Introduction

Leprosy, caused by Mycobacterium leprae and Mycobacterium lepromatosis, is a chronic infectious disease primarily affecting peripheral nerves, mucous membranes, eyes, and superficial skin nerves.1,2 Although highly infectious, the incidence is low due to the host’s immune response. Clinical forms are associated with immunity patterns. Leprosy initially manifests as indeterminate (IL), with spontaneous cure or progression to other clinical forms.3

According to Ridley and Jopling classification (1966), two prevalent poles, tuberculoid leprosy (TT) and lepromatous leprosy (LL), exhibit opposing immune response profiles. TT elicits a Th1 pattern, controlling bacterial growth, while LL does not see an effective cellular response, leading to multiple bacilli and skin lesions. Intermediate forms, including borderline-tuberculoid (BT), borderline-borderline (BB), and borderline-lepromatous (BL), reflect immunological instability and varying degrees of immune response.3,4

Recent evidence challenges the well-described Th1/Th2 dichotomy. New cell populations, including gamma-delta T-cells (Tγδ), invariant natural killer T cells (iNKT), double-negative T-cells, regulatory B-cells, and regulatory T-cells (Treg), play a role in leprosy’s immunopathogenesis.5–7 While the intricate function of these cells in leprosy has become increasingly evident, our focus narrows to Treg, which was first identified in 1995 and emerged as a key contributor to various aspects of immunoregulation.8 In leprosy, Treg (CD4+CD25+FoxP3+) cells influence Th1, Th17, Th9, and innate cells, modulating the immune response.5,6,9 However, the precise mechanisms remain unclear.

This study aims to analyse evidence on the role of regulatory T cells in leprosy, focusing on the frequency, expression, and functional characteristics of their subpopulations.

Methods

This integrative literature review aimed at gathering and synthesising the available scientific evidence on the guiding question “What is the role of Treg cells in Hansen’s disease?” Following the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), the review was conducted systematically, in an organised manner, and comprehensively, with the goal of providing a broader understanding of the subject.10

A literature search was conducted in the Medical Literature Analysis and Retrieval System Online (MEDLINE) database via PubMed and Virtual Health Library (BVS, Biblioteca Virtual da Saúde), using terms registered in the Medical Subject Headings (MeSH). The search strategy employed was: (“Leprosy” [MeSH Terms]) OR (“Mycobacterium leprae” [MeSH Terms]) AND (“T-Lymphocytes, Regulatory” [All Fields]).

The search for studies was conducted from March to July 2023, with an update in January 2024, covering different researches published over the last 11 years, from 2012 to 2023. Full-text articles available through open online access, published in English or Portuguese, were included. Studies that were not available in full, as well as case reports, reviews, response letters, editorials, duplicate studies, or those that did not align with the research question and purpose, were excluded from the study. The implementation of the inclusion and exclusion criteria has been outlined step by step, with the rationale for each stage illustrated in Figure 1.

Figure 1: PRISMA flow chart summarising the screening process.

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We initially identified 134 pertinent articles. Subsequently, pre-established inclusion and exclusion criteria were applied to select the most pertinent studies, resulting in a total of 18 articles. Next, 10 studies (55.6%) were excluded as they did not fit into the proposed theme.

In Virtual Health Library (VHL), the search resulted in the identification of 128 relevant articles. After applying pre-established inclusion and exclusion criteria, 29 articles were selected. Of these, 15 (51.7%) studies had already been included, and 10 (34.5%) did not address the proposed theme and were excluded. After reading the articles in full, 12 studies were selected, eight (8) from Medical Literature Analysis and Retrieval System Online/Pubmed and four (4) from BVS, composing the sample for this integrative review.

Results Characterisation of included studies

Out of the 12 selected articles, five were conducted in Brazil, four in India, one in China, one in Ethiopia, and one with volunteers from Ethiopia, Nepal, and the Netherlands. Most studies aimed to evaluate the presence or frequency of Treg cells in leprosy patients, both in skin lesions and in peripheral blood mononuclear cell (PBMC) culture assays. The Ridley and Jopling classification was used to classify leprosy in ten studies, while one study used the paucibacillary (PB) and multibacillary (MB) classification. In turn, another study used buffy-coat bags from blood donors for laboratory assays. Table 1 provides a summary of the selected studies.

