Lymphocyte Subset Imbalance in Cardiometabolic Diseases: Are T Cells the Missing Link?

TL;DR

This review explores how imbalances in lymphocyte subsets may contribute to cardiometabolic diseases and highlights their role in inflammation and tissue damage.

Contribution

The paper provides a comprehensive review of lymphocyte subset involvement in various cardiometabolic diseases, emphasizing their potential as therapeutic targets.

Findings

Lymphocyte subsets regulate inflammation and tissue damage in cardiometabolic diseases.

Immune system dysregulation contributes to atherosclerosis, diabetes, and hypertension.

Restoring immune homeostasis could offer novel therapeutic strategies for CVD.

Abstract

Cardiometabolic and cardiovascular diseases (CVDs) remain the leading cause of death worldwide, with well-established risk factors such as smoking, obesity, and diabetes contributing to plaque formation and chronic inflammation. However, emerging evidence suggests that the immune system plays a more significant role in the development and progression of CVD than previously thought. Specifically, the finely tuned regulation of lymphocyte subsets governs post-injury inflammation and tissue damage resolution and orchestrates the functions and activation of endothelial cells, cardiomyocytes, and fibroblasts in CVD-associated lesions (e.g., atherosclerotic plaques). A deeper understanding of the immune system’s involvement in CVD development and progression will provide new insights into disease biology and uncover novel therapeutic targets aimed at re-establishing immune homeostasis. In this review, we summarize the current state of knowledge on the distribution and involvement of lymphocyte subsets in CVD, including atherosclerosis, diabetes, hypertension, myocardial infarction, and stroke.

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1. Introduction

T and B lymphocytes, natural killer (NK) cells, and innate lymphoid cells play pivotal roles in orchestrating immune responses and maintaining immune homeostasis in healthy conditions, as they possess distinct phenotypic and functional activities, enabling appropriate responses to antigens and pathogens [1,2]. In recent decades, our knowledge of lymphocyte functions in immunity has significantly expanded, with innovative studies revealing their involvement in chronic inflammatory conditions, including cardiometabolic and cardiovascular diseases (CVDs) [3]. T lymphocytes represent 10–25% of circulating peripheral blood cells and are mainly divided into two major groups of CD4^+^ and CD8^+^ αβ T cells, along with a small population of γδ T cells (1–5%) and natural killer T (NKT) cells (0.01–0.5%) [2]. CD4^+^ T helper (Th) cells can differentiate into different subsets, each expressing distinct surface molecules, cytokines, and transcription factors, thereby regulating immune responses in diverse ways [4]. For example, Th1 cells promote immune responses against intracellular bacteria and viruses by activating cytotoxic CD8^+^ T cells through the secretion of pro-inflammatory cytokines, such as interferon-gamma (IFN-γ) [5]. Similarly, B cells and innate lymphoid cells (ILCs) have attracted significant attention due to their ability to shape inflammatory responses and influence tissue remodeling processes in physiological and pathological conditions [6].

Identifying and characterizing lymphocyte subsets rely on standard methodologies, including cell sorting, functional assays, and multiparametric flow cytometry (FCM), which is the most commonly used and widely employed technique [7]. Furthermore, FCM can be combined with cell sorting for specific cell subset isolation. Functional assays include measurements of cytokine levels, immune cell proliferation cytotoxic activity, and other effector functions in response to specific stimuli [8]. Molecular biology techniques, such as polymerase chain reaction (PCR) and gene expression analysis, are used to further characterize lymphocyte subsets based on gene expression profiles, up- or downregulation of specific signaling pathways, or specific genetic polymorphisms, which might affect and alter immune responses [9].

