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1.
Vaccine ; 39(15): 2074-2079, 2021 04 08.
Article in English | MEDLINE | ID: mdl-33726955

ABSTRACT

Following the 1892 cholera pandemic, Richard Pfeiffer, Director of the science section of Robert Koch's Institute for Hygiene in Berlin, began laboratory-based studies on the pathogenesis of the disease using an animal model. These investigations resulted in his discovery of bacterial endotoxin; recognition of the bacteriolytic properties of both animal and human immune sera; and identification of the specific nature of protective immune responses. His research led naturally from cholera to typhoid fever and in November 1896 Pfeiffer published the results of experimental studies on a typhoid vaccine. In September 1896 Almroth Wright, a professor of pathology in the British Army Medical School, published a short note entitled "Typhoid Vaccination". It was appended to a review on the use of styptics to control defective blood coagulation: his previous research studies had a physiological basis that stemmed from earlier studies on tissue fibrinogen. In December 1895, Wright had been commissioned by the Army Medical Department to develop a typhoid vaccine and he later admitted that such work began only after he had spoken with Pfeiffer. In January 1897 Wright published a further paper in which he claimed precedence over Pfeiffer in the introduction of anti-typhoid vaccination. This self-entitlement has subsequently been accepted, primarily because the British Army approved typhoid vaccination in 1914 at the beginning of the First World War. That time has been used as their starting point by many of Wright's biographers, but without any attempt to confirm Wright's claim to priority. This paper concludes Richard Pfeiffer, not Almroth Wright, provided the first account of human typhoid vaccination. It also provides early examples of laboratory-based responses to pandemic and epidemic infectious diseases.


Subject(s)
Cholera , Military Personnel , Typhoid Fever , Typhoid-Paratyphoid Vaccines , Animals , Berlin , Cholera/prevention & control , Humans , Male , Typhoid Fever/prevention & control , Vaccination
2.
J Transl Autoimmun ; 3: 100070, 2020.
Article in English | MEDLINE | ID: mdl-33294833

ABSTRACT

This article explores the possibility that the major autoimmune diseases come about because of the breakdown of T lymphocyte ignorance - that state in which antigen and lymphocyte have never come together in such a way as to induce tolerance or an immune response. By use of transgenic technique to place a foreign antigen/peptide in various mouse tissues the widespread occurrence of ignorance has been observed and information obtained on when it is likely to occur. Now, with the advent of tetramer technique to enrich specific T cells and the recognition of lymphocyte markers indicating whether or not antigen interaction has taken place, ignorance of genuine self-antigens is being examined in mouse and man. In the absence of thymic deletion it seems that tolerance to self-antigens is brought about either by T cell ignorance or T cell regulatory control. The initiating factor in these major diseases is likely to be a change in the condition of the antigen leading to tolerance failure. There is evidence that it is ignorance that breaks down in Type 1 diabetes and systemic lupus erythematosus. If this proves a general rule, it may be because ignorance is the tolerance mechanism most vulnerable to subversion.

3.
J Vis Exp ; (144)2019 02 28.
Article in English | MEDLINE | ID: mdl-30882785

ABSTRACT

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class I/metabolism , Lymphocyte Activation/physiology , Humans , Transfection
4.
BMC Res Notes ; 7: 662, 2014 Sep 20.
Article in English | MEDLINE | ID: mdl-25239080

ABSTRACT

BACKGROUND: It can be difficult to register a weak proliferative response of T lymphocytes to an antigen, particularly in a simple culture system of peripheral blood mononuclear cells (PBMC). Here we assess the usefulness of the cytokine IL-2 in amplifying such a response. METHODS: PBMC from healthy donors were cultured in the presence or absence of keyhole limpet haemocyanin (KLH), an antigen to which people have not been previously exposed. IL-2 was added from the beginning or on the fifth day of culture. Proliferation was determined by incorporation of tritiated thymidine at eight days. The recall antigen, tuberculin PPD, provided a positive control. RESULTS: IL-2 added at the beginning of culture can induce extremely high levels of proliferation even in the absence of antigen. However, when added on the fifth day it allowed the clear observation of a proliferative response to KLH that was barely detectable in its absence. Added late it was similarly able to boost low responses to PPD and to the mitogens lipopolysaccharide and poly(I:C), but it had no such effect with pokeweed mitogen. CONCLUSIONS: IL-2 added late in culture is highly effective in increasing the sensitivity of T lymphocyte proliferative assays.


