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In JoVE (1)
- A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
Other Publications (12)
- Neuromolecular Medicine
- Journal of Immunological Methods
- Journal of Virology
- Current Protocols in Immunology / Edited by John E. Coligan ... [et Al.]
- BMC Genomics
- Journal of Immunology (Baltimore, Md. : 1950)
- PloS One
- Journal of Virology
- Advances and Applications in Bioinformatics and Chemistry : AABC
- PloS One
- Science (New York, N.Y.)
- PloS One
Articles by Pamela J. Skinner in JoVE
JoVE General
A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
1Department of Microbiology, Medical School, University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University of Minnesota
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.
Other articles by Pamela J. Skinner on PubMed
Amino Acids in a Region of Ataxin-1 Outside of the Polyglutamine Tract Influence the Course of Disease in SCA1 Transgenic Mice
Spinocerebellar ataxia type 1 (SCA1) belongs to a family of polyglutamine induced neurodegenerative disorders. Transgenic mice that overexpress a mutant allele of the SCA1 gene develop a progressive ataxia and Purkinje cell pathology. In this report, the pathological importance of a segment of ataxin-1 previously shown to be important for protein-protein interactions was examined. While the absence of a 122 amino acid segment from the protein-protein interaction region of ataxin-1 did not effect the initiation of disease, its absence substantially suppressed the progression of disease in SCA1 transgenic mice. Thus, these data suggest that this region of ataxin-1 has a role in disease progression. Furthermore, these results provide evidence that ataxin-1-induced disease initiation and disease progression involve distinct molecular events.
In Situ Tetramer Staining
The development of MHC tetramer staining has opened the doors to multiple avenues of new research [Science 274 (1996) 94]. In this review, we will discuss the development and application of in situ MHC tetramer (IST) staining. We describe two independently developed IST staining methodologies and discuss current uses, limitations, future uses and the interesting biology revealed by the use of IST staining.
CD8+ T-lymphocyte Response to Major Immunodominant Epitopes After Vaginal Exposure to Simian Immunodeficiency Virus: Too Late and Too Little
In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8+ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4+ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4+ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8+ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4+ T lymphocytes in the GALT. Thus, the virus-specific CD8+ T-lymphocyte response is "too late and too little" to clear infection and prevent CD4+ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8+ T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of transmission.
In Situ Staining Using MHC Class I Tetramers
The invention of MHC-tetramer technology to label antigen-specific T cells has lead to a greatly enhanced understanding of T lymphocyte biology. This protocol describes the use of MHC class I tetramers to stain antigen-specific T cells in tissue sections. In situ tetramer staining (IST) can be used to determine the localization, abundance, and phenotype of antigen-specific T cells in native environments and in three-dimensional space. IST is broadly applicable because it can be used to stain essentially any antigen-specific T cell in any tissue for which MHC tetramers are available. IST can be combined with histochemistry and/or immunohistochemistry to permit visualization and characterization of antigen-specific T cells relative to other cell types in stained tissue sections. Thus, IST is a useful and valuable component of MHC-tetramer technology.
Gene Expression Alterations in Brains of Mice Infected with Three Strains of Scrapie
Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML.
CTL Fail to Accumulate at Sites of HIV-1 Replication in Lymphoid Tissue
The inability of HIV-1-specific CTL to fully suppress virus replication as well as the failure of administration of exogenous CTL to lower viral loads are not understood. To evaluate the hypothesis that these phenomena are due to a failure of CTL to localize at sites of HIV-1 replication, we assessed the distribution of HIV-1 RNA and HIV-1-specific CTL identified by HIV-1 peptide/HLA class I tetrameric complexes (tetramers) within lymph nodes of 14 HIV-1-infected individuals who were not receiving antiretroviral therapy. A median of 0.04% of follicular compared with 0.001% of extrafollicular CD4(+) cells were estimated to be producing HIV-1 RNA, a 40-fold difference (p = 0.0001). Tetramer-stained cells were detected by flow cytometry in disaggregated lymph node cells from 11 subjects and constituted a significantly higher fraction of CD8(+) cells in lymph node (mean, 2.15%) than in PBMC (mean, 1.52%; p = 0.02). In situ tetramer staining in three subjects' lymph nodes, in which high frequencies of tetramer-stained cells were detected, revealed that tetramer-stained cells were primarily concentrated in extrafollicular regions of lymph node and were largely absent within lymphoid follicles. These data confirm that HIV-1-specific CTL are abundant within lymphoid tissues, but fail to accumulate within lymphoid follicles where HIV-1 replication is concentrated, suggesting that lymphoid follicles may be immune-privileged sites. Mechanisms underlying the exclusion of CTL from lymphoid follicles as well as the role of lymphoid follicles in perpetuating other chronic pathogens merit further investigation.