CVD remains a leading cause of mortality worldwide, despite increasing awareness to reduce risk factors and the widespread utilization of medications for primary prevention and disease treatment. Emerging evidence identifies the immune system as a crucial player in the development and progression of all types of CVD. It regulates post-injury inflammation and tissue damage resolution through a fine-tuned crosstalk between immune cells, endothelial cells, cardiomyocytes, and fibroblasts. Indeed, exaggerated uncontrolled responses can either lead to excessive tissue damage or fibrosis [10]. This dual role of the immune system underscores the intricate involvement of these cells in the pathogenesis of CVD, which is still not fully understood. For example, CD4^+^ T cells are involved in inflammatory responses in atherosclerosis and hypertension, while CD8^+^ T cells are linked to tissue injury and repair mechanisms during myocardial infarction and stroke [11]. These dynamics highlight the therapeutic potential of targeting T-cell subsets in CVD. NKT cells could also modulate vascular inflammation and influence plaque composition; however, their exact roles, as well as how this regulation may shift in response to different stages of CVD progression, are still under investigation [12]. Recent studies have emphasized the need for comprehensive immune profiling in patients with CVD to better delineate how different lymphocyte populations contribute to disease pathogenesis. The CVD immunome should include specific markers associated with immune cell activation, differentiation, and tissue infiltration, as well as identify molecular pathways that regulate immune cell responses in cardiovascular tissues (Figure 1) [10].

In addition, this intricate relationship between CVD and the immune system is further underscored by the increased risk of cardiovascular complications and mortality observed in autoimmune diseases and Acquired Immune Deficiency Syndrome (AIDS) [13,14]. Despite encouraging preclinical data, a substantial translational effort is required to bridge the gap between bench and bedside and to realize comprehensive T cell characterization in CVD for future T cell-directed therapeutic strategy development.

In this review, we provide a comprehensive overview of lymphocyte contributions in CVD, with a focus on atherosclerosis, hypertension, diabetes, stroke, and myocardial diseases. We also summarize current FCM applications in characterizing and monitoring immune cell subsets in CVD, highlighting their potential as diagnostic biomarkers and therapeutic targets. Flow cytometry immunophenotyping could soon become a powerful diagnostic tool for enhancing the clinical management of CVD patients, improving disease monitoring, and identifying targetable cell populations with novel specific therapies. Indeed, bridging the gap between immunology and cardiology could represent the turning point for advancing our understanding of CVD and for fostering the development of more targeted and effective therapies for these patients.

2. FCM Immunophenotyping for T Cell Characterization

Flow cytometry is a powerful technique for immunophenotyping at the single-cell level. It detects scattered light and emitted fluorescence for rapid, precise, and simultaneous measurement of multiple cellular properties, such as size, granularity, and the expression of specific markers [15]. Immunophenotypic characterization of immune cell populations, including T cells, plays a fundamental role in understanding pathogenesis, progression, and response to treatment in immunological conditions, tumors, benign disorders, neurological and infectious diseases, and CVDs [16]. Therefore, accurate standardization of antibody panels and gating strategies is crucial for reliable and reproducible results, especially across laboratories and in routine clinical settings, including trials and research investigations [17].

The introduction of high throughput technologies has enabled the implementation of in-depth immunophenotyping, allowing up to 50 markers that can be simultaneously detected on a single cell, often in combination with molecular features [18]. Multiple standardized panels and gating strategies have been proposed for the identification and characterization of circulating and tissue-specific populations in humans and animals [19]. These panels are usually designed to identify multiple populations within a tube or to deeply characterize a single cell type using the same tube panel, especially for research purposes using technologies that allow the combination of up to 20 different fluorochromes or metals [20]. Despite the promise of full-spectrum flow cytometry, the practical application of >20-color panels in clinical practice remains impracticable, mainly due to the limited availability of diagnostic certified cytometers capable of detecting high numbers of fluorophores. Indeed, clinically-certified cytometers are typically equipped with three lasers, supporting the use of only 10–12 colors. This technological gap between advanced research technologies and clinical practical realities mirrors a diagnostic gap, as complete immunophenotyping of neoplastic cells usually requires multiple panels, also due to the limited number of available fluorophores and their spectral overlaps [21]. Addressing this challenge will require advancements in cytometer technology and the development of standardized, clinically applicable panels to bridge the gap between research and clinical practice.

