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J G Sissons

Publications and source records attributed to J G Sissons.

At least 19 recordsLinked to original sources
PubMed1999

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.

Carrier State↗
PubMed1999

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.

Amino Acid Sequence↗
PubMed1999

Large clonal expansions of human virus-specific memory cytotoxic T lymphocytes within the CD57+ CD28- CD8+ T-cell population.

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28- is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28- CD8+ T cells contain large oligoclonal T-cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus-specific memory cytotoxic T-lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV-infected subjects. In each subject, from the T-cell receptor (TCR) beta-chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus-specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus-specific CTL clonotypes in CD57+ CD28- CD8+ T cells. Using limiting dilution analysis, we found functional peptide-specific CTLp at high frequency in CD57+ CD28- cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28- CD8+ T cells.

CD28 Antigens↗
PubMed1999

The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo.

Human cytomegalovirus (HCMV)-specific CD8(+) cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vbeta segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

Amino Acid Sequence↗
PubMed1998

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection.

In HIV-1 infection, circulating HIV-1-specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV-1-specific CTL is capable of clonal expansion upon culture with IL-2 without exogenous antigen. The IL-2-expandable HIV-1-specific CTL precursor frequency was reduced in patients with advancing infection, although HIV-1-specific memory CTL could still be detected by stimulation in vitro with allele-specific HIV-1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL-2-expandable HIV-1-specific CTL precursor (CTLp) frequency without a decline in Epstein-Barr virus (EBV)-specific or allo-specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL-2-expandable HIV-specific CTL response, the requirements for in vitro generation of HIV-1-specific and EBV-specific effector CTL were examined. In the absence of exogenous IL-2 in limiting dilution, generation of EBV-specific CD8+ effector CTL was dependent upon help from CD4+ cells. CD4+ help for EBV-specific CD8+ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL-2, CD4+ cells could also provide help for the optimal generation of HIV-1 peptide-specific CD8+ CTL, because in vitro depletion of CD4+ cells prior to culture using stimulation with an MHC class I-restricted HIV-1 peptide reduced the peptide-specific CD8+ CTL response. We conclude that there is a decline in the IL-2-expandable HIV-1-specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4+ T cell help for in vivo antigen-activated CD8+ T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1996

The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Amino Acid Sequence↗
PubMed1994

The transcription factor YY1 binds to negative regulatory elements in the human cytomegalovirus major immediate early enhancer/promoter and mediates repression in non-permissive cells.

We have previously shown that repression of human cytomegalovirus (HCMV) major immediate early (IE) gene expression in non-permissive human teratocarcinoma (T2) cells is associated with a number of nuclear factors which bind to the imperfect dyad symmetry located in the modulator region upstream of the major IE enhancer as well as to the 21 bp repeat elements within the enhancer. Differentiation of T2 cells with retinoic acid (RA) results in a decrease in binding of some of these nuclear factors to these sites and deletion of these specific binding sites from major IE promoter/reporter constructs results in increased IE promoter activity in normally non-permissive cells. In this study, we demonstrate that the transcription factor YY1, which can negatively regulate the adeno-associated virus P5 promoter, directly binds to both the imperfect dyad symmetry and the 21 bp repeat elements in the HCMV major IE promoter/regulatory region and mediates repression of HCMV IE gene expression. This strongly suggests that YY1 plays an important role in regulating HCMV expression in non-permissive cells.

Antigens, Viral↗
PubMed1994

Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.

Bone Marrow↗
PubMed1993

Polymorphonuclear cells are not sites of persistence of human cytomegalovirus in healthy individuals.

Polymorphonuclear leukocytes (PMNL) have been shown to harbour human cytomegalovirus (HCMV) in viraemic patients, but to date PMNL of asymptomatic healthy subjects have not been examined directly to determine whether this is a normal site of HCMV persistence. Using the polymerase chain reaction (PCR), paired DNA samples prepared from adherent peripheral blood mononuclear cells (PBMC), which are known to be a site of persistence of HCMV, and PMNL of 10 healthy adults were analysed. All of seven individuals who were HCMV seropositive, and one of three who were seronegative gave a reproducible signal for HCMV DNA in their adherent PBMC, whereas none of the paired PMNL DNA samples gave a positive result. The remaining two seronegative subjects showed no HCMV DNA in either the PBMC or PMNL samples. In every case where PCR for HCMV was negative, PCR amplification of a control human gene was used to show there was no inability to amplify the DNA. We conclude that within the leukocyte population of normal asymptomatic HCMV carriers, PMNL do not appear to harbour persistent HCMV whereas adherent PBMC in the same subjects are a site of persistence.

Adult↗
PubMed1993

Heterogeneity amongst natural killer cells revealed by limiting dilution culture; selectivity against virus-infected and tumour cell targets.

