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Article ; Online: A flow-cytometry-based protocol using diverse cell types for detecting autoantibodies from human plasma and serum samples

Andrew Kam Ho Wong / Deanna A. Kulpa / Guido Silvestri / Cheryl L. Maier

STAR Protocols, Vol 2, Iss 4, Pp 100924- (2021)

2021

Abstract: Summary: Here, we describe a protocol for cell-based detection of autoantibodies from human plasma and serum samples using a standard flow cytometer. The protocol allows detection of autoantibodies against a wide array of extracellular antigens. Antigen ... ...

Abstract	Summary: Here, we describe a protocol for cell-based detection of autoantibodies from human plasma and serum samples using a standard flow cytometer. The protocol allows detection of autoantibodies against a wide array of extracellular antigens. Antigen coverage is limited to the cell types tested, and researchers will need to further determine if autoantibody-positive samples correlate with cytotoxic or clinical outcomes. This protocol is less expensive and faster to perform when compared to protein microarrays and requires no prior knowledge of potential targets.For complete details on the use and execution of this protocol, please refer to Wong et al. (2021).
Keywords	Antibody ; Cell Biology ; Cell-based Assays ; Flow Cytometry/Mass Cytometry ; Health Sciences ; Science (General) ; Q1-390
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Human T cells efficiently control RSV infection

Chandrav De / Raymond J. Pickles / Wenbo Yao / Baolin Liao / Allison Boone / Mingyu Choi / Diana M. Battaglia / Frederic B. Askin / Jason K. Whitmire / Guido Silvestri / J. Victor Garcia / Angela Wahl

JCI Insight, Vol 8, Iss

2023 Volume 11

Abstract: Respiratory syncytial virus (RSV) infection causes significant morbidity and mortality in infants, immunocompromised individuals, and older individuals. There is an urgent need for effective antivirals and vaccines for high-risk individuals. We used 2 ... ...

Abstract	Respiratory syncytial virus (RSV) infection causes significant morbidity and mortality in infants, immunocompromised individuals, and older individuals. There is an urgent need for effective antivirals and vaccines for high-risk individuals. We used 2 complementary in vivo models to analyze RSV-associated human lung pathology and human immune correlates of protection. RSV infection resulted in widespread human lung epithelial damage, a proinflammatory innate immune response, and elicited a natural adaptive human immune response that conferred protective immunity. We demonstrated a key role for human T cells in controlling RSV infection. Specifically, primed human CD8+ T cells or CD4+ T cells effectively and independently control RSV replication in human lung tissue in the absence of an RSV-specific antibody response. These preclinical data support the development of RSV vaccines, which also elicit effective T cell responses to improve RSV vaccine efficacy.
Keywords	Immunology ; Virology ; Medicine ; R
Language	English
Publishing date	2023-06-01T00:00:00Z
Publisher	American Society for Clinical investigation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Transient CD4+ T cell depletion during suppressive ART reduces the HIV reservoir in humanized mice

Lijun Ling / Chandrav De / Rae Ann Spagnuolo / Nurjahan Begum / Shane D. Falcinelli / Nancie M. Archin / Martina Kovarova / Guido Silvestri / Angela Wahl / David M. Margolis / J. Victor Garcia

PLoS Pathogens, Vol 19, Iss

2023 Volume 12

Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2023-12-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Transient CD4+ T cell depletion during suppressive ART reduces the HIV reservoir in humanized mice.

Lijun Ling / Chandrav De / Rae Ann Spagnuolo / Nurjahan Begum / Shane D Falcinelli / Nancie M Archin / Martina Kovarova / Guido Silvestri / Angela Wahl / David M Margolis / J Victor Garcia

PLoS Pathogens, Vol 19, Iss 12, p e

2023 Volume 1011824

Abstract: Lifelong treatment is required for people living with HIV as current antiretroviral therapy (ART) does not eradicate HIV infection. Latently infected cells are essentially indistinguishable from uninfected cells and cannot be depleted by currently ... ...