Allogeneic Lymphocytes Persist and Traffic in Feral MHC-matched Mauritian Cynomolgus Macaques
BACKGROUND: Thus far, live attenuated SIV has been the most successful method for vaccinating macaques against pathogenic SIV challenge; however, it is not clear what mechanisms are responsible for this protection. Adoptive transfer studies in mice have been integral to understanding live attenuated vaccine protection in models like Friend virus. Previous adoptive transfers in primates have failed as transferred cells are typically cleared within hours after transfer. METHODOLOGY/ PRINCIPAL FINDINGS: Here we describe adoptive transfer studies in Mauritian origin cynomolgus macaques (MCM), a non-human primate model with limited MHC diversity. Cells transferred between unrelated MHC-matched macaques persist for at least fourteen days but are rejected within 36 hours in MHC-mismatched macaques. Cells trafficked from the blood to peripheral lymphoid tissues within 12 hours of transfer. CONCLUSIONS/SIGNIFICANCE: MHC-matched MCM provide the first viable primate model for adoptive transfer studies. Because macaques infected with SIV are the best model for HIV/AIDS pathogenesis, we can now directly study the correlates of protective immune responses to AIDS viruses. For example, plasma viral loads following pathogenic SIV challenge are reduced by several orders of magnitude in macaques previously immunized with attenuated SIV. Adoptive transfer of lymphocyte subpopulations from vaccinated donors into SIV-naïve animals may define the immune mechanisms responsible for protection and guide future vaccine development.
With Minimal Systemic T-cell Expansion, CD8+ T Cells Mediate Protection of Rhesus Macaques Immunized with Attenuated Simian-human Immunodeficiency Virus SHIV89.6 from Vaginal Challenge with Simian Immunodeficiency Virus
The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus 89.6 (SHIV89.6) is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here, we definitively demonstrate the protective role of the SIV-specific CD8(+) T-cell response in SHIV-immunized monkeys by CD8(+) lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T-cell responses in blood, lymph nodes, and genital mucosa. While in the T-cell-intact SHIV-immunized animals, polyfunctional and degranulating SIV-specific CD8(+) T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 postchallenge, strikingly, expansion of SIV-specific CD8(+) T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV89.6 is primarily mediated by CD8(+) T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8(+) T cells can provide significant protection from vaginal SIV challenge.
Prion Disease Induced Alterations in Gene Expression in Spleen and Brain Prior to Clinical Symptoms
Prion diseases are fatal neurodegenerative disorders that affect animals and humans. There is a need to gain understanding of prion disease pathogenesis and to develop diagnostic assays to detect prion diseases prior to the onset of clinical symptoms. The goal of this study was to identify genes that show altered expression early in the disease process in the spleen and brain of prion disease-infected mice. Using Affymetrix microarrays, we identified 67 genes that showed increased expression in the brains of prion disease-infected mice prior to the onset of clinical symptoms. These genes function in many cellular processes including immunity, the endosome/lysosome system, hormone activity, and the cytoskeleton. We confirmed a subset of these gene expression alterations using other methods and determined the time course in which these changes occur. We also identified 14 genes showing altered expression prior to the onset of clinical symptoms in spleens of prion disease infected mice. Interestingly, four genes, Atp1b1, Gh, Anp32a, and Grn, were altered at the very early time of 46 days post-infection. These gene expression alterations provide insights into the molecular mechanisms underlying prion disease pathogenesis and may serve as surrogate markers for the early detection and diagnosis of prion disease.
Localized Populations of CD8 MHC Class I Tetramer SIV-specific T Cells in Lymphoid Follicles and Genital Epithelium
CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8(+). Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer(+)CD8(low/-) cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer(+)CD20(-) cells inside B cell follicles and that tetramer(+) cells did not stain with gammadelta-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer(+)CD8(low/-) cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium.
Visualizing Antigen-specific and Infected Cells in Situ Predicts Outcomes in Early Viral Infection
In the early stages of viral infection, outcomes depend on a race between expansion of infection and the immune response generated to contain it. We combined in situ tetramer staining with in situ hybridization to visualize, map, and quantify relationships between immune effector cells and their targets in tissues. In simian immunodeficiency virus infections in macaques and lymphocytic choriomeningitis virus infections in mice, the magnitude and timing of the establishment of an excess of effector cells versus targets were found to correlate with the extent of control and the infection outcome (i.e., control and clearance versus partial or poor control and persistent infection). This method highlights the importance of the location, timing, and magnitude of the immune response needed for a vaccine to be effective against agents of persistent infection, such as HIV-1.
GagCM9-specific CD8+ T Cells Expressing Limited Public TCR Clonotypes Do Not Suppress SIV Replication in Vivo
Several lines of evidence suggest that HIV/SIV-specific CD8(+) T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag(45-269)) that were subsequently infected with SIVsmE660. These seven Mamu-A*01(+) animals developed CD8(+) T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8(+) T cells could not control virus replication in vivo. GagCM9-specific CD8(+) T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8(+) T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20-250 GagCM9-specific CD8(+) T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8(+) T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8(+) T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8(+) T cell population elicited by vaccination and infection.
