Elite Controllers Display Higher Activation on Central Memory CD8 T Cells Than HIV Patients Successfully on HAART

Abstract

T cell activation plays an important role in driving CD4 depletion during the course of HIV infection. There is scarce information about activation of different T cell subsets in HIV⁺ individuals experiencing distinct disease progression. The activation of different CD4⁺ and CD8⁺ T cell subsets and its contribution to total T cell activation were examined measuring CD38 expression by flow cytometry in 120 HIV-infected individuals and 9 uninfected healthy controls. HIV-infected patients were divided into four groups: 11 elite controllers (EC), 14 viremic controllers (VC), 61 antiretroviral-naive typical progressors (TP), and 34 progressors with viral suppression (VS) under antiretroviral therapy. EC displayed significantly greater activation levels than VS, with a higher contribution of central memory subsets to the activation of total CD8 T cells (p = 0.002). The activation of central memory CD8⁺ T cells significantly correlated with viral load in TP regardless of CD4 counts. In contrast with VS, proviral load was undetectable in all EC. Compared to VS, EC display abnormal and higher activation levels of different CD8⁺ T cell subsets. Factors other than the size of the viral reservoir should explain the high level of activation of central memory CD8⁺ T cells characteristically seen in HIV⁺ individuals with spontaneous control of viral replication.

Introduction

L ong-term nonprogressors (LTNPs) represent approximately 5% of all chronically HIV-infected individuals. These individuals characteristically display high and stable CD4 counts along with low or undetectable plasma viremia for longer than 10 years in the absence of antiretroviral therapy.¹ To date, a subset of these subjects with undetectable plasma viremia using commercial assays (<50 HIV-RNA copies/ml) is known as elite controllers (EC) and may behave differently from viremic controllers (VC), who are those with detectable but low-level plasma HIV-RNA (always below 5000 copies/ml).^2,3

Research on LTNPs has attracted much attention given that these individuals provide a unique opportunity for identifying potential factors associated with spontaneous control of HIV replication. With very low or undetectable viral replication, there is very slow disease progression or a complete lack of disease progression in LTNPs. Multiple factors both from the host and the virus seem to result in the favorable outcome of LTNPs.^1,3

–6 Among host variables, HIV-specific T cell responses have received particular attention. In a pivotal study that simultaneously examined five functions of HIV-specific T cells, a polyfunctional response with high quality was demonstrated in LTNPs, which was not seen in subjects experiencing HIV disease progression.⁷ However, the HIV-specific T cell response does not fully explain the viral suppression observed in these patients.^8,9 Thus, factors underlying CD4 depletion in the face of low or undetectable HIV replication are yet to be defined.

Activation of the immune system has been a major focus of attention.^10
–12 It is now well established that generalized immune activation is a hallmark of HIV infection^13

–16 and that T cell activation is one of the main mechanisms driving CD4⁺ T cell depletion.^17,18 In the simian model of AIDS, T cell activation differentiates between pathogenic and nonpathogenic infection.^19
–21 T cell activation can be characterized by examining the surface expression of different proteins, among which CD38 has been one of the most extensively analyzed.²² CD38 expression on T cells is upregulated early during HIV infection and is a good surrogate marker of disease progression and CD4 depletion.^12,23 Interestingly, CD38 expression continues to steadily decline over time in subjects with undetectable viremia under antiretroviral therapy, suggesting a role for CD38 as a marker of residual HIV replication,²⁴ and is associated with the IL-7–CD127 system.²⁵

The comparison of T cell activation in EC and patients with undetectable viral load under antiretroviral therapy has shown that activation levels are significantly increased in the former group.²⁶ However, no studies so far have examined activation of different T cell populations, and especially of the T central memory (TCM) subset. Herein, we have assessed quantitative and qualitative activation features in different T cell subsets from a group of HIV⁺ subjects with spontaneous control of HIV replication (EC), taking as reference groups HIV⁺ individuals with undetectable viremia under antiretroviral therapy (viral suppression, VS), viremic controllers (VC), and antiretroviral-naive typical progressors (TP).

