Becca Asquith | Imperial College London (original) (raw)
Papers by Becca Asquith
PLOS One, 2008
HIV-1 escape from surveillance by cytotoxic T lymphocytes (CTL) is thought to cause at least tran... more HIV-1 escape from surveillance by cytotoxic T lymphocytes (CTL) is thought to cause at least transient weakening of immune control. However, the CTL response is highly adaptable and the long-term consequences of viral escape are not fully understood. The objective of this study was to address the question ''to what extent does HIV-1 escape from CTL contribute to HLA-associated AIDS progression?'' We combined an analysis of 21 escape events in longitudinally-studied HIV-1 infected people with a population-level analysis of the functional CTL response in 150 subjects (by IFNg ELISpot) and an analysis of the HIV-1 sequence database to quantify the contribution of escape to the HLA-associated rate of AIDS progression. We found that CTL responses restricted by protective HLA class I alleles, which are associated with slow progression to AIDS, recognised epitopes where escape variants had a weak evolutionary selective advantage (P = 0.008) and occurred infrequently (P = 0.017). Epitopes presented by protective HLA class I alleles were more likely to elicit a CTL response (P = 0.001) and less likely to contain sequence variation (P = 0.006). A third of between-individual variation in HLA-associated disease risk was predicted by the selective advantage of escape variants: a doubling in the evolutionary selective advantage was associated with a decrease in the AIDS-free period of 1.2 yrs. These results contribute to our understanding of what makes a CTL response protective and why some individuals progress to AIDS more rapidly than others. Citation: Asquith B (2008) The Evolutionary Selective Advantage of HIV-1 Escape Variants and the Contribution of Escape to the HLA-Associated Risk of AIDS Progression. PLoS ONE 3(10): e3486.
Physics Letters B, 1996
The restrictions imposed on the strong force in the 'non-commutative standard model' are examined... more The restrictions imposed on the strong force in the 'non-commutative standard model' are examined. It is concluded that given the framework of non-commutative geometry and assuming the electroweak sector of the standard model many details of the strong force can be explained including its vectorial nature. PACS-92: 11.15 Gauge field theories MSC-91: 81E13 Yang-Mills and other gauge theories sept 1995 DTP/95/49 CPT-95/P.3239 1 funded by PPARC r.e.asquith@dur.ac.uk
Trends in Immunology, 2009
Quantitative understanding of immunology requires the development of experimental and mathematica... more Quantitative understanding of immunology requires the development of experimental and mathematical techniques for estimation of rates of division and death of lymphocytes under different conditions. Here, we review the advantages and limitations of several labelling methods that are currently used to quantify turnover of lymphocytes in vivo. In addition to highlighting insights into lymphocyte kinetics which have recently been gained thanks to the development of novel techniques, we discuss important directions for future experimental and theoretical work in the field of lymphocyte turnover.
European Journal of Immunology, 2003
During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lympho... more During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lymphocytes are produced. To investigate the dynamics of clonal expansion, we measured cell proliferation during AIM using deuterated glucose to label DNA of dividing cells in vivo, analyzing cells according to CD4, CD8 and CD45 phenotype. The proportion of labeled CD8+CD45R0+ T lymphocytes was dramatically increased in AIM subjects compared to controls (mean 17.5 versus 2.8%/day; p<0.005), indicating very rapid proliferation. Labeling was also increased in CD4+CD45R0+ cells (7.1 versus 2.1%/day; p<0.01), but less so in CD45RA+ cells. Mathematical modeling, accounting for death of labeled cells and changing pool sizes, gave estimated proliferation rates in CD8+CD45R0+ cells of 11–130% of cells proliferating per day (mean 47%/day), equivalent to a doubling time of 1.5 days and an appearance rate in blood of about 5×109 cells/day (versus 7×107 cells/day in controls). Very rapid death rates were also observed amongst labeled cells (range 28–124, mean 57%/day),indicating very short survival times in the circulation. Thus, we have shown direct evidence for massive proliferation of CD8+CD45R0+ T lymphocytes in AIM and demonstrated that rapid cell division continues concurrently with greatly accelerated rates of cell disappearance.
