Background
Human immunodeficiency virus-1 (HIV-1) infection is mostly characterized by ongoing viral replication leading to a progressive decline of CD4+ T cells and eventually progression to clinical AIDS. Nevertheless, there is a small subset of HIV-1+ subjects who are able to spontaneously control HIV-RNA replication in plasma to levels below the limit of detection in the absence of antiretroviral therapy. They are called elite controllers (ECs) and represent less than 1% of the total of HIV-infected patients [
1]. ECs have been considered as a model of spontaneous functional cure and the role of host genetic factors, innate and adaptive immune responses, viral characteristics, and their interactions have been analyzed in several studies [
1‐
5].
However, ECs constitute a heterogeneous group in terms of genetic and immunologic characteristics [
6], and no single mechanism has been described as being responsible for controlling viral replication [
5‐
7]. These patients are also heterogeneous from the clinical point of view, since some of them show either virologic and/or immunologic progression [
8‐
11]. Studies with large cohorts of EC patients have reported that as many as 25% of them experience virological progression (losing the ability to maintain HIV replication control) [
11], and around 20% experience immunological progression (a decline in CD4 counts) even in the absence of virological progression [
8,
9], as we have also previously demonstrated in our cohort of HIV controllers of the Spanish AIDS Research Network (the ECRIS cohort) [
11]. However, the mechanisms accounting for this CD4 T-cell loss in ECs remain largely unknown.
Given the relevance of different alterations of T-cell homeostasis in the CD4 depletion observed in HIV patients with uncontrolled viral replication [
12‐
14], some of these same mechanisms might be operating in EC patients experiencing CD4 T-cell loss. In this regard, previous studies have shown that even though EC patients are able to suppress HIV replication to an undetectable level, they present different T-cell homeostasis disturbances compared to uninfected controls and/or to HIV non-controllers with suppression of HIV replication mediated by combination antiretroviral therapy (cART) [
15‐
20], suggesting that T-cell homeostasis disturbances are still operating in EC patients and could potentially be involved in the immunological progression.
To test this hypothesis, and with the final aim of finding predictors of immunological progression, herein we have comprehensively compared several important parameters of T-cell homeostasis in EC patients with long-lasting HIV replication control but experiencing immunological progression with those for EC patients showing no progression. The partial least-squares–class modeling (PLS-CM) methodology was applied as a statistical modeling technique to discriminate between the two groups of patients, and as a predictive model.
Discussion
The present study was designed to test the hypothesis that perturbations in immunological parameters related to T-cell homeostasis may be associated with CD4 T-cell loss in EC patients maintaining long-term undetectable levels of plasma HIV viremia. The main findings of our study are: (1) there is an important perturbation of T-cell homeostasis in EC patients despite long-term undetectable viremia; (2) based on the T-cell parameters analyzed, a PLS-CM statistical approach was able to discriminate and to predict, with high sensitivity and specificity, EC patients experiencing immunological progression from those EC patients with a stable CD4 count; and (3) immunological parameters associated with the loss of CD4 T cells in EC patients included mainly those related to CD8 T-cell homeostasis.
Despite the persistent control of HIV replication to undetectable levels, several alterations related to a state of systemic inflammation have been recognized in ECs compared to seronegative subjects and to HIV patients with cART-mediated control of HIV replication [
23‐
25], and this has been associated with an increased rate of morbidity and mortality in EC patients [
24‐
26]. Our study demonstrates that immunological alterations in EC patients are not limited to markers of systemic inflammation and extend to several cellular markers of T-cell homeostasis. Although the EC patients included in our study had maintained long-term undetectable plasma HIV viremia, they clearly departed from seronegative healthy subjects (HCs) regarding the distribution of T-cell subsets. As many as 93 immunological variables were necessary to discriminate between EC patients and HCs and many of these presented levels significantly different between ECs and HCs, suggesting there is an important perturbation of T-cell homeostasis in EC patients despite long-term undetectable viremia.
Different processes involved in T-cell homeostasis were altered in ECs compared to healthy subjects. Among them, naïve and RTE subsets, stem cell memory subsets, regulatory T cells, and expression of the costimulatory receptor CD28 were significantly decreased, whereas activation and expression of CD95 were significantly increased. Some of these perturbations have already been reported in different cohorts of ECs, such as alterations in naïve/memory subsets [
17,
19], increased activation [
15,
18,
27], increased senescence [
27], and decreased regulatory T cells [
28]. However, EC cohorts employed in these previous studies had varying degrees of HIV-associated immune suppression (as reflected in CD4 counts) and different cutoffs of plasma viremia, meaning that some alterations reported in these studies could be influenced by CD4 counts [
28] or low-level plasma HIV viremia [
27]. Since all EC patients included in our study had high CD4 counts at the time of the immunophenotypic analysis, our results demonstrate that perturbation of T-cell homeostasis is already present in ECs from the early stages of HIV disease. Moreover, our study is the first reporting alterations in ECs of some important parameters of T-cell homeostasis such as Tscm cells [
29,
30]. Interestingly Tscm cells were diminished in both CD4 and CD8 T cells and thus, the potential to repopulate other subsets of memory cells [
31] may be compromised in ECs at early stages of infection.
