Prostaglandin I₂ Therapy Promotes Regulatory T Cell Generation in Patients with Pulmonary Arterial Hypertension

To the Editor:

Pulmonary arterial hypertension (PAH) results from a narrowing of the vascular lumen because of pulmonary vascular remodeling, ultimately leading to an increase in pulmonary arterial pressure (1). PAH has several different etiologies; as such, the pathophysiological mechanisms that lead to PAH are multifaceted (2). Both inflammation and an abnormal immune response contribute to the development and exacerbation of PAH (3). T effector cells (Teffs)—through their production of IL-6, IL-2, IL-21, IL-4, IL-13, IL-17A, interferon IFN-γ, and tumor necrosis factor α—directly promote inflammation, pulmonary vascular remodeling, and increased right ventricular systolic pressure in models of PAH (3–12). Additionally, elevated numbers of cytotoxic CD8⁺ T cells are present in patients with PAH (6). It is important to note that regulatory T cells (Tregs), which are important for maintaining tolerance and immune homeostasis and express the canonical transcription factor Foxp3, are protective against the pathogenesis of PAH in animal models (13–15).

The production of prostaglandin I₂ (PGI₂) occurs largely in the mammalian vasculature, where it functions as a robust vasodilatory agent when bound to its G-protein–coupled receptor, IP (16). PGI₂ signaling has a significant impact on the cardiovascular system and results in several significant vasoprotective effects, including vasodilation, prevention of platelet aggregation, and prevention of smooth muscle proliferation (17). Several PGI₂ analogs exist and include treprostinil and epoprostenol, which are used therapeutically as treatments for PAH (18); however, their mechanism of action within patients with PAH is not fully understood.

Athymic rats are protected from the development of pulmonary hypertension (PH) when they are reconstituted with Tregs (19). This Treg reconstitution resulted in elevated plasma PGI₂ levels and cardiopulmonary expression of PGI₂ synthase (PTGIS) in rats undergoing models of PH (19). This effect was more profound in athymic female rats who had developed more severe PH because of the lower bioavailability of PGI₂; Tregs induced PGI₂ synthesis in these models (19). We recently demonstrated that PGI₂ signaling promotes the in vitro differentiation of mouse and human CD4 T cells to Tregs, as well as Treg function and stability in mouse models of allergic inflammation (20). Therefore, we hypothesized that PGI₂ therapy in patients with PAH would increase the percentage of peripheral blood Tregs and decrease the peripheral blood Teff:Treg ratio.

Methods

Subjects initiating PGI₂ analog therapy for PAH for clinical indications were invited to participate in a research study (Vanderbilt University Medical Center Institutional Review Board #170161). The subjects provided informed consent; then, peripheral blood was drawn before the onset of treatment, and a clinically scheduled return visit (typically 1–2 mo) after the onset of treatment. Peripheral blood mononuclear cells were isolated by using Lymphoprep, SepMate tubes (STEMCELL Technologies), and gradient centrifugation; they were frozen down in Bambanker freezing medium (Bulldog Bio). Participants with samples before and after initiation of treatment were included, and the samples were analyzed as a single batch by cytometry by time-of-flight. Participant samples were analyzed by the Cancer and Immunology Core at the Vanderbilt University Medical Center. The T cell–focused panel included the following markers: live/dead; from BioLegend: CD66b*, CD16*, CD8*, CD14*, CD4*, CD3*, CD19*, and CD27; from Abcam: CPT1a*; from Standard BioTools: CD45, CD45RO, CD127, CCR5, CD20, CCR4, CD134, ICOS, TCRgd, CXCR3, CD137, CCR7, CTLA4, Ki-67, CRTH2, CD95, CD44, CD38, CD25, CD45RA, CXCR5, CD57, CXCR4, HLA-DR, PD-1, CD56, and CD11b; and Foxp3*, a triclonal antibody sourced from BioLegend and eBioscience (asterisks denote a custom conjugate completed with the Maxpar X8 Antibody Labeling Kit; Standard BioTools). Teffs were identified as live cells that were CD45⁺CD3⁺CD4⁺Foxp3⁻. Treg cells were identified as live cells that were CD45⁺CD3⁺CD4⁺Foxp3⁺. Statistical significance was determined by paired t test.

Results

Twelve participants were enrolled and returned for follow-up visits before May 1, 2022, and thus were included in this analysis. Demographics for these individuals are shown in Table 1. All patients with connective tissue disease–associated PAH had scleroderma, and a single patient was on low-dose prednisone, which was discontinued by the follow-up visit. Patients who died before follow-up were not included in this study.

Table 1.