Journal of Theoretical Biology, 2000
Journal of Theoretical Biology, 2003
Fratricide between CD8 þ T lymphocytes is known to occur in HTLV-I and possibly HSV-1 and HIV-1 i... more Fratricide between CD8 þ T lymphocytes is known to occur in HTLV-I and possibly HSV-1 and HIV-1 infection. However it is not known what effect, if any, T-cell fratricide has on the course of infection. Here we present simple mathematical techniques to investigate T-cell fratricide with particular reference to HTLV-I infection. Using a general model we predict the qualitative and quantitative effect of fratricide on HTLV-I equilibrium proviral load. We also investigate the effect of fratricide on the probability of viral clearance.
Proceedings of The National Academy of Sciences, 2002
Lymphocyte homeostasis is the result of a critical balance between cell proliferation and death. ... more Lymphocyte homeostasis is the result of a critical balance between cell proliferation and death. Disruption of this subtle equilibrium can lead to the onset of leukemia, an increase in the number of lymphocytes being potentially due to both of these parameters. The relative importance of cell proliferation vs. apoptosis during pathogenesis induced by the primate T cell lymphotropic viruses and bovine leukemia virus (BLV) has been difficult to assess because of conflicting data from a range of in vitro and ex vivo experimental systems. Here, we aim to resolve this issue by measuring the rates of cell proliferation and death in the BLV-ovine system, an animal model of human T lymphotropic virus (HTLV-1). We use a method based on the i.v. injection of 5-bromodeoxyuridine into BLV-infected sheep. We show that B lymphocytes in BLV ؉ asymptomatic sheep proliferate significantly faster than in uninfected controls (average proliferation rate: 0.020 per day vs. 0.011 per day). In contrast, the rates of cell death were not significantly different between aleukemic BLV-infected and control sheep (average death rate 0.089 per day vs. 0.094 per day, respectively). We conclude that the increase in the number of B cells during BLVinduced lymphocytosis results from higher proliferation rates but is not due to a significant decrease in apoptosis, in contrast to data from in vitro (ex vivo) experiments. The imbalance created by the net increase in proliferation in the absence of compensating cell death reveals a complex mechanism of feedback regulation controlling homeostasis in the blood compartment. § To whom reprint requests should be addressed at
Journal of Infectious Diseases, 2004
Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spas... more 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.
Journal of Virology, 2003
Significantly higher frequencies of tumor necrosis factor alpha-and interleukin-2-secreting human... more Significantly higher frequencies of tumor necrosis factor alpha-and interleukin-2-secreting human Tlymphotropic 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.
British Journal of Haematology, 2008
Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been inten... more Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukaemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements indicate that B lymphocytes do divide at significant rates in CLL. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared with their normal counterparts. This report addressed directly this point and compared B-cell kinetics in CLL subjects and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-2H2-glucose into DNA. We confirmed that B cells proliferated at significant levels in CLL but found that the proliferation rates were reduced compared with healthy subjects (mean 0·47 vs. 1·31%/d respectively, P = 0·007), equivalent to an extended doubling time of circulating B cells (147 d vs. 53 d). In conclusion, CLL B cells proliferate at reduced levels compared with healthy controls. CLL is thus characterized by an aberrant B-cell kinetics with a decrease in cell turnover, an observation that may impact on elaboration of efficient therapeutic strategies.
Immunology and Cell Biology, 2007
Despite significant advances in our understanding of the immune response to persistent viruses li... more Despite significant advances in our understanding of the immune response to persistent viruses like human T-cell lymphotropic virus type I (HTLV-I), many important questions remain unanswered. Mathematical modelling enables us to interpret and synthesise diverse experimental data in new ways and thus can contribute to our understanding. Here, we review recent advances in mathematical modelling of HTLV-I infection and illustrate how mathematics has enabled us to identify factors that determine an individual's viral burden and risk of developing HTLV-I-associated diseases.
Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persi... more Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persistence of the human T-lymphotropic virus type 1 (HTLV-1), which is responsible for HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A major determinant of disease progression is increased peripheral blood proviral load (PVL), possibly via the accumulation of infected cells in the central nervous system (CNS) creating a damaging inflammatory response. Current therapeutic approaches that focus on reducing either cell proliferation, viral replication, or tissue invasion are still unsatisfactory. Contrasting with these inhibitory strategies, we evaluated the efficacy of a novel approach aimed, paradoxically, at activating viral gene expression to expose virus-positive cells to the host immune response. We used valproate (VPA), a histone deacetylase inhibitor that has been used for decades as a chronic, safe treatment for epileptic disorders. Based on in vitro and in vivo data, we provide evidence that transient activation of the latent viral reservoir causes its collapse, a process that may alleviate the condition of HAM/TSP. This represents the first such approach to treating HAM/TSP, using gene activation therapy to tilt the host-pathogen balance in favor of an existing antiviral response. This trial is registered at http://clinicaltrials.gov/as no. NCT00519181.
