Biology subjects

Arthos, J.

Publications and source records attributed to Arthos, J..

8 recordsLinked to original sources

bioRxiv2025

IL-6 is a Key Factor in the Formation of Gut Tissue Resident Memory T Cells from Naïve T cells

Tissue resident memory CD4+ T cells (TRMs) populate mucosal sites and play a critical role in local immune responses. Gut TRM cells persist for extended periods in the gut mucosa where they rapidly respond to invading pathogens and provide long lasting protection. This study investigates the factors that mediate differentiation of naive CD4+ T cells into cells presenting a gut TRM phenotype. Naive CD4+ T cells were cultured under conditions that mimicked mucosal environments. This included signaling through MAdCAM-1 in the presence of Retinoic Acid (RA) and TGF-{beta}. This combination of stimuli primed naive CD4+ T cells to adopt a TRM phenotype. However, to fully differentiate into TRMs an additional soluble factor provided by memory T cells was required. Our results identified IL-6 as a key factor that induces the expression of TRM -associated markers, including CD69, CD103 and CCR5. This unique combination of stimuli promoted TRM differentiation despite low level proliferation. TRM differentiation was mediated through JAK/STAT signaling, and antagonists that target JAK/STAT pathways suppressed MAdCAM-1 mediated TRM cell formation. Our findings revealed that MAdCAM-1 works together with TGF-{beta}, RA and IL-6 in this process. Such information may aid in the design of next generation adjuvants and effective mucosal vaccines. Additionally, each of these factors may be targeted to treat excessive gut inflammation associated with conditions like inflammatory bowel disease. Overall, these findings provide new strategies aimed at modulating immune responses to invading pathogens and identify therapeutic approaches toward regulating gut inflammation. Author SummaryImmunologists and vaccinologists have long been interested in strategies aimed at boosting immune responses in mucosal tissues where pathogens are first encountered. Gut Tissue resident memory T cells (TRMs) play a central role in gut homeostasis and immunity. They provide a rapid long-lasting protection against pathogens. TRMs must target harmful pathogens and at the same time tolerate harmless commensal bacteria. This balance between immunity and tolerance in the complex environment of gut tissues is essential to the maintenance of gut homeostasis. In this study we have identified key factors present in the gut tissue milieu that are involved in TRM differentiation from naive T cells. These findings point to new therapeutic approaches that can potentially target immune responses in gut tissues and may help in developing effective mucosal vaccines. Conversely, these factors may be targeted in excessive gut inflammation involved in conditions like inflammatory bowel disease (IBD). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/660664v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@14e6015org.highwire.dtl.DTLVardef@860b93org.highwire.dtl.DTLVardef@61f8c8org.highwire.dtl.DTLVardef@142b48_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

TGF-β blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-{beta} blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on ART were treated with four 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption (ATI). 7 out of 8 macaques rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with 64Cu-DOTA-F(ab)2-p7D3-probe. A decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk, and single-cell (sc)RNAseq revealed a shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrated that galunisertib, a clinical stage TGF-{beta} inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity. One-sentence summaryTGF-{beta} blockade drives an effector phenotype in immune cells leading to SIV latency reversal and enhanced immune responses in vivo.

immunology↗

bioRxiv2023

Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF- and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

immunology↗

bioRxiv2023

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/530526v1_ufig1.gif" ALT="Figure 1000"> View larger version (30K): org.highwire.dtl.DTLVardef@10e7728org.highwire.dtl.DTLVardef@b1078aorg.highwire.dtl.DTLVardef@65f874org.highwire.dtl.DTLVardef@9ab0e8_HPS_FORMAT_FIGEXP M_FIG C_FIG Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.

microbiology↗

bioRxiv2022

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo

Elevated levels of TGF-{beta}, a potent immunosuppressive factor, are present in HIV-1 infected individuals even after years of antiretroviral therapy (ART). TGF-{beta} plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent one of the main cellular reservoirs after long term ART and the low inducibility of the latent provirus constitutes one of the major obstacles to "kick and kill" cure strategies. We hypothesized that releasing cells from TGF-{beta}-driven signaling would promote latency reversal. To test our hypothesis, we compared ex vivo models of HIV-1 latency reactivation with and without TGF-{beta} and a TGF-{beta} type 1 receptor (TGFBR1) inhibitor, galunisertib. We also tested the effect of galunisertib in SIV infected, ART treated macaques by monitoring SIV envelope (env) protein expression via PET/CT using the Cu64-anti gp120 Fab (7D3) probe, along with plasma and tissue viral loads (VL). Exogenous TGF-1{beta} reduced HIV-1 reactivation in U1 and ACH2 latency models. Galunisertib increased HIV-1 latency reversal both in ex vivo models and in PBMC from HIV-1 infected, cART treated aviremic donors. In vivo, oral galunisertib promoted increased SIV env protein total standardized uptake values (SUVtot) in PET/CT images of tissues (gut and lymph nodes) of 5 out of 7 aviremic, long-term ART-treated, SIV-infected, macaques. This increase correlated with an increase in SIV RNA in gut tissue. Two out of 7 animals also exhibited increases in plasma viral load. Higher anti-SIV T cell responses and anti-SIV env antibody titers were detected after galunisertib treatment in most animals. In summary, our data suggest that blocking TGF-{beta} signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

immunology↗

bioRxiv2020

SARS-CoV-2 genomic and quasispecies analyses in cancer patients reveal relaxed intrahost virus evolution

Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (HCW; non-cancer controls) by deep sequencing and investigated the within-host viral quasispecies of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers (HCW) from the Brazilian Cancer Institute were collected in April-May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants (iSNVs) were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%). Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to HCW (p = 0.009). Intrahost genetic diversity in cancer patients was independent of SARS-CoV-2 Ct values, and was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Such a feature may explain, at least in part, the more adverse outcomes to which cancer/COVID-19 patients experience. Author SummaryCancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. In this study, phylogenetic and variation analysis of SARS-CoV-2 genomes from cancer patients and non-cancer healthcare workers at the Brazilian National Cancer Institute were characterized by deep sequencing. Viral genomes showed signatures characteristic of Brazilian viruses, consistent with the hypothesis of local, community transmission rather than virus importation from abroad. Despite most genomes in patients and healthcare workers belonging to the same lineage, intrahost variability was higher in cancer patients when compared to non-cancer counterparts. The intrahost genomic diversity analysis presented in our study highlights the relaxed evolution of SARS-CoV-2 in a vulnerable population of cancer patients. The high number of minor variations can result in the selection of immune escape variants, resistance to potential drugs, and/or increased pathogenicity. The impact of this higher intrahost variability over time warrants further investigation.

microbiology↗

bioRxiv2020

Signal Peptide of HIV-1 Envelope Functions to Modulate Glycosylation Impacting Exposure of V1V2 Epitopes

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by [~]90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including the conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex. Virus neutralization was also affected, especially by antibodies against the V2i, V2p and V2q epitopes. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 epitopes on Env. Author SummaryHIV-1 Env glycoprotein is produced by a precursor gp160 that has a signal peptide at its N-terminus. The SP is highly diverse among the HIV-1 isolates and no two SP are same. This study presents site-specific analyses of N-linked glycosylation on HIV-1 envelope glycoproteins from infectious viruses produced with different envelope signal peptides. We show that signal peptide swapping alters the envelope glycan shield, including the conserved N156 and N160 located in the V1V2 region on the trimer apex, to impact Env recognition and virus neutralization by antibodies, particularly those targeting the the V1V2 region. The data offer crucial insights into the role of signal peptide in the interplay between HIV-1 and antibodies and its potential utility to control Env glycosylation in the development of Env-based HIV-1 vaccine.

microbiology↗

bioRxiv2018

Protection against repeated vaginal SHIV challenges by a combination of VRC01 and an anti-α4β7 antibody

VRC01 protects macaques from vaginal SHIV infection after a single high-dose challenge. Infusion of a simianized anti-4{beta}7 mAb (Rh-4{beta}7) just prior to, and during repeated vaginal exposures to SIVmac251 partially protected macaques from vaginal SIV infection and rescued CD4+ T cells. To investigate the impact of combining VRC01 and Rh-4{beta}7 on SHIV infection, 3 groups of macaques were treated with a suboptimal dosing of VRC01 alone or in combination with Rh-4{beta}7 or with control antibodies prior to the initiation of weekly vaginal exposures to a high dose (1000TCID50) of SHIVAD8-EO. The combination Rh-4{beta}7-VRC01 significantly delayed SHIVAD8-EO vaginal infection. Following infection, VRC01-Rh-4{beta}7-treated macaques maintained higher CD4+ T cell counts and exhibited lower rectal SIV-DNA loads compared to the controls. Interestingly, VRC01-Rh-4{beta}7-treated macaques had less IL-17 producing cells in the blood and the gut during the acute phase of infection. Moreover, higher T cell responses to the V2-loop of the SHIVAD8- EO envelope in the VRC01-Rh-4{beta}7 group inversely correlated with set point viremia. The combination of suboptimal amounts of VRC01 and Rh-4{beta}7 delayed infection, altered anti-viral immune responses and minimized CD4+ T cell loss. Further exploration of the effect of combining bNAbs with Rh-4{beta}7 on SIV/HIV infection and anti-viral immune responses is warranted and may lead to novel preventive and therapeutic strategies.\n\nShort summaryA combination of VRC01 and Rh-4{beta}7 significantly delayed SHIV acquisition, protected CD4 counts, decreased gut viral load and modified the immune response to the virus.

immunology↗