We used intracellular cytokine staining to measure the response to optimized CTL epitopes in eight treatment-naive HIV-infected individuals. The ability to measure the response to multiple peptides at the same time allowed the use of a simplified screening protocol (1
). This allowed us to accurately screen CD8+
T-cell responses to 95 different epitopes with limited amounts of patient blood. This assay is similar in sensitivity to tetramer assays in both our hands and others' (3
). Mollet et al. (12
) and Murali-Krishna et al. (13
) have found similar results when comparing tetramer staining and intracellular cytokine staining using a slightly different intracellular staining method. In addition, intracellular IFN-γ staining measures a functional response to antigen that has an antiviral effect and is known to occur in activated CTL.
After complete viral suppression by HAART, we found that the median t1/2 of decay for HIV-specific CD8+ T-cell responses was 49.8 weeks in the five patients for whom results could be reported as number of responding cells per microliter. In the seven patients for whom t1/2s were calculated using data expressed as percent responding cells, the median t1/2 was 38.8 weeks (Table ). Most patients responded to more than one HIV-specific epitope, and the decay rates for different epitope responses in the same individual varied by a factor of 2.2 or less for each patient. The decay rate for the frequency of response to HIV-specific CD8+ T-cell epitopes was not related to the initial strength of the response and appeared to be more a function of the patient than of the antigen. Reexposure to measurable levels of virus resulted in a rapid increase in the frequency of HIV-specific CD8+ T cells in the one patient who discontinued therapy. The relatively slow decrease in the frequency of HIV-specific CD8+ T-cell response during therapy and the rapid increase during viral breakthrough suggest that the decrease in CD8+ T-cell response observed while patients are on HAART represents a normal response to withdrawal of antigen.
The difference between the median t1/2
that we report for percent responding cells, 38.8 weeks, and that reported by Ogg et al. (14
), 45 days, is most likely due to the length of time the patients were studied. Ogg et al. (14
) used data from the time of initiation of therapy to a median period of 100 days to calculate his t1/2
s. We used data from the time the patients achieved an undetectable viral load, usually within 112 days of starting therapy, to a median period of 78 weeks to calculate our t1/2
s. This suggests a rapid early decay of HIV-specific CD8+
T-cell response with initiation of HAART. Our data agree with this hypothesis. A rapid early decay is most apparent when our results are expressed as the absolute number of HIV-specific CD8+
T cells per microliter of blood (Fig. b). Using data from the first 8 weeks of therapy for patient 2, the calculated t1/2
for the response to peptide gp41 843–851 was 3.8 weeks, compared to 28.6 weeks for data obtained after an undetectable viral load was achieved. This is much closer to the t1/2
reported by Ogg et al. (14
). Unfortunately, our data do not include the number of early time points required to accurately measure an initial decay rate.
We have attempted to describe the rate of decline of HIV-specific CD8+ T lymphocytes in peripheral blood. Probably nothing measured in the blood reflects whole-body frequencies or number of responding cells. Data expressed as percentages of the total peripheral CD8+ T-cell count do not reflect changes in the total CD8+ cell count. Absolute numbers are subject to wide changes because of redistribution and may not reflect the true status of the immune system. We have reported our data as both responding cells per microliter of blood and percent responding cells whenever possible. Our data for all patients except one, patient 3, are similar when reported in either manner. In patient 3, an increase in the peripheral blood CD8+ pool during treatment resulted in an increased in the t1/2 for the response to peptide p24 311–319 from 28 weeks to >10 years when data were calculated as total number of responding cells per microliter. This illustrates the possible effect of changes in the peripheral CD8+ pool on kinetic analysis. Similarly, reporting CD8+ T-cell responses as percentage of responding cells may underestimate the rate of decay early in therapy when CD8+ numbers decrease.
Rinaldo et al. (17
) and Mollet et al. (12
) have also reported an increase in CD8+
T-cell responses to the CMV peptide epitope pp65 NLVPMVATV in patients on effective antiretroviral therapy. Clinical data showing immune reconstitution syndrome associated with CMV vitritis (6
) suggest that increased responsiveness to CMV starts soon after initiation of HAART in patients with CD4+
cell counts below 100 cells/μl. In our patients, the frequency of CMV-specific CD8+
T-cell responses increased with initiation of HAART even though there was no sign of CMV-specific end organ disease and CD4+
cell counts were 274 and 704 cells/μl, well above where immune reconstitution syndrome occurs.
These data show that in medically compliant patients who achieve an undetectable viral load, a measurable HIV-specific CD8+
T-cell response can persist for >2 years after the initiation of HAART. These data also indicate that CD8+
T-cell responses to different HIV epitopes all decay at approximately the same rate within an individual. This indicates that HIV-specific CD8+
T cells with different T-cell receptors recognizing different epitopes in different HIV proteins all respond similarly to the loss of viral antigen in vivo. The rate of decay appears to be constant throughout this study, without evidence of establishment of a new immunologic set point. Discontinuation of therapy results in a prompt increase in HIV-specific CD8+
T cells to levels similar to those seen before therapy. Our data do not allow us to address the reports of early fluctuation in CD8+
response seen by Ogg et al. (14
) in the first 2 weeks of therapy or the increased responsiveness seen by Mollet et al. (12
) in the first month of therapy. However, our data suggest that there is a lower rate of HIV-specific CD8+
T-cell decline after an undetectable viral load is achieved. Any estimate of the longevity of antigen-specific CD8+
T cells that relies heavily on data obtained prior to the clearance of viremia may underestimate the persistence of these cells because of a rapid initial decrease in HIV-specific CD8+
T cells with initiation of HAART. The original justification for structured treatment interruption in patients with chronic infection is based on the assumption that the CTL response to HIV drops rapidly with therapy. Our data demonstrate a slower decrease in HIV-specific CD8+
T-cell responses. This lower decay rate needs to be taken into consideration in the design of structured treatment interruption and therapeutic vaccination protocols.