T Cell Dynamics and The Response to HAART in a Cohort of HIV-1 Infected Elite Suppressors

Abstract

Elite controllers or suppressors (ES) are untreated HIV-1-infected patients who maintain undetectable viral loads. In this study, we show that most ES do not experience significant changes in T-cell counts over a 10 year period. Interestingly, treatment of an ES with HAART led to a marked decline in immune activation.

Introduction

While CD4⁺ T-cell depletion is the hallmark of progressive HIV-1 infection, the mechanisms involved in this depletion are still not fully understood. Some studies have shown that the HIV-1 viral load correlates with the rate of decay of CD4⁺ T cells [1,2] while others have shown a strong correlation between level of immune activation and the degree of CD4⁺ T cell depletion [3,4], possibly as a result of a type I interferon-mediated pathway [5].

Elite suppressors (ES) represent a rare group of HIV⁺ patients who maintain viral loads of <50 copies/ml. While these patients generally maintain high CD4 cell counts, cases of progressive CD4⁺ T-cell decline in ES have been described [6-9]. How frequently this phenomenon occurs is not known, and the dynamics of other T-cell subsets have not been adequately studied in these patients. Since many ES are infected with replication-competent virus [10], they serve as a model of immune control of HIV-1 infection. It is therefore important to understand the rate of change of various T-cell subsets in ES, especially if therapeutic vaccination of HIV-1 infected patients becomes a feasible goal.

We here present an analysis of the long-term T-cell dynamics in ES who have been followed for 10 or more years. We also present the case of one ES patient who was treated with HAART for a declining CD4 cell count despite undetectable viremia. The results stand in contrast to previous studies of patients with progressive disease and advance our understanding of the factors involved in T-cell dynamics in HIV-1 infection.

Methods

Participants

The subjects were followed at the Johns Hopkins Medical clinics for at least 10 years after their first positive HIV test. Elite Suppressors are defined as untreated HIV-1 seropositive patients who maintain viral loads of < 50 copies/ml. ES15 was treated with zidovudine monotherapy during the last trimester of a pregnancy despite having a viral load of <50 copies/ml. The remainder of the patients were antiretroviral-therapy naïve with the exception of ES26, who was started on a regimen of efavirenz, tenofovir, and emtricitabine for a progressive decline in CD4 cell counts. ES3 was successfully treated with ribavarin and pegylated interferon alpha for Hepatitis C virus infection. Since this treatment is known to affect CD4⁺ T cell counts, we excluded all T cell counts for a period of 1 year after the completion of therapy.

Measurements

HIV-1 viral loads and T-cell counts were obtained in the clinics as part of routine HIV-1 care. After informed consent was obtained, a retrospective analysis of T-cell counts was performed.

For immune-activation studies, informed consent was obtained before phlebotomy. The protocol was approved by the Johns Hopkins University School of Medicine Institutional Review Board. Peripheral-blood mononuclear cells were stained as previously described [9] and a tetramer containing HLA-B*57 and the HIV gag peptide KAFSPEVIPMF (Beckman Coulter) was used to identify HIV-specific CD8+ T cells. A total of 200,000 to 500,000 events were analyzed for each sample.

Statistical analysis

The absolute CD3 and CD8 cell counts were obtained by determining the ratio of the percentage of these cells to that of CD4+ T cells and then multiplying this ratio and the absolute CD4 cell count.

Statistical analyses were carried out with the R software package. We used ordinary and mixed linear models to assess changes in T-cell counts, making use of the packages lme4 [Bates and Maechler, http://lme4.r-forge.r-project.org/], “multcomp” [11], and RLRsim [12]. Residuals were approximately normal. All reported correlations are Spearman rank correlations.

Results

Figure 1A shows the rate of change of CD4+ and CD8+ T cells for each ES. We also fitted a mixed-effect model which allowed for both an overall rate of decline in CD4 cell count as well as patient-specific random variations around this rate. We found that the overall rate of decline was not significantly different from zero (P=0.113) while the random variations were ( P=10^-4). Although the CD4 cell counts declined for ES2, ES3, and ES4 (Figure 1A, Table 1), there was no overall trend for CD4 cell counts to decline in the patient population as a whole.

Figure 1.

A. Time course of CD4 counts and CD8 counts for eight untreated ES patients. The lines of best fit for changes in CD4 counts and CD8 counts are included with best fit parameters in Table 1.

B. Time course of CD4 count in ES26 (top), and percentage of cells that co-expressed CD38 and HLA-DR in HIV-seronegative individuals, the cohort of ES, and ES26 before and after the initiation of HAART (bottom).

Table 1.

Clinical characteristics and rate of T cell changes in a cohort of ES. Significant rates of change of T cellsare shown in bold.