Subject(s)
Antigens/immunology , Cell Proliferation , Immunologic Memory , Interleukin-2/physiology , Humans
5.
Arthritis Rheumatol ; 66(11): 2976-88, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25132672

ABSTRACT

OBJECTIVE: HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. METHODS: HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. RESULTS: We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. CONCLUSION: The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease.


Subject(s)
Endoplasmic Reticulum-Associated Degradation/physiology , Endoplasmic Reticulum/physiology , HLA-B27 Antigen/physiology , Membrane Proteins/physiology , Protein Folding , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/physiology , HeLa Cells , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/drug effects , RNA, Small Interfering/pharmacology , Regulatory Factor X Transcription Factors , Signal Transduction/physiology , Transcription Factors/antagonists & inhibitors , Transcription Factors/drug effects , Transcription Factors/physiology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/drug effects , Ubiquitin-Protein Ligases/physiology , X-Box Binding Protein 1
6.
J Exp Med ; 210(9): 1807-21, 2013 Aug 26.
Article in English | MEDLINE | ID: mdl-23940257

ABSTRACT

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide-MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4-MHCII interaction is generally weaker than CD8-MHCI.


Subject(s)
Epitopes/immunology , Histocompatibility Antigens Class II/metabolism , Histocompatibility Antigens Class I/metabolism , Peptides/metabolism , Receptors, Antigen, T-Cell/agonists , Receptors, Antigen, T-Cell/metabolism , Amino Acid Sequence , Animals , CD8-Positive T-Lymphocytes/immunology , CHO Cells , Computer Simulation , Cricetinae , Cricetulus , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/immunology , Humans , Kinetics , Lymphocyte Activation/immunology , Mice , Models, Molecular , Molecular Sequence Data , Ovalbumin/immunology , Peptides/chemistry , Peptides/immunology , Protein Binding/immunology , Receptors, Antigen, T-Cell/immunology
7.
Antioxid Redox Signal ; 15(3): 635-44, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21050141

ABSTRACT

Stable presentation of peptide epitope by major histocompatibility complex (MHC) class I molecules is a prerequisite for the efficient expansion of CD8(+) T cells. The construction of single-chain MHC class I molecules in which the peptide, ß(2)-microglobulin, and MHC heavy chain are all joined together via flexible linkers increases peptide-MHC stability. We have expressed two T cell epitopes that may be useful in leukemia treatment as single-chain MHC class I molecules, aiming to develop a system for the expansion of antigen-specific CD8(+) T cells in vitro. Disulfide trap versions of these single-chain MHC molecules were also created to improve anchoring of the peptides in the MHC molecule. Unexpectedly, we observed that soluble disulfide trap single-chain molecules expressed in eukaryotic cells were prone to homodimerization, depending on the binding affinity of the peptide epitope. The dimers were remarkably stable and efficiently recognized by conformation-specific antibodies, suggesting that they consisted of largely correctly folded molecules. However, dimerization was not observed when the disulfide trap molecules were expressed as full-length, transmembrane-anchored molecules. Our results further emphasize the importance of peptide binding affinity for the efficient folding of MHC class I molecules.


Subject(s)
Epitopes/metabolism , Histocompatibility Antigens Class I/metabolism , ATP-Binding Cassette Transporters/metabolism , Animals , Antibody Specificity/immunology , CD8-Positive T-Lymphocytes/immunology , CHO Cells , Cell Line , Cricetinae , Cricetulus , Epitopes/chemistry , HEK293 Cells , HLA-A2 Antigen/immunology , HLA-A2 Antigen/metabolism , Histocompatibility Antigens Class I/chemistry , Humans , Peptides/immunology , Peptides/metabolism , Protein Folding , Protein Multimerization , beta 2-Microglobulin/immunology , beta 2-Microglobulin/metabolism
8.
Antioxid Redox Signal ; 15(3): 645-55, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21126187