Table 1: Characteristics of the studies stratified by the year included in the integrative review (N=12)

Author Country Subjects (n) Sample/procedures and/or techniques Markers Main findings Palermo et al. (2012)16 Brazil BT/TT (12), BL/LL (16), and healthy household contacts (6). Skin lesions/Immunohistochemistry. In situ: CD25, FoxP3, CTLA-4, IL-10, TGF-β. The BL/LL patients exhibited a notably elevated expression of CD25+FoxP3+ Treg cells approximately double that observed in BT/TT patients. Moreover, the expression of CTLA-4 and IL-10, but not TGF-β, was higher in BL/LL patients. In the histopathological examination, Treg cells were identified within the infiltrate of vacuolated histiocytes in BL/LL patients, contrasting with the infrequent occurrence of Treg cells within granulomas in lesions of BT/TT patients. PBMC/ Cell culture with MLCwA.

- PBMC: CD4, CD25, Foxp3.

- Cytokines in cell culture supernatant: IL-10.

The frequency of CD4+CD25+FoxP3+ Treg cells was higher in BL/LL patients than in BT/TT patients and healthy household contacts. Additionally, a higher production of IL-10 was observed in the culture supernatant of BL/LL patients. Fernandes et al. (2013)20 Brazil PB (6), MB (6), and healthy household contacts (17). PBMC/ Cell culture with MLT. PBMC: CD3, CD4, CD8, CD25, FoxP3. The frequency of CD3+CD4+CD25highFoxP3+ and CD3+CD8+CD25highFoxP3+ Treg cells was higher in MB patients than in healthy household contacts, but not in PB patients. Additionally, a positive correlation was noted between CD4+CD25highFoxP3+ and CD8+CD25highFoxP3+ Treg cells with bacilloscopy index and the number of lesions. Saini et al. (2014)13 India BT (28), LL (28), healthy household contacts (7), and healthy individuals undergoing cosmetic surgery (4). Skin lesions/Immunohistochemistry. In situ: FoxP3, TGF-β, IL-10. The FoxP3+ cells exhibited a distinctive pattern, forming a circumscribed arrangement around tuberculoid granulomas and interspersed among epithelioid cells. In contrast, FoxP3+ cells were dispersed among foam cells within Virchowian granulomas. Furthermore, the frequency of cells positive for FoxP3+, IL-10+, and TGF-β+ was elevated in skin lesions of LL patients in comparison to BT patients. PBMC/ Cell culture with sonicated M. leprae antigen.

- PBMC: CD3, CD4, CD8, CD25, FoxP3.

- Intracytoplasmic cytokine: TGF-β.

- Cytokines in cell culture supernatant: TGF-β, IL-10.

The frequency of CD3+CD8+CD25+FoxP3+ was lower than CD3+CD4+CD25+FoxP3+ Treg cells in both forms of the disease and healthy individuals, and significantly higher in LL patients than in BT patients. Moreover, a heightened frequency of CD3+CD4+CD25+FoxP3+ Treg cells producing TGF-β after in vitro stimulation was observed in LL patients. The analysis of cytokines in the supernatant also revealed a significant increase in IL-10 and TGF-β in LL patients, in contrast to BT patients. qPCR for gene expression in PBMC and skin lesions. Gene expression in PBMC and skin lesions: FoxP3, TGF-b, IL-10. An upregulation in the gene expression of FoxP3, TGF-β, and IL-10 was observed in both PBMC and skin lesions of LL patients, in comparison to BT patients, healthy contacts, or the normal skin of individuals undergoing cosmetic surgery. *Kumar et al. (2014)12 India BT/TT (25), BL/LL (25), and healthy controls (10). PBMC/ Cell culture. PBMC: CD4, CD25, FoxP3, Ki-67+. The frequency of CD4+CD25+FoxP3+ Treg cells was higher in BL/LL patients compared to BT/TT and healthy controls. Additionally, a considerably higher number of Ki-67+CD4+CD25+ cells were observed in BL/LL patients compared to healthy controls and BT/TT. PCR for gene expression in PBMC. Gene expression in PBMC: STAT-5 No significant differences were observed in pSTAT5 expression in BL/LL patients compared to BT/TT or healthy controls. Immunoblot FoxP3 Immunoblot analysis confirmed the overexpression of FoxP3. †Bobosha et al. (2014)15 Ethiopia, Nepal, and Netherlands BT/TT (16), LL (40), endemic healthy controls (5), and non-endemic healthy controls (13). Skin lesions/Immunohistochemistry. In situ: FoxP3, CD68, CD163, CD39. The expression of M2 macrophages (CD68+CD163+) and FoxP3+ was higher in LL patients compared to BT/TT. Additionally, the expression of CD39+FoxP3+‡ was observed in only two out of ten LL patients. PBMC/ Cell culture with WCL.

