Background
Foxp3
+ regulatory T (Treg) cells play a pivotal role in the regulation of the immune response due to suppressive abilities. The frequency of Tregs was associated with an inadequate immunological response during HIV-1 infection [
1]. Natural regulatory T (nTreg) cells are produced in the thymus, while inducible regulatory T (iTreg) cells or adaptive Treg cells can be induced in vitro in the presence of IL-2 and TGF-β [
2]. Compared with Foxp3
+Helios
+, the traditional combination of CD25 and Foxp3 surface markers underestimates the proportion of Treg cells [
3]. Helios, one of the Ikaros zinc finger transcription factors, was reported to bind to the Foxp3 promoter and stabilize the suppressive function of Treg cells [
4]. In the periphery, Helios is expressed in approximately 70% of CD4
+Foxp3
+ human and mouse Treg cells [
5]. In HIV-1-infected individuals who were on combined antiretroviral therapy (cART), the frequency of memory Foxp3
+Helios
+ Treg cells was significantly higher than that of healthy controls, whereas the frequency of memory Foxp3
+Helios
− Treg cells was similar to that of healthy controls [
6]. Until now, Helios expression in T-regulatory cells has not been fully characterized in HIV-1-infected patients, especially during acute HIV-1 infection (AHI).
Monocytes can act as regulators of inflammation and HIV-related comorbidities [
7]. Monocytes are heterogeneous with different phenotypes and functions, and in 2010, the following three monocyte subsets were recommended by the Nomenclature Committee of the International Union of Immunological Societies: classical (CD14
++CD16
−), intermediate (CD14
++CD16
+), and non-classical (CD14
+CD16
++) subsets [
8]. Monocytes have many immunological functions, including antigen presentation, making them a link between the innate and adaptive immune systems [
9]. Monocytes were reported to control Treg cell differentiation. Activated monocytes can influence Treg cells through the production of soluble mediators, CD16
+ monocytes inhibit proliferation of Helios
+ Tregs through IL-12, whereas Helios
− Tregs are suppressed by CD16
− monocytes via TNF-α [
10]. The specific roles of three monocyte subsets on Treg cell differentiation have remained poorly understood in HIV-1-infected patients.
Programmed cell death protein 1 (PD-1) can be expressed on T cells, B cells and other cell types. PD-1 expression on T cells and its binding by PD ligand 1 (PD-L1) on antigen-presenting cells (APCs) is a major mechanism that results in T-cell exhaustion [
11‐
13]. PD-1 signaling may also occur independently of T-cell or B-cell antigen receptor signaling [
14]. PD-1 can be induced and expressed on human monocytes during HIV-1 infection [
15]. The triggering of PD-1 on monocytes was shown to induce IL-10 production and inhibit CD4 T-cell proliferation, which indicates that PD-1 on monocytes could modulate immune responses by inducing IL-10 production [
16]. The therapeutic potential of manipulating PD-1 and PD-1 ligands provides an impetus to understand the function of PD-1 expression on APCs [
17]. Limited data are available on PD-1 expression on three monocyte subsets and their association with Treg cells during HIV-1 infection.
Host immune responses during AHI can influence the establishment of the viral setpoint, which is a predictor of disease progression [
18]. We previously found that in acute HIV-1-infected patients, the frequency of the intermediate CD14
++CD16
+ monocyte subsets was positively associated with the frequency of IL-4, whereas this subset was positively associated with the frequency of IFN-γ and IL-4 producing CD4
+ T cells in chronic HIV-1-infected cART-naïve patients [
19]. In the present study, we explored Helios expression in Treg cells in HIV-1-infected patients, and we evaluated the association between Foxp3
+Helios
+ Treg cells and the levels of three monocyte subsets and their PD-1 expression.
Discussion
The specific roles of three monocyte subsets and their PD-1 expression on Treg cell differentiation have remained poorly understood in acute HIV-1-infected patients. In the present study, we characterized the perturbations of Foxp3+Helios+ Treg cells during HIV-1 infection. We found that in acute HIV-1-infected patients, the frequency of Foxp3+Helios+CD45RA+ Treg cells was inversely correlated with the frequency of intermediate CD14++CD16+ monocyte subsets, whereas it was positively correlated with PD-1 expression on intermediate CD14++CD16+ monocyte subsets.
