Biology subjects

Tremblay, C.

Publications and source records attributed to Tremblay, C..

29 records · Page 2Linked to original sources

bioRxiv2021

Immune checkpoint expression on HIV-specific CD4+ T cells and response to their blockade are dependent on lineage and function

BackgroundAntigen-specific T cell impairment is observed in chronic infections. CD4+ T cells are diverse in phenotype and function; how their different lineages are impacted by inhibitory immune checkpoints (IC) is unknown. MethodsWe examined IC expression and function in HIV-specific CD4+ T cells of viremic individuals prior to ART initiation and persons with spontaneous or therapy-induced viral suppression. We investigated IC patterns associated with exhaustion-related transcription factors and chemokine receptors using cytokine-independent activation-induced marker assays. We determined effector functions representative of TFH, TH1 and TH17/TH22 using ultra-sensitive RNA flow cytometric fluorescence in situ hybridization (FISH), and their response to IC blockade. FindingsThe dysfunction-related transcription factor TOX was elevated in HIV-specific CD4+ T cells of viremic patients, and its expression was associated with lineage differentiation. We observed a hierarchy of PD-1, TIGIT and CD200 expression associated with both infection status and effector profile. In vitro responsiveness to PD-L1 blockade varied with defined CD4+ T cell functions rather than IC expression levels: frequencies of cells with TH1- and TH17/TH22-, but not TFH-related functions, increased. Response to PD-L1 blockade was strongest in viremic participants and reduced after ART initiation. InterpretationOur data highlight a polarization-specific regulation of IC expression and differing sensitivities of antigen-specific Thelper subsets to PD-1-mediated inhibition. This heterogeneity may direct ICB efficacy on CD4+ T cells in HIV infection. FundingThis work was supported by the National Institutes of Health, the Canadian Institutes for Health Research, the Canada Foundation for Innovation and the Fonds de Recherche du Quebec-Sante. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSCombination antiretroviral therapy (ART) is highly effective in controlling HIV but requires life-long medication due to the latent viral reservoir, and does not restore suppressive immune responses. In particular, there is no generation of effective HIV-specific T cell responses, which are thought to play an important role in controlling HIV in the rare individuals who can spontaneously control the virus. Inhibitory immune checkpoints (IC) such as PD-1 contribute to T cell dysfunction and failure to control viral infections, including HIV, and IC blockade (ICB) represents a potential adjuvant to ART through restoration of T cell functions. While most studies have focused on CD8+ T cells, increasing evidence shows that the remarkable impact of ICB therapy in a subset of cancer patients is enhanced by functional CD4+ T cell help, which can be directly affected by ICB. While effective virus-specific CD4+ T cell responses are also thought to be important for immune control of HIV, these cells are highly heterogenous. How IC expression and function differs across CD4+ T cell lineages and the consequences of this diversity for IC blockade (ICB) strategies are still poorly understood. Added value of this studyTo compare various stages of immune dysfunction, we examined people living with HIV (PLWH) with different levels of viral control pre-ART (including elite controllers who spontaneously control virus) and followed a cohort longitudinally post-ART. We used a panel of assays to characterize HIV-specific CD4+ T cell subsets, including activation-induced marker (AIM) assays and flow cytometric detection of mRNAs coding for a wider variety of HIV-specific CD4+ T cell functions than what is detected by standard procedures. Our experiments indicate a hierarchy of IC (PD-1, TIGIT, CD200) expression on blood HIV-specific CD4+ T cells that depends not only on the persons infection status but also on expression of lineage differentiation markers and effector functions representative of CD4+ T cell subsets critical for antiviral responses (TFH, TH1 and TH17/TH22 cells). This hierarchy was also present in the putatively functional cells of elite controllers. We characterized the expression of the dysfunction-related transcription factor TOX, and saw that its association with the key IC PD-1 in the setting of viremia varied across CD4+ T cell polarizations. Response to blockade of the PD-1 pathway resulted in increased antiviral and mucosal-protective functions, but did not affect TFH-related functions. Response to ICB was most prominent in viremic patients, and subdued but not fully abrogated in the setting of viral suppression. Implications of all available evidenceThese results highlight a previously unrecognized impact of ICB on mucosal immunity-related CD4+ functions, which are known to be depleted upon HIV infection and not restored by ART, and strong links between IC expression patterns and HIV-specific CD4+ T cell differentiation. The impact of ICB on CD4+ T cells in HIV infection has primarily been studied in the context of viral reservoir reactivation, which are preferentially harbored in IC+ cells. Our work emphasizes the importance of considering the differentiation profile of the virus-specific CD4+ T cells in studies of ICB blockade, as it may direct ICB efficacy in HIV infection. This data may also have implications for CD4+ T cell help in other infectious and non-infectious chronic human diseases.

