Kit ligand (KitL) and its tyrosine kinase receptor c-kit are critical for germ cells, melanocytes, mastocytes, and hematopoietic stem cells. Alternative splicing of KitL generates membrane-bound KitL (mb-KitL) or soluble KitL, providing survival or cell migration, respectively. Here we analyzed whether c-kit can function both as an adhesion and signaling receptor to mb-KitL presented by the environmental niche. At contacts between fibroblasts and MC/9 mast cells, mb-KitL, and c-kit formed ligand/receptor clusters that formed stable complexes, which resisted dissociation by c-kit blocking mAbs and provided cell anchorage under physiological shear stresses. Clusters recruited tyrosine-phosphorylated proteins and induced spatially restricted F-actin polymerization. Mutational analysis of c-kit demonstrated kinase-independent mb-KitL/c-kit clustering, anchorage, F-actin polymerization, and Tyr567-dependent cluster phosphorylation. Kinase inhibition of c-kit by imatinib reduced cluster coalescence, but allowed cluster phosphorylation and F-actin polymerization, which required PI3K recruitment and a newly identified juxtamembrane residue. Synergies between integrin and c-kit-mediated spreading and adhesion of MC/9 cells were studied in vitro on immobilized-KitL/fibronectin surfaces. While c-kit blocking antibodies prevented spreading, imatinib blocked spreading induced by soluble- but not immobilized KitL. Thus, "mechanical" activation of c-kit provides signaling, niche-anchorage, and synergies with integrin-mediated adhesion, which is independent of kinase function and resistant to c-kit kinase inhibitors.-
8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME), an analogue of cAMP. Simultaneous measurements of intracellular calcium ([Ca 2þ ] i ) with Fura-2 and isometric force were made in RV trabeculae before and during cpTOME application. At 1.5 mM [Ca 2þ ] o , 10 mM cpTOME had no effect on the amplitude of [Ca 2þ ] i transients (0.7 5 0.1 to 0.7 5 0.1, P ¼ 0.35) or on twitch force (24.1 5 5.2 mN mm À2 to 22.80 5 4.62 mN mm À2 , P ¼ 0.20) (n = 7). However, at 0.5 mM [Ca 2þ ] o , cpTOME increased peak stress from 10.5 5 2.8 mN mm À2 to 15.0 5 2.7 mN mm À2 , P = 0.01 (n = 6), but without any change in [Ca 2þ ] i transients (P = 0.16). The force-[Ca 2þ ] i relationship of intact trabeculae exhibited increased myofilament Ca 2þ sensitivity with cpTOME at low [Ca 2þ ] o but not at 1.5 mM [Ca 2þ ] o . In isolated cells, cpTOME increased Ca 2þ spark frequency (Fluo-4) from 6.6 per 100 mm 3 s (n = 3) to 32.3 per 100 mm 3 s (n = 9), P = 0.05, with a reduction in the peak amplitude of the sparks. The latter result recapitulates the idea that changes in RyR sensitivity do not alter the amplitude of Ca 2þ transients (Eisner, 2009).
Introduction: Chimerism changes post hematopoietic cell transplant (HCT) can be an early indication of disease relapse. Graft immune cell fraction composition has been seen associated with clinical outcome of hematopoietic cell transplantation patients (Saliba et al., Haematologica February 2020). AlloSeq HCT (CareDx Inc., CA, USA) is the latest innovation in chimerism testing, an NGS-based chimerism assay that uses a targeted SNP panel and provides standardized workflow with automated data analyses. Objectives: We aimed to analyze graft immune cell composition using the AlloSeq HCT assay and compared it to the current standard laboratory method based on Short Tandem Repeat (STR) analysis. Methods: A total of 8 post HCT samples from peripheral blood were taken at the same time point for cellular subfraction and isolation of CD15+, CD33+, granulocytes (GR) and monocytes (MN) cells prior to genomic DNA (gDNA) extraction. For each recipient, donor gDNA was input as a reference sample. The kit-based test AlloSeq HCT was used to prepare libraries and sequenced on the Illumina MiSeq platform. AlloSeq Software performed the QC and data analysis for the evaluation of percent chimerism (median ± CI 95%). Results below limit of detection (LOD of 0.32% for AlloSeq HCT and 3% for STR) were excluded in the Mann-Whitney and Spearman correlation analyses. For inter-assay variation 3 subfraction samples with results along the dynamic range were repeated 2-3 times on AlloSeq HCT and the coefficient of variation (CV) was calculated. Results: CD3+ cells dominated graft cellular composition with a chimerism of 31.6% (10.4 to 44.3%), followed by MN cells at 10.0% (1.4 to 18.6%) and GR cells at 7.3% (0.56 to 13.9%), and CD15+ was the smallest fraction with 2.9% (-4.0 to 9.8%). Median levels of chimerism in each cellular subfraction of the grafts were not statistically significant different when comparing AlloSeq HCT and STR (p>0.5) (Fig. 1A). Results of percent of chimerism obtained for each cell fraction using both methods exhibited an overall correlation of r=0.96 (CI 95%: 0.88 to 0.98) (Fig. 1B). LOD results were reported in 5 with AlloSeq HCT compared to 9 with STR out of the total 26 subfraction tested (19% vs 35%). The inter-run CV was 1.7, 14.5 and 1.6 with a means percent chimerism of 0.8, 17.0 and 14.3, respectively. Conclusion: The immune cell composition of the graft can be characterized in clinical practice with NGS-based chimerism assay. Evaluation of AlloSeq HCT showed excellent assay performance and a larger dynamic range compared with STR, as well as increased sensitivity by reporting 4 cases where percent chimerism was undetectable by STR. NGS-based medicine shows great promise for better detection and definition of HCT graft with a streamlined workflow. Figure 1 Disclosures Kyle: CareDx: Current Employment. Casas:CareDx: Current Employment. Grskovic:CareDx: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding. Viard:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding.
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