 T cells were then used in a 4hr chromium release assay of NT2.5 tumor cells. Data shown is representative of 3 total experiments, and standard deviations for each point are less than 10% of the mean. P values from a Student’s t-Test are less than .02 for the points indicated. B. DCs generated in vivo with PTX enhance CD8+ T cell lysis. Tumor challenged _neu_-N mice were vaccinated with or without PTX as described in the Methods. Seven days post-vaccination, DCs were isolated from vaccine draining nodes, pulsed with 10ug of RNEU(420–429) (Upper panels) or used directly (Lower panels) to stimulate naïve C100 T cells for 5 days prior to Cr-51 release assay. Targets were peptide-pulsed T-2Dq (Left panels) or NT2.5 tumor cells (Right panels). T-2Dq cells pulsed with NP(118–126) were used as a specificity control. In experiments using T-2Dq as target cells, groups were tested by ANOVA and Kruskal-Wallace p < .02. In the experiment using freshly isolated DCs and T-2Dq targets, individual points from PTX + vaccine versus vaccine alone were compared by Student’s t-Test p<.002. For experiments using NT2.5 tumor cells as target, individual dilution points were compared by Student’s t-Test. The p values are shown on the graph. Each experiment was repeated 3 times with similar results. C. PTX treatment increases the frequency of antigen-specific CD8+ T cells in vivo. CD8+ T cells were isolated from FVB/N mice 9 days after vaccination with 3T3neuGM or PTX+3T3GM and tested for RNEU(420–429) reactivity by ICS as described in the Methods. Percent IFN-γ cells are shown. Data is representative of 2 experiments.

Figure 3. PTX matures DCs with enhanced ability to induce high avidity CD8+ T cells with higher lytic activity

Figure 4. TLR4 blockade abrogates the effect of PTX on DC development

DCs were cultured as described in the Methods with or without the addition of 10μg/ml anti-TLR4 until maturation with LPS on day 6. The p values shown compare PTX-treated, LPS matured DC’s vs PTX-treated, LPS matured DC’s incubated in the presence of TLR4 blocking antibody by paired Students t-Test. Data was pre-analyzed by ANOVA and Kruskal-Wallace. All eight columns and the last four columns were compared. For IL-12, CD86, and MHCII, overall ANOVA and Kruskal-Wallace scores were p<.0001 and <.002, respectively. For the last four columns the scores were ANOVA p<.0001 and Kruskal-Wallace p<.02. The experiment shown is representative of 3 repeated experiments.

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Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice - PubMed (original) (raw)