Previous studies have suggested that natural killer (NK) cells exhibit heterogeneous cytotoxicity towards different tumour cell targets. No studies have set out to determine whether different NK populations have relative selectivity for virus-infected cells. The aims of this study were to determine if this was the case for short-term clones, and whether there were differences in relative selectivity for particular target cells between clones with NK activity but with different surface phenotypes. Cells from different starting populations [whole peripheral blood lymphocytes (PBL), E-rosette positive or negative, CD16+ or CD3- cells] were grown in limiting dilution culture (LDC) with interleukin-2 (IL-2). The precursor frequency (NK-p) of cells proliferating and exhibiting NK activity towards various virus-infected or uninfected fibroblasts or tumour cell targets was determined by split-well analysis of the LDC. The relative NK-p were similar for different individuals, but were much lower for virus-infected fibroblasts than a tumour cell target. The pattern of cytotoxicity of 757 short-term clones, identified from the LDC, against four to five tumour and virus-infected target cells were analysed. We conclude that there was selective lysis of virus-infected cells by a proportion of NK clones which were predominantly PBL-derived (mainly CD3+). Twenty-six per cent of E(+)-derived clones lysed Molt4 cells only in the absence of phytohaemagglutinin (PHA), and a proportion of PBL- or E(+)-derived clones (up to 44%) lysed uninfected or virus-infected fibroblasts but not Molt4+PHA. Thus, under hese conditions lectin-induced cytotoxicity does not detect total potential cytotoxicity.

Adult↗
PubMed1992

Repression of human cytomegalovirus major immediate early gene expression in a monocytic cell line.

We have previously shown that a major site of persistence of human cytomegalovirus (HCMV) in healthy carriers is in peripheral blood monocytes. However, monocytes are difficult to infect in vitro with HCMV, and HCMV gene expression cannot be reproducibly detected in peripheral blood cells of healthy carriers. Here we show that the monocytic cell line THP1 is non-permissive for HCMV infection due to a block in expression of the HCMV major immediate early (IE) promoter. This repression is correlated with the presence of a differentiation-specific cellular factor which binds to the imperfect dyad symmetry and the 21 bp enhancer repeats of the major IE promoter regulatory region and which has characteristics of MBF1, a factor which we have previously defined in HCMV non-permissive, undifferentiated teratocarcinoma cells. Both differentiation of THP1 cells into macrophages, which results in a decrease in this factor, or deletion of the factor's binding sites from the IE promoter/enhancer lifts this repression and permits expression from the major IE promoter.

Antigens, Viral↗
PubMed1992

A 10-base-pair element of the human immunodeficiency virus type 1 long terminal repeat (LTR) is an absolute requirement for transactivation by the human cytomegalovirus 72-kilodalton IE1 protein but can be compensated for by other LTR regions in transactivation by the 80-kilodalton IE2 protein.

Transient gene expression studies have indicated that human cytomegalovirus (HCMV) specifically transactivates the human immunodeficiency virus (HIV) long terminal repeat (LTR). We show here, by a specific mutational analysis, that only the TATA box region is obligatory for transactivation of the HIV-1 LTR by HCMV. Similarly, this element is also sufficient for transactivation by either the HCMV 72-kDa major immediate-early 1 (IE1) or 80-kDa IE2 gene product independently. However, deletion of a 10-bp region from the minimal responsive element, 5' to the TATA box, dramatically reduced the level of HCMV 72-kDa IE1 or 80-kDa IE2 transactivation, indicating a crucial role for this element in transactivation. Whereas inclusion of the TAR element or Sp1 sites on this 10-bp-deleted minimal promoter had no effect on the removal of IE1 transactivation, TAR and Sp1 elements did compensate for the 10-bp element in transactivation by IE2 and HCMV. Consequently, the sequence requirements of the HIV-1 LTR for transactivation by HCMV can be reproduced by these IE1 and IE2 gene products of HCMV.

Base Sequence↗
PubMed1991

The 21bp repeat element of the human cytomegalovirus major immediate early enhancer is a negative regulator of gene expression in undifferentiated cells.

The major immediate early regulatory region of human cytomegalovirus (HCMV) has a complex set of DNA sites through which both cellular and viral factors coordinately regulate immediate early gene expression. In undifferentiated human teratocarcinoma (T2) cells we have previously shown that major immediate early gene expression is repressed by a differentiation specific nuclear factor MBF1, which binds to the imperfect dyad symmetry located upstream of the enhancer. However, upon differentiation MBF1 decreases resulting in immediate early gene expression. In this study we show, by mobility shift analysis that the same or similar factor(s) also binds to the 21bp repeat of the major immediate early enhancer. Deletion of this 21bp repeat from the immediate early enhancer expression vectors results in increased CAT expression in undifferentiated T2 cells, to levels similar to that in differentiated cells. Consequently, the 21bp repeat of the HCMV enhancer also acts to negatively regulate major immediate early enhancer function in non-permissive cells.

Base Sequence↗
PubMed1991

Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells.

We have used the nested polymerase chain reaction (PCR) combined with fluorescence-activated cell sorting to define sites of latency of human cytomegalovirus (HCMV) in the peripheral blood of healthy subjects. Peripheral blood mononuclear (PBM) cells were separated into T cell or non-T cell populations and monocytes, and were then analysed by PCR for the presence of HCMV DNA. In five of six seropositive subjects, HCMV was found predominantly in the non-T cell population. Further analysis suggested that the virus was present in adherent cells and CD14+ cells. In three of nine seronegative subjects we could demonstrate HCMV DNA, which we do not believe was due to contamination, reproducibly by PCR. In one of these seronegative subjects, HCMV DNA was present predominantly in the non-T cell fraction of PBM cells. No HCMV DNA was detectable in the remaining six seronegative subjects. We conclude that, within the PBM cells of normal asymptomatic seropositive and some seronegative subjects, HCMV is present predominantly in the monocyte fraction. In addition, the detection of HCMV sequences in seronegative subjects may indicate that infection with HCMV is more widespread than conventional seroepidemiology suggests.

Antibodies, Viral↗