All-trans-retinoic acid improves differentiation of myeloid cells and immune response in cancer patients

Abstract

Abnormal dendritic cell differentiation and accumulation of immature myeloid suppressor cells (ImC) is one of the major mechanisms of tumor escape. We tested the possibility of pharmacologic regulation of myeloid cell differentiation using all-trans-retinoic acid (ATRA). Eighteen patients with metastatic renal cell carcinoma were treated with ATRA followed by s.c. interleukin 2 (IL-2). Eight healthy individuals comprised a control group. As expected, the cancer patients had substantially elevated levels of ImC. We observed that ATRA dramatically reduced the number of ImC. This effect was observed only in patients with high plasma concentration of ATRA (>150 ng/mL), but not in patients with lower ATRA concentrations (<135 ng/mL). Effects of ATRA on the proportions of different dendritic cell populations were minor. However, ATRA significantly improved myeloid/lymphoid dendritic cell ratio and the ability of patients' mononuclear cells to stimulate allogeneic T cells. This effect was associated with significant improvement of tetanus-toxoid-specific T-cell response. During the IL-2 treatment, the ATRA effect was completely eliminated. To assess the role of IL-2, specimens from 15 patients with metastatic renal cell carcinoma who had been treated with i.v. IL-2 alone were analyzed. In this group also, IL-2 significantly reduced the number and function of dendritic cells as well as T-cell function. These data indicate that ATRA at effective concentrations eliminated ImC, improved myeloid/lymphoid dendritic cell ratio, dendritic cell function, and antigen-specific T-cell response. ATRA treatment did not result in significant toxicity and it could be tested in therapeutic combination with cancer vaccines.

Figure 1. ATRA pharmacokinetics

Patients with RCC were treated with indicated doses of ATRA and the level of the compounds was measured in plasma as described in Materials and Methods. Six patients had received 50 mg/m²/day dose, 6 patients – 100 mg/m²/day, and 6 patients – 150 mg/m²/day. ATRA concentrations were measured in 17 out of 18 treated patients. A. Plasma ATRA concentrations at different time points after administration of the single dose of the drug. Measurements were performed on day 1, day 4 and day 8 during the course of the treatment. B. The peak level of ATRA after first dose. Individual results and mean values in the groups receiving different doses are shown. C. The peaks of ATRA concentrations in all patients.

Figure 2. Effect of ATRA treatment on the proportion of ImC and DCs

MNC from cancer patients and healthy donors were stored in liquid nitrogen. All samples from the same patient or donor were thawed simultaneously and cells were incubated in complete culture medium overnight at 37°C and 5% CO₂. After that time cells were labeled with antibodies and analyzed by multicolor flow cytometry as described in Material and Methods. A, C, E – Proportion of ImC, DCs, and DC/ImC ratio in all 18 patients treated with ATRA. Control represent data from 8 healthy volunteers and donors. Mean and standard error are shown. ** - statistically significant differences from control group (P<0.05). Actual p values are shown in groups that statistically different (p<0.05) from pre-treatment level. B, D. Proportion of ImC and DCs in patients with different ATRA concentration in plasma. Nine patients had lower ATRA concentrations (<135 ng/ml) and 8 patients had higher ATRA concentrations (>150 ng/ml). ** - statistically significant differences from control group (P<0.05). # - statistically significant differenced from pre-treatment level (p<0.05).

Figure 3. Effect of ATRA treatment on the proportion of DC populations.

A–D. Results in patients with different levels of ATRA in plasma. All abbreviation as in Figure 2B and D.

Figure 4. Effect of ATRA treatment on DC function and antigen-specific immune response

A. MNC isolated from patients and donors were irradiated and cultured for 5 days in triplicates in U-bottom 96-well plates with 10⁵ allogeneic T cells at different ratios. ³[H]-thymidine (1 μCi) was added to each well 18 hr prior cell harvesting. Thymidine uptake was measured in liquid scintillation counter as described in Methods. The background values of T-cell proliferation were subtracted. Only one (1:1) ratio is shown. ** - statistically significant differences from control group (P<0.05). # - statistically significant differenced from pre-treatment level (p<0.05). B – D. MNC (2 × 10⁵/well) were incubated in triplicates with 0.1 μg/ml T-T (B), immobilized anti-CD3 antibody (C), or 5 μg/ml PHA (D) for 4 days. ³[H]-thymidine (1 μCi) was added to each well 18 hr prior cell harvesting. The background values (MNC incubated with medium alone) were subtracted. E. – The proportion of CD4⁺CD25⁺GITR⁺ T cells was evaluated. All abbreviations are as in Figure 2.

Figure 5. Effect of IL-2 on ImC and DCs

Fifteen patients with RCC were treated with IL-2 alone. Blood samples were collected prior the start of the treatment and after finish of one 4-week cycle of the therapy. Immunological parameters were evaluated as described above. Actual p values in two-sided paired Wilcoxon-Mann-Witney test are shown.