ABSTRACT

Stable major histocompatibility complex (MHC) class I molecules at the cell surface consist of three separate, noncovalently associated components: the class I heavy chain, the ß(2)-microglobulin light chain, and a presented peptide. These three components are assembled inside cells via complex pathways involving many other proteins that have been studied extensively. Correct formation of disulfide bonds in the endoplasmic reticulum is central to this process of MHC class I assembly. For a single specific peptide to be presented at the cell surface for possible immune recognition, between hundreds and thousands of peptide-containing precursor polypeptides are required, so the overall process is relatively inefficient. To increase the efficiency of antigen presentation by MHC class I molecules, and for possible therapeutic purposes, single-chain molecules have been developed in which the three, normally separate components have been joined together via flexible linker sequences in a single polypeptide chain. Remarkably, these single-chain MHC class I molecules fold up correctly, as judged by functional recognition by cells of the immune system, and more recently by X-ray crystallographic structural data. This review focuses on the interesting properties and potential of this new type of engineered MHC class I molecule.


Subject(s)
Histocompatibility Antigens Class I/immunology , Peptides/immunology , beta 2-Microglobulin/immunology , ATP-Binding Cassette Transporters/metabolism , CD8-Positive T-Lymphocytes/immunology , Disulfides/chemistry , Disulfides/immunology , Endoplasmic Reticulum/immunology , HLA-A2 Antigen/genetics , HLA-A2 Antigen/immunology , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Humans , Peptides/chemistry , Peptides/genetics , Protein Conformation , Protein Engineering , beta 2-Microglobulin/chemistry
9.
Trends Immunol ; 31(10): 363-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20832361

ABSTRACT

Major histocompatibility complex (MHC) class I molecules can be engineered as single chain trimers (SCTs) that sequentially incorporate all three subunits of the fully assembled proteins, namely peptide, ß2 microglobulin, and heavy chain. SCTs have been made with many different MHC-peptide complexes and are used as novel diagnostic and therapeutic reagents, as well as probes for diverse biological questions. Here, we review the recent and diverse applications of SCTs. These applications include new approaches to enumerate disease-related T cells, DNA vaccines, eliciting responses to pre-assembled MHC-peptide complexes, and unique probes of lymphocyte development and activation. Future applications of SCTs will be driven by their further engineering and the ever-expanding identification of disease-related peptides using chemical, genetic and computational approaches.


Subject(s)
Histocompatibility Antigens Class I/immunology , Animals , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Lymphocyte Activation , Lymphocytes/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Vaccines, DNA/immunology
10.
Eur J Immunol ; 40(7): 2050-9, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20432238

ABSTRACT

Size-dependent protein segregation at the cell-cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK-cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL-2 stimulated C57BL/6 mouse-derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK-cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H-2K(b), a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H-2K(b) resulted in decreased inhibition of both lysis and IFN-gamma production by NK cells. These results establish that small ligand dimensions are important for both NK-cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.


Subject(s)
H-2 Antigens/metabolism , Killer Cells, Natural/metabolism , Minor Histocompatibility Antigens/metabolism , NK Cell Lectin-Like Receptor Subfamily A/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Animals , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Cytotoxicity, Immunologic/genetics , H-2 Antigens/genetics , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Ligands , Mice , Mice, Inbred C57BL , Minor Histocompatibility Antigens/genetics , Mutagenesis, Insertional/genetics , Mutagenesis, Site-Directed , NK Cell Lectin-Like Receptor Subfamily A/genetics , Protein Binding/genetics , Transgenes/genetics
11.
J Biol Chem ; 284(38): 26096-105, 2009 Sep 18.
Article in English | MEDLINE | ID: mdl-19628870

ABSTRACT

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.