Materials and Methods

Study population

A total of 120 individuals with chronic HIV infection on regular follow-up at our institution were selected for this study. Twenty-five patients met the criteria for LTNPs, based on the demonstration of serologically proven HIV-1 infection lasting for over 10 years, CD4 counts always above 500 cells/μl, and lack of HIV-related symptoms in the absence of any antiretroviral therapy. All these individuals belonged to a well-characterized cohort of LTNPs, whose main demographic features have been reported elsewhere.⁵ Based on viral load values, these individuals were divided into two categories. First, 11 EC, who had always maintained plasma HIV-RNA <50 copies/ml. The second group was represented by 14 VC, who displayed persistent viremia but always below 2000 HIV-RNA copies/ml. The remaining patient population was comprised of 61 antiretroviral-naive TP and 34 progressors with VS under antiretroviral therapy. A group of nine uninfected healthy subjects was taken as control. Written informed consent for the examinations conducted in the study was obtained from all individuals, and the study protocol was evaluated and approved by the hospital Ethics Committee.

Clinical specimens

All studies were done in cryopreserved peripheral blood mononuclear cells (PBMCs). EDTA-anticoagulated blood was obtained by venipuncture. PBMCs were immediately isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) and frozen in fetal calf serum (FCS) plus 10% dimethyl sulfoxide (DMSO). Viability of thawed PBMCs was always greater than 85%. Some samples from patients were kindly provided by the HIV BioBank integrated in the Spanish AIDS Research Network (RIS) and by the spanish network of spontaneous controllers.

Flow cytometry

CD45RA and CD27 were used to define the differentiation status of CD4 and CD8 T cells. Accordingly, four subsets were defined: naive (TN: CD45RA⁺ CD27⁺), central memory (TCM: CD45RA⁻CD27⁺), effector memory (TEM: CD45RA⁻CD27⁻), and totally differentiated effector (TEMRA: CD45RA⁺ CD27⁻).

CD38 expression in these different T cell subsets was measured by using a five-color, modified version of a commercial quantitative flow cytometry assay (Cellquant CD38; Biocytex).²⁴ For this assay, in combination with CD38, the next panel of monoclonal antibodies was used in the flow cytometry analysis: CD45RA-ECD, CD27-PECy5, CD4-PECy7, and CD8-PE (Beckman Coulter). A minimum of 5000 CD4 or CD8^bright cells per sample was examined using an FC 500 cytometer (Beckman Coulter). CD38 expression was expressed as CD38 molecules/cell. Fig. 1 shows representative dot plots.

FIG. 1.

(A) Quantification standard contains four subpopulations of beads with known numbers of anti-CD38 antibody binding sites. (B) Quantitative expression of CD38 on different subsets of CD8⁺ T cells in EC.

HIV proviral load

For quantification of proviral HIV-DNA, aliquots of 10⁶ PBMCs were obtained at each patient's visit and cell pellets were stored frozen at −80°C. A modified real time polymerase chain reaction (PCR) method was then used.²⁷ The HIV proviral load was expressed as the number of HIV-DNA copies per μg of total cellular DNA, with a limit of detection of five copies/μg of DNA.

Plasma HIV-RNA

Viral load was measured using the branched DNA (bDNA) assay (Quantiplex v3.0, Bayer, Barcelona, Spain), which has a lower detection limit of 50 HIV-RNA copies/ml.

Statistical methods

All statistical analyses were performed using the SPSS version 15 software. The activation level of each CD4 and CD8 T cell subset as well as its contribution to the total activation of CD4 and CD8 T cells (expressed as the proportion of global activation due to that particular subset) were calculated. CD4 counts, viral load, and length of HIV infection are expressed as mean ± standard deviation. Mann–Whitney U or Kruskall–Wallis tests were used to compare the levels and contribution of activation for each CD4 and CD8 T cell subset in the different groups, and the Wilcoxon signed-rank test was used to compare these parameters within the same group of patients. The potential associations between the activation of different subsets of CD4 and CD8 T cells, plasma HIV-RNA, and CD4 counts were examined using Spearman or Pearson correlation coefficients, as appropriate.

Results

Study population

The main immunological and virological characteristics of the study population are recorded in Table 1.

