Eugene V Ravkov

University of Utah, Salt Lake City, Utah, United States

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Publications (9)48.97 Total impact

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    ABSTRACT: Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE. We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy. Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects. 17 epitopes restricted to DRB∗01:01, DRB1∗03:01, DRB1∗04:01, DRB1∗09:01, DQB1∗02:01, DQB1∗03:02 and DQB1∗05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13. The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.
    Human immunology 08/2013; · 2.55 Impact Factor
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    ABSTRACT: Cytomegalovirus (CMV) infection is one of the most important infectious complications of transplantation. Monitoring CMV-specific CD8 T cell immunity is useful for predicting active CMV infection and for directing targeted antiviral therapy. In this study, we examined four basic parameters for validation of CMV-specific tetramer staining and peptide stimulation assays that cover five most frequent HLA class I alleles. We also examined the potential use of CMV-specific CD8(+) T cell numbers and functional and cytolytic responses in two autologous HSCT recipients treated for multiple myeloma. The coefficient of variation (CV %) of the precision within assays was 3.1-24% for HLA-tetramer staining, 2.5-47% for IFN-γ, and 3.4-59.7% for CD107a/b production upon peptide stimulation. The precision between assays was 5-26% for tetramer staining, 4-24% for IFN-γ, and 5-48% for CD107a/b. The limit of detection was 0.1-0.23 cells/μL of blood for tetramer staining, 0-0.23 cell/μL for IFN-γ, and 0.11-0.98 cells/μL for CD107a/b. The assays were linear and specific. The reference interval with 95% confidence level was 0-18 cells/μL for tetramer staining, 0-2 cells/μL for IFN-γ, and 0-3 cells/μL for CD107a/b. Our results provide acceptable measures of test performance for CMV immune competence assays for the characterization of CD8(+) T cell responses posttransplant measured in the absolute cell count per μL of blood.
    Clinical and Developmental Immunology 01/2012; 2012:451059. · 3.06 Impact Factor
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    ABSTRACT: Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.
    Human immunology 12/2011; 73(4):426-31. · 2.55 Impact Factor
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    ABSTRACT: IL-2 provides a memory differentiation signal to CD8+ T cells during the primary response that impacts the ability of the subsequent memory pool to mount a successful recall response. In this study, we find that although primary effector CTL development is modestly decreased in the absence of IL-2, the persistence of short-term and long-term effector memory CD8+ T cells on pathogen clearance is greatly diminished. Furthermore, secondary challenge of CD8+ memory T cells lacking the high-avidity IL-2R results in a failure to repopulate the effector pool. The role of IL-2 in promoting effector differentiation is not shared with the highly related cytokine, IL-15. Although IL-15 supports the survival of effector CD8+ T cells after pathogen clearance, its absence does not impair either primary or secondary effector CTL differentiation, nor does it impact the differentiation of long-term effector memory CD8+ T cells. These findings indicate a unique role for IL-2, but not IL-15, in promoting the differentiation not only of primary effector CD8+ T cells, but also of CD8+ memory T cells capable of secondary effector differentiation.
    The Journal of Immunology 06/2010; 184(12):6719-30. · 5.52 Impact Factor
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    Eugene V Ravkov, Matthew A Williams
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    ABSTRACT: The parameters controlling the generation of robust CD4(+) T cell recall responses remain poorly defined. In this study, we compare recall responses by CD4(+) and CD8(+) memory T cells following rechallenge. Homologous rechallenge of mice immune to either lymphocytic choriomeningitis virus or Listeria monocytogenes results in robust CD8(+) T cell recall responses but poor boosting of CD4(+) T cell recall responses in the same host. In contrast, heterologous rechallenge with a pathogen sharing only a CD4(+) T cell epitope results in robust boosting of CD4(+) T cell recall responses. The disparity in CD4(+) and CD8(+) T cell recall responses cannot be attributed to competition for growth