Subject(s)
Histocompatibility Antigens Class I/immunology , Leukocyte Common Antigens/immunology , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , ZAP-70 Protein-Tyrosine Kinase/immunology , Animals , CD3 Complex/genetics , CD3 Complex/immunology , CD3 Complex/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/metabolism , Lymphocyte Activation/genetics , Mice , Mice, Knockout , Peptides , Phosphorylation/genetics , Phosphorylation/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/enzymology , ZAP-70 Protein-Tyrosine Kinase/genetics , ZAP-70 Protein-Tyrosine Kinase/metabolism
12.
J Immunol ; 182(8): 4565-71, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19342630

ABSTRACT

We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , H-2 Antigens/immunology , Nucleoproteins/immunology , Orthomyxoviridae/immunology , Animals , CHO Cells , Cricetinae , Cricetulus , Mice , Mice, Inbred C57BL , beta 2-Microglobulin/immunology
13.
J Immunol ; 174(2): 925-33, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15634915

ABSTRACT

We reported previously that different MHC class I molecules can compete with each other for cell surface expression in F(1) hybrid and MHC class I transgenic mice. In this study, we show that the competition also occurs in transfected cell lines, and investigate the mechanism. Cell surface expression of an endogenous class I molecule in Chinese hamster ovary (CHO) cells was strongly down-regulated when the mouse K(d) class I H chain was introduced by transfection. The competition occurred only after K(d) protein translation, not at the level of RNA, and localization studies of a CHO class I-GFP fusion showed that the presence of K(d) caused retention of the hamster class I molecule in the endoplasmic reticulum. The competition was not for beta(2)-microglobulin, because a single chain version of K(d) that included mouse beta(2)-microglobulin also had a similar effect. The competition was not for association with TAP and loading with peptide, because a mutant form of the K(d) class I H chain, not able to associate with TAP, caused the same down-regulation of hamster class I expression. Moreover, K(d) expression led to a similar level of competition in TAP2-negative CHO cells. Competition for cell surface expression was also found between different mouse class I H chains in transfected mouse cells, and this competition prevented association of the H chain with beta(2)-microglobulin. These unexpected new findings show that different class I H chains compete with each other at an early stage of the intracellular assembly pathway, independently of beta(2)-microglobulin and peptide.


Subject(s)
H-2 Antigens/biosynthesis , Peptides/chemistry , Protein Folding , Protein Subunits/biosynthesis , beta 2-Microglobulin/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/metabolism , Animals , Antigen Presentation/genetics , CHO Cells , Cell Line , Cell Line, Tumor , Cell Membrane/immunology , Cell Membrane/metabolism , Cricetinae , Down-Regulation/immunology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , H-2 Antigens/genetics , Mice , Mice, Transgenic , Protein Binding/genetics , Protein Binding/immunology , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/immunology , Protein Subunits/genetics , Protein Transport/genetics , Protein Transport/immunology , Transfection
14.
J Infect Dis ; 189(12): 2294-8, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15181578

ABSTRACT

Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We used interferon- gamma enzyme-linked immunospot assays with overlapping peptides spanning the entire HTLV-1 proteome to test whether the HTLV-1-specific CD8(+) T cells differed significantly in frequency or immunodominance hierarchy between patients with HAM/TSP and asymptomatic carriers and whether the frequency correlated with provirus load. Tax was the immunodominant target antigen. There was no significant qualitative or quantitative difference in the HTLV-1-specific CD8(+) T cell response between the 2 groups. Virus-specific CD8(+) T cell frequency alone does not indicate the effectiveness of the cytotoxic T lymphocyte response in controlling provirus load at equilibrium.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Carrier State/immunology , Human T-lymphotropic virus 1/immunology , Immunodominant Epitopes/immunology , Paraparesis, Tropical Spastic/immunology , Epitopes, T-Lymphocyte/immunology , HTLV-I Infections/immunology , HTLV-I Infections/virology , Humans , Interferon-gamma/biosynthesis , Peptides/immunology
15.
J Virol ; 77(17): 9716-22, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12915584

ABSTRACT

Significantly higher frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4(+) T cells were present in the peripheral blood mononuclear cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients than in those of asymptomatic carriers with similar provirus loads. The data suggest that HTLV-1-specific CD4(+) T cells play a role in the pathogenesis of HAM/TSP.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Human T-lymphotropic virus 1/immunology , Interleukin-2/biosynthesis , Paraparesis, Tropical Spastic/immunology , Tumor Necrosis Factor-alpha/biosynthesis , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/drug effects , Carrier State/immunology , Enterotoxins/pharmacology , Human T-lymphotropic virus 1/isolation & purification , Humans , In Vitro Techniques , Interferon-gamma/biosynthesis , Lymphotoxin-alpha/biosynthesis , Paraparesis, Tropical Spastic/etiology , Proviruses/immunology , Proviruses/isolation & purification , Tetradecanoylphorbol Acetate/pharmacology
16.
J Immunol ; 169(10): 5615-21, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12421940

