The authors would like to thank the NIAID VRC Flow Cytometry Core and the MHRP Flow Cytometry Core facilities for maintenance and operation of FACS instruments and sorting equipment; Maria Montero, Vishakha Sharma, Kaimei Song for expert technical assistance; Michael Piatak, Jr. (deceased) for assistance with SIV qPCR assay design; and Brandon Keele and Matthew Scarlotta for SIV isolate sequences. The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense. Research was conducted under an approved animal use protocol in an AAALAC accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.

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This work was supported by a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense (DOD). The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense. Research was conducted under an approved animal use protocol in an AAALACi accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.

The protocol described here, termed tSCEPTRE, integrates single-cell surface protein quantitation by multiparameter flow cytometry with quantitative single-cell mRNA expression by highly multiplexed RT-qPCR. The union of these two technologies enables high-content snapshots of the combined transcriptional and protein profile of single cells in a high-throughput format. We use the method to identify heretofore elusive cells infected with SIV in vivo, and describe differentially expressed host genes and proteins. ...

Many intracellular pathogens rely on host cell machinery to replicate, often altering host cell biology or targeting very specific subpopulations of host cells to maximize their chances of propagation. As a result, cell biological processes are commonly disrupted, with deleterious consequences for the overall health of the host. Understanding the interactions between viruses and the host cells in which they replicate will elucidate disease mechanisms that may aid in the development of improved therapies and strategies to prevent infection. Direct analytic tools that enable the study of host-pathogen interactions are essential toward this end. Single-cell analysis provides the only means to unambiguously attribute a cellular phenotype to a particular genotype, or infection status1. For example, pathogenic infections frequently induce both direct and indirect changes in host cells. Therefore, distinguishing infected cells from their uninfected counterparts is necessary to attribute host cell changes to either direct infection or secondary effects, such as generalized inflammation. Moreover, for many pathogens, like SIV and human immunodeficiency viruses (HIV), host cell infection proceeds through multiple stages, such as early, late, or latent, each of which may be characterized by distinct gene and protein expression profiles2,3,4,5. Bulk analyses of cell mixtures will fail to capture this heterogeneity6. By contrast, highly multiplexed single-cell analyses able to quantify the expression of both viral and host genes offer a means to resolve infection-specific cellular perturbations, including variations across infection stages. Further, analyzing host-pathogen interactions in physiologically relevant settings is critical for the identification of events that occur in infected organisms. Thus, methods that can be applied directly ex vivo are likely to best capture in vivo processes.
SIV and HIV target CD4+ T cells, in which they counteract host antiviral &#34;restriction&#34; factors and downregulate antigen presenting molecules to establish productive infection and avoid immune surveillance7,8,9,10,11. Without treatment, the infection results in massive loss of CD4+ T cells, ultimately culminating in acquired immunodeficiency syndrome (AIDS)12. In the setting of antiretroviral therapy, latently infected cell reservoirs persist for decades, posing a formidable barrier to curative strategies. Understanding the properties of in vivo HIV/SIV-infected cells has the potential to reveal host cell features instrumental in pathogenesis and persistence. However, this has been highly challenging, primarily due to the low frequency of infected cells and lack of reagents able to readily identify them. Cells that transcribe viral RNA, are estimated to be present at 0.01&#8211;1% of CD4+ T cells in blood and lymphoid tissue13,14,15. Under suppressive therapy, latently infected cells are even less frequent at 10-3&#8211;10-7 16,17,18. Viral protein staining assays that work well for studying in vitro infections, such as for intracellular Gag, are suboptimal due to background staining of 0.01&#8211;0.1%, similar to or greater than the frequency of infected cells13,14. Surface staining for Env protein using well-characterized SIV/HIV Env-specific monoclonal antibodies has also been proven to be difficult, likely for similar reasons. Recently, novel tools aim to improve the detection of cells expressing Gag by either incorporating assays specific for gag RNA or by using alternative imaging technologies14,15,19. However, such approaches remain limited in the number of quantitative measurements performed on each cell.
Here, we describe methodology that (1) identifies single virus-infected cells directly ex vivo by sensitive and specific viral gene quantitative qPCR and (2) quantifies the expression of up to 18 surface proteins and 96 genes for each infected (and uninfected) cell. This methodology combines single-cell surface protein measurement by FACS followed by immediate cell lysis and gene expression analysis using multiplexed targeted qPCR on the Biomark system. The integrated fluidic circuit (IFC) technology allows multiplexed quantitation of 96 genes from 96 samples simultaneously, accomplished by a matrix of 9,216 chambers in which the individual qPCR reactions are performed. The live cell FACS sorting records high-content protein abundance measurements while preserving the entire transcriptome for analysis performed immediately downstream. To identify virus-infected cells, assays specific for alternatively spliced and unspliced viral RNAs (vRNA) are included in the qPCR analysis, along with a panel of user-defined assays totaling up to 96 genes, the maximum number of assays currently accommodated in the IFC. The gene expression and protein information collected for each cell are linked by well position. We previously reported results from this analysis elsewhere20. Here, we provide more detailed methodological guidelines as well as further descriptive phenotyping of SIV-infected CD4+ T cells.
This approach, which we term tSCEPTRE, can be applied to the suspensions of any viable cell population reactive to fluorescently labeled antibodies and expressing a transcriptome compatible with available qPCR assays. For example, it can be used for characterizing differential gene and protein expression in rare cells or cells not readily distinguished by surface protein markers. The sample preparation relies on a standard staining protocol using commercially available antibodies. Cytometers with single-cell sorting capability are also commercially available, but additional biosafety precautions are required for processing infectious live cells. Recording the single-cell protein expression profile for each cell by well position, referred to herein as indexed sorting, is a common feature of commercially available FACS sorting software. Computational analysis of differentially expressed host genes among cell populations of interest is not described here, but references are provided to previously published methods.