Foxp3+Helios+ Treg cells regulate the immune response due to suppressive abilities. Monocytes are heterogeneous with subset-specific phenotypes and functions, and have been reported to control Treg cell differentiation. PD-1 signaling may also occur independently of T-cell or B-cell antigen receptor signaling, and the expression of PD-1 on monocytes could modulate immune responses. In the study, monocyte subsets and their PD-1 expression were found to have varying impacts on Treg-cell differentiation in HIV-1-infected patients. Therefore, understanding how monocyte subsets and their PD-1 expression influence Treg cells may aid in boosting or preventing pathological responses during HIV-1 infection.
The perturbations of Treg cells during AHI remain controversial, which is partially because of the gating strategy and the lack of consensus phenotypic markers that define Treg cells [
21,
22]. In our study, we found that in acute HIV-1-infected patients, the frequency of Foxp3
+Helios
+CD45RA
+ Treg cells was significantly higher than those of both HC and HIV-1-infected patients after cART. There are several possible causes for the Treg cell expansion, including enhanced Treg cell generation and increased cell survival [
23,
24]. Previous studies have shown that there is an inverse correlation between Treg cells frequency and CD4 T-cell counts [
25], which is consistent with our findings. In our study, the frequency of Foxp3
+Helios
+CD45RA
+ Treg cells in acute HIV-1-infected patients was significantly higher than those of HC and CHI&ART+, whereas CD4 T-cell counts of acute HIV-1-infected patients were lower than that of HC and CHI&ART+. Reduced Treg cell numbers are associated with a reduced suppression capacity of CD8
+ T cells, NK cells, and other immune cells [
26]. Systemic immune activation is a consequence of HIV-1 infection, and cART produces profound suppression of HIV replication, but fails to eliminate chronic immune activation completely. An increased proportion of Treg cells during chronic HIV-1 infection may be beneficial for controlling persistent hyperactivity [
27]. Due to the beneficial or detrimental roles of Tregs in HIV-1 infection, different therapeutic strategies will be considered to enhance the immune response or to downregulate global hyperactivity by pharmacologic manipulation of Tregs or transferring adoptive cells.
Monocytes are generally regarded as precursors of macrophages and dendritic cells (DCs), and DCs have the capacity to expand antigen-specific Tregs [
28]. The CD16
+ monocyte subset was positively correlated with IFN
− CD4
+ T cells, but negatively correlated with CD4
+CD25
hiFoxp3
+ Treg cells in immune thrombocytopenia [
29]. In this study, we found that the frequency of intermediate CD14
++CD16
+ monocytes was inversely correlated with the frequency of Foxp3
+Helios
+CD45RA
+ Treg cells in acute and chronic HIV-1-infected patients. Defining the influence of monocytes on Tregs will enable more precise targeting of immune cells to enhance defense against HIV-1 infection.
PD-1 and its related pathways are considered a central regulator of T cell exhaustion [
30], but the function of PD-1 expression on APCs requires further clarification. During
Listeria monocytogenes (LM) infection, PD-1 can be induced on splenic DCs, and PD-1 on DCs negatively regulates IL-12 and TNF-α production and inhibits innate immune responses [
31]. PD-1 expression on monocytes/macrophages in patients with sepsis is higher than that in HC. Moreover, PD-1 expression on monocytes is associated with cellular dysfunction [
32]. PD-1 signaling inhibits T-cell activation, but can promote induced regulatory T-cell development [
33]. In this study, we found that the expression of PD-1 on both intermediate CD14
++CD16
+ and non-classical CD14
+CD16
++ monocyte subsets was positively correlated with the frequency of Foxp3
+Helios
+CD45RA
+ Treg cells in HIV-1-infected patients. PD-1:PD-L1 interactions can limit effector T-cell responses, and induce regulatory T cells. Significant clinical activity has been shown in a variety of cancers by targeted therapy against PD-1/PD-L1 [
34]. Therapeutic targeting of the PD-1:PD-L1 pathway could be beneficial for the enhancement of immune responses against viral pathogens.
In this study, an in vitro stimulation system was used, in which PBMCs were stimulated with PMA/ionomycin. Monocyte and T-cell coculture systems and ex vivo or in vitro experiments will be performed in future studies to investigate the roles of monocyte subsets and their PD-1 expression on Treg differentiation. Monocytes are generally regarded as precursors of macrophages and DCs; therefore, it is of interest to characterize the modulation of CD4
+ Tregs by monocyte-derived DCs [
35,
36].
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