Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare, severe condition that predominantly affects children and adolescents (<18 years), typically occurring 2 to 6 weeks after the acute infection of Severe Acute Respiratory Syndrome COronaVirus 2 (SARS-CoV-2) (1, 2).

Notably, the age group most frequently affected by this syndrome is children aged 5–13 years, although some data show an even wider range (from 1.6 up to 20 years) (1–5).

MIS-C clinically presents with a multi-system involvement, featuring persistent fever, gastrointestinal, mucocutaneous, cardiovascular (e.g., hypotension, shock), and neurological symptoms, along with potential mild respiratory involvement and a diffuse maculopapular rash (1–5). Diagnosis is essentially based on clinical criteria, elevated inflammatory markers, multisystem involvement, and evidence of recent SARS-CoV-2 exposure (6).

Treatment aims to control inflammation, support organ function, and manage complications with immunomodulatory therapies (e.g., intravenous immunoglobulin, corticosteroids, biologics), anticoagulation for thrombotic risks, and supportive care (7–9). The prognosis for children with MIS-C is generally favorable, with the majority achieving complete recovery following timely therapeutic intervention. Nevertheless, a subset of patients experiencing severe disease may develop long-term cardiac complications (7–9). The overall mortality associated with MIS-C remains below 2% (1–5, 7–9).

The pathophysiology of MIS-C is incompletely understood, but it is believed to result from an exaggerated immune response to the SARS-CoV-2, characterized by a hyperinflammatory state and dysregulated immune response marked by elevated cytokines [such as Interleukin (IL)-6, IL-1β, Interferon (INF)-γ and Tumor Necrosis Factor (TNF)-α], inflammatory markers (such as CRP and ferritin) alongside an overactivation of the innate and adaptive immune systems (10–14).

Studies have revealed significant activation of T cells, as well as the presence of autoantibodies, suggesting an autoimmune-like component (10–13). The T cells that remained often exhibited signs of heightened activation, evidenced by increased expression of Human Leukocyte Antigen (HLA)-DR (15–17).

In light of the evidence of immune hyperactivation in MIS-C, it is of particular interest to investigate potential alterations in immune regulation associated with the syndrome.

Immune regulation achieves a balanced response through a complex network of cells and molecules that distinguish self from non-self (18).

Crucial to immune regulation, FoxP3⁺ regulatory T cells (Tregs) maintain immune homeostasis by controlling responses, preventing autoimmunity, and limiting overreactions to infections (19–21). Dysfunctional Tregs disrupt this balance, potentially causing autoimmune diseases through excessive immune activity or weakened immunity through over-suppression (19–27).

Within the framework of immune regulation, we previously identified a novel potential regulatory cell population, CD3⁺ CD56⁺ T cells (T_R3-56) (28).

In type 1 diabetes (T1D), lower T_R3–56 cell frequencies correlate with increased cytotoxic CD8⁺ T lymphocyte (CTL) activation, worse disease progression, impaired β-cell function, and diabetic ketoacidosis (28). T_R3–56 suppress CTL activity via reactive oxygen species reduction, but this mechanism is impaired in T1D, highlighting their role as key CTL regulators and potential T1D self-tolerance biomarkers (28). Similarly, T_R3–56 cells negatively correlate with CTLs in myelodysplastic syndromes (MDS) (29, 30) and may contribute to immune escape in chronic lymphocytic leukemia (CLL) (31). In COVID-19, T_R3–56 cells exhibit both regulatory and effector functions, modulating inflammation, potentially limiting tissue damage, and promoting immune balance (32).

An intriguing aspect of immune responses and immune regulation is the difference between male and female individuals (33). In this regard, sex-based variations in immune function are influenced by both sex hormones and the presence of two X chromosomes in females (33–36). Compared to males, females typically exhibit stronger innate and adaptive immune responses (33–36). Conversely, males tend to have higher infection-related mortality (33–36).

It is of some relevance that MIS-C exhibits gender-based differences, with male children showing a higher incidence and more severe outcomes than females (1–5, 7–9, 37). Males are at higher risk of developing MIS-C after SARS-CoV-2 infection and typically exhibit more severe symptoms, whereas females are less frequently affected and tend to present with milder clinical manifestations (37–39). In the context of previous coronavirus epidemics, such as Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome-Coronavirus (MERS-CoV), studies have shown that sex differences affect disease severity and clinical outcomes (37–39). The underlying mechanisms driving these gender differences are not fully understood but are thought to involve a combination of hormonal, immune, and genetic factors that differ between males and females (33–39).

In this scenario, we previously identified that MIS-C patients exhibit elevated levels of key cytokines, including IFN-γ, IL-6, IL-10, and TNF-α (12). Furthermore, dysregulation was observed in lymphocyte subpopulations, including NK cells, B cells, T cells, Th1, and Th17 cells, but no significant differences in Treg populations between MIS-C patients and healthy controls.

