Differential effects of Paclitaxel on dendritic cell function

Abstract

Background: The potential utility of dendritic cells (DC) as cancer vaccines has been established in early trials in human cancers. The concomitant administration of cytotoxic agents and DC vaccines has been previously avoided due to potential immune suppression by chemotherapeutics. Recent studies show that common chemotherapy agents positively influence adaptive and innate anti-tumour immune responses.

Results: We investigated the effects of paclitaxel on human DC biology in vitro. DCs appear to sustain a significant level of resistance to paclitaxel and maintain normal viability at concentrations of up to 100 micromol. In some cases this resistance against paclitaxel is significantly better than the level seen in tumour cell lines. Paclitaxel exposure led to a dose dependent increase in HLA class II expression equivalent to exposure to lipopolysaccharide (LPS), and a corresponding increase in proliferation of allogeneic T cells at the clinically relevant doses of paclitaxel. Increase in HLA-Class II expression induced by paclitaxel was not blocked by anti TLR-4 antibody. However, paclitaxel exposure reduced the endocytic capacity of DC but reduced the expression of key pro-inflammatory cytokines such as IL-12 and TNFalpha. Key morphological changes occurred when immature DC were cultured with 100 micromol paclitaxel. They became small rounded cells with stable microtubules, whereas there were little effects on LPS-matured DC.

Conclusions: The effect of paclitaxel on human monocyte derived DC is complex, but in the clinical context of patients receiving preloaded and matured DC vaccines, its immunostimulatory potential and resistance to direct cytotoxicity by paclitaxel would indicate potential advantages to co-administration with vaccines.

Figure 1

Paclitaxel inhibits mitochondrial activity in tumour cell lines and DC as assessed by MTS/PMS assay. Tumour cell lines MJT-3 (black squares), MCF7-pR (grey squares) and DC (white squares) were incubated with paclitaxel for 2 h before being washed and returned to culture for 48 h. Sensitivity was determined by the addition of MTS/PMS for triplicate samples ± SD. Each experiment was repeated 3 times and DC from 3 individual donors evaluated. Representative histograms from one experiment are shown. At lower doses (up to 0.5 μmol) of paclitaxel, the DC are more resistant than tumour cells (p value = 0.003). DC were more resistant overall than MCF7-pR.

Figure 2

(A) DC viability after exposure to paclitaxel. Day 7 DC were incubated with paclitaxel at 1-100 μM for 2 h, washed and returned to culture for up to 120 h (5 days). Viability was assessed by trypan blue (and propidium iodide (PI) dye exclusion, data not shown). Representative data from one of 3 individual donors are shown. No deleterious effect on survival was seen, (B). DC were exposed to paclitaxel at 1-100 μM for 2, 24 or 48 h and viability assessed at the end of 48 h by PI dye exclusion. Short-term exposure of DC to paclitaxel had no effect on viability, however, 100 μM paclitaxel for 48 h did induce a significant loss in viability (* P < 0.05).

Figure 3

Enhanced DC immunostimulatory capacity at low dose Paclitaxel. Day 7 DC were treated with paclitaxel for 2 hours before being co-cultured with allogeneic T cells at different cell ratios in a 5 day MLR. The proliferation of the T cells was determined by the incorporation of [³H] thymidine and the data shown are the mean S.I. ± SD of triplicate samples, and is representative of 3 independent experiments using DC from 3 different donors. (* P < 0.05).

Figure 4

DC exposed to paclitaxel have less endocytic function shown by uptake of FITC-conjugated dextran. Control and paclitaxel treated DC were incubated with FITC-dextran for 2 h at either 37°C (black bars) or 4°C (white bars). DC were washed with ice cold PBS to remove unbound FITC-dextran prior to FACS analysis. Combined data from 3 separate donor experiments are shown. Increasing doses of paclitaxel significantly reduced active endocytosis compared to no taxol exposure (P < 0.05 with 10 μmol taxol exposure).

Figure 5

Effects of Paclitaxel versus LPS on the ability of DC to produce inflammatory cytokines. DC treated with LPS or paclitaxel for 2 hours were extensively washed and returned to culture for 24 h. Supernatants were collected and analysed by CBA for the presence of A; IL-12; B, TNFα; C, IL-10; D, IL-1β; EIL - 8. Overall, paclitaxel appeared to reduce cytokine secretion by DC compared to LPS; significant increases of IL-1β secretion were seen at lower doses of paclitaxel.

Figure 6

Paclitaxel induced alterations to DC morphology and cytoskeletal organisation similar to LPS. DC were adhered to fibronectin-coated coverslips prior to treatment with LPS +/- Paclitaxel for 2 h. The cells were extensively washed before being stained for microtubule arrangements and analysis by microscopy. Representative figures from 4 experimental repeats are shown. A, non-treated DC; B; 1 μM paclitaxel; C, 100 μM paclitaxel; D, 1 μg/ml LPS. In keeping with its known action, exposure to paclitaxel resulted in microtubule stabilisation but marked changes in DC morphology reduction in cell size, rounding and loss of dendrites.

Figure 7

Upregulation of DC class II expression by paclitaxel is not mediated via TLR-4. DC were cultured for 2 h with LPS or paclitaxel in the presence or absence of anti-TLR-4 Abs prior to washing and reculture for a further 24 h. Anti-TLR4 antibody did reduce class II expression after exposure to LPS, but not to paclitaxel at high dose. Numbers in parenthesis represent MFI of class II detection. Data is representative of 3 independent experiments from 3 donors.