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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 others1997; Daenke et al., 1996). The role of this response iscontroversial. Some evidence indicates that a high frequencyof HTLV-I-specific CD8 + cells is pathogenic and causesthe inflammation associated with HAM/TSP (Bieganowskaet al., 1999; Greten et al., 1998; Hoger et al., 1997; Jacobson,2002); conversely, other data suggest that the HTLV-IspecificCD8 + cell response reduces proviral load and isassociated with a lower risk of disease (Bangham, 2000;Jeffery et al., 1999, 2000; Niewiesk et al., 1994). Thesecontradictory results may reflect the limitations of traditionalmeasures of CD8 + cell function: it is increasinglybeing realized that the size of the virus-specific CD8 + cellresponse is not the only factor determining its impact onthe virus in vivo (Rowland-Jones et al., 2001; van Baalenet al., 2002; Yang, 2003; Yang et al., 2003; Zhang et al.,2003). Other CD8 + cell attributes such as T-cell receptoravidity, specificity and cell maturation state, as well as targetcell attributes including efficiency of epitope processingand presentation and phenotype, will all affect the abilityof CD8 + cells to control a viral infection. Simply measuringspecific CD8 + cell frequency by tetramer analysis, orby using additional functional measures such as cytokineproduction, fails to account for this complexity. Chromiumrelease assays do not accurately reflect the complexity ofthe in vivo cytotoxic response, both because of the nonphysiologicalnature of the target cells and because of theusual requirement for prolonged culture in vitro. Even ifall key CD8 + cell attributes (frequency, avidity, phenotypeetc) are measured simultaneously it is still not possible tosynthesize this data into a single index of CD8 + cell antiviralefficacy. Thus, although great advances have been madein techniques to measure CD8 + cell function, all currentassays measure individual attributes in isolation andtherefore cannot capture the overall impact of the CD8 +lymphocyte response on long-term viral load.The failure of current assays of the CD8 + response isdemonstrated by the use of specific CD8 + cell frequency asa measure of in vivo CD8 + cell function in both HTLV-Iand HIV-1. It has been argued that if the specific CD8 +T-cell response is important in controlling persistent viralinfection, individuals with a large virus-specific CD8 + cellfrequency would be expected to have a low viral burden,that is, a negative correlation between virus-specific CD8 +cell frequency and viral burden would be observed.However, in HIV-1, virus-specific CD8 + cell frequency asmeasured by tetramers and/or IFNc ELISpot has shownpositive, negative and zero correlations with HIV-1 viralload (Betts et al., 2001; Ogg et al., 1998). In HTLV-I,similarly divergent results have been obtained (Goon et al.,2004; Kubota et al., 2000).The aim of this study was to develop a composite measureof CD8 + T-cell mediated antiviral efficacy that directlymeasures the combined impact of key determinants ofCD8 + cell function, including virus-specific cell frequency,cytolytic ability, scope of epitope recognition and efficiencyof antigen presentation. We achieved this by measuringthe rate at which naturally, endogenously infected cells werecleared by autologous CD8 + cells ex vivo. Having developedsuch an assay we use it to answer two questions: (i) howimportant is the CD8 + cell response in controlling HTLV-Iproviral burden? and (ii) what is the relationship betweenCD8 + cell efficacy and HAM/TSP?METHODSPatients. All patients attended the HTLV-I clinic at St Mary’sHospital, London and gave informed consent. The study wasapproved by the Local Research Ethics Committee of St Mary’sHospital NHS Trust. HTLV-I infection was confirmed by thepresence of antibodies to HTLV-I Gag and Env antigens in sera byWestern blot (HTLV blot 2?4; Genelabs). Diagnosis of HAM/TSPwas made following World Health Organization criteria. None ofthe patients were receiving antiviral or immunosuppressive therapyat the time of the study. Patient data are summarized in Table 1.CD8 + cell assay. CD8 + cells were positively selected from thawedcryopreserved PBMC using magnetic microbeads (Miltenyi Biotec).The CD8 + and CD8 2 fractions were then washed twice, resuspendedin standard culture medium (RPMI 1640 supplemented with10 % fetal calf serum, 2 mM L-glutamine, 100 IU penicillin ml 21and 100 mg streptomycin ml 21 ) and aliquotted (total volume 1 ml)into 5 ml round-bottomed, vented capped tubes at three to sixdifferent CD8 + : CD8 2 ratios (lower, including and higher thanthe subject’s normal ratio) depending on cell numbers available.No mitogens, cytokines or artificial peptides were added. Whenrequired, concanamycin A (CMA; Sigma) was added at a final concentrationof 20 nM. After 18 h culture at 37 uC, 5 % CO 2 , the cellswere washed in PBS, fixed for 20 min at room temperature in 2 %paraformaldehyde (pH 7?4; Sigma), washed then surface stainedfor CD4 and CD8 antigens by incubation at room temperature for20 min in PBS/7 % normal goat serum with relevant mAbs (15 mgPC5-conjugated anti-CD4 and ECD-conjugated anti-CD8 ml 21 ;Beckman Coulter). The cells were washed once and stained intracellularlyfor the HTLV-I early protein Tax (Lee et al., 1989), asdescribed by Hanon et al. (2000a), then analysed by flow cytometryon a Coulter EPICS XL. Thirty thousand events were routinelycollected. All assays were done in duplicate.The resulting data were analysed using non-linear regression. Theantiviral efficacy, i.e. the rate at which Tax + CD4 + cells were cleared,was estimated in each subject using the following model:dydt ~c{eyzðmodel1Þwhere y is the proportion of CD4 + cells expressing Tax (i.eTax + CD4 + cells/CD4 + cells), c is the rate of increase of Taxexpression, e is the CD8 + cell-mediated antiviral efficacy and z isthe proportion of lymphocytes that are CD8 + . This model wassolved analytically and fitted to the data using non-linear leastsquares regression, providing an estimate of the antiviral efficacy (e)in each individual. This model does not assume that CD8 + cellsreduce the proportion of Tax-expressing cells since e is free to benegative or zero as well as positive. At time zero the proportion ofCD4 + cells expressing Tax is usually below the detection limit. Inthe absence of CD8 + cells it then increases over time. We modelthis empirically using a constant rate of Tax expression (c); morecomplex Tax expression dynamics are discussed below.Quantification of HTLV-I proviral load. Cells were available tomeasure proviral load in 16 subjects. The HTLV-I proviral load andb-globin copy number of each sample was quantified using real-timequantitative PCR (primers as in Kwok et al., 1988; Seiki et al., 1985)2 Journal of General Virology 86

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