Publications by authors named "Ted H Hansen"

67 Publications

Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation.

Genome Med 2021 Apr 21;13(1):56. Epub 2021 Apr 21.

Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO, 63110, USA.

Background: Preclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation.

Methods: We developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor.

Results: Our data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ub) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor.

Conclusions: We have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.
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http://dx.doi.org/10.1186/s13073-021-00872-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059244PMC
April 2021

A herpesvirus encoded Qa-1 mimic inhibits natural killer cell cytotoxicity through CD94/NKG2A receptor engagement.

Elife 2018 12 21;7. Epub 2018 Dec 21.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, United States.

A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of 'missing-self' recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.
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http://dx.doi.org/10.7554/eLife.38667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320069PMC
December 2018

Immunodominant West Nile Virus T Cell Epitopes Are Fewer in Number and Fashionably Late.

J Immunol 2016 05 20;196(10):4263-73. Epub 2016 Apr 20.

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104;

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.
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http://dx.doi.org/10.4049/jimmunol.1501821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874531PMC
May 2016

TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells.

Eur J Immunol 2016 07 30;46(7):1600-14. Epub 2016 May 30.

Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, Oxfordshire, United Kingdom.

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1-mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1-mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1-mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B-cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B-cell lines, suggesting differential regulation in different cell types. APC activation and NF-κB signaling were critical for MR1-mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1-mediated MAIT cell activation. Overall, MR1-mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.
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http://dx.doi.org/10.1002/eji.201545969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297987PMC
July 2016

Functional Heterogeneity and Antimycobacterial Effects of Mouse Mucosal-Associated Invariant T Cells Specific for Riboflavin Metabolites.

J Immunol 2015 Jul 10;195(2):587-601. Epub 2015 Jun 10.

Division of Infectious Diseases, Allergy, and Immunology, Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, MO 63104; Department of Microbiology and Immunology, Edward A. Doisy Research Center, Saint Louis University School of Medicine, Saint Louis, MO 63104

Mucosal-associated invariant T (MAIT) cells have a semi-invariant TCR Vα-chain, and their optimal development is dependent upon commensal flora and expression of the nonpolymorphic MHC class I-like molecule MR1. MAIT cells are activated in an MR1-restricted manner by diverse strains of bacteria and yeast, suggesting a widely shared Ag. Recently, human and mouse MR1 were found to bind bacterial riboflavin metabolites (ribityllumazine [RL] Ags) capable of activating MAIT cells. In this study, we used MR1/RL tetramers to study MR1 dependency, subset heterogeneity, and protective effector functions important for tuberculosis immunity. Although tetramer(+) cells were detected in both MR1(+/+) and MR1(-/-) TCR Vα19i-transgenic (Tg) mice, MR1 expression resulted in significantly increased tetramer(+) cells coexpressing TCR Vβ6/8, NK1.1, CD44, and CD69 that displayed more robust in vitro responses to IL-12 plus IL-18 and RL Ag, indicating that MR1 is necessary for the optimal development of the classic murine MAIT cell memory/effector subset. In addition, tetramer(+) MAIT cells expressing CD4, CD8, or neither developing in MR1(+/+) Vα19i-Tg mice had disparate cytokine profiles in response to RL Ag. Therefore, murine MAIT cells are considerably more heterogeneous than previously thought. Most notably, after mycobacterial pulmonary infection, heterogeneous subsets of tetramer(+) Vα19i-Tg MAIT cells expressing CXCR3 and α4β1 were recruited into the lungs and afforded early protection. In addition, Vα19iCα(-/-)MR(+/+) mice were significantly better protected than were Vα19iCα(-/-)MR1(-/-), wild-type, and MR1(-/-) non-Tg mice. Overall, we demonstrate considerable functional diversity of MAIT cell responses, as well as that MR1-restricted MAIT cells are important for tuberculosis protective immunity.
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http://dx.doi.org/10.4049/jimmunol.1402545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490942PMC
July 2015

MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage.

J Exp Med 2014 Jul 21;211(8):1601-10. Epub 2014 Jul 21.

Division of Pulmonary and Critical Care Medicine, Department of Molecular Microbiology and Immunology, and Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239Division of Pulmonary and Critical Care Medicine, Department of Molecular Microbiology and Immunology, and Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239 Portland VA Medical Center, Portland, OR 97239

Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRβ chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.
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http://dx.doi.org/10.1084/jem.20140507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113934PMC
July 2014

A novel T-cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice.