- PBMC: CD3, CD4, CD8, CD25, FoxP3.

- Intracytoplasmic cytokine: IL-10.

The frequency of CD3+CD8+CD25+FoxP3+ Treg cells was significantly higher in LL patients compared to BT/TT. Likewise, there was an increase in the frequency of CD3+CD4+CD25+FoxP3+ Treg cells in LL patients; however, no significant difference was observed. There was no difference in the production of intracytoplasmic IL-10+CD4+FoxP3+ among the LL, BT/TT, and control groups. Parente et al. (2015)14 Brazil IL (9), TT (13), BT (26), BB (3), BL (8), LL (27), RR (8), ENL (2). Skin lesions/Immunohistochemistry. In situ: FoxP3. All samples indicated the presence of FoxP3+ positive cells, with no significant differences in the expression of this marker among different clinical forms. In BT/TT patients, FoxP3+ expression was observed within and around granulomas. Conversely, in BL/LL patients, a higher expression of FoxP3+ was noted in the diffuse infiltrate of macrophages, displaying a random distribution. Sadhu et al. (2016)6 India BT/TT (20), BL/LL (20), and healthy controls (10). PBMC/ Cell culture with WCL.

- PBMC: CD3, CD4, CD25, CCR4, FoxP3, PD-1/PDL-1

- Intracytoplasmic cytokines: IL-10.

The frequency of CD3+CD4+CD25+FoxP3+ Treg cells was five times higher in BL/LL patients compared to BT/TT and healthy controls. Similarly, there was an increase in the expression of CCR4+CD4+FoxP3+ and intracytoplasmic IL-10+CD4+FoxP3+ production in BL/LL patients. Furthermore, CD3+CD4+CD25+FoxP3+ Treg cells and monocytes from BL/LL patients expressed a higher frequency of PD-1 molecules and its ligand PD-L1, respectively. Yang et al. (2016)9 China TT (12), LL (13), and healthy controls (6). Skin lesions/Immunohistochemistry. In situ: IL-10, MHCII (HLA-DR+HLA-DQ). The analysis revealed higher expression of IL-10 with low expression of MHC class II in LL patients. PBMC/ Macrophage infection with live and dead M. leprae (Thai-53 strain)

- PBMC: FoxP3, CD163, MHCII (HLA-DR+HLA-DQ)

- Cytokines in cell culture supernatant: IL-10.

BL/LL patients exhibited significantly elevated levels of IL-10. Moreover, macrophages from both healthy individuals and those infected with M. leprae were associated with increased production of IL-10 and CD163 expression in culture supernatant after infection. Subsequently, phenotypic characteriation of IL-10-producing cells demonstrated heightened levels of FoxP3 expression. Lima et al. (2017)19 Brazil TT (12), LL (12), and healthy controls ​​(12). PBMC/Allogeneic PBMC proliferation inhibition assay

- PBMC: CD4, CD25, FoxP3, PD-1, CTLA-4

- Cytokines in cell culture supernatant:

IL-10 e TGF-β.

There was a significant increase in the expression of PD-1 and CTLA-4 in CD4+CD25+FoxP3+ Treg cells from LL patients compared to the TT and control groups. No significant differences were observed in the production of IL-10 or TGF-β. Additionally, it was noted that CD4+CD25+ T cells obtained from LL patients suppressed allogeneic proliferation in functional tests. ‡Negera et al. (2017)18 Ethiopia LL (31) and ENL (46). PBMC/ Cell culture. PBMC: CD3, CD4, CD8, CD25, FoxP3, CD127. A higher frequency of CD3+CD4+CD25+FoxP3+CD127-/lo Treg cells was observed in LL patients compared to the ENL group. On the other hand, there were no differences in the frequencies of CD3+CD8+CD25+FoxP3+CD127-/lo Treg cells between LL and ENL. LL patients also exhibited a lower ratio of CD4/CD8 cells compared to ENL. §Oliveira et al. (2021)17 Brazil Healthy blood donors (number of donors not specified). PBMC/mDCs infection with ML, MLMA, or MLSA.

