The CD4+ T-lymphocyte levels at baseline tended to differ according to plasma HIV RNA load (P = 0.07). Indeed, the mean CD4+ T-lymphocyte level was 734 × 106 cells/l (95% CI, 583–884) for those with an HIV RNA load of > 3.3 log10 copies/ml, 689 × 106 cells/l (95% CI, 546–833) for those with an HIV RNA load between 1.7 and 3.3 log10 copies/ml and 882 × 106 cells/l (95% CI, 773–991) for those with an undetectable HIV RNA (< 1.7 log10 copies/ml). The CD4+ T-lymphocyte count decline within the first month after treatment interruption was significantly steeper for the patients with a lower HIV RNA (P < 10−2) achieved under treatment: −23 × 106 cells/l per month (95% CI, −139 to 93), −86 × 106 cells/l (95% CI, −201 to 29) and −238 × 106 cells/l (95% CI, −323 to −153), respectively. During the period after M1, the CD4+ T-lymphocyte slopes significantly decreased in each group (all P < 10−3) but tended to be steeper in patients with the highest HIV RNA load (P = 0.15): −25 × 106 cells/l per month (95% CI, −33 to −16), −16 × 106 cells/l (95% CI, −24 to −7), −14 × 106 cells/l (95% CI, −20 to −7), respectively (Fig. 2b). In the multivariable model, the effect of HIV RNA load on CD4+ T-lymphocyte kinetics did not remain statistically significant (Table 3).
Other baseline variables associated with the CD4+ T-lymphocyte level at baseline and during follow-up included the CD8+ T-lymphocyte percentage, the percentage of activated CD4+ DR+ T-lymphocytes, myeloid and lymphoid dendritic cell levels (Table 3). The higher the CD4+ DR+ T-lymphocyte counts and dendritic cells levels (both types) were, the higher was the initial CD4+ T-lymphocyte level (P = 0.03, 0.002 and 0.04, respectively) and the steeper was the CD4+ T-lymphocyte count decline at M1 (P = 0.04, 0.02 and 0.01, respectively). The higher the CD8+ T-lymphocyte percentage was, the lower was the initial CD4+ T-lymphocyte level (P = 0.002) and the blunter was the CD4+ T-lymphocyte count decline at M1 (P = 0.03). Assuming that mobilization and/or the effect of some cell subsets is correlated to the viral load, we looked at the possible association of these cell subsets with the CD4+ T-lymphocyte count taking into account the HIV RNA level. After adjustment for the baseline HIV RNA level only, the effects of the CD4+ DR+ T-lymphocyte count and myeloid dendritic cells remained unchanged (variation of estimations less than 20%). However, the strength of the effect of the CD8+ T-lymphocyte percentage and the effect of the lymphoid dendritic cells on CD4+ dynamics was decreased (Table 3). In the full multivariable model, the effects which remained significant were: the effect of myeloid dendritic cells on the CD4+ baseline (+19.5 × 106 cells/l per 103 cells/ml higher, P = 0.001) and the first slope (−10.7 × 106 cells/l per month, P = 0.03), the effect of CD4+ DR+ T-lymphocyte count on the first CD4+ slope (−28 × 106 cells/l per month per 1% CD4+ DR+ higher, P = 0.05).
Immunological response to treatment interruption was also explored through modifications in activated cell subsets and dendritic cells (Table 2). Whereas the levels of CD4+ DR+ T-lymphocytes and of myeloid dendritic cells tended to be stable during the first month, the percentages of CD8+ and activated CD8+ DR+ T-lymphocytes subsequently increased with a median slope of 2.8%/month (IQR, 0.20–5.8) and 4.9%/month (IQR, 1.21–11.1), respectively. In the meantime, we observed a median decline in the lymphoid dendritic cell level of −1156 × 106 cells/l per month (IQR, −3853 to 1815).
The CD4+ T-lymphocyte count drop observed in the first month following treatment interruption was negatively correlated with the increases in HIV RNA load (r = −0.64, P < 10−4), the CD8+ T-lymphocyte percentage (r = −0.53, P < 10−4), the activated CD8+ DR+ T-lymphocyte percentage (r = −0.56, P < 10−4) and positively correlated with the decline in lymphoid dendritic cells (r = 0.61, P < 10−4).
The decline in lymphoid dendritic cells was itself negatively correlated with the HIV RNA load, the CD8+ T-lymphocyte percentage and the activated CD8+ DR+ T-lymphocyte percentage increases (r = −0.53, r = −0.32, r = −0.47, all P values < 0.05) indicating a link between the number of lymphoid dendritic cells and the viral load which in itself would foster CD8+ T-lymphocytes activation and proliferation. The first month slopes of HIV RNA load, activated CD4+ DR+, CD8+, activated CD8+ DR+ T-lymphocyte percentages and lymphoid dendritic cells did not seem to influence the CD4+ T-lymphocyte count slope significantly after 1 month until month 12 (P = 0.31, 0.40, 0.87, 0.75, 0.97, respectively) which probably represents a situation where all variables are in equilibrium.
In this study of the modifications in markers after long-term supervised treatment interruption in chronically HIV-1 infected patients, the change in CD4+ cell count took place in two phases: a first steep decrease in the first month (−142 × 106 cells/l on average), followed by a smooth decline (−17 × 106 cells/l on average) as recently reported in another study . According to baseline CD4+ and HIV RNA levels, our estimations of short term [9,16,23] and long term [11,12,16] CD4+ changes are similar to other studies as is also the case for HIV RNA [8,15,16].