Eur J Immunol 2014 Jul 3;44(7):1936-46. Epub 2014 May 3.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2D(b) , were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8(+) T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α(+) CD11c(+) dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b(+) CD11c(-) cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.
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http://dx.doi.org/10.1002/eji.201444450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107030PMC
July 2014

Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells.

J Exp Med 2013 Oct 7;210(11):2305-20. Epub 2013 Oct 7.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity; and 2 Department of Anatomy and Neuroscience; The University of Melbourne, Parkville, Victoria 3010, Australia.

Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH₂OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8α(+)CD8β(-/lo), implying predominant expression of CD8αα homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH₂OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in Vα19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-β repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.
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http://dx.doi.org/10.1084/jem.20130958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804952PMC
October 2013

CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner.

Eur J Immunol 2014 Jan 1;44(1):195-203. Epub 2013 Oct 1.

Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK.

CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-α chain 7.2 (Vα7.2)-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) Vα7.2(+) and CD161(++) Vα7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.
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http://dx.doi.org/10.1002/eji.201343509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947164PMC
January 2014

Decoupling the role of ubiquitination for the dislocation versus degradation of major histocompatibility complex (MHC) class I proteins during endoplasmic reticulum-associated degradation (ERAD).

J Biol Chem 2013 Aug 25;288(32):23295-306. Epub 2013 Jun 25.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Aberrantly or excessively expressed proteins in the endoplasmic reticulum are identified by quality control mechanisms and dislocated to the cytosol for proteasome-mediated, ubiquitin-dependent degradation by a process termed endoplasmic reticulum-associated degradation (ERAD). In addition to its role in degradation, ubiquitination has also been implicated in substrate dislocation, although whether direct ubiquitin conjugation of ERAD substrates is required for dislocation has been difficult to ascertain. An obstacle in probing the mechanism of quality control-induced ERAD is the paucity of ERAD substrates being dislocated and detected at any given time. To obviate this problem, we report here the use of a sensitive biotinylation system to probe the dislocation of major histocompatibility complex I (MHCI) heavy chain substrates in the absence of immune evasion proteins. Using this assay system the dislocation of MHCI heavy chains was found not to require potential ubiquitin conjugation sites in the cytoplasmic tail or Lys residues in the ectodomain. By contrast, dislocation of MHCI heavy chains did require deubiquitinating enzyme activity and rapid proteasome-mediated degradation required Lys residues in MHCI heavy chain ectodomain. These combined findings support the model that the endoplasmic reticulum quality control-induced dislocation of MHCI heavy chains may not require direct ubiquitination/deubiquitination as is required for proteasome-mediated degradation post dislocation.
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http://dx.doi.org/10.1074/jbc.M113.482018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743500PMC
August 2013

Antibody-peptide-MHC fusion conjugates target non-cognate T cells to kill tumour cells.

Cancer Immunol Immunother 2013 Jun 19;62(6):1093-105. Epub 2013 Apr 19.

Antibody and Vaccine Group MP88, Cancer Sciences Unit, Faculty of Medicine, General Hospital, University of Southampton, Tremona Road, Southampton SO16 6YD, UK.

Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT × Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.
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http://dx.doi.org/10.1007/s00262-013-1408-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869625PMC
June 2013

MAIT cell recognition of MR1 on bacterially infected and uninfected cells.

PLoS One 2013 14;8(1):e53789. Epub 2013 Jan 14.

Integrated Department of Immunology, National Jewish Health and University of Colorado School of Medicine, Denver, Colorado, United States of America.

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3544856PMC
July 2013

Cowpox virus employs a two-pronged strategy to outflank MHCI antigen presentation.

Mol Immunol 2013 Sep 10;55(2):156-8. Epub 2013 Jan 10.

Division of Immunobiology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, United States.