ABSTRACT

Mammalian cells express up to six different MHC class I alleles, many of which differ in terms of their interaction with components of the Ag presentation pathway and level of cell surface expression. However, it is often assumed in Ag presentation studies that class I alleles function independently of each other. We have compared cell surface expression levels and function of MHC class I molecules in F(1) hybrid mice with those in the homozygous parental strains. The level of cell surface expression of certain alleles in F(1) mice differed significantly from 50% of that found on the same cell type in the corresponding parental strain, suggesting allele-specific competition for cell surface expression, and not expression solely according to gene dosage. The strongest effect was observed in H-2(b) x H-2(k) F(1) mice, in which the H-2(b) class I molecules dominated over the H-2(k) class I molecules. The magnitude of H-2(k)-restricted CTL responses to influenza A virus infection was similar in the F(1) hybrid and parental H-2(k) mice. However, in H-2(k) mice expressing a K(b) transgene, cell surface levels of the endogenous class I molecules were down-regulated to a greater degree than in F(1) hybrid mice, and H-2(k)-restricted CTL responses against influenza A virus were greatly reduced, although the CTL repertoire was apparently present. Therefore, certain MHC class I molecules compete with each other for cell surface expression, and the resulting low cell surface expression of specific alleles can lead to a severe reduction in the ability to generate a CTL response.


Subject(s)
Alleles , Cytotoxicity, Immunologic/genetics , Gene Expression Regulation/immunology , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Influenza A virus/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Binding, Competitive/genetics , Binding, Competitive/immunology , Cell Membrane/genetics , Cell Membrane/immunology , Cell Membrane/metabolism , Crosses, Genetic , Dose-Response Relationship, Immunologic , Down-Regulation/genetics , Down-Regulation/immunology , Female , Gene Dosage , H-2 Antigens/biosynthesis , H-2 Antigens/genetics , Histocompatibility Antigen H-2D , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , T-Lymphocytes, Cytotoxic/metabolism
17.
J Gen Virol ; 83(Pt 3): 641-650, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11842259

ABSTRACT

Tax, the trans-activator of human T-lymphotropic virus type I (HTLV-I), is the dominant target antigen for cytotoxic T lymphocytes (CTLs) in the majority of infected individuals, although the reason for this immunodominance is not clear. Tax has been shown to associate physically with the proteasome, a protease that is responsible for the generation of the majority of major histocompatibility complex (MHC) class I ligands recognized by CTLs. This association could lead to the preferential targeting of Tax to the MHC class I pathway and account for its high immunogenicity. Here, the CTL response to Tax was investigated in mice by priming with a Tax expression vector and boosting with a Tax recombinant vaccinia virus (modified vaccinia virus Ankara strain). This approach led to the identification of a new H-2D(k)-restricted epitope in Tax, amino acid residues 38-46, sequence ARLHRHALL. Surprisingly, presentation of this epitope was found to be enhanced by the proteasome inhibitor lactacystin, although Tax was shown to associate with proteasomes in murine cells. The difficulties encountered in generating Tax-specific CTL responses and the results of enzyme-linked immunospot (ELISpot) analysis suggested that Tax is only poorly immunogenic for CTLs in mice. Therefore, the immunodominance of Tax in human CTL responses to HTLV-I is probably not due to an intrinsic property of the protein itself, such as an association with the proteasome, but instead may result from the fact that Tax is the predominant protein synthesized early after infection.


