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Clinical Trial
. 2018 Oct:63:282-291.
doi: 10.1016/j.intimp.2018.08.007. Epub 2018 Aug 16.

Targeting myeloid-derived suppressor cells using all-trans retinoic acid in melanoma patients treated with Ipilimumab

Richard P Tobin  1 , Kimberly R Jordan  2 , William A Robinson  3 , Dana Davis  4 , Virginia F Borges  5 , Rene Gonzalez  6 , Karl D Lewis  7 , Martin D McCarter  8
Affiliations
Clinical Trial

Targeting myeloid-derived suppressor cells using all-trans retinoic acid in melanoma patients treated with Ipilimumab

Richard P Tobin et al. Int Immunopharmacol. 2018 Oct.

Abstract

Background: Immune checkpoint inhibitors have improved overall survival rates for many cancers, yet the majority of patients do not respond to treatment and succumb to disease progression. One tumor-related mechanism limiting the efficacy of immunotherapies in melanoma is the recruitment and expansion of myeloid-derived suppressor cells (MDSCs). Therefore, targeting MDSCs in combination with immunotherapies is an attractive strategy to improve response rates and effectiveness.

Methods: We tested this strategy by designing a randomized phase II clinical trial treating advanced melanoma patients with either Ipilimumab monotherapy or Ipilimumab plus all-trans retinoic acid (ATRA). Clinicaltrails.gov identifier (NCT02403778). The frequency of circulating MDSCs and the activation of CD8(+) T cells was measured by flow cytometry. Expression of immunosuppressive genes was measured with quantitative real time-PCR. T cell suppressive functions were measured by mixed lymphocyte reaction.

Results: Here we show that in vitro treatment with ATRA decreases immunosuppressive function of MDSCs in mixed lymphocyte reactions. Additionally, ATRA reduces the expression of immunosuppressive genes including PD-L1, IL-10, and indoleamine 2,3‑dioxygenase by MDSCs. Furthermore, the addition of ATRA to standard of care Ipilimumab therapy appears safe, as ATRA did not increase the frequency of grade 3 or 4 adverse events. Finally, ATRA significantly decreased the frequency of circulating MDSCs compared to Ipilimumab treatment alone in advanced-stage melanoma patients.

Conclusions: These results illustrate the importance of MDSCs in immunotherapy resistance and provide evidence that targeting MDSCs in cancer patients may augment immunotherapeutic approaches.

Keywords: ATRA; Immunotherapy; Ipilimumab; MDSC; Melanoma; Randomized Clinical Trial.

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Conflict of interest statement

Competing interests

RG reports grants, personal fees and other from BMS, grants from Merck, grants and personal fees from Roche/Genentech, grants from Amgen, grants from Incyte, grants from Novartis, grants from Checkmate Pharmaceuticals, grants from Boston Biomedical, grants from Takeda/Millenium, grants from Syndax, grants from Reata, grants from Array Biopharma, grants from Dynavax, grants from Prometheus, grants from Eisai, grants from Celldex, outside the submitted work. KDL reports grants and personal fees from Roche/Genetech, outside the submitted work. All other authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.. Trial Design.
Schematic depiction of the clinical trial design implemented for this study.
Figure 2.
Figure 2.. ATRA reduces the expression of immunosuppressive genes in human MDSCs.
(A) The relative expression of genes associated with MDSCs function were measure by qRT-PCR. Gene expression was measured 96 hours post treatment with GM-CSF (20ng/mL) + IL-6 (20ng/mL) +/− either 2uM ATRA or the vehicle control DMSO. (B) Example histogram depicting the cell-surface expression of HLA-DR and quantification of the mean fluorescence intensity (MFI) of HLA-DR expression on MDSCs treated as in A. (C) Representative histograms depicting the cell-surface expression of CD33, CD11b, CD14 on in vitro generated MDSCs +/− 2uM ATRA or the vehicle control DMSO compared to the appropriate isotype (Iso) control. Quantification of cell surface expression of CD33, CD11b, CD14 on in vitro generated MDSCs +/− 2uM ATRA or the vehicle control DMSO. (D) Representative contour plots depicting the percentage of dividing (CFSE-diluting) T cells following allogeneic T cell activation via MLR stimulation. A 1:1 ratio of responder T cells:suppressor MDSCs cultured with or without the designated ATRA or vehicle (veh.) treated MDSCs. Error bars indicate standard error of the mean. Data are representative of 5 separate experiments. MDSCs were generated from a different donor for each replicate experiment, from a total of 5 separate donors. Error bars indicate standard error of the mean associated with biological replicates. * Denotes p < 0.05.
Figure 3.
Figure 3.. ATRA decreases frequency of circulating MDSCs in melanoma patients.
(A) Representative flow cytometric gating strategy for human MDSCs. (B and D) Frequency of MDSCs of the live CD45(+) lineage(-) (CD3, CD19, CD20, CD56) CD11b(+) population. (C and E) Frequency of MDSCs of all live CD45(+) cells. (F and G) Frequency HLA-DR(+) cells of CD45(+) lineage(-) CD11b(+) population. Data was analyzed at each of the blood draws described in Fig. 1 and compared throughout treatment to analyze the difference between the treatment groups or analyzed pre and post-treatment. (H) Ratio of the frequency of MDSCs to HLA-DR(+) cells. Error bars indicate standard error of the mean. For all graphs, Ipilimumab n = 4 and Ipilimumab + ATRA n = 4. * Denotes p < 0.05.
Figure 4.
Figure 4.. ATRA increases CD8+ T cell activation.
(A) Representative flow cytometric gating strategy showing contour plots analyzing intracellular cytokine staining in unstimulated cells or cells stimulated for 5 hours with PMA + ionomycin. CD8(+) T cells were identified by first gating on single, live, CD3(+)CD8(+) cells. (B) Comparison of the pre-treatment and post-treatment frequency of CD8(+)IFNγ (+)CD107a(+) T cells. (C) Correlation between the frequencies of MDSCs and CD107a(+)IFNγ(+)CD8(+) T cells. Error bars indicate standard error of the mean. * Denotes p < 0.05.
Figure 5.
Figure 5.. ATRA decreases the frequency of circulating eosinophils.
The absolute number of (A) white blood cells, (B) lymphocytes, (C) neutrophils, (D) neutrophil:lymphocyte ratio, (E) eosinophils, and (F) percentage of eosinophils was determined by standard clinical laboratory complete blood counts with automatic-differential. Error bars indicate standard error of the mean. * Denotes p < 0.05.
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