Smallpox decimated humanity for thousands of years before being eradicated by vaccination, a success facilitated by the fact that humans are the only host of variola virus. In contrast, other orthopoxviruses such as cowpox virus can infect a variety of mammalian species, although its dominant reservoir appears to be rodents. This difference in host specificity suggests that cowpox may have developed promiscuous immune evasion strategies to facilitate zoonosis. Recent experiments have established that cowpox can disrupt MHCI antigen presentation during viral infection of both human and murine cells, a process enabled by two unique proteins, CPXV012 and CPXV203. While CPXV012 inhibits antigenic peptide transport from the cytosol to the ER, CPXV203 blocks MHCI trafficking to the cell surface by exploiting the KDEL-receptor recycling pathway. Our recent investigations of CPXV203 reveal that it binds a diverse array of classical and non-classical MHCI proteins with dramatically increased affinities at the lower pH of the Golgi relative to the ER, thereby providing mechanistic insight into how it works synergistically with KDEL receptors to block MHCI surface expression. The strategy used by cowpox to both limit peptide supply and disrupt trafficking of fully assembled MHCI acts as a dual-edged sword that effectively disables adaptive immune surveillance of infected cells.
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http://dx.doi.org/10.1016/j.molimm.2012.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737064PMC
September 2013

Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection.

Blood 2013 Feb 18;121(6):951-61. Epub 2012 Dec 18.

National Institute for Health Research Biomedical Research Centre, University of Oxford, Oxford, UK.

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.
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http://dx.doi.org/10.1182/blood-2012-06-436436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567342PMC
February 2013

Structural mechanism of ER retrieval of MHC class I by cowpox.

PLoS Biol 2012 27;10(11):e1001432. Epub 2012 Nov 27.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.
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http://dx.doi.org/10.1371/journal.pbio.1001432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507924PMC
April 2013

A structural and molecular dynamics approach to understanding the peptide-receptive transition state of MHC-I molecules.

Mol Immunol 2013 Sep 28;55(2):123-5. Epub 2012 Nov 28.

Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases NIAID, NIH, Bethesda, MD 20892-1892, USA.

The mature conformation of major histocompatibility complex class I (MHC-I) proteins depends on the presence of bound peptides, permitting recognition at the cell surface by CD8(+) T lymphocytes. Newly synthesized MHC-I molecules in the endoplasmic reticulum are maintained in a peptide-receptive (PR) transition state by several chaperones until they are released concomitant with the loading of peptides. By determining the crystallographic structure of a region of an MHC-I molecule that is recognized by a unique monoclonal antibody and comparing this with docking and molecular dynamics simulations with the whole molecule, we demonstrate the movement of a hinged unit supporting the part of the binding groove that interacts with the amino terminal residues of the bound peptide. This unit contains a conserved 310 helix that flips from an exposed "open" position in the PR form to a "closed" position in the peptide-loaded (PL) mature molecule. These analyses indicate how this segment of the MHC-I molecule moves to help establish the A and B pockets critical for tight peptide binding and the stable structure required for antigen presentation and T cell recognition at the cell surface.
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http://dx.doi.org/10.1016/j.molimm.2012.10.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632263PMC
September 2013

Immunology: Vitamins prime immunity.

Nature 2012 Nov;491(7426):680-1

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http://dx.doi.org/10.1038/491680aDOI Listing
November 2012

Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.

Nat Immunol 2012 Nov 30;13(11):1118-28. Epub 2012 Sep 30.

Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York, USA.

We assessed gene expression in tissue macrophages from various mouse organs. The diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBP-α, Bach1 and CREG-1 were among the transcriptional regulators predicted to regulate these core macrophage-associated genes. The mRNA encoding other transcription factors, such as Gata6, was associated with single macrophage populations. We further identified how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells.
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http://dx.doi.org/10.1038/ni.2419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558276PMC
November 2012

Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination.

Proc Natl Acad Sci U S A 2012 Jul 16;109(31):12716-21. Epub 2012 Jul 16.

Department of Surgery, Washington University School of Medicine, St Louis, MO 63110, USA.

Activation of naïve cluster of differentiation (CD)8(+) cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8(+) T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8(+) T cells and requires CD8α(+)/CD103(+) DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8(+) T-cell responses to viral infection, tumors, and vaccines.
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http://dx.doi.org/10.1073/pnas.1203468109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411977PMC
July 2012

Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection.

Infect Immun 2012 Sep 9;80(9):3256-67. Epub 2012 Jul 9.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA.

Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection.
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http://dx.doi.org/10.1128/IAI.00279-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418730PMC
September 2012

The peptide-receptive transition state of MHC class I molecules: insight from structure and molecular dynamics.