Abstract	Lifelong treatment is required for people living with HIV as current antiretroviral therapy (ART) does not eradicate HIV infection. Latently infected cells are essentially indistinguishable from uninfected cells and cannot be depleted by currently available approaches. This study evaluated antibody mediated transient CD4+ T cell depletion as a strategy to reduce the latent HIV reservoir. Anti-CD4 antibodies effectively depleted CD4+ T cells in the peripheral blood and tissues of humanized mice. We then demonstrate that antibody-mediated CD4+ T cell depletion of HIV infected ART-suppressed animals results in substantial reductions in cell-associated viral RNA and DNA levels in peripheral blood cells over the course of anti-CD4 antibody treatment. Recovery of CD4+ T cells was observed in all tissues analyzed except for the lung 26 days after cessation of antibody treatment. After CD4+ T cell recovery, significantly lower levels of cell-associated viral RNA and DNA were detected in the tissues of anti-CD4 antibody-treated animals. Further, an 8.5-fold reduction in the levels of intact HIV proviral DNA and a 3.1-fold reduction in the number of latently infected cells were observed in anti-CD4-antibody-treated animals compared with controls. However, there was no delay in viral rebound when ART was discontinued in anti-CD4 antibody-treated animals following CD4+ T cell recovery compared with controls. Our results suggest that transient CD4+ T cell depletion, a long-standing clinical intervention that might have an acceptable safety profile, during suppressive ART can reduce the size of the HIV reservoir in humanized mice.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	570 ; 630
Language	English
Publishing date	2023-12-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: CD8+ lymphocyte control of SIV infection during antiretroviral therapy.

Youfang Cao / Emily K Cartwright / Guido Silvestri / Alan S Perelson

PLoS Pathogens, Vol 14, Iss 10, p e

2018 Volume 1007350

Abstract: CD8+ lymphocytes play an important role in suppressing in vivo viral replication in HIV infection. However, both the extent to which and the mechanisms by which CD8+ lymphocytes contribute to viral control are not completely understood. A recent ... ...

Abstract	CD8+ lymphocytes play an important role in suppressing in vivo viral replication in HIV infection. However, both the extent to which and the mechanisms by which CD8+ lymphocytes contribute to viral control are not completely understood. A recent experiment depleted CD8+ lymphocytes in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) on antiretroviral treatment (ART) to study the role of CD8+ lymphocytes. CD8+ lymphocytes depletion resulted in temporary plasma viremia in all studied RMs. Viral control was restored when CD8+ lymphocytes repopulated. We developed a viral dynamic model to fit the viral load (VL) data from the CD8 depletion experiment. We explicitly modeled the dynamics of the latent reservoir and the SIV-specific effector cell population including their exhaustion and their potential cytolytic and noncytolytic functions. We found that the latent reservoir significantly contributes to the size of the peak VL after CD8 depletion, while drug efficacy plays a lesser role. Our model suggests that the overall CD8+ lymphocyte cytolytic killing rate is dynamically changing depending on the levels of antigen-induced effector cell activation and exhaustion. Based on estimated parameters, our model suggests that before ART or without ART the overall CD8 cytolytic killing rate is small due to exhaustion. However, after the start of ART, the overall CD8 cytolytic killing rate increases due to an expansion of SIV-specific CD8 effector cells. Further, we estimate that the cytolytic killing rate can be significantly larger than the cytopathic death rate in some animals during the second phase of ART-induced viral decay. Lastly, our model provides a new explanation for the puzzling findings by Klatt et al. and Wong et al. that CD8 depletion done immediately before ART has no noticeable effect on the first phase viral decay slope seen after ART initiation Overall, by incorporating effector cells and their exhaustion, our model can explain the effects of CD8 depletion on VL during ART, reveals a ...
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2018-10-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Broad auto-reactive IgM responses are common in critically ill patients, including those with COVID-19

Andrew Kam Ho Wong / Isaac Woodhouse / Frank Schneider / Deanna A. Kulpa / Guido Silvestri / Cheryl L. Maier

Cell Reports Medicine, Vol 2, Iss 6, Pp 100321- (2021)

2021

Abstract: Summary: The pathogenesis of severe coronavirus disease 2019 (COVID-19) remains poorly understood. While several studies suggest that immune dysregulation plays a central role, the key mediators of this process are yet to be defined. Here, we demonstrate ...