- PBMC: CD4, CD25, FoxP3, IDO-1.

- Cytokines in cell culture supernatant:

IL-10.

There was a significant increase in the frequency of CD4+CD25+FoxP3+ cells after coculture with MLSA- or ML-stimulated mDCs. Additionally, MLSA stimulation induced an increase in IL-10 levels. Furthermore, IDO-1 inhibition significantly reduced Treg cell differentiation. Tarique et al. (2023)11 India BT/TT (20), BL/LL (20), and healthy controls (10). PBMC/ Cell culture with sonicated M. leprae.

- PBMC: CD3, CD4, CD25, FoxP3, PD-1.

- Intracytoplasmic cytokine: IL-10.

The frequency of CD4+CD25+FoxP3+ Treg cells and those expressing PD-1 was significantly higher in patients with BL/LL and BT/TT patients compared to healthy controls. Despite BL/LL patients exhibiting a higher frequency of these markers, no difference was observed between BL/LL and BT/TT patients. Furthermore, PD-1+ Tregs produced lower amounts of IL-10 in all patient groups as well as in healthy controls. A significant additional increase in suppression by PD-1+ Treg cells was observed when the anti-PD-1 monoclonal antibody was added to the cell culture in both groups of leprosy patients. Additionally, the overexpression of PD-1 positively correlated with disease severity and the bacilloscopy index among leprosy patients. PCR for gene expression in PBMC. Gene expression in PBMC: PD-1 PD-1 gene mRNA expression in PBMCs was higher in both BL/LL and BT/TT patients compared to healthy contacts, although BL/LL patients exhibited a particularly higher expression of PD-1. Analysis of gene expression in PBMC and skin lesions

Three studies employed the real-time polymerase chain reaction (RT-qPCR) technique to assess mRNA expression in PBMC or skin lesion samples from patients with leprosy.11–13 A high expression was observed for FoxP3, TGF-b, and IL-10 in PBMC and skin tissues.13 Furthermore, in studies exclusively concentrating on PBMC from individuals with BT/TT and BL/LL forms, Tarique et al.11 observed heightened expressions of PD-1, whereas Kumar et al.12 did not demonstrate significant differences in STAT5 expression, a transcription factor that regulates Treg cell expression.

Expression of Treg cells, cytokines, and other molecules in situ

Five studies conducted immunohistochemical analysis on skin lesion biopsies from leprosy patients classified according to Ridley & Jopling. Among these studies, Parente et al. recorded the highest number of patients (n=96) undergoing biopsy.14 The expression of Treg cells was assessed by immunohistochemistry using the markers FoxP3+ or CD25+FoxP3+.13–16 Other co-inhibitory molecules (CTLA-4, PD-1), cytokines (IL-10, TGF-β), and additional markers (CD68, CD163, CD39, MHCII) were also analysed.9,13,15,16

The studies revealed that patients exhibit Treg cells in skin lesions, but with occasional differences in expression and location. Patients affected by the BL/LL forms show a significantly larger infiltrate of Treg cells, which was diffuse in its distribution.9,13,16,17 Similarly, a higher expression of CTLA-4 and IL-10 was also observed in BL/LL patients.9,13,16 On the other hand, the expression of TGF-β was only evident in the study by Saini, Ramesh, and Nath,13 while in the study by Palermo et al.,16 no significant difference was observed between patients with the TT/BT and BL/LL forms. In TT/BT patients, the expression of Treg cells is lower, present within and around granulomas.9,13,14,16

Furthermore, the study by Bobosha et al.15 showed that the expression of FoxP3+ in skin lesions is likely induced by the presence of M2 macrophages (CD68+CD163+), as evidenced by the closer proximity of FoxP3+ cells in LL lesions compared to TT/BB lesions.

