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A functional CD8 cell assay reveals individual variation in CD8 cell ...

A functional CD8 cell assay reveals individual variation in CD8 cell ...

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%paper no. vir80766charlesworth ref: vir57918&B. Asquith and othersEffect of viral protein expression dynamics. The assumption,inherent in the basic model 1, that expression of the viral proteinTax increases at a constant rate over time is clearly an over simplification.We measured Tax expression in CD4 + cells over 18 h ineight of our subjects (five HAM/TSP and three ACs) and found nosystematic difference in the time course of Tax expression betweenHAM/TSP patients and ACs, or between subjects with a high or lowproviral load. We estimated the antiviral efficacy in five of thesesubjects – once with the basic model 1 and once taking detaileddynamics of Tax expression into account. The results from the twomethods were perfectly correlated, i.e. the rank order of the CD8 +cell antiviral efficacy was identical. Between-individual variation inthe dynamics of Tax expression was therefore unlikely to systematicallyaffect our results.Supporting information. Experimental data showing the rate ofclearance of autologous and allogenic target cells and an estimate ofthe number of infected cells cleared per HTLV-I-specific CD8 + cellper day are included in the Supplementary material in JGV Online.RESULTSDeveloping a composite measure of CD8 +cell-mediated antiviral efficacyThe CD8 + cell-mediated ‘antiviral efficacy’ was defined asthe rate of clearance of HTLV-I-infected Tax-expressingCD4 + cells by autologous CD8 + cells ex vivo. This CD8 +cell antiviral efficacy is calculated for the CD8 + cellpopulation as a whole, reflecting both the frequency andcytotoxicity of HTLV-I-specific CD8 + cells.CD4 + cells are the predominant infected cell populationin HTLV-I (Richardson et al., 1997; Yamano et al., 2004).In endogenously infected cells, the early viral protein Tax isusually undetectable immediately ex vivo, but increases aftershort-term culture (Hanon et al., 2000a). We quantified theCD8 + cell-mediated antiviral efficacy by measuring theproportion of CD4 + cells expressing Tax, after exposureto different numbers of autologous CD8 + cells. To dothis, PBMC were separated by CD8-positive selection intoCD8 + and CD8 2 cell fractions. Neither cell populationwas artificially expanded or stimulated ex vivo, and noexogenous cytokines or peptides were added. The cellpopulations were immediately recombined in differentproportions, to include CD8 + : CD8 2 ratios above, belowand at the physiological ratio for that individual. Cells werethen co-cultured for 18 h, after which the proportion ofTax + CD4 + cells was determined by flow cytometry. Aco-culture period of 18 h was chosen to provide sufficienttime for infected cells to express Tax (which normallypeaks at 6–12 h) and for Tax-expressing cells to be lysed.All assays were performed in duplicate.The resulting data – the proportion of Tax + CD4 + cellsas a function of CD8 + cell frequency – was analysedmathematically. The technique used was similar to thatused to analyse BrdU and deuterated glucose lymphocytelabelling data (Debacq et al., 2002; Mohri et al., 1998). Thatis, a simple mathematical model was formulated – in thiscase reflecting the expression of Tax by infected cells andFig. 1. CD8 + antiviral efficacy assay: an example. The figureshows an example of the antiviral efficacy assay. The proportionof CD4 + lymphocytes that were Tax + following 18 h co-culturewith different proportions of CD8 + lymphocytes was measured.The model (equation 1, Methods) was fitted to this data and inthis way the rate of clearance of Tax + CD4 + cells per day perCD8 + cell (antiviral efficacy) was estimated. This was repeatedin the same subject. Repeat 1, % observed data; dashed line,best theoretical fit. Repeat 2, m observed data; solid line, besttheoretical fit.the clearance of Tax expressing cells by CD8 + cells – andfitted to the data using non-linear regression. In this waywe estimated the CD8 + cell antiviral efficacy, that is the rateof CD8 + cell-mediated clearance of Tax-expressing cells.An example experiment is shown in Fig. 1. Since weestimated infected CD4 + cell survival as a function ofCD8 + cell frequency we automatically excluded CD8 + cellindependentdeath of infected cells (e.g. natural cell death).It was found that the frequency and mean fluorescenceintensity of Tax expression in CD4 + lymphocytes bothdecreased following co-culture with increasing numbers ofCD8 + cells (Fig. 2). Previous evidence (Hanon et al., 2000a,b) indicated this decrease was principally mediated byperforin-dependent cell lysis. To confirm this, we measuredthe change in antiviral efficacy in the presence and absenceof 20 nM of the perforin inhibitor, CMA (Kataoka et al.,1996). In samples from two unrelated subjects, treatmentwith CMA reduced the rate at which Tax + cells were clearedby CD8 + cells in each case by 80 % (data not shown),indicating that the major CD8 + cell-mediated antiviralpathway was perforin-dependent. We also compared therate at which autologous and allogeneic Tax-expressingCD4 + target cells were cleared by CD8 + cells from a rangeof HTLV-I-seropositive subjects. Since antiviral efficacy, asdefined here, is a measure of specific clearance of HTLV-Iinfectedcells, any non-HTLV-I-associated allogeneic killing4 Journal of General Virology 86

%paper no. vir80766charlesworth ref: vir57918&CD8 + cell response determines HTLV-I proviral loadFig. 3. Antiviral efficacy of the CD8 + cell response in 23HTLV-I-seropositive individuals. The CD8 + cell antiviral efficacywas measured in 23 HTLV-I-seropositive individuals, of whomnine were ACs (m) and 14 had HAM/TSP (%). There was nosignificant difference in antiviral efficacy between the twogroups.Fig. 2. Reduction in Tax expression with increasing CD8 + cellfrequency. Frequency and intensity of Tax expression in CD4 +cells decreases as a function of increasing CD8 + cell frequencyduring 18 h co-culture. Example data from subject HBE(see Table 1) are shown. CD8 + cell frequency: thin line, low(2?9 %); medium line, intermediate (17?6 %); thick line, high(33?9 %). Frequency of Tax + CD4 + cells (as a proportion ofCD4 + cells): 3?2 % (low CD8), 0?9 % (intermediate CD8),0?2 % (high CD8). Mean fluorescence intensity of Tax stainingin Tax + CD4 + cells: 43?3 (low CD8), 33?4 (intermediate CD8),26?7 (high CD8).does not contribute to the result. We found that HTLV-Iinfectedallogeneic target cells were cleared at a much lowerrate than autologous target cells, and that the rate ofclearance decreased as the extent of HLA class I mismatchincreased (see Supplementary material in JGV Online,Table S1). These results indicate that the dominantmechanism of reduction in Tax expression by CD8 + cellsis perforin-dependent and MHC class I-restricted; consistentwith classical CD8 + cell-mediated lysis of virallyinfected cells. However, our assay is not just restricted toperforin-dependent lysis, it will capture all CD8 + cellmediatedantiviral effects that result in a reduction of thenumber of Tax + cells in 18 h e.g. Fas-induced apoptosis.CD8 + cell-mediated antiviral effects that occur over alonger time, such as IFNc suppression of virus replication,will not be captured in this assay.Quantifying antiviral efficacy in anHTLV-I-infected cohortThis novel assay was applied to 23 HTLV-I-seropositiveindividuals, of whom nine were ACs and 14 had HAM/TSP. The results are summarized in Table 1. All assayswere done in duplicate and the results expressed as themean. The agreement between duplicate assays was good(Pearson’s correlation coefficient r=0?94, P

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