SummaryIntestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC(50)] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101.

IntroductionChronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the United States, with approximately 15 000 new cases and approximately 4500 deaths per year. 1 CLL is characterized by a B1 monoclonal lymphocyte immunophenotype with expression of the surface antigens CD19, CD5, CD20, CD23, and dim surface immunoglobulin G. The cell of origin of CLL is uncertain, but a gene expression pattern most similar to a mature memory B cell has been hypothesized. 2 In addition, CLL cells display disrupted apoptosis that is caused by both primary tumor features and codependent stromal elements. 3 Although many patients are asymptomatic at diagnosis, CLL is a progressive disease that in most patients eventually will require treatment. Once they become symptomatic, patients have a relatively short overall survival, ranging from 18 months to 6 years, with a 22.5% 10-year survival expectation. 4 Common treatments for CLL include alkylating chemotherapeutic drugs (such as chlorambucil and cyclophosphamide), purine analogs (such as fludarabine), and rituximab (used in combination with fludarabine, fludarabine and cyclophosphamide, or pentostatin and cyclophosphamide). Newer studies with either single-agent bendamustine or alemtuzumab have been shown to have improved response and progression-free survival over alkylator-based therapy. However, no current treatment option results in curative therapy, and all patients eventually relapse. This provides strong justification for developing additional types of therapies for CLL. Of particular interest are therapies that target signal transduction pathways essential to CLL cell survival mechanisms that are known to be aberrantly activated.One such pathway is the phosphoinositide 3-kinase (PI3K) pathway. The PI3K pathway is acknowledged as a key component of cell survival in many cancers, including CLL. It is activated by receptors, or the small guanosine triphosphatase Ras, and is made up of various classes of PI3K isoforms. 5 There are 3 classes of PI3K isoforms; however, only the class I isoforms phosphorylate inositol lipids to form second messenger phosphoinositides. Specifically, class I PI3K enzymes convert PtdIns(3,4)P 2 into PtdIns(3,4,5)P 3 , in the cell membrane that recruit, via binding to the amino-terminal pleckstrin homology domain, downstream signaling proteins such as Tec kinases, phosphatidylinositol-dependent kinase, Akt, integrin-linked kinase, and Rac guanine exchange factor. Class I isoforms are made up of 2 subsets (IA and IB). Class IA encompasses p110␣, p110␤, and p110␦ (catalytic domains), bound by p85, p50, or p55 (regulatory domains). Class IB is made up solely of the p110␥ (catalytic domain) bound by the regulatory domain p101. The p110␣ and p110␤ isoforms are ubiquitously expressed, and knock-out mice for both are embryonic lethal. 6 It is thought that this widespread functionality of PI3K signaling is at An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed ...

Two mechanisms have been proposed for regulating rolling velocities on selectins. These are (a) the intrinsic kinetics of bond dissociation, and (b) the reactive compliance, i.e., the susceptibility of the bond dissociation reaction to applied force. To determine which of these mechanisms explains the 7.5–11.5-fold faster rolling of leukocytes on L-selectin than on E- and P-selectins, we have compared the three selectins by examining the dissociation of transient tethers. We find that the intrinsic kinetics for tether bond dissociation are 7–10-fold more rapid for L-selectin than for E- and P-selectins, and are proportional to the rolling velocities through these selectins. The durations of pauses during rolling correspond to the duration of transient tethers on low density substrates. Moreover, applied force increases dissociation kinetics less for L-selectin than for E- and P-selectins, demonstrating that reactive compliance is not responsible for the faster rolling through L-selectin. Further measurements provide a biochemical and biophysical framework for understanding the molecular basis of rolling. Displacements of tethered cells during flow reversal, and measurements of the distance between successive pauses during rolling provide estimates of the length of a tether and the length of the adhesive contact zone, and suggest that rolling occurs with as few as two tethers per contact zone. Tether bond lifetime is an exponential function of the force on the bond, and the upper limit for the tether bond spring constant is of the same order of magnitude as the estimated elastic spring constant of the lectin–EGF unit. Shear uniquely enhances the rate of L-selectin transient tether formation, and conversion of tethers to rolling adhesions, providing further understanding of the shear threshold requirement for rolling through L-selectin.