Abstract	Summary: The pathogenesis of severe coronavirus disease 2019 (COVID-19) remains poorly understood. While several studies suggest that immune dysregulation plays a central role, the key mediators of this process are yet to be defined. Here, we demonstrate that plasma from a high proportion (93%) of critically ill COVID-19 patients, but not healthy controls, contains broadly auto-reactive immunoglobulin M (IgM) and less frequently auto-reactive IgG or IgA. Importantly, these auto-IgMs preferentially recognize primary human lung cells in vitro, including pulmonary endothelial and epithelial cells. By using a combination of flow cytometry, analytical proteome microarray technology, and lactose dehydrogenase (LDH)-release cytotoxicity assays, we identify high-affinity, complement-fixing, auto-reactive IgM directed against 260 candidate autoantigens, including numerous molecules preferentially expressed on the cellular membranes of pulmonary, vascular, gastrointestinal, and renal tissues. These findings suggest that broad IgM-mediated autoimmune reactivity may be involved in the pathogenesis of severe COVID-19, thereby identifying a potential target for therapeutic interventions.
Keywords	Medicine (General) ; R5-920
Subject code	610
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Limited induction of SARS-CoV-2–specific T cell responses in children with multisystem inflammatory syndrome compared with COVID-19

Vidisha Singh / Veronica Obregon-Perko / Stacey A. Lapp / Anna Marie Horner / Alyssa Brooks / Lisa Macoy / Laila Hussaini / Austin Lu / Theda Gibson / Guido Silvestri / Alba Grifoni / Daniela Weiskopf / Alessandro Sette / Evan J. Anderson / Christina A. Rostad / Ann Chahroudi

JCI Insight, Vol 7, Iss

2022 Volume 4

Abstract: Why multisystem inflammatory syndrome in children (MIS-C) develops after SARS-CoV-2 infection in a subset of children is unknown. We hypothesized that aberrant virus–specific T cell responses contribute to MIS-C pathogenesis. We quantified SARS-CoV-2– ... ...

Abstract	Why multisystem inflammatory syndrome in children (MIS-C) develops after SARS-CoV-2 infection in a subset of children is unknown. We hypothesized that aberrant virus–specific T cell responses contribute to MIS-C pathogenesis. We quantified SARS-CoV-2–reactive T cells, serologic responses against major viral proteins, and cytokine responses from plasma and peripheral blood mononuclear cells in children with convalescent COVID-19, in children with acute MIS-C, and in healthy controls. Children with MIS-C had significantly lower virus-specific CD4+ and CD8+ T cell responses to major SARS-CoV-2 antigens compared with children convalescing from COVID-19. Furthermore, T cell responses in participants with MIS-C were similar to or lower than those in healthy controls. Serologic responses against spike receptor binding domain (RBD), full-length spike, and nucleocapsid were similar among convalescent COVID-19 and MIS-C, suggesting functional B cell responses. Cytokine profiling demonstrated predominant Th1 polarization of CD4+ T cells from children with convalescent COVID-19 and MIS-C, although cytokine production was reduced in MIS-C. Our findings support a role for constrained induction of anti–SARS-CoV-2–specific T cells in the pathogenesis of MIS-C.
Keywords	COVID-19 ; Medicine ; R
Subject code	610
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	American Society for Clinical investigation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Vpr attenuates antiviral immune responses and is critical for full pathogenicity of SIVmac239 in rhesus macaques

iScience, Vol 26, Iss 12, Pp 108351- (2023)

2023

Abstract: Summary: The accessory viral protein R (Vpr) is encoded by all primate lentiviruses. Vpr counteracts DNA repair pathways, modulates viral immune sensing, and induces cell-cycle arrest in cell culture. However, its impact in vivo is controversial. Here, ... ...

