Low-dose cyclophosphamide synergizes with dendritic cell-based immunotherapy in antitumor activity

Joris D Veltman, Margaretha E H Lambers, Menno van Nimwegen, Sanne de Jong, Rudi W Hendriks, Henk C Hoogsteden, Joachim G J V Aerts, Joost P J J Hegmans, Joris D Veltman, Margaretha E H Lambers, Menno van Nimwegen, Sanne de Jong, Rudi W Hendriks, Henk C Hoogsteden, Joachim G J V Aerts, Joost P J J Hegmans

Abstract

Clinical immunotherapy trials like dendritic cell-based vaccinations are hampered by the tumor's offensive repertoire that suppresses the incoming effector cells. Regulatory T cells are instrumental in suppressing the function of cytotoxic T cells. We studied the effect of low-dose cyclophosphamide on the suppressive function of regulatory T cells and investigated if the success rate of dendritic cell immunotherapy could be improved. For this, mesothelioma tumor-bearing mice were treated with dendritic cell-based immunotherapy alone or in combination with low-dose of cyclophosphamide. Proportions of regulatory T cells and the cytotoxic T cell functions at different stages of disease were analyzed. We found that low-dose cyclophosphamide induced beneficial immunomodulatory effects by preventing the induction of Tregs, and as a consequence, cytotoxic T cell function was no longer affected. Addition of cyclophosphamide improved immunotherapy leading to an increased median and overall survival. Future studies are needed to address the usefulness of this combination treatment for mesothelioma patients.

Figures

Figure 1
Figure 1
Increase in the proportion of regulatory T cells in a murine model for mesothelioma. (a) Left: The transcription factor Foxp3, a hallmark of naturally arising CD4+CD25+ Tregs, was expressed inside the tumor (red staining). Cells were counterstained with haematoxylin. [Magnification 200×] Right: A higher magnification (400×) shows the appearance of Foxp3+ cells as indicated by arrows. (b) Tregs (blue staining) were occasionally present in the near vicinity of CD8+ cells (red staining) [Magnification 1000×]. (c) and (d) Percentage of Tregs (defined as CD4+CD25+Foxp3+ cells) from total CD4+ T cells in the peripheral blood and draining lymph nodes is significantly higher at day 10 in tumor-bearing mice compared to healthy mice as observed by flow cytometry. (e) A positive correlation was found between the total tumor weight and the percentage of Tregs, measured in the lymph nodes of tumor-bearing mice. Five mice were euthanized at day 10 (tumor weight varied from 0.1 gram to 1 gram) and three mice at stage with ill health (tumor weight 1.5 to 2.1 gram). Correlation coefficient of the trend line (R-squared) is 0.780 (P  value is  .0039).
Figure 2
Figure 2
Tregs inhibit CTL function in vitro. (a) Activated splenocytes from DC-treated mice were cocultured with CD4+CD25+Foxp3+ cells (purified from tumor-bearing mice) in different ratios. The percentage of intracellular IFN-γ of CD8+ cells was measured using flow cytometry. And (b) the percentage of intracellular IFN-γ and granzyme B expression was measured using FACS. A reduction in the IFN-γ and granzyme B expression was found especially in those fractions where increasing doses of Treg were added to the CD8+ T cells.
Figure 3
Figure 3
CTX influences lymphocyte subsets in peripheral blood and draining lymph nodes of tumor-bearing mice. (a) The percentage of CD19+ cells was significantly decreased (P < .05) in CTX-treated tumor-bearing mice. CD3+ cells were significantly increased (P = .0325) in the peripheral blood of CTX-treated tumor-bearing mice compared to untreated tumor-bearing mice. (b) CD19+ cells were significantly decreased (P < .05) while CD3+ cells were significantly increased (P < .05), while the percentage of CD4+CD25+Foxp3+ cells was significantly decreased (P < .05) in the splenocytes of CTX-treated mice compared to untreated tumor-bearing mice.
Figure 4
Figure 4
CTX combined with DC-based immunotherapy prolongs survival. Kaplan-Meier survival plot showing the effect of the combination of CTX and DC-based immunotherapy. Mice were given drinking water supplemented with 0.13 mg/ml CTX from day 3 till day 10 and day 14 till day 21. Tumor-lysate-pulsed DCs were given at day 12. Survival significantly improved when CTX and DC-based immunotherapy were combined compared to DC-based immunotherapy (P = .0035) or CTX administration alone (P = .0056). Each group contained 8 mice.
Figure 5
Figure 5
Long-period administration of CTX improves survival compared to pre- or post-immunotherapy treatment. Mice were divided into four groups (n = 6 per group) and inoculated with a lethal dose of AB1 tumor cells on day 0. Mice received low-dose CTX before (day 3 till 10) or after (day 14 till 21) immunotherapy or metronomic dosed CTX (day 3 till 10 and day 14 till 21). Groups 2, 3, and 4 were treated with DC-immunotherapy on day 12. Group 1 functioned as a tumor control group and did not receive any treatment. Administration of metronomic dosed CTX was not significantly better than CTX treatment before (P = .840) or after (P = .454) immunotherapy. However, the combination of CTX and immunotherapy was significantly better than no treatment (CTX before immunotherapy compared to untreated P = .0081, CTX after immunotherapy compared to untreated P = .0147, metronomic dosed CTX and immunotherapy compared to untreated P = .0018).

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