The objective of this current investigation was to elucidate the immunological mechanisms contributing to MIS-C, with a specific focus on immune cell dysregulation and the involvement of T_R3–56 cells in its pathogenesis. A detailed analysis of the immunological characteristics of our previously characterized MIS-C patient cohort (12) was conducted. The primary aims were to assess the frequency of T_R3–56 lymphocytes and their potential role in immune regulation in the context of MIS-C. Additionally, sex-based differences in immune profiles between male and female MIS-C patients were explored.

Our study aims to provide a deeper understanding of the immune mechanisms underlying MIS-C (1–5, 7–9, 11, 37), with a specific focus on immune cell dysregulation.

In this regard, we investigated the immunological characteristics of untreated MIS-C patients, with particular emphasis on the percentage of T_R3–56 lymphocytes and their potential role in immune regulation within the context of MIS-C.

Our previous analysis identified significant immunological differences between MIS-C patients and healthy controls, including alterations in both pro-inflammatory and anti-inflammatory cytokines (IFN-γ, IL-6, IL-10, and TNF-α) and modifications in lymphocyte, monocyte, and granulocyte levels, along with changes in key innate and specific lymphocyte subpopulations highlighting a distinct immune dysregulation compared to healthy individuals (12).

We recently identified the T_R3–56 cells as a novel regulatory T cell population in several immune-mediated diseases (28–32).

Here, we first highlighted that there are no statistically significant differences in the percentages of T_R3–56 lymphocytes and Treg (19–27) cells between MIS-C patients and healthy controls. This evidence suggests that immune dysregulation in MIS-C may not be driven by changes in the abundance of these regulatory cells but rather by their functional roles in the interactions within the immune network.

In our observations, a positive correlation indicates that as one variable increases, the other also increases (or both decrease together), whereas a negative correlation implies that as one variable increases, the other decreases. Understanding these patterns is essential for interpreting the observed relationships between immune cell subsets and clinical parameters in MIS-C, as they may reflect coordinated or opposing biological processes.

In this regard, we observed a positive correlation between T_R3–56 and Tregs in MIS-C patients, absent in controls. Such a noteworthy finding suggests a potential immunoregulatory function for T_R3–56 in the disease context, which could indicate either a compensatory mechanism to counteract inflammation or a hallmark of immune dysregulation specific to MIS-C. This insight may potentially contribute to a better understanding of immune alterations in MIS-C and could have implications for future diagnostic and therapeutic strategies targeting immune regulation.

Moreover, our findings reveal distinct correlation patterns of T_R3–56 cells with various immunological markers in MIS-C patients, providing novel insights into their potential role in immune modulation. These results highlight the importance of investigating functional dynamics beyond mere quantitative assessments to gain a deeper understanding of immune alterations in MIS-C.

In this regard, T_R3–56 lymphocytes show negative correlations in MIS-C patients with CD45RA⁺ naïve T cells, which are crucial for immune responses and differentiation (44). Conversely, T_R3–56 levels are positively correlated with CD45RO⁺ memory T cells (44).

These results suggest that T_R3–56 cells may influence early immune activation and naïve T lymphocyte balance in MIS-C, linking them to antigen-primed T cells and long-term immunity (44).

T_R3–56 also correlate with the activated Th1 cells (45) in MIS-C patients. This evidence suggests that T_R3–56 cells may be involved in balancing immune responses, as their correlation with Tregs implies a potential role in immune tolerance and suppression of excessive activation, while their association with activated Th1 cells points to a contribution in pro-inflammatory responses.

This dual interaction may indicate a regulatory role in maintaining immune homeostasis, with T_R3–56 cells potentially contributing to the fine-tuning of the balance between immune activation and suppression. Understanding this dynamic could provide insights into their role in immune-related diseases and inflammatory disorders.

This study aimed also to identify sex-based differences in MIS-C by analyzing immune activation and regulation in male and female patients.

Our findings confirm a male predominance in MIS-C, consistent with previous studies (1–5, 7–9, 11, 33–39). Moreover, disease severity was more frequently observed among males, who were more likely to present with moderate to severe clinical manifestations compared to females. However, our analysis indicates that these sex-related differences are not attributable to distinct severity patterns between males and females. Rather, the observed disparity is primarily explained by a higher overall incidence of MIS-C in males, resulting in a greater absolute number of male patients with severe presentations. Crucially, when disease severity was evaluated within each sex, no significant differences emerged in terms of clinical course or extent of organ involvement.

These findings suggest that male sex confers a greater susceptibility to developing MIS-C, but does not inherently predispose to a more severe disease phenotype. Collectively, our results support the notion of a sex-biased vulnerability to MIS-C, rather than a sex-specific variation in disease progression or severity.