Table 1.

Main Immunological and Virological Characteristics of the Study Population

	No.	CD4 counts (cells/μl)	Plasma HIV-RNA (log copies/ml)	Length of infection (years)	HCV coinfection (%)
EC	11	899 ± 78	1.69	11 ± 1	100
VC	14	831 ± 89	3.02^a,b ± 0.19	11 ± 1	83
VS	34	528^a,c ± 58	1.69	6^c ± 1	50
TP	61	388^a,c ± 44	4.54^a,b ± 0.08	5^a,c ± 1	38

p < 0.05 when compared to EC.

p < 0.05 when compared to VS.

p < 0.05 when compared to VC.

Similar differentiation stages of CD4 and CD8 T cells in both elite controller and patients successfully treated with HAART

CD45RA and CD27 expression allowed characterization of the differentiation stage of CD4⁺ and CD8⁺ T cells in the different groups of patients and uninfected controls. No significant differences were recognized in CD4⁺ T cells (Fig. 2A). The majority of CD4⁺ T cells were naive or TCM. By contrast, the majority of CD8⁺ T cells were naive or TEMRA followed by TEM and finally by TCM (Fig. 2B). The differentiation stage of CD4⁺ T cells was similar in HIV⁺ patients and uninfected controls. In contrast, naive CD8⁺ T cells were lower and TEMRA CD8⁺ T cells were higher in HIV⁺ patients than in controls. Interestingly, EC and VC displayed higher levels of naive CD8⁺ T cells than TP (p = 0.01 and p = 0.001, respectively), whereas no differences were found between VS and TP (Fig. 2B).

FIG. 2.

Differentiation stage of CD4⁺ T cells (A) and CD8⁺ T cells (B) in elite controllers (n = 11) , viremic controllers (n = 14) , patients successfully treated with HAART (n = 34) , typical progressors (n = 61) , and uninfected subject (n = 9) . Data are represented as median and IRQ. *p < 0.05 when different groups of HIV-infected patients are compared to uninfected controls. (p < 0.05): for the rest of the comparisons.

Similar activation levels of different subsets of CD4 T cells in both EC and patients successfully treated with HAART

Activation level (using CD38 as a surrogate marker) was quantitatively analyzed in total, TN, TCM, TEM, and TEMRA CD4 T cells and compared between the different study groups (Fig. 3A).

FIG. 3.

Activation levels on total and on different subsets of CD4⁺ T cells (A) and CD8⁺ T cells (B) in elite controllers (n = 11) , viremic controllers (n = 14) , patients successfully treated with HAART (n = 34) , typical progressors (n = 61) , and uninfected subjects (n = 9) . Data are represented as median and IRQ. *p < 0.05 when different groups of HIV-infected patients are compared to uninfected controls. (p < 0.05): for the rest of the comparisons.

TP and VC showed significantly increased levels of activation in total, TCM, TEM, and TEMRA CD4⁺ T cells in comparison with uninfected controls. There were no significant differences between TP and VC. EC showed normal activation levels of all different CD4⁺ T cell subsets, whereas VS showed higher activation levels in TEMRA than uninfected controls (p = 0.012). Interestingly, EC and VS showed similar levels of activation in all CD4⁺ T cell subsets (Fig. 3A). EC displayed lower CD4⁺ T cell activation levels than VC and TP (p = 0.025 and p = 0.009, respectively), which was mainly due to a lower activation of the TCM subset. A similar profile was noticed when comparing VS and TP. Finally, there was no influence of HCV status, HCV replication, viral load, or CD4 counts on activation in TP.

