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Changes in CD4 Cells During HIV Treatment Interruption
  “Changes in CD4+ T-Cell Differentiation Phenotype During Structured Treatment Interruption in Patients With Chronic HIV-1 Infection”
JAIDS Journal of Acquired Immune Deficiency Syndromes 2003; 34(5):475-481
Thomas H. Alexander, MD, MS; Gabriel M. Ortiz, MD, PhD; Melissa F. Wellons, MD, MS; Andrew Allen, PhD; Edward J. Grace II, BS; Becky Schweighardt, PhD; Jason Brancato, BS; Johan K. Sandberg, PhD; Scott N. Furlan, BA; G. Diego Miralles, MD; Douglas F. Nixon, MD, PhD; John A. Bartlett, MD
From the Duke University Medical Center, Durham, NC (Dr Alexander, Dr Allen, Mr Brancato, Dr Bartlett); Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA (Dr Ortiz, Mr Grace, Dr Schweighardt, Dr Sandberg, Mr Furlan, Dr Nixon); University of Alabama School of Medicine, Birmingham, AL (Dr Wellons); and Trimeris Pharmaceuticals, Durham, NC (Dr Miralles).

Markers of maturation and activation were measured on peripheral CD4+ T cells in chronically HIV-1-infected patients in a randomized, controlled pilot study of structured treatment interruption (STI).
Eight subjects underwent 2 cycles of 1 month off and 1 month on highly active antiretroviral therapy (HAART), followed by a final 3-month interruption. During STI, CD4+ T-cell percentage remained relatively stable in 4 of 8 subjects. The remaining 4 STI subjects had significant rapid decline in CD4+ T-cell percentage during STI, followed by return to pre-STI baseline while on HAART. Changes in overall CD4+ T-cell percentage corresponded with fluctuations in the CD45RA+CCR7+ naive and CD45RA-CCR7+ central memory subsets. Subjects with variable CD4+ T-cell percentages tended to have higher pre-HAART plasma HIV-1 RNA setpoints and experienced higher levels of plasma HIV-1 RNA rebound during STI.
These results suggest that interruptions should be avoided whenever possible in patients on HAART with high plasma HIV-1 RNA setpoints.
Patients with access to highly active antiretroviral drug treatment (HAART) have dramatic improvements in the clinical course of HIV disease. Despite these benefits, recent studies indicate that HAART alone is unable to completely eliminate viral reservoirs, even after decades of treatment. Structured treatment interruptions (STIs) have been investigated as a means of improving viral control by augmenting HIV-1-specific cellular immune responses with the additional potential benefits of reduced drug exposure and cost.
Results have been encouraging for patients who undergo STIs following HAART initiated during the acute infection period. However, the benefit of STIs in the setting of chronic infection is less clear. Preliminary data indicate that only a minority of chronically infected individuals are able to maintain viral suppression without HAART after STIs, even though most have increases in antiviral cellular immune responses. Furthermore, rapid declines in peripheral CD4+ T-cell counts have been observed during STI. These CD4+ T-cell declines are inversely proportional to increases in plasma HIV-1 RNA during STIs and are generally followed by return to preinterruption CD4+ T-cell levels after reinitiating HAART.
Evidence suggests that the initial rapid increase in CD4+ T-cell levels after initiation of HAART is due to release of cells previously sequestered in lymphoid tissue. The HAART-mediated inhibition of viral replication is followed by reduced expression of lymph node homing and activation markers on T cells and reduced expression of adhesion molecules in lymphoid tissue. Less is known about the rapid CD4+ T-cell declines following interruption of HAART. Potential mechanisms include T-cell redistribution to peripheral compartments, increased T-cell clearance due to turnover or death, or decreased T-cell production.
To characterize changes in CD4+ T-cell populations following interruption of HAART, we performed detailed immunophenotypic analysis of maturation and activation markers on peripheral blood mononuclear cells (PBMCs) in a group of chronically HIV-1-infected patients participating in a randomized, controlled pilot study of STIs.