J Immunol 2012 Aug 29;189(3):1391-9. Epub 2012 Jun 29.

Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I-restricted CD8(+) T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 3(10) helix that flips from an exposed "open" position in the PR transition state to a "closed" position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46-53 of murine H-2L(d) MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.
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http://dx.doi.org/10.4049/jimmunol.1200831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422668PMC
August 2012

Amino-terminal extended peptide single-chain trimers are potent synthetic agonists for memory human CD8+ T cells.

J Immunol 2012 Jun 9;188(12):5839-49. Epub 2012 May 9.

Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.

Upon Ag exposure, most memory T cells undergo restimulation-induced cell death. In this article, we describe a novel synthetic agonist, an N-terminal extended decamer peptide expressed as a single-chain trimer, the amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT), which preferentially promotes the growth of memory human CD8(+) T cells with minimal restimulation-induced cell death. Using CMV pp65 and melanoma gp100 Ags, we observe the in vitro numerical expansion of a clonally diverse polyfunctional population of Ag-specific CD8(+) T cells from healthy individuals and vaccinated melanoma patients, respectively. Memory CD8(+) T cells stimulated with AT-SCT presented on MHC class I/II-null cells show reduced cytokine production, slower kinetics of TCR downregulation, and decreased cell death compared with native nonamer MHC class I single-chain trimer (SCT)-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC complexes using fluorochrome-conjugated TCR multimers suggests that nonamer- and decamer-linked peptides may be anchored differently to the HLA-A2 peptide-binding groove. Our findings demonstrate that modified peptide-MHC structures, such as AT-SCT, can be engineered as T cell agonists to promote the growth and expansion of memory human CD8(+) T cells.
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http://dx.doi.org/10.4049/jimmunol.1103647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370155PMC
June 2012

Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor.

J Exp Med 2012 Apr 12;209(4):761-74. Epub 2012 Mar 12.

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia.

Mucosal-associated invariant T (MAIT) cells express a semiinvariant αβ T cell receptor (TCR) that binds MHC class I-like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vβ2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR-MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR β chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR-MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR-CD1d-antigen interaction, in which both the α and β chain of the NKT TCR is required for ligation above the F'-pocket of CD1d.
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http://dx.doi.org/10.1084/jem.20112095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328369PMC
April 2012

Newly discovered viral E3 ligase pK3 induces endoplasmic reticulum-associated degradation of class I major histocompatibility proteins and their membrane-bound chaperones.

J Biol Chem 2012 Apr 8;287(18):14467-79. Epub 2012 Mar 8.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Viral immune invasion proteins are highly effective probes for studying physiological pathways. We report here the characterization of a new viral ubiquitin ligase pK3 expressed by rodent herpesvirus Peru (RHVP) that establishes acute and latent infection in laboratory mice. Our findings show that pK3 binds directly and specifically to class I major histocompatibility proteins (MHCI) in a transmembrane-dependent manner. This binding results in the rapid degradation of the pK3/MHCI complex by a mechanism dependent upon catalytically active pK3. Subsequently, the rapid degradation of pK3/MHCI secondarily causes the slow degradation of membrane bound components of the MHCI peptide loading complex, tapasin, and transporter associated with antigen processing (TAP). Interestingly, this secondary event occurs by cellular endoplasmic reticulum-associated degradation. Cumulatively, our findings show pK3 uses a unique mechanism of substrate detection and degradation compared with other viral or cellular E3 ligases. More importantly, our findings reveal that in the absence of nascent MHCI proteins in the endoplasmic reticulum, the transmembrane proteins TAP and tapasin that facilitate peptide binding to MHCI proteins are degraded by cellular quality control mechanisms.
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http://dx.doi.org/10.1074/jbc.M111.325340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340253PMC
April 2012

Single chain MHC I trimer-based DNA vaccines for protection against Listeria monocytogenes infection.

Vaccine 2012 Mar 26;30(12):2178-86. Epub 2012 Jan 26.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, United States.