Proceedings of The Royal Society B: Biological Sciences, 2006
The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify ce... more The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep.
Journal of Virology, 2003
Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell pr... more Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day ؊1 versus 0.156 day ؊1 in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day ؊1 versus 0.0085 day ؊1 ). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virusinduced adult T-cell leukemia in humans.
PLOS Computational Biology, 2009
Theoretical methods for predicting CD8+ T-cell epitopes are an important tool in vaccine design a... more Theoretical methods for predicting CD8+ T-cell epitopes are an important tool in vaccine design and for enhancing our understanding of the cellular immune system. The most popular methods currently available produce binding affinity predictions across a range of MHC molecules. In comparing results between these MHC molecules, it is common practice to apply a normalization procedure known as rescaling, to correct for possible discrepancies between the allelic predictors. Using two of the most popular prediction software packages, NetCTL and NetMHC, we tested the hypothesis that rescaling removes genuine biological variation from the predicted affinities when comparing predictions across a number of MHC molecules. We found that removing the condition of rescaling improved the prediction software's performance both qualitatively, in terms of ranking epitopes, and quantitatively, in the accuracy of their binding affinity predictions. We suggest that there is biologically significant variation among class 1 MHC molecules and find that retention of this variation leads to significantly more accurate epitope prediction.
Journal of General Virology, 2005
The CD8 + lymphocyte response is the main component of host immunity, yet it is difficult to quan... more The CD8 + lymphocyte response is the main component of host immunity, yet it is difficult to quantify its contribution to the control of persistent viruses. Consequently, it remains controversial as to whether CD8 + cells have a biologically significant impact on viral burden and disease progression in infections such as human immunodeficiency virus-1 and human T-lymphotropic virus type I (HTLV-I). Experiments to ascertain the impact of CD8 + cells on viral burden based on CD8 + cell frequency or specificity alone give inconsistent results. Here, an alternative approach was developed that directly quantifies the impact of CD8 + lymphocytes on HTLV-I proviral burden by measuring the rate at which HTLV-I-infected CD4 + cells were cleared by autologous CD8 + cells ex vivo. It was demonstrated that CD8 + cells reduced the lifespan of infected CD4 + cells to 1 day, considerably shorter than the 30 day lifespan of uninfected cells in vivo. Furthermore, it was shown that HTLV-I-infected individuals vary considerably in the rate at which their CD8 + cells clear infected cells, and that this is a significant predictor of their HTLV-I proviral load. Forty to 50 % of between-individual variation in HTLV-I proviral load was explained by variation in the rate at which CD8 + cells cleared infected cells. This novel approach demonstrates that CD8 + cells are a major determinant of HTLV-I proviral load. This assay is applicable to quantifying the CD8 + cell response to other viruses and malignancies and may be of particular importance in assessing vaccines.
Journal of Virology, 2006
Journal of Virology, 2006
Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, ... more Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.
Immunology, 2007
Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. ... more Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium-enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24-hr intravenous infusion of 6,6-D2-glucose, CD3– CD16+ NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n = 5), deuterium enrichment was maximal ∼ 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (± standard deviation) proliferation rate was 4·3 ± 2·4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 ± 7·6 × 106 cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6·9 ± 4·0%/day; half-life (T½) < 10 days]. Healthy elderly subjects (n = 8) had lower proliferation and production rates (P = 2·5 ± 1·0%/day and 7·3 ± 3·7 × 106 cells/l/day, respectively; P = 0·04). Similar rates were seen in patients chronically infected with human T-cell lymphotropic virus type I (HTLV-I) (P = 3·2 ± 1·9%/day). In acute infectious mononucleosis (n = 5), NK cell numbers were increased but kinetics were unaffected (P = 2·8 ± 1·0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV-I infection and normalize rapidly following acute Epstein–Barr virus infection.