Standardized flow cytometry panels comprise 8 to 50 markers for comprehensively characterizing the steady-state phenotype of T cell subsets in human peripheral blood and tissues. These panels are designed to identify and characterize T cells, detailing their differentiation and activation status, co-inhibitory receptor expression, and T helper cell subsets, such as CD4^+^ naïve, TEMRA (terminally differentiated effector memory), TEM (effector memory), TSCM (stem cell memory), and TCM (central memory) cells [22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45]. For example, naïve T cells (TN) are characterized as CCR7^+^CD45RA^+^CD27^+^CD28^+^CD95^−^, whereas TSCM is CCR7^+^CD45RA^+^CD27^+^CD28^+^CD95^+^. Within the effector memory compartment, early TEM cells are identified as CCR7^+^CD45RA^−^CD27^+^CD28^+^CD95^+^; early-like TEM cells are CCR7^+^CD45RA^−^CD27^−^CD28^+^CD95^+^; intermediate TEM cells are CCR7^+^CD45RA^−^CD27^+^CD28^−^CD95^+^; and terminal TEM cells are CCR7^+^CD45RA^−^CD27^−^CD28^−^CD95^+^. In addition, TEMRA cells are also marked [38,39,40,41,42]. Standardized fluorochromes and clones for T cell identification and characterization, along with their relative optimized panels and gating strategies, are reported in Table 1.

A 50-color panel allows for deep characterization of all T cell subsets and their activation status for research purposes, while this high throughput approach is not possible in clinical settings. Therefore, 10-color panels are the most practical solution, enabling the identification of T cell populations and the evaluation of key activation markers. For CD4^+^ and CD8^+^ immunophenotyping, a panel composed of CCR7(FITC), PD-1(PE), CD45Ra (ECD), CD25 (PC5.5), CD38 (PC7), CD4 (APC), CD8 (APC700), CD3 (APC-750), CD19 (PB), and CD45 (KO) could be used for principal T cell subset characterization (Figure 2).

3. Atherosclerosis

Atherosclerosis is a chronic inflammatory condition of the arterial wall that remains a major cause of morbidity and mortality worldwide [46]. Chronic inflammation promotes smooth muscle cell proliferation, extracellular matrix deposition, and the formation of fibrous caps over lipid-rich plaques. However, fibrous caps can break under permanent inflammatory stress, and plaques can rupture or erode (unstable plaques), causing thrombosis and acute cardiovascular events, such as heart attacks or strokes [47]. Macrophages and lipid deposition roles in these events have been extensively investigated; conversely, T cells and NK cells are only currently under the spotlight, with studies showing their involvement in modulating the inflammatory environment within atherosclerotic plaques [48].

NK cells play a dual role in atherosclerosis, acting as both pro-inflammatory and anti-inflammatory agents. They can contribute to plaque instability by killing endothelial and foam cells, and by secreting pro-inflammatory cytokines, like IFN-γ and tumor necrosis factor-alpha (TNF-α), amplifying local inflammation and recruiting other immune cells, such as macrophages [49,50]. NK cells can also regulate immune responses by interacting with dendritic cells (DCs), macrophages, and T cells, to resolve inflammation and stabilize plaques [51].

T cells are now recognized as a major immune cell population in plaques, where they actively contribute to lesion development and progression [52]. T cells are recruited within plaques via endothelial interactions mediated by selectins, integrins, and chemokine signaling, contributing to inflammation through cytokine secretion and clonal expansion [53]. CD4^+^ T cells are a critical component of adaptive immune responses and are involved in various atherosclerotic processes. For example, CD4^+^ TCM maintains immunological memory, coordinates adaptive immune responses, interacts with antigen-presenting cells (e.g., DCs) within atherosclerotic plaques, and contributes to inflammation and immune activation [54]. CD4^+^ TEM participates in local immune response, by producing pro-inflammatory cytokines and interacting with other immune cells (e.g., macrophages) to regulate inflammation and tissue remodeling. In a mouse model, CD4^+^ TEMRA contributes to endothelial-directed cytotoxicity and a pro-inflammatory environment within atherosclerotic lesions, promoting tissue damage and plaque instability [55]. CD4^+^ T cells significantly influence the progression of atherosclerotic plaques, as demonstrated by crossing apoE^−/−^ mice, which are prone to atherosclerosis, with immunodeficient scid/scid mice. The transfer of CD4^+^ T cells from apoE^−/−^ mice into immunodeficient apoE^−/−^/scid/scid mice resulted in larger lesions [56].