Participant Demographics

Demographic	n or Median (IQR)
	(n = 12)
Age, mean (SD)	44.8 (13.5)
Sex, n
Female	10
Male	2
PAH group, n
Idiopathic PAH	5
Scleroderma PAH	4
Heritable PAH	3
Analog given, n
Intravenous epoprostenol	8
Intravenous treprostinil	2
Subcutaneous treprostinil	2
Received prior therapy for PAH, n	6
PDE5 inhibitor	6
Endothelin receptor agonist	4
Other prostanoid	1
Use of immunomodulatory agents, n	1
Systemic prednisone	1
Biologics	0
Hemodynamics, median (IQR)
Pulmonary vascular resistance, WU	18.1 (15.5–21.2)
Right atrial pressure, mm Hg	13.5 (11.7–19.0)
Pulmonary artery pressure, mm Hg	59.0 (56.5–69.5)
Follow-up epoprostenol dose, ng/kg/min	19.5 (18.0–20.2)
Follow-up treprostinil dose, ng/kg/min	43.2 (40.0–46.6)
Time between blood draws, d	65.0 (36.0–248.5)

Definition of abbreviations: IQR = interquartile range; PAH = pulmonary arterial hypertension; PDE5 = phosphodiesterase 5; WU = Wood units.

There was a significant increase in Treg percentage in participants at their follow-up visit (Figure 1A). Furthermore, there was a significant increase in the mean fluorescence intensity (MFI) of Foxp3 within the Treg population (Figure 1B). Moreover, there was a significant increase in the MFI of CD44 within the Treg population (Figure 1C). CD44 expression on Tregs promotes their suppressive functionality and correlates with Foxp3 expression (21, 22). There was no difference in expression on Tregs of CD25, CD27, CD38, CD95, CTLA4, PD1, or ICOS (data not shown). Furthermore, there was no change in memory status (CD45RO⁺) of the Tregs (data not shown). These data suggest that PGI₂ analog therapy promotes the differentiation and suppressive function of Tregs.

Figure 1.

Prostaglandin I₂ (PGI₂) signaling promotes regulatory T cell (Treg) polarization in participants treated with a PGI₂ analog for pulmonary arterial hypertension (PAH). Blue circles indicate participants with IPAH, red squares indicate participants with SPAH, black triangles indicate participants with HPAH. (A) Treg percentage in all participants. (B) Mean fluorescence intensity (MFI) (geometric mean) of Foxp3 within Tregs in all participants. (C) MFI (geometric mean) of CD44 within Tregs in all participants. (D) T effector cell (Teff):Treg ratio in all participants. Statistical significance was determined by paired t test. *P < 0.05 and **P < 0.01. HPAH = heritable PAH; IPAH = idiopathic PAH; SPAH = scleroderma PAH.

Notably, there was also a significant decrease in the Teff:Treg ratio present when the entire patient population was analyzed (Figure 1D), demonstrating that, after treatment, participants had fewer proinflammatory Teffs present for every antiinflammatory Treg.

Discussion

In this study, we show—to our knowledge, for the first time—that PGI₂ treatment increased the circulating Treg percentage, as well as the MFI of Foxp3 and CD44 on Tregs, and decreased the Teff:Treg ratio in a subpopulation of patients with PAH. These data are exciting and contribute to the growing understanding of the possible mechanisms by which PGI₂ analog treatment may result in the improvement in symptoms and quality of life in patients with severe PAH.

The impact of PGI₂ signaling in decreasing the Teff:Treg ratio, in addition to increasing the Treg percentage and the MFI of Foxp3 and CD44 on Treg, is important. Previously published data have demonstrated that there are fewer Tregs in the lungs of patients with PAH but, paradoxically, more Tregs in the peripheral circulation, although these circulating Tregs are less suppressive (23). Our data suggest that PGI₂ signaling is likely either increasing the functionality of the Tregs directly, thereby making the Tregs more suppressive, or it is increasing the total number of functional Tregs. Furthermore, it is interesting to speculate on the basis of previously published data demonstrating that Tregs promote the bioavailability of PGI₂ through interaction with cardiac vascular endothelial cells (19)—and on the basis of our previously published data showing that PGI₂ promotes Treg differentiation and function (20)—that a feed-forward mechanism may be occurring in our population of patients with PAH receiving PGI₂ infusion. In this circumstance, infusion of PGI₂ may be promoting the development and function of Tregs in patients with PAH, which are thereby further promoting endogenous PGI₂ synthesis, leading to further enhancement of Treg functionality and vasodilation and an improvement in the condition of the patient. Further ex vivo studies utilizing isolated Tregs to examine functional differences before and after the onset of PGI₂ therapy are an important future direction for research.

Furthermore, it is important to consider that the presence of an underlying autoimmune disorder may contribute to the efficacy of PGI₂ signaling–mediated immune augmentation in these patients with scleroderma PAH.

It is also important to note that a limitation of this study was the small sample size. Limited numbers of patients with PAH are placed on PGI₂ therapy each year: those who generally have severe disease. As such, the deaths of enrolled individuals are not uncommon, further limiting sample size. Follow-up validation studies in additional cohorts are necessary for confirmation of the results described herein.

In conclusion, these data support the use of PGI₂ analogs as antiinflammatory therapeutics that promote Treg function in humans, thus suggesting that drugs that augment this pathway may be used to treat multiple inflammatory diseases, including PAH.