To inhibit perforin-mediated cytotoxicity, V��9V��2 T cells were incubated with concanamycin A (CMA, 15 nM) for 30 min at 37��C before co-culture with target CICs, without further washing [27]. To block the relevant cytotoxic pathways, kinase inhibitor Palbociclib specific or isotype-control mAbs were used at 10 ��g/ml final concentration just before co-incubation assay [27]. Real-time Quantitative RT-PCR Total RNA was extracted with the ABI PRISM 6100 Nucleic Acid PrepStation (Applied Biosystems through Life Technologies) according to the manufacturer��s instructions. Random hexamers and an MMLV Reverse Transcriptase kit (Stratagene, La Jolla, CA) were used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7700 Sequence Detector (Applied Biosystems) with Applied Biosystems predesigned TaqMan Gene Expression Assays and reagents according to the manufacturer�� s instructions.
The following probes were used (identified by Applied Biosystems assay identification number): HLA-A, Hs01058806_g1; HLA-B, Hs00818803_g1; HLA-C, Hs00740298_g1; ICAM-1, Mm00516023_m1; CD155, Hs00197846_m1; CD112, Hs01071562_m1; MICA, Hs00741286_m1; MICB, Hs00792952_m1; ULBP-1, Mm01180648_m1; ULBP-2, Hs00607609_mH; ULBP-3, Hs00225909_m1; Fas (CD95), Hs00236330_m1; TNF-R1, Mm00441883_g1; DR4 (TRAIL-R1), Hs00269492_m1; DR5 (TRAIL-R2), Hs00366278_m1. For each sample, mRNA abundance was normalized to the amount of 18S rRNA. Statistics The two-tailed Student��s t test was used to compare significance of differences between groups. All values are expressed as mean �� standard deviation (SD).
Supporting Information Figure S1 A low number of colon CIC spheres retain the capacity to form a tumor when injected s.c. into immunodeficient mice. Subcutaneous tumor growth in NOD/SCID mice 10 weeks after injection of 2000 disaggregated cells from colon cancer spheres. One representative experiment of two performed with cells from different donors is shown. (TIF) Click here for additional data file.(611K, tif) Acknowledgments We thank Ruggero De Maria, Vaclav Horejsi, Marc Bonneville, Giovanni Triolo and Henning Walczak for providing us with the cell lines and reagents. Funding Statement This work has been supported by grants from the Italian Ministry for Instruction, University and Research (contract no.
2008L57JXW to FD), the Italian Ministry of Health (Progetto Ricerca Finalizzata 2007 ��Stem cells in different pathological conditions: innovative therapeutical approches�� to FD), Istituto Carfilzomib Superiore di Sanit�� Oncoproteomic Project 2007-527/B/3A/3 (to GS and FD) and the University of Palermo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Tim-3, a member of the novel T cell immunoglobulin and mucin domain (Tim) family, was originally identified as a negative immune regulator which is expressed on T helper 1 (Th1) cells, but not Th2 cells [1].