Subject(s)
Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Antigen Presentation/drug effects , Epitopes, T-Lymphocyte/immunology , Gene Products, tax/immunology , H-2 Antigens/immunology , Human T-lymphotropic virus 1/immunology , Multienzyme Complexes/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Cysteine Endopeptidases/metabolism , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Female , Gene Products, tax/chemistry , Gene Products, tax/genetics , Genetic Vectors/genetics , Histocompatibility Antigen H-2D , Human T-lymphotropic virus 1/genetics , Mice , Mice, Inbred CBA , Molecular Sequence Data , Multienzyme Complexes/metabolism , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Proteasome Endopeptidase Complex , Protein Binding , T-Lymphocytes, Cytotoxic/immunology , Vaccinia virus/genetics
18.
J Gen Virol ; 82(Pt 7): 1749-1755, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11413387

ABSTRACT

Influenza A virus infection of mice has been used extensively as a model to investigate the mechanisms of antigen presentation to cytotoxic T lymphocytes (CTL) and the phenomenon of immunodominance in antiviral CTL responses. The different virus-encoded epitopes that are recognized in H-2(b) and H-2(d) mice have been characterized and their relative immunodominance has been well-studied. In H-2(k) mice, four different K(k)-restricted influenza virus epitopes have been described, but the dominance hierarchy of these epitopes is unknown and there is also an uncharacterized D(k)-restricted response against the virus. In this study, a D(k)-restricted epitope derived from the influenza virus A/PR/8/34 polymerase protein PB1, corresponding to amino acid residues 349-357 (ARLGKGYMF), was identified. This peptide is the major epitope within the PB1 polymerase and is at least as dominant as any of the four K(k)-restricted epitopes that are recognized in CBA mice following primary influenza virus infection. The PB1 epitope is only the fourth D(k)-presented peptide to be reported and the sequence of this epitope confirms a D(k)-restricted peptide motif, consisting of arginine at position two, arginine or lysine at position five and a hydrophobic residue at the carboxy terminus.


Subject(s)
Epitopes, T-Lymphocyte/analysis , H-2 Antigens/immunology , Influenza A virus/immunology , Orthomyxoviridae Infections/virology , RNA-Dependent RNA Polymerase/immunology , Viral Proteins/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Dose-Response Relationship, Immunologic , Female , Histocompatibility Antigen H-2D , Immunodominant Epitopes/immunology , Influenza A virus/chemistry , Mice , Mice, Inbred CBA , Orthomyxoviridae Infections/immunology , Peptides/chemical synthesis , Peptides/immunology , Spleen/immunology , T-Lymphocytes, Cytotoxic
19.
J Gen Virol ; 82(Pt 5): 1069-1074, 2001 May.
Article in English | MEDLINE | ID: mdl-11297681

ABSTRACT

The influenza virus strains A/NT/60/68 and A/PR/8/34 both have an immunodominant D(b)-restricted epitope in their nucleoprotein (NP) at amino acid residues 366-374, with two amino acid differences between the epitopes. Cross-reactive cytotoxic T lymphocytes (CTLs) were generated by priming mice with the influenza virus A/NT/60/68 NP and restimulating in vitro with influenza virus A/PR/8/34. CTLs that gave high levels of specific lysis recognized target cells infected with either strain of influenza virus with similar efficiency. Surprisingly, when target cells were infected with recombinant vaccinia viruses (VV) expressing the two different NPs, presentation of the D(b)-restricted epitope from the A/NT/60/68 NP was extremely poor, whereas presentation of the equivalent epitope from the A/PR/8/34 NP was as efficient as in influenza virus-infected cells. This difference was observed in spite of the fact that the two NP sequences show 94% identity at the amino acid sequence level. Experiments with additional cross-reactive CTL cell lines which recognized target cells less efficiently revealed a similar difference in presentation between the two NP epitopes in influenza virus-infected cells and showed a difference in the efficiency of presentation of the D(b)-restricted epitope from the two NP molecules independent of VV infection. The results show that two equivalent epitopes in highly similar proteins are processed with very different efficiency, even though they are both immunodominant epitopes. They also suggest that the previously described inhibition of antigen presentation by VV is a general, non-specific effect, which is more apparent for epitopes that are processed and presented less efficiently.


Subject(s)
Antigen Presentation/immunology , Antigens, Viral/immunology , Epitopes, T-Lymphocyte/immunology , H-2 Antigens/immunology , Influenza A virus/immunology , Major Histocompatibility Complex/immunology , Nucleoproteins/immunology , Peptide Fragments/immunology , Viral Core Proteins/immunology , Animals , Cell Line , Cross Reactions , Female , Genetic Vectors/immunology , Histocompatibility Antigen H-2D , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , T-Lymphocytes, Cytotoxic/immunology , Vaccinia virus/immunology
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