scholarly journals Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences

2010 ◽  
Vol 207 (1) ◽  
pp. 61-75 ◽  
Author(s):  
Christoph T. Berger ◽  
Jonathan M. Carlson ◽  
Chanson J. Brumme ◽  
Kari L. Hartman ◽  
Zabrina L. Brumme ◽  
...  
Keyword(s):  
Immune Responses ◽  
Viral Replication ◽  
Ex Vivo ◽  
Natural Infection ◽  
Human Leukocyte ◽  
Population Level ◽  
Sequence Information ◽  
Viral Control

CD8+ cytotoxic T lymphocyte (CTL)–mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q < 0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03–restricted +2 frame–encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals STAT2-Dependent Immune Responses Ensure Host Survival despite the Presence of a Potent Viral Antagonist

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Vu Thuy Khanh Le-Trilling ◽  
Kerstin Wohlgemuth ◽  
Meike U. Rückborn ◽  
Andreja Jagnjic ◽  
Fabienne Maaßen ◽  
...  
Keyword(s):  
Immune Responses ◽  
Viral Replication ◽  
Mutual Influence ◽  
Type I ◽  
Mcmv Infection ◽  
General Importance ◽  
Interferon Antagonism ◽  
Deficient Mice

ABSTRACTA pathogen encounter induces interferons, which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, the host and pathogens are situated in a continuous arms race which shapes host evolution toward optimized immune responses and the pathogens toward enhanced immune-evasive properties. Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2, which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lackingM27and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pairin vitroandin vivo. In contrast to wild-type (wt) MCMV, ΔM27 mutant MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g., liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection. Taken together, the results of our study reveal the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary standoff situation with fatal consequences when the equilibrium is disturbed.IMPORTANCEThe host limits viral replication by the use of interferons (IFNs), which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g., cytomegaloviruses, Zika virus, dengue virus, and several paramyxoviruses). We analyzed infections caused by MCMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate its importance for the host and the virusin vitroandin vivo. The inability to counteract STAT2 directly translates into exaggerated IFN susceptibilityin vitroand pronounced attenuationin vivo. Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.

scholarly journals Development of A Novel Highly Selective TLR8 Agonist for Cancer Immunotherapy

2020 ◽  
Author(s):  
Yuxun Wang ◽  
Heping Yang ◽  
Huanping Li ◽  
Shuda Zhao ◽  
Yikun Zeng ◽  
...  
Keyword(s):  
Immune Responses ◽  
Drug Targets ◽  
Ex Vivo ◽  
Single Agent ◽  
Cancer Drug ◽  
Phase I Clinical Trials ◽  
Tlr Agonists ◽  
Favorable Safety

ABSTRACTToll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. The selectivity profile distinguished DN052 from all other TLR agonists currently in clinical development. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase I clinical trials.

scholarly journals Development of a novel TLR8 agonist for cancer immunotherapy

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Yuxun Wang ◽  
Heping Yang ◽  
Huanping Li ◽  
Shuda Zhao ◽  
Yikun Zeng ◽  
...  
Keyword(s):  
Immune Responses ◽  
Drug Targets ◽  
Ex Vivo ◽  
Phase 1 ◽  
Single Agent ◽  
Cancer Drug ◽  
Favorable Safety ◽  
Molecular Patterns

Abstract Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase 1 clinical trials.

scholarly journals Maturation of hematopoietic stem cells from prehematopoietic stem cells is accompanied by up-regulation of PD-L1

2017 ◽  
Vol 215 (2) ◽  
pp. 645-659 ◽  
Author(s):  
Joanna Tober ◽  
Marijke M.W. Maijenburg ◽  
Yan Li ◽  
Long Gao ◽  
Brandon K. Hadland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Responses ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Programmed Death

Hematopoietic stem cells (HSCs) mature from pre-HSCs that originate in the major arteries of the embryo. To identify HSCs from in vitro sources, it will be necessary to refine markers of HSCs matured ex vivo. We purified and compared the transcriptomes of pre-HSCs, HSCs matured ex vivo, and fetal liver HSCs. We found that HSC maturation in vivo or ex vivo is accompanied by the down-regulation of genes involved in embryonic development and vasculogenesis, and up-regulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that ex vivo–matured and fetal liver HSCs express programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on HSCs matured ex vivo. PD-L1 signaling is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for, HSC maturation.

scholarly journals Blood DCs activated with R848 and poly(I:C) induce antigen-specific immune responses against viral and tumor-associated antigens

2021 ◽  
Author(s):  
Gerulf Hänel ◽  
Caroline Angerer ◽  
Katja Petry ◽  
Felix S. Lichtenegger ◽  
Marion Subklewe
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Poly I:C ◽  
Tumor Associated Antigens ◽  
Cell Induction