Treg cells, cytokines, and other molecules in PBMC culture after in vitro stimulation

Out of the 12 included studies, 10 included in vitro cell cultures with PBMC to identify and evaluate the functional characteristics of Treg cells. For this purpose, blood samples were collected from leprosy patients, household contacts, and healthy controls from endemic and/or non-endemic regions. PBMC were isolated and cultured in vitro with culture medium (negative control), phytohemagglutinin (PHA; positive control), and antigens derived from M. leprae at 37°C, with an incubation period ranging from 18 to 144 hours across studies. Despite this, Kumar et al.12 and Negera et al.18 did not utilise any antigens derived from M. leprae. Subsequently, cell populations were immunophenotyped by flow cytometry, and other studies quantified cytokines in the cell culture supernatant by enzyme-linked immunosorbent assay (ELISA) or flow cytometry.6,9,11,13,15–17,19

Regardless of the stimulus used, PBMC from patients with MB or BL/LL present a higher frequency of CD4+CD25+FoxP3+ cells compared to those with TT/BT. This difference is also observed when comparing patients with BL/LL in type II leprosy reaction or erythema nodosum leprosum (ENL).6,11–13,16–18,20 In turn, Negera et al. demonstrated that during and after treatment, there is a decrease in CD4+CD25+FoxP3+ cells in patients with LL, while patients with LL and ENL show an increase in these cells.18

Furthermore, Treg cells were associated with increased intracytoplasmic production of TGF-β in the study conducted by Saini, Ramesh, and Nath, as well as IL-10 according to Sadhu et al. and Bobosha et al., on the other hand, demonstrated that there were no differences in intracytoplasmic IL-10 production among the leprosy groups (LL, BL/TT) and controls.6,13,15 Saini, Ramesh, and Nath also reported similar results, showing little to no production of TGF-β.13 Interestingly, Tarique et al. observed that increased PD-1 expression in Treg in patients with leprosy was associated with a reduction in IL-10 production, regardless of the clinical form of leprosy.11

The frequency of CD3+CD8+CD25+FoxP3+ Treg cells was also assessed in some studies.13,15,18,20 While these cells are more frequent in LL patients when compared to those with TT/BT or ENL, they are less abundant than CD3+CD4+CD25+FoxP3+ Treg cells. This observation suggests a predominant role of CD3+CD4+CD25+FoxP3+ Treg cells in the immunoregulation of MB patients. Negera et al.18 also demonstrated an increase in CD3+CD8+CD25+FoxP3+ Treg cells, following prednisone treatment in ENL patients, contrasting with a reduction in these cells observed in the LL group. These findings highlight distinct roles for the two subsets, with CD3+CD4+CD25+FoxP3+ cells likely playing a central regulatory role, while CD3+CD8+CD25+FoxP3+ cells may have a context-dependent function, particularly in response to therapeutic interventions.

In addition to the PBMC cell culture methodology, infection models and co-cultures were also performed.9,17,19 Monocyte-derived macrophages isolated from healthy volunteers, when infected with viable M. leprae (Thai-53 strain), exhibited elevated levels of IL-10. Phenotypic characterisation of the IL-10-producing cells showed high levels of FoxP3 expression. Oliveira et al.,17 demonstrated an increased frequency of CD4+CD25+FoxP3+ cells in co-culture assays using monocyte-derived dendritic cells (mDCs) and lymphocytes after stimulation with M. leprae soluble antigen (MLSA) and the cell membrane fraction antigen of M. leprae (MLMA). The study by Lima et al. showed that CD4+CD25+ T-cells obtained from LL patients had a greater capacity to inhibit the proliferation of allogeneic PBMC when co-cultured, compared to the same cells from TT patients.19 Moreover, similar to in situ assays, Sadhu et al. demonstrated that BL/LL patients exhibit higher expression of PD-1 and PD-L1 on Treg (CD4+FoxP3+) and antigen-presenting cells (APC), respectively, after in vitro stimulation with whole-cell lysate antigen (WCL).6Figure 2 illustrates a schematic diagram that highlights the role of Treg cells in Hansen’s disease, providing a visual representation of their involvement in the immune response within the context of the disease.

Figure 2: Treg cells in Hansen’s disease: (a) Visual representation of their role in the immune response; (b) Characteristics of Treg across the clinical forms of the disease.

