High susceptibility of c-KIT+CD34+ precursors to prolonged doxorubicin exposure interferes with Langerhans cell differentiation in a human cell line model

Abstract

As neoadjuvant and adjuvant chemotherapy schedules often consist of multiple treatment cycles over relatively long periods of time, it is important to know what effects protracted drug administration can have on the immune system. Here, we studied the long-term effects of doxorubicin on the capacity of dendritic cell (DC) precursors to differentiate into a particular DC subset, the Langerhans cells (LC). In order to achieve high telomerase activity as detected in hematological stem cells, precursor cells from the acute-myeloid leukemia (AML)-derived cell line MUTZ3 were stably transduced with human telomerase reverse transcriptase (hTERT) to facilitate their growth potential, while preventing growth, and drug-induced senescence, and preserving their unique capacity for cytokine-dependent DC and LC differentiation. The hTERT-MUTZ3 cells were selected with increasing concentrations of the anthracyclin doxorubicin. After 1-2 months of selection with 30-90 nM doxorubicin, the cells completely lost their capacity to differentiate into LC. This inhibition turned out to be reversible, as the cells slowly regained their capacity to differentiate after a 3- to 4-month drug-free period and with this became capable again of priming allogeneic T cells. Of note, the loss and gain of this capacity to differentiate coincided with the loss and gain of a subpopulation within the CD34(+) proliferative compartment with surface expression of the stem cell factor receptor (SCF-R/CD117/c-Kit). These data are in favor of cytostatic drug-free intervals before applying autologous DC-based vaccination protocols, as specific DC precursors may need time to recover from protracted chemotherapy treatment and re-emerge among the circulating CD34(+) hematopoietic stem and precursor cells.

Fig. 1

Telomerase expression and doxorubicin sensitivity of MUTZ3 and hTERT-MUTZ3 cells. a MUTZ3 progenitors and hTERT-MUTZ3 progenitors were analyzed for telomerase expression by RT-PCR. b MUTZ3 and hTERT-MUTZ3 progenitors were analyzed for their sensitivity to the anthracyclin doxorubicin by means of a XTT assay over 96 h (experiment representative of 3)

Fig. 2

Effects of hTERT introduction on MUTZ3-LC differentiation and proliferation. a Dot plots of flow cytometric analysis for the LC-typifying markers CD1a and Langerin on immature LC (iLC) cultures from MUTZ3 (iLC; left) and hTERT-MUTZ3 (hT-iLC; right). b Average percentages (+standard deviation) of CD14⁺, CD34⁺, CD1a⁺ and Langerin⁺ cells within iLC cultures from MUTZ3 or hTERT-MUTZ3 cells (n = 3). c Progenitor cell proliferation in the presence of 30 nM doxorubicin. Shown are the passage numbers against the expansion of the progenitor cells. MUTZ3 progenitor cells stopped proliferating after 9 passages in the presence of 30 nM doxorubicin, whereas hTERT-MUTZ3 cells could be cultured for over 160 passages (shown are data up to passage 50)

Fig. 3

Doxorubicin selection of progenitor cells hampers LC differentiation. a Output of viable hTERT-MUTZ3 and dox90+ hTERT-MUTZ3 cells after 10 days of LC differentiation b hTERT-MUTZ3 and dox90+ hTERT-MUTZ3 cells were differentiated into immature LC following standard culture protocols and were analyzed for phenotypic marker expression by flow cytometry on day 10. Shown are the percentages of CD1a⁺ (top left), Langerin⁺ (top middle) and CD40⁺ (top right) cells and the mean fluorescence intensity levels of CD86 (bottom left), CD54 (bottom middle), and HLA-DR (bottom right) in both the hTERT-MUTZ3 and dox90+ LC cultures (n = 3, P < 0.05)

Fig. 4

Doxorubicin-induced suppression of LC differentiation is reversible. hTERT-MUTZ3 cells, dox90+ cells, and dox90− cells (drug-free) were differentiated into immature LC for 10 days after 1, 3, and 4 months of drug depletion of the dox90− cells. a dotplots showing CD1a and Langerin expression on hT-iLC, dox90+ iLC, and dox90− iLC. Upon a drug-free period of at least 3 months, the dox90− cells regained their differentiation potential. b Percentages of cells expressing the DC maturation marker CD83 in cultures started 1, 3, and 4 months after drug depletion of the dox90− cells. c MLR was performed using hT-mLC, dox90+ mLC, and dox90− mLC [depleted for drugs for 1 month (left), 3 months (middle), and 4 months (right)], to analyze the T-cell stimulatory capacities of the mLC

Fig. 5

Doxorubicin exposure down-regulates SCF-R/c-Kit expression on CD34⁺ progenitors. a hTERT-MUTZ3, dox90+, and dox90− (4 months drug-free) progenitor cells were analyzed for the expression of the SCF-R/c-Kit. Continuous exposure to doxorubicin abrogated the expression of SCF-R/c-Kit on a subset of CD34⁺ progenitors. SCF-R/c-Kit expression returned upon drug depletion. b wtMUTZ3 cells were cultured in the presence of 0, 30, 100, or 300 nM doxorubicin for 4 days. SCF-R⁺ and SCF-R⁻ CD34⁺ cells were analyzed for their doxorubicin uptake. Left: percentages of SCF-R⁻ (white squares) and SCF-R⁺ (black squares) cells positive for doxorubicin after 4 days with the indicated drug concentrations. Right: doxorubicin uptake in SCF-R⁻ and SCF-R⁺ cells, determined by the mean fluorescence index (doxorubicin mean fluorescence intensity (FL-2 channel) of drug-exposed cells divided by the mean fluorescence intensity of non-exposed (0 nM) cells). *P < 0.05 (n = 3). c Percentages of SCF-R⁻ and SCF-R⁺ cells that died upon doxorubicin treatment, as determined by flowcytometric analysis with propidium iodide (PI). *P < 0.05 (n = 3)