AbstractMonocyte-derived Dendritic cells (DCs) have successfully been employed to induce immune responses against tumor-associated antigens in patients with various cancer entities. However, objective clinical responses have only been achieved in a minority of patients. Additionally, generation of GMP-compliant DCs requires time- and labor-intensive cell differentiation. In contrast, Blood DCs (BDCs) require only minimal ex vivo handling, as differentiation occurs in vivo resulting in potentially better functional capacities and survival. We aimed to identify a protocol for optimal in vitro activation of BDCs including the three subsets pDCs, cDC1s, and cDC2s. We evaluated several TLR ligand combinations and demonstrated that polyinosinic:polycytidylic acid [poly(I:C)] and R848, ligands for TLR3 and TLR7/8, respectively, constituted the optimal combination for inducing a positive co-stimulatory profile in all BDC subsets. In addition, TLR3 and TLR7/8 activation led to high secretion of IFN-α and IL-12p70. Simultaneous as opposed to separate tailored activation of pDCs and cDCs increased immunostimulatory capacities, suggesting that BDC subsets engage in synergistic cross-talk during activation. Stimulation of BDCs with this protocol resulted in enhanced migration, high NK-cell activation, and potent antigen-specific T-cell induction.We conclude that simultaneous activation of all BDC subsets with a combination of R848 + poly(I:C) generates highly immunostimulatory DCs. These results support further investigation and clinical testing, as standalone or in conjunction with other immunotherapeutic strategies including adoptive T-cell transfer and checkpoint inhibition.

scholarly journals Modeling the Ruminant Placenta-Pathogen Interactions in Apicomplexan Parasites: Current and Future Perspectives

2021 ◽  
Vol 7 ◽  
Author(s):  
Iván Pastor-Fernández ◽  
Esther Collantes-Fernández ◽  
Laura Jiménez-Pelayo ◽  
Luis Miguel Ortega-Mora ◽  
Pilar Horcajo
Keyword(s):  
Immune Responses ◽  
Fetal Death ◽  
Ex Vivo ◽  
Neospora Caninum ◽  
Economic Losses ◽  
Host Immune Responses ◽  
Partial View ◽  
And Function

Neospora caninum and Toxoplasma gondii are one of the main concerns of the livestock sector as they cause important economic losses in ruminants due to the reproductive failure. It is well-known that the interaction of these parasites with the placenta determines the course of infection, leading to fetal death or parasite transmission to the offspring. However, to advance the development of effective vaccines and treatments, there are still important gaps on knowledge on the placental host-parasite interactions that need to be addressed. Ruminant animal models are still an indispensable tool for providing a global view of the pathogenesis, lesions, and immune responses, but their utilization embraces important economic and ethics restrictions. Alternative in vitro systems based on caruncular and trophoblast cells, the key cellular components of placentomes, have emerged in the last years, but their use can only offer a partial view of the processes triggered after infection as they cannot mimic the complex placental architecture and neglect the activity of resident immune cells. These drawbacks could be solved using placental explants, broadly employed in human medicine, and able to preserve its cellular architecture and function. Despite the availability of such materials is constrained by their short shelf-life, the development of adequate cryopreservation protocols could expand their use for research purposes. Herein, we review and discuss existing (and potential) in vivo, in vitro, and ex vivo ruminant placental models that have proven useful to unravel the pathogenic mechanisms and the host immune responses responsible for fetal death (or protection) caused by neosporosis and toxoplasmosis.

scholarly journals Metabolic Syndrome and Viral Pathogenesis: Lessons from Influenza and Coronaviruses

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Maria Smith ◽  
Rebekah Honce ◽  
Stacey Schultz-Cherry
Keyword(s):  
Metabolic Syndrome ◽  
Immune Responses ◽  
Ex Vivo ◽  
Severe Disease ◽  
Viral Susceptibility ◽  
Metabolic Dysregulation ◽  
High Risk Populations

ABSTRACT Metabolic syndrome increases the risk of severe disease due to viral infection. Yet few studies have assessed the pathogenesis of respiratory viruses in high-risk populations. Here, we summarize how metabolic dysregulation impairs immune responses, and we define the role of metabolism during influenza virus and coronavirus infections. We also discuss the use of various in vitro, in vivo, and ex vivo models to elucidate the contributions of host factors to viral susceptibility, immunity, and disease severity.

scholarly journals OP0001 BARICITINIB, TOFACITINIB, UPADACITINIB, FILGOTINIB, AND CYTOKINE SIGNALING IN HUMAN LEUKOCYTE SUBPOPULATIONS: AN UPDATED EX-VIVO COMPARISON

