scholarly journals The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines

2010 ◽  
Vol 84 (6) ◽  
pp. 2648-2656 ◽  
Author(s):  
Ronen Borenstein ◽  
Haim Zeigerman ◽  
Niza Frenkel
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Restriction Enzymes ◽  
Artificial Chromosome ◽  
Reading Frame ◽  
Viral Genomes ◽  
Replicative Intermediates ◽  
T Cell Lines

ABSTRACT Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by ∼8- to 10-kb left and right direct repeats, DRL and DRR. We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short ∼2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained ∼8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained ∼2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DRL, including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DRR sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth “conquering” the population. The DR1 gene and the first DR6 exon are not required for propagation in culture.

scholarly journals Elevated secretion of reactive nitrogen and oxygen intermediates by inflammatory leukocytes in hyperacute experimental autoimmune encephalomyelitis: enhancement by the soluble products of encephalitogenic T cells.

1992 ◽  
Vol 176 (1) ◽  
pp. 303-307 ◽  
Author(s):  
J D MacMicking ◽  
D O Willenborg ◽  
M J Weidemann ◽  
K A Rockett ◽  
W B Cowden
Keyword(s):  
T Cell ◽  
Experimental Autoimmune Encephalomyelitis ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Reactive Nitrogen ◽  
Autoimmune Encephalomyelitis ◽  
Oxygen Intermediates ◽  
Peripheral Blood Mononuclear ◽  
T Cell Lines ◽  
Experimental Autoimmune

Perivascular lesions within the central nervous system (CNS) of rats with hyperacute experimental autoimmune encephalomyelitis (HEAE) contained large numbers of peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN), cells enzymatically capable of producing reactive nitrogen and oxygen intermediates (RNI and ROI), which, in excess, are mediators of tissue damage. PBMC and PMN isolated from the CNS and periphery of HEAE-affected rats secreted significantly (p less than 0.01-0.0001) elevated levels of ROI and RNI compared with that of similar cell populations from pertussis- and saline-treated control animals. Coincubation of systemically derived PBMC and PMN with antigen-stimulated myelin basic protein-specific T cell lines led to further increases in ROI and RNI output of between 15.3 and 83.1%, an effect that could be largely attributed to heat-labile, soluble products released by these T cell lines. Our studies suggest a putative neuropathological role for ROI and RNI in HEAE, which may be mediated via cytokines emanating from autoreactive T lymphocytes.

scholarly journals Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the Secreted Mycobacterium tuberculosisAntigen 85B Recognized by Antigen-Specific Human CD4+T-Cell Lines

2000 ◽  
Vol 68 (7) ◽  
pp. 3933-3940 ◽  
Author(s):  
Abu S. Mustafa ◽  
Fatema A. Shaban ◽  
Adnan T. Abal ◽  
Raja Al-Attiyah ◽  
Harald G. Wiker ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Mhc Class Ii ◽  
Mononuclear Cells ◽  
Restriction Analysis ◽  
T Cell Epitopes ◽  
Peptide Epitopes ◽  
Hla Dr ◽  
Ifn Γ ◽  
T Cell Lines

ABSTRACT Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+ T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1,DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

Characteristics of Interleukin 2 and Interleukin 4 Dependent T Cell Lines Infected with HTLV-1 in Vitro

1997 ◽  
Vol 10 (3) ◽  
pp. 189-194 ◽  
Author(s):  
B. Macchi ◽  
S. Grelli ◽  
C. Favalli ◽  
M. De Carli ◽  
Garaci ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
Normal Adult ◽  
Interleukin 4 ◽  
Human T Cell ◽  
T Cell Lines

