No abstract

Cancer Radiation therapy (RT) induces cardiovascular disease (CVD) even when the heart is shealed or not irradiated, but there is a paucity of available preventive measures for RT-induced CVD. Ionizing radiation (IR) induces senescence, which was originally discovered to suppress tumorigenesis by inducing cell cycle blockade and necrosis, and positioned IR as pro-senescence cancer therapy. IR-induced senescence cells secrete cytokines, growth factors, and reactive oxygen species (ROS), becoming so called senescence associated secretory phenotype (SASP), and we hypothesize that SASP induction in immune cells cause CVD after RT. Although the involvement of DNA damage response (DDR), efferocytosis, and clonal hematopoiesis drivers (CHD) to SASP induction has been suggested, the exact mechanisms through which RT induces SASP in a specific cell type remains unclear. We characterize most of the major human immune cell lineages in a single assay using mass cytometery (CyTOF). We generated a CyTOF panel which includes antibodies against various senescence phenotype, DDR, efferocytosis, and CHD. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 month after RT from 16 thoracic cancer patients. First, we found the frequency of only B cell subtype was decreased after RT. Second, we obtained 138 functional profiling subsets by unsupervised clustering with our antibody set, and found that T-bet expression was increased in the largest B cell subset of naïve B Cell (CD27 - ) Ki67 lo CD38 lo DNMT3a hi after RT, which showed the good correlation with p-p90RSK expression in the samples from pre-RT and post-RT. Lastly, the significant increase of CD38 expression in the subsets of naïve B cell (CD27 - ) and CD8 + T cell (EMRA) was detected. These data suggest the unique response of naïve B cell (CD27-) to RT with the increase of CD38 expression, and T-bet in the subset of B Cell (CD27 - ) Ki67 lo CD38 lo DNMT3a hi , and also the potential role of p90RSK activation in IR-induced T-bet expression. T-bet plays a role in developing the age-associated B cell (ABC), and the increase of CD38 expression promotes aging-related events. Therefore, the induction of T-bet and CD38 in naïve B (CD27 - ) cell after RT supports the novel role of naïve B cell in IR-induced SASP and subsequent CVD.

Mast cell (MC) is an unique immune cell displaying wide variety of functions. Recent break through concluded MC generation is mostly provided by embryonic Yolk Sac (YS) and AGM, and post-natal BM HSC does not supply MC (HSC-independent) as our last presentation. Because YS erythro-myeloid progenitor (EMP) provide earliest tissue resident macrophage (brain microglia) and MC together as a common progenitor, and one report shows part of tissue resident macrophages are marked by IL-7 receptor (IL-7R) as an exception of lymphoid essential lineage marking, we hypothesized that MC could be also marked by IL-7R. We tested by utilizing IL-7Rcre/flox-dTom model and found that newborn to young peritoneal cavity (PerC) and skin MCs, surprisingly, exhibited more than 90% dTom positivity (90.8 ± 3.1%). IL-7R protein was not expressed on the mouse PerC, skin MCs and E12.5 Fetal Liver MC progenitors. To confirm functional IL-7R involvement for the MC development, we measured MCs in IL-7Rcre/+(Het) and IL-7Rcre/cre (KO) mice. Despite marked reductions of T/B cell counts, PerC MC count was comparable (WT: 2.4 ± 1.6 vs KO: 4.1 ± 2.3 × 10e4), suggesting IL-7R is temporarily expressed in early EMP stage in an only short period. We also tested MC differentiation from the adult BM dTom-negative Lin−Sca+Kit+ (LSK) cells. MC production was comparable between Het and KO and interestingly enough, both WT/KO LSK derived MCs showed least dTom positivity (3.4± 2.5 vs 4.7 ± 3.1 %) whereas more than 70% of macrophages turned dTom positive, suggesting embryonic and adult MC differentiation used different program respectively. Consistently, %dTom in PerC MC was reduced (79.2 ± 8.1 %) in old mice (> 1year), indicating that post-natal de novo MC production is minimum without IL-7R use. Supported by R01AI121197-01A1 NIAID R01AI147685-01 NIAID

Recent progress in developmental immunology has revealed the hematopoietic stem cell (HSC)-independent ontogeny for the various hematopoietic lineages, including mast cells (MC). Adult bone marrow (BM) HSCs fail to generate MC in both lineage tracing and transplantation assays. However, it is unclear whether fetal liver (FL) HSCs can generate MC in vivo. We investigated the MC potential of HSCs from FL and the aorta-gonad-mesonephros (AGM) in lineage tracing and transplantation assays. We employed different genetic mouse models that allowed us to identify the Tomato+ progeny of hematopoietic precursors (Runx1mer-cre-mer), endothelial cells (EC, Cdh5ERT2Cre) and HSCs (Fgd5ERT2Cre) by injecting tamoxifen (TAM) at different embryonic days starting at E7.5. TdTomato (Tom)+ HSCs and MCs were quantified after birth. In addition, we transplanted HSCs from AGM (11.5), FL (E12.5 and E15.5), and adult BM (>6 wks) into irradiated mice. Results: The EC-labeling at E7.5 efficiently generated Tomato+ MC, but not HSCs (similar results were obtained for the Runx1mer-cre-mer model). EC-labeling at E9.5 made equivalent Tom+ labeling efficiency in MC and HSCs (~85%). However, E11.5 EC mostly failed to mark MC, but still generated Tom+ HSCs (~25%). HSC-labeling at E14 or postnatal day 2 efficiently marked HSCs, but not MC. Collectively, our results indicate that active MC production terminates before E12 and that HSCs after E14 lack MC potential. Our transplantation study also shows lack of MC repopulation by HSCs after E15. However, E11.5 AGM and E12.5 FL HSCs repopulated MC, revealing a transient MC production potential of HSCs. Conclusion: MC production by hemogenic ECs terminates before E12 and early HSCs produce MC within a limited time window.