EC presented higher levels of activation in different subsets of CD8 T cells than patients treated with HAART

In contrast to CD4⁺ T cells, activation was increased in all CD8⁺ T cell subsets of HIV⁺ patients compared to uninfected controls. Only VS displayed normal activation levels (Fig. 3B). Although activation in EC was not completely normalized, this group displayed significantly lower levels in all T cell subsets (but naive cells) when compared to TP (p = 0.0001 for TCM and TEM; p = 0.04 for TEMRA). Interestingly, EC showed higher levels of activation in total CD8⁺ T cells than VS (p = 0.031). This was mainly mediated by an increased activation of TN and TCM subsets (p = 0.04 and p = 0.002, respectively) regardless of HCV status. Furthermore, no significant differences were observed in levels of activation on different CD8⁺ T cell subsets when EC with detectable and EC with undetectable HCV replication were compared (data not shown)

Finally, in TP, potential associations between levels of activation in all subsets of CD8 T cells and viral load as well as CD4 counts were analyzed. Interestingly, the activation level in TCM CD8 T cells was significantly associated with viral load regardless of CD4 counts, HCV status, and length of HIV infection (r = 0.55, p = 0.001; Fig. 4A).

FIG. 4.

Significant associations between viral load and activation level in TCM CD8⁺ T cells (A) or contribution of TCM CD8⁺ T cells to the activation in total CD8⁺ T cells (B) in typical progressors. Association between proviral DNA and activation level in TCM CD8⁺ T cells (C) in patients successfully treated with HAART.

Contribution of different subsets to activation of total CD4⁺ and CD8⁺ T cells

Another approach we used when analyzing activation of CD4 and CD8 T cells was to calculate the contribution (expressed as proportion) of each subset to the activation level observed in the global population of CD4 or CD8 T cells (Fig. 5).

FIG. 5.

Contribution of different subsets to the activation of total CD4⁺ T cells (A) and CD8⁺ T cells (B) in elite controllers (n = 11) , viremic controllers (n = 14) , patients successfully treated with HAART (n = 34) , typical progressors (n = 61) , and uninfected subjects (n = 9) . Data are represented as median and IRQ. *p < 0.05 when different groups of HIV-infected patients are compared to uninfected controls. (p < 0.05): for the rest of the comparisons.

The order in the level of contribution of distinct T cell subsets (TN, TCM, TEM, and TEMRA) to the total CD4⁺ T cell activation was quite similar in all study groups, with TN and TCM contributing more than TEM and TEMRA (Fig. 5A). When the contribution of each subset was compared between groups, TP showed a higher contribution of TEM (p = 0.001) and TEMRA (p = 0.001) and a lower contribution of TN (p = 0.001) cells as compared to uninfected controls. Likewise, EC showed a higher contribution of TEM (p = 0.001) and TEMRA (p = 0.008) than uninfected controls, whereas in VS only the contribution of TEMRA was significantly increased (p = 0.03). No significant differences were observed in the contribution of distinct T cell subsets between EC, VC, and VS.

Activation of CD8⁺ T cells in uninfected controls was mostly mediated by TN cells with a much lower contribution for the rest of the subsets, whereas in HIV⁺ patients the contribution of the different subsets was more equally distributed, especially in TP (Fig. 4B). In this group, the contribution of the TEMRA, TEM, and TCM subsets was increased whereas the contribution of TN was decreased with respect to uninfected controls (p < 0.001 for all comparisons). Similar alterations were observed in EC, VC, and VS, with only slight differences between them. These abnormalities were more pronounced in TP and less recognizable in EC (Fig. 5B), except for TCM subset for which the contribution was significantly higher in EC compared to VS (p = 0.024).

In TP, the potential associations between plasma viremia and CD4 count, with the contributions of different subsets to the activation level of total CD4⁺ and CD8⁺ T cells, were analyzed. The only significant association found was between the contribution of TCM to the activation level of total CD8⁺ T cells with plasma viral load independently of CD4 counts, HCV status, and length of HIV infection (r = 0.39, p = 0.025; Fig. 4B).

EC present lower proviral DNA load than patients successfully treated with HAART

Since activation levels differed in some CD8⁺ T cell subsets when comparing EC and VS, and there was a close correlation between viral load and activation in untreated HIV⁺ patients, differences in proviral HIV-DNA were expected between EC and VS. Interestingly, proviral load was undetectable in all EC using our assay (lower limit of detection <5 DNA proviral copies/μg). In contrast, all 29 except 2 tested VS showed detectable proviral DNA [median value, 17 (132) copies/μg]. Moreover, as shown in Fig. 4C, a direct correlation between proviral HIV-DNA amount and activation of TCM CD8⁺ T cells was found in VS, regardless of CD4 counts, HCV status, and length of HIV infection (r = 0.59, p = 0.001).