	Age when initial CD4 count obtained	Years Since first HIV+ positive test	Initial CD4 count	HLA- A	HLA- B	Rate of CD3 change cells/year (95% CI)	Rate of CD4 change cells/year (95% CI)	Rate of CD8 change cells/year (95% CI)
ES2	42	21	1002	02,31	51,57	-38.4 (-88.5, 11.6)	-53.2 (-71.7, -34.7)	7.3 (-41.7, 56.3)
ES3	44	18	1603	25,68	51,57	-56.8 (-98.9, -14.8)	-27.4 (-50.8, -8.9)	-28.1 (-48.9, -7.3)
ES4	40	13	1015	02,30	08,44	-77.7 (-131.1, -24.3)	-28.4 (-53.5, -3.3)	-45.4 (-106.2, 15.4)
ES5	41	19	1735	23,68	57,58	-9.4 (-32.2, 13.4)	-10.3 (-22.7, 2.0)	15.6 (-2.0, 33.2)
ES6	39	17	1056	23	15,57	-46.3 (-97.2, 4.7)	-25.5 (-56.4, 5.4)	-19.8 (-37.8, -1.7)
ES7	42	15	1015	30,32	57,81	11.5 (-32.2, 55.3)	3.9 (-21.8, 29.7)	7.0 (-12.8, 26.9)
ES13	53	16	782	03,23	15,35	12.6 (-2.6, 27.7)	6.2 (-3.0, 15.3)	22.7 (10.0, 35.3)
ES15	27	13	867	30,55	07,57	7.3 (-35.0, 49.7)	13.7 (-9.9, 37.3)	-23.5 (-58.5, 11.5)
Mean	41	16.5	1134
ES26	31	10	553	02,03	35/57	NA	-24.9 (-34.0, -15.9)	NA

NA: Not available

In patients with progressive disease, depletion of CD4⁺ T cells is usually accompanied by an increase in CD8 cell counts, so that the CD3 count remains roughly unchanged—a phenomenon previously referred to as blind T cell homeostasis [13]. We thus analyzed CD8⁺ T cells in our cohort of ES (Figure 1A). This subset of cells was not measured routinely after 2005, so less data is available for these cells. As in the case of CD4⁺ T cells, a mixed-effect model revealed no evidence for an overall trend of CD8 cell counts to either increase or decrease in the patient population as a whole (P=0.721), but found significant differences among patients (P=0.017).

Prior studies have shown a high degree of immune activation on CD8⁺ T cells of ES who have significant decline in CD4⁺ T cells [6, 8, 9]. We thus determined if there was a correlation between the degree of activation (measured as co-expression of HLA-DR and CD38) of CD4 and CD8⁺ T cells and the rate of decline of CD4⁺ T cells and CD8⁺ T cells. We found no relationship between the level of activation of CD4⁺ or CD8⁺ T cells and the rate of change of either T-cell subset over time. But the small number of patients limited the statistical power of this analysis.

ES26 is an HLA-B*5701⁺ female who experienced a progressive decline in CD4⁺ T cells over a 9 year period (Figure 1B). A single copy assay [14], performed prior to the initiation of HAART, revealed a viral load of 7 copies/ml. After 3 months of therapy, a repeat single copy assay gave a measurement of < 1 copy/ml. We compared the level of CD38 and HLA-DR co-expression on bulk CD4⁺ and CD8⁺ T cells as well as CD8⁺ T cells specific for an immundominant Gag peptide before and after the initiation of HAART. As shown in Figure 1B, the baseline level of immune activation on CD4⁺ and CD8⁺ T cells was much higher than that seen on cells from seronegative donors and the other ES in our cohort. After a year of treatment the percentage of CD8⁺ T cells specific for the immunodominant peptide decreased from 1.4% to 0.6%, and the level of immune activation declined markedly on all three cell types even though her CD4 cell count did not increase.

A defective nef gene was present in one prior report of an ES with a declining CD4⁺ T cell count [6]. We therefore analyzed this gene in ES26 and found no evidence of a deletion. We also analyzed the reverse transcriptase gene in this patient after a year of HAART to rule out the acquisition of drug resistance. No known drug resistance mutations to any of the drugs in her regimen were detected (data not shown).

Discussion

Our study shows that while the majority of ES do not experience significant changes in T-cell counts over a 10 year period, a gradual decline in CD4 cell counts is seen in some patients. Our mixed-model analysis of the entire cohort found no significant change in the CD4 cell count of the cohort as a whole. This is in marked contrast to the results from a recent study that looked at 41 patients who had been defined as long-term non-progressors [LTNPs] at baseline based on CD4 cell counts [15]. 11 years after they had initially met LTNP criteria, all 41 patients had developed progressive disease with declining CD4 cells counts and increasing levels of viremia that required the initiation of HAART. Thus the clinical course of most ES may be very different from that of LTNPs.

The only study looking at the response to HAART in an ES showed no significant difference in the level of immune activation on CD8⁺ T cells after 3 months of treatment [6]. In our study, we followed a patient for a year after HAART was initiated and while there was not a significant increase in her CD4 cell count, there was a marked decrease in the percentage of total CD8⁺ T cells and HIV-specific CD8⁺ T cells that co-expressed the HLA-DR and CD38 activation markers. This observation suggests that HAART can reduce the immune activation that has been observed in ES and may therefore have long-term benefit in ES with declining CD4 T-cell counts.

Ours is the first analysis of long-term T-cell dynamics in ES. It appears that while there is a slow rate of CD4⁺ T-cell depletion over long periods of time in some patients, the majority of ES do not experience a significant decline of CD4⁺ T cells after more than 10 years of infection. These results may have implications for the utility of a therapeutic vaccination for HIV-1 infection and suggest that if such a vaccine could reduce the viral load to <50 copies/ml, the majority of subjects would experience only very low rates of CD4 cell decline.