Although multiple mechanisms have been proposed to account for the loss of CD4 T cells in HIV-infected patients, few have been validated to occur in vivo. Identification of the HIV protease-specific Caspase 8 cleavage product, Casp8p41, in samples from HIV-infected patients is evidence of the occurrence of this cleavage effect in vivo. Moreover, demonstrating that the Casp8p41 level correlates with CD4 T cell count, and that changes in Casp8p41 content are inversely correlated with CD4 T cell count change, argue for a pathogenic role of Casp8p41 in CD4 T cell depletion during HIV disease. This model does not exclude the importance of other mechanisms of CD4 T cell loss during HIV infection. Rather, these data confirm only that Casp8p41 is present in vivo, which ultimately may be a useful predictor of CD4 T cell loss. It should be noted that our longitudinal study was restricted to patients who initiated antiretroviral therapy; it is unknown whether Casp8p41 has similar predictive use in patients who are not receiving antiretroviral therapy.
Another potential limitation of our study is the low percentage of memory CD4 T cells that stained for Casp8p41. This low frequency is, in our experience, a function of sensitivity. First, the percentage of HIV-infected cells in peripheral blood lymphocyte has been previously determined to be from 0.3% to 2%–4%. Second, Casp8p41-expressing cells undergo apoptosis within minutes to hours after expression of the protein. In our staining protocol, we excluded dead cells. Therefore, the circulating HIV-infected cells expressing Casp8p41 that were available to be measured are the small fraction that are expressing the protein but have not yet died. Finally, the clinical samples used in this study were frozen, which in our experience decreases the measured number of Casp8p410-expressing cells, compared with freshly processed samples (data not shown).
Our analysis was performed by determining the proportion of cells containing Casp8p41, as opposed to the absolute number of Casp8p41-containing cells, because the latter approach would predictably be biased by the total CD4 T cell count. By using the proportion of cells containing Casp8p41 we have de facto measured the cells that (1) have previously been infected by HIV and (2) would be predicted to die because of Casp8p41-dependent mechanisms. In fact, our results support this prediction, because patients with a higher proportion of Casp8p41-containing cells subsequently have a greater rate of CD4 T cell loss. Our study was unable to determine the effect on Casp8p41 content of a protease inhibitor–based antiviral regimen compared with a regimen not based on a protease inhibitor because of the small number of patients in both cohorts receiving a regimen not based on a protease inhibitor. Early in the era of HIV therapy, viral load assays were shown to predict CD4 cell loss; however, baseline HIV RNA levels predict an individual person’s CD4 T cell loss only minimally [14
]. It will be of interest to formally compare the relative abilities of viral load and Casp8p41 level to predict within-person CD4 T cell loss in a larger cohort of patients.
It is well established that host factors affect the pace of HIV disease progression. These factors include the Δ32 mutation of CCR5, TRIM5α, CCL3LI gene duplication, PD-1, APOBEC 3G, and certain HLA types, including B*57 and B*27. Interindividual variations in Casp8p41 production, as shown by our study, might occur as a consequence of variable caspase 8 expression levels or polymorphisms within caspase 8. Alternately, altered HIV protease substrate recognition and/or cleavage, which often results from acquisition of drug resistance mutations in the protease gene, would predictably affect Casp8p41 production by HIV-infected cells. Determining whether failure of antiretroviral therapy resulting from acquired resistance to protease inhibitors is associated with relatively attenuated increases in Casp8p41 content and blunted CD4 T cell losses will be of great interest.