Subject selection and study design have been reported previously.10 Briefly, 12 patients on HAART with documented chronic HIV-1 infection were recruited from the Duke University Adult Infectious Diseases Clinic. All had CD4+ T-cell counts >400 cells/[mu]L for at least 6 months prior to entry and plasma HIV RNA levels <400 copies/mL for at least 3 months prior to entry. All patients provided written informed consent as approved by the Duke University Institutional Review Board. Cryopreserved PBMCs from 8 documented seronegative individuals were obtained from the Stanford Blood Bank for comparison.
Study Design
Patients were randomly assigned to undergo STI (STI group) or to continue their previous HAART regimen (on-HAART controls). Eight subjects in the STI group underwent 2 cycles of 1-month treatment interruptions (weeks 0-4 and 9-12) followed by 1 month on HAART (weeks 5-8 and 13-16). The second cycle was followed by a final 3-month interruption (weeks 17-28). The 4 subjects in the on-HAART control group remained on HAART for the entire 7-month study period. One subject in the on-HAART control group subsequently asked to undergo STI and was included in the STI group as a nonrandomized subject.
Peripheral blood samples were obtained weekly during STI and every other week while on HAART. PBMCs and plasma were isolated and cryopreserved for later analysis. The study protocol called for resumption of HAART if CD4+ T cells dropped below 200 cells/[mu]L at any time, plasma HIV RNA was >100,000 copies/mL during the final 3-month STI, or a patient withdrew consent.
Subject Characteristics
Subject demographics, HIV-1-specific cellular immune responses, and impact on control of plasma HIV RNA during STIs were reported previously. The median age of the 12 HIV-1-infected subjects was 47 years. The median time on HAART was 2.7 years (minimum 1.6) and plasma HIV RNA was suppressed to <400 copies/mL for a minimum of 1.1 years before enrollment (median 2.0).
Adverse Events
Three subjects randomized to the STI group did not complete the full 28-week protocol. One subject withdrew from the study after developing a rash with hives upon restarting nevirapine-containing HAART at week 6 and was excluded from the analysis. Another subject met the criterion for HAART resumption at week 22 based on plasma HIV-1 RNA level of 103,641 copies/mL after presenting with fatigue, sinus congestion, headache, and myalgias. A third subject elected to resume HAART at week 20 after presenting with respiratory symptoms and lymphadenopathy. Eight subjects (7 randomized and 1 nonrandomized) completed at least 20 weeks of STI.
CD4+ T-Cell Maturation Subsets in HIV-1-Infected Subjects on HAART Compared With Uninfected Controls
CD4+ T-cell maturation subsets were identified using CD45RA and CCR7 expression as described by Sallusto et al. There were no significant differences between on-HAART pre-STI baseline of the STI group and the on-HAART controls for any markers (data not shown). These baseline values were pooled for comparison to HIV-1-seronegative (HIVneg) controls. The HIV-1-seropositive subjects tended to have a higher percentage of CD45RA+CCR7+ naive cells and a significantly lower percentage of CD45RA-CCR7+ central memory cells. The CD45RA+CCR7- subset was significantly larger in the HIV-positive group.
2 Groups With Different Responses to STIs
Changes in CD4+ T-cell percentages followed 2 distinct patterns in the STI group. Four subjects had a significant decline in CD4+ T-cell percentage during STI (variable group), while the 4 remaining STI subjects had relatively stable CD4+ T-cell percentages throughout the study period (stable group). Significant CD4+ T-cell declines were defined as a mean decrease over the 3 interruptions of at least 5% from pre-STI baseline (i.e., 20% CD4+ T-cells at baseline) declining to 15%. Changes in the variable group occurred very rapidly during the 2nd and 3rd interruptions; the median change from pre-STI baseline CD4+ T-cell percentage in the variable group was -1.3 at week 8 (on HAART, just before second interruption) and -7.0 at week 9 (1 week into the second interruption). Percentages of both naive and central memory cells tended to decline during STI in the variable group, with recovery toward baseline while on HAART.