To circumvent limitations of poor antigen presentation and immunogenicity of DNA vaccines that target induction of CD8(+) T cell immunity, we have generated single chain MHC I trimers (MHC I SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, β2-microglobulin, and heavy chain of a MHC class I molecule connected by flexible linkers. Because of its pre-assembled nature, the SCT presents enhanced expression and presentation of the antigenic peptide/MHC complexes at the cell surface. Furthermore, DNA vaccination with a plasmid DNA encoding an SCT incorporating an immunodominant viral epitope elicited protective CD8(+) T cell responses against lethal virus infection. To extend these findings, here we tested the efficacy of SCT DNA vaccines against bacterial infections. In a mouse infection model of Listeria monocytogenes, the SCT DNA vaccine encoding H-2K(d) and the immunodominant peptide LLO 91-99 generated functional primary and memory peptide-specific CD8(+) T cells that confer partial protection against L. monocytogenes infection. DNA immunization of K(d)/LLO(91-99) SCTs generated functional memory CD8(+) T cells independently of CD4(+) T cells, although the expression of cognate or non-cognate CD4(+) helper T cell epitopes further enhanced the protective efficacy of SCTs. Our study further demonstrates that the SCT serves as a potent platform for DNA vaccines against various infectious diseases.
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http://dx.doi.org/10.1016/j.vaccine.2012.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288962PMC
March 2012

Specific mutation of a gammaherpesvirus-expressed antigen in response to CD8 T cell selection in vivo.

J Virol 2012 Mar 14;86(5):2887-93. Epub 2011 Dec 14.

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.
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http://dx.doi.org/10.1128/JVI.06101-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302239PMC
March 2012

Ubiquitination of substrates by esterification.

Traffic 2012 Jan 13;13(1):19-24. Epub 2011 Sep 13.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Post-translational modification by ubiquitination determines intracellular location and fate of numerous proteins, thus impacting a diverse array of physiologic functions. Past dogma has been that ubiquitin was only coupled to substrates by isopeptide bonds to internal lysine residues or less frequently peptide bonds to the N-terminus. Enigmatically, however, several proteins lacking lysines had been reported to retain ubiquitin-dependent fates. Resolution of this paradox was afforded by recent observations that ubiquitination of substrates can also occur on cysteine or serine and threonine residues by thio- or oxy-ester bond formation, respectively (collectively called esterification). Although chemically possible, these bonds were considered too labile to be of physiological relevance. In this review we discuss recent evidence for the ubiquitination of protein substrates by esterification and speculate on its mechanism and its physiological importance.
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http://dx.doi.org/10.1111/j.1600-0854.2011.01269.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973488PMC
January 2012

Which lymphoid cells express MHC class II antigens; are TCRs encoded within the MHC?

Authors:
Ted H Hansen

J Immunol 2011 Sep;187(5):2043-4

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.

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http://dx.doi.org/10.4049/jimmunol.1101982DOI Listing
September 2011

Endogenous MHC-related protein 1 is transiently expressed on the plasma membrane in a conformation that activates mucosal-associated invariant T cells.

J Immunol 2011 Apr 14;186(8):4744-50. Epub 2011 Mar 14.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

The development of mucosal-associated invariant T (MAIT) cells is dependent upon the class Ib molecule MHC-related protein 1 (MR1), commensal bacteria, and a thymus. Furthermore, recent studies have implicated MR1 presentation to MAIT cells in bacteria recognition, although the mechanism remains undefined. Surprisingly, however, surface expression of MR1 has been difficult to detect serologically, despite ubiquitous detection of MR1 transcripts and intracellular protein. In this article, we define a unique mAb capable of stabilizing endogenous mouse MR1 at the cell surface, resulting in enhanced mouse MAIT cell activation. Our results demonstrated that under basal conditions, endogenous MR1 transiently visits the cell surface, thus reconciling the aforementioned serologic and functional studies. Furthermore, using this approach, double-positive thymocytes, macrophages, and dendritic cells were identified as potential APCs for MAIT cell development and activation. Based on this pattern of MR1 expression, it is intriguing to speculate that constitutive expression of MR1 may be detrimental for maintenance of immune homeostasis in the gut and/or detection of pathogenic bacteria in mucosal tissues.
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http://dx.doi.org/10.4049/jimmunol.1003254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618670PMC
April 2011

Cancer genome sequencing and its implications for personalized cancer vaccines.

Cancers (Basel) 2011 Nov 25;3(4):4191-211. Epub 2011 Nov 25.

Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.

New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.
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http://dx.doi.org/10.3390/cancers3044191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763418PMC
November 2011