The most relevant findings of our study are those related to T-cell parameters associated with loss of immunological control in ECs. Using a set of 77 different immunological variables, the PLS model was able to discriminate, with high sensitivity and specificity, between EC patients showing a significant CD4 decline (cases) and those with stable CD4 counts (controls). The immunological variables were assessed when both groups of EC patients presented similar high levels of CD4 counts (prior to CD4 T-cell loss in EC cases) and thus, the differences found cannot be attributed to different degrees of HIV-associated immune suppression, but rather they are characteristic of some EC subjects and likely involved in the decline of CD4 T cells observed in EC cases. Since no functional methods were employed in this study, we can speculate only about the potential mechanisms involved in the differences observed in the extent of T-cell homeostasis disturbance between EC cases and EC controls. Besides viral factors, such as the higher levels of residual viral replication in EC cases [
10], several host-related factors could contribute to the CD4 T-cell loss and T-cell homeostasis disturbance observed in EC cases. Among them are defective thymic function [
17], lymphoid tissue fibrosis [
32], genetic variation in different genes related to immune response [
33,
34], and a cytokine-driven inflammatory milieu [
35].
Interestingly, the two-sample non-parametric test (Mann–Whitney U test) was not able to detect subtle but important differences between cases and controls for many of the immunological variables that were detected by the PLS model. PLS-CM is an approach that is best suited to extracting these differences employing latent factors and building a predictive model in situations with a small sample size and many predictive variables. In agreement with this higher sensitivity of PLS modeling to detect differences among a large panel of variables, the immune parameters that had significant differences by the two-sample test were those showing the highest loadings in the PLS model.
The factors involved in the immunological progression observed in some ECs that maintain virological control are largely unknown and studies addressing this issue are scarce, with only a few investigating potential associated immunological factors [
10,
15,
36,
37]. In some of these studies, T-cell activation was shown to be associated with disease progression in ECs using a cross-sectional design [
15,
36]. The other two studies addressing the association of T-cell activation with CD4 evolution in EC patients and using a longitudinal follow-up period similar to our study [
10,
37] found discordant results. One reported increased activation associated with immunologic progression [
10] but the other did not [
37]. Differences in the criteria used to define immunological progression may explain the discordant results. Moreover, both studies analyzed T-cell activation when CD4 counts were significantly different between EC patients showing immunological progression and those maintaining stable CD4 counts, which is in clear contrast with our study.
Our results show that several T-cell subsets showed significant differences between EC cases and controls and interestingly they were mainly related to CD8 cell homeostasis. Naïve and RTE subsets of CD8 cells were diminished in EC cases, suggesting either a defect in thymic output or an increased rate of transition of naïve cells to more differentiated subsets. Since a deficit in thymic output would be reflected in the RTE subset of both CD8 and CD4 compartments, our findings more strongly support an increased rate of differentiation. This, together with the finding of increased levels of effector CD8 cells in EC cases, supports the higher levels of residual HIV replication in EC cases. Unfortunately, we did not measure plasma HIV viremia with ultrasensitive assays and thus, we can only speculate on this possibility. However, in support of this hypothesis, previous studies have found higher levels of residual viremia in EC patients showing CD4 depletion [
9,
10,
38]. Although levels of effector CD8 cells were higher in EC cases, CD8 T cells showed increased levels of the CD28-CD57+ subset, a phenotype that has been associated with replicative senescence [
39]. CD8 T cells expressing this senescent phenotype have lower cytotoxic potential [
40,
41] and produce high amounts of inflammatory cytokines such as IL6 and TNFα [
39], promoting an inflammatory state that could contribute to the CD4 depletion observed in EC cases. An interesting finding was that naïve CD8 cells of EC cases also have increased levels of senescence, suggesting that they have undergone an antigen-independent expansion without differentiation to memory cells in an attempt to maintain CD8 T-cell homeostasis. Lastly, regarding alterations of CD4 T-cell subsets, EC cases had increased levels of expression of the cell exhaustion-associated marker PD1 in total CD4 cells and in central memory subset. Since overexpression of PD1 has been associated with a lower ability to reconstitute CD4 T cells in HIV patients with cART-mediated suppression of viral replication [
42,
43], this increased expression of PD1 in EC cases could be another factor contributing to CD4 depletion.
Finally, there are some caveats that deserve comment. First, studies employing EC patients are very limited in sample size given the very low frequency of this exceptional population of individuals among the whole population of HIV-infected patients. However, in our study, we addressed the potential bias of a small sample size by studying a very homogeneous study population. Also, for the data analyses we employed the PLS regression, which is a statistical technique for performing multivariate calibration, especially when there are more variables than samples. Second, although the objective of our study had no such scope, it could have been interesting to include a group of HIV-normal progressors to assess whether the underlying mechanisms of the loss of immune control in EC patients are comparable to those underlying the non-immunological control in normal HIV+ progressors patients, as well as to evaluate the power of the PLS model to discriminate between both phenomena.