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. 2012 Feb 15;188(4):1592-9.
doi: 10.4049/jimmunol.1101304. Epub 2012 Jan 9.

Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells (V体育官网)

Yuko Shirota  1 , Hidekazu Shirota, Dennis M Klinman
Affiliations

Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells

Yuko Shirota et al. J Immunol. .

V体育官网入口 - Abstract

Immunostimulatory CpG oligonucleotides (ODN) activate cells that express TLR9 and have been shown to improve the host's response to tumor Ags VSports手机版. Unfortunately, the immunosuppressive microenvironment that surrounds many cancers inhibits Ag-specific cellular responses and thus interferes with CpG-mediated immunotherapy. Myeloid-derived suppressor cells (MDSC) represent an important constituent of this immunosuppressive milieu. Large numbers of MDSC are present in and near tumor sites where they inhibit the activity of Ag-specific T and NK cells. Current studies indicate that the delivery of CpG ODN directly into the tumor bed reduces the immunosuppressive activity of monocytic (CD11b(+), Ly6G(-), Ly6C(high)) MDSC. Monocytic MDSC express TLR9 and respond to CpG stimulation by 1) losing their ability to suppress T cell function, 2) producing Th1 cytokines, and 3) differentiating into macrophages with tumoricidal capability. These findings provide insight into a novel mechanism by which CpG ODN contribute to tumor regression, and they support intratumoral injection as the optimal route for their delivery. .