immunology↗

bioRxiv2021

Covid-19 vaccine immunogenicity in people living with HIV-1

IntroductionCOVID-19 vaccine efficacy has been evaluated in large clinical trials and in real-world situation. Although they have proven to be very effective in the general population, little is known about their efficacy in immunocompromised patients. HIV-infected individuals response to vaccine may vary according to the type of vaccine and their level of immunosuppression. We evaluated immunogenicity of an mRNA anti-SARS CoV-2 vaccine in HIV-positive individuals. MethodsHIV-positive individuals (n=121) were recruited from HIV clinics in Montreal and stratified according to their CD4 counts. A control group of 20 health care workers naive to SARS CoV-2 was used. The participants Anti-RBD IgG responses were measured by ELISA at baseline and 3 to 4 weeks after receiving the first dose of an mRNA vaccine). ResultsEleven of 121 participants had anti-COVID-19 antibodies at baseline, and a further 4 had incomplete data for the analysis. Mean anti-RBD IgG responses were similar between between the HIV negative control group (n=20) and the combined HIV+ group (n=106) (p = 0.72). However, these responses were significantly lower in the group with <250 CD4 cells/mm3. (p<0.0001). Increasing age was independently associated with decreased immunogenicity. ConclusionHIV-positive individuals with CD4 counts over 250 cells/mm3 have an anti-RBD IgG response similar to the general population. However, HIV-positive individuals with the lowest CD4 counts (<250 cells/mm3) have a weaker response. These data would support the hypothesis that a booster dose might be needed in this subgroup of HIV-positive individuals, depending on their response to the second dose.

immunology↗

bioRxiv2021

A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses

The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.

immunology↗

bioRxiv2021

Impact of cobas PCR Media Freezing on SARS-CoV-2 Viral RNA Integrity and Whole Genome Sequencing Analyses

SARS-CoV-2 whole genome sequencing is an important molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swab samples and shipment to reference laboratories is usually required since RNA sequencing is rarely available in routine clinical microbiology laboratories where initial diagnosis and support to outbreak investigations occur. The cobas PCR Media transport medium developed by Roche facilitates high throughput analyses on cobas multianalyzer PCR platforms. There is no data on the stability of SARS-CoV-2 RNA after freezing and thawing of clinical samples in this transport medium, but potential denaturing of the molecular template could impair test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral whole genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Genomic coverage and sequencing depth did not significantly differ between fresh and frozen samples (n=10). For samples with lower viral inoculum and PCR cycle threshold above 30, sequencing quality scores and detection of single nucleotide polymorphisms did not differ either. Freezing of cobas PCR Media does not negatively affect the quality of SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories. Those results support secondary use of diagnostic nasopharyngeal swab material for viral sequencing without requirement for additional clinical samples.

molecular biology↗

bioRxiv2021

Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent individuals up to 8 months post-symptom onset

Functional and lasting immune responses to the novel coronavirus (SARS-CoV-2) are currently under intense investigation as antibody titers in plasma have been shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we sought to determine the presence of SARS-CoV-2-specific memory B cells in COVID-19 convalescent patients. In this study, we report on the evolution of the overall humoral immune responses on 101 blood samples obtained from 32 COVID-19 convalescent patients between 16 and 233 days post-symptom onset. Our observations indicate that anti-Spike and anti-RBD IgM in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity in convalescent plasma declines rapidly compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which increase over time, and the number of IgG+ memory B cells which remain stable thereafter for up to 8 months after symptoms onset. With the recent approval of highly effective vaccines for COVID-19, data on the persistence of immune responses are of central importance. Even though overall circulating SARS-CoV-2 Spike-specific antibodies contract over time during convalescence, we demonstrate that RBD-specific B cells increase and persist up to 8 months post symptom onset. We also observe modest increases in RBD-specific IgG+ memory B cells and importantly, detectable IgG and sustained Fc-effector activity in plasma over the 8-month period. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for the prevention of secondary infections, vaccine efficacy and herd immunity against COVID-19.

microbiology↗

bioRxiv2020

Deregulation of brain endothelial CD2AP in Alzheimer's disease impairs Reelin-mediated neurovascular coupling