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is a lymphotropic retrovirus. Cells infected with HTLV-1 in vitro, when maintained in interleukin-2 (IL-2) can be immortalized, remaining for a long time strictly dependent on IL-2 addition. In this study we have compared the effect of interleukin-4 (IL-4) and interleukin-2 on HTLV-1 infection of cord blood or normal adult mononuclear cells. The results showed that either cultures of cord blood or normal adult T cells are susceptible to HTLV-1 infection in presence of IL-4 as well as IL-2. Moreover HTLV-1 infected cells in the presence of IL-4 survived only for a limited length of time in culture, while those grown in IL-2 showed the characteristics of immortalized cell lines. Moreover the profile of cytokine production showed a different pattern in HTLV-1 infected cell lines maintained in IL-4 or IL-2. This suggests that the lymphokines differently modulate retrovirus infection in vitro.

scholarly journals Synthetic Peptides Identify Promiscuous Human Th1 Cell Epitopes of the Secreted Mycobacterial Antigen MPB70

2003 ◽  
Vol 71 (4) ◽  
pp. 1953-1960 ◽  
Author(s):  
Raja Al-Attiyah ◽  
Fatema A. Shaban ◽  
Harald G. Wiker ◽  
Fredrik Oftung ◽  
Abu S. Mustafa
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Synthetic Peptides ◽  
Mononuclear Cells ◽  
Mycobacterial Antigen ◽  
Th1 Cell ◽  
Cell Responses ◽  
Ifn Γ ◽  
T Cell Lines ◽  
Mycobacterial Antigens

ABSTRACT MPB70 is a secreted protein of Mycobacterium bovis and Mycobacterium tuberculosis which stimulates both cellular and humoral immune responses during infection with bovine and human tubercle bacilli. In addition, vaccination with MPB70 has been shown to induce Th1 cell responses and protection in animal models of tuberculosis. The present study was carried out to map the dominant human Th1 cell epitopes of MPB70 in relation to major histocompatibility complex (MHC) class II restriction in healthy subjects showing strong T-cell responses to complex mycobacterial antigens. Peripheral blood mononuclear cells (PBMC) from HLA-DR-typed donors were tested with complex mycobacterial antigens (whole-cell M. tuberculosis and M. tuberculosis culture filtrates), with MPB70 purified from the culture filtrate of M. bovis BCG Tokyo, and with 13 synthetic peptides (25-mers overlapping by 10 residues) covering the sequence of MPB70. The donors that responded to the complex antigens and MPB70 also responded to the cocktail of synthetic MPB70 peptides. Testing of PBMC with individual peptides showed that peptides p5 (amino acids [aa] 61 to 85), p6 (aa 76 to 100), p8 (aa 106 to 130), p12 (aa 166 to 190), and p13 (aa 181 to 193) were most frequently recognized in proliferation and gamma interferon (IFN-γ) assays. Testing of antigen-specific CD4+ T-cell lines with the individual peptides of MPB70 confirmed that peptides p8, p12, and p13 contain immunodominant Th1 cell epitopes of MPB70. MHC restriction analysis with HLA-typed donors showed that MPB70 and its immunodominant peptides were presented to T cells promiscuously. The T-cell lines responding to MPB70 and peptides p8, p12, and p13 in IFN-γ assays mediated antigen-peptide-specific cytotoxic activity against monocytes/macrophages pulsed with the whole-protein antigen or the peptides. In conclusion, the promiscuous recognition of MPB70 and its immunodominant peptide defined epitopes (aa 106 to 130 and 166 to 193) by IFN-γ-producing Th1 cells supports possible application of this secreted antigen to subunit vaccine design.

scholarly journals Bovine γδ T-Cell Responses to the Intracellular Protozoan Parasite Theileria parva

1999 ◽  
Vol 67 (5) ◽  
pp. 2241-2249 ◽  
Author(s):  
Claudia A. Daubenberger ◽  
Evans L. N. Taracha ◽  
Laima Gaidulis ◽  
William C. Davis ◽  
Declan J. McKeever
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Γδ T Cells ◽  
Theileria Parva ◽  
Γδ T Cell ◽  
Mhc Molecules ◽  
T Cell Lines

ABSTRACT T cells bearing the γδ antigen receptor (γδ T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that γδ T cells are involved in immune responses against Theileria parva. γδ T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of γδ T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the γδ T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the γδ T-cell lines and clones. These γδ T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of γδ T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine γδ T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.

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