CpG-ODN was synthesized at Shanghai Sangon Biological Engineering Technology Services Co Ltd (Sangon, Shanghai, China). CpG-ODN sequences were 5’-TCG TCG TTT TCG GCGCGC CG-3’ (regular letters represent phosphorothioate, bold and italic letters represent phosphodiester). The compound was diluted with phosphate buffer saline (PBS) to a final concentration 250 μg/ml and stored at 4 ℃.
Male C57BL/6 (20~22 g), 10~12 weeks’ old were purchased from SLAC laboratory animals Co. Ltd (SLAC, Shanghai, China), and were kept under standard laboratory conditions (a temperature of 23 ± 2 ℃ with 24 h cycles of fresh air and 12 h light/dark cycle). Food and water were sterilized by 60Co γ rays and high pressure, respectively.
Carbon ions radiation（CIR）
Mice was placed inside a specially designed and well-ventilated acrylic container with dimensions of 8.0 cm ×3.5 cm ×3.5 cm. the acrylic container was placed into the beam path at the entrance plateau throughout the exposure. Whole-body radiation of mice was performed using CIR at initial energy of 250 MeV/u and the average LET of 31.3 keV/μm. Each mouse received 5 Gy of radiation at a dose rate of 0.8 Gy/min. CIR was supplied by Heavy Ion Research Facility at Institute of Modern physics, Chinese Academy of Sciences (HIRF, Lanzhou, China). The acquisition of data (preset beams converted by doses of radiation) was automatically calculated and controlled by a microcomputer.
Treatment and Ethics approval
Mice were treated with either 50 μg CpG-ODN (250 μg/ml) or 0.2 ml PBS via intraperitoneal (i.p) injection. CpG-ODN was given 30 min, 24 h and 48 h after CIR. The effective dosage and the time points of CpG-ODN treatment in this study was selected based on data from previous study  Mice were randomly divided into four groups as follows: a normal group (the non-irradiated mice with PBS treatment), a CpG-ODN group (the non-irradiated mice with CpG-ODN treatment), a CIR control group (the irradiated mice with PBS treatment) and a CIR plus CpG-ODN group (the irradiated mice with CpG-ODN treatment). All studies were performed under the guidelines and protocols of the Institutional Animals Care and Use Committee of the Lanzhou General Hospital of PLA (IACUC approval#15010936) according to the Guide for Care and Use of Laboratory Animals published by US NIH (publication No 96~101).
Irradiated mice were observed to monitor survival for 30 days after CIR. Moribund mice were euthanized. On 31th day, surviving mice were euthanized by cervical dislocation. Data were expressed as percentage survival. 28 mice per groups were used for the experiments. The Mean Survival Time (MST) was calculated by Kaplan-Meier method.
The peripheral blood was obtained from halothane-anesthetized mice and collected in tubes containing heparin, then mice were killed humanely by cervical dislocation. Whole blood samples were evaluated by automated hematology system (CA-700NVET, STAC Group, China). White blood cell (WBC), lymphocytes, granulocyte, and monocytes counts were determined. T cells and B cells were examined by immunofluorescence . The specific method is: 65 μl of 10 % formaldehyde was added to surplus 100 μl of blood and incubated for 10 min at room temperature, followed by the addition of 1ml Triton X-100 (diluted in PBS) to obtain 0.1 final concentrations. After 30 min of incubation at room temperature, 1ml cold wash buffer was added. Samples were centrifuged, suspended in 1ml 50 % methanol diluted in PBS (stock stored at -20 °C), and incubated at 4 °C for a minimum of 10 min. All cells were stained blue by 4', 6-diamidino-2-phenylindole (DAPI, Sigma, USA). Rabbit polyclonal antibodies of CD3 (cat: 100203) and CD19 (cat: 115507) from Bio Legend were used to identify T cells and B cells respectively. These samples were examined and quantified under fluorescent microscope (Nikon, Japan).
Bone marrow cell count and bone marrow histological examination
two femurs were removed at the times of euthanasia. The bone marrow cells within one femur were flushed with PBS, and the number of live bone marrow cells were determined by a cell counter analyzer system (CASY, Innovates, Germany). The other femur was fixed in 4 % paraformaldehyde for 24 h, treated with a formic acid sodium citrate decalcification solution for 5 days, embedded in paraffin and stained with Hematoxylin and Eosin (H&E).
Mice were weighted and then euthanized by cervical dislocation. The whole thymus and spleen were rapidly excised, their masses were measured. Relative organ index was calculated: relative organ index= organ mass (mg)/body mass (g).
TUNEL assay and γ-H2AX assay
As description in M. Leonor Fernández-Murga's article  whole thymus and spleen were fixed for 48 h in 10 % neutral buffered formalin, 5-μm section was prepared and stained with H&E. Apoptotic cells were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining. 5-μm section was deparaffinized, permeabilized with proteinase K (20 μg/ml, Sigma) at 23 ℃ and rinsed 4 additional times with distilled water followed by incubation with a solution made up of TdT (1 μg/200 μl of mix solution), bovine serum albumin (1mg/ml), and biotin-16-dUTP (1 nmol/50μl of mix solution) in TdT buffer. Sections were then incubated in saline citrate for 15 min, rinsed and incubated with 2 % bovine serum albumin at 23 ℃. Endogenous peroxidase activity was blocked by incubating the tissue sections with 3 % H2O2. The labeled DNA fragments were detected with peroxidase-conjugated antibody elicited against biotin. Slides were developed with DBA (diaminobenzidine tetrahydrochloride), and stained with hematoxylin. Double strand breaks (DSB) were investigated by γ-H2AX immunohistochemistry. 5-μm section was prepared. The primary antibodies used were mouse monoclonal anti-γ-H2AX (1:100 Santa Cruz Santiago USA) at 4 ℃ overnight in humidified incubator. Unbound antibody was removed by several washing and goat anti-mouse antibody (1:100 Santa Cruz Santiago USA) was applied as a secondary labeled antibody for 30 min at room temperature. Slides were developed with DBA and stained with hematoxylin.
For the survival data, the difference in 30 days’ survival between was analyzed by Kaplan-Meier plots. For other data, the difference between CIR control group and CIR plus CpG-ODN group was analyzed by the two-sided, non-paired Student’s t-test. Data are expressed as mean ± standard error mean (SEM). The software analysis program SPSS 13.0 (release 12.0K; SPSS Inc. Chicago, USA) was used. All differences were considered significant if p was less than 0.05.