The variable group tended to have a greater increase in plasma HIV-1 RNA during STI. Median plasma HIV-1 RNA was 10 times higher in the variable group at week 20 (4.95 vs. 3.95 log10 copies/mL, P = 0.1143, Wilcoxon rank sum exact test). The increased viremia corresponded with increased CD4+ T-cell activation, as measured by CD38 expression. The median increase from baseline in percent CD4+CD38+ T cells at week 20 was 14.4% in the variable group and 7.6% in the stable group (P = 0.48, Wilcoxon rank sum exact test). The increased activation occurred primarily in CD45RA-CCR7+ central memory cells.
All subjects in the stable group maintained relatively stable CD4+ T-cell percentages through week 28 (median change from baseline -0.4% at week 28, range -0.5% to -0.1%). Two of the 4 subjects in the variable group did not complete the final STI. They restarted HAART before week 28 after presenting with symptoms as described earlier. Of the 2 subjects in the variable group completing the study, one returned to baseline CD4+ T-cell percentage by week 28 (-11.2% at week 20, +0.4% at week 28). The other also showed a trend toward recovery of CD4+ T-cell percentage (-8.0% at week 20, -3.3% at week 28).
Predictors of CD4+ T-Cell Changes During STI
Pre-HAART plasma HIV-1 RNA levels were available for 6 of 8 STI subjects. Pre-HAART plasma HIV-1 RNA correlated strongly with peak plasma HIV-1 RNA during STI at week 20 (R = 0.94, P = 0.0048, Spearman rank correlation). Subjects experiencing rapid CD4+ T-cell decline during STIs tended to have higher pre-HAART plasma HIV-1 RNA levels, although the difference was not significant (median 5.025 vs. 3.685 log copies/mL, P = 0.22, Wilcoxon rank sum exact test). Both groups had similar CD4+ T-cell nadirs before initiation of HAART (median 275 cells/[mu]L in the variable group vs. 318 cells/[mu]L in the stable group, P = 0.56, Wilcoxon rank sum exact test). Pre-HAART CD4+ T-cell nadir did not correlate strongly with week 20 CD4+ T-cell decline (R = 0.60, P = 0.1195, Spearman rank correlation). No significant phenotypic predictors of CD4+ T-cell decline were identified. Both groups had similar pre-STI baseline CD4+ T-cell percentages (mean of 26.1% in both groups) with similar distribution among maturation subsets (data not shown). Viral coreceptor usage did not differ among groups; each group had 3 subjects with virus using R5 and 1 subject with R5/X4 mixed co-receptor use.
To date, STI has not proven successful in patients with chronic HIV-1 disease due to a lack of control of plasma HIV-1 RNA rebound, rapid drops in CD4+ T-cell counts in some subjects, and the appearance of drug resistance mutations following multiple interruptions. The impact of these virologic and immunologic changes on disease progression is unknown. To gain an insight into the immunologic effects of STI, markers of maturation and activation were quantified on CD4+ T cells from chronically HIV-1-infected patients participating in a randomized pilot study of STIs.
The HIV-1-seropositive subjects were first compared with HIV-1-seronegative controls to gain a better understanding of the impact of HIV-1 infection on CD4+ T-cell maturation subsets in the setting of suppressive HAART. The maturation of CD4+ T cells appears to be skewed in HIV-positive subjects on HAART relative to uninfected controls. The most striking difference is the presence of a significant number of CD45RA+CCR7-cells in HIV-infected individuals. Sallusto et al found this subset to be absent among CD4+ T cells in healthy individuals. CD45RA+CCR7- cells have been shown in CD8+ T cells to represent a stage of terminal differentiation, but they have not been characterized in CD4+ T cells. A recent study in pediatric patients found that higher levels of CD4+CD45RA+CCR7- T cells are associated with dysfunctional HIV-1-specific CD8+ T-cell response (Sandberg J, personal communication). The differences observed in naive and central memory cells may also be related to factors other than HIV-1 infection; although exact demographics were not available for the HIV-1-seronegative controls, they are believed to represent an older population. Older individuals would be expected to have fewer naive CD4+ T cells due to decreased thymic output.