On the other hand, in humans, CD8^+^ TCM contributes to chronic inflammatory and plaque progression by producing pro-inflammatory cytokines, like IFN-γ and TNF-α [57,58]. CD8^+^ TEM is involved in local immune response within atherosclerotic lesions, by releasing pro-inflammatory cytokines, participating in cytotoxic responses, and influencing the inflammatory plaque environment. CD8^+^ TEMRA cells have more cytotoxic and pro-inflammatory activities than CD8^+^ TCM, and their presence within atherosclerotic lesions is related to plaque destabilization and rupture [59]. CD8^+^ TEMRA lymphocytes also contribute to local inflammation and exacerbate tissue damage within the arterial wall. Furthermore, elevated frequencies of TSCM cells have been observed in Treg lineage tracker-ApoE^−/−^ mice, suggesting that increased TSCM levels could represent potential hallmarks of advanced atherosclerosis in murine models [60].

The balance and interplay between these T cell subsets in atherosclerosis influence local immune responses, inflammation, plaque stability, and disease progression, and are still under investigation. Indeed, while CD8^+^ TCM cells could maintain surveillance and memory functions in the context of chronic inflammation, CD8^+^ TEMRA lymphocytes are the drivers for plaque destabilization and thrombosis [61]. Therefore, targeting specific CD4^+^ and/or CD8^+^ T cell populations could offer potential therapeutic strategies to modulate immune responses, attenuate inflammation, and mitigate atherosclerosis progression and associated cardiovascular risks [11].

4. Hypertension

Hypertension and inflammation are strictly interconnected, as inflammation can contribute to the development and progression of hypertension, and hypertension, in turn, can lead to a chronic inflammatory status. Individuals with hypertension often have elevated levels of pro-inflammatory markers, such as C-reactive protein (CRP) [62], interleukin-6 (IL-6), TNF-α, and reactive oxygen species (ROS) [63,64]. In this condition, subjects experience a long-lasting chronic low-grade inflammation that concurs with endothelial dysfunction, vascular remodeling, and increased vascular tone. Endothelial dysfunction, a hallmark of hypertension, is characterized by impaired vasodilation, increased oxidative stress, and pro-thrombotic status. It promotes vasoconstriction and vascular inflammation, as well as predisposes individuals to hypertensive vascular complications [65,66]. Moreover, inflammation involves several immune cell subsets, such as T lymphocytes, macrophages, and DCs, which infiltrate blood vessels and target tissues, promote inflammation and tissue damage, induce antigen presentation, cytokine release, and immune cell recruitment, and contribute to vascular inflammation and hypertension [67]. In addition, the renin–angiotensin–aldosterone system (RAAS), a key regulator of blood pressure, is intertwined with inflammatory pathways. Angiotensin II, a major RAAS component, exerts pro-inflammatory effects, contributing to vascular inflammation and hypertension. In turn, inflammatory mediators can induce RAAS activity [68].

Anti-inflammatory strategies, such as lifestyle modifications, dietary interventions, pharmacological agents targeting inflammation, and antihypertensive medications with anti-inflammatory properties, have shown promising beneficial effects in mitigating hypertension and associated vascular damage [69].

CD8^+^ T cell subsets can potentially be involved in hypertension-related vascular dysfunction and inflammation; however, the specific roles of each CD8^+^ T cell subset are still unclear. Immune-mediated processes involving CD8^+^ T cells could contribute to endothelial dysfunction, vascular remodeling, and hypertension-related end-organ damage through pro-inflammatory and cytotoxic mechanisms [70]. Longitudinal studies, mechanistic investigations, and clinical trials are needed to elucidate the specific roles of CD8^+^ T cell subsets in the development, progression, and complications of hypertension. In particular, circulating interleukin (IL)-17A-producing CD4^+^ T cells are increased in hypertensive patients, as well as in IFN-γ-producing CD4^+^ and CD8^+^ T cells, independently from angiotensin II levels [71].

Unrevealing the complex interplay between hypertension and inflammation is critical for developing personalized treatment strategies that simultaneously target both conditions. Anti-inflammatory therapeutic approaches could benefit blood pressure control, vascular health, and overall cardiovascular outcomes in hypertensive individuals [72]. Moreover, CD8^+^ T cells could also contribute to these pathogenetic mechanisms. Thus, immunomodulatory agents could improve the clinical management of patients with hypertension [70].