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1-2
Author(s):  
I. Mcinnes ◽  
G. Rocha ◽  
R. E. Higgs ◽  
D. Dairaghi ◽  
T. Wehrman ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Leukocyte ◽  
Parent Drug ◽  
Cell Types ◽  
Vertical Line ◽  
Cytokine Signaling ◽  
Cell Type ◽  
Research Support

Background:everal JAKi are now used for the treatment of RA; approved doses include baricitinib (bari) 2- and/or 4-mg QD, tofacitinib (tofa) 5-mg BID, upadacitinib (upa) 15-mg QD. The JAK selectivity these agents is proposed to vary across the class.Objectives:In vitro cellular pharmacology of bari to tofa, upa, and filgotinib (filgo) were compared.Methods:PBMCs from 6 healthy donors were incubated with the JAKis over a 7- to 8-point concentration range. Following cytokine stimulation, levels of pSTAT were measured and IC50 calculated in gated leukocyte subpopulations. Therapeutic dose relevance was assessed using calculated mean concentration-time (CT) profiles over 24 hours for bari 2- and 4-mg QD; tofa 5- and 10-mg BID; upa 15- and 30-mg QD; filgo 100- and 200-mg QD. Average daily % inhibition of pSTAT (%SI) was calculated for each JAKi, cytokine, and cell type; filgo %SI integrated parent drug + metabolite.Results:The cytokines did not signal in all cell types (Figure 1). When signaling was detected, IC50 and %SI for a particular JAKi were generally similar across cell types, with dose-dependent inhibition (Figures 1 & 2). Based on IC50s, upa was most and filgo/metabolite least potent across JAK2/2 or JAK2/TYK2-dependent (IL-3, GM-CSF, G-CSF), JAK1/3-dependent (IL-2, 4, 15, 21), and JAK1/2/TYK2 dependent (IL-6 & 10, IFN-α & γ) signaling pathways.Figure 1.IC50 values (nM) for baricitinib, tofacitinib, upadacitinib, filgotinib (parent and metabolite) in cytokine-stimulated human PBMC preparations. *p<0.01, **p<0.001, ***p<0.0001 vs. bari.Incorporating CT profiles, no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Comparing bari 4-mg to tofa 5-mg BID, upa 15-mg QD, and filgo 100-mg QD, %SI of JAK2/2 or JAK2/TYK2-dependent cytokines was highest with bari 4-mg and upa. Inhibition of JAK1/2/TYK2 cytokines was highest with bari 4-mg. Inhibition of JAK2/2 or JAK2/TYK2, and of JAK1/3-dependent cytokines was lowest for filgo 100-mg QD. For bari 2-mg QD vs. these other JAKi doses, %SI of JAK2/2 or JAK2/TYK2 was highest with upa followed by bari 2-mg. The highest inhibitors of the JAK1/2/TYK2-dependent cytokines varied by cytokine / cell type though consistently included upa. Inhibition of JAK1/3 was highest for upa and tofa. Across comparisons, filgo 100-mg QD showed the least %SI of JAK2/2 or JAK2/TYK2-dependent, and of JAK1/3-dependent cytokines. Filgo reached the highest levels of %SI among agents only for 200-mg QD vs. lower doses of the other JAKi (for selected JAK1/2/TYK2-dependent cytokines).Conclusion:JAKis display different in vitro pharmacologic profiles which, coupled to their in vivo pharmacokinetics, suggest they modulate distinct cytokine pathways to differing degrees and durations over 24 hours. Ex vivo whole cell assays seem distinct from cell free kinase inhibition assays in determining the overall cytokine modulatory potential of members of the JAKi class.References:[1]McInnes IB, et al. Arthritis Res Ther. 2019 Aug 2;21(1):183Figure 2.Baricitinib, tofacitinib, upadacitinib, filgotinib: calculated average percent daily STAT inhibition for selected cytokines. -p<0.01, --p<0.001, ---p<0.0001 significantly lower compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”). +p<0.01, ++p<0.001, +++p<0.0001 significantly higher compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”).Disclosure of Interests:Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Guilherme Rocha Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Richard E Higgs Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Daniel Dairaghi Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Thomas Wehrman Shareholder of: An insignificant amount in AbbVie as part of a larger portfolio, Consultant of: Primity Bio Inc. works with many pharmaceutical and biotech companies and provides CRO services., Evan Wang Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Zhang Xin Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Jorge Ross Terres Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Terence Rooney Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UCB

scholarly journals TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Salik Hussain ◽  
Collin G Johnson ◽  
Joseph Sciurba ◽  
Xianglin Meng ◽  
Vandy P Stober ◽  
...  
Keyword(s):  
Immune Responses ◽  
Ex Vivo ◽  
Dominant Negative ◽  
Tlr4 Signaling ◽  
In Vivo Animal Models ◽  
Molecular Patterns ◽  
Myd88 Pathway ◽  
Tlr4 Receptor

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.

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