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Discussion

Homeostasis is maintained through a balanced immune response, and Treg cells play a central role in preventing tissue damage. Conversely, in chronic infections, the induction of exhaustion mechanisms and inhibition of the immune response by Treg cells can favor the persistence of the disease. The mechanisms of immunoregulation by Treg cells have been demonstrated in several infectious and autoimmune diseases such as tuberculosis, leishmaniasis, hepatitis B, systemic lupus erythematosus, rheumatoid arthritis, and Kawasaki disease.21–26

In leprosy, the initial findings were published by Nath et al.27 Since then, numerous studies have been conducted to better characterise subpopulations of Treg cells and understand their role in disease development. Although Treg cells are present in all clinical forms of leprosy, a higher frequency is observed in MB patients, particularly those with the BL or LL forms. Otherwise, PB patients with the BT or TT forms and/or healthy controls exhibit a lower frequency of Treg cells after in vitro stimulation with different antigens of M. leprae. It is inferred that the higher concentration of Treg in peripheral blood is related to the presence of these cells in lesions, which may be associated with the clinical manifestation of the disease. Curiously, the increased expression of CCR4, a chemokine receptor related to the recruitment of Treg cells, and the cytokines IL-10 and TGF-b, both intracytoplasmic and in the culture supernatant, strongly suggest an active role of these cells in the infectious process.

In all studies that analysed IL-10 production, except for Bobosha et al.,15 it was possible to identify elevated levels of this cytokine concomitant with a higher frequency of Treg cells, suggesting that these cells represent an important source of IL-10. The production of IL-10 by Treg cells, in addition to inhibiting and/or reducing the synthesis of pro-inflammatory cytokines by Th1 cells (IL-2, IL-12, IFN-γ, and TNF), also promotes a reduction in IL-17 by Th17 cells.6,9,13 It was reported that in vitro inhibition of TGF-b and IL-10 promoted a reactivation of the Th17-mediated immune response in BL/LL patients, and decreased suppressive function of Treg cells, through lower expression of Foxp3.6 Thus, it is plausible to consider that the high frequency of Treg cells and cytokines, especially IL-10, act by suppressing effector Th1 and Th17 cells, which are associated with the milder clinical form of the disease (TT) and reactive episodes. Additionally, these cells participate in granuloma formation, limit the growth and multiplication of bacilli, and contribute to the acute phase in reactive states of leprosy.28

The production of IL-10 seems to modulate the shift of macrophages from M1(inflammatory) to M2 (anti-inflammatory).9 Recently, it was also demonstrated that anti-inflammatory macrophages (CD163+) exert a suppressive effect on Th1 responses and that these cells can be found in the vicinity of FoxP3+ lesions from LL patients, compared to lesions from BT/TT patients.17 In this review, three of the eight studies that assessed IL-10 production showed the identification of the cellular origin of IL-10.6,11,15 Then, considering the Th2 profile in MB forms,5 IL-10 might be secreted by several subpopulations of CD4+ T and innate cells.

Interestingly, naïve T-lymphocytes (CD45RA+) co-cultivated in the presence of macrophages infected with viable or heat-inactivated M. leprae produced significant levels of IL-10 and IFN-g, respectively. These results suggest that the presence of viable bacilli is a necessary condition for the differentiation of naïve T lymphocytes into Treg cells (FoxP3+) producing IL-10.9 Treg cells were also observed when PBMC were cultured with other infectious agents such as live M. bovis BCG, suggesting that the stimulation of Treg cells may play a crucial role in regulating the immune response in infections, including those caused by M. bovis BCG.29,30 The MLSA also induced a significant increase in the concentration of IL-10 when compared to other stimuli.17 Taken together, it is plausible to consider that local stimuli, cytokine gradients in the inflammatory microenvironment, antigenic nature, expression of receptors and co-stimulatory or co-inhibitory molecules, as well as types of macrophages, contribute to the evolution and severity of leprosy.