<table><tbody><tr><td> &lt;p&gt;&lt;strong&gt;RNA extraction and PCR reagents and consumables&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;Genemate 96-Well Semi-Skirted PCR Plate&lt;/p&gt; </td><td> &lt;p&gt;BioExpress/VWR&lt;/p&gt; </td><td> &lt;p&gt;T-3060-1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Adhesive PCR Plate Seals&lt;/p&gt; </td><td> &lt;p&gt;ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;AB0558&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Armadillo 384-well PCR Plate&lt;/p&gt; </td><td> &lt;p&gt;ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;AB2384&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;MicroAmp Optical Adhesive Film&lt;/p&gt; </td><td> &lt;p&gt;Applied Biosystems/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;4311971&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;DEPC Water&lt;/p&gt; </td><td> &lt;p&gt;Quality Biological&lt;/p&gt; </td><td> &lt;p&gt;351-068-101&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Glass Distilled Water&lt;/p&gt; </td><td> &lt;p&gt;Teknova&lt;/p&gt; </td><td> &lt;p&gt;W3345&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Superscript III Platinum One-Step qRT-PCR Kit&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;11732088&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;SUPERase-In Rnase Inhibitor&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;AM2696&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Platinum Taq&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;10966034&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;dNTP Mix&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;18427088&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;ROX Reference Dye (if separate from kit)&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;12223012&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;DNA Suspension Buffer&lt;/p&gt; </td><td> &lt;p&gt;Teknova&lt;/p&gt; </td><td> &lt;p&gt;T0223&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;RNAqueous kit&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;AM1931&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;TaqMan gene expression assays not listed in Table 2&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;CD6&lt;/p&gt; </td><td> &lt;p&gt;Applied Biosystems/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;Hs00198752_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TLR3&lt;/p&gt; </td><td> &lt;p&gt;Applied Biosystems/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;Hs1551078_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;Biomark reagents&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;Control Line Fluid Kit&lt;/p&gt; </td><td> &lt;p&gt;Fluidigm&lt;/p&gt; </td><td> &lt;p&gt;89000021&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan Universal PCR Mix&lt;/p&gt; </td><td> &lt;p&gt;Applied Biosystems/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;4304437&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Assay Loading Reagent&lt;/p&gt; </td><td> &lt;p&gt;Fluidigm&lt;/p&gt; </td><td> &lt;p&gt;85000736&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sample Loading Reagent&lt;/p&gt; </td><td> &lt;p&gt;Fluidigm&lt;/p&gt; </td><td> &lt;p&gt;85000735&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Dynamic Array 96.96 (chip)&lt;/p&gt; </td><td> &lt;p&gt;Fluidigm&lt;/p&gt; </td><td> &lt;p&gt;BMK-M-96.96&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;FACS reagents&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;SPHERO COMPtrol Goat anti-mouse (lambda)&lt;/p&gt; </td><td> &lt;p&gt;Spherotech Inc.&lt;/p&gt; </td><td> &lt;p&gt;CMIgP-30-5H&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;CompBeads Anti-Mouse Ig,k&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;51-90-9001229&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;5 ml Polystyrene tube with strainer cap&lt;/p&gt; </td><td> &lt;p&gt;FALCON&lt;/p&gt; </td><td> &lt;p&gt;352235&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Aqua Live/Dead stain&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;L34976&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:800&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD3 BV650 clone SP34-2&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;563916&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:40&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD4 BV786 clone L200&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;563914&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:20&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD8 BUV496 clone