5-Azacytidine treatment sensitizes tumor cells to T-cell mediated cytotoxicity and modulates NK cells in patients with myeloid malignancies

Abstract

Treatment with the demethylating agent 5-Azacytidine leads to prolonged survival for patients with myelodysplastic syndrome, and the demethylation induces upregulation of cancer-testis antigens. Cancer-testis antigens are well-known targets for immune recognition in cancer, and the immune system may have a role in this treatment regimen. We show here that 5-Azacytidine treatment leads to increased T-cell recognition of tumor cells. T-cell responses against a large panel of cancer-testis antigens were detected before treatment, and these responses were further induced upon initiation of treatment. These characteristics point to an ideal combination of 5-Azacytidine and immune therapy to preferentially boost T-cell responses against cancer-testis antigens. To initiate such combination therapy, essential knowledge is required about the general immune modulatory effect of 5-Azacytidine. We therefore examined potential treatment effects on both immune stimulatory (CD8 and CD4 T cells and Natural Killer (NK) cells) and immune inhibitory cell subsets (myeloid-derived suppressor cells and regulatory T cells). We observed a minor decrease and modulation of NK cells, but for all other populations no effects could be detected. Together, these data support a strategy for combining 5-Azacytidine treatment with immune therapy for potential clinical benefit.

Figure 1

Enhanced direct ex vivo cytotoxicity in patients treated with 5-Azacytidine. CD8 T-cell reactivity upon co-culture with CD34 myeloid blasts is depicted, measured by CD107a expression on the CD8 T cells. T cells and myeloid blasts were isolated from a first cycle sample (pre-treatment) and from a late cycle (4th-6th cycle) sample, separated and co-cultured in four combinations. (a) First cycle T cells against first and late cycle myeloid blasts. (b) Late cycle T cells against first and late cycle myeloid blasts. (c) First and late cycle T cells against first cycle myeloid blasts. (d) First and late cycle T cells against late cycle myeloid blast cells. Note that AZA 14 is only included in (c, d). The frequency of CD107a T cells are given in percentage of CD8 T cells. Significance is indicated by *P<0.05.

Figure 2

Cancer testis antigen (CTA)-specific T cells in the peripheral blood of patients. Detection of CTA- or viral-specific T cells in PBMCs by MHC multimers, expressed in percentage of CD8 T cells. (a) The sum of CTA-specific T cells as measured in peripheral blood at different time points during treatment. Eight patients were tested, results from the six patients with detectable responses are shown. First cycle represents a sample obtained before treatment. The following responses were found for each patient: AZA 1 (SART-3_WLE, SART-3_QIR, Sp17_ILD), AZA 2 (MAGE-A2_LVH, MAGE-A2_KMV, TAG-1_SLG), AZA 4 (MAGE-A2_LVH, MAGE-A2_LVQ, NY-ESO-1_QLS), AZA 5 (MAGE-A1_EAD), AZA 12 (MAGE-A2_LVH, MAGE-A2_KMV, CDCA1_KLA, TAG-1_SLG, NY-ESO-1_SLL, MAGE-A1_EAD) and AZA 16 (MAGE-A2_LVH, MAGE-A2_KMV, GnTV_VLP, TAG-1_SLG). (b) The frequency of individual CTA-specific T cells detected after an in vitro peptide pre-stimulation was performed at different time points during treatment. (c) The sum of virus-specific T cells detected over the course of treatment. The following responses were detected: AZA 1 (EBV_RLR, EBV_RLR, FLU_ILR), AZA 2 (EBV_GLC, FLU_ILR), AZA 4 (EBV_GLC, EBV_YVL, FLU_GIL), AZA 5 (FLU_BP-VSD), AZA 12 (CMV_VTE, CMV_YSE, CMV_NLV, FLU_GIL), AZA 14 (CMV_YSE, CMV_VTE, FLU_BP-VSD), AZA 16 (CMV_NLV, EBV_GLC) and AZA 17 (CMV_YSE, CMV_VTE, FLU_BP-VSD). MHC-multimer-specific T cells are given in percentage of CD8 cells. Significance is indicated by *P<0.05.

Figure 3

General immune effector cells are not affected by 5-Azacytidine treatment in vivo. The number and reactivity of NK cells and CD8 and CD4 T cells are shown. First cycle represents a sample obtained before treatment. (a, c, e) absolute peripheral blood counts of CD8 and CD4 T cells and CD3⁻CD56⁺CD16^+/− NK cells, respectively. (b, d, f) Expression of CD107a on CD8 and CD4 T cells and NK cells, respectively, in response to SEB (for T cells) or to K562 cells (for NK cells).

Figure 4

Functional capabilities of NK cells affected by 5-Azacytidine in vivo and in vitro. The number and functional activity of NK cells, when further divided in subsets and measured over a longer treatment period, and the in vivo and in vitro impact of 5-Azacytidine on NK-cell functionality. (a) Absolute peripheral blood counts of CD3⁻CD56⁺CD16⁺ NK cells, P=0.061 for first versus late cycle. (b) Absolute peripheral blood counts of NK cells with the inhibitory phenotype CD3⁻CD56⁺CD16⁺CD158b⁺. (c) Absolute peripheral blood counts of NK cells with the activating phenotype CD3⁻CD56⁺CD16⁺CD158d⁺. (d) NK cell-mediated killing of K562 cells after 5-Azacytidine addition, either once, or every 24 h (circles, 5-Aza continuous addition) or only at the initiation of the 72 h culturing period (triangles, 5-Aza one addition). Analyses were performed on two healthy donors (black and gray symbols, respectively). K562 killing was determined by a flow cytometry-based NK cell-killing capacity assay. NK cell-mediated killing of K562 cells was compared to a negative control with no effector cells present and killing of a HLA-A3 transduced K562 line. Counts are given in 10⁶ cells/l of blood. First cycle represents samples obtained before treatment. Significance is indicated by *P<0.05.

Figure 5

Inhibitory cell subsets are not affected by 5-Azacytidine treatment in vivo. Analyses of Tregs and monocytic MDSCs during 5-Azacytidine treatment are shown. (a) Absolute peripheral blood counts of CD4⁺CD25⁺CD127⁻FOXP3⁺CD49d⁻ Tregs over the course of treatment. (b) Absolute peripheral blood counts of CD3⁻CD19⁻CD56⁻HLA-DR⁻CD33⁺CD11b⁺CD14^highCD15^low monocytic MDSCs over the course of treatment. Counts are given in 10⁶ cells/l of blood. First cycle represents samples obtained before treatment.