Overall, 20 of the 30 study subjects (67%) had CD8 DP T cells that were HIV provirus-positive before HAART. Although 13 (43%) had detectable infected CD8 SP T cells, proviral loads were exceptionally low in this subset, approaching the sensitivity of the assay [mean, 2 copies/106 cells; 95% confidence interval (CI), 1–3]. The mean CD4 T cell proviral load for the 30 subjects (4098 copies/106 cells; 95% CI, 1575–6620) was significantly higher than that of CD8 DP T cells (258 copies/106 cells; 95% CI, 71–446) (Z = −4.659, P < 0.001).
There was a significant negative correlation between both CD4 and CD8 DP T cell pre-HAART proviral loads and CD4 cell count in the 30 subjects (CD4 cells: rs = −0.406, P = 0.026; CD8 DP: rs = −0.467, P = 0.009; Fig. 2a,b). The significant relationship between CD8 DP proviral load and CD4 cell count was not affected by the removal of PCR-negative samples (n = 20; rs = −0.495; P = 0.026). CD4 T cell proviral load significantly correlated with plasma viral load (n = 29; rs = 0.384; P = 0.040) (Fig. 2c), whereas no such relationship was true for CD8 DP T cell proviral load with (n = 29; rs = 0.090; P = 0.642) and without (n = 20; rs = −0.008; P = 0.974) the inclusion of PCR-negative samples (Fig. 2d).
HIV-positive CD4 T cells were detected during HAART in all 12 study subjects tested (Fig. 3a). Samples from three subjects yielded insufficient cells to estimate CD8 DP T cell proviral loads precisely during HAART and were excluded from the analysis (Table 1). In the remaining nine subjects, four had CD8 DP T cells in which HIV-1 proviral DNA was detectable (Fig. 3b). Although the lower limit of detection for the other five were low (< 3, < 11, < 15, < 22 and < 43 copies/106 cells), this does not preclude a level of infection below assay sensitivity. Where indicated, values halfway between the negative cut-off and zero have, therefore, been used for these PCR-negative CD8 DP T cell samples. The ratio of CD4 T cell proviral load to CD8 DP T cell proviral load pre-HAART and the corresponding ratio during HAART were strongly correlated (n = 9; rs = 0.891; P = 0.001), whereas there was no significant correlation between paired CD4 and CD8 DP T cell proviral loads pre-HAART (n = 30; rs = 0.251; P = 0.181) and during HAART (n = 9; rs = 0.033; P = 0.932). Only one study subject (WT02) had CD8 SP T cells that were HIV positive during HAART (7 copies/106 cells) although CD8 DP T cells from this sample had a considerably higher proviral load (182 copies/106 cells).
The median percentage reduction in proviral load was similar between CD4 T cells (64%; n = 12; 95% CI, 56–82) and CD8 DP T cells (82%; n = 9; 95% CI, 68–90). Moreover, there were no significant differences between the values for all eight paired samples (Z = −1.362; P = 0.173) and for just the four paired samples where CD8 DP T cell proviral load was positive during HAART (Z = −0.730; P = 0.465). Both T cell subsets showed a significant reduction in mean proviral load following therapy (CD4 T cells: n = 12; Z = −3.621; P < 0.001; CD8 DP T cells: n = 9; Z = −2.666; P = 0.008), irrespective of whether the detection limit or half its value was used as the proviral load for PCR-negative CD8 DP T cells (statistics identical).
The mean rate of HIV-1 DNA decay per month was very similar for both T cell subsets (CD4 cells: 0.071 log10 copies/106 cells; 95% CI, 0.035–0.107; CD8 DP: 0.077 log10 copies/106 cells; 95% CI, 0.047–0.108; Fig. 4). Although the true value for CD8 DP T cells may differ slightly from this value owing to the use of lower detection limits in its estimation, the mean rate of decay of HIV-1 proviral load for the four CD8 DP T cell PCR-positive samples (0.078 log10 copies/106 cells; 95% CI, 0.011–0.144) was almost identical to that estimated including the ‘negative’ samples. Individual values of CD8 DP proviral decay showed a significant negative correlation (albeit marginal) with the time to complete plasma virus suppression (< 50 copies/ml) (n = 9; rs = −0.667; P = 0.050) whereas no such relationship was true for CD4 T cell decay (n = 12; rs = −0.168; P = 0.602).