RPA-T8&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;564804&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:10&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD28 BV711 clone CD28.2&lt;/p&gt; </td><td> &lt;p&gt;Biolegend&lt;/p&gt; </td><td> &lt;p&gt;302948&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:20&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD95 BUV737 clone DX2&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;564710&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:10&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD14 BV510 clone M5E2&lt;/p&gt; </td><td> &lt;p&gt;Biolegend&lt;/p&gt; </td><td> &lt;p&gt;301842&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:83&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD16 BV510 clone 3G8&lt;/p&gt; </td><td> &lt;p&gt;Biolegend&lt;/p&gt; </td><td> &lt;p&gt;302048&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:167&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD20 BV510 clone 2H7&lt;/p&gt; </td><td> &lt;p&gt;Biolegend&lt;/p&gt; </td><td> &lt;p&gt;302340&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:37&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-CD38-R PE clone OKT10&lt;/p&gt; </td><td> &lt;p&gt;NHP reagent recource&lt;/p&gt; </td><td> &lt;p&gt;N/A&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:100&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human CD69 BUV395 clone FN50&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;564364&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:10&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human HLA-DR APC-H7 clone G46-6&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td> &lt;p&gt;561358&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:20&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse Anti-Human ICOS Alexa Fluor 700 clone C398.4A&lt;/p&gt; </td><td> &lt;p&gt;Biolegend&lt;/p&gt; </td><td> &lt;p&gt;313528&lt;/p&gt; </td><td> &lt;p&gt;dilute 1:80&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;&lt;strong&gt;Instruments&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;BioPrptect Containment Enclosure&lt;/p&gt; </td><td> &lt;p&gt;Baker&lt;/p&gt; </td><td></td><td></td></tr><tr><td> &lt;p&gt;BD FACS Aria&lt;/p&gt; </td><td> &lt;p&gt;BD Biosciences&lt;/p&gt; </td><td></td><td></td></tr><tr><td> &lt;p&gt;ProtoFlex Dual 96-well PCR system&lt;/p&gt; </td><td> &lt;p&gt;Applied Biosystems/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;4484076&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Quant Studio 6 qPCR instrument&lt;/p&gt; </td><td> &lt;p&gt;Applied Biosystems/ThermoFisher&lt;/p&gt; </td><td> &lt;p&gt;4485694&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;IFC controller HX&lt;/p&gt; </td><td> &lt;p&gt;Fluidigm&lt;/p&gt; </td><td> &lt;p&gt;IFC-HX&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Biomark HD&lt;/p&gt; </td><td> &lt;p&gt;Fluidigm&lt;/p&gt; </td><td> &lt;p&gt;BMKHD-BMKHD&lt;/p&gt; </td><td></td></tr></tbody></table>

single-cell quantitation of mrna and surface protein expression in simian immunodeficiency virus-infected cd4+ t cells isolated from rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

The workflow for the entire protocol is depicted in Figure 1. It consists of two variations defined by the number of cells sorted: either limiting dilution or as single cells, as described in the text. Examples of primer-probe qualification analyses on 2-fold serial RNA dilutions are shown in Figure 2. The gating strategy to identify potential SIV+ cells is shown in Figure 3. A successful,...

Watch this Scientific Journal Video about Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques at JoVE.c

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

single-cell-quantitation-mrna-surface-protein-expression-simian

Research

JoVE Journal

Immunology and Infection

10.6K Views.  Walter Reed Army Institute of Research. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van gastheer-pathogen interacties, zoals wat zijn de unieke kenmerken van de productief geïnfecteerde cellen, welke gastheer cofactoren virussen toestaan om productieve infectie vast te stellen, en hoe deze cellen te richten in therapeutische interventies. Het belangrijkste voordeel van deze techniek is dat het kwantitatieve maatregelen biedt voor zowel eiwit als mRNA binnen enkele cellen, en de meeste reagenti...

Video: Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Biology

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting ...

Bioengineering

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4⁺ T Cells Isolated from Rhesus macaques

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