PLOS One, 2008
HIV-1 escape from surveillance by cytotoxic T lymphocytes (CTL) is thought to cause at least tran... more HIV-1 escape from surveillance by cytotoxic T lymphocytes (CTL) is thought to cause at least transient weakening of immune control. However, the CTL response is highly adaptable and the long-term consequences of viral escape are not fully understood. The objective of this study was to address the question ''to what extent does HIV-1 escape from CTL contribute to HLA-associated AIDS progression?'' We combined an analysis of 21 escape events in longitudinally-studied HIV-1 infected people with a population-level analysis of the functional CTL response in 150 subjects (by IFNg ELISpot) and an analysis of the HIV-1 sequence database to quantify the contribution of escape to the HLA-associated rate of AIDS progression. We found that CTL responses restricted by protective HLA class I alleles, which are associated with slow progression to AIDS, recognised epitopes where escape variants had a weak evolutionary selective advantage (P = 0.008) and occurred infrequently (P = 0.017). Epitopes presented by protective HLA class I alleles were more likely to elicit a CTL response (P = 0.001) and less likely to contain sequence variation (P = 0.006). A third of between-individual variation in HLA-associated disease risk was predicted by the selective advantage of escape variants: a doubling in the evolutionary selective advantage was associated with a decrease in the AIDS-free period of 1.2 yrs. These results contribute to our understanding of what makes a CTL response protective and why some individuals progress to AIDS more rapidly than others. Citation: Asquith B (2008) The Evolutionary Selective Advantage of HIV-1 Escape Variants and the Contribution of Escape to the HLA-Associated Risk of AIDS Progression. PLoS ONE 3(10): e3486.
Physics Letters B, 1996
The restrictions imposed on the strong force in the 'non-commutative standard model' are examined... more The restrictions imposed on the strong force in the 'non-commutative standard model' are examined. It is concluded that given the framework of non-commutative geometry and assuming the electroweak sector of the standard model many details of the strong force can be explained including its vectorial nature. PACS-92: 11.15 Gauge field theories MSC-91: 81E13 Yang-Mills and other gauge theories sept 1995 DTP/95/49 CPT-95/P.3239 1 funded by PPARC r.e.asquith@dur.ac.uk
Trends in Immunology, 2009
Quantitative understanding of immunology requires the development of experimental and mathematica... more Quantitative understanding of immunology requires the development of experimental and mathematical techniques for estimation of rates of division and death of lymphocytes under different conditions. Here, we review the advantages and limitations of several labelling methods that are currently used to quantify turnover of lymphocytes in vivo. In addition to highlighting insights into lymphocyte kinetics which have recently been gained thanks to the development of novel techniques, we discuss important directions for future experimental and theoretical work in the field of lymphocyte turnover.
European Journal of Immunology, 2003
During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lympho... more During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lymphocytes are produced. To investigate the dynamics of clonal expansion, we measured cell proliferation during AIM using deuterated glucose to label DNA of dividing cells in vivo, analyzing cells according to CD4, CD8 and CD45 phenotype. The proportion of labeled CD8+CD45R0+ T lymphocytes was dramatically increased in AIM subjects compared to controls (mean 17.5 versus 2.8%/day; p<0.005), indicating very rapid proliferation. Labeling was also increased in CD4+CD45R0+ cells (7.1 versus 2.1%/day; p<0.01), but less so in CD45RA+ cells. Mathematical modeling, accounting for death of labeled cells and changing pool sizes, gave estimated proliferation rates in CD8+CD45R0+ cells of 11–130% of cells proliferating per day (mean 47%/day), equivalent to a doubling time of 1.5 days and an appearance rate in blood of about 5×109 cells/day (versus 7×107 cells/day in controls). Very rapid death rates were also observed amongst labeled cells (range 28–124, mean 57%/day),indicating very short survival times in the circulation. Thus, we have shown direct evidence for massive proliferation of CD8+CD45R0+ T lymphocytes in AIM and demonstrated that rapid cell division continues concurrently with greatly accelerated rates of cell disappearance.