Abstract	Summary: The accessory viral protein R (Vpr) is encoded by all primate lentiviruses. Vpr counteracts DNA repair pathways, modulates viral immune sensing, and induces cell-cycle arrest in cell culture. However, its impact in vivo is controversial. Here, we show that deletion of vpr is associated with delayed viral replication kinetics, rapid innate immune activation, development and maintenance of strong B and T cell responses, and increased neutralizing activity against SIVmac239 in rhesus macaques. All wild-type SIVmac239-infected animals maintained high viral loads, and five of six developed fatal immunodeficiency during ∼80 weeks of follow-up. Lack of Vpr was associated with better preservation of CD4+ T cells, lower viral loads, and an attenuated clinical course of infection in most animals. Our results show that Vpr contributes to efficient viral immune evasion and the full pathogenic potential of SIVmac in vivo. Inhibition of Vpr may improve humoral immune control of viral replication.
Keywords	Immunology ; Immunity ; Immune response ; Science ; Q
Subject code	570
Language	English
Publishing date	2023-12-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Innate, non-cytolytic CD8+ T cell-mediated suppression of HIV replication by MHC-independent inhibition of virus transcription.

Michelle Zanoni / David Palesch / Claudia Pinacchio / Maura Statzu / Gregory K Tharp / Mirko Paiardini / Ann Chahroudi / Steven E Bosinger / Jack Yoon / Bryan Cox / Guido Silvestri / Deanna A Kulpa

PLoS Pathogens, Vol 16, Iss 9, p e

2020 Volume 1008821

Abstract: MHC-I-restricted, virus-specific cytotoxic CD8+ T cells (CTLs) may control human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication via the recognition and killing of productively infected CD4+ T cells. Several studies in ... ...

Abstract	MHC-I-restricted, virus-specific cytotoxic CD8+ T cells (CTLs) may control human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication via the recognition and killing of productively infected CD4+ T cells. Several studies in SIV-infected macaques suggest that CD8+ T cells may also decrease virus production by suppressing viral transcription. Here, we show that non-HIV-specific, TCR-activated non-cytolytic CD8+ T cells suppress HIV transcription via a virus- and MHC-independent immunoregulatory mechanism that modulates CD4+ T cell proliferation and activation. We also demonstrate that this CD8+ T cell-mediated effect promotes the survival of infected CD4+ T cells harboring integrated, inducible virus. Finally, we used RNA sequencing and secretome analyses to identify candidate cellular pathways that are involved in the virus-silencing mediated by these CD8+ T cells. This study characterizes a previously undescribed mechanism of immune-mediated HIV silencing that may be involved in the establishment and maintenance of the reservoir under antiretroviral therapy and therefore represent a major obstacle to HIV eradication.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2020-09-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: IL-21 and IFNα therapy rescues terminally differentiated NK cells and limits SIV reservoir in ART-treated macaques

Justin Harper / Nicolas Huot / Luca Micci / Gregory Tharp / Colin King / Philippe Rascle / Neeta Shenvi / Hong Wang / Cristin Galardi / Amit A. Upadhyay / Francois Villinger / Jeffrey Lifson / Guido Silvestri / Kirk Easley / Beatrice Jacquelin / Steven Bosinger / Michaela Müller-Trutwin / Mirko Paiardini

Nature Communications, Vol 12, Iss 1, Pp 1-

2021 Volume 12

Abstract: Infection of African green monkeys with SIV is associated with reduced pathogenicity. Here the authors explore the requirement of differentiated NK cell populations in a pathogenic Rhesus macaque model of SIV infection and show administration of IL-21 ... ...

Abstract	Infection of African green monkeys with SIV is associated with reduced pathogenicity. Here the authors explore the requirement of differentiated NK cell populations in a pathogenic Rhesus macaque model of SIV infection and show administration of IL-21 and IFNα rescues terminally differentiated NK cells, similarly to what found in African green monkeys, and limits the SIV reservoir in antiretroviral therapy treated macaques.
Keywords	Science ; Q
Language	English
Publishing date	2021-05-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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