Considering the limitations of in vitro models, despite their advantage for understanding Treg cell behaviour, it is recommended to perform in situ analyses of lesions microenvironment in skin biopsies. Consistent with the results from cell culture, lesions of patients with LL exhibit a higher infiltrate of Treg cells (FoxP3+) than those with the TT form. In contrast, the presence of Treg cells in TT form was rare, and when present, they were found within granulomas.13–16 As observed in studies involving peripheral blood culture, the expression of IL-10, but not TGF-b, was significantly higher in lesions of patients with LL.16 Similarly, Yang et al. showed higher expression of IL-10 with low expression of MHC class II in biopsy samples from patients with LL.9 Nonetheless, it was demonstrated that patients with the LL form exhibited higher expression of TGF-b and IL-10 in skin lesions, which was confirmed through gene expression analysis using the RT-qPCR. PBMC stimulated with heat-inactivated M. leprae sonicated antigens showed increased levels of IL-10 and TGF-b, along with higher production of these cytokines in the supernatant.13

Although TGF-b is an immunoregulatory cytokine, divergent results have been observed in studies assessing its in situ expression, raising questions about the origin and role of this cytokine in leprosy. The reduced and/or absence of TGF-b in studies may indicate a subset of induced Treg cells known as type 1 regulatory T cells (Tr1) at the infection site, characterised by high IL-10 production and low TGF-b production.31

In conclusion, the results suggest that, unlike TGF-b, IL-10 is the most studied cytokine directly related to Treg, regulating the immune response and thus making the individual more susceptible to M. leprae infection. Although some results are contradictory regarding TGF-b production, more studies are needed, as only four studies have evaluated the in vitro and/or in situ production of this cytokine. Contrarily, none of the studies assessed IL-35 in skin lesions or blood, an anti-inflammatory cytokine discovered in 2007 and produced by a wide range of Treg cells.7 As IL-35 can suppress T0cell proliferation and the functions of effector Th1 and Th17 cells, its role in various clinical manifestations of leprosy should be explored in future studies.32

Another effector mechanism of Treg, besides cytokine release, involves the expression of co-inhibitory molecules, as CTLA-4 or PD-1, that control T-cell effector functions, reducing cytokine production and/or cell proliferation.7 CTLA-4 is a membrane protein that competes with CD28 (a positive co-stimulator) for binding to B7-1, B7-2 receptors on APC, induces an immunosuppressive environment, and is therefore linked to the inactivation of subsets of effector and memory CD4+ T-cells.33 Furthermore, Tarique et al.11 demonstrated that PD-1+ Treg from leprosy patients produced less IL-10 compared to PD-1neg Treg. This finding underscores the intricate and diverse nature of immune regulatory mechanisms, highlighting notable variations in cellular responses.

The presence of co-inhibitory molecules in situ and in vivo demonstrates that Treg participate through different mechanisms, and the inhibition of these molecules allows the restoration of IFN-g and IL-17.6 These cytokines belong to the Th1 and Th17 profiles, respectively, and contribute significantly to enhanced control of bacterial multiplication.3,28 In summary, the presence of co-inhibitory molecules creates an environment of cellular inactivation in the host, favouring the survival and multiplication of bacilli, as observed in patients with the LL form of the disease.

The vast majority of identified Treg cells guiding the discussion in the study were phenotypically characterised as CD3+CD4+CD25+FoxP3+. However, a small population of CD3+CD8+CD25+FoxP3+ cells with suppressor activity has emerged, presenting significant implications in autoimmune diseases and the inflammatory process.13,15 Data presented in some studies have shown that the frequency of this CD8+ subpopulation with suppressor activity is lower than that of CD4+ cells.13,15,18,20 Nevertheless, similar to CD4+ cells, this subpopulation is elevated in BL/LL patients compared to BT/TT and ENL. Despite being less numerous, these cells should be considered in the regulation of the immune response, and therefore, studies evaluating the viability and function of these cells are necessary.

Finally, it is evident that Treg cells play a crucial role in the immunopathogenesis of leprosy, particularly in shaping the immune response across the clinical spectrum of the disease. The higher frequency and expression of CD3+CD4+CD25+FoxP3+ Treg cells in multibacillary forms (BL/LL) compared to paucibacillary forms (TT/BT) suggest their involvement in modulating immune tolerance and enabling bacterial persistence. Despite these findings, the heterogeneity in study designs, patient populations, and methodologies underscores the need for standardised approaches in future research. It is important that future studies focus on longitudinal analyses of Treg cell dynamics before and after treatment, as well as their interactions with specific cytokines and other immune cells. Identifying patterns of immune responses regulated by Treg cells may offer a promising strategy for expanding the therapeutic arsenal against leprosy. This includes the potential development of immunosuppressive agents targeting inhibitory molecules secreted by these cells, such as CTLA-4, to modulate cellula