Journal of Theoretical Biology, 2000
Journal of Theoretical Biology, 2003
Fratricide between CD8 þ T lymphocytes is known to occur in HTLV-I and possibly HSV-1 and HIV-1 i... more Fratricide between CD8 þ T lymphocytes is known to occur in HTLV-I and possibly HSV-1 and HIV-1 infection. However it is not known what effect, if any, T-cell fratricide has on the course of infection. Here we present simple mathematical techniques to investigate T-cell fratricide with particular reference to HTLV-I infection. Using a general model we predict the qualitative and quantitative effect of fratricide on HTLV-I equilibrium proviral load. We also investigate the effect of fratricide on the probability of viral clearance.
Proceedings of The National Academy of Sciences, 2002
Lymphocyte homeostasis is the result of a critical balance between cell proliferation and death. ... more Lymphocyte homeostasis is the result of a critical balance between cell proliferation and death. Disruption of this subtle equilibrium can lead to the onset of leukemia, an increase in the number of lymphocytes being potentially due to both of these parameters. The relative importance of cell proliferation vs. apoptosis during pathogenesis induced by the primate T cell lymphotropic viruses and bovine leukemia virus (BLV) has been difficult to assess because of conflicting data from a range of in vitro and ex vivo experimental systems. Here, we aim to resolve this issue by measuring the rates of cell proliferation and death in the BLV-ovine system, an animal model of human T lymphotropic virus (HTLV-1). We use a method based on the i.v. injection of 5-bromodeoxyuridine into BLV-infected sheep. We show that B lymphocytes in BLV ؉ asymptomatic sheep proliferate significantly faster than in uninfected controls (average proliferation rate: 0.020 per day vs. 0.011 per day). In contrast, the rates of cell death were not significantly different between aleukemic BLV-infected and control sheep (average death rate 0.089 per day vs. 0.094 per day, respectively). We conclude that the increase in the number of B cells during BLVinduced lymphocytosis results from higher proliferation rates but is not due to a significant decrease in apoptosis, in contrast to data from in vitro (ex vivo) experiments. The imbalance created by the net increase in proliferation in the absence of compensating cell death reveals a complex mechanism of feedback regulation controlling homeostasis in the blood compartment. § To whom reprint requests should be addressed at
Journal of Infectious Diseases, 2004
Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spas... more 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.
Journal of Virology, 2003
Significantly higher frequencies of tumor necrosis factor alpha-and interleukin-2-secreting human... more Significantly higher frequencies of tumor necrosis factor alpha-and interleukin-2-secreting human Tlymphotropic 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.
British Journal of Haematology, 2008
Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been inten... more Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukaemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements indicate that B lymphocytes do divide at significant rates in CLL. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared with their normal counterparts. This report addressed directly this point and compared B-cell kinetics in CLL subjects and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-2H2-glucose into DNA. We confirmed that B cells proliferated at significant levels in CLL but found that the proliferation rates were reduced compared with healthy subjects (mean 0·47 vs. 1·31%/d respectively, P = 0·007), equivalent to an extended doubling time of circulating B cells (147 d vs. 53 d). In conclusion, CLL B cells proliferate at reduced levels compared with healthy controls. CLL is thus characterized by an aberrant B-cell kinetics with a decrease in cell turnover, an observation that may impact on elaboration of efficient therapeutic strategies.
Immunology and Cell Biology, 2007
Despite significant advances in our understanding of the immune response to persistent viruses li... more Despite significant advances in our understanding of the immune response to persistent viruses like human T-cell lymphotropic virus type I (HTLV-I), many important questions remain unanswered. Mathematical modelling enables us to interpret and synthesise diverse experimental data in new ways and thus can contribute to our understanding. Here, we review recent advances in mathematical modelling of HTLV-I infection and illustrate how mathematics has enabled us to identify factors that determine an individual's viral burden and risk of developing HTLV-I-associated diseases.
Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persi... more Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persistence of the human T-lymphotropic virus type 1 (HTLV-1), which is responsible for HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A major determinant of disease progression is increased peripheral blood proviral load (PVL), possibly via the accumulation of infected cells in the central nervous system (CNS) creating a damaging inflammatory response. Current therapeutic approaches that focus on reducing either cell proliferation, viral replication, or tissue invasion are still unsatisfactory. Contrasting with these inhibitory strategies, we evaluated the efficacy of a novel approach aimed, paradoxically, at activating viral gene expression to expose virus-positive cells to the host immune response. We used valproate (VPA), a histone deacetylase inhibitor that has been used for decades as a chronic, safe treatment for epileptic disorders. Based on in vitro and in vivo data, we provide evidence that transient activation of the latent viral reservoir causes its collapse, a process that may alleviate the condition of HAM/TSP. This represents the first such approach to treating HAM/TSP, using gene activation therapy to tilt the host-pathogen balance in favor of an existing antiviral response. This trial is registered at http://clinicaltrials.gov/as no. NCT00519181.