5. Diabetes

Recent advancements in the immunological understanding of type 1 diabetes (T1D) are leading to the development of novel therapeutic strategies that could potentially offer effective and safe cures for this disorder [73]. In T1D, the destruction of insulin-producing cells is a consequence of an autoimmune response, with antigen-specific CD8^+^ T lymphocytes playing a pivotal role in this autoimmune response against the pancreas [74]. At disease onset, naïve CD4^+^CD8^+^ T cells are increased, while effector memory subsets and T regulatory cells (Tregs) are decreased. Therefore, the percentage and absolute count of TEM CD4^+^ cells in late-stage disease are the most promising biomarkers of diabetes progression; a therapeutic combination of polyclonal Tregs with IL-2 or rapamycin could improve the survival and functionality of Tregs [75]. Other strategies have investigated combinatorial regimens with systemic therapies, such as anti-CD3, anti-CD20, anti-inflammatory agents, and antigen-specific agents, ultimately leading to antigen-specific Treg induction through the use of tolerogenic peptides and other immune modulators [76].

Type 2 diabetes mellitus (T2DM) is one of the principal risk factors for cardiometabolic disease development and circulating frequencies of immune cells are altered in individuals with diabetes, with a significantly increased risk of complications, including cardiovascular disease and peripheral neuropathy [77]. Moreover, these alterations are associated with insulin sensitivity, glycemic control, and lipid levels. Indeed, T2DM is currently considered a chronic low-grade subclinical inflammatory disease, often associated with metabolic syndrome, excessive nutrient intake, and obesity [78]. Peripheral leukocyte counts are higher compared to T1D and healthy subjects. In particular, T cells isolated from peripheral blood and adipose tissue of T2DM patients exhibit a proinflammatory phenotype, contributing to insulin resistance, pancreatic islet destruction, and disease progression, although their potential role remains controversial [79]. In humans, CD4^+^ T cells also play a pathogenetic role in obesity and insulin resistance; naïve CD4^+^ T cells and Tregs are reduced [80,81] while the number of NK cells is increased. However, NK cells in T2DM are dysfunctional, with low expression of the activation receptors NKG2D and NKp46 and reduced degranulation capacity [82]. Conversely, NKT cells are increased and produce high levels of IFN-γ, IL-4, and IL-17. Additionally, circulating and adipose tissue-resident ILCs, which are augmented, release high levels of IFN- γ [12].

6. Stroke

Stroke is the second major cause of death worldwide, after cardiac ischemia, and can result in permanent disability [83]. Innate and adaptive immune responses are actively involved in its pathogenesis; however, their exact roles in stroke recovery remain unclear [84]. During a stroke, neutrophils and monocytes rapidly recruit to the damaged brain tissue, trigger inflammatory responses, and promote debris clearance. Other immune cells, including macrophages, microglia, and T and B cells, are involved in the inflammatory cascade after a stroke [85]. Microglia, the brain-resident immune cells, are activated within minutes after an ischemic event and can release both pro- and anti-inflammatory cytokines, orchestrating immune responses to prevent long-term tissue damage [86,87,88]. Indeed, while inflammation is necessary for tissue repair, excessive responses can exacerbate damage [89], resulting in blood–brain barrier (BBB) disruption, and leading to increased neuronal death and impaired neurogenesis [90]. Moreover, chronic inflammation can extend into subacute and chronic phases of stroke recovery, contributing to post-stroke cognitive decline and further impairing functional outcomes. For instance, elevated levels of pro-inflammatory cytokines, such as TNF-α and IL-6, are associated with worse outcomes [91,92].

CD4^+^ T cells can influence post-stroke inflammation [93]; Th1 cells produce IFN-γ and other cytokines and promote inflammation, potentially aggravating brain injury, while Th2 cells mediate anti-inflammatory effects through IL-4 and IL-13 secretion, favoring tissue repair [94]. Tregs are also critical in controlling excessive inflammation and promoting recovery [95,96] by releasing anti-inflammatory cytokines, such as IL-10 and transforming growth factor (TGF)-β. Antigen-specific CD8^+^ T cells can recognize and kill neurons, contributing to further neuronal loss [97,98]. B lymphocytes might contribute to neuroinflammation through antibody production, although their exact role in brain injury during a stroke has not been well-established [16].

