Ril 27

To induce MHC mismatched acute GvHD, BALB/c mice received lethal irradiation (800 cGy) from a Cesium-137 irradiator, split into 2 doses in a 3 h interval to minimize gastrointestinal (GI) toxicity and were intravenously transferred with 6 × 10⁶ T-cell-depleted bone marrow (TCD-BM) cells isolated from C57BL/6 mice and 0.7 × 10⁶ MACS-purified CD3⁺ T cells from Thy1.1 mice. Sorted Tregs (from CD45.1⁺ Foxp3^GFP mice) were stimulated with plate-bound anti-CD3 (clone 2C11, 2 μg/ml), soluble anti-CD28 (clone 37.51, 2 μg/ml), IL-2 (100 U/ml) in the presence or absence of rIL-27 (10 ng/ml, R&D system, Minneapolis, MN) for 24 h prior to transfer into BALB/c recipients on the same day of GvHD induction. Survival was monitored for 60 days. GvHD severity was assessed twice a week by scoring system that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity as previously reported (31 (link)). Each parameter received a score of 0 (minimum) to 2 (maximum) (maximum index = 10).
For graft-versus-leukemia (GvL) experiments, 2 x 10⁴ luciferase-expressing A20 lymphoma cells were intravenously injected together with the donor graft as indicated. Tumor growth was measured on day 7 and 14 post-transfer using an IVIS SpectrumCT in vivo imaging system as previously described (32 (link)). For all experiments, a 30 s exposure time was used.

The following mAbs were produced in our laboratory: A6136 (IgM) and 6A4 (IgG1) (anti-HLA class I-A, -B, -C and HLA-E), FST24 (IgG2b, anti-HLA-II), A6/220 (IgM, anti-CD56), BAB281 (IgG1) and KL247 (IgM) (anti-NKp46), AZ20 (IgG1) and F252 (IgM) (anti-NKp30), BAT221 (IgG1, anti-NKG2D), KRA236 (IgG1) and F5 (IgM) (anti-DNAM-1), BAM195 (IgG1, anti-MICA), M5A10 (IgG1, anti-PVR), U191 (IgM, anti-Nectin-2), c227 (IgG1, anti-CD69). MAB1380 (IgG2a, anti-ULBP1), MAB163903 (IgG2A, anti-ULBP2), MAB1517 (IgG2A, anti-ULBP3) and M475 (IgG2B, anti-ULBP4) mAbs were purchased from R&D System Inc., (Minneapolis, MN, USA); anti-CD107a-PE and anti-CD56-PC5 mAbs were purchased from Becton Dickinson. Anti-PD-L1.3.1 (IgG1, anti-PD-L1) and anti-PD-L2 (IgG1, anti-PD-L2) were generated in D. Olive's lab.
Human recombinant cytokines were purchased from PeproTech (rIL-15, rIFN-γ and rTNFα) MBL International (rIL-18) and R&D Systems (rIL-27).

Peripheral blood mononuclear cells were stained for CD11b, CD33, CD14, HLA-DR; CD11b⁺CD33⁺CD14⁺HLA DR^-/lo MDSC and CD11b⁺CD33⁺CD14⁺HLA DR^+/hi monocytes were isolated using Beckman Coulter MoFlo flow cytometer; sorted cells were >90% positive (Supplemental Figure 1). Isolated cells (50, 000 – 80, 000) were cultured in RPMI 1640 (Gibco) and 10% human serum (MP Biomedicals) at 37⁰C and 5% CO₂ in the presence or absence of, M tuberculosis Erdman (Erdman) or M tuberculosis H37Rv whole cellular lysate (WCL) (10 µg/ml) (BEI resources) for 24 hrs. Supernatants and cells in Trizol were stored at -80°C for cytokine measurement and RNA purification, respectively. For some experiments, cells were cultured in the absence or presence of recombinant IL-27 (rIL-27) (10 ng/ml; R&D Systems).

C57BL/6 mice were injected with zymosan (1 mg in 1 mL PBS) intraperitoneally (i.p) with or without co-injection of rIL-27 (200 ng, R&D Systems). After 24 h, blood or 5 mL peritoneal lavage (PBS) was obtained.