Becca Asquith

Fratricide among CD8+ T Lymphocytes Naturally Infected with Human T Cell Lymphotropic Virus Type I

Infection and gene expression by the human T lymphotropic virus type I (HTLV-I) in vivo have been thought to be confined to CD4(+) T lymphocytes. We show here that, in natural HTLV-I infection, a significant proportion of CD8(+) T lymphocytes are infected by HTLV-I. Interestingly, HTLV-I-specific but not Epstein-Barr virus-specific CD8(+) T lymphocytes were shown to be infected. Furthermore, HTLV-I protein expression in naturally infected CD8(+) T lymphocytes renders them susceptible to fratricide mediated by autologous HTLV-I-specific CD8(+) T lymphocytes. Fratricide among virus-specific CTLs could impair the immune control of HTLV-I and possibly other lymphotropic viruses.

In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection

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.

Measurement and modeling of human T cell kinetics

The ability to measure, describe and interpret T cell kinetics is pivotal in understanding normal lymphocyte homeostasis and diseases that affect T cell numbers. Following in vivo labeling of dividing cells with 6,6‐D2‐glucose in eight healthy volunteers, peripheral blood T cells were sorted by CD4, CD8 and CD45 phenotype. Enrichment of deuterium in DNA was measured by gas chromatography‐mass spectrometry. A novel model of T cell kinetics, allowing for heterogeneity within T cell pools, was used to analyze data on acquisition and loss of label and calculate proliferation and disappearance rates for each subpopulation. Proliferation rates for CD45RO+CD8+ cells and CD45RO+CD4+ cells were 5.1% and 2.7% /day, respectively (equivalent doubling times: 14 and 26 days). CD45RA+CD8+ lymphocytes and CD45RA+CD4+ lymphocytes had slower proliferation rates, 0.5% and 0.6% / day, respectively (doubling time about 4 months). Disappearance rates of labeled cells were similar for all cell types (7%–12% / day) and exceeded corresponding proliferation rates. This disparity may be understood conceptually in terms of either phenotypic heterogeneity (rapid versus slow turnover pools), or history (recently divided cells are more likely to die). The new kinetic model fits the data closely and avoids the need to postulate a large external source of lymphocytes to maintain equilibrium.

Lymphocyte kinetics: the interpretation of labelling data

DNA labelling provides an exciting tool for elucidating the in vivo dynamics of lymphocytes. However, the kinetics of label incorporation and loss are complex and results can depend on the method of interpretation. Here we describe two approaches to interpreting labelling data. Both seek to explain the common observation that the estimated death rate of lymphocytes is higher than their estimated proliferation rate. In the first approach, an additional source of lymphocytes is postulated. In the second, it is maintained that lymphocyte heterogeneity is sufficient to account for the observation. We explain why we favour the second approach, arguing that the addition of a large source of lymphocytes is unnecessary and difficult to reconcile with what is currently known about lymphocyte physiology. We discuss how the choice of model can affect data interpretation.

In vivo T lymphocyte dynamics in humans and the impact of human T-lymphotropic virus 1 infection

Human T-lymphotropic virus type 1 (HTLV-1) is a persistent CD4+ T-lymphotropic retrovirus. Most HTLV-1-infected individuals remain asymptomatic, but a proportion develop adult T cell leukemia or inflammatory disease. It is not fully understood how HTLV-1 persists despite a strong immune response or what determines the risk of HTLV-1-associated diseases. Until recently, it has been difficult to quantify lymphocyte kinetics in humans in vivo. Here, we used deuterated glucose labeling to quantify in vivo lymphocyte dynamics in HTLV-1-infected individuals. We then used these results to address four questions. (i) What is the impact of HTLV-1 infection on lymphocyte dynamics? (ii) How does HTLV-1 persist? (iii) What is the extent of HTLV-1 expression in vivo? (iv) What features of lymphocyte kinetics are associated with HTLV-1-associated myelopathy/tropical spastic paraparesis? We found that CD4+CD45RO+ and CD8+CD45RO+ T lymphocyte proliferation was elevated in HTLV-1-infected subjects compared with controls, with an extra 1012 lymphocytes produced per year in an HTLV-1-infected subject. The in vivo proliferation rate of CD4+CD45RO+ cells also correlated with ex vivo viral expression. Finally, the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with significantly increased CD4+CD45RO+ cell proliferation. We suggest that there is persistent viral gene expression in vivo, which is necessary for the maintenance of the proviral load and determines HTLV-1-associated myelopathy/tropical spastic paraparesis risk.