Novel therapeutic approaches include immune response targeting [99], which involves inhibiting neutrophil and M1 macrophage infiltration, inducing anti-inflammatory Tregs and M2 macrophages, and regulating cytokine production, to limit secondary damage and promote neuroprotection. Other therapies aim to maintain BBB integrity and recovery to reduce leukocyte infiltration and mitigate the detrimental effects of inflammation [100].

7. Myocardial Diseases

The mammalian heart is composed of striated myocytes, which enable rhythmic contractions throughout the lifespan, and includes both resident and circulating immune cells [101]. It plays a pivotal role in maintaining cardiac homeostasis and facilitating adaptive responses to injury.

Macrophages are the majority of resident immune cells in cardiac tissue, accounting for ~40% of CD45^+^ cells, and express CD64, CD163, MERTK (MER proto-oncogene tyrosine kinase), LYVE1 (lymphatic vessel endothelial hyaluronan receptor 1), and MHC class II [102]. Following ischemic myocardial injury, both resident and monocyte-derived macrophages rapidly engulf endogenous debris, and CD14^+^CD16^−^ monocyte frequencies are associated with adverse cardiac remodeling and function [102].

Lymphocytes also play an important role in myocardial diseases, with T cells being essential for maintaining cardiac homeostasis and mediating the heart’s response to injury [103]. T cells are involved in fetal development and functions of the heart, including protecting against infections and other insults, cardiac remodeling (a process by which the heart adapts to injury), and blood vessel formation [104]. Indirect evidence of the involvement of T cells in maintaining cardiac functions involves the development of myocarditis under immune checkpoint inhibitors (ICIs) for cancer treatment, likely induced by programmed cell death (PD)-1 activation of antigen-specific T cells against PD-1 ligand (PD-L1)-expressing cardiac cells [105]. During ICI-induced myocarditis, CD8^+^ TEMRA cells expand and express CXC motif chemokine receptor 3 (CXCR3), which directs them to the site of inflammation, such as the heart. In addition, Tregs exert anti-inflammatory effects through the secretion of immunosuppressive cytokines, such as TGF-β and IL-10, and influence antigen-presenting cells and CD8^+^ effector T lymphocytes, leading to decreased inflammation and mitigation of immune reactions [106]. During myocardial injury, Tregs limit inflammation, reduce fibrosis, and improve cardiac function by modulating effector T cells and macrophage activities [106]. Because of their role in promoting the resolution of inflammation and supporting tissue repair, Tregs represent a potential therapeutic target in the treatment of myocardial diseases, such as ischemic injury and heart failure.

B cell roles in myocardial diseases are less studied, and most of the available data are derived from murine model studies. In the myocardial infarction model, CD19^+^ B cells are increased in the myocardium and pericardial adipose tissue following ischemia, peaking between 5 and 7 days after injury, and their depletion results in improved cardiac function [107]. B lymphocytes negatively influence left ventricular (LV) remodeling by recruiting Ly6C^+^ monocytes from the bone marrow [108]. Simultaneously, intracardiac injections of bone marrow-derived B cells protect the myocardium from cardiomyocyte apoptosis, highlighting the dual role of this immune cell population. Indeed, B cells can limit inflammation and promote tissue repair by producing regulatory cytokines, such as IL-10 [109]. However, under chronic inflammatory conditions, these cells exacerbate myocardial damage by promoting autoimmunity through heart-specific autoantibody production or by driving maladaptive immune responses [108]. In human hearts, B cells are present at similar levels to CD4^+^ and CD8^+^ T cells; however, in the mice model, during acute myocardial infarction, a temporary decrease in circulating B cells is observed, followed by an increase 24 h after reperfusion [109].

NK cells are another immune cell population involved in myocardial homeostasis and response to injury, especially after ischemic events [110]. NK cells can produce large amounts of pro-inflammatory cytokines, such as IFN-γ, contributing to adverse remodeling after myocardial infarction [111]. However, there is discordant evidence suggesting that NK cells might play a role in limiting excessive inflammation and promoting tissue healing [111].

