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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 othersFig. 4. Proviral load plotted against antiviral efficacy for sevenACs and nine HAM/TSP patients. A negative correlationbetween proviral load and antiviral efficacy was observed inboth ACs and HAM/TSP patients (P=0?03 and 0?04, respectively;Spearman’s rank correlation two-tailed test). Furthermore,for a given rate of clearance the proviral load was lower in ACsthan in HAM/TSP patients (P=0?03; permutation two-tailedtest). %, HAM/TSP patients. m, ACs. Eight AC data pointsfrom seven individuals are illustrated. One subject (HS – seeTable 1) appears twice because blood samples from two differenttime points with two different proviral loads were available.Including HS twice did not alter our conclusions. We found asignificant negative correlation between proviral load and antiviralefficacy if only the first point or only the second point wasincluded [excluding first HS point: r s =”1,0

%paper no. vir80766charlesworth ref: vir57918&CD8 + cell response determines HTLV-I proviral loadremains controversial and there are many conflicting data.In particular, in HTLV-I it is not known if CD8 + cells playa biologically significant role in controlling proviral loador even whether a strong CD8 + cell response protectsfrom or causes HAM/TSP. Part of the difficulty lies in thecurrent assays of the CD8 + lymphocyte response (Yang,2003). The efficacy of the CD8 + lymphocyte response isa function of many factors including the frequency of thevirus-specific CD8 + cells, the number and identity ofepitopes recognized, the lytic capacity of specific cells,their activation status and phenotype and also the efficiencywith which target cells express and present antigen.Techniques exist to assay these attributes separately. However,it is difficult to determine the relative importanceof each attribute and to integrate these data to ascertainthe composite effectiveness of the CD8 + lymphocyte response.Furthermore, these techniques are often highly nonphysiological.For example, exogenous addition of artificialpeptides is common in chromium release, ELISpot andLYSISpot (Snyder et al., 2003) assays, and does not allowfor natural processing and presentation of viral proteins inthe context of productive infection.The CD8 + cell assay described here has the advantage thatit measures clearance of natural target cells (a subject’sHTLV-I-infected CD4 + lymphocytes) by the relevanteffectors (autologous CD8 + cells), and naturally processedviral peptides are presented in the appropriate context.The assay excludes the effects of any redundant CD8 + cellclones directed against viral epitopes that have undergonemutation and are no longer present in the viral genome;such CD8 + cell clones remain detectable by tetramerstaining, LYSISpot or ELISpot. The assay also avoids a biastowards immunodominant epitopes, which are not necessarilythe most important epitopes for the control of viralinfection in vivo. Thus, in contrast to previous measures ofthe CD8 + response this approach provides a physiologicalindex of the overall impact of CD8 + cells on virally infectedcells and allows direct comparison of the efficacy of theCD8 + cell response between individuals.Inevitable disadvantages of this assay are that peripheralblood cells may not be representative of the majority oflymphocytes and that ex vivo cell behaviour may not reflectin vivo behaviour. Both these differences will be manifestedas a reduced correlation between proviral load and CD8 +cell efficacy, i.e. our analysis will err on the side of caution.We have developed this assay in the context of HTLV-Iinfection but it could be adapted to measure the efficacy ofthe CD8 + lymphocyte response to a range of targets –allogeneic tissue, leukaemic cells or alternative pathogeninfectedcells. It could be particularly important in assessingthe efficacy of vaccines designed to elicit a protective CD8 +cell response (Pantaleo & Koup, 2004).The most commonly used measure of the ‘strength’ of theantiviral response is the frequency of virus-specific cells.Previous studies (Kubota et al., 2000; Wodarz et al., 2001)have failed to find a consistent relationship between proviralload and HTLV-I-specific CD8 + cell frequency in both ACand HAM/TSP subject groups. When correlations havebeen observed they have tended to be positive, which hasbeen interpreted as indicating that the CD8 + cell responsepassively reflects, rather than controls, proviral load.In contrast, using the assay presented here, we found asignificant negative correlation between proviral load andCD8 + cell antiviral efficacy in both ACs and HAM/TSPpatients. Indeed, antiviral efficacy is a much better predictorof proviral load than HTLV-I-specific CD8 + cell frequency.Quantifying this relationship we found that within the ACand HAM/TSP groups 40–50 % of the between-individualvariation in proviral load was attributable to variation inthe antiviral efficacy. That is, the rate at which infectedcells were cleared by CD8 + cells was a major determinantof long-term viral burden. Other possible determinantsinclude the antibody response, the strain of infecting virusand the route of infection.We also explored the relationship between CD8 + cell antiviralefficacy and clinical outcome. We found no differencein antiviral efficacy between HAM/TSP patients and ACs.However, there was no significant difference in proviralload between the HAM/TSP and AC groups in our cohort,and as the protective effect of the CD8 + response ishypothesized to operate through a reduction in proviralload (Asquith & Bangham, 2000; Jeffery et al., 1999, 2000;Niewiesk et al., 1994; Vine et al., 2004), our study did nothave the power to detect such a protective effect. We suggestthat given that a low CD8 + cell antiviral efficacy issignificantly associated with a high proviral load and giventhat, in large population studies, a high viral load isassociated with HAM/TSP (Nagai et al., 1998) then it islikely that in a larger patient cohort we would observe anassociation between low CD8 + cell antiviral efficacy andHAM/TSP. Interestingly, we did find an unexpected relationshipbetween CD8 + cell antiviral efficacy and clinicalstatus in that there was a clear separation of the antiviralefficacy – proviral load curves between the HAM/TSPsubjects and the ACs (Fig. 4). So at any given antiviralefficacy, HAM/TSP patients had a significantly higher proviralload than ACs. Therefore, within our patient groupthe combination of proviral load and antiviral efficacytogether was a significantly better predictor of disease statusthan proviral load alone. This suggests that there is anextra factor in addition to CD8 + cell antiviral efficacy thatdetermines an individual’s proviral load and moreoverthat this factor differs systematically between HAM/TSPpatients and ACs.Several observations have led to the suggestion that theCD8 + lymphocyte response has negligible benefit in HTLV-I infection: proviral loads can be extremely high, suggestinga breakdown of immune control; there appears to belittle presentation of viral peptide by infected cells in vivo,and individuals with a high proviral load tend to have ahigh HTLV-I-specific CD8 + cell frequency (Bieganowskaet al., 1999; Kubota et al., 2000). However, these argumentshttp://vir.sgmjournals.org 7

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