HLA Class I Binding of HBZ Determines Outcome in HTLV-1 Infection

CD8+ T cells can exert both protective and harmful effects on the virus-infected host. However, there is no systematic method to identify the attributes of a protective CD8+ T cell response. Here, we combine theory and experiment to identify and quantify the contribution of all HLA class I alleles to host protection against infection with a given pathogen. In 432 HTLV-1-infected individuals we show that individuals with HLA class I alleles that strongly bind the HTLV-1 protein HBZ had a lower proviral load and were more likely to be asymptomatic. We also show that in general, across all HTLV-1 proteins, CD8+ T cell effectiveness is strongly determined by protein specificity and produce a ranked list of the proteins targeted by the most effective CD8+ T cell response through to the least effective CD8+ T cell response. We conclude that CD8+ T cells play an important role in the control of HTLV-1 and that CD8+ cells specific to HBZ, not the immunodominant protein Tax, are the most effective. We suggest that HBZ plays a central role in HTLV-1 persistence. This approach is applicable to all pathogens, even where data are sparse, to identify simultaneously the HLA Class I alleles and the epitopes responsible for a protective CD8+ T cell response.

The Role of CTLs in Persistent Viral Infection: Cytolytic Gene Expression in CD8 Lymphocytes Distinguishes between Individuals with a High or Low Proviral Load of Human T Cell Lymphotropic Virus Type 1

The proviral load in human T cell lymphotropic virus type 1 (HTLV-1) infection is typically constant in each infected host, but varies by >1000-fold between hosts and is strongly correlated with the risk of HTLV-1-associated inflammatory disease. However, the factors that determine an individual’s HTLV-1 proviral load remain uncertain. Experimental evidence from studies of host genetics, viral genetics, and lymphocyte function and theoretical considerations suggest that a major determinant of the equilibrium proviral load is the CD8+ T cell response to HTLV-1. In this study, we tested the hypothesis that the gene expression profile in circulating CD8+ and CD4+ lymphocytes distinguishes between individuals with a low proviral load of HTLV-1 and those with a high proviral load. We show that circulating CD8+ lymphocytes from individuals with a low HTLV-1 proviral load overexpressed a core group of nine genes with strong functional coherence: eight of the nine genes encode granzymes or other proteins involved in cell-mediated lysis or Ag recognition. We conclude that successful suppression of the HTLV-1 proviral load is associated with strong cytotoxic CD8+ lymphocyte activity in the peripheral blood.

Human Cell Lymphotropic Virus (HTLV) Type-l-Specific CD8+ Cells: Frequency and Immunodominance Hierarchy

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.

HTLV-1: persistence and pathogenesis.

ContentsIntroduction. . 131HTLV-1 pathogenesis 132HTLV-1 and disease. . . 132ATL 132HAM/TSP . . 132Determinants of proviral load 132Integration and clonal expansion . . 132CD8þ T cell lysis 133The immune response to HTLV-1. . . 133Determinants of protective immunity 133Immunodominance 134‘‘Innate’’ receptors enhance the HLA class I-restricted response 135Persistence in the face of a strong immune response 136Immunogenicity of HTLV-I-infected cells 136Slow clearance of Tax-expressing cells in vivo 137Concluding remarks 137Is HBZ a viable target for HTLV-I vaccine design? 137Why is CTL killing low in vivo? 137How do KIRs affect the outcome of HTLV-1 infection? . . 137References . . . 137

Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester (CFSE).

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.