Two groups with distinct patterns of response to STI were identified, retrospectively. Subjects in the stable group maintained stable CD4+ T-cell percentage during STI, whereas subjects in the variable group had significant decline in CD4+ T-cell percentage during STI with return to baseline while on HAART. Absolute T-lymphocyte counts were not available for this study, and the decreases in CD4+ T-cell percentage may reflect a true decrease in the number of peripheral CD4+ T cells, an increase in the number of CD8+ T cells, or a combination of both. Both the naive and central memory subsets fluctuated dramatically in the variable group during STI. This finding contrasts with changes seen in the setting of initiation of HAART in previously untreated individuals, where the rapid increase in peripheral CD4+ T cells is primarily due to release of CD45RA- memory cells previously sequestered in lymphoid tissue. The reason for the possible decline in naive CD4+ T cells during STI is unclear. Increases in markers of activation during STI tended to be greater in the variable group, but the percentage of CD38+ cells in peripheral blood increased mainly in the CD45RA-CCR7+ central memory compartment. CD45RA+CCR7+ naive cells may be preferentially sequestered in lymph tissue in the presence of increased antigenic stimulation during STI, may be preferentially destroyed, or may convert to a CD45RA-CCR7+ phenotype.
A complex relationship exists between plasma HIV-1 RNA and CD4+ T-cell setpoints. Pre-HAART plasma HIV-1 RNA setpoint has been shown previously to be predictive of plasma HIV-1 RNA level during treatment interruption, and our data confirm this finding. A greater increase in plasma HIV-1 RNA during STI resulted in a greater decrease in CD4+ T-cell percentage. Therefore, pre-HAART plasma HIV-1 RNA setpoint may be a useful predictor of CD4+ T-cell response during interruption of therapy.
The pattern of CD4+ T-cell changes during STI contrasts with the stable total and naive CD4+ T-cell numbers seen in subjects with virologic relapse while on HAART. It is intriguing that viremia due to interruption of HAART has different effects on CD4+ T cells than viremia occurring while on HAART. These differences may be due to relatively lower plasma HIV RNA levels in patients with relapse on HAART, or attenuated viral fitness associated with resistance mutations resulting in less compromise of thymic production, cellular destruction, and redistribution.
Several important limitations should be considered when interpreting these results. First, the sample size is very small. The analysis was performed on samples obtained during a small pilot study of STIs and was intended to generate hypotheses regarding the effect of STI on CD4+ T cells. A second potential limitation is the use of cyropreserved PBMCs. Although only cyropreserved PBMCs were available for analysis in this study, a recent trial validated the use of frozen cells by demonstrating comparable results for naive, central memory, and effector memory T cells identified in cryopreserved PBMCs and fresh whole blood. Finally, the comparison of groups identified post hoc increases the risk of finding differences due to chance.
The duration of repeated interruptions may be important in determining the effect on CD4+ T cells. CD4+ T-cell declines occurred very rapidly during the last 2 interruptions in this study. Studies of shorter interruptions (i.e., 1 week off, 1 week on) have not found similar declines. When such rapid changes in CD4+ T-cell levels do occur, they may be accompanied by AIDS-defining clinical events.34, 35 Two of 4 subjects in this study with large declines in CD4+ T-cell percentage did not complete the 3rd interruption after presenting with clinical symptoms. Because of the potential for large declines in CD4+ T cells, interruptions should be avoided in patients on HAART with high plasma HIV-1 RNA setpoints.
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