Time-resolved assessment of single-cell protein secretion by sequencing.

Secreted proteins play critical roles in cellular communication. Methods enabling concurrent measurement of cellular protein secretion, phenotypes and transcriptomes are still unavailable. Here we describe time-resolved assessment of protein secretion from single cells by sequencing (TRAPS-seq). Released proteins are trapped onto the cell surface and probed by oligonucleotide-barcoded antibodies before being simultaneously sequenced with transcriptomes in single cells. We demonstrate that TRAPS-seq helps unravel the phenotypic and transcriptional determinants of the secretion of pleiotropic TH1 cytokines (IFNγ, IL-2 and TNF) in activated T cells. In addition, we show that TRAPS-seq can be used to track the secretion of multiple cytokines over time, uncovering unique molecular signatures that govern the dynamics of single-cell cytokine secretions. Our results revealed that early central memory T cells with CD45RA expression (TCMRA) are important in both the production and maintenance of polyfunctional cytokines. TRAPS-seq presents a unique tool for seamless integration of secretomics measurements with multi-omics profiling in single cells.

α4ß7+ CD4+ Effector/Effector Memory T Cells Differentiate into Productively and Latently Infected Central Memory T Cells by Transforming Growth Factor ß1 during HIV-1 Infection.

HIV-1 transmission occurs mainly through mucosal tissues. During mucosal transmission, HIV-1 preferentially infects α4ß7+ gut-homing CCR7- CD4+ effector/effector memory T cells (TEM) and results in massive depletion of these cells and other subsets of TEM in gut-associated lymphoid tissues. However, besides being eliminated by HIV-1, the role of TEM during the early stage of infection remains inconclusive. Here, using in vitro-induced α4ß7+ gut-homing TEM (α4ß7+ TEM), we found that α4ß7+ TEM differentiated into CCR7+ CD4+ central memory T cells (TCM). This differentiation was HIV-1 independent but was inhibited by SB431542, a specific transforming growth factor ß (TGF-ß) receptor I kinase inhibitor. Consistently, TEM-to-TCM differentiation was observed in α4ß7+ TEM stimulated with TGF-ß1 (TGF-ß). The TCM properties of the TGF-ß-induced TEM-derived TCM (α4ß7+ TCM) were confirmed by their enhanced CCL19 chemotaxis and the downregulation of surface CCR7 upon T cell activation in vitro Importantly, the effect of TGF-ß on TCM differentiation also held in TEM directly isolated from peripheral blood. To investigate the significance of the TGF-ß-dependent TEM-to-TCM differentiation in HIV/AIDS pathogenesis, we observed that both productively and latently infected α4ß7+ TCM could differentiate from α4ß7+ TEM in the presence of TGF-ß during HIV-1 infection. Collectively, this study not only provides a new insight for the plasticity of TEM but also suggests that the TGF-ß-dependent TEM-to-TCM differentiation is a previously unrecognized mechanism for the formation of latently infected TCM after HIV-1 infection.IMPORTANCE HIV-1 is the causative agent of HIV/AIDS, which has led to millions of deaths in the past 30 years. Although the implementation of highly active antiretroviral therapy has remarkably reduced the HIV-1-related morbidity and mortality, HIV-1 is not eradicated in treated patients due to the presence of latent reservoirs. Besides, the pathogenesis in CD4 T cells early after infection still remains elusive. Immediately after HIV-1 mucosal infection, CD4 T cells are preferentially infected and depleted. However, in addition to being depleted, the other roles of the CD4 T cells, especially the effector/effector memory T cells (TEM), in disease progression are not completely understood. The significance of this study is in revealing a novel mechanism for the formation of latently HIV-1-infected central memory CD4 T cells, a major latent reservoir from CD4 TEM after infection. Our findings suggest previously unrecognized roles of CD4 TEM in HIV-1 pathogenesis.

Identification, sorting and profiling of functional killer cells via the capture of fluorescent target-cell lysate.

Assays for assessing cell-mediated cytotoxicity are largely target-cell-centric and cannot identify and isolate subpopulations of cytotoxic effector cells. Here we describe an assay compatible with flow cytometry for the accurate identification and sorting of functional killer-cell subpopulations in co-cultures. The assay, which we named PAINTKiller (for 'proximity affinity intracellular transfer identification of killer cells'), relies on the detection of an intracellular fluorescent protein 'painted' by a lysed cell on the surface of the lysing cytotoxic cell (specifically, on cell lysis the intracellular fluorescein derivative carboxyfluorescein succinimidyl ester is captured on the surface of the natural killer cell by an antibody for anti-fluorescein isothiocyanate linked to an antibody for the pan-leucocyte surface receptor CD45). The assay can be integrated with single-cell RNA sequencing for the analysis of molecular pathways associated with cell cytotoxicity and may be used to uncover correlates of functional immune responses.