Genetic variations in CD2-associated protein (CD2AP) predispose to Alzheimers disease (AD) but the underlying mechanisms remain unknown. Here, we show that a cerebrovascular loss of CD2AP is associated with cognitive decline in AD and that genetic downregulation of CD2AP in brain endothelial cells impairs memory function in two distinct mouse models. Mice with reduced CD2AP in brain microvessels display decreased resting cerebral blood flow, impaired functional hyperemia and vasomotion. In brain endothelial cells, CD2AP regulates the levels and signaling of ApoE receptor 2 elicited by Reelin glycoprotein. Activation of the CD2AP-ApoER2 pathway with Reelin mitigates the toxic effects of A{beta} on resting blood flow and vasomotion of brain vessels depleted of CD2AP. Thus, we demonstrate that deregulation of CD2AP perturbs specific functions and segments of the cerebral microvasculature and propose that targeting CD2AP molecular partners may offer refined therapeutic strategies for the treatment of AD.

neuroscience↗

bioRxiv2020

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labelling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

cell biology↗

bioRxiv2020

Free-fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice

ObjectiveMaintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty-acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (, {beta}, and {delta} cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting {delta} cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. MethodsGlucose tolerance tests were performed in mice after administration of the selective GPR120 agonist Compound A. Insulin, glucagon and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous ({omega}-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knockout of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in , {beta} and {delta} cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. ResultsAcute exposure to Compound A increased glucose tolerance and circulating insulin and glucagon levels in vivo. Endogenous and/or pharmacological and GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in {delta} cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in {delta} cells but increased these signals in and {beta} cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and was partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. ConclusionsInhibitory GPR120 signaling in {delta} cells contributes to both insulin and glucagon secretion in part via mitigating somatostatin release.

physiology↗

bioRxiv2020

Repurposing beta3-adrenergic receptor agonists for Alzheimer's disease: Beneficial effects on recognition memory and amyloid pathology in a mouse model.

ABSTRACTOld age, the most important risk factor for Alzheimer’s disease (AD), is associated with thermoregulatory deficits. Brown adipose tissue (BAT) is the main thermogenic driver in mammals and its stimulation, through β3-adrenergic receptor (β3AR) agonists or cold acclimation, counteracts metabolic deficits in rodents and humans. Studies in animal models show that AD neuropathology leads to thermoregulatory deficits and cold-induced tau hyperphosphorylation is prevented by BAT stimulation through cold acclimation. Since metabolic disorders and AD share strong pathogenic links, we hypothesized that BAT stimulation through a β3AR agonist could exert benefits in AD as well.Here, we show that β3AR agonist administration (CL-316,243, 1 mg/kg/day i.p., from 15 to 16 months of age) decreased body weight and improved peripheral glucose metabolism and BAT thermogenesis in both non-transgenic and 3xTg-AD mice. One-month treatment with a β3AR agonist increased recognition index by 19% in 16-month-old 3xTg-AD mice compared to pre-treatment (14-month-old). Locomotion, anxiety and tau pathology were not modified. Finally, insoluble Aβ42/Aβ40 ratio was decreased by 27% in the hippocampus of CL-316,243-injected 3xTg-AD mice.Overall, our results indicate that β3AR stimulation reverses memory deficits and shifts downward the insoluble Aβ42/Aβ40 ratio in 16-month-old 3xTg-AD mice. As β3AR agonists are being clinically developed for metabolic disorders, repurposing them in AD could be a valuable therapeutic strategy.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Integrated morphometric, molecular, and clinical characterization of Parkinson's disease pathology

Individuals with Parkinsons disease present with a complex clinical phenotype, encompassing sleep, motor, cognitive, and affective disturbances. However, characterizations of PD are typically made for the "average" patient, ignoring patient heterogeneity and obscuring important individual differences. Modern large-scale data sharing efforts provide a unique opportunity to precisely investigate individual patient characteristics, but there exists no analytic framework for comprehensively integrating data modalities. Here we apply an unsupervised learning method--similarity network fusion--to objectively integrate MRI morphometry, dopamine active transporter binding, protein assays, and clinical measurements from n = 186 individuals with de novo Parkinsons disease from the Parkinsons Progression Markers Initiative. We show that multimodal fusion captures inter-dependencies among data modalities that would otherwise be overlooked by field standard techniques like data concatenation. We then examine how patient subgroups derived from fused data map onto clinical phenotypes, and how neuroimaging data is critical to this delineation. Finally, we identify a compact set of phenotypic axes that span the patient population, demonstrating that this continuous, low-dimensional projection of individual patients presents a more parsimonious representation of heterogeneity in the sample compared to discrete biotypes. Altogether, these findings showcase the potential of similarity network fusion for combining multimodal data in heterogeneous patient populations.

neuroscience↗

bioRxiv2018

Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells

Holo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cells refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

cell biology↗