Discussion

Alterations in the differentiation stage and increased activation of T cells are a hallmark of HIV infection,^13

–16 and most studies have linked these alterations to HIV replication and to the degree of immunosuppression as indicated by CD4 counts.^16,28 However, very few studies have tested the existence of these perturbations in HIV patients who have spontaneously maintained high levels of CD4 counts and undetectable levels of viral replication along the course of infection. To test this we analyzed levels and activation status of several T cell subsets in a group of EC. We also analyzed a group of HAART-induced virally suppressed patients, to determine if T cell perturbations were similar irrespectively of the mechanism involved in the suppression of viral replication (spontaneous or HAART induced). Moreover, untreated patients with high levels of viral replication, VC with low viral replication, and HIV-seronegative subjects were included as reference groups.

Different phenotypic markers have been used in distinct studies for analyzing the differentiation stage of T cells and at present, there is no consensus on what markers are best associated with the different differentiation stages.²⁹ We have analyzed the differentiation stage of CD4⁺ and CD8⁺ T cells based on CD45RA and CD27 expression, phenotypic markers that have been validated to define different T cells subsets in distinct differentiation steps in seminal studies.^30,31 In this way, T cells can be classified into four different subsets: CD45RA⁺ CD27⁺(naive), CD45RA⁻CD27⁺(memory), CD45RA⁻CD27⁻ (effector memory), and CD45RA⁺ CD27⁻ (totally differentiated effector) T cells.

In our study, the differentiation stage of CD4⁺ T cells was normal and similar in all groups of HIV-infected patients, which is not surprising given that an imbalance of CD4 T cell subsets is associated with the degree of immunosupression¹⁶ and that all patients groups analyzed presented relatively high CD4 counts. In contrast, alterations in the differentiation stage of CD8⁺ T cells were observed in all patients groups. Levels of TN CD8⁺ T cells were decreased and levels of TEMRA CD8⁺ T cells were increased in all groups compared to uninfected controls, meaning that even in the setting of high CD4 counts and undetectable viral replication the perturbations of CD8⁺ T cell subsets persist, supporting the notion that CD8⁺ T cell subset alterations are a highly sensitive marker of HIV infection. Interestingly, EC and VC presented higher levels of naive CD8⁺ cells than VS, suggesting a more preserved CD8⁺ T cell compartment in patients with partial or complete control of HIV replication. The fact that EC and VC have always maintained low viral replication and high CD4 counts, while VS were exposed to high virus replication and variable degrees of immunosuppression before therapy, may explain this result. Furthermore, this result is compatible with the hypothesis of a higher level of ongoing viral replication in VS than in EC. In agreement with this, higher levels of proviral HIV-DNA were observed in VS than in EC patients.

One of the most interesting finding of our study pertains to the activation of T cell subsets in patients spontaneously controlling viral replication. Regarding CD4⁺ T cells, activation was significantly increased in all subsets (except naive cells in which CD38 is constitutively expressed) of TP and VC. Of note, the level of increase was similar in TP and VC despite the much higher levels of plasma viremia in TP than in VC, suggesting that other factors independent of viral replication must be involved in driving CD4 activation.³² In contrast to TP and VC, EC patients presented normal levels of activation in all subsets of CD4⁺ T cells and VS showed an increased activation only in the TEMRA subset.

Increases in CD8⁺ T cells activation were more pronounced in all groups of patients and for all subsets. As for CD4⁺ T cells, TP and VC presented the highest increases followed by EC and VS patients. In contrast to CD4⁺ cells, however, activation of TCM subset was significantly higher in TP than in VC, suggesting a direct relationship to viral replication, which was confirmed by the significant correlation we found between the activation level of this subset and plasma viremia in the TP group, which is in agreement with several previous studies.^25,27 VS patients presented the lowest levels of activation with values very similar to uninfected controls, except for TEM cells that still presented significant increases. Interestingly, EC patients presented higher levels of activation than VS, especially in the TCM subset.