factors or APCs, as robust CD4(+) and CD8(+) T cell recall responses can be simultaneously induced following rechallenge with peptide-pulsed dendritic cells. Instead, CD4(+) T cell recall responses are dependent on the duration of the secondary challenge. Increasing the rechallenge dose results in more potent boosting of CD4(+) T cell recall responses and artificially limiting the duration of secondary infection following heterologous rechallenge adversely impacts the magnitude of CD4(+) T cell, but not CD8(+) T cell, recall responses. These findings suggest that rapid pathogen clearance by secondary CTL following homologous rechallenge prevents optimal boosting of CD4(+) T cell responses and therefore have important practical implications in the design of vaccination and boosting strategies aimed at promoting CD4(+) T cell-mediated protection.
    The Journal of Immunology 09/2009; 183(4):2382-9. · 5.52 Impact Factor
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    ABSTRACT: Requirements for CD4+ T cell memory differentiation were analyzed with adoptively transferred SMARTA T cell receptor (TCR) transgenic cells specific for alymphocytic choriomeningitis virus (LCMV) epitope. LCMV-induced effector and memory differentiation of SMARTA cells mimicked the endogenous CD4+ T cell response. In contrast, infection with a recombinant Listeria expressing the LCMV epitope, although resulting initially in massive SMARTA expansion, led to loss of effector function and rapid cell death characterized by high expression of the apoptosis regulator Bim. Defective memory differentiation was seen after stimulation of naive but not memory SMARTA cells, was independent of precursor frequency, and correlated with a lower TCR avidity compared to endogenous responders. In addition, long-lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4+ T cell memory differentiation and longevity depend on the strength of the TCR signal during the primary response.
    Immunity 05/2008; 28(4):533-45. · 19.80 Impact Factor
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    ABSTRACT: Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.
    Journal of Immunological Methods 02/2007; 319(1-2):13-20. · 2.23 Impact Factor
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    ABSTRACT: CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.
    Journal of Immunological Methods 06/2006; 312(1-2):34-9. · 2.23 Impact Factor
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    Eugene V Ravkov, Christy M Myrick, John D Altman
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    ABSTRACT: Memory T cells exhibit a high degree of heterogeneity in terms of their phenotype and functional characteristics. It has been proposed that the CCR7 chemokine receptor divides memory T cell populations into central memory T cells and effector memory T cells with distinct functions in secondary immune responses. We were interested whether this hypothesis holds true in experiments performed on Ag-specific CD8(+) T cells. To identify CCR7(+) cells, we engineered a fluorescent ligand for CCR7; results with the new CC chemokine ligand 19 chemotetramer were verified by staining with a CCR7 mAb. Staining with the CC chemokine ligand 19 chemotetramer reveals two subsets within CCR7(+) cells: a CCR7(int) population containing memory cells and a CCR7(high) population containing naive T cells. Phenotypic analysis of MHC class I/peptide tetramer-positive cells revealed that HLA-A2-restricted CMV-specific CD8 T cells exhibit the lowest percentage of CCR7(+) cells (0.5-5%), while HLA-A2-restricted flu- and HLA-B8-restricted EBV-specific CD8 T cells showed the highest (45-70%). Intracellular staining of unstimulated cells revealed that both CCR7(int)- and CCR7(-)-specific CD8 T cells exhibit a detectable level of perforin. Both CCR7(int) and CCR7(-) Ag-specific CD8(+) T cells produced IFN-gamma and TNF-alpha following short-term peptide stimulation. Therefore, our finding that CCR7(+)CD8(+) T cells are able to exert immediate effector functions requires a substantial revision to the central and effector memory hypothesis.
    The Journal of Immunology 04/2003; 170(5):2461-8. · 5.52 Impact Factor

Publication Stats

279 Citations
48.97 Total Impact Points

Institutions

  • 2009–2013
    • University of Utah
      • Department of Pathology
      Salt Lake City, Utah, United States
  • 2007
    • Wisconsin National Primate Research Center
      Madison, Wisconsin, United States
  • 2003
    • Emory University
      Atlanta, Georgia, United States