DCs are professional antigen-presenting cells that populate the myocardium, where they can uptake, process, and present cardiac antigens to T cells after tissue damage, such as under injury or infections. During myocardial infarction or myocarditis, DCs can drive protective immune responses or contribute to pathological inflammation and cardiac dysfunction, based on predominant stimuli [112].

Fibroblasts actively participate in immune response within the myocardium. This is due to their crosstalk with macrophages and T cells, which finely modulate cardiac remodeling processes, resulting in functional recovery or heart failure. Therapeutic targeting of signaling pathways involved in fibroblast-immune cell crosstalk, such as TGF-β or IL-1 downstream, could offer new treatment approaches to prevent maladaptive fibrosis in myocardial diseases [113].

8. CVD Pharmacological Treatment and Imbalance of Lymphocyte Subsets

While there is growing evidence of the pivotal role of the immune system in cardiometabolic disease, there are few immune-based therapies for CVD treatment, with a limited number of clinical trials investigating the role of Tregs in atherosclerosis, such as LeukoCAPE-2 trial, NCT03105427, an observational case-only study. In this study, Tregs were studied as predictors of cardiovascular risk in two patient groups: those with a known CVD history undergoing major non-cardiac surgery, and those who have undergone cardiovascular surgery [114]. Results from the LeukoCAPE-2 trial indicate that preoperative Treg levels can highly and specifically increase preoperative risk assessment in combination with other well-established cardiac biomarkers, especially in intermediate-risk patients for major adverse cardiac and cerebrovascular events (MACCE) [115]. Conversely, in the CANTO study, IL-1β neutralization was investigated in CVD patients, showing a significant reduction in non-fatal myocardial infarction, non-fatal stroke, or cardiovascular death, although the mechanism remains unclear [116].

Clinical data supporting the key role of T-cell modulation in cardiovascular disease are still emerging. One randomized trial investigated canakinumab (IL-1β monoclonal antibody inhibiting the activity of Tregs) in patients with a history of myocardial infarction, demonstrating a significant reduction in the recurrence of cardiovascular events [115]. A randomized clinical trial involving patients with stable ischemic heart disease and acute coronary syndrome used low-dose IL-2. Aldesleukin increased the average circulating Treg levels, alleviated endothelial dysfunction, and reduced the formation of atherosclerotic plaques, favoring cardiovascular health [114,117].

Moreover, numerous studies have investigated the roles of different lymphocyte subsets in CVD across various tissues and species, as detailed and summarized in Table 2.

9. Conclusions

Comprehensive lymphocyte subset immunophenotyping in clinical settings represents a long-standing area of interest, yet its clinical application remains limited due to the following substantial technical and logistical challenges: (i) the rarity of certain subpopulations, which reduces the sensitivity and specificity of FCM for their quantification and requires optimized antibody panels, rigorous gating strategies, and large sample volumes, which are not always available, especially in pediatric or critically ill patients; (ii) inter-operator and inter-laboratory variabilities, such as sample handling, staining protocols, and instrument calibration, which introduce discrepancies and, thus, reduce results comparability; (iii) pre-analytical variables, such as sample storage conditions and processing times, which add other variations that affect rare subpopulation quantification. Therefore, protocol standardization is essential to establish international consensus criteria for processing, analysis, and reporting, ensuring more reliable and comparable results, especially in clinical settings, including CVD.

Despite these challenges, integrating immune profiling into the diagnostic and therapeutic landscape of CVD holds great promise. A better understanding of the intricate crosstalk between immune cells and disease pathology could enhance prognostic accuracy and lead to the development of targeted therapies, such as selective modulation of Tregs. Furthermore, identifying immune-based biomarkers for early disease detection and therapy response prediction represents a promising frontier. While animal models have significantly contributed to advancing methodological approaches, their species-specific differences warrant careful extrapolation of findings to human disease. Consequently, refining cytometric techniques and identifying biomarkers will be pivotal in overcoming current limitations and unlocking the full potential of lymphocyte subpopulation analysis for improving the clinical management of CVD.