Interestingly, the second slope of CD4+ T-lymphocyte decline was not correlated with the first and no factor but baseline HIV RNA influenced this second slope. As a consequence, the CD4+ level at the end of the 12-month follow-up was mainly determined by the baseline level and CD4+ modification during the first month. This finding implies that the management of supervised treatment interruption in chronically infected patients should be re-evaluated at 1 month. Furthermore, the decline in CD4+ during the first period may be anticipated, as it was steeper in patients with higher baseline CD4+ and lower HIV RNA levels. This result is in agreement with all previously published studies [9,11,13] but one . Moreover, for a given CD4+ level at the time of treatment interruption, the decline was less pronounced in patients with a CD4+ nadir > 350 × 106 cells/l. This effect is much greater than the baseline HIV RNA effect. In fact, patients with lower baseline HIV RNA presented higher baseline CD4+ and steeper short-term decline leading to comparable CD4+ levels at 1 month (Fig. 2b). However, those with a CD4+ nadir < 350 × 106 cells/l presented similar baseline CD4+ but their short-term decline was greater (−134 × 106 cells/l per month lower). Therefore, when antiretroviral treatment interruption is under consideration, the decision should take into account the CD4+ nadir. Moreover, this result underlines that, in addition to poor CD4+ T-lymphocyte restoration under treatment [24,25], the CD4+ decline after treatment interruption is greater in patients having already experienced a more pronounced HIV-CD4+ cell depletion, although this finding is in discrepancy with the study published by Tebas et al. . The importance of the CD4+ nadir on CD4+ T-lymphocyte kinetics after treatment interruption in chronically infected patients has also been reported recently  and is likely to reflect the extend of T-lymphocyte repertoire contraction and the difficulty associated with restoring sufficient diversity after HAART initiation. Thus, this information should be integrated in the decision-making process on when to initiate antiretroviral treatment as reaching a value of less than 350 × 106 cells/l may compromise the immune response after subsequent treatment interruption.
One of the aims of our study was to analyse the dynamic and the links potentially existing between several immunological factors particularly in the first month after treatment interruption. As expected, a higher proportion of activated CD4+ T-lymphocytes at baseline was associated with a steeper decrease in the CD4+ T-lymphocyte count, the half-life of such cells being very short . The adjustment for plasma HIV RNA level modified slightly the effect of activated CD4+ T-lymphocyte count reflecting the fact that the activation process is not only mediated through antigenic stimulation . If the activated CD8 DR+ T-lymphocyte count at baseline did not significantly influence the change in CD4+ T-lymphocyte count, the dynamics of the CD8+ DR+ T-lymphocyte count was positively correlated with the viral RNA load increase and the CD4+ T-lymphocyte count decrease as has been reported in patients undergoing antiretroviral treatment . A higher percentage of the CD8+ T-lymphocyte count, as a surrogate marker for the cytotoxic T-lymphocyte antiviral response, was associated with a slower decline in CD4+ T-lymphocyte count and seems to be closely associated with the magnitude of antigenic stimulation measured by the plasma HIV RNA level as its effect on CD4+ T-lymphocyte was not independent. This result is in agreement with those reported in treated patients where the partial antigenic stimulation due to intermittent viremia or persistent low levels of HIV RNA leads to an effective HIV-specific T-cell response . This idea was first described in the context of acute infection . However, some authors have claimed that, although interrupting drug therapy can boost the HIV-specific CD8+ T-cell response, the immune response is limited because of a lack of cytotoxic T-cell diversity, leading to poor control of virus production [30,31]. In our study, we observed high levels of HIV viral load despite an increase in activated CD8+ T cells. The effects of the different dendritic precursor cells on CD4+ cell count modification were similar. However, this effect seems to be associated partly with HIV RNA load for lymphoid but not for myeloid dendritic cells. The dynamics of each differed: myeloid dendritic cell counts were stable whereas lymphoid dendritic cell counts tended to decrease in association with CD4+ T lymphocytes and in negative correlation with the HIV RNA load slope. In summary, although the influences of baseline myeloid and lymphoid dendritic cells on changes in the CD4+ T-lymphocyte count were comparable, the mechanism through which they are involved is more directly linked to the magnitude of the antigenic stimulation for the lymphoid subset than for myeloid cells. This is in agreement with a negative correlation between lymphoid and HIV RNA load reported elsewhere . Therefore, the decrease in the number of circulating lymphoid dendritic cells with disease progression, which is presumably associated with a decrease in IFN alpha production, should contribute to the lack of HIV virus control . Taken together, these data suggest that lymphoid DC are directly involved in the antiviral response probably through secretion of interferon-alpha and through the specific CD8 T-cell response , whereas myeloid DC seem to be more involved in the dissemination of the infection probably because of their unique DC-Sign expression.
In conclusion, treatment interruption in patients with a CD4+ T-lymphocyte count > 400 × 106 cells/l is followed by a substantial increase in plasma HIV RNA load and a decline in the CD4+ T-lymphocyte count within the first month. Thereafter, there is a nearly steady-state situation in those patients who do not resume their therapy during the 12-month follow-up. The nadir of the CD4+ T-lymphocyte count before treatment interruption exerted a major influence on the level of CD4+ T-lymphocyte count reached after 1 month and therefore on the probability of resuming therapy. This information should become one of the criteria taken into consideration when deciding on treatment interruption, at least in patients above 400 × 106 CD4+ T-lymphocytes/l and should stimulate further research to evaluate strategies allowing at further increase in the CD4+ cell count before interruption. The monitoring of DC cells should be considered on longer follow up to increase our understanding of the complex relationship between the virus and different DC subsets in vivo.
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