Rebecca Liu

CyTOF supports efficient detection of immune cell subsets from small samples.

Analysis of immune cell states is paramount to our understanding of the pathogenesis of a broad range of human diseases. Immunologists rely on fluorescence cytometry for cellular analysis, and while detection of 8 markers is now well established, the overlap of fluorescent signals limits efficiency. Mass cytometry or CyTOF (Cytometry by Time-Of-Flight) is a new technology for multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry and can routinely detect as many as 40 markers per sample. This technology provides tremendous detail for cellular analysis of multiple cell populations simultaneously and is a powerful technique for translational investigations. Here we present reproducible detection of immune cell subsets starting with as few as 10,000 cells. Our study provides methods to employ CyTOF for small samples, which is especially relevant for investigation of limited patient biopsies in translational and clinical research.

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

Significance Complement activation contributes to host defense and immunopathology. We recently discovered that membrane attack complexes (MAC), the terminal effector mechanisms of complement, activate proinflammatory functions in human endothelial cells (ECs) via noncanonical NF-ΚB signaling. Here we elucidate the initial steps of how MACs activate this pathway. MACs formed on the surface of human ECs are rapidly internalized via clathrin-mediated endocytosis into Rab5+ endosomes, which subsequently recruit activated Akt in a Rab5-dependent manner. Akt recruitment results in NIK protein stabilization on the surface of the endosome within 30 min, initiating noncanonical NF-ΚB signaling. MAC internalization in ECs lining human coronary arteries in vivo similarly activates noncanonical NF-ΚB signaling. Our findings suggest new therapeutic targets for controlling complement-mediated inflammation. Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB–inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5+endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC+ endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt+NIK+ signalosome on Rab5+ endosomes.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell–cell interactions of circulating leukocytes with IL-1– or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17–induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17–activated PCs, but not ECs, induce pertussis toxin–sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17–activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17–treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17–activated PCs can modulate neutrophil functions within the perivascular tissue space.

Antigen Presentation by Vascular Cells

Antigen presentation by cells of the vessel wall may initiate rapid and localized memory immune responses in peripheral tissues. Peptide antigens displayed on major histocompatibility complex (MHC) molecules on the surface of endothelial cells (ECs) can be recognized by T cell receptors on circulating effector memory T cells (TEM), triggering both transendothelial migration and activation. The array of co-stimulatory receptors, adhesion molecules, and cytokines expressed by ECs serves to modulate T cell activation responses. While the effects of these interactions vary among species, vascular beds, and vascular segments within the same tissue, they are capable of triggering allograft rejection without direct involvement of professional antigen-presenting cells and may play a similar role in host defense against infections and in autoimmunity. Once across the endothelium, extravasating TEM then contact mural cells of the vessel wall, including pericytes or vascular smooth muscle cells, which may also present antigens and provide signals that further regulate T cell responses. Collectively, these interactions provide an unexplored opportunity in which targeting of vascular cells can be used to modulate immune responses. In organ transplantation, targeting ECs with siRNA to reduce expression of MHC molecules may additionally mitigate perioperative injuries by preformed alloantibodies, further reducing the risk of graft rejection. Similarly, genetic manipulation of vascular cells to minimize antigen-dependent responses can be used to increase perfusion of tissue engineered organs without triggering rejection.

Interferon- γ converts human microvascular pericytes into negative regulators of alloimmunity through induction of indoleamine 2,3-dioxygenase 1

Early acute rejection of human allografts is mediated by circulating alloreactive host effector memory T cells (TEM). TEM infiltration typically occurs across graft postcapillary venules and involves sequential interactions with graft-derived endothelial cells (ECs) and pericytes (PCs). While the role of ECs in allograft rejection has been extensively studied, contributions of PCs to this process are largely unknown. This study aimed to characterize the effects and mechanisms of interactions between human PCs and allogeneic TEM. We report that unstimulated PCs, like ECs, can directly present alloantigen to TEM, but while IFN-γ-activated ECs (γ-ECs) show increased ability to stimulate alloreactive T cells, IFN-γ-activated PCs (γ-PCs) instead suppress TEM proliferation but not cytokine production or signaling. RNA sequencing analysis of PCs, γ-PCs, ECs, and γ-ECs reveal induction of indoleamine 2,3-dioxygenase 1 (IDO1) in γ-PCs to significantly higher levels than in γ-ECs that correlates with tryptophan depletion in vitro. Consistently, shRNA knockdown of IDO1 markedly reduces γ-PC-mediated immunoregulatory effects. Furthermore, human PCs express IDO1 in a skin allograft rejection humanized mouse model and in human renal allografts with acute T cell-mediated rejection. We conclude that immunosuppressive properties of human PCs are not intrinsic but instead result from IFN-γ-induced IDO1-mediated tryptophan depletion.

Medical students as primary care providers: a novel curriculum enhancing understanding of chronic disease management.

The traditional model of medical education has been criticized for providing a fragmented learning experience that fails to engage students in patient-centered care.1-3 Because students rotate between specialties each month of their clinical year, they develop only brief therapeutic relationships with individual patients, rarely assume ownership of patient care, and are unable to contribute to chronic disease management or appreciate the long-term effects of medical interventions. We thus present a newly structured clerkship in which medical students, overseen by preceptors, function as long-term primary care providers (PCPs) for a personal panel of patients. Moreover, because the clerkship is integrated into the operation of our institution’s Primary Care Center (PCC), our model has several unique features that facilitate implementation of medical student education in the outpatient setting and the delivery of high-quality patient care. Clinic mission Wednesday Evening Clinic (WEC) is a student-staffed clinic recently integrated into the PCC of the Yale-New Haven Hospital (YNHH). It was established with the mission of providing an extensive primary care experience for senior Yale medical students, in which they manage their own cohort of approximately 30 adult patients as PCPs under supervision of attending physicians. Each year, up to 15 Yale medical students beyond their third year are selected to participate in WEC weekly for one year or more. Medical students take ownership of multiple aspects of patients’ care, including general health maintenance, chronic disease management, age-appropriate screening, counseling, and patient-care coordination. WEC primarily serves an underserved patient population mostly insured by Medicaid and Medicare.

A subset of hypertrophic cardiomyopathy patients is predisposed to angulated septum

Background Hypertrophic cardiomyopathy (HCM) is a complex genetic disease with marked morphofunctional heterogeneity. Some HCM patients develop obstructive symptoms later in life, long after cessation of hypertrophic progression. The mechanism underlying this phenomenon is poorly understood. It is known that aorto-septal angulation progresses with age. However, the relationship between age, septal angulation, and HCM subtype has not been explored. In this present study, we examined the relationship between age, aorto-septal angulation and subtypes of HCM. Methods Control normal subjects (n=19) and consecutive HCM patients with apical, concentric, and septal subtypes (n=53) were identified from the MRI database at YaleNew Haven Hospital. Angulated septal angle, the angle between the right septal surface and anterior aortic wall during end systole and diastole, was measured blindly by two readers. Disagreements between two reads >10˚ were excluded. In addition, we further age stratified our cohort of septal subtype (above or below 40 years) to explore differences in the pattern of aorto-septal angle over age. Results Patients with septal, but not apical or concentric subtypes, exhibit more acute angulated septum versus controls in end-systole (p=0.008, 0.326, and 0.167, respectively). The acuity of this angle increased with age in controls (p=0.0009). Interestingly, HCM patients with septal hypertrophy deviated from this pattern, demonstrating early angulation without progression over age (p=0.918). The associations above remained significant in end-diastole.