Although this issue has previously been described,^26,33 ours is the first study showing differences in activation of CD8⁺ T cell subsets between patients spontaneously controlling viral replication and HAART-treated patients. Although a recent study did find similar levels of activation when comparing EC and patients suppressed with HAART,³⁴ it must be noted that in the mentioned study activation was measured specifically in HIV-specific CD4 and CD8 T cells whereas we measured activation on total CD4 and CD8 T cells. From this observation we can conclude that the higher activation we observed in EC is not mediated by a higher activation of HIV-specific T cells. Some EC present progressive CD4⁺ T cell loss despite having undetectable viral load and this has been associated with high levels of activation on total CD8⁺ T cells.²⁶

The inverse correlation between activation of TCM CD8⁺ T cells and plasma viremia that we observed in TP suggests the existence of higher residual viremia, indicating a higher level of viral reservoirs in EC than in VS. Recent studies have found evidence of persistent low level viremia in patients with spontaneous control of HIV replication,^35,36 and we had previously suggested a role for CD38 as a marker of residual HIV replication.²⁴ In line with this hypothesis, in patients treated with HAART proviral DNA load was directly and significantly correlated with the level of activation on TCM CD8⁺ T cells. On the basis of this, higher levels of proviral DNA load in EC should be expected. To our surprise, however, EC presented a lower level of proviral DNA load than VS. This supports the role for other factors different from virus replication driving CD8⁺ T cell activation in EC.

Recent studies suggest that higher levels of HIV-specific CD8⁺ T cells,^33,37,38 low levels of regulatory T cells,³⁹ the existence of microbial translocation,²⁶ and the presence of other coinfections (CMV, EBV, or HCV⁴⁰) could be the mechanisms responsible for the increased activation of CD8⁺ T cells of EC. Regarding this last point, in our study all EC present HCV coinfection (as proven by HCV serostatus), suggesting that this fact could be underlying the high level of activation observed in CM CD8⁺ T cells. However, two observations in our study argue against this hypothesis. First, activation of CM CD8⁺ T cells was similar in TP patients with HCV coinfection compared to those without (data not shown), suggesting that the presence of HCV coinfection does not seem to have an influence on the quantity and quality of CM CD8 T cell activation. Second, in the EC group we did not find differences in T cell activation when comparing those with detectable HCV replication and those without. Nonetheless, a more detailed study is required to verify these findings.

In summary, our results demonstrate the existence of some T cell dysfunctions, especially increased activation of central memory CD8⁺ T cells, in HIV patients with spontaneous control of HIV replication as compared to patients with HAART-induced viral suppression. These alterations do not seem to be explained by higher levels of HIV reservoirs, and thus new studies with large cohorts of EC are required to elucidate the pathways that drive the high activation observed in these patients. Moreover, because spontaneous control of viral replication is not necessarily a benign clinical condition,^26,41,42 the search for new therapies aimed at normalizing activation of T cells is warranted.

Footnotes

Acknowledgments

We would like to thank Sara Lozano, Almudena Cascajero, and Celia Ballesteros for excellent technical assistance and Dr. Juan Gonzalez-Lahoz for his continuous support. This work was supported in part by grants from FIPSE, Fundacioń IES, FIS (ISCIII-RETIC R006/006), and the European NEAT (LSHP-CT-2006.037570).

Author Disclosure Statement

No competing financial interests exist.

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Elite Controllers Display Higher Activation on Central Memory CD8 T Cells Than HIV Patients Successfully on HAART

Abstract

Introduction

Materials and Methods

Study population

Clinical specimens

Flow cytometry

HIV proviral load

Plasma HIV-RNA

Statistical methods

Results

Study population

Similar differentiation stages of CD4 and CD8 T cells in both elite controller and patients successfully treated with HAART

Similar activation levels of different subsets of CD4 T cells in both EC and patients successfully treated with HAART

EC presented higher levels of activation in different subsets of CD8 T cells than patients treated with HAART

Contribution of different subsets to activation of total CD4+ and CD8+ T cells

EC present lower proviral DNA load than patients successfully treated with HAART

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Contribution of different subsets to activation of total CD4⁺ and CD8⁺ T cells