Cell Granularity Reflects Immune Cell Function and Enables Selection of Lymphocytes with Superior Attributes for Immunotherapy.

In keeping with the rule of "form follows function", morphological aspects of a cell can reflect its role. Here, it is shown that the cellular granularity of a lymphocyte, represented by its intrinsic side scatter (SSC), is a potent indicator of its cell state and function. The granularity of a lymphocyte increases from naïve to terminal effector state. High-throughput cell-sorting yields a SSChigh population that can mediate immediate effector functions, and a highly prolific SSClow population that can give rise to the replenishment of the memory pool. CAR-T cells derived from the younger SSClow population possess desirable attributes for immunotherapy, manifested by increased naïve-like cells and stem cell memory (TSCM )-like cells together with a balanced CD4/CD8 ratio, as well as enhanced target-killing in vitro and in vivo. Altogether, lymphocyte segregation based on biophysical properties is an effective approach for label-free selection of cells that share collective functions and can have important applications for cell-based immunotherapies.

Vedolizumab-mediated integrin α4ß7 blockade does not control HIV-1SF162 rebound after combination antiretroviral therapy interruption in humanized mice.

OBJECTIVE: The combined combination antiretroviral therapy (cART) and anti-α4ß7 RM-Act-1 antibody therapy allows macaques to durably control simian immunodeficiency virus (SIV) rebound after withdrawal of the interventions. Here, we aimed to investigate whether vedolizumab (VDZ), a clinical-grade humanized anti-α4ß7 antibody, would have similar effects in controlling live HIV-1 infection in humanized mice. DESIGN AND METHODS: The integrin α4ß7 blockade by VDZ was evaluated on human primary memory CD4+ T (MEMT) cells and retinoic acid-induced gut-homing α4ß7+MEMT cells (α4ß7+MEMT) in vitro. The antiretroviral activity of VDZ was determined using pseudotyped R5-tropic HIV-1SF162, which displays binding activity to α4ß7. The preventive and immunotherapeutic efficacies of VDZ were further investigated in humanized mice using the live HIV-1SF162 strain compared with RM-Act-1. RESULTS: VDZ effectively and dose-dependently blocked the binding of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1), the native ligand of α4ß7, to α4ß7+MEMT cells. HIV-1SF162 not only displayed binding capacity to α4ß7-expressing cells, but also showed an increased infectivity in retinoic acid-induced α4ß7+MEMT cells pretreated with VDZ. Moreover, VDZ failed to prevent live HIV-1SF162 infection and did not reduce the peak viral load when administrated prior to viral challenge in humanized mice. Lastly, in immunotherapeutic settings, the withdrawal of combined cART with either VDZ or RM-Act-1 resulted in an uncontrolled HIV-1SF162 rebound in humanized mice, whereas the α4ß7 molecules remained significantly blocked on circulating CD4+ T cells. CONCLUSION: VDZ neither prevents nor controls HIV-1SF162 infection both in vitro and in humanized mice. Our findings have significant implications to the clinical application of VDZ in HIV-1 preventive and immunotherapeutic interventions.

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection.

Newly emerging viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome CoVs (MERS-CoV), and H7N9, cause fatal acute lung injury (ALI) by driving hypercytokinemia and aggressive inflammation through mechanisms that remain elusive. In SARS-CoV/macaque models, we determined that anti-spike IgG (S-IgG), in productively infected lungs, causes severe ALI by skewing inflammation-resolving response. Alveolar macrophages underwent functional polarization in acutely infected macaques, demonstrating simultaneously both proinflammatory and wound-healing characteristics. The presence of S-IgG prior to viral clearance, however, abrogated wound-healing responses and promoted MCP1 and IL-8 production and proinflammatory monocyte/macrophage recruitment and accumulation. Critically, patients who eventually died of SARS (hereafter referred to as deceased patients) displayed similarly accumulated pulmonary proinflammatory, absence of wound-healing macrophages, and faster neutralizing antibody responses. Their sera enhanced SARS-CoV-induced MCP1 and IL-8 production by human monocyte-derived wound-healing macrophages, whereas blockade of FcγR reduced such effects. Our findings reveal a mechanism responsible for virus-mediated ALI, define a pathological consequence of viral specific antibody response, and provide a potential target for treatment of SARS-CoV or other virus-mediated lung injury.