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Figures

Figure 1
Figure 1
Intra-tumoral injection of CpG ODN impacts tumor immunity. 105 CT26 colon cancer cells were injected s.c. into BALB/c mice on day 0. These formed solid tumors that reached an average diameter of >1 cm by day 14. A-C) On days 14 and 15, mice were injected i.p. or intra-tumorally with 200 ug of CpG or control ODN, and tumor size monitored. C) Mice were also injected i.p. with anti-CD8, anti-asialo GM1 (NK cell) or control Ab on days 12, 14, 17 and 20. Data represent the mean + SE of 5-8 mice/group from 2 independent experiments. * p < 0.05 compared with the untreated (tumor alone) group ** p < 0.01 compared with the untreated (tumor alone) group
Figure 2
Figure 2
Effect of intra-tumoral CpG ODN on CD11b+ cells. A) Mice were treated as described in Fig. 1. The number of tumor infiltrating CD11b+ cells that expressed Ly6c and Ly6g was determined on day 17 by FACS. A) Representative results from one mouse/group and B) mean + SD from 5 independently analyzed mice/group, showing Ly6c and Ly6g expressing cells as a percentage of all tumor infiltrating CD45+ lymphocytes. C) The frequency of CD11b, Ly6c double positive cells as a percentage of CD45+ tumor infiltrating cells was examined 2 - 5 days post CpG administration. Mean + SD from 4-7 independently analyzed mice/group. * p < 0.01 versus untreated group
Figure 3
Figure 3
Monocytic MDSC express TLR9 and respond to CpG ODN. Spleens were removed from CT26 tumor bearing mice and CD11b+, Ly6c+, Gr- 1intermediate or CD11b, Ly6g+, Gr-1hi MDSC were FACS sorted to >98% purity. (A) TLR9 mRNA levels were determined by RT-PCR in comparison to the RAW 264.7 macrophage cell line (positive control) and EL4 thymoma cell line (negative control). (B) 105 FACS sorted MDSC were cultured with 1 uM CpG or control ODN for 24 hr. Culture supernatants were assayed for IL-12 levels by ELISA. Data represents the mean + SD from 3 independent experiments. (C) MDSC isolated independently from the spleen or tumor of CT26 tumor bearing mice were cultured with 1 uM CpG or control ODN for 8 h. Brefeldin A was added during the final 4 h of incubation. Cells were stained to identify CD11b+, Ly6c+, Gr-1intermediate mMDSC for the presence of intracytoplasmic IL-12. All experiments were repeated three times with similar results. * p < 0.01 versus untreated group
Figure 4
Figure 4
CpG ODN treatment inhibits the suppressive activity of monocytic MDSC A,B) mMDSC from the spleen of CT26 tumor bearing mice were isolated by MACS (final purity 90 - 95%). These cells were cultured for 3 h with 1 uM CpG or control ODN and then washed. 5 × 105 ODN-pulsed MDSC were cultured for 3 days with 5 × 105 CFSE-labeled CD8+ HA-specific Tg T cells plus 106 mitomycin C treated spleen cells (as APCs) in the presence of 0.1 ug/ml HA peptide. CD8 T cell proliferation was monitored by CFSE dilution. A) Representative example, B) mean + SD (N = 5 independent MDSC preparations in 2 independent experiments). C) Mice bearing 1.5 cm diameter CT26 tumors were injected i.p. with 300 ug of CpG or control ODN. Three hours later, spleens were removed and Ly6chigh, Gr-1intermediate MDSC isolated by MACS sorting. These MDSC were co-cultured with CFSE-labeled CD8+ HA-specific Tg T cells plus HA peptide for 3 days, as described above. The proliferation of CD8+ T cells was monitored by CFSE dilution. Results represent the mean + SD of 4-7 independent mice/group studied in 3 independent experiments. * p < 0.01 versus CpG-treated MDSC.
Figure 5
Figure 5
CpG ODN treatment down-regulates the production of NO and arginase by mMDSC. mMDSC purified by MACS sorting were cultured with HA-specific Tg CD8+ T cells plus 106 mitomycin C treated spleen cells (as APCs) in the presence of HA peptide for 1 day as described in Fig 4. NO levels in culture supernatants were quantified using the Griess reagent. mMDSC purified by MACS were pulsed with 1 uM CpG or control ODN for 36 to 48 hr and analyzed B) for arginase 1 mRNA levels by real time qPCR and C) for expression of Ly6c and arginase I by flow cytometry. Results represent the mean + SD of results from 5 independent MDSC preparations. * p < 0.01 versus untreated MDSC group
Figure 6
Figure 6
Differentiation of monocytic MDSC. mMDSC were MACS purified as described in Fig 4. The cells were incubated with 1 uM CpG or control ODN for 48 hr and then analyzed for the expression of Ly6c and F4/80 by flow cytometry. Representative results are shown by dot plot (A) and histogram (B) (shadow; untreated, dotted line; control ODN, solid line; CpG ODN). The mean + SD in MFI from 4 independent experiments is shown in (C). D) MDSC were pulsed with CpG or control ODN for 3 hr, washed extensively, and then transferred to the upper well of a transwell plate. Untreated MDSC were added to the lower well of the same plate. After 48 hr, both cell populatons were analyzed for the expression of Ly6c and F4/80 by flow cytometry. Experiments were repeated three times with similar results. * p < 0.01 versus untreated group.
Figure 7
Figure 7
Effect of TLR ligands on monocytic MDSC A) Spleens were removed from CT26 tumor bearing mice. mMDSC (identified as CD11b, Ly6c+, Gr-1intermediate) were FACS sorted to >98% purity. TLR 2, 3, 4, 7 and 8 mRNA levels were determined by RT-PCR in comparison to the RAW 264.7 macrophage cell line (positive control) and EL4 thymoma cell line (negative control). B,C) Purified mMDSC were cultured with 1 uM ODN, 1 ug/ml peptideglycan (PGN), 30 ug/ml Poly(I:C), 1 ug/ml MPL 10ug/ml Imiquimod. B) Culture supernatants were assayed after 24 hr for IL-6, TNFα, IL-12 and IL-10 levels by ELISA (all values in ng/ml). Data represents the mean + SD from 3 independent experiments. C) CD11b+cells stimulated for 48 hr were analyzed for the expression of Ly-6c and F4/80 by flow cytometry. Experiments were repeated three times with similar results.
Figure 8
Figure 8
Effect of CpG treated monocytic MDSC on tumor growth. 105 (A) or 3×104 (B) CT26 colon cancer cells were injected s.c. into BALB/c mice. mMDSC were MACS purified from other donor mice as described in Fig 4, pulsed with CpG or control ODN for 3 hr and then washed extensively. 2 × 106 ODN-treated MDSC were injected into the tumor site on days 0 and 2 (A) or into established tumors on days 12 and 13 (B). Data show tumor size in 6 - 7 mice/group from 2 independent experiments. C) ODN-pulsed MDSC were mixed with CT26 target cells at various E:T ratios in vitro. Specific lysis was determined by quantitative measurements of LDH. Results are the mean from 3 independently studied cell populations/group. * p < 0.01 compared with the untreated treatment group or untreated MDSC.
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