Notably, male MIS-C patients exhibit significantly higher percentages of T_R3–56 cells than females Interestingly, a positive correlation between T_R3–56 and Tregs is observed in males but not in females. These findings suggest a potential sex-based difference in the immune response to MIS-C, with T_R3–56 cells playing a more prominent role in males and may indicate a sex-specific immunoregulatory mechanism that warrants further investigation.

Male MIS-C patients show higher percentages of HLA-DR⁺ activated T (15–17, 45), and CD45RO⁺ memory T cells. The higher memory T cell levels in males correspond to a greater presence of CD45RA⁺ naïve T cells in females, highlighting potential sex-based differences in immune dynamics within MIS-C.

These findings suggest that males may have a more activated immune profile, whereas females might maintain a larger naïve T cell pool, potentially influencing disease progression and response to treatment.

We observed also a higher TNF-α production in male than in female MIS-C patients. Since such cytokine is a key mediator of inflammation (48), its increased levels in males could be associated with heightened immune activation, tissue damage, or a more severe clinical course.

These findings align with the observed greater activation of T cell subsets in males (15–17, 45), reinforcing the hypothesis that sex-related immune mechanisms influence MIS-C pathophysiology.

Sex-specific correlations suggest distinct immunoregulatory roles of T_R3–56 cells. In males, their negative association with CD45RA⁺ naïve T cells and positive correlations with Tregs and activated Th1 cells point to involvement in immune differentiation and modulation of Th1-driven responses. In females, the negative correlation with naïve T cells and positive association with memory T cells imply a shift toward immune memory. Moreover, T_R3–56 cells in males appear to contribute to balancing activation and regulation within a pro-inflammatory milieu, while in females, Treg correlations indicate stronger homeostatic control, potentially influencing immune adaptation and disease course in a sex-dependent manner. These data highlight sex-related variations in immune adaptation, which could influence disease progression and immune responses in MIS-C.

Moreover, we examined age-related differences in both male and female children to assess their potential impact on immune responses in MIS-C disease.

MIS-C patients were divided into two age groups: younger (Group 1) and older (Group 2) children, with males outnumbering females in both groups. In both age groups, a higher proportion of children exhibited moderate to severe clinical conditions, with males being more frequently affected than females. This trend was particularly evident in younger patients.

Group 1 patients exhibited a greater percentage of CD8+ T effector cells than Group 2. Such occurrence may point to a vigorous immune reaction, potentially utilizing T cell cytotoxicity to eradicate infected cells. This dual-edged adaptive response, while potentially beneficial for pathogen elimination and moderating inflammation, could also contribute to the tissue pathology described in MIS-C. The observed increment in CD45RA⁺ naïve T cells could be a sign of their engagement and activation triggered by SARS-CoV-2 infection or the consequent inflammatory environment in Group 1 patients.

Significant sex-based differences in lymphocyte populations were observed in Group 1 patients. Specifically, males exhibited a higher percentage of T, CD8⁺ T, T_R3-56, HLA-DR₊ activated T, and CD45RO⁺ memory T lymphocytes, and a lower percentage of B and CD45RA⁺ naïve T lymphocytes when compared to females. These results suggest that males show a T cell-dominant, activated/memory profile, while females have relatively higher B cell and naïve T cell percentages.

The consistently higher TNF-α production in males across both Group 1 and 2 indicates a potentially stronger inflammatory response in males than in females, contributing to the systemic inflammation characteristic of MIS-C.

The elevated percentages of HLA-DR⁺ activated T and CD45RO⁺ memory T cells, along with a decreased percentage of CD45RA⁺ naïve T cells in males of both MIS-C groups, suggest a more pronounced adaptive immune response, possibly reflecting prior antigen exposure or the development of robust immune memory.

The increase in T_R3–56 cells in Group 1 and Treg cells in Group 2 suggests that males may be differentially regulating their immune responses compared to females, likely maintaining a distinct balance between immune activation and control.

In addition, males with greater disease severity consistently appeared to present a trend with higher T_R3–56 cell percentages than females. This observation suggests a potential sex-related difference in immune response, warranting further investigation into the role of T_R3–56 cells in MIS-C pathophysiology.

The higher percentages of CD8⁺ T cells and HLA-DR⁺ activated T cells in males, particularly in younger MIS-C patients (Group 1), suggest a more robust cytotoxic immune response, possibly driven by a heightened immune activation or a response to SARS-CoV-2 infection. The increased percentage of CD45RO⁺ memory T cells in younger males indicates that male MIS-C patients may exhibit a more pronounced adaptive immune response than in females, potentially due to earlier antigen exposure or stronger immune memory development. A decrease in B cells suggests a shift toward a more T-cell-mediated immune response in younger males compared to females in Group 1.

