Summary Intrinsic immunosuppression is a major obstacle for a successful cancer therapy. Mechanisms how immunosuppression is induced and regulated in humans are ill-defined. A micro-environmental component that might prevent anti-tumor immunity is the presence of dying tumor cells, which is abundant following conventional cancer ablation methods such as chemo- or radiotherapy. Shedding of apoptotic debris and/or secretion of factors to the tumor bed or draining lymph nodes thus might have a profound impact on professional phagocytes such as DC and subsequent priming of lymphocytes. In this study, we exposed human DC to supernatants of living, apoptotic or necrotic human breast cancer cells and co-cultured them with autologous T cells. Priming with apoptotic debris prevented DC from establishing cytotoxicity towards living human tumor cells by inducing a regulatory T cell population, defined by co-expression of CD39 and CD69. Immunosuppression via Treg was transferable and required the release of sphingosine-1-phosphate (S1P) from apoptotic cells, acting via S1P receptor 4 on DC to induce IL-27 secretion. We propose that CD69-expression on CD39+ Treg enables them to interact with CD73-expressing CD8+ T cells to generate adenosine, thereby suppressing cytotoxicity. These findings aid the understanding how dying tumor cells limit anti-tumor immunity.

IntroductionMammalian hematopoiesis in the BM is regulated among others by the oxygen (O 2 ) availability. O 2 concentrations in the BM range from anoxia to 6% opposed to 4%-14% in well-oxygenated tissues, including the blood. 1,2 Recent data indicate that O 2 gradients within the BM participate in keeping hematopoietic stem cells (HSCs) in a low-replicating pluripotent state. HSCs are located in an extremely hypoxic niche as demonstrated by dye-perfusion and engraftment studies. 3,4 Hypoxia-inducible factors 1-3 (HIF-1-HIF-3) are stabilized by a low pO 2 to induce adaptive gene expression. They are heterodimers consisting of distinct O 2 -sensitive ␣-subunits and a stable common ␤-subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT). 5 HIF-1 and HIF-2 were recently connected to HSC biology. In their hypoxic niche, HIF-1 maintains HSC quiescence, 6 whereas HIF-2 maintains their self-renewing capacity. 7 HIF-1 also affects embryonic hematopoiesis as demonstrated by defective myeloid and erythroid progenitor formation in HIF-1␣ Ϫ/Ϫ as well as ARNT Ϫ/Ϫ embryos. 8 In adult hematopoiesis, HIF-1 is essential for B-cell progenitor proliferation and mature B-cell subclass differentiation. 9 However, its involvement in mononuclear phagocyte development is unknown. Previous findings indicated defective development of human plasmacytoid dendritic cells (pDCs) under hypoxia in vitro. 10 Therefore, we asked whether HIF-1 regulates DC lineage differentiation in mice. Methods AnimalsHIF-1␣ fl/fl or HIF-2␣ fl/fl mice 11,12 were bred with LysM-Cre transgenic mice 13 in the C57BL/6 background. Age-matched C57BL/6 wild-type (WT) mice were controls. ID2 Ϫ/Ϫ mice and their respective WT control were in the NMRI background. 14 The guidelines of the Hessian animal care and use committee were followed. DC generation from BMFor DC generation in vitro, 2 ϫ 10 6 total BM cells/mL in RPMI 1640 with 10% FCS and 200 ng/mL recombinant murine fms-related tyrosine kinase 3-ligand (Flt3-L, PeproTech) were cultured in 6-well Ultra-Low attachment plates (Corning) for up to 9 days 15 at various O 2 levels as indicated, using a InVivo 2 400 hypoxia workstation (Ruskinn Technologies). Alternatively, cells were cultured with 100M dimethyloxallyl glycine (DMOG, from Biomol). Sorted monocyte/DC progenitors/common DC progenitors (MDPs/ CDPs; 10 4 cells/well) were cultured with 200 ng/mL Flt3-L in 24-well Ultra-Low attachment plates. Flow cytometry and cell sortingBM cells, spleen, or whole blood cells were stained with fluorochromeconjugated antibodies and analyzed on a LSRII/Fortessa flow cytometer (BD Biosciences). MDP/CDP were sorted from lineage Ϫ cell-enriched BM (lineage cell depletion kit and AutoMACS cell separator from Miltenyi Biotec) using a FACSAria III cell sorter (BD Biosciences). For details (antibodies, surface markers, intracellular transcription factor staining procedures), see supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). pDCs were i...

Recent genome-wide surveys have revealed age-related genetic mosaicism and established a link between clonal mosaicism and risk of hematological and solid-tumor cancers (Laurie et al, 2012, Nat Genet, 44:642; Jacobs et al, 2012, Nat Genet, 44:651; Forsberg et al, 2012, AJHG, 90:217). Existing methods to identify chromosomal aberrations leading to mosaic allelic imbalance (AI) rely on the detection of increased variance of the estimated within-sample allele frequencies at heterozygous loci, ignoring the dependence among allele frequencies in regions of an AI-inducing event, such as a deletion or copy-neutral loss of heterozygosity (LOH), expected from the loss or gain of a chunk of an inherited chromosome. As a result, they break down when the proportion of DNA from an aberrant source is below 10-15%. Here we present a suite of new methods (hapLOH, J-LOH, hapLOHseq) that harness the important information contained in the germline haplotypes to help distinguish signal from noise, accommodate multiple samples of different tumor purities (to deconvolute aberration type and tumor purity) and infer co-occurrence of aberrations in heterogeneous mixtures. For example, our initial method, hapLOH (Vattathil & Scheet, 2013, Gen Res 23:152), can identify 10 Mb regions with just 4% of the signal coming from the tumor genome in data simulated from a series of experiments from a 317K SNP microarray. Application of hapLOH to data analyzed by Laurie et al (2012) indicates that we have substantially greater sensitivity to detect clonal mosaicism, particularly events harbored in a small proportion of cells (< 5-10%). We identify more than twice as many mosaic events, demonstrating the potential role for our methods to aid in profiling cancer risk. Our suite of tools is designed for both microarray and next-generation sequencing data and is available at scheet.org. Citation Format: Paul Scheet, Rui Xia, F. Anthony San Lucas, Christina Hahn, Jerry Fowler, Selina Vattathil. Novel computational methods reveal subtle clonal mosaicism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5320. doi:10.1158/1538-7445.AM2014-5320