Regulating Innate and Adaptive Immunity for Controlling SIV Infection by 25-Hydroxycholesterol.

Persistent inflammation and extensive immune activation have been associated with HIV-1/SIV pathogenesis. Previously, we reported that cholesterol-25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25-HC) had a broad antiviral activity in inhibiting Zika, Ebola, and HIV-1 infection. However, the underlying immunological mechanism of CH25H and 25-HC in inhibiting viral infection remains poorly understood. We report here that 25-HC effectively regulates immune responses for controlling viral infection. CH25H expression was interferon-dependent and induced by SIV infection in monkey-derived macrophages and PBMC cells, and 25-HC inhibited SIV infection both in permissive cell lines and primary monkey lymphocytes. 25-HC also strongly inhibited bacterial lipopolysaccharide (LPS)-stimulated inflammation and restricted mitogen-stimulated proliferation in primary monkey lymphocytes. Strikingly, 25-HC promoted SIV-specific IFN-γ-producing cellular responses, but selectively suppressed proinflammatory CD4+ T lymphocytes secreting IL-2 and TNF-α cytokines in vaccinated mice. In addition, 25-HC had no significant immunosuppressive effects on cytotoxic CD8+ T lymphocytes or antibody-producing B lymphocytes. Collectively, 25-HC modulated both innate and adaptive immune responses toward inhibiting HIV/SIV infection. This study provides insights into improving vaccination and immunotherapy regimes against HIV-1 infection.

Tandem bispecific neutralizing antibody eliminates HIV-1 infection in humanized mice.

The discovery of an HIV-1 cure remains a medical challenge because the virus rebounds quickly after the cessation of combination antiretroviral therapy (cART). Here, we investigate the potential of an engineered tandem bispecific broadly neutralizing antibody (bs-bnAb) as an innovative product for HIV-1 prophylactic and therapeutic interventions. We discovered that by preserving 2 single-chain variable fragment (scFv) binding domains of each parental bnAb, a single gene-encoded tandem bs-bnAb, BiIA-SG, displayed substantially improved breadth and potency. BiIA-SG neutralized all 124 HIV-1-pseudotyped viruses tested, including global subtypes/recombinant forms, transmitted/founder viruses, variants not susceptible to parental bnAbs and to many other bnAbs with an average IC50 value of 0.073 µg/ml (range < 0.001-1.03 µg/ml). In humanized mice, an injection of BiIA-SG conferred sterile protection when administered prior to challenges with diverse live HIV-1 stains. Moreover, whereas BiIA-SG delayed viral rebound in a short-term therapeutic setting when combined with cART, a single injection of adeno-associated virus-transferred (AAV-transferred) BiIA-SG gene resulted dose-dependently in prolonged in vivo expression of BiIA-SG, which was associated with complete viremia control and subsequent elimination of infected cells in humanized mice. These results warrant the clinical development of BiIA-SG as a promising bs-bnAb-based biomedical intervention for the prevention and treatment of HIV-1 infection.

Vaccine Engineering with Dual-Functional Mineral Shell: A Promising Strategy to Overcome Preexisting Immunity.

Dual-functional biomineral-vaccine core-shell nanohybrids are obtained using recombinant adenovirus serotype 5 (rAd5) as templates, which efficiently masks the neutralizing epitope of vaccines and preserve their original immunogenicity. The versatile vaccine hybrid can evade the preexisting anti-Ad5 immunity, leading to boosted multifunctional antigen-specific cytokine-secreting T cell responses and presenting promising applications of vaccine-material hybrid for the rational design of vaccines.

Aflatoxin B1 negatively regulates Wnt/ß-catenin signaling pathway through activating miR-33a.

MicroRNAs are known to play an important role in modulating gene expression in various diseases including cancers and cardiovascular disorders, but only a few of them are associated with the pathology of aflatoxin B1 (AFB1), a potent mycotoxin. Here, we discovered a novel regulatory network between AFB1, miR-33a and ß-catenin in human carcinoma cells. The level of miR-33a was up-regulated in hepatocellular carcinoma (HCC) cells treated with AFB1, while in the same cells causing the decrease in ß-catenin expression when treated at their IC50 values. miR-33a, specifically miR-33a-5p, was demonstrated to down-regulate the expression of ß-catenin, affect the ß-catenin pathway, and inhibit cell growth. Also, by employing a luciferase assay, we found that miR-33a down-regulated ß-catenin by directly binding to the 3'-UTR of ß-catenin. These results suggested that AFB1 might down-regulate ß-catenin by up-regulating miR-33a. This understanding opens new lines of thought in the potential role of miR-33a in the clinical therapy of cancer.