Finally, we assessed whether differences existed in the correlations between T_R3–56 cells with cytokines and with the main effector cells of the adaptive immune response in male and female from Groups 1 and 2.

In males from Group 1, T_R3–56 cells show a positive correlation with CD8⁺ T cells, memory T cells, activated Th1 lymphocytes, and Tregs, indicating a potential link to an activated and sustained memory T cell response, along with a regulatory immune profile. Conversely, T_R3–56 cells negatively correlate with CD45RA⁺ naive T cells, B cells, and the CD4/CD8 T cell ratio, suggesting a shift away from these immune cell effectors.

In the Group 2, T_R3–56 cells correlate positively with inflammatory markers such as IFN-γ, TNF-α, Th1, and activated Th1 lymphocytes, indicating an association with a pro-inflammatory, Th1-polarized immune response (45, 48).

In females from Group 1, T_R3–56 cells positively correlate with IFN-γ, memory T cells, and Th17 lymphocytes (45, 48), while negatively correlating with B cells and Th1 lymphocytes. This evidence suggests that T_R3–56 cells play a role in balancing inflammation, shaping memory, and modulating Th17 responses to support immune tolerance, while suppressing B and Th1 cell activity.

In females from Group 2, T_R3–56 cells correlate positively with cytotoxic CD8+ T cells, memory T cells, and Th1 lymphocytes, suggesting the persistence of antigen-primed T cells.

However, T_R3–56 cells negatively correlate with naive T cells, Tregs, and the CD4/CD8 T cell ratio, which may indicate a more differentiated, antigen-experienced immune phenotype with reduced immunosuppressive activity. Additionally, negative correlations with inflammatory cytokines such as TNF-α, IL-6, IL-10, and IL-17 (48) suggest that T_R3–56 cells may contribute to a more controlled immune environment by limiting excessive inflammatory signaling.

These findings highlight the complex role of T_R3–56 cells in shaping immune responses, with distinct patterns observed between sexes and age groups.

In males, T_R3–56 cells appear to be actively involved in both immune activation and regulation. Specifically, these cells interact with Tregs and activated Th1 cells, which may contribute to a heightened inflammatory response as well as immune regulation. This dual role could be critical in modulating the immune system during MIS-C, potentially influencing the severity and progression of the disease.

In contrast, in females, T_R3–56 cells seem to primarily support immune memory and regulatory responses, with an emphasis on controlling inflammation. These cells may play a more suppressive role, helping to maintain immune tolerance by modulating Th17 responses and limiting excessive activation of inflammatory pathways. This function could be crucial in preventing the excessive immune response that characterizes MIS-C, particularly in the more regulated immune environment observed in females.

Taken in all, our findings underscore the complexity of the immune response in MIS-C, emphasizing the relevance of understanding the differential roles of T_R3–56 cells. The distinct functions of these cells in males and females highlight a potential sex-specific immunological framework that may contribute to differences in disease susceptibility, severity, and progression. Given the observed sex-based disparities in immune profiles, further research into the mechanisms underlying these variations appears to be essential. This could lead to the development of targeted, sex-specific therapeutic strategies that more effectively address the unique immune responses in each group.

Moreover, these results point to the potential for T_R3–56 cells to serve as biomarkers or therapeutic targets in MIS-C, offering new avenues for intervention. Further studies exploring the precise signaling pathways and molecular interactions involving T_R3–56 cells could provide deeper insights into their role in immune dysregulation and disease manifestation. Ultimately, understanding how these cells influence immune responses in MIS-C could pave the way for more tailored, effective treatments that account for both sex-based differences and the unique immune mechanisms involved in this complex inflammatory syndrome.

The study did not assess the functional aspects of the analyzed cells, so the hypotheses drawn from immunophenotypic profiles lack proof of concept. Additionally, due to practical constraints, long-term follow-up of pediatric MIS-C patients was not feasible, limiting the ability to track changes or outcomes over an extended period. These factors should be considered when interpreting the findings of this study.

Moreover, a limitation of our study is the lack of SARS-CoV-2 variant characterization, which restricts the generalizability of our findings and highlights the need for future research accounting for variant-specific effects on MIS-C.

Another limitation of the present study is the arbitrary selection of age 9 as the cut-off for patient stratification. Although this choice is grounded in developmental and immunological hypotheses -recognizing that puberty-related hormonal changes, particularly in females, typically begin around this age and may influence immune function and disease susceptibility - it remains a pragmatic threshold rather than a definitive biological boundary. This arbitrary selection may impact the interpretation of puberty-related sex differences in MIS-C and should be considered when generalizing the findings.

Finally, a limitation of this study is the relatively small size of the control group, which included only 13 pediatric healthy subjects. However, the enrollment of a larger number of healthy children was constrained by the critical emergency situation during the COVID-19 pandemic, which limited access and availability for study participation.