Proceedings of The Royal Society B: Biological Sciences, 2006
The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify ce... more The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep.
Journal of Virology, 2003
Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell pr... more Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day ؊1 versus 0.156 day ؊1 in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day ؊1 versus 0.0085 day ؊1 ). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virusinduced adult T-cell leukemia in humans.
PLOS Computational Biology, 2009
Theoretical methods for predicting CD8+ T-cell epitopes are an important tool in vaccine design a... more Theoretical methods for predicting CD8+ T-cell epitopes are an important tool in vaccine design and for enhancing our understanding of the cellular immune system. The most popular methods currently available produce binding affinity predictions across a range of MHC molecules. In comparing results between these MHC molecules, it is common practice to apply a normalization procedure known as rescaling, to correct for possible discrepancies between the allelic predictors. Using two of the most popular prediction software packages, NetCTL and NetMHC, we tested the hypothesis that rescaling removes genuine biological variation from the predicted affinities when comparing predictions across a number of MHC molecules. We found that removing the condition of rescaling improved the prediction software's performance both qualitatively, in terms of ranking epitopes, and quantitatively, in the accuracy of their binding affinity predictions. We suggest that there is biologically significant variation among class 1 MHC molecules and find that retention of this variation leads to significantly more accurate epitope prediction.
Journal of General Virology, 2005
The CD8 + lymphocyte response is the main component of host immunity, yet it is difficult to quan... more The CD8 + lymphocyte response is the main component of host immunity, yet it is difficult to quantify its contribution to the control of persistent viruses. Consequently, it remains controversial as to whether CD8 + cells have a biologically significant impact on viral burden and disease progression in infections such as human immunodeficiency virus-1 and human T-lymphotropic virus type I (HTLV-I). Experiments to ascertain the impact of CD8 + cells on viral burden based on CD8 + cell frequency or specificity alone give inconsistent results. Here, an alternative approach was developed that directly quantifies the impact of CD8 + lymphocytes on HTLV-I proviral burden by measuring the rate at which HTLV-I-infected CD4 + cells were cleared by autologous CD8 + cells ex vivo. It was demonstrated that CD8 + cells reduced the lifespan of infected CD4 + cells to 1 day, considerably shorter than the 30 day lifespan of uninfected cells in vivo. Furthermore, it was shown that HTLV-I-infected individuals vary considerably in the rate at which their CD8 + cells clear infected cells, and that this is a significant predictor of their HTLV-I proviral load. Forty to 50 % of between-individual variation in HTLV-I proviral load was explained by variation in the rate at which CD8 + cells cleared infected cells. This novel approach demonstrates that CD8 + cells are a major determinant of HTLV-I proviral load. This assay is applicable to quantifying the CD8 + cell response to other viruses and malignancies and may be of particular importance in assessing vaccines.
Journal of Virology, 2006
Journal of Virology, 2006
Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, ... more Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.
Immunology, 2007
Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. ... more Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium-enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24-hr intravenous infusion of 6,6-D2-glucose, CD3– CD16+ NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n = 5), deuterium enrichment was maximal ∼ 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (± standard deviation) proliferation rate was 4·3 ± 2·4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 ± 7·6 × 106 cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6·9 ± 4·0%/day; half-life (T½) < 10 days]. Healthy elderly subjects (n = 8) had lower proliferation and production rates (P = 2·5 ± 1·0%/day and 7·3 ± 3·7 × 106 cells/l/day, respectively; P = 0·04). Similar rates were seen in patients chronically infected with human T-cell lymphotropic virus type I (HTLV-I) (P = 3·2 ± 1·9%/day). In acute infectious mononucleosis (n = 5), NK cell numbers were increased but kinetics were unaffected (P = 2·8 ± 1·0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV-I infection and normalize rapidly following acute Epstein–Barr virus infection.