Before the onset of HAART, CD8 DP cells represented an average of 2.18% of CD8 lymphocytes (n = 29; 95% CI, 1.75–2.60). After HAART, this value had fallen significantly (n = 11; Z = −2.934; P = 0.003) to 0.64% (n = 12; 95% CI, 0.33–0.94) (Fig. 3c).
CD4 and CD8 DP T cells showed significant overall reduction in the numbers of large cells following HAART (CD8 DP cells: n = 8; Z = −2.240; P = 0.025; CD4 cells: n = 9; Z = −2.192; P = 0.028; Fig. 5). No significant correlation was found between the percentage reduction in HIV-1 proviral load and the reduction in the percentage of large cells of the same subset (CD4: n = 9; rs = −0.317; P = 0.406; CD8 DP: n = 6; rs = −0.314; P = 0.554).
Heterogeneity in both plasma viral load and the number of productively infected cells prior to the start of HAART influences the kinetics of HIV-1 DNA decay. Moreover, decay kinetics are biphasic, with a variable time period for first-phase clearance [33,34], and combined analyses of decay rates may show no significant trend in the temporal reduction of HIV-1 DNA during therapy [35,20]. All study subjects here, however, showed reductions in proviral load for both cell types, and mean proviral loads were significantly reduced after the 6–13 months on therapy. Values derived for individual study subjects likely represent an average rate covering both the initial rapid decay during the first few months of therapy and the slower second phase . Importantly, CD8 T cell subsets were highly pure, with negligible CD4 T cell contamination (Table 3). In addition, only 7 of 17 study subjects tested had HIV-1-positive CD14+CD3− monocytes (with very low proviral loads, average 5 copies/106 cells; results not shown) ensuring that CD8 T cell proviral loads are not obscured by contamination with other infected cell types. Our finding of a significant reduction in large CD4 and CD8 DP T cells during HAART most probably represents clearance of activated, acutely infected cells , as the increased size of T cells has been shown to be a sensitive corollary of activation .
Although the kinetics of HIV-1 clearance from CD4 T cells by HAART has been studied [28,36,37], this is the first study of the rate of clearance of HIV-1-infected CD8 T cells. HAART has previously been shown to reduce numbers of activated CD8 T cells [38,39], an observation confirmed here by the significant reduction in numbers of CD8 DP T cells (as well as CD4 T cells) in peripheral blood following HAART. The action of HAART on HIV-1 replication appears to be similar for both CD4 T cells and CD8 DP T cells. HIV-1-infected CD8 DP T cells were cleared at a similar rate to that of infected CD4 T cells, while the relative level of infection of CD8 DP T cells to that of CD4 T cells remained unaltered. Despite this similarity, it is interesting to note that the time taken to suppress viral replication below detectable levels (< 50 copies/ml in peripheral blood) correlated significantly with the rate of decay of HIV-1-infected CD8 DP T cells, but not with the decay of infected CD4 T cells. This indicates that it is the removal of HIV-1 infection from the CD8 T cell compartment which is crucial to the speed of successful suppression of viral replication. The small sample size of this study, coupled with the similar structure of most HAART regimens, prevents any further analysis based upon specific drugs/regimens.
HAART undoubtedly reduces the activation status of the immune system, observed here by a decrease in the numbers of circulating CD8 DP T cells. Moreover, it appears that HAART leads to a preferential clearance of HIV-1-infected large, activated T cells (both CD4 and CD8). Both of these mechanisms contribute to a reduction in the CD8-specific HIV-1 reservoir. Nonetheless, prior to HAART, activated CD8 T cells may enter the resting pool . Although the majority of effector T cells undergo apoptosis, a minority (∼10%) persist to form memory cells, many of which can be maintained for long periods of time . This situation provides a means for the establishment of a CD8-based HIV-1 reservoir that is not readily cleared by therapy. The lack of complete clearance of HIV-1-infected CD8 DP T cells found here provides further support for a pathway that creates an ideal situation for HIV-1 to establish a CD8 T cell reservoir analogous to the well-characterized reservoir in CD4 memory T cells. CD8 T cells should, therefore, be considered a potentially significant long-term reservoir for HIV-1, particularly given their significant contribution to the replication population of HIV-1 in untreated individuals .
The authors would like to thank the staff of the Regional Infectious Diseases Unit, Western General Hospital, Edinburgh for their invaluable help in collection of blood samples and provision of clinical data. We also thank Alison Hardie for assistance with the processing of samples, Shonna Johnston for FACS sorting of cells and Fraser Lewis for statistical advice.
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